Summary

We invite you to participate in this Forum Discussion with Yong Shen (Sun Health Research Institute). This discussion will not be hosted via our live discussion software. Instead, we will develop written exchanges between our participants and Yong Shen. Take advantage of this slower format to formulate questions, comments, and replies at your leisure! Contact us with questions, suggested answers to our bullet point issues, critiques, or kudos. Tom Fagan, who is temporarily filling in for Gabrielle Strobel, Managing Editor, will forward them to Yong Shen and in this way mediate your conversation. We will post comments on the site as we receive them.
  

 

Suggested questions for discussion:

  • It used to be apoptosis or necrosis. Now there is paraptosis. What is this?
  • Given the maze of pathways, the crosstalk and overlap, (link to diagram), how do we find a non-redundant nexus that would make a good drug target?
  • Are there drug targets?
  • What's holding drug developers back at this point?
  • What are the 3 most important research questions that academic scientists should tackle to encourage renewed therapeutic programs by industry?
  • Are cell death pathways in early AD different from the ones that kill neurons in advanced AD?
  • Animal research these days focuses on pre-plaque toxicity, even pre-tangle changes in tau. How does this translate to death pathways in human disease?
  • Apoptosis is sometimes dismissed as irrelevant to AD because it is a fast process, whereas the death of a neuron in AD is deemed slow. Is this still a legitimate argument?
  • In light of present research, where has the debate moved? What are the big questions now?
  • DNA repair seems to be an Achilles heel of the embattled neuron. How does it lead to cell death, and how can we study DNA repair in adult neurons, in vivo?

Background

Background Text
By Yong Shen.

Cell Death: Time to Push it Out of the Doldrums

A physiological process during development, neuron death clearly becomes harmful in mature brains under injured or diseased conditions such as Alzheimer's, Parkinson's, or other neurodegenerative diseases. And indeed, neuronal death generated tremendous enthusiasm in the early 1990s after some researchers discovered caspase enzymes and others made inroads into the mechanisms of excitotoxicity.

But the effort quickly bogged down. Experimental drugs targeting both caspases and the NMDA receptor flopped in clinical trials, cooling industry interest. The basic science parsing out death pathways became complex and seemingly impenetrable. Meanwhile, AD researchers were challenging cell death aficionados: Is it even apoptosis, or is it necrosis or perhaps a third beast, that drive neuron death in AD? Apoptosis is but the endgame, why bother keeping a sickly neuron alive but unwell? Show us the key pathways! How do they get turned on by plaques or tangles? And are there any good drug targets in there? Adding insult to injury, a growing focus on synaptic dysfunction as a cause for early cognitive symptoms did its part to push neuron death out of the limelight.

It's changing now, with signs that the science of neurodegenerative cell death it getting a second wind. We argue that despite all else, it is still neuronal loss in the brain that leads to the major clinical symptoms of these disorders, and that the study of death mechanisms in AD deserves renewed attention. The field has come a long way: Academia now has the tools (such as specific markers for capase activation, to name but one) and the knowledge to interest drug developers in new approaches. Here we lay out the major evidence-based models for neuronal loss that have been worked out in recent years. There are myriad ways for an adult neuron to die, and the mechanisms described are not mutually exclusive. At this point, we believe that Aβ misfolding, free radicals, and inflammation will prove to play the most active role in AD upstream of caspases.

We invite everyone to proffer their opinion, challenge us on our contention that it's time to push the field back into a stiff breeze, or fill in the picture we describe here!

Too Much Excitement: Toxicity via Glutamate Receptor and Calcium Imbalance

Abundant evidence suggests roles for excitotoxicity both in acute disorders, such as stroke and traumatic brain injury, and chronic age-related diseases such as Alzheimer's and Parkinson's (Driscoll and Gerstbrein, 2003; Zhang et al., 2002) although a first wave of clinical trials of anti-excitotoxic drugs have failed. Thus, a better understanding of the excitotoxic process and new efforts that approach delivery and specificity in a different way are needed for the development of novel therapeutics for neurodegenerative disorders.

Excitotoxic neuron death involves an overload of intracellular calcium, oxygen radical production, and engagement of programmed cell death cascades. (Abramov et al., 2004; Dong-Gyu et al., 2004; Ray et al., 2003; Furukawa et al., 2003). A number of conditions can activate glutamate receptors, such as when a neuron has reduced levels of oxygen or glucose, increased oxidative stress (Schubert and Piasecki 2001), is exposed to toxins or other pathogenic agents, or when it carries a disease-causing genetic mutation. All of these varied conditions have been reported in AD or PD. Once activated, glutamate receptors can trigger cell death. In particular, Ca2+ entry through the NMDA subtype of glutamate receptors has the power to determine whether neurons survive or die. Interestingly, too much NMDA receptor activity is harmful to neurons, but so is too little. Is this a case of too much or too little Ca(2+) influx causing cell death, or do other factors play a role, such as receptor location or receptor-associated proteins? Understanding the mechanisms behind this dichotomous signaling is an important area of molecular neuroscience with direct clinical implications (Viviani et al., 2003).

Excitotoxic cascades are initiated in postsynaptic dendrites, where they may either cause local degeneration or may propagate the signals to the cell body, resulting in neuron death. Neurons possess an array of anti-excitotoxic mechanisms, including neurotrophic signaling pathways, intrinsic stress-response pathways, and survival proteins such as protein chaperones, calcium-binding proteins, and inhibitor of apoptosis proteins (Zhang et al., 2002).

Protein Misfolding: Does It Wreak Havoc on the Mitochondrial Membrane?

Recently, a body of evidence has grown to suggest that abnormal interactions and misfolding of proteins in the nervous system may be important pathogenic events preceding neurodegeneration. Protein misfolding may be at play in Alzheimer's and Parkinson's diseases, and dementia with Lewy bodies (DLB) (Cohen and Kelly, 2003; Dawson and Dawson, 2003; Forloni et al., 2002; Hashimoto et al., 2003; Kudo et al., 2002; Lee et al., 2003; Thompson and Barrow, 2002). Aggregated or soluble misfolded proteins could be neurotoxic through a variety of mechanisms, but these remain largely unknown.

In AD, misfolded Aβ accumulates in the neuronal endoplasmic reticulum (ER) and extracellularly as plaques. In general, the ER performs the synthesis, posttranslational modification, and proper folding of proteins. A variety of conditions can create ER stress, causing unfolding or misfolding proteins to accumulate in the ER. Three mechanisms for dealing with this accumulation, known collectively as the unfolded protein response (UPR), are transcriptional induction of stress response genes, a general translational attenuation to reduce the burden on the ER, and degradation of misfolding proteins. Kudo et al. recently reported a new mechanism by which PS1 mutations may interfere with the sensing of ER stress (Kudo et al., 2002), but why proteins misfold in sporadic AD is unclear. In PD and DLB, α-synuclein accumulates abnormally in neuronal cell bodies, axons, and synapses. Furthermore, in DLB, Aβ 42 may promote this a-synuclein accumulation.

