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Neuronal Degenerative Mechanisms as Clues to Pathogenesis and Treatment of Alzheimer's Disease

RALPH A. NIXON

Laboratories for Molecular Neuroscience. Mailman Research Center, McLean Hospital, Han'ard Medical School, 115 Mill Street, Belmont, MA 02178

ALZHEIMER'S Disease (AD) has multiple primary, etiologies but shares characteristic neuropathological end points and possibly one or more common pathogenetic pathways. In many individuals, a single factor, such as inheritance of a defective gene, may be a major determinant of life-time risk for Alzheimer's disease (28), but other factors can significantly influence the onset and course of the disease even in individuals with the same genetic background (5). The identification and molecular characterization of these ge- netic and environmental risk/protective factors continues, with strong justification, to be a major research strategy toward iden- tifying ways to prevent Alzheimer's disease. These approaches have been considered by a number of other contributors to this symposium monograph and are not discussed here. With an epi- demic rise in the incidence of Alzheimer's disease looming, how- ever, the urgency to develop even partial therapeutic solutions calls for consideration of additional pragmatic approaches toward slowing progression of the disease. The approach discussed here is based on the growing evidence that clinical severity and behavioral decline in Alzheimer patients are closely related to the progressive functional compromise of specific neuron groups, as reflected structurally in neurofibrillary tangle development (2), extensive synaptic loss (14,48), and, ultimately, the fall-out of neurons (31). Therefore, irrespective of primary, inciting event(s), preventing irreversible neuronal injury and enhancing the survival abilities of the remaining neurons should be a useful avenue for therapeutic intervention.

Discussed in this article is the need to accelerate efforts toward defining the cell and molecular biology of specific patterns of neuronal degeneration and irreversible cell inju~' to achieve sev- eral immediate goals. One goal is to distinguish molecular features of the Alzheimer degenerative pattern shared more generally by neurons undergoing degeneration (final common pathways) from features that point toward additional proximal pathogenetic events and may be more specific therapeutic targets. In the short term. it is reasonable to expect that even agents that target final common pathways of cell injury may prove to be clinically useful. A second goal is to develop criteria by which to judge whether a particular degenerative pattern induced experimentally by a putative etio- logic factor is indeed, relevant to Alzheimer's disease. Third, fundamental information about general and specific features of neurodegenerative patterns is also essential to create in vitro and in vivo systems of cell injury, that accurately portray the Alzheimer pattern and enable rational drug screening and preclinical evalua- tion. Finally, the same information is required to differentiate cellular responses during the degenerative process that are mal- adaptive from those that are compensato~'--a distinction crucial to predicting the efficacy of therapeutic agents.

Therapies aimed at preventing neuronal degenerative processes and cell death in Alzheimer's disease are certainly not a novel idea: various pharmacological agents are currently being exploited for this purpose. Less well developed, however, is the notion of approaching this goal from a basic understanding of how cells die in different disease states, As discussed next. the fundamental nature of this process in Alzheimer's disease and most other hu- man neiarodegenerative diseases remains unclear. Moreover, de- spite its central importance to clinical progression, the phenome- nology of neuronal degeneration and cell death is rarely a specific focus or organizing theme for presentations at major conferences on Alzheimer's disease. The tendency to subsume this topic under the heading of a particular putative etiologic agent or. more often, under many different headings, may be impeding the process of consolidating the existing knowledge and of stimulating synthesis of new ideas among investigators working on different aspects of this general problem.

A full understanding of neuronal degenerative mechanisms is likely to be beyond the time frame of the 5-5. 10--10 initiative proposed by Khachaturian (24); however, proportionately, so little of the collective attention in the Alzheimer field has centered on the pathobiology of neuronal degenerative mechanisms that prog- ress toward this goal is likely to be relatively rapid. Answers to even some of the initial basic questions about final common path- ways might fundamentally influence the direction of drug devel- opment. The identification of a specific genetic program that con- trols developmental cell death in C. elegans (19) and possibly in Drosophila (51) are recent examples of how a previously bewil- dering array of biochemical events can now be better understood as a cascade triggered by the induction of a specific set of genes. The bcl-2 proto-oncogene product, which has been shown to block the apoptotic cell death induced by various stimuli (39), is another example of unexpected and welcomed reductionism in understand- ing cell death processes. How these genetic controls relate to mammalian neurons and Alzheimer's disease is unknown, but an- swers to this or similar questions may be of great immediate im- portance. If, indeed, there is a limited repertoire of ways by which cells die, these final common pathways can be defined and tar- geted as a first step in therapy and point research toward anteced- ent disease-specific triggers of these final events. A significant opportunity may exist for crossfertilization from other fields of basic medicine if there is overlap between these mechanisms in neuronal and nonneuronal cells: agents now being developed to treat conditions involving the death of nonneuronal cells may be instructive or even prove to be therapeutic in Alzheimer disease. Initial information about the patterns of cell death in other neuro- degenerative diseases may also hold clues to molecular mecha-

