An Assessment of the Chances of Antiapoptotic Drug Therapy in Patients with Neurodegenerative Disorders

Chapter 21

An Assessment of the Chances of Antiapoptotic Drug Therapy in Patients with Neurodegenerative Disorders Peter C. Waldmeier APOPTOSIS AND OTHER MODES OF CELL DEATH A bit of history Developmental biologists and histologists had been well aware of physiological, i.e. somehow regulated cell deaths, since the mid-nineteenth century, but the mechanisms involved did not get much attention (for a review of the history of programmed cell death and apoptosis see (1)). In the 1950s the interest focused on the role of the newly discovered lysosomes (2) in cell death, and it was only in the 1960s, when studies of metamorphosis in insects suggested that cell death followed a sequence of controlled steps, it was realized that this implied genetic determination. The term “programmed cell death” (PCD) was created (3), and it was found that this process could be affected by drugs (4). The term “apoptosis” (from Greek απóπτωση, approximately meaning shedding or decline) was coined by Kerr and coworkers (5) to describe a type of active, organized cell death characterized by chromatin condensation, cell shrinking, membrane blebbing and finally phagocytosis of the rest by neighboring cells. The impact of these findings which today are considered as seminal was initially meagre, as judged from the number of publications found in Medline under the search term apopto$ between 1972 and 1990 (Fig. 1). This changed after 1990, probably as a consequence of a small number of discoveries within a short period of time: the demonstration that apoptosis is characterized by a specific pattern of internucleosomal DNA degradation (6), which provided a simple means for identification and quantification; the identification of bcl-2 as an antiapoptosis gene (7) and of the tumor suppressor protein p53 (8,9) and c-myc (10,11) as proapoptotic regulators; the identification of Fas-Apo-1as a cell surface death-transducing receptor (12,13); the discovery of the caspases (14); and the realization that mitochondria are key players in life/death decisions (15). Since 1994, there has been a rather steady growth in the number of publications, the rate of annual increase amounting to about 1200. Although a majority of publications relate to roles of apoptosis in immunology and cancer, a pretty constant fraction of about 15% of these papers also respond to the search term (neuro$ or nerv$), indicating that they at least in part deal with apoptosis in neurons. Close to 10–20% of this fraction of papers explicitly refer to chronic and acute neurodegenerative conditions, respectively (Fig. 1). Probably the first paper in this Oxidative Stress and Neurodegenerative Disorders Edited by G. Ali Qureshi and S. Hassan Parvez

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Fig. 1. Number of publications per year relating to apoptosis and various disease areas. Note: Medline was searched for keywords or combinations thereof as indicated in the legends. ∗ cND means chronic ND and comprises AD, PD, ALS, HD and MS; † aND means acute ND and comprises the subheadings and/or keywords stroke, cerebrovascular accidents, spinal cord injury, spinal injury, spinal cord trauma, spinal trauma, craniocerebral trauma, (traumatic) brain injury and brain trauma; ∗∗ “Cardiovasc.” comprises the subheadings/keywords heart, heart diseases, cardio$ and cardiovascular. diseases.

respect suggests that 1-methyl-4-phenylpyridinium, the active metabolite of the dopaminergic neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes apoptosis of cerebellar granule cells, and that “neurodegenerative diseases (ND) may result from inappropriate activation of PCD by apoptosis” (16). It soon appeared obvious that programmed processes for the clean and controlled removal of single cells in response to cellular damage, at that time thought to be predominantly represented by apoptosis, could

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provide therapeutic targets for a variety of diseases, including neurodegenerative conditions (17–19). These might be consequences of erroneously induced apoptosis (death by mistake) or apoptosis induced as a response to serious damage, in which case its prevention might buy time for self-repair (20). The term apoptosis has often been used as a synonym for PCD, and until recently, cell death was often considered as either necrotic or apoptotic, although it was pointed out early on that PCD should be considered as a genetically determined sequence of events not necessarily leading to a morphology of apoptosis (21). It may therefore be appropriate to briefly review the status.

Necrosis Pure necrosis or oncosis is a nonregulated, accidental form of death in which the cell has no active role. Morphological changes consist of condensation (and finally disappearance) of chromatin into small, irregular clumps without clear changes in distribution, sometimes abnormally swollen mitochondria and local membrane disruption followed by disintegration of organelles and membranes. Cells lyse and cause substantial inflammation, compromising previously unaffected neighboring cells (see e.g. (22)). Thus, necrosis is the “maximum damage” form of cell death and usually hits groups of cell located together.

Programmed cell death (PCD) PCD comprises all forms of active, genetically controlled forms of cell death occurring in sequential steps, including apoptosis, programmed necrosis and autophagic cell death. Apoptosis Apoptosis is a form of PCD associated with a particular morphology (5). It is often dependent on activation of caspases, a family of cysteinyl-aspartate-cleaving proteases many of which are part of an evolutionarily conserved cleanup machinery. Caspases -1, -4, -5 and -11 are involved in inflammatory cytokine production and probably not in propagation of death signals. Others have physiological functions in addition to their role in cell death. Caspases are synthesized as proenzymes and converted into mature proteases through activation of complex signaling cascades by stress stimuli via intrinsic and/or extrinsic pathways, depending on whether the signal originates from inside or outside the affected cell. Initiator caspases (-2, -8, -9, -10 and -14), generally upstream of the point of no return in the sequence of events leading to cell death, are activated by oligomerizationinduced autoprocessing. Effector caspases (-3, -6 and -7), which make the death process irreversible by destroying essential proteins, are activated by other proteases including initiator caspases. External signals are mediated by the death receptor (extrinsic) pathway (comprising TNF receptor-1, TRAIL receptors-1 and -2, APO-3, FAS) via activation of caspase-8, which directly activates the effector caspase-3, but some cell types can also trigger the distal part of the intrinsic (mitochondrial) pathway by truncating the

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proapoptotic Bcl-2 family member Bid (tBid); this amplification loop can be necessary to achieve apoptosis via the extrinsic pathway. Internal cellular distress signals triggered e.g. by oxidative or DNA damage, trophic factor deprivation etc. are sensed by a subset of Bcl-2 family proteins such as Bax, Bad, Bak, Bid etc., which contain the BH3 domain only, are proapoptotic and activate the intrinsic pathway. Mobilized by post-translational modification, they translocate to mitochondria and trigger mitochondrial outer membrane permeabilization (MOMP), leading to the release of cytochrome c which in concert with Apaf-1 activates procaspase-9, under normal circumstances sufficient to quickly dispose of a cell in a caspase-dependent manner. MOMP however, releases other harmful proteins like apoptosis-inducing factor (AIF), endonuclease G (EndoG) or the serine protease HtrA2/Omi from the mitochondrial intermembranal space, which can lead to caspaseindependent cell death with an apoptosis-like morphology if caspases are disabled. This explains why caspase inhibitors are often quite inefficient against cell death mediated by the intrinsic pathway, and suggests that the actual commitment point to cell death mediated by the intrinsic pathway is MOMP. On the other hand, MOMP can be prevented by antiapoptotic Bcl-2 family members under the control of pro-survival pathways, which stabilize the mitochondrial membrane and/or sequester their proapoptotic cousins. Distress signals like reactive oxygen species (ROS) or elevated Ca2+ levels can also induce the mitochondrial permeability transition (MPT), involving the opening of a pore at the junction of outer and inner membranes. Beyond giving rise to the release of cytochrome C, AIF, EndoG etc., this leads to large amplitude swelling of the affected mitochondria, loss of mitochondrial membrane potential and uncoupling of oxidative phosphorylation. If this happens to enough mitochondria in a cell, ATP production may be compromised to such an extent that apoptosis (which requires energy) cannot be completed, and necrosis ensues. Stress to the endoplasmatic reticulum (ER) by disturbed glycosylation, misfolded proteins, perturbed Ca2+ homeostasis or glucose deprivation can cause apoptotic or necrotic cell death through the unfolded protein response which may cause repression of Bcl-2 transcription, increased cytosolic Ca2+ , which may trigger dephosphorylation of Bad and/or activate the MPT, or activate caspase-12 (for which, however, no active human ortholog is known) normally localized at the cytosolic side of the ER. For more in-depth reviews, the reader is referred to e.g. (23–26).