The central event leading to synaptic and neuronal loss in these diseases is not completely clear yet; however, recent advances in the field suggest that damage might result from the conversion of nontoxic monomers to toxic oligomers and protofibrils. The mechanisms by which misfolded Aβ and α -synuclein might lead to synapse loss are under intense investigation. Here are some ideas: Protein aggregates could cause damage directly by derailing intracellular trafficking in neurons. Several lines of evidence support the possibility that Aβ peptide and α -synuclein might interact to cause mitochondrial and plasma membrane damage upon translocation of protofibrils to the membranes. Accumulation of Aβ and α -synuclein oligomers in the mitochondrial membrane might result in the release of cytochrome C, with subsequent activation of the apoptosis cascade. Conversely, the oxidative stress and mitochondrial dysfunction associated with AD and PD may also lead to increased membrane permeability and cytochrome C release, which further promotes Aβ and α -synuclein oligomerization and neurodegeneration. In short, the translocation of misfolded proteins to the mitochondrial membrane might play an important role in either triggering or perpetuating neurodegeneration. Insight obtained from the characterization of this process may be applied to the role of mitochondrial dysfunction in other neurodegenerative disorders, including AD. New evidence may also provide a rationale for the mitochondrial membrane as a target for therapy in a variety of neurodegenerative diseases.

Apoptosis and Caspase Activation: When, How, Why?

Clearly, adult brain neurons are particularly vulnerable to degeneration by apoptosis, at least in culture. And the inducers that activate the apoptotic program in vitro (e.g. Aβ , oxidative damage, low energy metabolism) are present in the Alzheimer's disease (AD) brain. This suggests the possibility that apoptosis may be one of the mechanisms contributing to neuronal loss in this disease. Indeed, some neurons in vulnerable regions of the AD brain show evidence of DNA damage, nuclear apoptotic bodies, chromatin condensation, and the induction of select genes characteristic of apoptosis in cell culture and animal models. Strangely, however, neighboring neurons appear normal, so this is not a homogenous process across a given tissue area, (Clement et al., 2003). Still, this data suggests the existence of apoptosis in the AD brain, a hypothesis that is consistent with evolving research in one of the regulatory functions of the presenilin genes (Gamliel et al., 2003; Hashimoto-Gotoh et al., 2003; Terro et al., 2002; Yu et al., 2001).

How Important is Apoptosis in AD?

The majority of neurons in vulnerable regions in early and mild AD cases show DNA damage, yet in most tissues cells in full-fledged apoptosis disappear within hours to days. Thus it seems unlikely that DNA damage by itself would signify terminal apoptosis. Instead, the presence of extensive DNA damage suggests an acceleration of damage, faulty repair processes, loss of protective mechanisms, or an activation and arrest of aspects of the apoptotic program. It is sometimes said that DNA damage is an artifact of postmortem delay or agonal state, but this is unlikely. Protective mechanisms for neurons probably are at work as these cells are non-dividing and essential. In this context it is interesting to note that the (apoptosis brake) Bcl-2 is upregulated in most neurons with DNA damage, as is at least one DNA repair enzyme (Boland and Campbell, 2003; Romero et al., 2003; Suh et al., 2003). Thus it appears as if neurons are for a period locked in a struggle between degeneration and repair until one side wins out. As research advances, it will be critical to reduce the stimuli that cause the neuronal damage and discover the key intervention points to assist neurons in the repair processes.

Microglia: A Deadly Embrace

In apoptosis, the regulated triggering of a proteolytic caspase cascade is quickly followed by efficient removal of cell corpses. Microglia contribute to the elimination of dead cells. In developmental apoptosis, it is also the microglia that promote the death of neurons engaged in synaptogenesis (Marin-Teva et al., 2004). In this study, selective elimination of microglia strongly reduced apoptosis of Purkinje cells in cerebellar slices. Sixty percent of dying neurons expressing activated caspase-3 were engulfed or contacted by spreading processes from microglial cells, and superoxide ions produced by microglial respiratory bursts played a major role in this Purkinje cell death. The study illustrates a mammalian form of cell death promoted by microglia engulfment, and it connects the execution of neuron death to the scavenging of dead cells (Ahmadi et al., 2003).

Death Receptors Signal Neuron's Demise

Death receptors are cell surface receptors that transmit apoptotic signals delivered by specific ligands (Agerman et al., 2000). These receptors belong to a subgroup of the tumor necrosis factor receptor (TNFR) superfamily and are characterized by a so-called death domain (DD) that resides in the cytoplasmic region. In addition to DR3, DR4, DR5, and DR6, other members of this subgroup are TNFRI, Fas, the ectodysplasin receptor (EDAR), and the p75 neurotrophin factor receptor (p75NTR) (Aloyz et al., 1998; Ashkenazi et al., 1998). Death domain sequences vary slightly between receptors, but all are highly homologous and capable of protein-protein interactions. Death domains recruit intracellular adaptor proteins that also contain a death domain, such as Fas-associated death domain protein (FADD), TNFR-associated death domain protein (TRADD), and receptor-interacting protein (RIP).

In AD brains, TNFRI has shown increased expression and is related to the apoptotic process. Recent studies on apoptotic protease-activating factor-1 (Apaf-1)-transgenic mice (Cecconi et al., 1998; Li et al., 1997; Yoshida et al., 1998) demonstrate that activation of Apaf-1 induces obvious abnormality in tissues where cellular development depends on apoptosis (Cregan et al., 2002). This observation suggests a critical role of Apaf-1 in apoptotic cell death. Apaf-1 is a member of the protein family that contains a caspase recruitment domain and regulates apoptosis.

The activation of NF-κ B translocation causes apoptosis in vitro (Inohara et al., 1999; McCarthy et al., 1998; Ogura et al., 2001; Thome et al., 1998). Another NF-κB related mechanism of apoptosis is TNFRI. TNFRI and its intracellular mediators may activate common pathways that lead to degradation of I- B , which relocates NF-κB from the cytoplasm into the nucleus (Hsu et al., 1995; Miyamoto et al., 1994). The translocation of NF-κB might play an anti-apoptotic role in various cells (Guo et al., 1998; Kaltschmidt et al., 1999) and an apoptotic role in neuronal cells (Schneider et al., 1999; Lipton, 1997; de Erausquin et al., 2003; Straus et al., 2000; Yang et al. 2002). This discrepancy may be due to different cell types or the involvement of distinct receptors in neurons. In AD, Aβ peptide has been shown to activate NF-κB (Akama et al., 1998; Ghribi et al., 2001; Kuner et al., 1998). A recent report demonstrated that the toxicity induced by soluble Aβ40 correlates with its association to the cell membrane (Mathews et al., 2002; Morishima-Kawashima et al., 1998).

