Online newspaper of professor Yasser Metwally

The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

February 25, 2009 — Neuropathic pain associated with diabetic peripheral neuropathy (DPN) is a common debilitating complication of diabetes mellitus with unmet therapeutic needs. Pregabalin is a recently introduced a2-d subunit ligand at the voltage- sensitive calcium channel that has shown good efficacy with a tolerable side-effect profile in the treatment of PDN. According to the Australian datasheet, 1438 patients have been studied receiving either placebo (460) or pregabalin (978). The dose ranged from 75 to 600 mg daily. The number needed to treat averaged from the published data is 3.8 patients for the two higher doses. Pregabalin has a linearly increased plasma concentration with dose escalation and achieves analgesia as early as 1 day after initiating therapy. As its mechanism of action is different from other anticonvulsants and antidepressants, and interactions with other drugs are unlikely from the pharmacokinetic profile, combining pregabalin with other agents that are effective in DPN is a clinically safe option and should result in improved pain control.

Pregabalin (Lyrica®) was licensed in 2004 in the USA and more than 60 countries for use in postherpetic neuralgia and neuropathic pain associated with diabetic peripheral neuropathy. In the early 1990s, inspired from the knowledge gained about the working mechanism of gabapentin in modulating the voltage-sensitive calcium channel, a new stereo-selective a2d-ligand has been developed by Pfizer laboratories (Surrey, UK).[1] Compared with its predecessor gabapentin, pregabalin has a similar safety profile of low drug interaction and renal excretion without hepatic metabolism, but shows improved pharmacokinetic properties such as achieving efficacy in 1 week and a linear plasma concentration with increasing dose, which makes it a good choice for the growing elderly population suffering from diabetic neuropathy (DN). A shorter time span to decide if the drug is effective for a particular patient is a clinical advantage in the treatment of neuropathic pain conditions.

The last sales figures published by Pfizer stated an increase of 52% to a total of US$614 million in the second quarter of 2008 compared with the prior year quarter.[101] In the last annual report, Lyrica was the latest drug to reach the one billion sales barrier and has been used in more than 4 million patients worldwide, mainly for the treatment of neuropathic pain conditions. Pregabalin has been explored as a treatment for fibromyalgia[2] and received US FDA approval in June 2007 for this indication.[102] Other indications include central neuropathic pain in spinal cord injury[3] and perioperative analgesia,[4] and it can be considered as one of the most commercially successful pain medications to date.[103]

• Pathophysiology of Painful Diabetic Neuropathy

Diabetic neuropathy is a common complication of diabetes, which can affect somatic nerves, sensory nerves and nerves of the autonomic system.[104] One consequence of damage to sensory fibers is the lost ability to feel pressure on the skin of the feet. This is the most concerning negative symptom found in patients with DN. The subsequent development of pressure sores with concomitant infection is common.

Weakness in any extremity can develop if motor fibers are affected. Small, unmyelinated fibers and thin myelinated fibers are responsible for the transmission of painful stimuli. In the case of damage to these sensory nerve fibers, the patient can develop pain with an often burning character and intermittent electric shock-like symptoms. On clinical examination, the patient reports pain to nonpainful stimuli, such as touching the skin, known as allodynia. Many patients also experience unpleasant nonpainful abnormal sensations in the affected limbs (dysesthesias). These pathological changes in the periphery with parallel alterations in processing of incoming sensory information at the spinal cord level make up the condition of painful diabetic neuropathy (PDN).[5] A transition between different stages of DN can be observed and the degree of nerve pain can fluctuate accordingly.

There are different theories about the pathophysiology of DN: one theory postulates microvascular disease in the vasa nervorum (the blood supply of a nerve) of peripheral nerves, which leads to malnutrition and lack of oxygen, with subsequent destruction of the axons of the different nerve fibers in a peripheral nerve.[6] This explains the mixed picture of DN and the effects on the different nerve fiber populations. Vascular damage is accelerated by poor glucose control and elevated blood pressure and high cholesterol (particularly triglycerides), which are both very common concomitant symptoms in diabetic patients.[7,8] Another theory postulates direct damage of the nerve fibers by radicals and subsequent cell death in the dorsal root ganglion as the reason for the development of painful DN.[9-11] The third theory for the pathophysiological mechanism concentrates on mitochondrial damage as a cause for neuronal damage.[12] Standard treatments to optimize glycemic control and early treatment of secondary diseases such as hypertension and lipid lowering are very important to reduce the likelihood of neuropathy.[13] At present, there is no therapy that directly reduces the progression of nerve damage due to diabetes.[14]

Altered glucose homeostasis makes the already changed processing of pain impulses in the dorsal horn described by the gate theory even more vulnerable to the increased production of substance P in damaged Aß fibers and results in higher transmission rates of painful stimuli to the brain. Rewiring in the dorsal horn, also known as sprouting, has long been believed to play a pivotal role in peripheral nerve injury,[15] but data presented during a recent conference have debated this sprouting.[16]

Persistent stimulation of spinal cord neurons leads to activation of NMDA receptors, which leads to extended depolarization resulting in increased sodium and calcium influx, and potassium efflux. Subsequent impulses from pain fibers cause much larger postsynaptic potentials that lead to a state of central spinal sensitization.[17]

The last neurological consequence of nerve fiber damage that will be discussed here is the accumulation of sodium channels at the injury site and along the axon, which propagates the generation of ectopic impulses and the development of hyperexcitability.[18]

All treatments for PDN are symptomatic and the most commonly used agents are anticonvulsants and antidepressants, with opioids and topical agents playing a lesser role.[19] Duloxetine and pregabalin are the only agents licensed for the treatment of PDN.

• Epidemiology

• Incidence/Prevalence

Diabetes is a very common disease with a prevalence of 7.8% in the USA.[105] The prevalence for Australia in 2000 was 7.4%, which had doubled from 1981 and again increased over the last 6 years to 8%.[20] In the UK, according to a health survey performed in 2003, the prevalence was 4.3% in men and 3.4% in women.[21] Of patients with diabetes mellitus, it is estimated that 50% develop PDN sometime during their lifetime.[22]

• Diagnosis

The American Academy of Neurology emphasizes the importance of a careful medical history, complemented by neurological examination and neurophysiological studies (conduction velocity, quantitative sensory testing and quantitative autonomic function testing). The ‘diabetic neuropathy symptom score’ involves asking patients about their unsteadiness on walking, presence of pain, paraesthesias or numbness. The maximum score is four, score of one or higher is diagnostic for neuropathy. A sensitive physical examination test is to apply a 128-Hz tuning fork to the bony prominence at the base of the big toe.

• Treatment Guidelines

The American Diabetes Assocation emphasizes stabilization of glycemic control and the use of tricyclic antidepressants (TCAs) followed by anticonvulsants and opioids.[23]

Guidelines from the British National Institute of Clinical Excellence recommend simple analgesics (paracetamol or aspirin) as the first step, followed by low-to-medium doses of TCAs, with an explanation to the patient that they are used as a treatment trial. The third step should be gabapentin titrated to the maximum tolerated dose or at least 1800 mg/day. The newer drugs pregabalin and duloxetine are not yet included in this guideline as they are considered to need further postmarketing evaluation. Opioid treatment should be reserved for multidisciplinary pain clinics.[106]

American guidelines published by the Mayo Foundation for Medical Education and Research recommend the use of duloxetine, oxycodone SR, pregabalin or a TCA as a first-line treatment option as they are supported by more than two randomized controlled trials. Drugs with different modes of action can be combined to achieve better pain relief and lesser side effects (multimodal treatment).[22]

• Evidence for Current Therapies

Currently, only pregabalin and duloxetine are specifically licensed for the treatment of DN. Gabapentin is licensed in the UK for the treatment of peripheral neuropathic pain. Without an official license, but commonly used for the treatment of neuropathic pain and PDN, are TCAs, venlafaxine, carbamazepine and other anticonvulsants. The Cochrane collaboration has published a review about the use of anticonvulsants and antidepressants in neuropathic pain.[24,25] The number of patients that need to be treated with a drug to achieve 50% pain relief is a commonly used term in evidence-based medicine and is called the number needed to treat (NNT). A compilation of the NNT data from this review for the different drugs used in the treatment of PDN are listed in Table 1 .[26-33]

Table 1. Number Needed to Treat Data for Drugs Used to Treat Painful Diabetic Neuropathy

The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

February 23, 2009 — Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are sporadic four-repeat tauopathies that have characteristic clinical and neuropathologic profiles, but also share overlapping features. Both present with various clinical phenotypes, but while the clinical diagnostic criteria of the classical and most frequent presentation of PSP have optimal specificity, the diagnostic accuracy of the various CBD phenotypes is suboptimal. On the other hand, neuropathologic diagnostic criteria for PSP and CBD have been validated and have appropriate accuracy. This article discusses the similarities and differences between these two disorders in clinical manifestations, neuroimaging and neuropathology and provides a perspective on possible future developments that may help in their diagnosis and treatment.

Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are both closely related sporadic parkinsonian disorders with tau pathology that share many clinical features, such as rapid disease progression, poor levodopa response, eye movement abnormalities, cognitive impairment, pyramidal signs and dystonia.[1-3] These similarities may cause difficulty in their clinical differentiation.[4-6] Research criteria have been defined and validated for PSP and in most instances allow for adequate clinical diagnosis of the most common phenotype of this disorder – recently named the Richardson phenotype.[7] PSP patients with the Richardson phenotype usually present with severe postural instability and falls, and eventually develop a vertical supranuclear gaze palsy that allows its diagnosis. Some of the other PSP phenotypes, such as PSP-parkinsonism (PSP-P), are harder to diagnose in life.[7]

There are no validated clinical diagnostic criteria for CBD and there are no biological markers that can distinguish premortem between the two disorders.[8,9] CBD classically presents with the corticobasal syndrome (CBS) and lateralized cognitive features, such as unilateral apraxia, cortical sensory deficits or aphasia, and unilateral motor symptoms such as dystonia or myoclonus.[10] However, this phenotype is not very specific. In fact, in a series from the Mayo Clinic updated from previous publications, of 34 patients clinically diagnosed with CBS, neuropathologic investigation confirmed this diagnosis in only 18 cases (53%); in the remaining 16, the most frequent pathologic diagnosis was PSP.[10,11] Not surprisingly, in the cortices of PSP patients presenting with CBS there is increased tau burden.[12] Moreover, among 32 cases diagnosed pathologically with CBD in the Mayo Clinic series, the most frequent clinical diagnosis besides CBD was PSP. Dementia and primary progressive nonfluent aphasia resembling frontal lobe dementia have been reported in autopsy-proven CBD.[13] Neuroimaging methods, such as brain parenchyma sonography, MRI, single-photon emission computed tomography (SPECT) and PET, may improve diagnostic accuracy, but characteristic findings may be lacking in the early stages. Furthermore, patients participating in neuroimaging studies have rarely been confirmed by neuropathology.[14,15] On the other hand, neuropathologic research diagnostic criteria have been validated for both disorders,[16,17] and there are features that allow the differentiation of PSP (i.e., tufted astrocytes and specific distribution) from CBD (i.e., astrocytic plaques and specific distribution).

• Molecular Genetics & Mechanisms

The human tau gene locus is unique and located over 100 kb on the long arm of chromosome 17q21 and contains 16 exons.[18] Alternative splicing of exons 2, 3 and 10 allows for six isoforms that differ from each other by the presence of either three-repeat (3R) or four-repeat (4R) regions in the carboxy-terminal (C-terminal) part of the molecule and the absence or presence of one or two inserts (29 or 58 amino acids) in the amino-terminal (N-terminal) part.[19] Each of these isoforms is likely to have particular physiological roles since they are differentially expressed during development. Only one tau isoform, characterized by 3R and no N-terminal inserts, is present during fetal stages, while the six isoforms (with one or two N-terminal inserts and 3- or 4R) are expressed during adulthood. The tau protein is expressed predominantly in the neurons of the peripheral nervous system and CNS where it has a role in building and stabilizing microtubules, neuronal polarity and signal transduction.[20] The tau isoforms may be differentially distributed in neuronal subpopulations and bind microtubules through repetitive regions in their C-terminal region. It has been demonstrated that adult tau isoforms with 4R (R1-R4) are more efficient at promoting microtubule assembly than the fetal isoform with 3R (R1, R3 and R4).[21] Phosphorylation of tau is regulated by a host of kinases and phosphorylated tau proteins are less effective than nonphosphorylated tau proteins on microtubule polymerization. Glycogen synthase kinase 3 is a tau kinase that is able to phosphorylate both non-Ser/Thr-Pro sites and Ser/Thr-Pro sites. Protein kinase N, a Ser/Thr kinase when activated, phosphorylates tau resulting in disruption of microtubule organization. Hyperphosphorylation of the tau protein can result in the self-assembly of intraneuronal tangles of tau filaments, which are involved in the pathogenesis of tauopathies such as PSP and CBD.[21,22]

The chromosomal region containing the tau gene has been shown to evolve into two major haplotypes, H1 and H2, which are defined by linkage disequilibrium between several polymorphisms over the entire gene. The more common haplotype, H1, is over-represented in patients with PSP. However the H1/H1 haplotype does not influence the age of onset, severity or survival in patients with PSP.[23] Williams et al. found that the effect of the H1/H1 susceptibility genotype appeared stronger in the typical PSP-Richardson syndrome phenotype than in the PSP-P phenotype.[7] Melquist et al. recently identified additional genetic loci involved in conferring the risk of PSP through a pooling-based genome wide association study of more than 500,000 single nucleotide polymorphisms.[24] The H1 haplotype was strongly detected by this methodology, as was a second major locus on chromosome 11p12-p11 that showed evidence of association at allelic, genotypic and haplotypic levels, and was narrowed to a single haplotype block containing the DNA damage-binding protein 2 and lysosomal acid phosphatase 2 genes. DNA damage and lysosomal dysfunction have been implicated in aging and neurodegenerative processes and both genes could be viable candidates for conferring the risk of developing PSP.[24] H1 haplotypes have also been associated with CBD in pathologically confirmed cases.[25] There are rare reports of familial PSP and CBD with more than one affected member.[26,27] Identifying the functional basis of the H1 haplotype association provides the most promising prospect for understanding the etiopathogenesis of PSP and CBD.

• Characteristic Clinical Profiles

• Progressive Supranuclear Palsy

The typical form of PSP – the Richardson phenotype – presents in middle- to late-life with postural instability and falls, frontal cognitive impairment, axial parkinsonism and ocular motor disturbances.[7] One of the earliest complaints is early postural instability and falls, and patients eventually develop a peculiar wide-based, slow and unsteady gait.[4] The parkinsonism in PSP is characterized by axial rather than limb involvement, symmetric extremity involvement, as well as its lack of or only transient benefit from levodopa therapy, distinguishing these patients from those with other parkinsonian disorders, such as Parkinson’s disease (PD). Patients with PSP usually develop early dysarthria and dysphagia. However, what defines and allows one to make the diagnosis of this disease is the presence of vertical supranuclear gaze palsy, which is later followed by horizontal gaze abnormalities. Slowing of vertical saccades precede the development of the supranuclear gaze palsy and allows an earlier diagnosis. Most PSP patients develop marked cognitive deficits and personality changes that are suggestive of frontal lobe dysfunction, leading to the pattern of ’subcortical dementia’.[28] Cognitive deficits in PSP progress consistently; executive functions are the earliest and most severely affected and help differentiate PSP from other parkinsonian disorders.[29] Attention and memory, although also impaired, are less severely affected and are usually related to the frontal deficits (i.e., encoding new information is preserved, but retrieval is affected, in contrast with what occurs in Alzheimer’s disease). Apraxia is not a major disturbance in PSP and its extent is related to the dementia.[30] In PSP, the apraxia is not unilateral, as in CBS, but is bilateral and does not affect activities of daily living. PSP patients also exhibit behavioral symptoms. A study of 61 PSP patients showed that the majority suffered from continuous apathy, mostly in the moderate-to-severe range, and a third exhibited moderate-to-severe disinhibition.[31] However, in contrast to what has been reported in PD and CBS, depression is infrequent and mild. While the Richardson phenotype is usually easy to diagnose using standardized diagnostic criteria, presentations with parkinsonism without oculomotor disturbances, isolated dementia, freezing of gait or lateralized presentations, are a diagnostic challenge. Golbe et al. recently devised and validated a rating scale for PSP.[32] In 162 patients with PSP it was sensitive to disease progression and a good independent predictor of survival.

• Corticobasal Degeneration

Corticobasal degeneration can manifest with a CBS characterized by lateralized motor or cognitive problems, or with a dementia syndrome characterized by bilateral parkinsonism and global cognitive disturbances.[4,33] However, the CBS phenotype may have various underlying pathologies, the most common (60%) being CBD.[34] Rarely, presentations mimicking the Richardson phenotype, or more commonly with early severe frontal dementia have also being described and are challenging to diagnose in life.[33,35] Patients with a CBS characterized by unilateral motor (unilateral parkinsonism, dystonia or myoclonus) and cognitive disturbances (aphasia, unilateral ideomotor apraxia, alien limb phenomena and cortical sensory deficits, such as hemivisual or hemisensory neglect) may also eventually develop a full-blown dementia with frontal deficits.[4]

There are other clinical features that help in differentiation between the two disorders. Patients with CBS show a severe ideomotor apraxia, which is not usually observed in patients with PSP, and systematic assessment of ideomotor apraxia facilitates the differential diagnosis between the two.[36] On the other hand, frontal deficits in patients with CBS are not as severe as those found in PSP.[29] PSP patients with the Richardson phenotype have decreased spontaneous speech and reduced fluency that manifests as a type of progressive nonfluent aphasia (dynamic aphasia), but the speech disturbances in CBS are due to a language disturbance rather than frontal

dysfunction.[37-39] Language impairment in CBS at baseline has been found to be more impaired in cognitive than motor-onset CBS, but there was no correlation between the side of atrophy or motor impairment.[40] Additionally, the oculomotor disturbances usually differ; CBS patients have difficulties initiating horizontal and vertical saccades, typical of an oculomotor apraxia, whereas PSP patients with the Richardson phenotype develop slow vertical saccades but normal saccade latency.[41] Assessment of orofacial praxis may help in the diagnosis. Patients with CBS are significantly more impaired in demonstrating multiple sequential gestures than those with PSP. This deficit in CBS is likely related to simultaneous lesions of the parietal lobe and the supplementary motor area.[42]

A clinicopathologic study, which retrospectively examined the clinical features of patients pathologically diagnosed with PSP and CBD, identified two sets of predictors (models) for CBD patients using logistic regression analysis.[43] One set consisted of cognitive disturbances at onset, and instability with falls at the first clinic visit (suggestive of PSP), and the other set consisted of asymmetric parkinsonism, limb dystonia, cognitive disturbances at symptom onset and speech disturbances (suggestive of CBD). Patients with PSP often had severe postural instability at onset, symmetric parkinsonism, vertical supranuclear gaze palsy, speech and frontal lobe-type features. In contrast with PSP, the diagnosis of CBD in life remains a major challenge.