Programmed necrosis The idea that apoptosis can derail into necrosis for shortage of ATP has been around for a while, and has never been seriously contested. It implies that the process, dubbed necrapoptosis, begins as apoptosis, with the very same pathways involved, and must therefore be seen as a variant of apoptosis with a different outcome. As an entity of its own, the concept of programmed necrosis has led a modest existence. Based on data suggesting that caspase-independent necrotic cell death elicited in the presence of caspase inhibitors or antiapoptotic Bcl-2 family members can be prevented by antioxidants or by eliminating the activity of the protein kinase RIP, it has been regarded as a mere tissue culture phenomenon.

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A number of recent findings, however, suggest that programmed necrosis may well play a role in (patho)physiological settings. Thus, TNFα, which normally induces apoptosis, causes necrotic cell death in cells infected with a virus encoding an antiapoptotic protein, through initiation of signaling cascades downstream of its receptor, TNFR-2 (27). Moreover, stimulation of the Fas/TNFR family in the absence of apoptotic signaling can trigger a nonapoptotic form of cell death showing hallmarks of necrosis and autophagy, called necroptosis by the authors, which could be inhibited by a small molecule, necrostatin-1. Necrostatin-1 also had protective effects in a mouse model of delayed ischemic brain injury, suggesting pathophysiological relevance of the process (28). Programmed necrosis triggered by DNA damage was found to be initiated by the DNA repair enzyme poly(ADP-ribose)-polymerase (PARP), but only in rapidly proliferating cells, which depend much more on glycolysis as the source for their ATP than vegetative cells which can do with oxidative phosphorylation and aminoacid catabolism. In such cells, enhanced polyADP-ribosylation results in rapid depletion of NAD and thus inhibition of glycosylation which in turn leads to ATP depletion and programmed necrosis (29). Thus it is probably justified to consider the concept of programmed necrosis seriously, in particular in situations when apoptosis is inhibited, a condition obviously relevant when patients with neurodegenerative diseases (ND) are treated with antiapoptotic drugs. It has been argued that ongoing apoptosis might actively suppress necrosis because activated caspases cleave proteins involved in programmed necrosis, for example RIP and PARP (30–32). Inhibition of apoptosis might thus result in activation of programmed necrosis, a safety catch to make sure that damaged cells can commit suicide even in the presence of roadblocks (33).

Autophagy Autophagy is an evolutionarily conserved mechanism allowing eukaryotic cells to survive in conditions of nutrient restriction by catabolically producing ATP. When nutrients are not in shortage, it plays an important role in bulk removal and recycling of proteins, targeting long-lived proteins and entire organelles, for example mitochondria to the lysosome; in contrast, shorter-lived regulatory proteins are disposed of by the ubiquitin-proteasome system (34). Of the three known forms of autophagy (macroautophagy, microautophagy and chaperone-mediated autophagy), macroautophagy as the major inducible form for turnover of cytoplasmic components is the focus of our interest in connection with ND. It involves four discrete steps: induction and formation of autophagic vacuoles or autophagosomes, consisting of double-membrane vesicles sequestering portions of cytoplasm, which dock to and fuse with lysosomes, and finally are broken down proteolytically (35). Much has been learned about the mechanisms of regulation of autophagy recently. Very briefly, phosphatidylinositol 3- kinases (PI3K) control autophagy at different levels: class I PI3K inhibit sequestration of cytoplasm through activation of the inhibitor of autophagy, target of rapamycin (TOR). Starvation, by reducing the levels of intercellular growth factors, reduces class I PI3K signaling and thus induces autophagy. Class III PI3K positively control the formation of the sequestering membrane via the autophagy regulator Atg6 or Beclin1 (for more details on the regulation of autophagy see e.g. (36–38)).

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Despite the recognition that autophagy is primarily a survival mechanism putting the cell in a state of hibernation, a role in cell death is now discussed (25,38,39), not least because of the description of autophagic morphology in various ND. It is not clear whether its role in dying cells is protective or deleterious, and whether and under what circumstances it may turn into autophagic death (34). Death of autophagic cells typically occurs if apoptosis is blocked, and autophagy may precede apoptosis, representing a cellular salvage attempt which can derail into apoptosis, autophagic cell death or perhaps even necrosis if its capacity is exceeded. There appears to be crosstalk between autophagy/autophagic cell death and apoptosis at multiple levels. Thus, caspase activation can inhibit autophagy by proteolysis of regulatory factors like RIP (38). Also, induction of MOMP or MPT below the threshold for apoptosis induction results in autophagic sequestration of damaged mitochondria, probably as a cytoprotective mechanism. If such removal of mitochondria occurs at a higher rate and in conditions where apoptosis is inhibited, it may result in autophagic or necrotic cell death (40,41). Death-associated protein kinase (DAPk) causes apoptosis in a caspase-dependent manner, probably through induction of MOMP, because Bcl-2 prevents it. On the other hand, DAPk is essential for the induction of autophagic cell death in several paradigms (39,41).

PCD is the last segment of a damage control system The different routes to cell death and their interrelations are simplistically depicted in Fig. 2. The take-home message from our little tour d’horizon is that, with the exception of plain, catastrophic necrosis, cell death is highly regulated and can take several routes

recovery

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Fig. 2. Cell-fate decision tree.

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depending on causative factors and cellular conditions. The goal is limitation of damage. The routes not only share certain elements, but are also plastic in the sense that switches between (some of the) different routes can occur if the conditions change. Examples of such changes in conditions are impairment of energy metabolism, dysfunction of signaling pathways or of the execution machinery. A possible scenario after infliction of damage is that cells first try to control and repair (DNA repair systems, antioxidant defenses, autophagy, etc.). If this is not possible or fails underway, apoptosis is attempted; if that fails, programmed necrosis, autophagic death or plain necrosis ensue. To quote Lockshin & Zakeri (34): “When the death of a cell is inevitable, it will take any available route to death”. In a given tissue, both threshold and outcome may vary for different cells of the same type and probably even more so for cells of different types. These considerations should be borne in mind when we come to the discussion of the clinical results obtained with the first antiapoptotic drugs that were tested.

EVIDENCE FOR A ROLE OF PCD IN ND Chronic ND Conclusive pathophysiological evidence for a prominent role of apoptosis or other forms of PCD in human ND is not easy to come by. Since corresponding data have to be gathered from patient’s postmortem brain tissue, they generally represent single snapshots of whatever parameter that is measured at a very late time in the course of the disease. We have little possibility to figure out whether what we see is representative of the mechanisms involved in the demise of neurons earlier on in the course of the disease, or rather for those involved in killing the more resistant ones about to die at the moment of the patient’s death, or on the contrary, they reflect countermeasures of the hardy survivors. In this situation, it appears wise to resort to animal models, where one can follow the time courses of such events and thus gain access to data which one could hardly obtain from patient’s postmortem material. But this approach implies that the pathways to apoptosis (or PCD, to be precise) that neurons take in such models match those in the corresponding disease. And how do we ascertain that in the absence of suitable pathophysiological data from patients? These reflections should be considered when we look at the evidence for a role of apoptosis or PCD in ND. Parkinson’s disease (PD) In the early days of the search for markers of apoptosis in postmortem patient brain tissue, a favorite technique was TdT-mediated dUTP-biotin 3 nick-end-labeling (TUNEL), now known to be an unreliable marker for apoptosis when used alone, one important confounding factor being antimortem hypoxia (42,43). Accordingly, the use of the TUNEL technique originally caused considerable confusion with respect to the question whether or not dopamine (DA) neurons die by apoptosis in the Parkinsonian substantia nigra. Some authors found increased TUNEL staining in PD DA neurons (44), some did not (45,46), others only in glia (47), and still others found it in both PD and control tissue (48,49).