Although one study has demonstrated that knocking out both TNFRI and TNFRII can increase neurodegeneration (Bruce et al., 1996), the specific contributions of each TNF receptor subtype to neuronal cell death has not been clearly identified. We demonstrated that TNF- -induced neuronal cell death is mediated by NF-κB translocation and dependent on TNFRI dependent (Yang et al., 2002). More recently, we explored whether Aβ-induced neuronal apoptosis is related to TNFRI and its unique signal transduction pathway. By using a gene targeting approach, we provide novel evidence that Aβ40 increases Apaf-1 expression and neuronal NF-κB translocation. Our results suggest that, upon treatment with Aβ40, Apaf-1 activates NF-κB via binding the DD of TNFRI with high affinity, and that this eventually leads to neuronal death. Furthermore, our binding results suggest that death receptors not only interact with their own ligands but also bind other offensive molecules such as Aβ , which activates their death cascade (Li et al., 2004). Importantly, this finding is supported by a recent study (Del Villar and Miller, 2004; See ARF news story) conducted in AD brains. Del Villar and Miller found TNFR-associated death domain (TRADD) protein in AD hippocampus. Interestingly, they found that TNF death receptor binding protein, DENN/MADD is significantly reduced, which promotes neuron death. This result suggests that the TNF death receptor binding protein DENN/MADD may play a critical checkpoint role during the neurondegenerative process.

A Brief Note on Parkinson's.

The primary neuropathological feature is this disease is a massive loss of dopaminergic neurons in the substantia nigra, and TNFRI levels are increased in both brain and cerebrospinal fluid (Boka et al., 1994; Hunot et al., 1997), as well as on circulating T-lymphocytes (Bongioanni et al., 1997). These data indicate an association between TNF and neurodegeneration in PD. Indirect evidence for TNF-induced toxicity in dopamine neurons may be reflected in the general neurotoxicity of ceramide in primary mesencephalic cultures. Ceramide can enhance activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-α . TNF-α also can elevate levels of endogenous ceramide and activate the intrinsic cell death pathway. Transgenic mice devoid of both TNFRI and TNFRII, moreover, are resistant to the dopaminergic neurotoxicity caused by 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (Sriram et al., 1997; Tatton et al., 2003).

Akt and Neuron Death

In peripheral systems, including the immune system, phosphoinositide 3-kinase (PI 3-kinase) and its downstream serine-threonine kinase effector, Akt, provide a potent stimulus for cell proliferation, growth, and survival. (Akt is also called Protein Kinase B.) In the past 10 years, exciting studies have demonstrated that Akt is actively involved in neuron death in the brain (Brunet et al., 2001; La Spada and Taylor, Neuron, 38, 681-685, 2003; Yuan and Yankner, 2000). For example, Huda Zoghbi's group recently reported that Akt phosphorylation can regulate ataxin-1 association with 14-3-3, which mediates neurodegeneration in a Drosophila model of spinocerebellar ataxia type 1 (SCA1) (Chen et al., 2003, see ARF related news story). This finding provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.

Interestingly, erythropoietin appears to protect neurons against excitatory neurotoxicity by preventing decreased phosphorylation levels of ERK1/2 and Akt and by enhancing neurotrophin-associated signaling pathways (Dzietko et al., 2004). Furthermore, it was shown that glutamate inactivates Akt involved in the pro-survival actions of IGF-I, and uncoupling of IGF-I signaling from Akt by glutamate may contribute to excitotoxic neuronal injury (Garcia-Galloway et al., 2003).

Fyn and Neuron Death

Fyn belongs to the Src family of tyrosine kinases and is more ubiquitously expressed than other members. Pioneering work on Fyn in AD was reported by Shirazi and Wood, 1993 and by Bill Klein's group (Zhang et al., 1996, Lambert et al., 1998). The latter found that Aβ upregulates the stable association of focal adhesion kinase (FAK) with Fyn. Aberrant Fyn activity due to A β-evoked association with FAK could play a role in neuronal degeneration and also cause anomalies in synaptic plasticity.

Actually, Lambert and colleagues also found that, in Fyn knockout mice, soluble Aβ, which they called Aβ -derived diffusible ligands (ADDLs), provoked neurological dysfunction before cellular neurodegeneration was apparent. Moreover, despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, suggesting an immediate effect on signal transduction. Their results on impaired synaptic plasticity and associated memory dysfunction may play a role in early stage Alzheimer's disease.

Recently, Lennart Mucke's group deepened Fyn's implication in synaptic damage in AD by looking at reductions in hippocampal levels of synaptophysin-immunoreactive presynaptic terminals, or SIPTs, by Aβ (Chin et al., May, J Neurosci. 2004). Aβ did reduce SIPT, in correlation with hippocampal Aβ levels, in hAPP/fyn+/+, but not in hAPP/fyn knockout mice. This suggests that Fyn provides a link between Aβ and SIPTs. Furthermore, overexpression of Fyn exacerbated SIPT reductions in hAPP mice. In contrast, axonal sprouting in the hippocampus of hAPP mice was unaffected. We conclude that Fyn-dependent pathways are critical in AD-related synaptotoxicity.

Fyn kinase also plays an important role during myelination and has been shown to promote morphological differentiation of cultured oligodendrocytes. (Klein et al., J Neurosci., 1992). Specifically, these investigators found that Fyn binds to the cytoskeletal proteins Tau and α -Tubulin in oligodendrocytes. Tau interacts with the Fyn SH3 domain, whereas α -Tubulin binds to the Fyn SH2 and SH3 domains. This effect is caused by interference with the Fyn-Tau-microtubuli cascade rather than inactivation of the kinase. Because ligation of the cell adhesion molecule F3 on oligodendrocytes leads to activation of Fyn kinase localized in rafts, these findings suggest that recruitment of Tau and Tubulin to activated Fyn kinase in rafts is a step in the initiation of myelination.

DNA Repair and Death

DNA double-strand breaks are one serious form of DNA damage. Left unrepaired, they can lead to cell death. When repaired faultily, they contribute to chromosomal aberrations and genomic instability. Cells deficient in repairing DNA double-strand breaks have an increased level of spontaneous chromosomal aberrations. Experimental modulation of the level of molecular oxygen and its reactive metabolites has demonstrated that oxygen metabolism is a major source of genomic instability (Love, 1999). However, the cause and natural function of such breaks remains a mystery. In a broader context, there is now renewed speculation that DNA recombination might be occurring during neuronal development, similar to DNA recombination of antibody and T cell receptor genes in developing lymphocyte (Chen, 2000; Culmsee et al., 2001; Gilmore et al., 2000). If this is true, the target gene(s) of recombination and their significance remain to be determined.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme, activated by DNA strand breaks to participate in DNA repair. Overactivation of PARP by cellular insults depletes its substrate NAD(+) and then ATP, leading to an energy deficit and cell death (Eliasson et al., 1997; Ha and Snyder, 2000; Sheline et al., 2003). This mechanism appears to be prominent in vascular stroke and other neurodegenerative processes in which PARP gene deletion and PARP-inhibiting drugs provide major protection. Neuron death associated with excessive PARP-1 activation appears to be predominantly necrotic, while apoptosis is associated with PARP-1 cleavage, which may conserve energy needed for the apoptotic process. Novel forms of PARP derived from distinct genes and lacking classic DNA-binding domains may have non-nuclear functions, perhaps linked to cellular energy dynamics. (Fonnum F, Lock, 2004, see also related ARF Live Discussion )