• Neuroimaging

• Magnetic Resonance Imaging

In PSP, atrophy of the midbrain tegmentum and dilation of the third ventricle, are the most important features. Atrophy of the corpus callosum, anterior cingulate cortex and superior cerebellar peduncle, visualized by MRI, has been reported to correlate with disease duration in PSP.[44] The ‘morning glory’ sign, which is a concavity of the lateral margin of the tegmentum, suggesting atrophy of the midbrain, has been proposed to relate to the vertical supranuclear gaze palsy observed in this disorder.[45] In patients with CBS, asymmetric frontoparietal atrophy is usually observed, which could be the basis of focal signs.[46] In CBS cases that demonstrate the alien limb syndrome, MRI scans may reveal focal abnormalities in the corresponding leg area of the homunculus.[47] 3D imaging and hemispheric volumetry shows most cortical regions are less atrophic in PSP than in patients with CBS. The parietal lobe, paracentral regions, anterior middle frontal lobe and posterior inferior frontal lobe are significantly more atrophic in CBS than in PSP, whereas the brainstem is significantly more atrophic in PSP.[48] Oba et al. measured the areas of the midbrain tegmentum and the pons on mid-sagittal MRI and evaluated the ratio of the area of the midbrain to the area of the pons in patients with PSP, PD, multiple system atrophy (MSA-P) and age-matched normal control subjects.[49] The average midbrain area of the patients with PSP (56 mm2) was significantly smaller than that of the patients with PD (103.0 mm2), MSA-P (97.2 mm2) and the age-matched control group (117.7 mm2). The ratio of the area of the midbrain to the area of the pons in the patients with PSP (0.124) was significantly smaller than that in those with PD (0.208), MSA-P (0.266) and in normal control subjects (0.237). Use of the ratio allowed differentiation between the PSP group and the MSA-P group. The area of the midbrain on mid-sagittal MRI can differentiate PSP from PD, MSA-P and normal aging. A recent study established a mathematical model based on 3D MRI to differentiate between PSP and CBD. Using MRI-based volumetry, the pattern of atrophic changes in 33 patients diagnosed as PSP and 18 patients diagnosed as CBD by strict clinical criteria was compared with 22 age-matched controls. Postmortem confirmation was obtained in eight PSP and seven CBD cases. A significant reduction in average brain, brainstem, midbrain and frontal gray matter volumes was seen in patients with PSP, whereas patients with CBD showed atrophy of the parietal cortex and corpus callosum. The model allowed correct antemortem prediction of the diagnosis in 95% of controls as well as in 76% of all PSP and 83% of all CBD patients.[50] Paviour et al. evaluated the volume of the superior cerebellar peduncles (SCPs) on MRI prospectively in PSP, MSA-P, PD and healthy controls.[51] The mean SCP volume, corrected for total intracranial volume, was significantly lower in patients with PSP than in patients with MSA-P and PD. Neuroradiologic rating correctly identified PSP cases based on the presence of SCP atrophy with a sensitivity of 74% and a specificity of 94%.

• Single Photon Emission Computed Tomography

There are differences between patients with PSP and those with CBS on SPECT with the dopamine marker ß-CIT. Striatal binding of ß-CIT is significantly reduced in PSP (-60%) and CBS (-35%), as compared with normal controls.[52] Asymmetry of striatal ß-CIT binding was significantly increased in patients with CBS. Putamen:caudate nucleus ratios were significantly reduced in patients with PSP, but not in those with CBS.[52] Postsynaptic D2-receptor imaging with IBZM SPECT has shown significant reductions in D2-receptor binding in patients with PSP and CBS.[53]

• Positron Emission Tomography

PET imaging with 18F-fluorodeoxyglucose has shown bilateral frontal hypometabolism in PSP, whereas in the CBS the abnormality is typically asymmetric.[54] PET scanning using a postsynaptic D2-receptor ligand fluorine-18 desmethoxyfallypride has shown that patients with MSA-P and PSP have decreased postsynaptic tracer binding.[55] PET scans have also revealed a pattern of increased microglial activation in PSP patients involving cortical and subcortical regions that correspond well with the known distribution of neuropathological changes and may help in characterizing the underlying disease activity in PSP in vivo.[56]

• Brain Parenchyma Sonography

A recent study evaluated patients with PSP and CBS using a transcranial color-coded, phased-array, ultrasound system equipped with a 2.5-MHz transducer through preauricular acoustic bone windows using a standardized protocol. In 88% of eight CBS patients, marked hyperechogenicity of the substantia nigra was seen, but this was not observed in any of the 11 PSP patients.[15] This significant finding showed that CBS has a positive predictive value of 100%. Marked dilatation of the third ventricle (width > 10 mm) was found in 83% of 12 PSP patients, but in none of the CBS patients. Brain parenchyma sonography (BPS) measurements of ventricle widths closely matched MRI measurements. The presence of at least one of the BPS findings – marked SN hyperechogenicity and third-ventricle width less than 10 mm – indicated CBS with a sensitivity of 100%, a specificity of 83% and a positive predictive value of 80%.[15] If the CBS patients participating in these studies are eventually confirmed by neuropathology to have CBD, and future BPS studies reconfirm these findings, this technique could be a useful noninvasive and accurate antemortem method to differentiate between these disorders.

• Neuropathology

The neuropathologic diagnostic criteria for both PSP and CBD have been validated. In PSP, gross examination of the brain shows midbrain atrophy. PSP is characterized by neuronal loss, gliosis and abundant neurofibrillary tangles and neuropil threads in the striatum, especially in the globus pallidus interna, subthalamic nucleus, substantia nigra, oculomotor complex, reticular formation, periaqueductal gray, superior colliculi, basis pontis, dentate nucleus and prefrontal cortex.[16] The cerebellar dentate nucleus may show degeneration. There is a uniform presence of tau-positive cortical lesions, especially in the precentral and angular gyrus, primarily affecting the deep cortical layers. Tau-positive glial inclusions – tufted astrocytes – are a consistent pathologic finding. Coiled bodies, which are tau deposits in oligodendrocytes found in the white matter, are also widely distributed. Early pathology is evident primarily in the pedunculopontine nuclei, perhaps explaining the early postural instability and falls. The pontine nucleus raphe interpositus and deep pontine nuclei are also affected. The neuropathology of CBD includes prominent atrophy of the frontoparietal cortex, particularly peri-Rolandic regions, as well as depigmentation of the substantia nigra. In affected regions, there is neuronal loss, gliosis and prominent glial and neuronal intracytoplasmic filamentous tau-immunoreactive pathology. Achromatic ballooned neurons, which are most numerous in cortical and limbic regions, are strongly immunoreactive for phosphorylated neurofilaments and B-crystallin but variably positive for tau.[57] The glial tau pathology consists of characteristic astrocytic plaques, as well as numerous tau-immunoreactive inclusions in gray and white matter in astrocytes and oligodendrocytes (coiled bodies). Perhaps the most striking feature of CBD is the extensive accumulation of tau-immunoreactive cell processes throughout both the gray and white matter. Another recent advance has been the development of tau antibodies that recognize each of the six human tau isoforms. These antibodies can stain corresponding recombinant tau isoforms in tau lesions in an isoform-specific manner on western blot. Using immunohistochemistry of tau isoforms in autopsied brains, attempts have been made to distinguish between diseases with 3R- and 4R-tau using monoclonal antibodies specific for these isoforms of tau. Yoshida used two monoclonal antibodies, RD3 and RD4, and a polyclonal antibody for exon 10 that effectively distinguished between 3R and 4R tau.[58] In PSP and CBD, both neuronal and glial tau accumulation demonstrated 4R-tau in the cerebral cortices. However, in the basal ganglia and brainstem, neuronal and glial inclusions were occasionally immunopositive for 3R-tau in addition to 4R-tau, suggesting that the isoform composition may be heterogenous and may have a spectrum in individual cases, and that the cellular isoform composition may differ in various brain regions. Williams et al., in their study of 103 pathologically confirmed cases of PSP, found significant differences in the isoform composition of insoluble tangle-tau isolated from the basal pons. In the typical phenotype of PSP-Richardson syndrome – the mean 4R-tau:3R-tau repeat tau ratio was 2.84 and in PSP-P it was 1.63.[7] Ueno et al. recently developed and characterized two novel conformation-sensitive antibodies, T3R and T4R to the 3R and 4R-tau isoforms, respectively.[59] Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. Despite the high similarity of the antigens, there was no crossreactivity between T3R and T4R. These features may enable these antibodies to act as novel indicators that will allow observation and evaluation of conformational changes in each distinct isoform of tau.

Arai et al. analyzed the immunoblots of sarkosyl-insoluble brain extracts of patients with CBD and PSP and found distinctive patterns of tau fragments.[60] A 33kDa band predominated in the low molecular weight tau fragments in PSP, whereas two closely related bands of approximately 37kDa predominated in CBD. These results suggest that, despite the identical composition of tau isoforms, different proteolytic processing of abnormal tau takes place in these two diseases. Such a biochemical divergence may be related to the neuropathological features of these diseases.[61]

Epidemiology & Pathogenesis

Progressive supranuclear palsy is the most frequent parkinsonian disorder after PD. Its prevalence increases with age.[62] The average annual incidence rate for PSP between the ages of 50 to 99 years is 5.3 new cases per 100,000 person-years, and the age-adjusted prevalence rate is 6.0 to 6.4.[63]

The incidence and prevalence of CBD are unknown, but it was estimated that approximately 4.9 per 100,000 people have CBS in the USA and 1.7 per 100,000 in Japan.[64,65]

• Prognosis & Complications

In general, these disorders progress steadily. In PSP, most patients eventually require a wheelchair and a feeding tube; speech may become unintelligible, palilalic or mute. In a study of the progression of PSP in patients selected from the research and clinical files of seven medical centers involving tertiary centers of Austria, England, France and the USA, the median survival time was 5.6 years with a range from 2 to 16.6 years.[29] Onset of falls during the first year, early dysphagia and incontinence predict a shorter survival time. Age at onset, gender and early onset of dementia, have no effect on prognosis of survival. Pneumonia was the most common immediate cause of death.[29]

In CBD, the condition is slow in progression and relentless. In one series of pathologically confirmed cases, dysarthria occurred 40 months after disease onset and dysphagia at a median of 64 months, in contrast to 84 months and 130 months in PD.[66] The early dementia phenotype of CBD, characterized by a severe frontal dementia followed by bilateral parkinsonism, has a shorter survival than the CBS variant presenting with unilateral motor as well as cognitive disturbances, which is harder to recognize clinically. Medications do not appear to affect the natural progression of the neurodegenerative process, and the disease usually progresses to death within 6 to 9 years.[4,33]

• Management

In PSP and CBS, trials with dopaminergic drugs and cholinesterase inhibitors have failed to show benefit. The myoclonus in CBS has been treated with benzodiazepines and sodium valproate with varying degrees of success. Botulinum toxin injections are useful for focal dystonias that may occur in these disorders. Depression can be treated with selective serotonin-reuptake inhibitors.

Palliative therapies include physical, occupational, speech and swallowing therapies for these patients’ gait instability, dysarthria and dysphagia.[67]

• Conclusion

At present, only PSP can be easily diagnosed in life when patients present with the Richardson phenotype. Only 60% of patients with CBS may have CBD as an underlying pathology. The fact that PSP is the second most common underlying pathology in patients with CBS makes the differentiation between the two even more challenging. PSP and CBD also share a common genetic haplotype link and are characterized by abnormal accumulation of 4R-tau in neurons and glia. Advances in neuroimaging studies have added more information in trying to reach an accurate diagnosis in these two disorders. The final diagnosis in cases with mixed features or with a dementia presentation is often made postmortem. Both these tauopathies are relentlessly progressive disorders with median survival times of approximately 5-8 years, and the management is mostly supportive and symptomatic. There is a pressing need to identify the basic mechanisms of abnormal tau accumulation and develop appropriate interventional strategies.

• Future Perspective

In the future, movement disorder specialists are likely to develop and validate clinical diagnostic criteria for CBD and a clinical rating scale instrument using objective parameters of apraxia, eye movements, focal cognitive impairments (supplemented by neuropsychological testing) to help with the diagnosis and rate of progression. Advances in neuroimaging are likely to further help in the differentiation between the two disorders.

Development of PET or SPECT ligands for the different tau isoforms may help in following the progression of the diseases or their responses to therapeutic interventions. Future intensive research at cellular levels may identify the molecular mechanisms involved in the abnormal tau accumulation (i.e., glycogen synthase kinase 3-ß), and may lead to development of therapeutic agents that can halt or reverse this process. Preclinical testing of therapeutic options in existing tau transgenic models will accelerate this search.

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26. Ros R, Gómez Garre P, Hirano M et al.: Genetic linkage of autosomal dominant progressive supranuclear palsy to 1q31.1. Ann. Neurol. 57, 634-641(2005).

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30. Soliveri P, Piacentini S, Girotti F: Limb apraxia and cognitive impairment in progressive supranuclear palsy. Neurocase 11, 263-267 (2005).

31. Aarsland D, Litvan I, Larsen JP: Neuropsychiatric symptoms of patients with progressive supranuclear palsy and Parkinson’s disease. J. Neuropsychiatry Clin. Neurosci. 13, 42-49 (2001).

32. Golbe LI, Ohman-Strickland PA: A clinical rating scale for progressive supranuclear palsy. Brain 130, 1552-1565 (2007).

33. Wenning GK, Litvan I, Jankovic J et al.: Natural history and survival of 14 patients with corticobasal degeneration confirmed at postmortem examination. J. Neurol. Neurosurg. Psychiatry 64, 184-189 (1998).

34. Boeve B, Maraganore DM, Parisi JE et al.: Pathologic heterogeneity in clinically diagnosed corticobasal degeneration. Neurology 53, 795-800 (1999).

35. Shiozawa M, Fukutani Y, Sasaki K et al.: Corticobasal degeneration: an autopsy case clinically diagnosed as progressive supranuclear palsy. Clin. Neuropathol. 19, 192-199 (2000).

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38. Robinson G, Shallice T, Cipolotti L: Dynamic aphasia in progressive supranuclear palsy: a deficit in generating a fluent sequence of novel thought. Neuropsychologia 44, 1344-1360 (2006).

39. Frattali CM, Grafman J, Patronas N et al.: Language disturbances in corticobasal degeneration. Neurology 54, 990-992 (2000).

40. McMonagle P, Blair M, Kertesz A: Corticobasal degeneration and progressive aphasia. Neurology 67, 1444-1451 (2006).

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42. Ozsancak C, Auzou P, Dujardin K et al.: Orofacial apraxia in corticobasal degeneration, progressive supranuclear palsy, multiple system atrophy and Parkinson’s disease. J. Neurol. 251, 1317-1323 (2004).

43. Litvan I, Agid Y, Goetz C et al.: Accuracy of the clinical diagnosis of corticobasal degeneration: a clinicopathological study. Neurology 48, 119-125 (1997).

44. Tsuboi Y, Slowinski J, Josephs KA et al.: Atrophy of superior cerebellar peduncle in progressive supranuclear palsy. Neurology 60, 1766-1769 (2003).

45. Adachi M, Kawanami T, Ohshima H et al.: Morning glory sign: a particular MR finding in progressive supranuclear palsy. Magn. Reson. Med. Sci. 3, 125-132 (2004).

46. Arai K: MRI of progressive supranuclear palsy, corticobasal degeneration and multiple system atrophy. J. Neurol. 253(Suppl. 3), 25-29 (2006).

47. Hu WT, Josephs KA, Ahlskog JE et al.: MRI correlates of alien leg-like phenomenon in corticobasal degeneration. Mov. Disord. 20, 870-873 (2005).

48. Taki M, Ishii K, Fukuda T et al.: Evaluation of cortical atrophy between progressive supranuclear palsy and corticobasal degeneration by hemispheric surface display of MR images. Am. J. Neuroradiol. 5, 709-714 (2004).

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68. Metwally, MYM: Textbook of neuroimaging, A CD-ROM publication, (Metwally, MYM editor) WEB-CD agency for electronic publication, version 10.1a January 2009 [Click to have a look at the home page]

The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

February 21, 2009 — The benefits of surgery for the management of low-grade gliomas have been difficult to determine from the literature. This difficulty might be explained by the inconsistency of the published data, and also by advances in both neuroimaging and neurosurgical techniques, which have made surgical intervention a safer and more viable option than it has been in the past, making the earlier studies less applicable to modern care. In this article, we critically analyze the utility of surgery in the management of low-grade gliomas, including the value of observation without surgical intervention, the relative risks and benefits of biopsy versus craniotomy and resection, and recent advances that have made surgery safer and gross total resection a more realistic proposition. As we will discuss, the literature provides modest evidence that surgery leads to improved outcomes through a reduction in tumor burden. As a result of advances in surgical techniques, the time might now be right to effectively and accurately assess the influence of aggressive surgical resection on the prognosis of low-grade gliomas.

Although their name might imply otherwise, low-grade gliomas result in considerable morbidity and inevitable death. The benefits of various interventions–including fractionated radiotherapy, chemotherapy and surgical resection–have all been disputed, but the role of surgical intervention is particularly controversial. This debate stems in part from the inconsistency of results from published reports regarding the benefits of surgical intervention, and is complicated further by advances in neuroimaging and neurosurgical techniques that have made surgical resection a safe and viable option for an increasing number of patients.

In this post, we will critically analyze the utility of surgery in the management of low-grade gliomas, including the role of observation without surgical intervention, the debate regarding biopsy versus resection, and the recent surgical advances that have made surgery for low-grade gliomas increasingly safe and widely available.

• Epidemiology

The phrase ‘low-grade gliomas’ encompasses the entire spectrum of WHO grade I and II gliomas, which includes ependymomas, pilocytic astrocytomas, pleomorphic xanthoastrocytomas, diffuse astrocytomas, oligodendrogliomas, and mixed gliomas. Although arbitrarily clustered under the umbrella low-grade glioma nomenclature, this heterogeneous group of tumors is clinically, histologically and molecularly diverse, and is, therefore, not generally studied or discussed as a single entity. For example, WHO grade I lesions, which include pilocytic astrocytomas and gangliogliomas, are, unlike WHO grade II lesions, well circumscribed and noninfiltrative, and complete surgical resection is universally considered curative.[1] In this post, we will limit our discussion to WHO grade II diffuse astrocytomas, oligodendrogliomas and oligoastrocytomas, all of which have similar invasive and malignant potential.