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In the studies supporting occurrence of apoptosis in PD, including one in which the presence of apoptotic features was assessed by morphological criteria (50) 5–6% of the DA neurons in PD tissue and 1–2% in control brains were found to be apoptotic as defined by TUNEL staining at the time of death. Considering the slow progression rate of the disease, these high rates suggest a perimortem phenomenon related to hypoxia due to the patients’ agonal state. The higher percentage in PD brains may indicate an increased vulnerability of these DA neurons (51). A more recent study using in situ end labeling (ISEL) and staining for chromatin condensation to identify apoptotic neuronal nuclei in PD substantia nigra, established clearly higher numbers of melanized neurons showing apoptotic features than in controls (52), along with increased caspase-3, bax immunoreactivity and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein implicated in apoptosis (53). This author also pointed out that there is little evidence to support the assumption of a very short survival time of apoptotic neurons in situ in the human brain underlying the argument that the observed frequency of apoptotic neurons in PD brain was too high to be real. Moreover, the probability of detecting apoptotic cells depend on the lifetime of the chosen marker which may differ from the survival time of the corresponding cell, a further reason why argumenting with numbers is doubtful. Increased abundance of apoptotic markers like bax, activated caspase-3 and caspase-8, and p53 in suffering melanized neurons in PD substantia nigra (54–57) and evidence for an activation of the fas signaling pathway (58,59) support the idea of an involvement of apoptotic processes in the death of nigral DA neurons in PD (51,60) . Evidence supporting a role for apoptosis has also been derived from animal models of PD, particularly the MPTP model (see e.g. (20,60,61)) . Although this is appealing because it allows to look into the death processes temporally and in much more detail, it assumes that the routes to, and pathways involved in, DAergic cell death are a true reflection of what happens in the PD brain. Proof of this is neither available nor easy to come by. It should also be noted that DA neurons showing clear features of autophagy, but not necrotic neurons were found in the substantia nigra of PD patients (50). Autophagosomes in such tissue appear to contain damaged mitochondria (62), compatible with the idea that DAergic neurons affected in PD try to remove such organelles and that autophagy may represent a salvage attempt. Such neurons likely end up dead anyway, one way or the other. Summing up this section, it is likely that apoptosis does occur in nigral DA neurons at or around the time of death of PD patients. Whether this is representative for most or all of them at any stage of the disease is an unsolved question, which will be discussed in another section below. Alzheimer’s disease (AD) TUNEL staining was sought and found in AD brain tissue (63–68), in neurons, astrocytes or microglia, associated with plaques or tangles or neither; however, Stadelmann et al. (69) pointed out that cells displaying DNA fragmentation rarely co-label for apoptosisspecific proteins or display typical apoptotic morphology. These authors suggested that

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the increased rate of neuronal DNA fragmentation in AD patients reflects metabolic disturbances in the premortem period, and that cell destruction is mediated through necrosis rather than apoptosis. There are many studies reporting activated caspases (caspases-1, -2L, -3, -5, -6, -7, -8, -9; (70–76)) often co-localizing with plaques and/or tangles or sometimes preceding them, in neurons, astrocytes and microglia. Activated caspase-3, in particular, was found to be sequestered in granulovacuolar degeneration (GVD) granules rather than being associated with plaques and tangles (77,78) , and co-localized in GVDs with a caspase-cleavage product of amyloid precursor protein (APP), in contrast to activated caspase-8, which was found in the cytoplasm but not in GVD granules (79). As to the apparently restricted occurrence of caspase-3 in GVD granules, it should be noted that this caspase rapidly degrades itself (80), and cysteine proteases in general are easily oxidatively inactivated (81). More evidence for caspase activation in AD brain tissue comes from the detection of caspase-cleaved fragments of actin (82), fodrin (70), APP (83) or AMPA receptor subunits (84) in synapses, plaques and tangles. Caspases-3 (85), -6(74) and -9(72) have been implicated in the cleavage of tau protein and tangle pathology (86,87). Regarding alterations in Bcl-2 family proteins in AD brain tissue, a less number of papers were published in the late1990s, some of them reporting upregulation of proapoptotic family members like Bax, Bak or Bad, associated with plaques and/or tangles (88,89) membranous fractions (90) tangle-free neurons (91) or dystrophic neurites (92), others finding no change (93,94). Antiapoptotic Bcl-2 family members, mostly Bcl-2 itself, occasionally Bcl-XL or Bcl-w, were reported upregulated in affected cortical and/or hippocampal areas in parallel with increasing disease severity (95) associated with plaques and/or tangles (96,97), or rather with glia but not plaques and tangles (92), with mitochondria (96) and membranous fractions which may include mitochondria (90). However, there are also the inevitable papers that found nothing (93,94). The few who have looked have also found p53 upregulated in neurons and/or glia (98,99). Finally, upregulation of Fas and/or Fas ligand has been fairly consistently reported in AD neurons and/or astrocytes (76,99–101), and a role of the TNFα-pathway has also been suggested (102,103). Thus, it appears that while there is ample evidence for the occurrence of molecular events associated with apoptosis in AD brain, there is very little evidence for completion of the process. Many of the affected neurons, fighting for survival, seem to abort apoptosis (104). The abundant occurrence of autophagic vacuoles (AVs) in neurites and terminals may be an element in this; their accumulation in immature forms indicates impairment of their transport and processing, thus impeding the suspected protective function of autophagy and promoting neuronal degeneration (105). It is likely that the mode of death of the affected neurons is highly individual, depending on the circumstances. Some may die by apoptosis, some by other forms of PCD, some by autophagic death and some even by necrosis. Amyotrophic lateral sclerosis (ALS) The evidence for and against occurrence and importance of PCD and/or apoptosis in ALS has been repeatedly summarized over the last few years, ever expanding the picture by

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incorporating new information (20,106–110). As usual for ND, a considerable amount of information comes from cellular and animal models, in this case from cells or mice expressing a mutant form of superoxide dismutase-1(SOD1) associated with familial ALS. Nevertheless, the view has been expressed that, since experimental data from human cases (as far as available) by and large correlate with those from the models, and clinical and pathological manifestations of all forms of ALS are quite similar, a common pathological mechanism may be implicated in both familial and sporadic forms of the disease (110). Briefly (for references see the above reviews), the search for internucleosomal DNA breaks using the TUNEL or ISEL techniques has yielded controversial results, as have morphological studies applying classical criteria for apoptosis. In human spinal cord tissue, elevated caspase-1 and -9 activities were found; increased caspase-3 activity was found in spinal cord and motor cortex in one study but not another. Several studies reported upregulation of caspase-1 preceding that of caspase-3 in spinal cords of SOD1-transgenic mice; others found that activation of caspase-1 truncated Bid, leading to cytochrome c release and subsequent activation of caspase-9, -3, -7 and -8. Proapoptotic Bcl-2 family members like Bax, Bad, Bak, Bid and harakiri appear generally increased in spinal cords of ALS patients and/or transgenic mice, whereas the antiapoptotic Bcl-2 and Bcl-XL are either decreased or unchanged. In transgenic animals, these changes are not present before they become symptomatic, and become more obvious with progression of the disease process. Overexpression of Bcl-2 in SOD1transgenic mice delayed disease onset and prolonged survival, without altering disease duration. Interestingly, mutant SOD1 seems to bind more avidly to Bcl-2 than its wild-type counterpart. There is also some evidence for a role of p53 in sporadic ALS, such as increased immunoreactivity and functionality in motor cortex and spinal cord. On the other hand, knocking out p53 did not have beneficial effects in G93A SOD1-transgenic mice, suggesting that in these mice, p53 is not crucially involved in cell death. Of note, this may be different in another SOD1-transgenic mouse based on the G86R mutation, in which activation of p53 was found. Other findings implicating apoptotic processes in ALS include expression of the LeY carbohydrate antigen, a marker of apoptosis, in cervical cord motor neurons of ALS patients, presence of fractin produced by caspase-3 mediated cleavage of β-actin, in spinal cords of SOD1-transgenic mice and expression of prostate apoptosis response-4 protein (Par-4) in spinal cords of ALS patients and SOD1-transgenic mice. Taken together, these findings suggest an involvement of the intrinsic mitochondrial pathway in motor neuron death in these animal models and perhaps also sporadic ALS. As Przedborski states in his recent review (109), “while degenerating neurons in human ALS and its experimental models do exhibit some features reminiscent of apoptosis, most dying cells cannot be confidently labeled as typically apoptotic”, but “the currently available evidence indicates that PCD is in play in ALS”. Although there is also some evidence of upregulation of elements of the death-receptor mediated, extrinsic apoptotic pathway, this may relate to the neuroinflammatory component of ALS, and it is unclear whether, for example, TNF has a pathogenic or protective role in this disease (111).