The Cell Cycle: Trying to Divide, Dying Instead

Terminally differentiated neurons in the normal brain cannot divide, and yet, accumulating evidence has suggested that certain degenerative diseases, they do try. Abortive cell cycle events play a major role in the loss of neurons in advanced Alzheimer's disease. It is currently unknown, however, whether the same is true in early disease. In Alzheimer's disease, regulators from every phase of the cell cycle are upregulated in affected neurons, leading to successful DNA replication but unsuccessful mitosis (see related ARF discussion). The end point of this nonproductive cycle of division is death. Elucidating the details of this cascade may lead to novel strategies for curbing the onset and progression of degenerative diseases. Immunocytochemistry shows that a significant percentage of hippocampal neurons in mild cognitive impairment (MCI) express three cell cycle-related proteins, proliferating cell nuclear antigen, cyclin D, and cyclin B (Yang et al., 2003). The percentage is similar to that found in AD cases but significantly higher than in normal controls. In entorhinal cortex, the density of cell cycle-positive neurons was greater in MCI than in AD. These findings support the hypothesis that both the mechanism of cell loss (a cell cycle-induced death) and the rate of cell loss (a slow atrophy over several months) are identical at all stages of the AD disease process.

With regard to Parkinson's, Lee et al., recently reported (2003) that over-expression of α -synuclein led to enhanced proliferation and an enrichment of neurons in the S phase of the cell cycle. This was associated with increased accumulation of the mitotic factor cyclin B and phosphorylation of ERK1/2, key molecules in proliferation signaling. Immunohistochemical studies on postmortem brains revealed intense cyclin B immunoreactivity in Lewy bodies in cases of dementia with Lewy bodies (DLB) and to a lesser extent in PD. These findings suggest that elevated expression of α -synuclein may cause changes in cell cycle regulators through ERK activation, leading to the death of postmitotic neurons. Cyclin B is also ectopically expressed in Lewy bodies.

Recently, Harvey et al., (2003) reported that mutations in the Drosophila gene hpo result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo can interact physically and functionally with Sav and Wts, and regulates DIAP1 levels. Thus, Hpo links Sav and Wts to a key regulator of apoptosis during development. Clarifying these mechanisms of developmental cell growth versus death may provide hints for the prevention of pathological neuronal death.

In summary, there are myriad reasons and myriad ways for a neuron to die. The reasons range from the good, i.e. developmental pruning, to the bad, i.e. succumbing to insults such as oxidative radicals or amyloid. The pathways that have been studied overlap at many levels, and staying on top of this developing web of interactions is a challenge. Although protein misfolding/misassembly and aggregation currently look like critical and early suspects in AD pathogenesis, it is also true that apoptosis contributes to neuronal death in important ways. Extrapolating the wealth of data gained from the typical cultured postnatal neuron to the mature in-vivo neuron has been difficult. This is in part because differentiated neurons have different signal transduction systems active and running than do immature ones. Partly it is because neurons embedded in their tissue environment can rally powerful protective mechanisms to counter disease insults, mechanisms that are not available to the cultured cell. Postmortem analysis and tissue slice analysis can bridge some of this knowledge gap. We need to get a better grip on what drives this yin-yan balance for neuronal survival. Apoptosis inhibitors should be investigated but used with caution as they might carry a risk of cancer.

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  1. Yong Shen does an excellent job of summarizing some of the
    important data supporting an important role for cell death in a number
    of neurodegenerative diseases. Clearly, there are a myriad of possible
    scenarios that may lead to neuronal cell death, and some of these
    mechanisms may overlap with one another. Shen illustrates exceptionally well the vast number of pathways that may lead to the stimulation of cell death pathways, including apoptosis.

    With regard to Alzheimer's disease, I would like to address one topic mentioned in the text, namely: Is apoptosis irrelevant because
    it is a late-stage event in AD?

    Most scientists would agree that the earliest known step in the cascade
    of events leading to neuronal cell death in AD is the formation of toxic
    Ab fibrils. Several studies have supported the hypothesis that Ab may
    induce neuronal cell death by activation of multiple pathways of
    apoptosis; this includes the receptor-mediated pathway and the mitochondrial
    pathway involving reactive oxygen species [2, 3, 5]. However, it is likely that, due to the arsenal of anti-apoptotic mechanisms neurons possess, the activation of apoptosis and eventual neuronal cell death may be separated in time. We have proposed that during this protracted battle, caspases may
    continue to be active at low levels and cleave critical cellular
    proteins that may actually drive the pathology observed in AD [7].

    One potential target for caspase-mediated cleavage may be
    the microtubule-associated protein, tau. Well known in AD circles
    as being the protein whose hyperphosphorylation leads to
    neurofibrillary tangle formation, recent evidence suggests that tau is a
    target for caspase-3-mediated cleavage. Three recent studies now
    suggest that caspase-3 cleavage of tau occurs in the AD brain, is a
    relatively early event (preceding PHF formation), and may enhance
    filament formation in vitro [1, 4, 6]. Furthermore, in the study by
    Gamblin et al., the authors demonstrate that the link between caspase
    activation and cleavage of tau is Ab. They demonstrate
    in primary cultured cortical neurons that application of Ab induces
    caspase activation and subsequent cleavage of tau. The cleavage of tau
    by caspase-3 leads to enhanced filament formation in vitro [1].

    Supporting these findings is a study currently in press by Rissman et
    al. (This paper will be released in the July 1st JCI. Due to embargo, I cannot reveal details at this point.) Taken together, these studies now show is that caspase activation may in fact be a proximal event that leads to neurofibrillary tangle formation in AD.

    Apoptosis in AD has long been criticized for being a late-stage event, but these studies support the idea that inhibition of caspases may be a viable target for drug development in treating AD. Yes, the morphological features of apoptosis probably do occur late in the disease, but the activation of caspases and cleavage of critical proteins may occur earlier and contribute to the downstream pathology observed in AD. It is noteworthy that numerous other neurodegenerative diseases, including Pick's disease, dementia with Lewy bodies, Progressive Supranuclear Palsy (PSP), Parkinson's and corticobasal degeneration (CBD), are all associated with the abnormal
    intracellular accumulations either of the tau protein or of
    alpha-synuclein. We have examined whether caspase-cleavage of tau is a
    common denominator in these diseases and found the answer to be yes
    (Unpublished observations). Thus, caspase activation and accumulation of
    cleaved tau may represent a common pathway leading to neurofibrillary
    lesions in these neurodegenerative diseases.