Although low-grade gliomas are less common than malignant gliomas (of which there are ~8,000 – 10,000 cases per year) and far less common than brain metastases (~75,000 – 150,000 cases per year), they are far from rare. Approximately 2,000 – 3,000 low-grade gliomas are diagnosed in the US every year, accounting for nearly 15% of all primary brain tumors.[2] According to the Central Brain Tumor Registry of the United States (CBTRUS), the various histological classes of low-grade glioma have incidences varying between 0.10 and 0.46 per 100,000 people, and a cumulative incidence of approximately 0.9 per 100,000 people.[2]

Despite the preponderance of astrocytomas, there has been an apparent increase in recent years in the incidence of pure oligodendrogliomas and of mixed oligoastrocytomas. This increase might be partly attributable to an increased tendency for neuropathologists to seek out such diagnoses, in view of the relatively favorable prognosis conveyed by an oligodendroglial component.[3] Low-grade gliomas are particularly prevalent among white people and among men, and the highest incidence is in people between 35 and 44 years of age. Low-grade astrocytomas in adults are most commonly located in the cerebral hemispheres, with a predilection for ’secondary’ functional areas such as the supplementary motor area and the insular lobe.[4] Oligodendrogliomas are most commonly seen along the cerebral convexity in subcortical areas, particularly in the frontal lobe, but sporadic reports of posterior fossa oligodendrogliomas exist.

• Signs, Symptoms and Prognosis

Low-grade gliomas produce signs and symptoms of disease through three main mechanisms: direct infiltration and destruction of neurons; local pressure from edema, hemorrhage and tumor mass; and intracranial hypertension owing to mass effect or ventricular obstruction. In many cases, the insidious onset of symptoms related to low-grade gliomas might delay the correct diagnosis for many years. When patients become symptomatic, seizure is the most common presenting sign, occurring in up to 80% of cases.[5,6] Other less common modes of presentation include headache, lethargy and personality changes. The patient’s presenting symptoms and imaging characteristics can critically affect management; both neurosurgeons and neuro-oncologists are likely to recommend surgical intervention for patients presenting with signs and symptoms attributable to mass effect or intracranial hypertension.

Although the effect of surgery on low-grade glioma prognosis is a topic for debate, several factors are recognized to be associated with poor prognosis, including, but not limited to, advanced age at time of diagnosis,[3,5-7] larger tumor size on presentation (before surgical intervention),[6,8-10] and rapid tumor growth rate.[11] Favorable prognostic factors include presentation with seizure (as opposed to with an abnormal neurological examination), and having an oligodendroglial component, a 1p and 19q chromosomal deletion, or a tumor that does not cross the midline.

• Is There a Role for Observation Without Surgery?

Historically, expectant management has been considered to be a plausible management paradigm for patients who have lesions with typical features of low-grade glioma, especially those with minimal symptoms or seizures that are well controlled with antiepileptic drugs.[12] Expectant management can potentially provide patients with an overall improved quality of life (QOL) for the duration of disease by not exposing them at the time of diagnosis to the morbidity and mortality risks associated with biopsy or with craniotomy and resection. Imaging characteristics considered to be typical of low-grade glioma include low attenuation on CT scanning, and T1 shortening, T2 prolongation, and absence of contrast enhancement on MRI (Figure 1).[13] The diagnostic value of these ‘typical’ imaging characteristics has, however, come under scrutiny. Kondziolka and colleagues found that imaging-based diagnosis of low-grade glioma was accurate in only 50% of patients (on the basis of comparison with stereotactic biopsy results).[13] Likewise, Scott and colleagues found that, in a series of 314 patients, grading gliomas on the basis of imaging characteristics alone underestimated the degree of malignancy in one-third of cases.[14] Furthermore, although contrast enhancement on MRI has long been considered a sign of malignancy, recent studies of chemotherapeutics for low-grade glioma have reported contrast enhancement in up to 60% of low-grade gliomas.[15] The use of magnetic resonance spectroscopy and perfusion-weighted imaging has markedly increased the accuracy and sensitivity of imaging-based diagnosis and grading of gliomas, but these techniques are still associated with significant error rates and are not used in routine clinical neuroimaging.[16-18]

Figure 1. Typical MRI scan of a low-grade glioma, histopathologically defined as a WHO grade II oligodendroglioma. (A) T1 sequence demonstrating T1 shortening in the right frontal lobe. (B) T2 sequence demonstrating T2 prolongation (hyperintensity) at the site of the glioma. (C) Contrast-enhanced imaging of the glioma showing no marked contrast enhancement. Although these images are considered ‘typical’, numerous studies have questioned the reliability and accuracy of these imaging characteristics for the diagnosis of low-grade glioma. (Click to enlarge figure)

The preponderance of studies that contradict the notion of a pathognomonic imaging profile for low-grade glioma that could be elucidated using standard imaging sequences suggests that tissue acquisition remains mandatory for the accurate diagnosis, prognostication and management of these tumors. In an era when management regimens are predicated on the basis of tumor cytogenetics, tissue-based diagnosis carries increasing prognostic and therapeutic implications and the absence of such evaluation inevitably limits the scope of therapeutic interventions.

In addition to misdiagnosis, expectant management carries other risks, including malignant degeneration, interval tumor growth that makes subsequent resection more difficult or impossible, and development of an irreversible neurological deficit.[19] Despite these theoretical risks, a comparison between initial conservative management and immediate surgical resection by Recht and colleagues reported that the timing of intervention did not affect rates of malignant transformation, overall survival (OS), or QOL.[20] Similarly, Reijneveld and co-workers found that, although patients with low-grade gliomas experienced significant cognitive disturbances and impairments in QOL, a ‘wait-and-see’ policy did not worsen these afflictions.[21] Furthermore, van Veelen et al. reported that the 5-year survival was identical (63%) in patients who underwent surgery immediately on diagnosis and those who underwent surgery at the time of clinical or radiographic progression.[22] Despite the consistent finding that the timing of tissue diagnosis is not critical, it is important to stress that the studies performed on this topic to date have all been retrospective, which inevitably introduces considerable selection bias and limits interpretation and applicability.

• Biopsy or Resection?

Both stereotactic biopsy and open biopsy provide an opportunity to characterize the histology and cytogenetics of a suspected low-grade glioma without the morbidity and mortality risks associated with a large craniotomy. In general, biopsy has been advocated in the setting of tumors located adjacent or deep to eloquent cortices, which would not be amenable to gross total resection (GTR).[23] Whereas stereotactic biopsies are preferred for patients with deeper lesions, open biopsies are reserved for those with superficial lesions that are easily accessible via a small craniotomy and for those with lesions immediately adjacent to eloquent cortices, which should be mapped before biopsy. Craniotomy and resection, on the other hand, have traditionally been reserved for patients with appreciable mass effect from low-grade glioma (which results in increased intracranial pressure or neurological deficit) and for those with symptomatic epilepsy that is refractory to antiepileptic therapy.[22,24]

In general, the diagnostic yield of stereotactic biopsy is high (90 – 95%).[25-27] Several studies have, however, raised concerns regarding the reliability and accuracy of biopsy-based diagnoses.[23,26,28,29] Aker and colleagues scrutinized results from 23 patients who initially underwent stereotactic biopsy and subsequently required craniotomy, and reported that the accuracy of histological diagnosis made on the basis of a stereotactic biopsy sample was only 83%.[26] In three of the four misdiagnosed cases, a biopsy-based diagnosis of low-grade glioma was changed to anaplastic astrocytoma, granulomatous cerebritis, or dysembryoblastic neuro epithelial tumor. Similarly, McGirt and co-workers reported 79% accuracy of diagnosis in 23 patients who underwent biopsy followed within 60 days by resection.[28] Even more concerning, Jackson and colleagues at the MD Anderson Cancer Center, Houston, TX, reported accuracy rates inferior to those reported by McGirt et al., ranging between 51% and 62%.[23] Accuracy rates depended on the expertise of the interpreting neuro pathologists, highlighting the need for expert neuropathology consultation when using stereotactic biopsy in order to obtain the best possible accuracy.[23] Despite discrepancies between histopathology results from stereotactic biopsy and craniotomy, some groups have argued that appropriate treatments are still administered in 93 – 96% of cases, owing to similarities in treatment paradigms for gliomas of different grades.[28,30] Such viewpoints are, however, becoming less valid as clinical studies further stratify treatment paradigms according to tumor grade and cytogenetics. Moreover, prognostication and patient counseling is inevitably adversely affected by misdiagnosis.

When assessing the value and role of biopsy in the management of low-grade gliomas, it is important to consider not only the yield and accuracy of biopsy, but also the risks associated with the procedure. Despite its reputation for safety, stereotactic biopsy is not without risk. To quantify the morbidity and mortality of the procedure, Hall reviewed 17 series comprising a total of 7,471 stereotactic brain biopsies.[27] He reported an overall morbidity rate of 3.5% and a mortality rate of 0.7%, which were largely related to procedure-induced hemorrhage. Risk factors for biopsy-associated morbidity include basal ganglia lesions, thalamic lesions, diabetes, and hyperglycemia on the day of surgery.[31]

In contrast to biopsy, surgical resection minimizes the likelihood of sampling error by providing considerably more tissue for histopathological analysis, but it inherently exposes the patient to greater risk. Enthusiasm for surgical resection has grown in light of advances in neuroimaging and neurosurgical techniques that have made surgical resection safer and more accessible, and on the basis of numerous reports on the benefits of GTR with respect to both symptom control and prolongation of survival and time to progression. Although not curative, GTR theoretically provides multiple benefits, including reduction in the number of cells at risk of undergoing genetic events that result in malignant transformation, increased efficacy of adjuvant therapy, and symptomatic relief owing to reduced mass effect. Surgical resection has been particularly effective at controlling medically refractory seizures, resulting in near-seizure-free status, or a substantial reduction in seizure frequency and intensity, in nearly all patients.[32,33]

Although large preoperative tumor size (diameter > 5 – 6 cm) is consistently identified as a poor prognostic factor for survival,[3,6] the effect on clinical outcome of reduced tumor size remains controversial, and several studies have produced inconsistent results (Table 1). Keles and colleagues critically reviewed the literature with respect to the effect of extent of resection on outcomes in low-grade glioma and found that a preponderance of modern studies support extensive resection over biopsy alone.[19] Leighton and co-workers, who retrospectively studied 167 consecutive patients treated at the London Regional Cancer Centre, ON, Canada, for low-grade glioma over a period of 16 years found, on both univariate and multivariate analysis, that minimal postoperative residual tumor was associated with significantly improved 5-year OS (82% vs 64%; P = 0.008 on univariate analysis, P = 0.006 on multivariate analysis).[7] Other groups have likewise reported, in retrospective series, that aggressive surgical resection can prolong the time to tumor recurrence and reduce the malignant transformation rate.[5,8,34] Lo and colleagues specifically queried whether patients who received postoperative radiation therapy had benefited from extensive resections.[35] Of the 65 patients who received postoperative radiation therapy, the 12 patients who had GTR (as assessed by postoperative imaging) had significantly longer 10-year OS than those who had subtotal resection or biopsy only (90% vs 41.4%; P = 0.001).

Table 1. Large Studies Investigating the Effect of Surgical Resection on Outcomes of Low-grade Glioma (Click to enlarge table)

The results from prospective trials on resection and postoperative radiation therapy, however, are not so compelling. For example, a prospective intergroup American trial, designed primarily to compare the effects of low-dose versus high-dose radiation therapy, reported, on univariate analysis, a significant 5-year OS advantage in patients who had GTR compared with those who had subtotal resection or biopsy alone (88% vs 56% and 71%, respectively; P = 0.0151).[3] This trial also reported prolonged progression-free survival (PFS) in patients who had GTR compared with those who did not (hazard ratio 0.44, P = 0.0138 on univariate analysis).[3] Pignatti and colleagues and Karim and co-workers both retrospectively studied prospectively collected data from European Organisation for Research and Treatment of Cancer trials (22844 and 22845, and 22844 only, respectively) and found, on univariate analysis, that extensive resection (i.e. > 90%) was associated with longer OS.[6,10] Despite consistent findings on univariate analysis, on multi variate analysis the benefits of extensive resection were marginal at best in both studies. The failure of prospective trials to identify a benefit on multivariate analysis seriously challenges the role of extensive surgery in the management of low-grade gliomas. It is important to point out, however, that these studies were not primarily intended to assess the effect of extensive resection, and were, therefore, not designed or powered to reliably evaluate this factor. For example, in most studies, the extent of resection was gauged on the basis of the ’surgeon’s impression’, which is an unreliable assessment. Further complicating the interpretation of the literature are several other studies that contradict the finding that the difference in OS or PFS among patients relates to the extent of resection.[19,36,37]

The effect of surgical resection on QOL, as well as on survival, must be considered. Teixidor and colleagues recently studied the consequence of surgical resection on higher cognitive functions, specifically verbal working memory, as this function can dramatically affect a patient’s QOL.[38] As in previous studies, the authors found that most patients with low-grade glioma had pre operative cognitive deficits.[21,38] Although surgery can induce further deficits, these exacerbations are transient in most patients. Patients generally recover pre operative cognitive function, or even improve upon it, within 3 months after surgery.

Despite the preponderance of modern studies favoring GTR, there is a paucity of class I evidence supporting or refuting radical tumor removal. Moreover, the available statistical evidence in both retrospective and prospective reports published to date is adulterated by numerous inherent limitations, including patient-selection bias, treatment-selection bias, and inconsistent and inaccurate means of asses sing the extent of resection. The effect of selection bias in particular should not be underestimated. Unfortunately, surgical selection criteria are rarely, if ever, reported (Table 1). Patients with polar or lobar tumors with sharp borders are more likely to undergo and benefit from aggressive resection than those with less well defined lesions. By contrast, patients with probable low-grade glioma who are fully functional and never undergo treatment are not enrolled in surgical series. Tumor histopathology might also introduce selection bias; oligodendrogliomas–which often have sharp radiographic borders and are, therefore, more amenable to extensive resection than are fibrillary astrocytomas–have a better natural history than astrocytomas, and might thereby introduce a considerable potential selection bias into a surgical series. For each study, therefore, it is important to consider the patient and tumor characteristics of both patients who were included in the analysis and those who were excluded. Notwithstanding the effect of selection bias, the lack of a consistent means of measuring the extent of resection probably adversely affects the interpretation of the literature (Table 1). In some studies, the extent of resection has been assessed on the basis of the surgeon’s impression alone, whereas in others it has been assessed by use of inferior imaging modalities such as CT or by objective evaluation of postoperative T2-weighted or fluidattenuated inversion recovery (FLAIR) MRI. The definition of ‘extensive’ resection has also been variable, with some studies reporting and evaluating the absolute volume of residual tumor[8] and others considering the percentage of the preoperative tumor volume that remained after surgery.[6,22]

Notably, a couple of recent studies suggest a possible role for neoadjuvant chemotherapy before surgical resection.[39,40] In both reported cases, although contralateral involvement via the corpus callosum originally prohibited GTR, chemotherapy was able to shrink the tumors to such an extent as to make GTR possible. Studies of temozolomide for the treatment of low-grade glioma suggest that significant tumor shrinkage is possible with chemotherapy alone.[15,41] It is unclear, however, how often chemotherapy can shrink a tumor enough to make GTR possible. The effect of the timing of surgery can and will be appreciated only when a prospective trial is performed, as was performed by van den Bent et al. to assess the timing of radiation therapy for the management of low-grade glioma.[42]

• Advances in Neurosurgery

Advances in neurosurgical technology, including the introduction of preoperative and intraoperative brain mapping techniques and intraoperative image guidance, have made surgery for low-grade glioma increasingly safe and accessible. Perhaps more importantly, pre operative functional brain mapping has empowered surgeons to operate on patients who previously might not have been considered suitable candidates for surgery.

Historically, eloquent cortices have been identified on the basis of anatomical landmarks. In the context of brain tumors, however, these landmarks can be difficult to delineate, and this approach to identification cannot account for individual variability or plasticity-related changes. Preoperative brain mapping techniques, including functional MRI (fMRI) and magnetic source imaging (MSI), have, therefore, revolutionized the assessment of patients with brain tumors. Preoperative maps help determine the need for intraoperative mapping, the best approach (or ‘corridor’) to tumor resection in order to spare eloquent cortices, and the limits of resection. Moreover, the use of preoperative maps can make intraoperative mapping safer and more efficient.

Blood-oxygen-level-dependent (BOLD) fMRI,[43] a functional neuroimaging technique that maps the brain by detecting perfusionrelated changes that are coupled to cognitive tasks (and, therefore, to neuronal activity), has become the predominant functional neuroimaging technique (Figure 2). Recent technological advances, including increasing the magnetic resonance field strength, improved sequence and task design and selection, and superior analytical techniques, have made fMRI increasingly sensitive and reliable.[44-49] Increasing the field strength provides improved signal-to-noise ratio and spatial resolution, and more-rapid imaging–a particularly important consideration in patients with low-grade glioma who might not tolerate prolonged imaging sessions. FitzGerald and colleagues found that fMRI was highly sensitive (81 – 92%), but not very specific (0 – 53%), for intraoperative identification of essential language areas.50 The low specificity (i.e. fMRI showed activation of areas that were not essential for linguistic tasks) suggests that fMRI could direct the surgeon to areas of interest so that it might be determined whether activated cortices are ‘essential’ or ‘nonessential’–thereby obviating the need to map the entirety of the exposed cortex, but still necessitating intraoperative cortical mapping to guide resection. Nevertheless, the relationship between the use of fMRI maps and clinical outcomes remains uncharacterized. Moreover, the question of whether perfusion-related responses are adversely affected by an adjacent tumor has not yet been answered.

