Online newspaper of professor Yasser Metwally

The author: Professor Yasser Metwally

www.yassermetwally.com

INTRODUCTION

The mechanisms underlying disruption of physiologic cell-death programs are slowly being revealed. Information gleaned from genetic profiles soon may allow physicians to delay onset of many silent, age-related diseases. While development of actual cures could prove elusive, simply postponing clinical onset would have an impressive effect on the goal of healthy aging.Both age-related neurodegenerative diseases and cancer involve disruption of physiologic cell-death programs. Neural cell death is abnormally accelerated in such neurodegenerative diseases as those of the Alzheimer’s, Huntington’s, Parkinson’s, Kennedy’s (X-linked spinal and bulbar muscular atrophy), and Machado-Joseph (Azorean) types. In cancer pathogenesis, normal mechanisms of cell death are blocked, leaving cell proliferation unchecked. The link between these two processes–and the possibility of manipulating that link for medical benefit–has recently become the focus of a dynamic area of aging research.

Shifting the Cellular Apostat

Apoptosis (falling [ptosis] away [apo]) is probably the most common–and certainly the best understood–of the programs of cell death. The central set of cysteine-aspartyl proteases or caspases that drive the process are instrumental in the vast majority of apoptotic events that occur during normal embryonic development, as was dramatically illustrated in studies of the nematode Caenorhabditis elegans conducted by Robert Horvitz and coworkers. Elimination of the caspase homologue led to complete cessation of the 131 programmatic cell deaths that normally occur during development of that organism. The role of caspase-driven apoptotic events in human pathogenesis is less clear. However, recent evidence supports the theory that caspase cleavage of mutant proteins may represent an important signaling event in the initiation of cell death in a variety of degenerative conditions.

Caspases are highly specific proteases; until recently, only about 20 of their substrates had been identified. Over the past several years, however, a remarkable number of gene products associated with neurodegeneration have proved to be caspase substrates (Table 1). In 1995, researchers at the Burnham Institute reported that mutants of copper/zinc superoxide dismutase (CuZnSOD) associated with familial amyotrophic lateral sclerosis were proapototic in cultured neural cells, while wild-type CuZnSOD had an antiapoptotic effect. Since then, similar results have been obtained by various groups for amyloid precursor protein mutants, presenilin mutants, and a number of the polyglutamine expansion diseases, including Huntington’s, Kennedy’s, and Machado-Joseph. Although a direct relationship to pathogenesis has not been shown, the fact that so many neurodegeneration-associated mutants display proapoptotic phenotypes in cultured cells is probably not coincidental.

Table 1. Gene Products Associated with Neurodegeneration

Table 1. (Click of table to magnify)

? = Unknown

Negative Signal Transduction. A few years ago, my colleagues and I described a novel form of proapoptotic signal transduction–negative signal transduction–that hinges on the failure of certain receptors to bind with their ligands. At the time, it was already well established that survival of developing cells (and to a lesser extent mature cells) is mediated by the presence of such stimuli as trophic factors, cytokines, or hormones, or by adhesion or electrical activity. However, the assumption had been that survival was the norm and that cell death merely reflected miscommunication of the appropriate signal. Our studies demonstrated something closer to an on/off switch, in which expression of the receptor in the absence of associated ligand actively shifts the cellular apostat in the direction of apoptosis, while ligand binding blocks the effect. Using site-directed mutagenesis, we have since identified specific regions or addiction/dependence domains (ADDs) within the receptors that mediate this process. Synthesis of these domains produces a peptide that is clearly proapoptotic.

Dependence Receptors. The first of the dependence receptors to be described–so named because they confer a cellular state of dependence on, or addiction to, their ligands–was the common neurotrophin receptor designated p75NTR. Mice bred to lack this receptor demonstrate an approximately 50% increase in number and 30% increase in somatic volume of cholinergic medial septal and diagonal band neurons. Since these neurons are known to undergo age-related atrophy and degeneration in both mice and humans, the p75NTR receptor may prove to be a key player in age-associated cognitive decline. Both the neuronal and the cognitive changes can be blocked by specific trophic factors that bind to p75NTR.

Another interesting example is the androgen receptor, generally identified with cells of the reproductive tract but also expressed by motor neurons and a variety of other cell types. When expressed in the presence of testosterone, the androgen receptor has transcriptional activity; if testosterone is absent, the same receptor can induce apoptosis. As described by Lisa M. Ellerby, the addiction/dependence domain of the androgen receptor is situated near the amino terminus and includes a polyglutamine sequence that has already been linked to motor neuron death in a number of neurodegenerative diseases (Figure 1).

Recent work has shown that many ADDs are activated by caspase cleavage, and the polyglutamine portion of the androgen receptor provides a good example. The number of glutamines in the sequence is the genetic signal that determines whether or not apoptosis is induced. Healthy persons have 11 to 33 glutamines in the sequence. Above that number, the motor neurons become vulnerable to degeneration. Below it (fewer than about 11 glutamines in the sequence), men are at risk of metastatic prostate cancer. Our hypothesis is that glutamine expansion enhances cellular dependence, so that, in the case of the androgen receptor, apoptosis may occur even in the presence of physiologic concentrations of testosterone. In contrast, an insufficient number of glutamines creates a milieu in which cell death is curtailed. We currently are investigating whether other hormones, such as estrogen and prolactin, have similar dependence receptor functions, in the hope of clarifying the connection between age-related changes in hormone secretion and disease pathogenesis.