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nisms in A!zheimer disease. The discover' of superoxide dismu- tase mutations promoting free radical production in certain fornls of familial amyotrophic lateral sclerosis 141~, for example, has renewed the interest in whether or not free radicals are an impor- tant factor in other late-onset diseases, including Alzheimer's dis- ease {l I.

Drug discovery will only be as successful as drug screening systems are accurate in modeling pathologic events relevant to the cause of neuronal degeneration. The detailed information on de- generative mechanisms needs to be applied to the development of cell systems and experimental paradigms used as standard models of Alzheimer-relevant cell injury. Experimentally induced neuro- pathologic states are proposed as models of AD pathogenesis based on relatively few criteria, which have still unestablished pathogenetic relevance. Features such as diffuse [3-amyloid depo- sition, reappearance of developmentally regulated phosphorylation events on cytoskeletal proteins, or neuron degeneration without regard to the specific pattern, may, by themselves, not be specific enough pathologic end points on which to base experimental mod- els for Alzheimer's drug discovery. Additional information about whether cell death in Alzheimer's disease occurs by apoptotic or necrotic mechanisms, over minutes or weeks, and in a dying-back or perikary'al pattern, may turn out to be additional useful criteria in evaluating suitable screening systems. Moreover, before in vitro data can be reliably extrapolated in a clinically useful way. the basis for the great differences in vulnerability of the same neuron to a given toxic agent in vitro and in vivo, as in the case of [3-amyloid. must be more fully understood. In this regard, given that aging of the brain is a well-established universal risk factor for Alzheimer's disease, it will be important to know, for example, whether or not mature or aging neuronal systems may need to be used to model features of Alzheimer's disease and to evaluate potential therapeutic agents.

PROBING NEURONAL DEGENERATIVE MECHANISMS FOR CLUES TO ALZHEIMER'S PATHOGENESIS

The neuronal degenerative marker, neurofibrillary tangles, has long been the popular basecamp from which to hunt for more proximal intracellular metabolic disturbances in Alzheimer's dis- ease. The resultant advances have been significant in revealing the importance of tau and identifying specific protein kinases and phosphatases that may regulate tan function and be putative targets for Alzheimer's therapeutics. The present repertoire of intracellu- lar markers used to conceptualize and model the intracellular pa- thology of Alzheimer's disease, however, remains very limited. Equivalent effort should now be devoted to revisiting the cellular pathology of Alzheimer's disease and systematically applying the newly available molecular and immunologic probes for specific organellar systems to optimally prepared autopsy or biopsy human brain tissue. Cytoskeletal abnormalities are likely to be only one of a number of markers of more primary" cellular disturbance, which may be uncovered by probing the degenerative mechanism in Alz- heimer brain in a systematic way.

One case in point are recently identified disturbances of the endosomal-lysosomal system in at-risk populations of neurons in Alzheimer brain 17.8,33,34). Lysosomal system activation in the natural neuronal cell death occurring in several invertebrate sys- tems during development ( 13,491 originally fueled a popular as- sumption that lysosomal system alterations are commonly linked to the phenomcnology of cell death; however, later studies sug- gested that. in mammalian neurons, prominent endosomal- lysosomal system changes are a distinctive cellular response to certain types of metabolic compromise rather than part of a final common pathway to irreversible cell injury (32).