Figure 2. Functional MRI scan for preoperative planning. Rather than relying on anatomical landmarks for functional localization, functional MRI has enabled surgeons to precisely localize brain function and to use this information to plan for safer resection of low-grade glioma. (A) Axial, (B) sagittal and (C) coronal T1 MRI scans with overlying red areas of activation demonstrate the regions of the brain activated during right-hand motor activity. These images can be fused to create a three-dimensional rendering of functional anatomy to guide the surgeon and make surgery safer. (Click to enlarge figure)

MSI fuses magnetoencephalography and MRI, projecting the extrapolated source localization of the magnetoencephalographic signals onto a co-registered anatomical MRI scan. As in other mapping techniques, MSI maps are generated by comparing ‘activity’ during a resting state with that detected during the performance of a specific task. Like fMRI, MSI correlates well with intraoperative maps.[51,52] Moreover, MSI can be used to identify the 25 – 46% of patients with gliomas who are at risk of neurological deficit from surgical intervention.[53-55] Patients with MSI signals within 5 mm of a tumor are consider ed to be at high risk of surgery-induced neurological deficit, those with activations between 6 mm and 10 mm from the tumor are slated for subtotal resection, and those with localizations greater than 10 mm from the tumor are deemed candidates for GTR.[54]

Diffusion tensor imaging (DTI) is an MRI technique that creates probabilistic maps of white matter pathways on the basis of voxel anisotropy, a measure of the preferential direction of diffusion of water. DTI can identify white matter pathways that have been either infiltrated or displaced by tumors. Preclinical studies suggest that DTI will have an increasingly important role in the planning of glioma surgery; the technique can identify white matter pathways that should be spared during surgery in order to ensure that functional cortices (as identified by other techniques) are not ‘disconnected’ from their respective projection areas.[56] Kamada and colleagues verified that preoperative cortical and subcortical maps generated by a combination of fMRI, MSI and DTI are predictive and can be confirmed by intraoperative cortical and subcortical stimulation mapping.[57]

In the operating room, technological advances have made cortical and subcortical stimulation mapping easier to implement, making these techniques a more routine part of neurosurgery. In view of its reliability in predicting postoperative outcomes, stimulation mapping is considered to be the gold standard for neurosurgical brain mapping. In this approach, the brain is mapped by either activating the tissue of interest, as in motor mapping, or by temporarily inducing deficits, as in language mapping. To evaluate the effect of stimulation mapping on the efficacy and safety of surgery for low-grade glioma, Duffau and colleagues compared a retrospective series of 100 patients who were operated on without guidance from intra operative mapping (between 1985 and 1996) and a prospective series of 122 patients who were operated on with concurrent use of electrocortical stimulation mapping (between 1996 and 2003).[58] The latter group had a significantly lower rate of severe permanent deficits (6.5% vs 17%), a higher rate of GTR and subtotal resections (25% and 51% vs 6% and 37%, respectively), and a survival advantage, compared with the former group.[58] Moreover, the authors found that the definition of ‘operability’ changed considerably between the two series: the percentage of tumors operated on within eloquent cortices increased from 35% to 62%. Stimulation of subcortical white matter tracts has also become an increasingly important adjunct to intraoperative mapping, permitting the surgeon to spare descending pathways and predict postoperative morbidity.[59] Patients in whom subcortical motor tracts are identified in the resected areas are significantly more likely to experience temporary or permanent motor deficits than those in whom sub cortical motor pathways cannot be identified in these areas.[60]

In addition to stimulation mapping, advanced intraoperative image guidance techniques can now provide a highly accurate real-time three-dimensional ‘road map’ to an individual patient’s brain, which enables surgeons to operate more safely in the face of distorted anatomy. These road maps are created by registering anatomical or artificial landmarks (fiducials) on a high-resolution preoperative MRI scan of the patient’s head and pairing them with corresponding positions on the patient’s head after it is positioned for surgery. Current frameless stereotactic neuronavigation devices use optical triangulation or low-frequency electromagnetic fields to determine the location of an instrument within the operative field. Intraoperative image guidance provides ongoing feedback with respect to regional anatomical relationships, location of normal tissues that must be preserved, and extent of resection. This feedback during surgery can be particularly useful for low-grade glioma, in which the delineation between tumor and surrounding white matter is indistinct. Lumenta et al. described a series of 40 patients (21 of whom had glial neoplasms) who underwent resection of deep-seated intracerebral lesions guided by neuronavigation and cortical mapping. Complete resection was possible in all but three of the patients, and permanent neurological deficits were only noted in two.[61] Although excellent for initial planning, the reliability of maps from preoperative MRI for intraoperative guidance decreases during surgery owing to ‘brain shift’, a phenomenon attributable to tumor removal, ongoing cerebrospinal fluid loss, and brain edema.[62,63]

The limitations of intraoperative image guidance have been partially overcome with the advent of intraoperative magnetic resonance systems that provide real-time imaging of the brain in its intraoperative position. Black and colleagues highlighted the important contribution of intraoperative MRI to successful tumor resection with the observation that, in over one-third of cases, the surgeon’s judgment of the extent of GTR was deemed incorrect by intraoperative MRI, thus prompting further surgical resection.[64] Similarly, Wirtz et al. found that implementation of intraoperative MRI resulted in a significantly higher rate of GTR (67% compared with 38% without the use of intraoperative MRI), which was associated with a significant increase in survival times.[65] Claus and co-workers found that patients who underwent surgical resection of low-grade gliomas with concurrent intraoperative MRI guidance had better 1-year, 2-year and 5-year survival rates than those reported in the CBTRUS.[66] The authors also suggested a possible association between GTR using intraoperative MRI guidance and prolonged PFS and OS (although this correlation did not achieve significance). Further studies of this topic are warranted, especially in light of the ongoing interest in defining the effect of GTR on outcomes in low-grade glioma.

Notwithstanding the technological advances that have made surgical resection a viable option in a greater number of patients with low-grade glioma, it is still recognized that GTR might not be possible in all cases. The radiographic appearance of a low-grade glioma necessarily dictates its amenability to resection. Low-grade gliomas might involve critical structures, including the corpus callosum, thalamus or basal ganglia, might be well circumscribed or in filtrative, and might be unilateral or bilateral. Predictors of incomplete tumor resection include diffuse tumor margin on T2-weighted MRI, oligodendroglioma or oligoastrocytoma histopathological tumor type, large tumor volume, and tumor involving the corpus callosum, corticospinal tract, insular lobe, middle cerebral artery, motor cortex, optic radiation, visual cortex, or basal ganglia.[67] In general, unilateral, well-circumscribed, cortexbased lesions are ideal candidates for resection. Nonetheless, recent reports suggest that tumors that were once considered unresectable, owing to involvement of the corpus callosum, insula or eloquent language cortices, can now undergo resective procedures without the risk of sequelae being increased.[68,69]

• Future Trials

Given the advances in modern neurosurgical and neuroimaging techniques, which permit safer surgical resections and more-accurate assessment of the extent of resection than was previously possible, neurosurgeons and neuro-oncologists might finally be in a position to accurately explore the extent to which aggressive resection improves the prognosis of low-grade glioma.

Planning a trial to compare conservative and aggressive surgical management is complex and poses several challenges. The trial should ideally be randomized, but ethical concerns have limited this approach. A randomized trial was proposed to the American College of Surgeons Oncology Group (ACOSOG), in which patients with incidental low-grade glioma (or those well controlled with a single antiepileptic agent after a single seizure) would be randomly allocated to either biopsy or surgical resection. This trial was, however, rejected over concerns that the literature might already contain sufficient evidence to support resection. Instead of a randomized design, a trial must, therefore, be designed with a prospective cohort construction. To obtain adequate accrual and follow-up, such a trial would require the enrollment of at least 1,100 patients, who would have to be observed for approximately 10 years (or until death).[70] In light of the low incidence of low-grade glioma (2,000 – 3,000 cases per year in the US), enrollment of a sufficient number of patients would necessitate multi-institutional–if not multinational–cooperation. To reduce institutional bias, data must be collected and analyzed at a central location. To ensure the validity of the trial, the extent of resection would have to be evaluated objectively by unbiased investigators (i.e. investigators not involved with patient care and blinded to patient outcomes) who would use volumetric measurements from T2-weighted or FLAIR MRI sequences. Primary end points should include OS, PFS, and QOL (including assessments of neuro cognitive function). Pre-planned statistical analyses should account for known prognostic factors, including age, preoperative tumor size, and histopathological subtype (including 1p and 19q deletion status). Moreover, analyses should take into account factors that limit the success of GTR, including diffuse tumor margin on T2-weighted MRI and involvement of eloquent areas (e.g. visual cortex and motor cortex). Finally, analyses must account for adjunctive therapies, including fractionated radiotherapy and chemotherapy. Interim analyses should be planned in case either biopsy or surgical resection proves to be superior on early analysis. Clearly, the coordination, data collection and statistical analysis that would be involved in such a trial would be technically difficult, but this method might be the only means of definitively answering the question regarding the role of surgical resection in the management of low-grade glioma.

• Conclusions

Although a ‘wait-and-see’ policy might not adversely affect patients with low-grade glioma, the emergence of novel therapeutic strategies has increased the importance of obtaining tissue for both histological and cytogenetic characterization, so as to optimize patient management. Although the benefit of tissue acquisition by either biopsy or cytoreductive surgery seems indisputable, the precise role of surgical resection— especially in patients without mass effect or symptoms of intracranial hypertension— remains unclear. The literature provides modest evidence that surgery improves patient outcomes by reducing tumor burden. Nonetheless, it is not possible to devise a prescribed treatment algorithm on the basis of the current level of evidence. Until prospective trials have been conducted and analyzed, neuro-oncologists and neuro surgeons must continue to individualize the evaluation and treatment of low-grade glioma on the basis of symptomatology, patient and tumor characteristics, and patient preference.

Key Points

1. Approximately 2,000 – 3,000 low-grade gliomas are diagnosed in the US every year, resulting in considerable morbidity and inevitable death
2. Management options for low-grade glioma include observation, radiotherapy, chemotherapy and surgical resection; the role of surgical resection is particularly controversial
3. The literature provides modest evidence that surgery improves outcomes by reducing tumor burden, but there is a paucity of class I evidence
4. Advances in neurosurgical and neuroimaging techniques have made surgical resection of low-grade gliomas a safe option for an increasing number of patients
5. In light of recent advances, the time might be right to effectively and accurately assess the effect of aggressive surgical resection on the prognosis of low-grade glioma

---

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The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

February 21, 2009 — The term neurodegenerative disease refers to the principal pathology associated with disorders such as amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease and Parkinson’s disease, and it is presumed that neurodegeneration results in the clinical findings seen in patients with these diseases. Decades of pathological and physiological studies have focused on neuronal abnormalities in these disorders, but it is becoming increasingly evident that astrocytes are also important players in these and other neurological disorders. Our understanding of the normative biology of astrocytes has been aided by the development of animal models in which astrocyte-specific proteins and pathways have been manipulated, and mouse models of neurodegenerative diseases have also revealed astrocyte-specific pathologies that contribute to neurodegeneration. These models have led to the development of targeted therapies for pathways in which astrocytes participate, and this research should ultimately influence the clinical treatment of neurodegenerative disorders.

Until relatively recently, astrocytes, along with other cells of the glial lineage such as oligodendrocytes and microglia, were believed to be structural cells, the main function of which was to hold neurons together. It is now known, however, that astrocytes serve many housekeeping functions, including maintenance of the extracellular environment and stabilization of cell–cell communications in the CNS. The function of astrocytes in regulating cerebral blood flow and maintaining synaptic function is becoming increasingly recognized as being of paramount importance in the maintenance of the neuronal environment. Astrocytes are also central to the maintenance of neuronal metabolism and neurotransmitter synthesis. Understanding these functions has allowed a refocusing with regard to the role of astrocytes in neurodegenerative diseases, which has led to astrocyte-specific analyses with potential for drug discovery.

• Cellular Functions of Astrocytes

We will begin by highlighting a subset of the many cellular functions of astrocytes, focusing specifically on those functions that have the most relevance to neurodegeneration (Figure 1). Other astrocytic functions, which are beyond the scope of this Review, include the regulation of cell volume, structural support, and the release of neurotransmitters other than glutamate.

Figure 1. Normal Functions of Astrocytes. (1)Astrocyte functions include modulation of synaptic function via glutamate transporters, which convey glutamate from the synaptic cleft into the cell.[1] (2)Communication between astrocytes occurs via ATP release and binding to purine receptors on adjacent astrocytes.[14] ATP binding results in phospholipase C activation, with subsequent downstream activation of inositol trisphosphate, resulting in calcium mobilization. (3)Gap junctions contribute to an astrocyte syncytium for the exchange of small molecules and cell–cell communication.[14] Metabolic functions include (4) the replenishment of neuronal glutamate via the glutamate–glutamine cycle, and (5) the transport of glucose from the vasculature.[1] (6)The regulation of blood flow is modulated by astrocyte end-feet apposing blood vessels, with vasodilation being mediated through release of vasoactive substances.[3,4] (7)Glutamate release might occur following elevations in intracellular calcium and the activation of other factors related to prostaglandins.[12] (8)Glutamate release through hemichannels can be induced in vitro through lowering of extracellular calcium.[13] (9)Glutamate binding to metabotropic glutamate receptors activates intracellular calcium, leading to the release of vasodilatory substances.[4] Abbreviations: Gln, glutamine; Glu, glutamate; IP3, inositol trisphosphate; PLC, phospholipase C(Click to enlarge figure).

• Amino Acid, Nutrient and Ion Metabolism in the Brain

Astrocytes are central to the catabolism of selected amino acids in the brain, as well as to the synthesis of new amino acids. The production of longer carbon backbones in the brain can only occur in astrocytes, owing to the selective localization of pyruvate carboxylase, the only brain enzyme capable of replenishing molecular intermediates for other metabolic reactions.

Astrocytes transport various nutrient and metabolic precursors to neurons via the malate–aspartate shuttle. One of the most important metabolic links between neurons and astrocytes, however, is the glutamate–glutamine shuttle. Astrocytes transport the vast majority of extracellular glutamate (especially neurotransmitter pools) and convert it to glutamine. This glutamine is shuttled back to presynaptic terminals, and is critical for the synthesis of the neurotransmitter glutamate. Astrocytes also convert glucose to lactic acid, which is subsequently taken up into neurons and converted to pyruvate for energy metabolism.[1]

Astrocytes have an important role in the regulation of ion concentrations in the intracellular and extracellular spaces in the brain. Carbon dioxide is produced by neurons following the oxidative metabolism of pyruvate. Astrocytes regulate acid–base balance via carbonic anhydrase, which converts carbon dioxide and water to hydrogen ions and bicarbonate ions. Extracellular potassium also accumulates from neural activity,[2] and buffering of potassium occurs through potassium channels expressed by astrocytes at synapses and at end-foot processes around capillaries.

• Coupling of Neuronal Activity and Cerebral Blood Flow

Evidence is accumulating that astrocytes have an important function in cerebrovascular regulation. Astrocytic processes have end-feet with contact to the brain vasculature, and they envelop neuronal synapses. The relationship between neurons, astrocytes and blood vessels makes astrocytes a central element that can modulate neuronal activity and cerebral blood flow. In vitro and in vivo studies of cortical tissue indicate that synaptic release of glutamate activates metabotropic glutamate receptors on astrocytes. These receptors trigger the release of arachidonic acid metabolites,[3] leading to a localized increase in calcium at astrocyte end-feet, which results in dilation of nearby arterioles.[4]

• Modulation of Excitatory Synaptic Transmission

Glutamate is the primary excitatory neurotransmitter in the CNS, and its activity is carefully regulated by both neuronal and glial influences. The majority of synaptic and perisynaptic glutamate regulation occurs through glutamate transporters. In addition to the tightly coupled synaptic relationship between neuronal synapses and astrocytes, astrocyte-to-astrocyte transmission through gap junctions, as well as paracrine release of ATP, might modulate synaptic biology.

Regulation of Glutamate Transport. Glutamate transport is a sodium- and potassium-coupled process that is capable of concentrating intracellular glutamate more than 10,000-fold compared with the extracellular environment. The glutamate transporters GLAST and GLT1 (EAAT1 and EAAT2 in humans; also known as EAA1 and EAA2) are localized primarily on astrocyte membranes.[5-8] Antisense knockdown studies showed that these two glial transporters are responsible for over 80% of glutamate uptake in the brain,[9] an observation that was later confirmed in GLT1 (Slc1a2)-null mice.[10]

Release of Glutamate. Although glutamate is the primary neuronal excitatory neurotransmitter in the brain, evidence also exists for an astrocytic role in glutamate release. In vitro preparations have demonstrated astrocyte-specific glutamate release via exocytosis.[11,12] Certain conditions, such as low levels of extracellular calcium, can trigger glutamate release through a separate mechanism, namely hemichannels—a single cell’s contribution to a gap junction.[13] These findings are intriguing in that they suggest an additional layer of fine-tuning of the perisynaptic environment modulated by astrocytes.

Propagation of Glutamatergic Transmission via the Astrocyte Network. Current evidence indicates that propagation of glutamate transmission through the astrocyte syncytium occurs through two prominent calcium-mediated mechanisms: one involving gap junctions and the other involving paracrine release of ATP. Activation of metabotropic glutamate receptors on astrocytes following neuronal release of glutamate results in the activation of an inositol trisphosphate (IP3) pathway, which induces calcium release from intracellular stores. This calcium can then be transferred to the adjacent astrocyte through connexin 43 (Cx43) gap junctions, thereby producing a calcium wave through an astrocyte syncytium. IP3 also activates ATP release through Cx43 hemichannels. This ATP release acts in a paracrine fashion, activating purine receptors on adjacent astrocytes. This activation results in IP3 production, more ATP release and intracellular calcium mobilization through a feed-forward mechanism.[14]

• Astrocyte-Specific Pathology: Lessons From Mouse Models

Transgenic and knockout mice have been valuable in helping us to understand the pathophysiology of neurodegenerative disorders. Transgenic models for specific neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Huntington’s disease (HD) and Parkinson’s disease (PD) have been developed, and these models are reviewed later in this article. Some examples of mouse models in which astrocyte protein function is altered are detailed in Table 1 .

Table 1. Mouse Models With Altered Astrocyte Protein Function (Click to enlarge table).

Abbreviations: GFAP, glial fibrillary acidic protein; Vim, vimentin.

• Manipulations of the Astrocytic Structural Protein Glial Fibrillary Acidic Protein

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is known to be localized to astrocytes, although its precise contributions to astroglial physiology and function are not clear. The upregulation of GFAP following injury and astrogliosis has been a long-standing pathological observation. Deletion of GFAP in mice did not result in any specific pathology in the absence of injury, but some modest abnormalities were observed following specific injury paradigms.[15] Interestingly, the overexpression of human wild-type GFAP in mice produced pathology consistent with that observed in Alexander disease.[16] The deletion of both GFAP and vimentin, another intermediate filament protein, resulted in more-marked pathology following injury, including a greater loss of neuronal synapses, although it also led to improved regenerative potential.[17] These findings suggest that the significance of GFAP is magnified following injury in which cellular stability might be affected, but that other intermediate filaments have overlapping functions with GFAP.