Alzheimer’s Disease. The list of caspase substrates also includes a number of Alzheimer’s disease mutants. In collaboration with a team from the University of California, San Diego, headed by Edward H. Koo, our group recently observed that amyloid precursor protein (APP)–the parent molecule of amyloid beta-protein, which is the main constituent of senile plaques–is cleaved by caspases and produces a fragment consisting of its carboxy terminus, which independently induces a cell death program. This second cytotoxic peptide, along with its associated active caspase, is seen only in the brains of Alzheimer’s patients.

It is highly questionable, however, whether inhibiting caspase cleavage of APP would be an effective strategy for prevention of Alzheimer’s disease. Amyloid beta-protein, while clearly neurotoxic, may not be the only cause of neuronal and synapse loss in the neocortex. It may not even be the main cause. Other proposed mediators include oxidative damage, inflammation, mitochondrial dysfunction, and apolipoprotein E. Even the mode of cell death that occurs in these patients is controversial, with apoptosis thought to be only one mechanism involved.

Dependence Receptors and Cancer. How does the concept of cellular dependence apply to cancer pathogenesis? The best-studied example concerns the candidate tumor-suppressor gene DCC (deleted in colorectal cancer), which encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC gene expression is one of a number of features associated with malignant cell proliferation. Reestablishment of DCC gene expression suppresses tumorigenicity. DCC’s mechanism of action is, however, unknown.

Several years ago, a team led by Patrick Mehlen demonstrated that DCC functions as a dependence receptor, inducing apoptosis in the absence of ligand binding, blocking it when engaged by netrin-1. Moreover, DCC was revealed to be a caspase substrate. Mutation of caspase cleavage entirely suppresses the proapoptotic effect of the gene. These results strongly suggest that DCC may function as a tumor-suppressor or metastasis-suppressor gene by inducing apoptosis in settings where ligand is unavailable–for example, when tumor growth extends beyond the local blood supply. Loss of DCC function, on the other hand, may enhance survival of cells outside the region of ligand availability.

The assumption that DCC is a dependence receptor is compatible with a number of previously established findings: 1) In some tumors, DCC expression is associated with increased cell death. 2) DCC deletion tends to be a late event in the development of colorectal cancer. 3) DCC deletion tends to be associated with invasive and metastatic tumors. It has not been established whether netrin-1 or another ligand, such as heparin, is the agent normally responsible for blocking DCC-induced apoptosis in vivo.

Novel Treatment Strategies

The fact that gene products associated with neurodegeneration are overrepresented as caspase substrates suggests that inhibition of caspase cleavage may be an effective intervention for this group of disorders. Simply by examining the polyglutamine sequence or another portion of the genetic fingerprint, tomorrow’s clinicians may be able to identify silent abnormalities in time to intervene effectively.

With respect to suppression of tumor cell proliferation, a team led by H. Michael Ellerby is working on novel chimeric peptides that can induce a program of cell death in the angiogenic endothelial cells that supply tumors. Each chimeric peptide consists of two functional domains: a homing motif designed to guide the peptide to targeted cells in tumor blood vessels and allow its internalization, and a proapoptotic sequence designed to be nontoxic outside cells but toxic (via disruption of mitochondrial membranes) when internalized into targeted cells (Figure 2). This research was based on prior work by Erkki Ruoslahti and Renata Pasqualini showing that the endothelial cells of angiogenic vessels are stamped with specific molecular addresses, so that different peptides will automatically home in on different regions. Both in tissue culture and in the mouse model, these hunter-killer peptides went directly to the angiogenic vessels of malignant tumors and shut them down, suggesting that proapoptotic peptides represent a potentially valuable new class of anticancer agents.

Presumably, the same concept could be applied in reverse, using supportive molecules to modulate neurodegenerative disease pathways. Early intervention would be vital, since preventing the loss of neurons, however difficult, would be an easier task than replacing them.

Looking Ahead to Healthy Aging

I believe that this area of research ultimately will revolutionize the way in which medical science approaches patient management. Historically, physicians have always tended to approach age-related diseases reactively–we wait until something goes wrong and then attempt to fix it. The cloning of the human genome and other recent breakthroughs have made such thinking obsolete. Rather than waiting until a patient has reached mid-life or is already in poor health before discussing preventive management, planning for healthy aging should begin at birth. Within this century, information gleaned from patients’ genetic profiles may permit physicians to stave off progression of common degenerative diseases for years, if not indefinitely. While development of actual cures could prove elusive–in the case of the neurodegenerative diseases, therapeutic options are currently few–simply postponing clinical onset could have an impressive effect on mortality and allow the oldest segment of our population to enjoy a much longer period of productivity.

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Referenecs

1. Bredesen DE et al. p75^NTR and the concept of cellular dependence: Seeing how the other half die. Cell Death Diff 5:365, 1998

2. Ellerby HM et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nature Med 5:1032, 1999

3. Ellerby LM et al. Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity. J Biol Chem 274:8730, 1999

4. Ellerby LM et al. Kennedy’s disease: Caspase cleavage of the androgen receptor is a crucial event in cytotoxicity. J Neurochem 72:185, 1999

5. Lu DC et al. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nature Med 6:397, 2000

6. Mehlen P et al. The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis. Nature 395:801, 1998

7. Rabizadeh S et al. Induction of apoptosis by the low-affinity NGF receptor. Science 261:345, 1993

8. Sperandio S, de Belle I, Bredesen DE. An alternative, nonapoptotic form of programmed cell death. Proc Natl Acad Sci USA 97:14376, 2000