Alzheimer's disease is one of a few human neuropathologic states in which the endosomal-lysosomal system is known to be prominently activated ~321. and certain features of this disturbance are highly characteristic of an Alzheimer pattern of neurodegen- eration (7). Underscoring a main point of this commentary, inves- tigations of end-stage lysosomal abnormalities have pointed the way to identifying antecedent endosomal-lysosomal alterations appearing relatively early in the compromise of neurons (8,33,34). Lysosomal hydrolases are exclusively intracellular enzymes in normal tissues; however, they are abundant in an abnormal extra- cellular location associated with diffuse and mature senile plaques in Alzheimer's disease and in certain other conditions where [3-amyloid accumulates (3,9.10,50). The principal sources of ex- tracellular hydrolases are degenerating neurons and their processes which massively accumulate hydrolase-laden secondary lyso- somes and residual bodies before the neurons degenerate (7,11, 30). One of these lysosomal hydrolases, cathepsin D, was recently shown to be markedly increased in cerebrospinal fluid of Alzhei- mer's patients but not individuals with Huntington's disease and various other neurologic diseases, demonstrating that the release of these intracellular enzymes from compromised cells is an active ongoing process (26). The persistence of acid hydrolases and hy- drolase-containing vesicular compartments in the extracellular space has, to our knowledge, only been observed in Alzheimer's disease and other conditions associated with [3-amyloidogenesis. By analogy to rheumatoid arthritis, we speculate that the release of these proteases could represent a possible stimdlus for the induc- tion of acute-phase reactants (38) and immune responses (16) in senile plaques that may account for the possible therapeutic effects of anti-inflammatory agents in Alzheimer's disease (40). If, as we believe, the release of lysosomal hydrolases is one of the triggers of these events, specific lysosomal hydrolase inhibitors that act in the extracellular space and are not taken up by cells may also warrant further investigation as therapeutic agents.

The lysosomal abnormalities described above are only the end- stage of a process that begins much earlier in a high percentage of neurons and are clearly not related to the mechanism of irrevers- ible cell injury. These early abnormalities involve a marked ab- normal upregulation of the endosomal-lysosomal system that can be identified in the majority of otherwise normal-appearing neu- rons within populations that are at risk to degenerate early or late in the disease course (6,35). Upregulation is evidenced by in- creased cathepsin gene expression, increased levels of hydrolase enzymes, and accumulation of late endosomes and secondary ly- sosomes in the perikaryon and proximal dendrites in the absence of atrophic, chromatolytic, or neurofibrillary changes (8,33,34). Be- cause late endosomes and lysosomes are sites of convergence of the endocytic (heterophagic) and autophagic pathways (15,20), upregulation of hydrolase synthesis and lysosome biogenesis may reflect either accelerated endocytosis or autophagy or both. Al- though their triggering mechanisms are not well understood, au- tophagy and endocytosis are responses expected in cells chroni- cally attempting to regenerate, catabolize injured membranes, and synthesize new membranes. The extensive axon terminal degen- eration and dendritic dystrophy of membranes in Alzheimer dis- ease, which may precede morphologic changes in neuronal perikarya, implies the breakdown and replacement of large areas of membrane surface and accelerated membrane trafficking to and from the periphery. This possibility is supported by a growing number of reported membrane alterations in Alzheimer disease involving cholesterol and other lipids (22,27,36,53), lipolytic en- zymes (18,36), and various membrane proteins (4). These consid- erations support the view that research on membrane pathobiology in Alzheimer's disease, which is minimal at present, should be expanded.

NEURONAL DEGENERATING MECHANISMS AND TREATMENT $63

Interestingly, early abnormalities of the endosomal-lysosomal system are likely to impact on pathogenetically significant mole- cules in Alzheimer's disease such as Apolipoprotein E (apoE) and amyloid precursor protein. The influence of the apoE genotype on risk for Alzheimer's disease {12,46) may involve the role of this protein in cholesterol transport (26.43), and the requirement of endosomal-lysosomal system activity in this function (52). ApoE synthesis in astrocytes and uptake by neurons are markedly up- regulated in response to neuronal injury (29.37.50L presumably to provide cholesterol for new membrane synthesis and regenerative processes (26). Similarly. the levels of 13-amyloid precursor pro- tein (I3-APP) in cells are regulated partly by endocytosis, and a major route of amyloid precursor protein processing and degrada- tion is through the endosomal-lysosomal pathway (for review, see ref. 21). The 13-amyloid peptide, AI3, is believed to be generated within a post-Golgi compartment (21), and several cathepsins are among the candidates for this process (25,44,47). I3-APP is also metabolized in lysosomes to smaller fragments containing the complete A~ domain (17,45) which represent an additional poten- tial source of AI3, especially under pathologic conditions when the function of the lysosomal system may be disturbed (7,9,23,47). Because APP mutations account for only a small percentage of Alzheimer's cases (28), early activation or late dysregulation of the endosomal-lysosomal system could be mechanisms to explain both the delayed phenotypic expression of APP mutations and increased production of A~. in the absence of APP mutations.