• Disruption of Astroglial Synaptic Regulation: Glutamate Transporter Knockout Mice

One of the pivotal functions of astrocytes is the maintenance of glutamate homeostasis in the CNS, and particularly at the synapse. In mice that are null for the sodium-dependent glutamate transporter GLT1, less than 5% of glutamate transport is preserved. Phenotypically, this mechanism is manifested in the development of seizures accompanied by a reduction in survival in animals after birth. These mice were more prone to acute brain injury, and to an increase in seizure activity and neuronal loss induced by the ?-aminobutyric acid (GABA) antagonist pentylenetetrazole, primarily in the CA1 region of the hippocampus.[18] The susceptibility to neural injury was subsequently highlighted by data documenting baseline elevations in hippocampal CA1 glutamate levels in microdialysate—as well as an abnormal rise in glutamate—in this model following ischemic injury.[19] By contrast, spontaneous seizures were not observed in GLAST (Slc1a3) knockout mice, but appeared to require a second ‘insult’. More-severe episodes of pentylenetetrazole-induced seizure activity were observed in GLAST knockout mice when compared with wild-type mice.[20]

Although no mutations have been observed in the coding sequence of the EAAT2 (SLC1A2) gene in humans, loss of EAAT2 protein is seen in neurodegeneration. Mutations in the EAAT2 promoter are associated with increased serum glutamate and worse outcomes following cerebrovascular insults, and one particular polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke.[21]

• Ablation of Reactive Astrocytes: GFAP-TK Mice

In addition to cellular hypertrophy and upregulation of specific genes associated with reactive astrocytosis, astrocyte proliferation is also observed following some insults. The GFAP-TK mouse is a transgenic model in which dividing, reactive astrocytes that emerge after CNS injury can be selectively ablated by the administration of ganciclovir. In a stab-and-crush model of spinal cord injury, this ablation of reactive astrocytes resulted in a combination of local tissue disruption, leukocyte infiltration, neuronal and oligodendrocyte death, and motor impairment. These findings indicate that astrocyte proliferation is protective in spinal cord injury, and they demonstrate that the process of proliferation has distinct consequences besides, or in addition to, cellular hypertrophy and upregulation of astrocyte-specific genes.[22]

• Loss of Astrocyte–Astrocyte Communications: Connexin 43 Knockout Mice

Astrocytes have a unique form of intercellular syncytial communication with neighboring astrocytes through gap junctions, which are primarily composed of Cx43 protein.[23] Studying the role of Cx43 in adult animals has proved to be difficult, because Cx43 null mice die at birth, probably from cardiopulmonary insufficiency.[24] Cx43 heterozygous mice have, therefore, been used for in vivo studies of this protein. These mice showed a significantly increased stroke volume compared with wild-type mice following middle cerebral artery occlusion.[25] Future studies using this model might help to elucidate the function of Cx43 in neurodegenerative disorders.

• Astrocytes in Neurodegenerative Disease

The potential effects of astrocyte dysfunction in neurodegenerative diseases are summarized in Figure 2.

Figure 2. Potential Astrocyte Dysfunction in Neurodegenerative Diseases. (1)Impairment of glutamate transporters, through reduced expression, aberrant RNA synthesis or altered function, results in increased synaptic glutamate and excitotoxicity.[64] (2)Amyloid-ß could potentially increase the amount of ATP released by astrocytes, as well as interacting with gap junctions to alter calcium signaling and glial communication.[43] (3)Upregulation of glial fibrillary acidic protein is a consistent pathological feature in neurodegenerative diseases, although the significance of this observation is not completely understood.[15] (4)Nitric oxide stimulates the release of glutathione from astrocytes to neurons, thereby increasing neuronal antioxidant reserves and limiting oxidative damage to neuronal mitochondria.[59] (5)Mutations in glial fibrillary acidic protein associated with Alexander disease result in the development of Rosenthal fibers, and lead to disordered intermediate neurofilament organization.[65] (6)Neurons surrounded by mutant astrocytes develop protein aggregates and axonal pathology, and are more susceptible to cell death in several neurodegenerative disease models, including amyotrophic lateral sclerosis, Alzheimer’s disease and Huntington’s disease.[36,47,56] (7)Mutant huntingtin expressed in astrocytes forms intranuclear aggregates and also influences neuronal cell death in vitro.[56] (8)Amyloid-ß42-positive material is seen within activated astrocytes in the tissues of individuals with Alzheimer’s disease, and is abundant in regions with the most active Alzheimer’s disease pathology.[40] (9)Accumulation of mutant superoxide dismutase 1 is also observed in astrocytes.[68] (10)Finally, communication between abnormal or injured astrocytes might affect the biology and function of surrounding normal astrocytes, through either hemichannels or soluble mediators.[25] Abbreviations: Aß, amyloid-ß; GFAP, glial fibrillary acidic protein; GSH, glutathione. (Click to enlarge figure).

• Amyotrophic Lateral Sclerosis

ALS is the most common form of adult motor neuron disease. This condition is characterized by progressive degeneration of the upper motor neurons in the cortex and the lower motor neurons in the brainstem and spinal cord. Approximately 95% of reported cases of ALS are apparently sporadic. The remaining 5% of ALS patients inherit the disease, and this is classified as familial ALS. In 1993, chromosome-21-linked familial ALS was found to be associated with mutations in SOD1, the gene that encodes copper–zinc superoxide dismutase;[26] since then, mutations in several other genes, including those encoding dynactin, alsin and senataxin, have been implicated in familial ALS.

Human Studies. Common to familial and sporadic ALS is the loss of the astrocyte glutamate transporter EAAT2. Studies of the EAAT2 transporter in tissue from individuals with sporadic ALS showed a marked loss of up to 95% of astroglial EAAT2 protein expression and activity in affected areas of the CNS.[27] A clue to a possible mechanism for EAAT2 reduction or dysfunction was provided by the finding of aberrant EAAT2 RNA species, which has been implicated in multiple neurodegenerative diseases. The production of truncated EAAT2 protein results in reduced function, and the retention of normal EAAT2 protein within the cytoplasm.[28] The significance of these aberrant EAAT2 RNA species continues to be debated, however, as they have also been found in some normal controls.[29,30]

Animal Models. Transgenic mouse and rat models carrying several SOD1 mutations have been generated; forms of SOD1 (SODC) protein with the point mutations G93A, G37R and G85R all produce reliable motor neuron degeneration when they are overexpressed in transgenic mice.[31-33] At present, these mouse lines are the most reliable and accurate animal models of ALS, and they have been used extensively in an attempt to understand how mutant SOD1 (mSOD1) causes cell death. As with the human disease, motor neurons are selectively affected, although this selectivity is not absolute—small interneurons also degenerate in the mouse models.

In both human tissue and transgenic models of ALS, there is abundant evidence that astroglial abnormalities and physiological dysfunction precede clinical disease. These changes include reactive astrocytosis that can be seen many months before motor neuron degeneration (G85R),[33] and loss of glutamate transport and GLT1 protein expression before the onset of clinical disease or overt motor neuron degeneration.[34] Is the reduction in GLT1 protein in astrocytes significant? Guo and colleagues addressed this question by overexpressing the EAAT2 protein in astrocytes in the mSOD1 mouse model, and demonstrated an increase in motor neuron survival and a delay in disease onset; similar outcomes are seen with drugs that increase GLT1 expression.[35] This evidence indicates that EAAT2 expressed in astrocytes—and probably also glutamate—influences the timing of disease onset and motor neuron survival.[35] Other changes associated with ALS include increased expression of various proteins in astrocytes, including inducible nitric oxide synthase (iNOS), the copper chaperone CCS, and metallothioneins. Pathologically, early cytosolic proteinaceous aggregates have been found in spinal cord astrocytes from all of the mSOD1 mouse lines examined to date.[26]

Until relatively recently, it was not clear whether the pathological changes observed in astrocytes (and other cells) in the mSOD1 mouse model were in response to initial neuronal injury, or whether mSOD1 in non-neuronal cells (i.e. astrocytes) could influence disease. In chimeric mice, expression of mSOD1 in neurons or motor neurons alone was not sufficient to lead to neuronal death—there had to be concomitant expression in glia. Furthermore, the chimeric wild-type–mSOD1 animals lived longer than nonchimeric mSOD1 mice, to a degree that was proportional to the percentage of wild-type cells present.[36] Another powerful observation from this study was that wild-type motor neurons appeared to undergo degeneration, and developed ubiquitinated inclusions, when surrounded by mSOD1-expressing astroglia. Interestingly, the delivery of small interfering RNA (siRNA) targeting human SOD1 to motor neurons by injection into the muscles of mSOD1 mice and allowing retrograde transport resulted in a delay in (but not complete sparing from) disease onset, and prolongation of survival.[37,38] These findings, although noted to indicate a potential for therapeutic interventions, also highlight the fact that mSOD1 mice still eventually develop disease, and again emphasize a role for other cell types besides motor neurons in ALS pathogenesis.

• Alzheimer’s Disease and the ‘Tauopathies’

AD is characterized clinically by cognitive loss in two or more domains, including memory, language, calculations, orientation and judgment; the loss must be of sufficient severity to cause social or occupational disability. These clinical features are the result of neuronal death and dysfunction in the cerebral cortex, entorhinal area, hippocampus, ventral striatum and basal forebrain, eventually resulting in severe dementia. Pathologically, the two hallmark findings of the disorder are neurofibrillary tangles and amyloid plaques.[39]

Human Studies. In tissue from individuals with AD, activated astrocytes were closely associated with amyloid plaques in the molecular layer of the cerebral cortex.[40] Astrocytes might be activated by human amyloid-ß (Aß),[41] indicating a correlation between this protein and subsequent alterations in astrocyte function. Astrocytes also accumulate neuron-derived amyloid material resulting from local neurodegeneration. Once substantial accumulation of this debris occurs, the astrocytes themselves might undergo cell death, resulting in the formation of GFAP+ amyloid plaques.[42]In vitro analyses also indicate that treatment of astrocytes with Aß results in an increase in calcium-wave signaling between these cells.[43] In cells expressing the familial AD presenilin 1 (PSEN1) mutation, calcium oscillations in astrocytes were found to occur at lower ATP and glutamate concentrations than in wild-type astrocytes.[44] These data support a model in which calcium signaling between astrocytes is altered by the disease process, which might, in ways that are not fully understood, contribute to dysfunction or death of neurons.

Animal Models. One of the hallmark features of AD and other ‘tauopathies’ is the accumulation of tau protein in neurons and glia.[45,46] This pattern contrasts markedly with the normal CNS distribution, in which tau is expressed predominantly in axons, and is only expressed at low levels in oligodendrocytes and astrocytes. To assess the contribution of astrocytes to tauopathies, transgenic mice were generated in which the tau protein was expressed selectively in astrocytes. In these mice, there was abundant astrocyte tau pathology associated with neuronal staining of phosphorylated neurofilament epitopes, axon degeneration, and inclusion formation, all of which indicated neuron injury; however, no significant neuronal loss was observed.[47] In an extension of these initial observations, investigators developed transgenic mice overexpressing the tauP301L mutation—which is linked to frontotemporal dementia and parkinsonism (FTDP) in humans—in astrocytes. These mice developed neuromuscular abnormalities with loss of strength. The astrocyte tau pathology was also associated with a reduction in expression and function of the astrocyte-specific glutamate transporters GLT1 and GLAST.[48] The selective tau expression in astrocytes in these models provides more evidence of an astrocyte-mediated effect in models of dementia.

• Huntington’s Disease

HD is a fatal autosomal dominant neurodegenerative disease that results from an expanded DNA segment containing a polymorphic trinucleotide CAG repeat in the gene that encodes the protein huntingtin. The most striking neuropathological changes are gross atrophy of the caudate nucleus and putamen, with concomitant marked neuronal loss and astrogliosis. A selective vulnerability of medium-sized striatal spiny projection neurons with a relative sparing of spiny striatal interneurons is also seen.[49]

Human Studies. As with other neurodegenerative disorders, astrocytosis is observed in affected regions of the brain of patients with HD. The huntingtin protein co-localizes with these reactive astrocytes in specific regions.[50] In a small study of three brains from individuals with HD analyzed postmortem, EAAT2 messenger RNA was reduced in the neostriatum, and the degree of reduction correlated with disease severity. The losses were found to be particularly prominent in the putamen, and less so in the caudate.[51] Other studies have failed to find significant changes in glutamate transport in the brains of patients with HD,[52] and more-conclusive implications for the biology of glutamate transporters in human HD are awaited.

Glutamate excitotoxicity in HD has been hypothesized to result from the failure of astrocytic functions that require cell–cell coupling to maintain their syncytial network and contribute to metabolic homeostasis. Alterations in gap junction expression or uncoupling of gap junctions between astrocytes would cause astrocytes to lose their ability to maintain a proper neuronal environment. In the caudate nucleus (a region with prominent HD pathology), however, Cx43 density was increased with HD, and became located in patches and accompanied by increased GFAP expression.[53] This observation raises several questions, such as why is there an increase in Cx43 expression?; could this increase result in enhanced astrocyte coupling in an attempt to provide an increased spatial buffer capacity?; and is this a neuroprotective response by astroglia?

Animal Models. In a transgenic model of HD, expression of the polyglutamine repeat protein results in a movement disorder with neuronal pathology. A reduction in the messenger RNA levels of GLT1 in the striatum and cortex of these mice was observed, and this was accompanied by a decrease in glutamate uptake. Because downregulation of GLT1 in denervated regions would normally be expected, as described above in experimental models of denervation, the authors were careful to note that the changes in GLT1 expression occurred before any neurodegeneration, thereby potentially implicating GLT1 in part of a neuronal death cascade.[54] Similar findings have been reported in the R6/2 transgenic mouse, which expresses an N-terminal fragment of mutant huntingtin.[55]

Mutant huntingtin protein is known to aggregate in the neurons of transgenic mouse models of HD. Recent evidence, however, indicates that mutant huntingtin is also present in the nuclei of astrocytes, a phenomenon that becomes more robust with age and corresponds with a downregulation of glutamate transporters in these cells. A potential cause-and-effect relationship implicating astrocytes in the neurotoxicity observed in HD was noted following the observation in a neuron–glial co-culture that wild-type glial cells protected neurons against mutant huntingtin-mediated neurotoxicity, whereas glial cells expressing mutant huntingtin increased neuronal vulnerability.[56] Taken together, these observations indicate that mutant huntingtin in glial cells can contribute to neuronal dysfunction and excitotoxicity in HD brains through disorders of astroglial biology.

• Parkinson’s Disease

PD is the second most prevalent neurodegenerative disease, after AD. PD is estimated to affect about 1 million Americans, or about 1% of the population over 60 years of age. PD is caused by the disruption of dopaminergic neurotransmission in the basal ganglia. On pathological examination, the numbers of dopaminergic neurons in the substantia nigra are markedly reduced, and Lewy bodies (cytoplasmic inclusions) are present in the residual dopaminergic neurons.[57] The focus has always been on the loss of these dopamanergic neurons and subsequent depletion of dopamine, but a role for non-neuronal cells in producing neuropathological or neuroprotective functions in PD is becoming increasingly recognized.

Human Studies. The studies that have been carried out to date appear to support a neuroprotective role for astrocytes in PD. From pathological examinations, an increase in the number of astrocytes as well as in GFAP expression is observed in PD, as with other neurodegenerative disorders.[58] The pathological evidence indirectly indicates that antioxidant pathways might contribute to this neuroprotective effect, because in control brains the density of glutathione-peroxidase-positive cells was higher in the vicinity of the dopaminergic cell groups known to be resistant to the pathological process of PD. The increase in glutathione-peroxidase-containing cells was inversely correlated with the severity of dopaminergic cell loss in the respective cell groups in patients with PD. The quantity of glutathione-peroxidase-containing cells, therefore, might be critical for a protective effect against oxidative stress.[59] Conversely, the presence of synuclein-positive astrocytes in pathological samples has been shown to correlate with nigral neuronal cell death.[60]

Animal Models. What is the timing of astrocytosis in animal models of PD? In a PD model generated by lesioning the brain with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), it appears that astrocytosis occurs after the death of dopaminergic neurons, and that this response remains elevated even after most dopaminergic neurons have died.[61] A more rapid response consisting of increased GFAP immunoreactivity as early as 1 hour following the injection of 6-hydroxydopamine into the nigrostriatal dopamine bundle has been observed, indicating a more direct effect of this compound on astrocytosis. Several pathways for this neuroprotection have been implicated, including the increased activation of astrocytes and neuroprotection in 6-hydroxydopamine models following infusion of interleukin-1ß (a cytokine released by activated microglia) into the substantia nigra.[62]

Nitric oxide production and glutathione depletion also appear as consistent features in human PD. The release of glutathione represents another pathway by which astrocytes might be neuroprotective in PD models. Glutathione production appears to be increased by exposure of astrocytes to nitric oxide, and the increase in glutathione release by astrocytes might increase its availability to neurons, thereby making them less susceptible to reactive nitrogen species. This pattern is consistent with the data in PD patients, in whom glutathione-containing cells are in regions with preserved dopaminergic neurons.[63]

Evidence regarding regulation of glutamate transporter expression and function in PD has been somewhat mixed, with downregulation of glutamate transporters being reported in some studies and upregulation being reported in others. The differences in these studies might be related to the methods by which the lesions were induced.[64]

• Alexander Disease

Alexander disease is a neurodegenerative disorder that is seen predominantly among the pediatric population. The disorder is associated with a prominent leukoencephalopathy, seizures, and death in the first decade of life. Adult forms have also been described, with ataxia and spasticity. As described above, some of the first suggestions that astrocyte-specific proteins (notably GFAP) were part of the disease pathogenesis came from GFAP overexpression in mouse models. Pioneering work from Brenner, Messing and colleagues subsequently demonstrated GFAP mutations in patients with the disorder.[65] The patients with Alexander disease were heterozygous for the mutations, and none of the parents of these patients carried similar mutations. These data indicate that Alexander disease arises from new mutations in GFAP, and that these mutations act in a dominant fashion. Furthermore, the Rosenthal fibers that are an important pathological observation in Alexander disease place this disease among others, including ALS, AD and PD, in which protein aggregation is a prominent feature.[66]

• Astrocytes as Potential Therapeutic Targets

One focus of therapeutics in neurodegenerative disease has been replacement strategies for neurotransmitters, such as levodopa (a dopamine precursor) for PD, or memantine (a glutamate receptor [N-methyl-D-aspartic acid] antagonist), for the treatment of AD. A large body of work has been devoted to neuroprotective strategies, with enormous basic science and clinical efforts devoted to treatments that are effective in ‘neuronal’ cultures. With our increased understanding of the relationship between neurons and glia, however, targeted therapeutics for pathways in which astrocytes have a prominent position, as outlined in this Review, need to be developed. It is also likely that therapeutics that are effective at neuroprotection also enhance astrocyte–neuron interactions, but this possibility has not been fully investigated.