    What needs to happen next in the field to further test this
    hypothesis? Animal models of AD where caspase inhibitors are employed
    would be a logical next step to prove that caspases are a suitable
    target for drug therapy.
    Will caspase inhibition prevent neurofibrillary lesions? Will neuronal cell death be prevented? And finally, will neuronal function and behavior be improved following caspase inhibition in such animal models? Until studies such as these are undertaken, the role of apoptosis in AD will continue to be a controversial and much-debated subject [8].

    References:

    1. Gamblin, T.C., Chen, F., Zambrano, A., Abraha, A., Lagalwar, S.,
    Guillozet, A.L., Lu, M., Fu, Y., Garcia-Sierra, F., LaPointe, N.,
    Miller, R., Berry, R.W., Binder, L.I. and Cryns, V.L., Caspase cleavage
    of tau: linking amyloid and neurofibrillary tangles in Alzheimer's
    disease, Proc Natl Acad Sci U S A, 100 (2003) 10032-7. Abstract

    2. Ivins, K.J., Thornton, P.L., Rohn, T.T. and Cotman, C.W.,
    Neuronal apoptosis induced by beta-amyloid is mediated by caspase-8,
    Neurobiol Dis, 6 (1999) 440-9. Abstract

    3. Miranda, S., Opazo, C., Larrondo, L.F., Munoz, F.J., Ruiz, F.,
    Leighton, F. and Inestrosa, N.C., The role of oxidative stress in the
    toxicity induced by amyloid beta- peptide in Alzheimer's disease, Prog
    Neurobiol, 62 (2000) 633-48. Abstract

    4. Rissman, R.A., Poon, W.W., Blurton-Jones, M., Oddo, S., Reidun,
    T., Vitek, M.P., LaFerla, F.M., Rohn, T.T. and Cotman, C.W.,
    Caspase-cleavage of tau is an early event in Alzheimer's disease
    pathology, J. Clin. Invest., In Press (2004).

    5. Rohn, T.T., Head, E., Nesse, W.H., Cotman, C.W. and Cribbs,
    D.H., Activation of caspase-8 in the Alzheimer's disease brain,
    Neurobiol Dis, 8 (2001) 1006-16. Abstract

    6. Rohn, T.T., Rissman, R.A., Davis, M.C., Kim, Y.-E., Cotman, C.
    and Head, E., Caspase-9 Activation and caspase cleavage of tau in the
    Alzheimer's disease brain, Neurobiol Dis, 11 (2002) 341-354. Abstract

    7. Rohn, T.T., Rissman, R.A., Head, E. and Cotman, C.W., Caspase
    activation in the Alzheimer's disease brain: tortuous and torturous,
    Drug News Perspect, 15 (2002) 549-557. Abstract

    8. Roth, K.A., Caspases, apoptosis, and Alzheimer disease:
    causation, correlation, and confusion, J Neuropathol Exp Neurol, 60
    (2001) 829-38. Abstract

  2. I agree with Troy that there may be different routes of anti-apoptotic mechanisms and neuronal apoptosis. The recent studies on caspase-mediated cleavage of MAP or tau are quite interesting. Troy, do you think this would be a normal physiological process or a pathological process?

    Besides the possibility of continuous low-grade caspase expression, I think the roles of cyclin-dependent kinase 5 (Cdk5) in the brain are critical, particularly under pathological conditions. Li-Huei Tsai's group has found that p25, a truncated form of p35, accumulates in neurons in the brains of patients with Alzheimer's disease (Patrick et al.,1999; Lee et al., 2000, see also recent ARF news update). This finding might indicate a relatively slow neurodegenerative process as the p25/Cdk5 complex hyperphosphorylates tau, which reduces tau's ability to associate with microtubules and also induces cytoskeletal disruption, morphological degeneration and apoptosis. This degenerative process seems to be mediated by amyloid.

    Regarding the use of caspase inhibitors in AD animal models, a more straightforward way to prove the concept might be to use gene targeting to knock out caspases in AD mice. For example, one could ask whether cross-breeding mutant tau-transgenic mice with caspase knockout mice would prevent neurofibrillary tangle damage. The Tau P301L-JNPL3 mouse may be a good candidate, since that strain was reported to induce apoptosis (in oligodendrocytes) and activate caspase-3 ( Zehr et al., 2004).
    To study further whether the longest human tau isoform (T40) is involved in neurodegeneration, and whether a caspase mediates its role, Akihiko Takashima's R406W transgenic mice might be suitable. Recently, Virginia Lee's group developed a R406W mutant strain (Zhang et al., 2004 ). These mice have axonal degeneration, which I think could be mediated by caspases. Therefore, it would be worthwhile crossing either of these strains with a caspase 3 or 8 k.o. strain to see if that reduces axonal degeneration in vivo.

  3. Yong Shen asks whether I consider caspase-mediated cleavage of MAP
    or tau a normal physiological process or a pathological process. This is an excellent question. We recently published data indicating a role for caspase-mediated cleavage of tau in neuronal
    cell dispersal and migration (Rohn et al., 2004). In this paper we report an action of caspase-3 involving cell
    dispersion that is independent of cell death. Upon plating PC12 cells, we found a transient activation of caspase-3 (within a 24-hour window) was required for these cells to disperse properly. Additional experiments showed that tau was in fact a target for caspase cleavage. A restructuring of the cytoskeleton would be expected for cells to be able to migrate and disperse. However, this activity of caspase-3 was not required for PC12 cells to differentiate into a neuronal phenotype. Our work supports other recent studies demonstrating that caspases may have additional functions beyond those described for apoptosis (Zermati et al., 2001); (Fernando et al., 2002). So, my answer to Yong's question would be that the cleavage of tau may serve as a physiological role or process under normal conditions, but if caspase activation is not turned off, then it may convert to a pathological
    process as observed in AD.

    A second excellent question Yong raises regards kinases, such
    as Cdk5, and hyper-phosphorylation of tau. How does this fit in with
    the role of caspase-mediated cleavage of tau in AD? I would like to comment on this based on our report due out in JCI, but have to defer this issue until its embargo has lifted. I will just say that caspase cleavage and hyper-phosphorylation of tau may not be two independent events, but serial
     events.

    Finally, Yong responded to my comments on how can we demonstrate in animal models that caspase activation is important for driving AD pathology? He suggests crossing caspase-3 or caspase-8 knockout mice with a mouse AD model. My understanding is that most of the caspase knockouts are lethal, which would prohibit doing such a cross. I have submitted a grant that circumvents this problem using the new AD mouse developed by Frank Laferla's group (Oddo et al., 2003). My proposal involves
    crossing this mouse with a Tg mouse that specifically over-expresses
    Bcl-2 in neurons of the CNS. If funded, I think this would provide an
    excellent opportunity to test this hypothesis. At the same time, I would encourage researchers to test some of the other ideas presented
    by Dr. Shen. In my mind, it is clear we have reached a "the proof is in
    the pudding" stage regarding caspase activation and apoptosis in AD.
    What we really need to do now is prove or disprove these ideas directly
    using animal models as described above before drug companies will become
    interested in picking up the ball.