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Multiple sclerosis (MS) The pathogenesis of MS is considered to consist of an inflammatory and a neurodegenerative phase. Inflammation begins with migration of activated T cells across the blood–brain barrier and subsequent stimulation of microglia and astrocytes, and induction of antibody production by plasma cells, resulting in axon demyelination and finally axon loss. In the neurodegenerative phase, excessive release of glutamate from microglia, macrophages and lymphocytes may cause necrotic damage to oligodendrocytes (ODCs) and axons. Moreover, demyelination makes neurons more susceptible to apoptotic stimuli (for review see e.g. (112)). Four fundamentally different patterns of demyelination were reported, suggesting pathogenetic heterogeneity. Two of these patterns displayed features of autoimmune reactions with lesions resembling those found in the experimental autoimmune encephalomyelitis animal model, the other two were suggestive of primary ODC dystrophy rather than autoimmunity, one of them (pattern III) exhibiting clear signs of apoptosis. These patterns were heterogenous between and homogenous within patients (113). A recent report described profuse ODC apoptosis characterized by chromatin condensation, but little DNA fragmentation and caspase-3 activation, in newly forming lesions in patients dying during or shortly after onset of a fatal relapse (114). The authors suggested that autoimmunity and inflammation might be secondary to massive ODC apoptosis, which in turn may represent a very early stage of most, if not all lesions causing acute exacerbations of MS, a view that remained not uncontested (115) but certainly calls for further investigation of the matter (116,117). Although ODC apoptosis in MS is not a new concept, little is known about the pathways that might be involved. Little evidence for a role of caspases exists except for some cell culture data; involvement of excitotoxicity and oxidative stress may imply the intrinsic pathway, but occurrence of death signaling molecules like p75 and fas may indicate a role of the extrinsic pathway (116,117). Thus, to what extent, and how apoptosis may be involved in MS awaits clarification. Huntington’s disease (HD) In the end-stage of HD, up to 95% of the GABAergic medium spiny neurons in the striatum, the primary area of neuronal degeneration in this disease, have disappeared (118). Those that survive exhibit loss of dendrites, dendritic spines and synaptic connections (119). The role of apoptosis in the death of these neurons has been implicated from the demonstration of DNA fragmentation by the TUNEL method in HD striata (45,119,120), the extent of which was even found to correlate with the number of CAG repeats (121). There has been much less disagreement about whether or not there is positive TUNEL staining than for example in PD. The problems of the TUNEL technique, and the fact that two groups could not clearly demonstrate DNA laddering by gel electrophoresis in the corresponding tissue samples (45,120) are reflected in the skepticism regarding a role of apoptosis in cell death per se in a recent review (122). A very recent study reported increased TUNEL staining along with the occurrence of neurons exhibiting typical apoptotic morphology, increased cytoplasmic expression of Bcl-2, Bax and PARP, but very weak caspase-3 immunoreactivity (123), suggesting an involvement of caspase-independent apoptosis. On the other hand, the presence of activated caspases (caspase-8 (124); caspases-2,-6 and-7, caspase-3 only in astrocytes (125); caspase-9 (126))

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was demonstrated in HD striatum, and caspases were thought to be involved in the processing of mutant huntingtin (mut htt) to toxic N-terminal fragments and wild-type huntingtin (127–129). It has therefore been argued that apoptotic processes, rather than being a direct cause of neuronal death in HD, function in the generation of the toxic fragments (122). While induction of apoptosis by mut htt can be observed in culture of transfected cells or cells derived from transgenic animals, most transgenic mouse models expressing fragments of or full-length mut htt, or knock-in models exhibit little or no neuronal loss, and if they do, only very late in the disease. Correspondingly, evidence for a role of apoptosis in neuronal death in these model is poor (122). They do display, however, caspase activation (130); in one of the best investigated transgenic models, the R6/2 mouse, activation of caspases preceded proapoptotic changes in Bcl-2 family members (131). Very recently, p53 was shown to be upregulated in HD striatal and cortical tissue, in striatum of transgenic mice and in cell expressing mut htt; mut htt was also found to bind to p53, probably stabilizing it and promoting its transcriptional and mitochondria-disturbing actions (132). The interesting possibility emerges that an association of mut htt with p53 might be a crucial link in a feedback loop promoting mitochondrial damage, release of caspases, generation of toxic fragments and transcriptional alterations in a slowly but steadily increasing manner. Of note, evidence for the occurrence of autophagy has been found in HD brain tissue (for review see e.g.(35)). It has been suggested that mut htt aggregates stimulate clearance of the harmful protein by autophagy through sequestration of mTOR, probably representing a defensive mechanism; even in the presence of aggregates, autophagy can be enhanced by pharmacological inhibition of this kinase (133).

Acute neurodegenerative conditions Stroke Since most stroke patients either die acutely or survive for a relatively long time, it is difficult to investigate early pathophysiological events in human postmortem brain tissue. Therefore, most of the information on the pathophysiology of ischemic stroke comes from studying the effects of focal cerebral ischemia induced by permanent or transient middle cerebral artery occlusion (MCAO) in laboratory animals. Severity and duration of local cerebral blood flow (CBF) reduction determine infarct size, with good correspondence between animals and humans. In the area of the most severe CBF reduction (the ischemic core), collapse of energy metabolism and ion homeostasis lead to loss of cellular integrity, resulting in cell death commonly thought of as necrotic. However, core neurons, in contrast to astrocytes, do not display the full panel of necrotic features but also present elements characteristic for apoptosis, particularly activation of certain caspases. In the surrounding penumbra, which early after stroke onset can amount to about half of the final infarct area, residual blood supply by collaterals prevents immediate necrosis. In this area, a wave of damage by excitotoxicity, from glutamate released locally as a consequence of hypoxic depolarization or diffusion from the ischemic core, free radical production

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and tissue acidosis propagates away from the ischemic core, causing necrosis, PCD and inflammation (134–138). PCD and inflammation begin hours after ischemia onset and continue for days, which makes them attractive as therapeutic targets because they may allow for a longer time window to treat after stroke onset, to rescue at least part of the penumbral tissue. The few studies in human tissue, while indicating the occurrence of some early biochemical features of PCD, have not provided conclusive evidence for an important role of apoptosis in neuronal death, and it has been suggested that apoptosis may play a smaller role in human than in experimental stroke in animals (for review see e.g. (20,138)). Evidence from rodent MCAO models implicates both the intrinsic and the extrinsic pathways; activation of inflammatory caspases -1 and -11 and of caspase-8, the latter probably mediated by glial release of TNFα and upregulation of fas ligand and receptor, occurs early in the core, followed by activation of caspases-9 and -3 in the penumbra (134,137). Transgenic mice overexpressing antiapoptotic Bcl-2 or Bcl-XL, and p53, Bid, caspase-1 and -3 knockout or fas-deficient mice exhibit decreased infarct size after MCAO; conversely, Bcl-2 knockout mice but also mice lacking both TNF receptors showed increased infarct size (135,136). The p53 inhibitor pifithrin afforded protection in a transient MCAO model (139). Although protective effects of (mostly nonselective) caspase inhibitors in permanent or transient MCAO or global ischemia models have been reported, the potential therapeutic value of caspase inhibition in stroke is disputed, especially as far as lasting protection is concerned (134,140). In conclusion, apoptotic or PCD processes seem to be involved in the development of neuronal damage in stroke, particularly in the penumbra and after reperfusion, but what potential for therapeutic intervention this offers is still unclear. Traumatic brain injury (TBI) The pathophysiological mechanisms leading to cell loss and tissue destruction in TBI and stroke appear to have many similarities, including excitotoxicity, oxidative stress, radical formation and inflammation. Progression of damage in both TBI and stroke was observed in both gray and white matter structures, implicating delayed neuronal death along with progressing axonal damage which may end up in secondary axotomy by wallerian-like degeneration (141). Following the primary insult in human and experimental TBI, cell death in the brain exhibits both necrotic and apoptotic features, the relative extents probably depending on its severity and on the energetic situation (i.e. ATP availability) in individual cells. In humans, apoptotic features including DNA fragmentation, activation of caspases-1, -3, -8 (along with Fas upregulation), upregulation of Bax, Bcl-2 and p53 were seen, though not in all patients and not by all authors; patients with higher Bcl-2 levels in CSF or excised brain tissue had a better outcome (20,142–144). Similar and additional evidence for an involvement of apoptotic processes comes from a variety of experimental models which reproduce different aspects of the inherently heterogeneous human TBI (145). This includes caspase-9 activation, cytochrome c release, nuclear translocation of AIF and bid cleavage in affected brain tissue. Caspase-3 inhibition and Bcl-2 overexpression reduced tissue damage, but functional improvement by the former is controversial and not seen by the latter; interestingly p53-deficient mice showed as much damage as