One example of recent efforts to influence astrocyte function in neurodegenerative disease was a clinical trial of ceftriaxone, a third-generation cephalosporin, in ALS. This antibiotic was demonstrated to increase astroglial gene expression and glutamate transporter expression and function in several in vitro and in vivo models, as well as in transgenic models of chronic neurodegeneration (mSOD1 mouse models).[67]

Future directions might also include modalities to examine astrocyte function in vivo. In vivo imaging of astrocyte–astrocyte communication (e.g. calcium waves) has already been carried out in living rodents. Tools to examine astrocyte function (e.g. PET imaging ligands) will also be valuable for humans, and are already being developed. With the development of animal models for understanding astrocyte biology and pathways involved in astrocyte dysfunction, new paradigms for the modulation of astroglial function might emerge as therapeutic strategies.

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The author: Professor Yasser Metwally

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INTRODUCTION

February 20, 2009 — B cells have a fundamental role in the pathogenesis of various autoimmune neurological disorders, not only as precursors of antibody-producing cells, but also as important regulators of the T-cell activation process through their participation in antigen presentation, cytokine production, and formation of ectopic germinal centers in the intermeningeal spaces. Two B-cell trophic factors—BAFF (B-cell-activating factor) and APRIL (a proliferation-inducing ligand)—and their receptors are strongly upregulated in many immunological disorders of the CNS and PNS, and these molecules contribute to clonal expansion of B cells in situ. The availability of monoclonal antibodies or fusion proteins against B-cell surface molecules and trophic factors provides a rational approach to the treatment of autoimmune neurological diseases. This post reviews the role of B cells in autoimmune neurological disorders and summarizes the experience to date with rituximab, a B-cell-depleting monoclonal antibody against CD20, for the treatment of relapsing-remitting multiple sclerosis, autoimmune neuropathies, neuromyelitis optica, paraneoplastic neurological disorders, myasthenia gravis, and inflammatory myopathies. It is expected that ongoing controlled trials will establish the efficacy and long-term safety profile of anti-B-cell agents in several autoimmune neurological disorders, as well as exploring the possibility of a safe and synergistic effect with other immunosuppressants or immunomodulators.

During the past three decades, investigations into neuroimmunological diseases of the CNS—and to a lesser degree the PNS—have centered predominantly on the roles of activated, cytotoxic and immunoregulatory T cells rather than B cells. The decision to focus on T cells can probably be attributed to the long-standing observation that the main lymphocytic subset within the lesions in the two most common autoimmune disorders, multiple sclerosis (MS) and Guillain-Barré syndrome, are dominated by T-cell infiltrates. In addition, myelin-specific T cells are responsible for disease transfer in the respective animal models for these conditions. The contribution of activated B cells to these disorders has traditionally been viewed as a secondary consequence of the breakdown of T-cell tolerance. Over the past few years, however, compelling data on the roles of B cells as sensors, coordinators and regulators of the immune response[1] have strengthened the view that B cells and autoantibodies are fundamental for activating T cells and/or mediating tissue injury in several disorders of the CNS and PNS. The observation that B-cell depletion is an effective therapy in autoimmune disorders such as rheumatoid arthritis has provided the impetus to explore the functions of B cells in neurological diseases, and has triggered an interest in conducting clinical trials in this area.

This Review focuses on B-cell homeostasis, addresses the roles of B-cell functions in autoimmune neurological disorders, and summarizes the experience to date with anti-B-cell therapies, in particular the B-cell-depleting monoclonal antibody rituximab.

• Roles of B Cells in the Immune Response: Neurological Aspects

In the context of autoimmune neurological disorders, B cells have traditionally been associated with the production of autoantibodies from plasma cells, the end products of B-cell differentiation.[2-5] In several neurological diseases, including myasthenia gravis and certain neuropathies, the autoantibodies are pathogenetic, exerting a direct effect on self antigens either by functioning as neutralizing antibodies or by activating and fixing complement on the targeted tissues (Figure 1A). Autoantibodies and immune complexes can also activate Fc receptors on macrophages or dendritic cells, leading to the production of cytokines, which cause further tissue injury (Figure 1A). In most autoimmune neurological disorders, however, the autoantibodies are directed against cytosolic antigens and might not be directly involved in tissue injury. In such cases, B cells might still participate in the autoimmune process through antibody-independent mechanisms that include antigen presentation, costimulation, cytokine production, and coordination of T-cell functions (Figure 1A-D).[4,6]

Figure 1. B-cell functions in neurological disorders. The figure shows the four main functions of B cells through which they contribute to the pathology of immune-mediated neurological conditions. (A) Production of antibodies that cause tissue damage either via complement activation or antibody-dependent-cell mediated cytotoxicity. (B) Acting as antigen-presenting cells, which results in clonal expansion of cytotoxic T cells and cytokine production. (C) Production of proinflammatory cytokines, such as IL-6, TNF and IL-10, which activate macrophages and T cells and enhance tissue damage. (D) De novo formation and maintenance of ectopic germinal centers in the intermeningeal spaces (neolymphogenesis). Abbreviations: IL = interleukin; LTßR = lymphotoxin-ß receptor; LTß = lymphotoxin-ß; TNF = tumor necrosis factor. (Click to enlarge figure)

A proof-of-principle that activated B cells are fundamental for coordinating T-cell functions was provided by the observation that B-cell-depleted mice exhibit a dramatic decrease in numbers of CD4+ and CD8+ T cells, and a tenfold inhibition of memory CD8+ T cells.[7,8] An important function of B cells is their ability to present antigenic peptides, in the context of major histocompatibility complex class II molecules on their surface, to the T-cell receptors of CD4+ cells, leading to expansion of antigen-specific T cells (Figure 1B).[1-6] B cells are 100-1,000 times more potent in antigen presentation than are the other antigen-presenting cells, such as macrophages or dendritic cells,[9] and they are especially effective at presenting low concentrations of antigen. Activated B cells are also as efficient as T cells at producing cytokines—most notably interleukins (IL-1, IL-4, IL-6, IL-10, IL-12, IL-23 and IL-16), tumor necrosis factor (TNF) and the chemokines macrophage inflammatory protein 1a (MIP1a) and MIP1ß.[10,11] These inflammatory mediators modulate the migration of dendritic cells, activate macrophages, exert a regulatory role on T-cell functions, and provide feedback stimulatory signals for further B-cell activation (Figure 1C). Some cytokines might theoretically exert an inhibitory role in the immune process, although this has not been clearly established in human diseases.

An additional role of B cells that is relevant to neurology is their involvement in de novo formation and maintenance of ectopic lymphoid structures, a process termed neolymphogenesis.[6] This is accomplished through the actions of ß-lymphotoxin, a TNF family member molecule that is expressed on the surface of B cells (Figure 1D). Ectopic follicular structures are found in the meningeal compartment in patients with various neuroinflammatory conditions, as discussed below. The multiple contributions of B cells to the complexity of the autoimmune process make B cells attractive targets for therapeutic interventions that extend beyond the traditional effects on antibody production.

• B-cell Maturation and Homeostasis

B lymphocytes arise from hematopoietic stem cells in the bone marrow. These cells mature independently of an antigen first into pro-B cells, then into pre-B cells and immature B cells (Figure 2).[4,5] They subsequently enter the antigen-dependent phase in the peripheral lymphoid tissues, where mature-but-naive B cells, after encountering their antigen in the extrafollicular regions of the lymphoid organs, become activated B cells and migrate to the follicular regions. From here, they exit to differentiate into memory B cells, late plasmablasts and plasma cells (Figure 2).[1-6] Specific markers, such as CD20, CD27, BAFF-R (B-cell-activating factor receptor), CD38 and CD138, identify the transitional phases of B cells from stem cells to plasma cells (Figure 2).

Figure 2. Maturation of B cells. The B-cell maturation process involves two phases of differentiation-an antigen-independent process in the bone marrow, and an antigen-dependent process that occurs not only in the lymphoid tissue but also in the brain. Specific CD (cluster of differentiation) markers such as CD20, CD27, BAFF-R, CD38 and CD138 are helpful for distinguishing the transitional phases, including stem cells, memory B cells and plasma cells, through which B cells pass during maturation. Abbreviation: BAFF-R = B-cell-activating factor receptor.(Click to enlarge figure)

The memory B cells, late plasmablasts and long-lived plasma cells migrate not only to the bone marrow, spleen and lymphoid tissues, but also to the brain, where they transform into antibody-secreting cells after encountering their antigen (Figure 3).[12] Interactions between the homeostatic chemokines CXC-chemokine ligand (CXCL) 13, CXCL10 and CXCL12 secreted from the endothelial cell wall and their respective receptors on B cells[3,13] are fundamental for B-cell homeostasis not only within the lymphoid follicles but also within the brain. These molecules are upregulated in the brains of patients with MS, allowing the recruitment and transmigration of antibody-producing B cells into the brain.[14,15] B-cell transmigration into the brain is also facilitated by the adhesion molecules very late antigen-4 (VLA-4; also known as integrin a-4 or ITA4) and lymphocyte function-associated antigen-1 (LFA-1; also known as integrin a-L or ITAL) and their counter-receptors vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) on the endothelial cells.[13] In secondary progressive MS, activated B cells form germinal centers not only in the lymphoid tissues but also within the intermeningeal spaces, where they undergo the same stages of differentiation as in the periphery (Figure 3).[16,17] Within these structures, which are observed in 41.4% of patients with secondary progressive MS,[17] B cells generate inflammatory mediators that can stimulate plasma cells for in situ production of immunoglobulins. The production of intrathecal immunoglobulins (i.e. the life-long persistent oligoclonal bands) in all forms of MS indicates a central role for activated B cells and plasma cells in this disease.

Figure 3. Activated B cells in the circulation and brain. Activated B cells-including memory B cells and plasmablasts-from the circulation migrate, after encountering their antigen, to the bone marrow and lymphoid tissue, where they are transformed into ASCs. These cells can also enter the CNS compartment and transform into ASCs within the brain. B-cell recruitment and transmigration depends on interactions between molecules such as CXCR5 and VLA-4 expressed on B cells and molecules such as CXCL13 and VCAM1, respectively, which are expressed by the endothelial cell walls of both the lymphoid tissue and the meningeal spaces, where germinal centers are formed. The upregulation of BAFF and APRIL and the secretion of these molecules by astrocytes within the brain promote the in situ clonal expansion of B cells. Abbreviations: APRIL = a proliferation-inducing ligand; ASC = antibody-secreting cell; B = B cell; BAFF = B-cell-activating factor; BAFF-R = B-cell-activating factor receptor; BCMA = B-cell-maturation antigen; CXCL = CXC-chemokine ligand; CXCR = CXC-chemokine receptor; ICAM1 = intercellular adhesion molecule 1; LFA-1 = lymphocyte function-associated antigen-1; TACI = transmembrane activator and calcium modulator and cyclophilin ligand interactor; VCAM1 = vascular cell adhesion molecule 1; VLA-4 = very late antigen-4..(Click to enlarge figure)

Two members of the TNF family, BAFF (B-cell-activating factor) and APRIL (a proliferation-inducing ligand), have emerged as crucial factors for B-cell survival, differentiation, germinal center formation and immunoglobulin production.[3,18,19] BAFF and APRIL are produced by monocytes, macrophages and dendritic cells, and they circulate in trimeric forms. They bind to B cells through three different receptors ( Table 1 ): BAFF-R, BCMA (B-cell-maturation antigen) and TACI (transmembrane activator and calcium modulator and cytophilin ligand interactor). Levels of BAFF-R and APRIL mRNA are increased in the monocytes and B cells of patients with MS[20] and in the muscles of patients with inflammatory myopathies (Raju R and Dalakas MC, unpublished data). In MS lesions, BAFF and APRIL are produced by astrocytes, and they promote the in situ survival and clonal expansion of B cells (Figure 3).[21,22] Agents that target BAFF or APRIL might, therefore, exert therapeutic effects in various neurological disorders by suppressing B-cell proliferation.

Table 1. B-cell Trophic Factors and Their Receptors in Autoimmune Neurological Disorders

B-cell trophic factor	Receptors	Functions of trophic factor	Evidence for roles in neurological disorders
BAFF	BAFF-R BCMA TACI	Promotes B-cell survival and differentiation, sustains germinal centers, and enhances immunoglobulin production Promotes lymphocyte maturation and immunoglobulin production Stimulates plasma cell survival and IgM production	Upregulated in the brain of patients with MS; produced by astocytes in vitro; BAFF mRNA and BAFF-R are upregulated in the brains of patients with MS; BAFF mRNA is increased in the muscles of patients with inclusion body myositis
APRIL	TACI BCMA	Stimulates plasma cell survival and IgM production	TACI mRNA is upregulated in the brain and monocytes of patients with MS; TACI is produced by astrocytes of patients with MS

Abbreviations: APRIL = a proliferation-inducing ligand; BAFF = B-cell-activating factor; BAFF-R = BAFF receptor; BCMA = B-cell-maturation antigen; MS = multiple sclerosis; TACI = transmembrane activator and calcium modulator cytophilin ligand interactor.

• Anti-B-cell Therapy in Neurology: Present and Future

The evidence that B cells have a role in autoimmune neurological disorders is summarized in Box 1 . Various immunomodulatory drugs that are currently used in neurology, such as intravenous immunoglobulin (IVIg), alemtuzumab, cyclophosphamide, mitoxanthrone and natalizumab, can affect some aspects of B-cell function that are relevant to the pathogenesis of neurological disease. New monoclonal antibodies or fusion proteins that specifically target B-cell survival or proliferation are, however, now becoming available.

Box 1. Observations Supporting a Role for B Cells in the Pathogenesis of Autoimmune Neurological Disorders

B cells are clonally expanded within the CNS, producing intrathecal immunoglobulin (IgG), in various CNS disorders such as multiple sclerosis (MS), paraneoplastic CNS disorders and stiff-person syndrome B cells, plasma cells, myelin-specific IgG and complement are present in the active and chronic plaques of MS IgGs specific for myelin oligodendrocyte glycoprotein and myelin basic protein are detected in the brains of individuals with MS Memory B cells, along with early, late or short-lived plasmablasts, are detected in the cerebrospinal fluid and ectopic germinal centers in the meninges of patients with MS B cells are required for disease induction by antigenic peptides in some experimental autoimmune encephalomyelitis and experimental autoimmune neuritis models, a requirement consistent with the unique ability of B cells to recognize antigenic conformation B cells have a role in the regulation of CNS inflammation Autoantibodies against glycolipids and glycoproteins can induce demyelination within the PNS T-cell-dependent B-cell activation leads to production of pathologic autoantibodies in myasthenia gravis Several antibody-mediated neurological disorders have been successfully treated using plasmapheresis or intravenous immunoglobulin, which remove autoantibodies or modify the idiotypic repertoire New therapeutic monoclonal antibodies such as rituximab that deplete B cells can result in clinical improvement when used in certain CNS or PNS disorders

• Agents Affecting B-cell Survival

Drugs that target BAFF or APRIL, or their receptors BAFF-R, TACI or BCMA, affect B cell survival and differentiation, resulting in reduced numbers of mature B cells in the lymphoid tissues and the circulation (Figure 4).[19] Blockade of BAFF-R, TACI or BCMA in mouse models of systemic lupus erythematosus (SLE) not only reduces antibody titers, but also improves animal survival.[6,23,24] The targeting of BAFF, BAFF-R and APRIL is of therapeutic interest in the neurological context, because these molecules are upregulated in the tissues of patients with autoimmune diseases. A number of agents are currently in phase I-II clinical trials in rheumatoid arthritis and SLE.[1,2,3-6,24] Agents that target BAFF include belimumab, a humanized monoclonal antibody against soluble BAFF, and the BAFF antagonist AMG G23. BR3-Fc is directed against BAFF-R, resulting in blockade of BAFF binding and, subsequently, B-cell reduction. BCMA-IgG is directed against APRIL. The TACI-IgG fusion protein neutralizes BAFF, APRIL and BAFF-APRIL heterodimers. Anti-lymphotoxin-ß receptor disrupts the architecture in the ectopic germinal centers.

Figure 4. Monoclonal antibodies or fusion proteins against B-cell targets. The figure highlights several B-cell molecules and their receptors, which are targeted by nine different monoclonal antibodies or fusion proteins currently in phase I-III clinical trials. Abbreviations: APRIL = a proliferation-inducing ligand; BAFF = B-cell-activating factor; BAFF-R = B-cell-activating factor receptor; BCMA = B-cell-maturation antigen; CTLA4 = cytotoxic T-lymphocyte antigen 4; LTßR = lymphotoxin-ß receptor; LTßR-Ig = anti-lymphotoxin-ß receptor antibody; MHC-II = major histocompatibility complex class II; TACI = transmembrane activator and calcium modulator and cyclophilin ligand interactor; TCR = T-cell receptor...(Click to enlarge figure)

• Agents Causing B-cell Depletion

Drugs directed against the CD20 or CD22 B-cell-surface glycoproteins can coat B cells and thereby cause their depletion (Figure 4). These drugs include: epratuzumab, which blocks CD22 survival signals on immature and mature B cells, as well as on pro-B and pre-B cells; rituximab, which is directed against the CD20 molecule; and occrelizumab, the humanized version of rituximab. In contrast to agents against trophic factors and their receptors, as mentioned above, these drugs deplete B cells but not the antibody-producing plasma cells.[1-6]

• Anti B-cell Agents in Neurology: The Role of Rituximab

Among all the aforementioned agents, only two, BCMA-IgG and rituximab, have been used in autoimmune neurological disorders. In mice with experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG), BCMA-IgG prevented disease development, improved the strength of already weak animals, depleted the CD19+ B cells in the blood, spleen and lymph nodes, reduced anti-MOG-specific IgG antibody titers, and suppressed inflammation and ongoing demyelination in the brain and spinal cord.[25]

Rituximab is a chimeric mouse-human monoclonal antibody consisting of human IgG1 and kappa constant regions and a mouse variable region. It was derived from a hybridoma directed at human CD20, a 297-amino-acid transmembrane phosphoprotein that is present on all cells of the B-cell lineage except for stem cells, pro-B cells and plasma cells (Figure 2).[1-6] In contrast to BAFF and APRIL, CD20 is not secreted, and it is not shed or endocytosed when exposed to rituximab.[26] The function of CD20 is unclear—it is thought to be involved in B-cell activation and proliferation,[1-6] although CD20 knockout mice do not exhibit B-cell deficits.[1-6,26] Rituximab is approved for the treatment of rheumatoid arthritis,[27] and, as outlined in the sections that follow, its use is currently being explored in a number of autoimmune neurological disorders in which B cells have a role.