  4. Yong Shen should be congratulated on this comprehensive and interesting review; it contains several novel connections and ideas. However, I was somewhat disappointed by one passage. Despite a great deal of publication and discussion, misunderstandings about NF-κB are still common. So the author can be forgiven for this misleading statement: "The activation of NF-κB translocation causes apoptosis in vitro." None of the references cited, including the Yang et al. (2002) paper, provides anything other than a correlation between conditions that cause cell death and the activation of NF-κB. This is likely to be because NF-κB is induced in a compensatory fashion. The McCarthy reference that was cited even includes the following statement arguing for a disconnection between NF-κB and cell death: "Mutational analysis revealed the pro-apoptotic function of RIP2 to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-κB activation."

    There are now scores of reports showing that NF-κB activation actually promotes cell survival, as we first reported (Barger et al., 1995, see also Wang et al., 1998 , Wu et al., 1998 ). Much of the confusion about NF-κB comes from analysis of culture lysates or tissue homogenates that contain non-neuronal cell types. The transcription factor is associated with inflammatory reactions in microglia and astrocytes, which can participate in a "sign change" that results in neurotoxicity, primarily through the release of excitotoxins; (see further below). But even in the activated microglia themselves, NF-κB probably promotes survival (e.g., Lu et al., 2002). There are new reports suggesting subtleties that arise from differences in the components of the "NF-κB" being activated, namely, a distinction between RelA- and c-Rel-containing complexes (Pizzi et al., 2002), but this paper appears to suffer the old problem of glial contamination, as well. Taken together, the more rigorous and well-controlled studies generally show anti-apoptotic, pro-survival roles for NF-κB.

    On a separate note, I was gratified to see in Yong Shen's Alzforum statement a section on excitotoxicity. I would like to draw a link between that topic and inflammatory activation of microglia. A significant number of papers have reported the release of glutamate receptor ligands by activated microglia. Glutamate itself is released, apparently through the exchange for cystine through the Xc transport system (Barger & Basile, 2001). Quinolinic acid is another candidate for such ligands. At least in microglia-neuron cocultures, these ligands for glutamate receptors activate the NMDA receptor to a degree that causes substantial excitotoxicity. In vivo, the high capacity for glutamate uptake by astrocytes may help to attenuate this effect, unless they are themselves compromised by ischemia or other pathological conditions. More recently, we determined that D-serine, an agonist at the "glycine" site of the NMDA receptor, is also released by microglia treated with Aβ (Wu et al., 2004, J. Neuroinflammation 1:2). Because the uptake mechanisms for D-serine are poorly characterized, it is unclear that this agonist could be cleared as effectively as glutamate. Furthermore, it may potentiate glutamate effects to the extent that even small elevations of the latter could become excitotoxic.

  5. Reply by Yong Shen

    I am glad Steve Barger brought up the controversy on whether ND-κB causes or counteracts apoptosis. This question in itself is interesting enough to attract more rigorous approaches for definitive study. Most of the studies that found NF-κB to be anti-apoptotic are conducted not only in non-neuronal cells, but also in immune or tumor-related cells. For example, the study by Wu et al., 1998 , was conducted in Jurkat cells. The experiments performed by Wang et al., 1998 were in H710801, a fibrosarcoma cell line. The paper from Pizzi et al., 2002, particularly the key evidence (Figs. 1-3), was done by using cerebellar granule cells. On the other hand, most of the evidence suggesting that NF-κB is involved in apoptosis is from cortical neurons (Schneider et al., Nat Med. 1999; Lipton, Nat Med., 1997), hippocampal neurons (Yang et al. (2002); Li et al., 2004), or midbrain neurons (de Erausquin et al., 2003).

    I believe that the experimental evidence from both sides is correct. It seems that functions of NF-κB are cell type-dependent. Even within the brain, consistent with recent publications (Wu et al., 2004, J. Neuroinflammation 1:2; Pizzi et al., 2002), the responses of NF-κB to neurons and microglia are completely different. Which molecules mediate this discrepancy, and what mechanisms are behind this remains unclear.

    What would be good ways to evaluate NF-kappa B? I think we need to look at both NF-κB binding activity as well as NF-κB translocation, because this is what (NF-κB) naturally does. Many studies demonstrate that IKKalpha can cause degradation of IkappaBalpha by phosphorylation, and result in the release of cytoplasmic NF-κB, which is then able to translocate into the nucleus (Miyamoto et al., 1994; Verma et al., 1995; Baeuerle and Baltimore, 1996). Probably p65 binds certain types of nuclear protein and-depending on the cell type or/and pathological conditions-turns on or off apoptotic genes or anti-apoptotic genes.

    Lastly, Steve draws a link between that topic and inflammatory activation of microglia. This is a great idea. Clearance and uptake of glutamate by microglia is critical. Appropriate glial activation may help maintain neuronal functions, including synaptic transmission. For example, Roger Nicoll's group (Luscher et al, 1998) found that glial cell responses (in this case glial glutamate transporter currents measured with whole-cell recording) are very sensitive to release of glutamate yet remained constant during LTP by directly measuring changes in the synaptic release of glutamate. Moreover, Robert Malenka's group (Malenka et al., 2003) found that glial TNFa enhances neuron synaptic plasticity in young neurons.

  6. Yong pointed out in his reply posted 6/24/2004 that the role of cyclin-dependent kinase 5 (Cdk5) in the pathology and death of neurons is important. Identified over a decade ago as tau-phosphorylating entities purified from brain lysates, Cdk5 and GSK3 have emerged as the two major kinases that contribute to tau hyperphosphorylation and other tau-associated pathologies in animal models of Alzheimer's disease.

    Intriguingly, calpain-mediated cleavage of the 'physiological' cdk5 activator p35 into the 'pathological' cdk5 activator p25 results in upregulation of cdk5 activity and an increased tendency for phosphorylating substrates implicated in neurodegeneration, such as tau. Accordingly, in AD and other neurodegenerative diseases, p25/cdk5 is thought to be an important link between excitotoxicity/calcium (calpain is Ca-dependent) and the downstream pathological processes that result in cell death.

    In vivo studies strongly support this view. Karen Duff's group has recently shown that p25 enhances the progression of neurofibrillary pathology in transgenic mice expressing the FTDP-mutant tau (Noble et al., 2003). In addition to tau hyperphosphorylation, p25 can cause cytoskeleton disruption, morphological degeneration, and apoptosis in cultured neurons. An inducible p25 transgenic mouse model that expresses p25 in the postnatal forebrain exhibits severe brain atrophy, up to 40 percent loss of total neurons in the brain, and tau-associated pathology (Cruz et al., 2003). While phosphorylation of tau (and other microtubule-associated proteins) by Cdk5 may contribute to cytoskeleton disruption and neuronal loss, recent studies suggest additional mechanisms by which the p25/Cdk5 kinase induces cell death.