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their wildtype counterparts despite reports of protection against excitotoxic and ischemic insults (142,144). Therefore, similar to stroke, although apoptotic processes seem to contribute to cell death and tissue damage in TBI, in is unclear to what extent antiapoptotic therapy may be useful in this condition. Spinal cord injury (SCI) Mechanical trauma to the primary site of injury in SCI shears neuronal and endothelial cell membranes, causing a hemorrhagic zone of necrosis predominantly in gray matter. Axons located near the gray matter suffer greater injury than those farther away, and myelinated axons are more vulnerable than unmyelinated ones. The initial insult then triggers pathophysiological mechanisms causing the propagation of a wave of secondary injury through the surrounding tissue. Vascular damage and secondary reactions like vasospasms, thrombosis and neurogenic shock limit blood supply, compromising energy metabolism, which in turn triggers necrotic mechanisms. Pro-inflammatory cytokines are released by microglial immediately after injury and later on by infiltrating leukocytes, inducing a host of inflammatory mediators including ROS, nitric oxide synthase, proteases, etc. via the NFκB pathway. Extracellular glutamate rises as a result of compromised re-uptake, increased release due to neuronal activity and cell disruption, initiating waves of excitotoxicity, which also hit glia. ODCs are particularly vulnerable because their ionotropic AMPA and kainate receptors are more penetrable to calcium, and because of their poor calcium buffering capacity. Finally, tissue reperfusion causes increased ROS from various sources, contributing to cellular damage. Apoptosis accompanies necrosis to varying extents, depending on conditions, during an initial phase of secondary injury. In the second phase, apoptosis occurs predominantly in white matter and particularly involves ODCs. Areas of ODC apoptosis strongly correlate with sites of wallerian degeneration, suggesting that it is due at least in part to loss of trophic support from degenerating axons (for reviews see e.g. (20,146–149)). The longer-term events including delayed ODC death bear much resemblance to the situation in MS (147). In human SCI, evidence for the occurrence of apoptosis includes apoptotic morphology, TUNEL staining and activated caspase-3 around the lesion epicenter and in ODCs (for this and the following, see the reviews quoted above; only newer work is separately quoted). Several animal models addressing different aspects of SCI which, despite limitations, are considered relevant to human SCI (150) have been used to investigate the role of apoptosis. TUNEL staining, chromatin condensation and DNA laddering were seen in neurons and glia early on at the lesion center and later farther away. P53, Bcl-2 and Bax expression occurring in microglia, ODCs and astrocytes, but not in neurons, spreading from the lesion site for days after spinal cord transection, and cytochrome c release and activation of caspases -3 and -9 along with protective effects of inhibitors of caspase-3 (151,152) and -9 (153), a pan caspase inhibitor (152), local overexpression of Bcl-2 or exogenous administration of Bcl-XL protein (154) implicate an involvement of the intrinsic pathway. Protective effects of cyclosporin A may implicate MPT caused by glutamate excitoxoxicity, mediated by intracellular calcium increases directly or through activation of calcineurin, which dephosphorylates Bad. On the other hand, increased Fas and p75NTR expression

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by ODCs and increased expression of FasL and TNFα by activated microglia in SCI, along with activation of caspase-8 (152), also suggest an involvement of the extrinsic pathway in ODC death, rescue from which is considered to have a considerable therapeutic value (149).

DEVELOPMENT OF ANTIAPOPTOTIC DRUGS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES (ND) By the mid-nineties of the last century, the identification of apoptotic mechanisms and signaling pathways had progressed to an extent (18,155–162) which seemed to allow the definition of some strategic intervention sites to inhibit apoptosis in ND. Inhibitors of caspases (163), p53 (164) and the JNK pathway (165) were developed and their antineurodegenerative potential evaluated in cellular and animal models (20,166). Conversely, targets related to apoptosis were searched for and found for compounds with known neurorescuing properties like certain propargylamines (167). The mixed lineage kinase inhibitor and functional JNK inhibitor CEP-1347 (Table 1; (168); a staurosporin derivative) and the GAPDH ligand TCH346 (Table 1; (169,170); a propargylamine derivative), were among the first ones to be further developed with the aim to evaluate them clinically in ND. Concerns that problems might arise due to increasing the survival of unwanted cells, such as promotion of cancer, maintaining dysfunctional or malfunctional cells, or interference with turnover of proliferating cells, were not supported by extensive safety studies.

THE FIRST TWO ANTIAPOPTOTIC COMPOUNDS FAILED IN CLINICAL TRIALS Thus, CEP-1347 and TCH346 were clinically evaluated in PD as their primary indication. Although there were also historical reasons for this choice in the case of TCH346 (designed to share the neurorescuing/antiapoptotic properties of deprenyl without monoamine oxidase inhibition and metabolism to amphetamine-like compounds (170)), the principal reason was their effectiveness in mouse and primate MPTP models (see Table 1), the strong belief that the pathophysiology observed in MPTP models is relevant for PD (20,171,172), and the conviction that, of all chronic ND, PD is the one with the best evidence for a relevant role of apoptosis. The results of large and well-controlled clinical studies in PD, and PD and ALS, respectively, for CEP-1347 and TCH346, are now known. Unfortunately, both compounds proved plainly ineffective (http://www.prnewswire.com/egi-bin/stories.pl?ACCT=104& STORY=/www/story/05-11-2005/0003595559&EDATE=) (http://www.alsa.org/news/ article.cfm?id=579&CFID=544070&CFTOKEW=43868217) (173). The fact that two compounds addressing quite different targets within the apoptotic signaling system not just missed to reach significance, but failed the clinical test without even a faint signal of efficacy, one of the compounds (TCH346) also in a second indication (ALS), calls for a reconsideration of the antiapoptotic strategy, and for reflections on implications

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Table 1. Neuroprotective profiles of CEP-1347 and TCH346 CEP-1347

TCH346

In vitro/cellular systems; rescues • Chick primary neurons (dorsal root/sympathetic ganglion, ciliary, motor) from trophic withdrawal • Rat sympathetic and motor neurons, and differentiated PC12 cells from trophic withdrawal, UV irradiation and oxidative stress • Also maintains metabolic activity and growth in rat sympathetic motor neurons after trophic withdrawal • SH-SY5Y cells from MPP+ toxicity • Cerebellar granule cells from K+ deprivation (short-lived effect only) • Rat cortical and sympathetic neurons, and PC12 cells from β-amyloid toxicity • Organotypic rat cochlear explants from aminoglycoside toxicity Active concentration range ≈ 10−7 –10−5 M

• PC 12 cells from toxicity by • Trophic withdrawal • β-amyloid toxicity • Rotenone • Lactacystin (proteasome inhibitor) • Rat cortical neurons from • NMDA excitotoxicity • Kainate excitotoxicity • Cerebellar granule cells from toxicity by cytosine arabinoside (ara C) • Rat oligodendrocytes from AMPA excitotoxicity • Rat embryonic mesencephalic DAergic cells from toxicity by MPP+/MPTP • Human neuroblastoma (PAJU) cells from toxicity by • Rotenone • GAPDH overexpression Active concentration range ≈ 10−12 –10−5 M, with a maximum at ≈ 10−9 M

Prevents neurodegeneration in the following in vivo animal models After local administration • Developmental apoptosis of motor neurons in chick embryos and neonatal rats • Cholinergic hypoglossal motor neurons after transaction After systemic administration • Cholinergic neurons after excitotoxic lesion of the nucleus basalis • Medial septal cholinergic neurons after fimbria-fornix transection in rats • MPTP model in mice • MPTP model in monkeys Active dose range ≈ 0.03–3 mg/kg p.o. or s.c.

• Unilateral carotid occlusion/transient hypoxia model • Facial motor neuron axotomy model • Progressive motor neuronopathy mice • MPTP model in mice • 6-OHDA models in rats • MPTP model in monkeys

Active dose range ≈ 0.003–0.3 mg/kg s.c.

Sources of information: CEP-1347 (168,174,175) and TCH346 (169,170,176)

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of these unexpected and quite disappointing results for the future development of antineurodegenerative drugs in general, and for drugs interfering with PCD in particular.