• Rituximab in Autoimmune Neurological Disorders

Multiple Sclerosis. In MS, B cells and antibodies are involved to varying degrees at different stages of the disease and in different subgroups of MS. In the I-IV classification of Lucchineti, for example, pattern II is characterized by prominent lymphocytic and macrophagic infiltrates, complement activation and deposits of immunoglobulins.[28] Additional evidence supporting an antibody-mediated process in patients with MS includes accumulations of clonally expanded B cells in the MS plaques; intrathecal production of IgG bands from oligoclonal populations of B cells; autoantibodies against MOG in actively demyelinating lesions; ectopic lymphoid tissue in the intermeningeal spaces; and upregulation of BAFF and APRIL.[17,21,22,28-31]

In patients with MS, 24 weeks of treatment with rituximab was shown to deplete B cells from the cerebrospinal fluid (CSF) and to suppress B-cell activation, but it did not affect the intrathecal synthesis of oligoclonal IgG bands derived from long-lived plasma cells.[32] In a phase II, controlled, multicenter clinical trial of 104 patients with relapsing-remitting MS, a 58% relative reduction in the proportion of patients who experienced a relapse was noted after 24 weeks of therapy. A significant reduction in the mean number of gadolinium-enhancing MRI lesions (the study’s primary end point), was also observed in the treated patients compared with the placebo group (P <0.0001).[33] In an open-label trial of 26 patients treated with two courses of rituximab 6 months apart and followed for up to 72 weeks after commencement of treatment, reductions in relapses and MRI lesions were noted, suggesting long-lasting benefit.[34] These very encouraging results and the excellent tolerance of the drug has led to phase III trials in relapsing-remitting and primary progressive forms of MS, which are currently ongoing.

Neuromyelitis Optica. Neuromyelitis optica (NMO) is an inflammatory CNS disorder that affects the optic nerves and the spinal cord. It typically presents with myelitis and optic neuritis, and is characterized by varying degrees of sensory motor disturbances, bladder-bowel dysfunction and visual loss. In NMO, autoantibodies, collectively termed NMO-Ig, bind to cerebral microvessels.[35] The main target antigen of these autoantibodies is the aquaporin-4 water channel. NMO-Ig is derived from peripheral B cells, activates complement, and has been implicated in the induction of inflammatory demyelination and necrosis in the endothelial cells of the spinal cord.[35] Patients with NMO experience frequent relapses, and the disease is associated with high morbidity. Some acute flare-ups can respond to plasmapheresis, although the disease responds poorly to immunotherapies overall.

In an open-label study, six out of eight patients with NMO became relapse-free after a year of rituximab treatment, with a decline in relapse rate from 26 to zero attacks per year. In addition, seven of the patients showed a substantial improvement in their Expanded Disability Status Scale (EDSS) score.[36] In a retrospective review of 34 patients from two different centers, rituximab significantly lowered the relapse rate compared with pretreatment data, and stabilized or improved the EDSS scores in 91% of the patients.[37,38]

Paraneoplastic Neurological Disorders. Patients with paraneoplastic neurological disorders have circulating antibodies against a variety of antigens that are expressed in both brain and cancer cells. There is evidence that B cells, plasma cells and cytotoxic T cells cross the blood-brain barrier, and antibodies are synthesized intrathecally. In paraneoplastic opsoclonus-myoclonus, the number of clonally expanded B cells within the CSF correlates with clinical severity.[39] Rituximab, as an add-on therapy to IVIg or adrenocorticotropic hormone (ACTH), improved the ataxia severity scores, ameliorated myoclonus and reduced the rate of clinical relapse in 81% of 16 children with opsoclonus-myoclonus, and selectively reduced the numbers of clonally expanded B cells in the CSF.[40] In addition, the required ACTH dose was reduced by 51% after rituximab treatment.

Chronic Autoimmune Neuropathies. The chronic autoimmune neuropathies include a spectrum of predominantly demyelinating neuropathies, the most common of which are chronic inflammatory demyelinating neuropathy (CIDP), multifocal motor neuropathy (MMN) and IgM anti-myelin-associated glycoprotein (IgM-MAG) neuropathies. Evidence for a role for B cells in the pathogenesis of these conditions includes the deposition of immunoglobulins and complement on the patients’ nerves, and the presence of complement-fixing antibodies against MAG and gangliosides.[41,42] In an open series of 21 patients with IgM antibodies to gangliosides, rituximab improved symptoms in 61% of the patients 6 months after therapy, and the benefits were maintained for up to 2 years with repeated infusions.[43] The IgM antibody titers dropped by 36% in the first year and by 57% in the second. Rituximab has also been reported to be effective in some patients with MMN or CIDP.[44,45] In another study, rituximab improved the symptoms in six out of nine patients with IgM-MAG neuropathy, and reduced IgM-MAG titers by a mean of 52% from baseline,[46] prompting a placebo-controlled study. In 26 randomized patients, rituximab significantly improved disability scores and reduced IgM-MAG titers after 8 months.[47] Rituximab is the first drug to demonstrate efficacy in a randomized trial in this particular neuropathy.

Stiff-person Syndrome. Stiff-person syndrome (SPS) is a rare but often misdiagnosed CNS disorder that is clinically characterized by stiffness and rigidity in the limbs and paraspinal muscles, intermittent superimposed muscle spasms, and heightened sensitivity to external stimuli. The majority of patients with SPS have antibodies against glutamic acid decarboxylase (GAD) or GABARAP, a linker protein responsible for ?-aminobutyric acid receptor clustering.[48] Anti-GAD antibodies are synthesized intrathecally, and oligoclonal bands are commonly detected in the CSF.[49] In one case report, rituximab was effective at reducing stiffness and increasing mobility 2 months after the treatment was initiated, and resulted in disappearance of GAD antibodies and normalization of the electromyogram.[50] The first double-blind controlled study using rituximab to treat SPS has now been completed at the NIH, and the results are currently being analyzed.

Inflammatory Myopathies. There are three main subsets of inflammatory myopathies: polymyositis, dermatomyositis and inclusion body myositis. In all these forms of the disease, B cells and plasma cells are present in the muscle tissues, and in dermatomyositis immunoglobulins are deposited on endomysial capillaries.[51] In 10 patients with polymyositis or dermatomyositis who had responded poorly to current therapies, rituximab increased or normalized muscle strength in 8 cases. Serum levels of creatine kinase and the required prednisone dose were concurrently reduced.[52,53] A multicenter NIH-sponsored clinical trial of rituximab is now ongoing for the treatment of polymyositis and dermatomyositis. The drug has not yet been tested in inclusion body myositis.

Myasthenia Gravis. Myasthenia gravis is a prototypic B-cell-mediated autoimmune disease caused by pathogenetic antibodies against the muscle acetylcholine receptors. Evidence from around 20 case reports suggests that rituximab is effective in most patients, but a controlled study has not yet been done.[54-57]

• Effect of Rituximab on Circulating B Cells, Autoantibodies and Immunoglobulin Levels

In general, 1 month after rituximab infusion, circulating B cells become undetectable, and their numbers remain low for at least 6 months. The cells start reappearing slowly thereafter, but even after 10 months their numbers remain below baseline.[3] The circulating memory CD20+CD27+ B cells are also depleted, and their levels remain low until month 8 (Figure 5). The B cells in the follicular splenic regions are preferentially affected, being depleted by 90%, compared with 25% depletion of marginal-zone B cells.[6] The germinal-center B cells are resistant to rituximab, even though they express CD20, possibly reflecting an inability of the antibody to access the intravascular spaces within the lymphoid tissues, or different sensitivities of B cells according to the local lymphoid microenvironment.[6] Stem cells in the bone marrow that do not express CD20 are also spared, thereby allowing the generation of new naive B cells.[58]

Figure 5. Kinetics of CD20+CD27+ memory B cells in rituximab-treated patients. The figure highlights several B-cell molecules and their receptors, which are targeted by nine different monoclonal antibodies or fusion proteins currently in phase I-III clinical trials. Abbreviations: APRIL = a proliferation-inducing ligand; BAFF = B-cell-activating factor; BAFF-R = B-cell-activating factor receptor; BCMA = B-cell-maturation antigen; CTLA4 = cytotoxic T-lymphocyte antigen 4; LTßR = lymphotoxin-ß receptor; LTßR-Ig = anti-lymphotoxin-ß receptor antibody; MHC-II = major histocompatibility complex class II; TACI = transmembrane activator and calcium modulator and cyclophilin ligand interactor; TCR = T-cell receptor..(Click to enlarge figure)

Rituximab is not expected to affect the levels of antibodies produced by plasma cells, although some reductions in these levels have been noted. In rheumatoid arthritis, for example, the titers of rheumatoid factor were shown to decrease two-to-threefold,[58,59] and in IgM-MAG neuropathy by 30-50%, after treatment with rituximab.[45,47] Such reductions can probably be attributed to depletion of CD27+ memory B cells, the precursors of short-lived plasma cells.[4] As the CD27+ memory B cells reappear, so do the short-lived plasma cells.[60] Given that the reconstituting B cells are naive cells with a new and diverse immunoglobulin rearrangement pattern,[4,58,59] it might take some time for them to be restimulated by the original antigen, hence the slow re-emergence of serum antibody titers. Antibody titers might therefore fall after rituximab treatment, and rebound slowly at a rate controlled by the replenishment of memory and short-lived plasma cells.[60] Interestingly, after several years of treatment, the antibody titers against anamnestic antigens, such as tetanus toxoid, remain stable.[59] This finding suggests that rituximab might have differential effects on ‘autoreactive’ B cells and their corresponding short-lived plasma cells, compared with ‘non-self-reactive’ B cells and their corresponding longer lived plasma cells, which are responsible for post-vaccination responses.[6] A recent study supports different roles for B cells and longer lived plasma cells in protective immunity.[61]

• Dosing, Tolerance, Safety and Combination Therapy

Rituximab can be administered intravenously at a dose of 375 mg/m2, given weekly for 4 weeks, or in two 1 g infusions, given at fortnightly intervals (total 2 g). The average half-life of the drug after completion of an infusion is 21 days. The infusions can be repeated after 6-12 months, at a point when B cells start rebounding or when the patient has relapsed. The drug is generally very well tolerated, although mild hypotension can be observed in some patients, necessitating the discontinuation of antihypertensive drugs on the day of the infusions. Anaphylactic or skin reactions can occur in rare cases, but these respond to intravenous methylprednisolone. Premedication with antihistamine is desirable to prevent the occurrence of such reactions.

Rituximab has been used in combination with other immunosuppressants, such as corticosteroids, mycophenolate, cyclophosphamide, azathioprine or methotrexate,[6,62] for the treatment of vasculitis and rheumatoid arthritis, without additional complications. This experience differs from that with natalizumab, which requires discontinuation of the other immunosuppressants for 2-3 months before initiating therapy.[63] It remains to be determined whether combination therapy will be more effective than monotherapy in difficult neurological cases. Rituximab has been also used effectively in some cases of pediatric SLE in two infusions of 750 mg/m2 administered 2 weeks apart, either alone or in combination with corticosteroids and cyclophosphamide,[1] suggesting that it can be used in children with difficult autoimmune neurological disorders.

The resistance of long-lived plasma cells to rituximab probably explains its excellent safety profile, the absence of infections, and the patients’ retained ability to produce immunoglobulins and mount an antibody response against anamnestic antigens. In spite of the apparent plasticity of the immune system, which enables it to compensate for the peripherally depleted B cells, vigilance is still required to guard against the possibility of infections in patients receiving repeated doses or concurrent immunosuppressants.[3] Such infections might not be limited to common bacterial or viral agents, but might also extend to agents that cause latent infections, such as JC virus or herpesviruses, as has been experienced with natalizumab.[63] Rare cases of progressive multifocal leukoencephalopathy have been reported in patients with SLE receiving rituximab, although a cause-and-effect relationship has not been established.[64]

A consistent observation in many series is the elevation of BAFF levels after rituximab treatment, probably as an inherent compensatory mechanism to drive B-cell production.[1-6] Theoretically, combining rituximab with one of the agents against BCMA or TACI-IgG, which reduce the survival of BAFF-dependent, immunoglobulin-producing, long-lived plasma cells, might have a prolonged effect on B cells and autoantibody levels. Such a combination therapy might be attractive in the future, in view of increased levels of BAFF in autoimmune neurological disorders.

• Modes of Action of Rituximab

Rituximab depletes B cells through three mechanisms (Figure 6): antibody-dependent cellular cytotoxicity, whereby antibody-coated cells bind to the Fc receptors of macrophages or natural killer cells; activating the membrane attack complex on B cells (complement-dependent cytotoxicity); and inducing apoptosis by changing the lipid raft environment on the CD20+ B-cell membrane.[1-6]

Figure 6. Mechanisms of action of rituximab. Rituximab induces cell death through three mechanisms. (A) Antibody-dependent cell-mediated-cytotoxicity. Rituximab recruits macrophages and natural killer cells by binding to their Fc? receptors. (B) Complement-mediated cytotoxicity. Rituximab activates complement and generates membrane attack complexes. (C) Induction of apoptosis..(Click to enlarge figure)

The most impressive observation in all published series, and from our own experience, is the long-lasting benefit of rituximab, sometimes exceeding 6-8 months after therapy. The degree of clinical response, however, varies from patient to patient, probably reflecting the varying degree of contribution of B cells to the autoimmune process, as discussed earlier. It is difficult to ascertain which of the B-cell functions depicted in Figure 1 is primarily influenced by the drug and is responsible for the noted benefit. Diminished production of pathogenetic autoantibodies might be a contributing factor, but this decrease in production might be insufficient to be clinically meaningful.[27,59] The effects of rituximab on other B-cell functions—effects that include blockade of costimulatory molecules required for clonal expansion of T cells, inhibition of the antigen-presenting role of B cells, suppression of the cytokine network, inhibition of immune complexes, and induction of immunoregulatory T cells—might be more important in explaining the noted clinical benefit. Accordingly, rituximab might be beneficial not only in antibody-mediated disorders of the CNS and PNS, but also in other autoimmune diseases where both B cells and T cells contribute to disease pathogenesis.

• Conclusions and Future Prospects

Anti-B-cell therapy, in particular treatment with rituximab, is a promising approach for immunotherapy of neurological diseases, and it has the potential to produce long-lasting benefits. The reported excellent tolerance of rituximab administered in combination with other immunosuppressants is an important advantage, but close monitoring will be required to promptly identify any long-term sequelae or unforeseen adverse effects. Newer monoclonal antibodies designed to target B-cell survival factors might prove to be even more effective than rituximab, because they can also affect the production of immunoglobulin and antibodies by plasma cells. The B cell is an attractive target for immunotherapeutic interventions, and controlled trials with rituximab and other new agents that work through similar mechanisms are warranted for the treatment of neurological disorders.

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65. Metwally, MYM: Textbook of neuroimaging, A CD-ROM publication, (Metwally, MYM editor) WEB-CD agency for electronic publication, version 10.1a January 2009 [Click to have a look at the home page]

The author: Professor Yasser Metwally

http://yassermetwally.com

INTRODUCTION

February 16, 2009 — Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine repeat expansion within the huntingtin protein. HD is characterized by problems with movement, cognition and behavioral functioning, and there is currently no effective treatment. Although multiple pathologic mechanisms have been proposed, the exact mechanism by which mutant huntingtin causes neuronal dysfunction is not known. Recent studies demonstrating altered messenger RNA expression point to transcriptional dysregulation as a central mechanism. The control of eukaryotic gene expression depends on the modification of histone proteins associated with specific genes, with histone acetylation playing a crucial role. Studies in numerous HD models have shown that mutant huntingtin alters histone acetyltransferase activity, and indicate that aberrant activity of this enzyme might be an underlying mechanism of transcriptional dysregulation in HD. Furthermore, recent studies have shown a therapeutic role for histone deacetylase inhibitors in a number of HD models. In this review, we summarize the current state of knowledge regarding the status of histones in HD. In addition, we discuss how these histone modifications not only lead to pathogenesis, but might also provide a novel therapeutic strategy for treating this devastating disease.

Huntington’s disease (HD) is an autosomal dominant neurological disease that afflicts around one in 10,000 people.[1] HD onset typically occurs in young adult life, and the disease is invariably fatal. Patients initially develop small choreic movements that later intensify in amplitude and frequency, causing an impairment of daily activities. Although the movement disorder is typically chorea, other types of movement disorder, such as dystonia, rigidity, myoclonus and athetosis, can also be seen. Psychiatric disturbances, the most common of which are apathy, depression, obsessive–compulsive disorder, psychotic disorders, substance abuse and paranoia, are often the most disabling symptoms for patients with HD. Cognitive disorders, on the other hand, are among the most difficult symptoms to treat. They appear later in the course of the disease, manifesting as dementia and difficulties in executive functioning. Weight loss is also a common feature in HD, implying the existence of a generalized metabolic defect. To date, there are no effective treatments to cure the disease or to slow its progression.

Huntingtin (Htt), the protein that is defective in HD, is expressed throughout the body, yet HD principally affects the brain. Although HD affects a number of regions, including the cortex, thalamus and subthalamic nucleus, the striatum is the most severely affected region.[2] Within the striatum, the medium spiny projection neurons are preferentially targeted by the disease, whereas interneurons are relatively spared.[3] The preferential loss of medium spiny neurons and sparing of interneuron populations indicates that neurodegeneration in HD is cell-type-specific. The factors that render striatal projection neurons more susceptible to damage are, however, currently unknown.

Transcriptional dysregulation has been proposed to have an important role in the pathology of HD, but the mechanisms that cause disruption of gene expression remain unknown. The control of eukaryotic gene expression depends on the modification of histone proteins associated with specific genes, with histone acetylation playing a crucial role. Acetylation of histones at specific residues increases gene transcription; conversely, histone deacetylation represses transcription. Recent studies in numerous HD models have demonstrated a potential therapeutic role for histone deacetylase (HDAC) inhibitors in the treatment of polyglutamine diseases. In this review, we summarize these studies, and discuss how histone modifications might provide a novel therapeutic approach for treating HD.