    One potential downstream cdk5 target mediating apoptotic events may be MEF2, a pro-survival transcription factor (Gong et al., 2003). Phosphorylation of Ser444 in the trans-activation domain of MEF2 leads to inhibition of MEF2 function in neurons. Ser444 phosphorylation is induced by oxidative stress and excitotoxicity. MEF2 mutations resistant to Cdk5 phosphorylation protect neurons from Cdk5-induced and neurotoxin-induced apoptosis. Another potential target of p25/Cdk5 is the NR2A subunit of NMDA receptors (Li et al., 2001; Wang et al., 2003). Phosphorylation of Ser1232 of NR2A by Cdk5 is induced by ischemic injury of the brain (Wang et al., 2003). Remarkably, expression of a NR2A mutant resistant to Cdk5 phosphorylation can prevent the death of CA1 neurons from ischemic injury.

    As discussed by Yong, there are numerous ways for a neuron to die; the same idea applies in p25/cdk5-mediated cell death. Perhaps different Cdk5 substrates contribute to cell death depending on the type of insult or physiological context. Alternatively, these substrates could all become phosphorylated to orchestrate the process of neuronal death. It is likely that additional cdk5 substrates will be identified in the future that contribute to p25/cdk5- mediated cell death.

    In the inducible p25 transgenic mice, induction of caspase 3 is observed that coincides with neuronal loss. It was reported recently that inhibition of Cdk5 is protective in necrotic and apoptotic paradigms of neuronal cell death and prevents mitochondrial dysfunction (Weishaupt et al., 2003). Thus, the neuronal death induced by p25/Cdk5 is likely to operate via the mitochondria-dependent mechanism. As neurodegeneration progresses rapidly in the inducible p25 transgenic mice, a rescue of neuronal loss should be relatively easy to detect. Crossing these mice with various caspase mutant mouse strains should help reveal the identity of the caspase family member(s) that mediate(s) p25/Cdk5- induced neuronal death. This information has clear implications in the therapeutic intervention of AD and ischemic brain injury. We have initiated a collaboration with Dr. Junying Yuan at Harvard Medical School to perform this experiment.

    References:

    Cruz, J.C. et al. (2003) Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles. Neuron 40 (3), 471-483. Abstract

    Gong, X. et al. (2003) Cdk5-mediated inhibition of the protective effects of transcription factor MEF2 in neurotoxicity-induced apoptosis. Neuron 38 (1), 33-46. Abstract

    Li, B.S. et al. (2001) Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc Natl Acad Sci U S A 98 (22), 12742-12747. Abstract

    Noble, W. et al. (2003) Cdk5 is a key factor in tau aggregation and tangle formation in vivo. Neuron 38 (4), 555-565. Abstract

    Wang, J. et al. (2003) Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors. Nat Neurosci 6 (10), 1039-1047. Abstract

    Weishaupt, J.H. et al. (2003) Inhibition of CDK5 is protective in necrotic and apoptotic paradigms of neuronal cell death and prevents mitochondrial dysfunction. Mol Cell Neurosci 24 (2), 489-502. Abstract

  7. Don't Lock the Barn Door After the Horse is Gone

    Yong Shen presents a comprehensive review of molecular phenomena related to neuron death in AD. Yet I suggest that his major contention deals with an event that is not one that leads to the major clinical symptoms but is, rather, the final straw. "We argue that despite all else, it is still neuronal loss in the brain that leads to the major clinical symptoms of these disorders, and that the study of death mechanisms in AD deserves renewed attention," Shen writes. However, just as the death of the individual with AD does not constitute the major clinical symptom of the disease, so is the death of AD affected neurons not a major contributor to the clinical symptomatology of AD. Neuron death is the latecomer in the cellular cascade of AD - the culmination of a series of events that consume decades prior to the death of that cell (e.g. Morsch et al., 1999). It is the series of events that precede neuron death that are central to the cognitive and mnemonic losses that define clinical AD.
    And loss and dysfunction of synapses are vital consequences and players in this cascade that precedes death of a neuron.

    Synapses are the means by which the nervous system transmits, processes, and stores information - all functions that are disrupted in AD. Although a synapse cannot exist once its parent neuron has died, it is clear that a synapse can cease to exist or function normally while its parent neuron remains alive, though sick. The argument here, then, is that synapse loss and dysfunction are the critical events in the deficits of AD, and that neuron death is a late event that administers the coup de grace by depleting the few synapses remaining associated
    with that neuron, whose postsynaptic dendrites have long since regressed (e.g. Buell and Coleman, 1979; Braak and Braak, 1997).

    What is the evidence in support of this contention? For over a decade, it has been known that synapse density is a significant correlate of cognitive and mnemonic deficits in AD while neuron density is not (e.g. Terry et al., 1991). The fact that synapses may be lost independently of neuron death has been demonstrated by the reduction by about half of the synapse/neuron ratio in AD (e.g. Bertoni-Freddari et al., 1996) as well as by the demonstration of reduced synaptophysin transcript in living, tangle-bearing neurons (Callahan et al., 2002). In addition to frank loss of synapses in AD, there is an additional decrement in functional capacity of existing synapses, as has been demonstrated by decreased
    expression of transcripts related to trafficking of synaptic vesicles
    even when transcripts related to other aspects of synaptic structure, such as PSD-95, remain unaffected (e.g. Yao et al., 2003). It seems probable that altered capacities of existing synapses may be a significant factor, since the correlation of about 0.75 between synapse density and cognitive status yields an r squared that accounts for only about 50 percent of the variance in cognition and memory (DeKosky and Scheff, 1990; Terry et al., 1991; Scheff and Price, 2003 for review).

    Thus, the
    loss and dysfunction of synapses appears to have multiple facets. These are:
    1) diminished functional capacity of existing synapses.
    2) Loss of synapses by still-living but affected neurons and, finally, 3) loss of synapses associated with the death of neurons.

    It remains to be formally determined whether synapse loss and dysfunction in AD is a way station in the cellular progression to neuron death, or whether synapse loss and dysfunction can, and does, take place through molecular cascades independent of the processes leading to neuron death, or indeed whether neuronal death processes are a consequence of local events at the synapse which may then feed back to the soma (e.g. Eberwine et al., 2001; Job and Eberwine, 2001).