Possible reasons Some of the issues addressed below were already recognized as possible stumble-blocks at the time it was decided to develop the compounds, and continued to be discussed throughout the development. However, the scientific knowledge base to judge the justification of these concerns was simply not available, and thus awareness that risk is inherent to novel approaches and confidence in the concept of inhibiting apoptosis prevailed. Now, with hindsight, i.e. knowing the negative outcome of the trials, such issues must be readdressed. The question whether apoptosis is involved at all in PD was discussed above; if it were not, the reason for the clinical inefficacy of CEP-1347 and TCH346 is obvious and needs no further contemplation. Although the mode of death of neurons in this disease may not predominantly fulfill the classical morphological criteria of apoptosis, there is little evidence that it is plainly necrotic, and an involvement of some form of PCD is likely. Since there is considerable overlap and cross-talk between the different forms of PCD, at least some impact of inhibiting apoptotic pathways might have been expected. Assuming, and there are no reasons not to, that drug exposure and clinical trial design in the CEP-1347 and TCH346 studies were adequate, we shall focus on the following, not mutually exclusive issues: • Is protection from consequences of acute toxin exposure (as in PD animal models) a different matter than protection from ongoing damage? • Are death pathways redundant, i.e. can cell death proceed by another programmed route if one pathway is blocked, or even eventually by plain necrosis? • Do all DA neurons die the same way, are only some of them rescuable by inhibition of apoptosis and were those already dead when treatment began? • Are the pathways targeted by CEP-1347 and TCH346 involved in only a minor proportion of cell death? • Did the drugs in fact rescue (some) cells, but not maintain them in a functional state? • Do our animal models reflect the pathophysiology of neuronal death in ND (and particularly in PD) appropriately? Acute damage as in animal models vs. persistent, ongoing damage as in disease The current view on the pathogenesis of sporadic PD is that an etiological factor or a combination of such factors, external to or from within healthy DAergic neurons, triggering multiple transcriptional and biochemical events which interact with each other, disturb the homeostasis, and ultimately kill the affected neuron (61). The relentless progression of the disease suggests that whatever the causative factors are, they continue hitting DAergic neurons more or less continuously over time. The loss of such neurons in the substantia nigra of PD patients shows a regional pattern (177), suggesting that some of them are more vulnerable than others, or that the causative factors exhibit regional differences.

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Quite generally, animal models of PD do not reflect this situation of continuous assault, certainly not the older ones like medial forebrain bundle transections or 6-OHDA, but neither do the acute nor most so-called chronic versions of the MPTP model (178); rather, they represent multiple toxin administrations over a limited period of time. The more recently developed chronic rotenone infusion model (179) seems to induce nonspecific CNS and systemic toxicity and may be of doubtful value as a disease model (180,181). Whether transgenic animal models based on rare inherited forms of PD will provide more faithful models of the disease remains to be seen. Although α-synuclein-null mice, mice (over)-expressing wildtype or mutant α-synuclein etc. (182), parkin- null mice (183,184) and DJ-1-null mice (185,186) all exhibit various alteration in the DAergic systems or/and (motor) behavioral deficits, loss of nigral DA neurons is not observed even at quite an old age. Despite temporally limited exposure to the toxin, the presence of activated microglia in the substantia nigra may suggest that degeneration of DAergic neurons caused by MPTP in humans (187,188) or monkeys (189), or by inflammatory lipopolysaccharide injections in rats (190) can progress long after the original cause has disappeared. This might indicate that such insults can induce self-perpetuating processes which might serve as the continuously present noxae alluded to above. However, evidence of recovery after cessation of MPTP treatment in most animal species including nonhuman primates(178) argues against the existence of such a disease triggering effect in these models. For a valid disease progression model for PD, it might after all be irrelevant whether damage is brought about by an acute insult in many DAergic neurons at the same time, or sequentially by a chronic insult in one after the other, as long as the processes and involved pathways leading to cell death are the same as in the disease. On one hand, we know little about these pathways. It is not even clear whether PD is a single disease entity (191) or represents a group of disorders due to nigrostriatal degeneration resulting from diverse underlying mechanisms and pathologies (192), or whether different triggers of disease may funnel into a common final pathway (193). On the other hand, DAergic neurons are likely to be sick for an extended period of time in PD (194); during their fight for survival and according to changes in homeostasis, they may be able to change their route to death and thus circumvent roadblocks in some pathways imposed by drugs. Time may be too short to allow this in a more acute situation as in the animal models. Dying by necrosis if apoptosis is blocked, or redundancy of death pathways? Changing death routes underway is not an unlikely scenario. In cellular models, it is common that cells die by necrosis if the execution of apoptosis is blocked e.g. by caspase inhibitors (55,195). Conversely, initiation of apoptosis might actively suppress programmed necrosis because activated caspases inactivate proteins required for this process (33). Also, the energy status of a cell can influence outcome, because the execution of apoptosis requires energy. In the absence of sufficient ATP, necrosis may ensue, and cellular features of apoptosis, necrosis and autophagy often coexist (196). In PD SN tissue for example, DA neurons showing apoptotic features were seen along with neurons exhibiting

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signs of autophagic degeneration (50), which may represent a (futile) rescue attempt (33). Astrocytosis and microglial activation in PD SN tissue is well-documented (190,197), may be a consequence of apoptosis, programmed necrosis or necrosis, and/or contribute to and propagate further cell death by providing stimuli that differ from, and engage different pathways than, those that originally started the process. On the other hand, they may also provide protection by releasing trophic factors (198). It was recently reported that microglia chronically exposed to apoptotic neurons change their expression and release pattern towards immunomodulatory and neuroprotective factors at the expense of proinflammatory molecules (199). The relevance of this with respect to PD is questionable in view of the marked upregulation of proinflammatory factors (190,197), unless such an adaptive ability of microglia subsided with age. If that did indeed occur, it might be a factor contributing to disease progression. Thus, there are many ways how cells initially routed towards apoptosis could change directions, only to end up dead anyway. Whether and in what way treatment with compounds like CEP-1347 or TCH346 would affect such routing processes is not known. In cellular or animals models, the time-course of cell death may have been too rapid for such phenomena to occur, but the conditions may be quite different in the diseased human brain in general and in the PD brain in particular, where cells may be in a suffering state for quite a long time (194). Whether damaged cells or neurons routed towards apoptosis and exposed to agents interfering with an initially preferred pathway can circumvent the roadblock within the apoptotic signaling system (for diagrammatic depictions see e.g. (20,200,201)), if given enough time and if the drive to die is strong enough, has not yet been addressed experimentally to our knowledge. It would hardly be surprising if they could. Can only a fraction of DA neurons be rescued by antiapoptotic treatment, and were these already dead when treatment started? The loss of nigral DAergic neurons is critical for a large part of PD symptomatology, thought to start when putaminal DA levels are reduced by about 70–80% and the loss of DAergic cell bodies in the substantia nigra pars compacta (SNpc) is of the order of 60% (see e.g. (60,202)). The observation that the loss of terminals exceeds that of cell bodies suggests a retrograde degenerative process beginning at the nerve endings (60), in the degeneration of which apoptosis-related processes involving mitochondria may participate (203). Some nigral DA neurons appear to be more vulnerable than others, the magnitude of neuronal loss decreasing in the order ventrolateral > ventromedial > dorsomedial part. This pattern is opposite to the much smaller loss during aging or in other basal ganglia diseases (177) and suggest that the degenerative process involved in PD does not represent some form of accelerated aging. Loss of DAergic neurons appears to begin sequentially and proceeds caudorostrally through 5 pockets called nigrosomes which contain ∼40% of SNpc DAergic neurons and stain poorly for calbindin; a parallel, but lesser caudorostral loss was observed in the other, calbindin-rich SNpc areas called matrix (204,205). This regional pattern of nigral cell loss corresponds to that of the extent of DA depletion in the respective striatal projection areas (206). Of note, it is to some extent mimicked by MPTP, a feature that has contributed to the reputation of this model (207); at least with