AREAS OF NEW RESEARCH FOR FUTURE EMPHASIS

The foregoing discussion illustrates one example of how the application of newer probes of biochemical and organellar func- tion to Alzheimer's degenerative events may help to identify po- tential new targets for drug intervention. Many fundamental ques- tions remain, however, about the nature of neuronal cell death under any condition, and particularly in the late-onset variety that is relevant to many neurodegenerative diseases. For example, we need to know whether cells of different types, or even the same type, die through the same final common pathway or whether multiple pathways exist. The question of whether young cells die by a different sequence of events than old cells has barely been addressed experimentally. Remarkably little is known about whether slow death implies a different biochemical program than acute death or whether various toxic agents induce cell death by fundamentally different mechanisms. How important are cell-cell interactions in modifying the cell death process? Answers to all of these questions are critical to understanding what the distinctive morphologies of degenerating cells in Alzheimer's disease are tell- ing us about the specific mode of cell death, how closely AD models reflect the essential nature of this process, and how optimal the current systems are for screening therapeutic agents.

Some goals that might be considered in a research plan to target neuronal degenerative mechanisms are briefly listed next. Al- though some have relatively long time frames, initial data in these areas are likely to have immediate application to the objectives of

the 5-5, 10--10 plan. particularly if the research is given more emphasis.

1. Integrate current information on molecular and structural fea- tures of cell injury' that establish operating criteria for recog- nizing specific modes of degeneration and final common path- ways to cell death {e.g., apoptosis, programmed cell death, necrosis, etc.). Determine the importance of differences in spe- cies, neuron type, stage of maturation, and rate of degeneration as influences on particular patterns of cell death.

"3 =. Identify more sensitive markers of incipient and late cell injury', including the immediate antecedents to irreversible cell injury' le.g., mechanisms of DNA damage and repair, activation of specific protein degradative mechanisms, synaptic loss, dying back neuropathic patterns, etc.). This includes markers that discriminate the compensator,.,' responses of a degenerating cell from steps in the cell-death mechanism itself.

3. Using the criteria developed, define the specific pattern of cell degeneration in Alzheimer's disease in relation to other dis- eases and other degenerative patterns and evaluate existing pu- tative etiologic factors on the basis of the specific patterns of degeneration they induce.

4. Develop genetic and reproducible biochemical models of neu- rodegeneration, particularly of the delayed or late-onset type that incorporate a broader array of features characteristic of the Alzheimer degenerative pattern. Devise additional methods for quantifying cell degeneration in vivo le.g., automated methods for quantifying cell death, synaptic loss, endosomal-lysosomal system upregulationl.

5. Identify agents that specifically interrupt either the steps in the final common pathways to neurodegeneration or their imme- diately antecedent triggers. Possible steps within these path- ways may involve calcium regulation, activation of specific proteases or protein kinases/phosphatases, changes in efficacy of free radical generators and scavengers, and the induction of cell death genes.

Obviously, this addresses only one of many areas where more concentrated attention should prove useful in responding to the mandate of the 5-5, 10-10 plan. Other needs complementary to the ones described here include the development of systems to deliver agents across the blood-brain barrier, to sustain long-term administration of agents to the brain, and to target compounds to specific cell populations. Moreover. many of the goals empha- sized in this article intersect with the goals of research recom- mended by other participants in this workshop.

. * C K N O W L E D G E M E N T S

I am grateful to Anne Cataldo for helpful discussions and Johanne Khan for assistance in manuscript preparation, Studies from this laborato~' described in this article have been supported by grants from the National Institute on Aging I AG08278, AG05134 and AG 10916 and from the Anna and Seymour Gitenstein Foundation.

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