• The Huntington’s Disease Mutation

HD is caused by a mutation in the IT15 gene on the short arm of chromosome 4.[4] This gene, which was subsequently renamed HD, consists of 67 exons that encode Htt, a 350-kD protein of 3,144 amino acids. The mutation is an expansion of the cytosine–adenine–guanine (CAG) trinucleotide repeat in exon 1, which codes for a polyglutamine moiety in the Htt protein. Normal individuals have CAG repeat lengths of 7–34. The CAG repeat is expanded and unstable in HD patients, with repeat length inversely correlating with age of disease onset. Repeat lengths of more than 40 glutamines produce HD, and repeats of over 70 glutamines invariably cause juvenile onset.[5]

When the HD mutation was eventually identified as a CAG trinucleotide repeat expansion, HD joined a novel class of neurodegenerative disease, the polyglutamine diseases.[6] In all of these disorders, expansion of the CAG repeat occurs within the coding region of the gene, so the CAG repeat is translated into a polyglutamine stretch. Polyglutamine diseases are all autosomal dominant or sex-linked dominant diseases, usually with adult onset, and are characterized by progressive neurodegeneration of selected neuronal populations. In both humans and transgenic mouse models, these disorders are characterized pathologically by polyglutamine protein-containing intracellular inclusions. Given the remarkable similarities between the various polyglutamine disorders, the polyglutamine moiety is strongly implicated as the portion of the protein that makes the greatest contribution to disease pathogenesis.

• Pathogenic Mechanisms

The genetic mutation that causes HD was discovered in 1993, but the mechanism by which mutant Htt causes neuronal dysfunction is still not known. Several theories have been proposed to explain the mechanism of HD pathogenesis. Excitotoxicity has been proposed as a pathogenic mechanism, on the basis of the observation that infusion of glutamate-receptor agonists into the brain leads to neuronal death and a phenotype similar to HD.[7] Mitochondrial dysfunction is another leading hypothesis,[8] as numerous deficits in mitochondrial metabolism have been reported in the brains of patients with HD at postmortem examination. Caspases, a family of proteases that are involved in apoptotic cell death, show increased activity in HD, and are also thought to participate in HD pathogenesis.[9] Furthermore, caspases can generate amino-terminal (N-terminal) fragments of Htt that can enter the nucleus and form nuclear inclusions.[9,10] Beside caspases, other types of proteolytic cleavage have been identified, involving calpain,[11] AUTOPHAGY,[12,13] proteasomes[14,15] or an aspartyl protease.[16] Nuclear inclusions formed by mutant Htt sequester proteins such as transcription factors, heat-shock proteins and proteasome subunits, thereby preventing these proteins from reaching their necessary point of action.[17]

In addition to the formation of nuclear inclusions, mutant Htt can also form toxic ß-sheet-rich aggregates through the misfolding of the expanded polyglutamine stretch.[18,19] This aggregation process appears to be associated with pathogenesis in HD. Polyglutamine expansion interferes with proteasome function,[14,15] and polyglutamine proteins might disrupt the global balance of protein-folding control.[20]

Autophagy has emerged as a mechanism of HD pathogenesis on the basis that mutant Htt accumulation activates the endosomal–lysosomal system and contributes to Htt proteolysis and autophagic cell death.[21] The role of autophagy is unclear, however. Inhibition of mTOR (mammalian target of rapamycin; also known as FRAP1) activates autophagy and attenuates Htt-induced toxicity,[13] indicating that autophagy might in fact be helpful as a clearance mechanism.

• Transcriptional Dysregulation in Huntington’s Disease

Recent studies have provided strong evidence that transcriptional dysregulation is an important underlying mechanism in HD pathogenesis.[22,23] Transcriptional dysregulation is an early event in HD pathology, and is observed across multiple HD models.[24] The polyglutamine repeats in the N-terminal region of Htt protein gives it structural similarities to known transcription factors,[25,26] and repeat expansion leads to aberrant cleavage by caspases.[10] The cleaved fragments gain access to the nucleus and form nuclear aggregates that might disrupt transcription. Mutant Htt interacts with numerous transcription factors[24] including CREB-binding protein (CBP),[27,28] TATA-binding protein (TBP),[29] p53[27] and Sp1,[30] raising the possibility that Htt nuclear aggregates cause transcriptional dysregulation by sequestering transcription factors.

Humans with HD and transgenic mouse models of HD show downregulation of specific genes at the level of messenger RNA (mRNA) expression.[31,32,33] The R6 mouse lines in particular have provided evidence for transcriptional dysregulation.[34] These mice were created by inserting exon 1 of the human HD gene containing 150 polyglutamine repeats, under the control of the human HD promoter, into the mouse genome. Alterations of neurotransmitter receptor protein levels, as well as of mRNA levels, have been reported in the R6/2 mouse line.[32] Both of these changes occurred before the onset of abnormal symptoms. This finding is in agreement with observations in patients in the early stages of HD, in that the pathologic changes appear to precede the appearance of disease symptoms. PET studies of gene-positive but clinically asymptomatic patients in the early stages of HD demonstrate that expression of dopamine D1 and D2 receptors is decreased before the onset of symptoms.[31,35] Therefore, neuronal dysfunction predates the appearance of neurological symptomatology in HD. In very early-grade HD cases (grade 0), there are profound reductions in the expression levels of cannabinoid CB1, dopamine D2 and adenosine A2a receptors.[36]

Widespread screening of mRNA levels, using DNA microarrays, in the brains of R6/2 mice confirmed the finding that specific genes are downregulated in this HD mouse model.[37] The transcripts affected are important for neuronal function, and they include genes involved in neurotransmitter signaling, calcium metabolism and transcription.[38,39,40] Decreases in mRNA levels were more common than increases, and the set of genes that were decreased at 12 weeks of age was larger than the set of genes that were affected at 6 weeks, indicating a progressive effect.

Gene expression studies have also been carried out in other HD mouse models. The HD-N171-82Q transgenic mouse model expresses a COMPLEMENTARY DNA that encodes a 171-amino-acid N-terminal fragment of Htt exon 1 containing 82 CAG repeats. The changes in gene expression in HD-N171-82Q mice are similar to those observed in R6/2 mice.[41] The mRNA changes observed in the R6/2 and HD-N171-82Q mice are of significance, given that they recapitulate the gene expression changes that occur in the human HD brain.[24] Expression profiling of human HD cases shows that gene changes are most pronounced in the striatum, with the motor cortex affected to a lesser degree, and the cerebellum showing the fewest gene changes.

R6/2 and HD-N171-82Q mice are fragment models; that is, they express truncated portions of Htt. Transgenic mouse models expressing full-length versions of Htt, such as the YAC72 mice, demonstrate fewer gene changes, indicating that increasing the length of Htt reduces the severity of polyglutamine-induced gene changes.[42]

Mutant Htt interacts with numerous transcription factors, providing a mechanism through which this protein can interfere with normal transcriptional activity.[24] For example, soluble mutant exon 1 Htt protein has been shown to interact with the polyQ-containing domain of CBP.[43] Recently, Zhai and colleagues have shown that mutant versions of N-terminal Htt can dissociate components of the transcriptional complex on gene promoters.[44] Transcriptional dysregulation has also been proposed as a disease mechanism in other polyglutamine diseases. In fact, the androgen receptor, which is mutated in spinobulbar muscular atrophy or Kennedy’s disease, and the TATA box binding protein (TBP), which is mutated in spinocerebellar ataxia type 17 (SCA-17), are themselves transcription factors. Ataxin-7, the protein mutated in SCA-7, is a component of the mammalian STAGA (SPT3-TAF9-ADA-GCN5 acetyltransferase) transcription coactivator complex, and it interacts directly with the GCN5 histone acetyltransferase.[45] Taken together, these results indicate that transcriptional dysregulation is likely to be an important mechanism in the pathogenesis not only of HD, but also of other polyglutamine disorders.

If downregulation of the expression of key molecules at the mRNA level underlies the neuronal dysfunction in HD, correction of these transcriptional abnormalities has great potential as a novel therapeutic approach. To harness this potential, however, it will be necessary to understand the mechanisms that cause selective downregulation of target genes.

• Histone Modifications

Regulation of gene expression is accomplished through the action of transcription factors that alter chromatin structure through the recruitment of histone-modifying enzymes. The N-terminal tails of the core histones (H2A, H2B, H3 and H4) are strongly basic, and contain specific amino-acid residues that are sites for several post-translational modifications, including acetylation, methylation, phosphorylation, ubiquitination and SUMOYLATION (Figure 1). These modifications can affect processes such as transcription, mitosis and chromosome stability.[46] In general, acetylation of lysine residues corresponds to transcriptionally active chromatin, whereas methylation of lysine and arginine residues leads to transcriptional repression. For example, acetylation of lysine 9 and 14 on histone H3 (AcK9K14H3) correlates with active chromatin and leads to transcription, whereas methylation of lysine 9 on histone H3 (MeK9H3) is a marker of silenced genes. Phosphorylation of serines also correlates with transcriptional activation, because it precedes acetylation at lysine residues. Many combinations of covalent modifications are possible on the core histones, and these precise patterns of histone modification seem to be a key factor in turning specific genes on or off. This system of gene control has been termed the ‘histone code’ ( Table 1 ).[46]

Table 1. Known Mammalian Histone Modifications (Click to enlarge table)

Figure 1. Known modifications of human histone H3. Acetylation and methylation at lysine residues are mutually exclusive, and lysine residues can be monomethylated, dimethylated or trimethylated.(Click to enlarge figure)

aa = amino acid; K = lysine; R = arginine; S = serine; T = threonine.

Histone acetylation and deacetylation are modulated by the interplay between histone acetyltransferases (HATs) and HDACs, which work in concert to modify chromatin structure and regulate transcription.[47] In simple terms, HAT activity leads to increases in gene transcription by creating a more open conformation of chromatin, whereas HDACs remove acetyl groups, leading to gene repression through condensation of chromatin (Figure 2). In reality, however, the relationship between histone acetylation and gene transcription is likely to be more complex. An emerging view is that histone acetylation not only governs the relaxation of chromatin, but also regulates the recruitment of specific transcriptional regulatory complexes. Therefore, rather than directly correlating with activation of gene expression, histone acetylation might render chromatin ‘transcription-competent’. Abnormalities of histone acetylation have been associated with a number of human cancers; for example, in leukemia and non-Hodgkin’s lymphoma, mutations activate and target HDACs.[48] In human gastrointestinal cancers, histone acetylation is globally reduced.[49]

Figure 2. Schematic representation of histone acetylation. Acetylation of core histone proteins through the activity of histone acetyl transferase proteins or treatment with histone deacetylase inhibitors leads to a more open conformation of chromatin, and therefore a transcriptionally active state. Removal of acetyl groups by histone deacetylases leads to the formation of repressed chromatin, which corresponds with transcriptional repression. Similarly, mutant huntingtin (Htt) expression is believed to drive the formation of repressed chromatin by decreasing acetylation of histones..(Click to enlarge figure)

HAT = histone acetyl transferase; HDAC = histone deacetylase.

A diverse group of molecules that can inhibit HDACs has been developed. HDAC inhibitors have been shown to act selectively on gene expression, and are inducers of cell growth, arrest, differentiation and death both in vitro and in vivo.[47] HDAC inhibitors increase acetylation of histones, thereby increasing transcription of genes that have been silenced. HDAC inhibitors promote growth arrest by inducing the expression of tumor suppressor genes, and are commonly used as anticancer drugs.[50,51,52] Recent studies in yeast, cell culture, Drosophila and mouse models of polyglutamine disease indicate that HDAC inhibitors might be useful as therapeutic agents in HD ( Table 2 ).[53,54,55,56,57,58]

Table 2. Summary of Histone Deacetylase Inhibitor Studies in Huntington’s Disease..(Click to enlarge table)

Although the overall status of histone modification is largely unknown in HD, recent studies support the idea that histone acetylation is important in HD pathogenesis. Histones have been shown to be hypoacetylated in R6/2[56,57] and HD-N171-82Q[58] mouse models, as well as in PC12 CELL LINES.[55,59] To date, there is no evidence that Htt directly interacts with histone proteins, although Htt aggregates have been found to contain histones H3 and H4.[60]

• Histone Deacetylase Inhibitors in Huntington’s Disease

Many Htt-interacting proteins possess histone-modifying activity. CBP contains an acetyltransferase domain and is a coactivator at a number of promoters.[27] CBP and other transcription factors have been shown to be sequestered into Htt aggregates in transgenic mice and in brains from individuals with HD.[27,28] Furthermore, overexpression of CBP decreases polyglutamine-induced cell death.[28] Steffan and colleagues[55] demonstrated that Htt exon 1 with 51 glutamines containing the polyproline domain (Httex1p 51Q) directly binds to the acetyltransferase domain of CBP and p300/CBP-associated factor (P/CAF). In vitro studies have shown that in the presence of mutant Htt, the acetylation of histone H4 is decreased, leading to the conclusion that the direct interaction of Htt with the acetyltransferase domains inhibits acetylation of histone proteins. In addition, expression of Httex1p 20Q or Httex1p 103Q in PC12 cells causes a global hypoacetylation of histones, an effect that is reversed by the presence of the HDAC inhibitors sodium butyrate, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Moreover, in transgenic Drosophila that express Httex1p 93Q, a decrease in RHABDOMERE degeneration and early adult death are observed if the flies have been reared on SAHA or sodium butyrate. Genetically reducing the activity of HDACs by a partial loss of Sin3A decreases rhabdomere degeneration in mutant flies.[55] These results indicated that reduced acetyltransferase activity might be an important component of polyglutamine pathogenesis in vivo, and paved the way for HDAC inhibitor studies in transgenic mouse models of HD.

Following the findings in Drosophila, two groups investigated the effects of HDAC inhibitors in R6/2 mice. Ferrante and colleagues[56] investigated the effects of sodium butyrate, whereas Hockly and colleagues[57] tested the effects of SAHA on the clinical and neuropathological phenotype of R6/2 mice. Sodium butyrate treatment significantly prolonged the survival of R6/2 mice. Both sodium butyrate and SAHA improved performance on a ROTAROD TEST, and were neuroprotective in that there was a decrease in gross brain atrophy and ventricular enlargement in the sodium-butyrate-treated mice,[56] and a reduction in neuronal atrophy in the striatum of SAHA-treated mice.[57] There was, however, no effect on Htt aggregates. In both studies, administration of HDAC inhibitors corrected global hypoacetylation of histones. In sodium-butyrate-treated R6/2 mice, there was an increase in Sp1 acetylation, but no change in basal levels of Sp1 was detected. Sodium butyrate also provided protection against 3-nitropropionic-acid (3-NP)-induced striatal damage in R6/2 mice. 3-NP is a mitochondrial toxin that specifically targets complex II of the electron transport chain. Striatal administration of 3-NP produces neuronal damage similar to that observed in the brains of individuals with HD, and 3-NP striatal lesioning has been used as a toxin model of HD. Microarray analysis of sodium-butyrate-treated R6/2 striatum demonstrates a selective change in gene expression, but there was no uniform correction of genes downregulated by mutant Htt.[56]

Gardian and colleagues[58] administered the HDAC inhibitor phenylbutyrate after the onset of symptoms in another transgenic mouse model, HD-N171-82Q. Phenylbutyrate administered to HD-N171-82Q mice at 75 days of age increased survival, and decreased striatal atrophy and ventricular enlargement. There was no effect on motor performance, weight loss or Htt aggregate formation. Histone H3 and H4 acetylation was increased in the striatum following phenylbutyrate treatment, and there was a decrease in methylation of histone H3. Microarray analysis of the phenylbutyrate-treated HD-N171-82Q striatum demonstrated that some genes were upregulated and others were downregulated. Although phenylbutyrate did not improve the expression of mutant-Htt-downregulated genes, it is promising that transgenic mice showed an overall improvement in their condition, given that treatment began after the onset of symptoms.

The therapeutic mechanism of HDAC inhibitors is not clear. The simplest mechanism that can be envisaged is one in which administration of HDAC inhibitors corrects the downregulation of specific genes caused by mutant Htt. Limited studies of HDAC inhibitor treatment of transgenic mice, however, indicate that this is not the case. An alternative possibility is that the benefits of HDAC inhibitors derive from global increased gene expression. If this is the case, HDAC inhibitors might be beneficial in other neurodegenerative conditions beyond HD. Indeed, HDAC inhibitors have also shown promise in transgenic mouse models of amyotrophic lateral sclerosis,[61] schizophrenia[62] and ischemia.[63]

Despite this early promise, further molecular definition of the mechanisms that underlie transcriptional dysregulation is required. Although some investigators have observed general hypoacetylation of histones in R6/2 mice, and downregulation of specific genes has been well described, there remains the issue of whether histones that are specifically associated with those genes are hypoacetylated. Using the molecularly specific technique of chromatin immunoprecipitation (ChIP), we have recently demonstrated that hypoacetylation of histones is associated with downregulated genes, whereas histones associated with genes that are expressed at a normal level are acetylated to the same degree as in wild-type mice (Sadri-Vakili et al., unpublished data). Defining the exact histone alterations in HD will undoubtedly prove valuable in designing effective treatments.

There are three classes of HDACs: class I and class II HDACs have been classified on the basis of sequence similarities, whereas class III HDACs are a group of NAD-dependent deacetylase enzymes related to the yeast Sir2 protein. The role of each individual HDAC in regulating gene expression is not known. Some HDACs deacetylate other proteins in addition to histones. In addition, there are tissue-specific and cell-type-specific differences in HDAC expression, localization and targets. Therefore, an individual HDAC inhibitor might have different effects in different biological systems.[64] HDAC inhibitors are toxic and can induce cell cycle arrest by increasing the transcription of p21 and p53, so it will be important to target individual HDACs to minimize cytotoxicity.[65,66] Other side effects are associated with HDAC inhibitor therapy; for example, prolonged TSA therapy enhances chromosomal instability, leading to defective centromeres and abnormal chromosomal segregation.[67] In addition, HDAC inhibitors can promote tumor development in some cases.[64] Therefore, it will be vital to determine the toxicity of these compounds.

Many HDAC inhibitors are currently approved for use in phase I and phase II human clinical trials.[66] There is extensive experience with the use of phenylbutyrate in patients for treatment of urea cycle disorders, sickle cell anemia, thalassemia minor and cystic fibrosis.[68,69,70] Phenylbutyrate is also being tested in phase I and II clinical trials for the treatment of cancer, and has shown minimal clinical side effects.[64,71,72] Phase I clinical trials with SAHA have shown that it is well tolerated. In addition, SAHA induces histone acetylation, and has anti-tumor activity.[73] On the basis of these promising results, phenylbutyrate clinical trials in HD patients have now commenced.[60]

• Conclusions

Currently, HD remains a fatal untreatable disease. Recent advances in understanding the underlying pathologic mechanisms, however, have provided hints to the development of effective therapies. Specifically, recent evidence points strongly to transcriptional dysregulation as an important mechanism. The alteration of gene expression is likely to occur as a consequence of abnormal histone modifications, including hypoacetylation. The use of HDAC inhibitors and other therapies that target gene transcription is an exciting development in the field of HD therapeutics. There are strong indications that HDAC inhibitors might be of therapeutic benefit in HD, but their precise mechanism of action has yet to be determined. 

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