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respect to this toxin, the ability of DAergic neurons to express calbindin does not appear to be responsible for that pattern (208). Given the long period of time over which DAergic neurons die in PD on the one hand and the differential pattern of loss on the other, one may wonder about what determines which of them die earlier and which later. Does the pathogenic factor reach some of them earlier than others for whatever reason? Are some of them better equipped to resist, because they have better intrinsic defenses or better assistance from neighboring cells, e.g. by way of trophic support? Is there a hierarchy in this, so that e.g. functionally more important neurons (if that exists) are privileged and die later? Does any of this affect the pathways engaged or the mode chosen for the route to death? Do they all die the same way, or do death modes or pathways in those that die earlier differ from those that die later? It is not known whether the indices of apoptotic processes observed in postmortem brain samples of PD patients deceased in the end-stage of their disease (20,209) are representative for those neurons that died earlier. Neither is it known whether those that showed signs of autophagy (50) are just in an earlier stage of degeneration and would finally have died by apoptosis, or rather by autophagic cell death or even necrosis, perhaps giving rise to the inflammatory signs observed in PD brain tissue (190,197), or whether they were the hardier ones that managed to hold on for longer. There is also the question how long such “sick” states of DA neurons might last. One may also wonder whether continuing neuronal degeneration is the only factor in disease progression and in the transition from the presymptomatic to the symptomatic state. If it were possible to entirely stop DA neuronal degeneration immediately before a patient becomes symptomatic, would symptoms develop more slowly, or not at all? Is symptomatology and disease progression exclusively a consequence of progressive degeneration of the last third of DA neurons, i.e. the exhaustion of their redundancy? Or is this last third functionally more important than the two-thirds that died earlier? Or is it perhaps also a reflection of progressive exhaustion of mechanisms which compensated for the loss of the first two-thirds? If the majority of DA neurons that die in the presymptomatic phase of PD do so by apoptosis, and if their preservation matters in keeping the nigrostriatal system at least partly functional, it seems possible that beginning treatment with the antiapoptotic compounds CEP-1347 and TCH346 well in the presymptomatic phase might have yielded a better result. Even if true this has no practical value in the absence of simple means to identify patients in presymptomatic stages. Did CEP-1347 and TCH346 target the wrong pathways? After MPTP intoxication, damage through generation of reactive oxygen species, perturbation of energy metabolism and calcium homeostasis etc. activates the JNK and p53 pathway(s), resulting in recruitment of the intrinsic (mitochondrial) PCD pathway. In Fas exon 9 knockout mice, which lack most of the death domain, preservation from MPTP toxicity of DAergic cell bodies in the SNpc, but not of their terminals in the striatum is observed (210). However, these mice still retain most of the intracellular domain as well as Fas expression on the cell surface; Fas-deficient lymphoproliferative mice are more

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susceptible to MPTP (211), suggesting that Fas has neuroprotective properties independent of the death domain. This may explain the protection of Fas exon 9 knockout mice without invoking a role of the extrinsic pathway in MPTP-mediated DAergic toxicity. Data from TNFR1, TNFR2 or double-knockout mice suggest only minor effects of TNFα on DA metabolism or survival of DA neurons after MPTP lesion, but no participation in cell death (212). TNFα protein was reported to be increased in striatum and substantia nigra after 6-OHDA lesions (213). However, others found only short-lasting elevations of mRNA of TNFα, but not of TNFR1, in contrast to a much longer-lasting increase in bax mRNA, after this toxin (214). Caspase-8, a downstream effector of death receptor activation, is activated along with caspase-9 in response to 6-OHDA or MPTP exposure of DAergic cells in culture or in vivo (215,216). Although an early event preceding cell death (55), caspase-8 activation may occur secondarily via activation of caspase-9 (215) and thus be mediated by the intrinsic pathway. It has been concluded that there is little evidence of recruitment of the extrinsic pathway in the MPTP model (209), and this is likely also true for the 6-OHDA model. While there is considerable evidence for mitochondrial disturbance and related activation of the intrinsic pathway (43,217,218) from the literature on samples from PD patients, some arguments for a participation of the extrinsic pathway also exist. Nigrostriatal tissue, ventricular and lumbar CSF contain increased levels of TNFα, along with other cytokines, and levels of TNFR1 and soluble Fas were increased in nigrostriatal tissue (219). Fas and Fas ligand immunoreactivities were reduced in both caudate/putamen and SNpc (220). Selective vulnerability of nigral DA neurons in patient SNpc correlated with the decrease of the percentage of DA neurons immunopositive for Fas-associated protein with a death domain (FADD), a proximal adaptor protein for the TNF receptor family death pathway (221), and an increased proportion of SNpc DA neurons exhibited caspase-8 activation (55). Such findings led to the suggestion that DA neurons expressing the TNFR1 transduction pathway are particularly degeneration-prone in PD (194). Considering the mechanism of action of TCH346, it is unlikely that this compound interferes with the extrinsic pathway. As for CEP-1347, stimulation of death receptors does lead to activation of the JNK pathway which is inhibited by this mixed lineage kinase inhibitor, but the apoptosis-inducing effects of this JNK activation appears to be mediated by the intrinsic pathway (222,223). Nevertheless, by inhibiting this branch, CEP-1347 might be expected to attenuate death-receptor mediated apoptosis in some cell types. Neurons rescued, but not functional? The observation that reductions of DA striatal levels are more marked than losses of DA cell bodies in the SNpc, both in PD and in different versions of the MPTP model, gave rise to the view that DA neurons are degenerating retrogradely from the terminals to the soma (dying back) (60). It is thus conceivable that preventing neurons from dying by apoptosis only preserves somata with axonal stumps, resulting in dysfunctional cells unable to sustain DAergic neurotransmission. TCH346 indeed only rescued tyrosine-hydroxylase-positive (DA) somata in the SNpc, but did not preserve striatal DA levels in the mouse MPTP model. On the other hand, it

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prevented behavioral deficits caused by intranigrally as well as intrastriatally administered 6-OHDA (224,225), and also prevented deterioration of motor performance and loss of 18 F-DOPA uptake in rhesus monkeys systemically lesioned with MPTP (176). CEP-1347 attenuated loss of nigral DAergic somata and partially preserved DA terminals in a mouse MPTP model (226) and showed similar protective effects including prevention of motor deterioration in a primate MPTP model (227). Reduced levels of TH mRNA and protein were found in surviving PD DA cell bodies (228). Moreover, in the PD SNpc but not in controls, there appears to be a pool of melanized neurons that do not stain for tyrosine hydroxylase (TH) as large as about 20% of the total of surviving melanized neurons (229). They might be damaged former DAergic neurons, not yet degenerated, but no longer operating as functional DAergic neurons (194). If CEP-1347 and TCH346 just kept such neurons alive, an accumulation of nonfunctional cells might result, explaining their clinical failure. However, in monkeys subacutely or chronically lesioned with MPTP a similar reduction of TH content was seen (230), suggesting the occurrence of a similar phenomenon as in PD. It is not known whether CEP-1347 or TCH346 affected this reduction of TH content per cell in the respective monkey MPTP experiments (176,227), but both compounds clearly attenuated the development of motor disability. Thus, irrespective of reservations with respect to the validity of PD animal models, one can argue that if DA neurons are preserved by these compounds, at least some of them are in a functional state. Nonfunctionality of preserved DA neurons is therefore not a likely explanation for the clinical failure of CEP-1347 and TCH346.

Are chronic ND the wrong diseases? As follows from the section on the evidence for a role of PCD in ND, there is currently no reason to think that apoptosis may play a more critical role in the progression of chronic ND other than PD or ALS. From the consideration of the possible reasons for clinical failure of CEP-1347 and TCH346, none appears to grossly differ in terms of relevance in any other chronic ND. Therefore, it seems daring to expect a better outcome for these compounds e.g. in AD or HD. If ODC apoptosis plays such an important role in MS as recently proposed (114), this may offer a therapeutic opportunity for antiapoptotic drugs, not least because it represents a quasi-acute situation in which adaptive processes have a lesser chance to circumvent their effects. Whether this holds for CEP-1347 or TCH346 may also depend on the relative roles of the intrinsic vs. the extrinsic pathway which are not known yet. One might speculate that particularly delayed apoptosis triggered by acute insults such as stroke, TBI or SCI might be easier to prevent pharmacologically, because cells are exposed to the noxious stimuli for a shorter period of time and may have less opportunity for adaptive pathway switching. However, although protective effects of antiapoptotic treatments (mostly caspase inhibitors) in animal models have been reported, the therapeutic implications for corresponding human conditions are unknown. Moreover, stroke and trauma clinical trials, due to the inherent inter-patient variability regarding site, extent, nature and severity of injury etc., are among the most challenging tasks in drug development, and not

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something that one would take on without an extremely thorough preclinical evaluation of a compound, including demonstration of functional improvement and an appropriate time-window after the insult for the initiation of treatment. Apoptosis appears to be involved in several degenerative ophthalmological conditions, including glaucoma, macular degeneration, retinitis pigmentosa and diabetic retinopathy (231–237). Depending on the condition, either the intrinsic or the extrinsic pathway or both may be involved. Eye diseases may therefore represent a therapeutic area in which the potential of antiapoptotic drugs could be explored. The cardiovascular area also may offer opportunities for antiapoptotic drugs. Apoptosis, besides necrosis, has been found to contribute to cardiomyocyte death after myocardial infarction (238,239), likely to be involved in reperfusion injury (240,241) and loss of myocytes in heart failure (242,243). A potential for inhibiting apoptosis in these conditions is recognized (244,245), and a caspase inhibitor appears to be in development for the treatment of myocardial infarction (244). Other possible applications of antiapoptotic drugs include ischemia/reperfusion injury in other organs (246–249), sepsis, acute liver failure, systemic inflammatory response syndrome or rheumatoid arthritis (244,245). Finally, protection of normal tissue from damaging effects of chemo- and radiotherapy of tumor cells might be an application specific for compounds interfering with the p53 pathway, which is inactivated in many tumors. Alternatively, since in some tissues p53 is protective by inducing growth arrest and preventing premature entrance into mitosis and death from mitotic catastrophe, p53 inhibition can sensitize such tissue to chemo- and radiotherapy (164). It is quite possible that antiapoptotic drugs, in the end, may find an application in an area other than neurodegenerative diseases.

The problem with the animal models The fact that there is no neuroprotective, disease-progression slowing pharmacotherapy for any ND means that no such treatment has been identified by the existing animal models which in turn means that none of these models is validated. It can be objected that ALS and riluzole are an exception to this, because the latter was reported effective in the SOD1transgenic G93A mouse model and is the only compound registered for the treatment of ALS. However, its clinical effect is limited both in patients and in the mouse model, and one may argue that one swallow does not make a summer. Our models are mostly built to reproduce symptoms of diseases rather than the pathophysiology that leads to the symptoms. That can probably be best illustrated with the MPTP model of PD, which reflects parkinsonian motor deficits quite well in primates (but less well in mice). Better yet, it reflects the loss of nigral DA neurons, even to some extent regional differences, and it inhibits mitochondrial complex I, which fits with the reported reduction of complex I activity in PD. Also, microglial activation is observed both in PD and after MPTP, although in the latter case it correlates poorly regionally with areas of neurodegeneration (250). Some of the changes in apoptosis-related parameters in animal brain after MPTP treatment qualitatively resemble those seen in postmortem PD brain tissue (20,171,251). The clinical failure of CEP-1347 and TCH346, two compounds with quite

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different antiapoptotic modes of action, painfully demonstrates that qualitative similarities are not enough, and that, although the MPTP models may be adequate static models of DA deficiency, they do not appropriately reflect the dynamic processes that cause and modify it. The latter even limits their usefulness for the development of symptomatic treatments (252), and the former makes them unreliable for finding disease-modifying drugs. It must be feared that the situation is not better with respect to models for other chronic ND. Relating to AD, APP transgenic, presenilin transgenic or tau transgenic mice all reproduce parts of AD pathology, but none of them show the full picture (253,254). Whether combination of different genetic mutations in double or multiple transgenic animals will result in a true reflection of AD pathophysiology or only generate static state models as in the case of MPTP for PD remains to be seen. The few compounds so far found effective in SOD1-transgenic mice that were clinically evaluated have all failed with the exception of riluzole (255). However, since their effects in the mice were statistically significant but not overwhelming, it may be prudent to await the results of clinical trials with minocyclin and celecoxib before extending doubts on this model. With respect to HD, the various transgenic and knockin mouse models show many similarities in phenotype and neuropathology, with differences in age of onset and rate of progression depending on fragment length, number of CAG repeats and protein expression levels; in contrast to the human disease, they all show very little and only late neuronal loss (122,256). Choice of an appropriate one among these models appears feasible for developing compounds that interfere with expression or disposal of mut htt or generation of toxic fragments; for compounds designed to interfere with toxicity in general and apoptotic processes, it is essential that pathophysiology in model and disease are at least similar. As long as it is not known in detail how mut htt damages and kills striatal medium spiny neurons in HD, how can an appropriate model be selected? Even in an acute condition like stroke, where an experimental situation can be created that is thought to approximate that in patients, it has been claimed that there are marked differences in gene activation between models and humans (257). If confirmed, it raises similar questions about model validity as for the chronic ND. In short, our knowledge about the pathophysiology of each and every ND is not complete enough to allow creation of, or selection among the existing, animal models which are relevant for the development of disease modifying drugs in general, and antiapoptotic compounds in particular.

CONCLUSIONS Some evidence for the occurrence of molecular events associated with apoptosis has been found in practically every chronic or acute ND, although in many if not most cases the observed morphology did not meet stringent criteria for apoptotic cell death, and elements of (programmed) necrosis and/or autophagy have also been described. The evidence for occurrence if not a role of apoptosis or PCD in PD, at least in part via the intrinsic pathway, appears to be most solid of all chronic ND, perhaps followed by ALS. The failures in clinical trials in PD of two compounds, and of one of them in ALS, targeting the intrinsic

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pathway at two different levels, are therefore clearly disappointing with respect to the therapeutic prospects of antiapoptotic drugs in chronic ND. They suggest that the MPTP model, the primary animal model used for developing antiparkinsonian drugs including those designed to slow disease progression, does not reflect the pathophysiology of the disease to an extent that would make it a reliable predictor of therapeutic efficacy. The quite limited knowledge of the pathophysiologies, especially of the early stages, of most if not all chronic ND makes it utterly difficult to create pathophysiologically relevant animal models with predictive value. On the other hand, models that mimick certain aspects of diseases like AD or HD are already available, but their predictive value with respect to therapeutic efficacy is not known. At first glance, the models for acute ND are closer to clinical reality, and so the chances of finding effective drugs may appear better. However, these models need first to be put to test regarding their therapeutic predictiveness, and this is a challenging task in view of the difficulties of such clinical trials due to the inherent variability of the insults and their consequences. Thus, it appears that currently, the possibilities to preclinically evaluate candidate drugs for ND with a reasonable chance for success are quite limited.

Can we do better? What can be done, then, to enhance the probability of success of future drug candidates for the treatment of ND? In the long term, efforts ought to be made to improve our knowledge of the pathophysiologies of ND’s, with an emphasis on the dynamics of their progression. A prerequisite for this would be organized collection of CNS tissue from patients that have died at different stages of, and from causes unrelated to their respective diseases, to achieve coverage of a significant temporal portion of the disease course. Obviously, capturing the very early, i.e. presymptomatic phases is difficult if not impossible except perhaps for entirely genetically determined ND, which however are so rare that acquisition of sufficient samples to meaningfully cover a significant stretch of disease course may take ages. Nevertheless, analysis of such ‘serial’ samples might provide insight into the sequence of pathophysiological events in contrast to the static, end-of-disease snapshots that are currently available. Such knowledge might in the end allow generation and calibration of meaningful animal models with a certain degree of predictivity. A more serendipitous, though no less ambitious way to proceed would be to seriously invest into development of surrogate markers able to predict therapeutic outcome in patients. They would allow quick evaluation of therapeutic potential of a larger number of compounds picked on the basis of current animal models, a shotgun approach in the hope of finding one or a few that are effective. A clearly effective compound in one or the other ND may be used as a tool to unravel at least some aspects of its pathophysiology and provide a basis for improvement. However, the case of riluzole in ALS, which as yet has not led to further pharmacotherapeutic progress, demonstrates that one compound alone may not be enough to achieve a significant leap forward. Great caution is warranted with respect to extrapolation of pathophysiological data from animal models to the human disease they are supposed to represent. To use

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such data as a basis for drug target selection has a quality of self-fulfilling prophecy and can only be successful if they are verified and validated in patients. Otherwise, the result will be disappointment, as the cases of CEP-1346 and TCH346 have sadly demonstrated.

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