    It follows that studies that focus on neuron death as the end point may or may not address synapse loss and dysfunction. Such studies may address synaptic deficits if, by chance, they addresses a critical step in the very earliest stages of the cascade to death of the neuron. A focus on neuron death will, unfortunately, fail to address the AD clinical phenotype if it addresses molecules participating in the late stages of the obvious end point of death of a neuron. Likewise, if type 1 and
    type 2 synaptic deficits (above) are related to cascades distinct from those leading to neuron death.
    Studies of neuron death are appropriate in their own right and have yielded many valuable insights. At the same time it is vital to place neuron death in proper perspective when dealing with the clinical phenotype of AD and other neurodegenerative diseases. That perspective is as an end game that is the consequence of a long series of events that have had major consequences for the clinical phenotype of AD well in advance of neuron death (Coleman et al., 2004).

    References:

    Bertoni-Freddari C. Fattoretti P. Casoli T. Caselli U. Meier-Ruge W.
    Deterioration threshold of synaptic morphology in aging and senile
    dementia of Alzheimer's type. Analytical & Quantitative Cytology &
    Histology. 18(3):209-13, 1996. Abstract

    Braak E. Braak H. Alzheimer's disease: transiently developing
    dendritic changes in pyramidal cells of sector CA1 of the Ammon's horn.
    Acta Neuropathologica. 93(4):323-5, 1997. Abstract

    Buell SJ. Coleman PD. Dendritic growth in the aged human brain and
    failure of growth in senile dementia. Science. 206(4420):854-6, 1979. Abstract

    Callahan LM. Vaules WA. Coleman PD. Progressive reduction of
    synaptophysin message in single neurons in Alzheimer disease. Journal
    of Neuropathology & Experimental Neurology. 61(5):384-95, 2002. Abstract

    Coleman, P.D. Federoff HJ and Kurlan R Neurology, 2004, In Press.

    DeKosky ST. Scheff SW. Synapse loss in frontal cortex biopsies in
    Alzheimer's disease: correlation with cognitive severity. Annals of
    Neurology. 27(5):457-64, 1990. Abstract

    Eberwine J. Miyashiro K. Kacharmina JE. Job C. Local translation of
    classes of mRNAs that are targeted to neuronal dendrites. Proceedings
    of the National Academy of Sciences of the United States of America.
    98(13):7080-5, 2001. Abstract

    Job C. Eberwine J. Identification of sites for exponential translation
    in living dendrites. Proceedings of the National Academy of Sciences
    of the United States of America. 98(23):13037-42, 2001. Abstract

    Morsch R. Simon W. Coleman PD. Neurons may live for decades with
    neurofibrillary tangles. Journal of Neuropathology & Experimental
    Neurology. 58(2):188-97, 1999. Abstract

    Scheff SW. Price DA. Synaptic pathology in Alzheimer's disease: a
    review of ultrastructural studies.
    Neurobiology of Aging. 24(8):1029-46, 2003. Abstract

    Terry RD. Masliah E. Salmon DP. Butters N. DeTeresa R. Hill R. Hansen
    LA. Katzman R. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is
    the major correlate of cognitive impairment. Annals of Neurology. 30(4):572-80, 1991. Abstract

    Yao PJ. Zhu M. Pyun EI. Brooks AI. Therianos S. Meyers VE. Coleman PD.
    Defects in expression of genes related to synaptic vesicle trafficking in frontal cortex of
    Alzheimer's disease. Neurobiology of Disease. 12(2):97-109, 2003. Abstract

  8. Yong Shen's review of cell death in Alzheimer's disease (AD) poses a number of interesting questions about the role of cell death, particularly apoptotic cell death, in human neurodegenerative disease. Shen states "despite all else, it is still neuronal loss in the brain that leads to the major clinical symptoms of these disorders." This assessment is challenged in Paul Coleman's comments, who argues, and I concur, that the critical event in AD pathogenesis is synaptic dysfunction, not neuron loss. Several investigators had originally reported that caspase-dependent neuron apoptosis was prominent in the AD brain and that unregulated apoptosis caused many neurodegenerative diseases (reviewed in Roth, Caspases, apoptosis and Alzheimer's disease: causation, correlation and confusion. JNEN 2001; 60:829-838). This hypothesis has been challenged on several fronts and has largely been revised to the proposal that apoptosis-associated molecules, particularly caspases, cause AD through mechanisms that do not acutely lead to apoptosis. This is the view presented by Troy Rohn, who proposes that caspases, through chronic low-level activation, cleave important cellular proteins that drive AD pathogenesis. If this hypothesis is correct, caspase inhibition should be a primary therapeutic objective in the pharmacological treatment of AD patients.

    There are at least three major issues that require additional investigation before caspase inhibitors can be considered for the treatment of AD: i) a role for caspases in selective axonal or synaptic degeneration, independent of apoptotic death, has not been proven, ii) the specific caspase or caspases involved in AD are unknown, and iii) most importantly, concern that caspase inhibition will trigger other, non-apoptotic forms of neuron death.

    It is clear from a variety of studies that caspases play physiological roles in processes other than cell death. Cytokine processing and cell cycle regulation (Woo et al., 2003) are two additional functions of some caspases, and the work of Rohn and others suggest that caspase-3 may be involved in tau and/or Aβ cleavage in the AD brain (Rissman et al., 2004). However, caspases have not yet been convincingly shown to be directly involved in AD-associated synaptic pathology. This will certainly be a prime objective of many laboratories in the future.

    It will be critical to establish a link between specific caspases and AD pathogenesis. The difficulties involved with doing this, however, are substantial, as a brief review of caspase-12 and AD will illustrate. In rat and mouse cells, caspase-12 was found to be localized to endoplasmic reticulum (ER) and to regulate cell death in response to ER stress (Nakagawa et al., 2000). Using both in vivo and in vitro models, several investigators implicated caspase-12 in Aβ responsiveness and increased sensitivity to DNA damage in cells containing presenilin-1 mutations (Nakagawa et al., 2000, Chan et al., 2002). This led to the speculation that caspase-12 may be important in the pathogenesis of AD and that the human orthologue of caspase-12 may be a therapeutic target for the treatment of AD. Unfortunately, this speculation proved incorrect as the human caspase-12 was recently found to lack caspase catalytic activity, be uninvolved in ER stress responsiveness in human cells, and to be unassociated with AD (Saleh et al., 2004). Similarly, species and strain-specific effects of other caspases, including caspase-three are well-known (Leonard et al., 2002). Thus, extreme caution is required before extrapolating studies of rodent AD models to humans (Roth, 2001).

    Finally, there is significant potential for caspase inhibitors to do more harm than good to AD patients. Under many experimental conditions, inhibition of caspases does not prevent death of cells exposed to apoptotic stimuli. Rather, caspase inhibition prevents the morphological changes that characterize apoptosis but not death per se (Roth et al., 2000). Non-apoptotic, autophagic cell death is observed in several neurodegenerative diseases and this type of cell death is unaffected by caspase inhibition (Zaidi et al., 2001). The complexities of cell death regulation are illustrated by studies of caspase-8. Caspase-8 is important for triggering apoptotic death through the extrinsic apoptotic death pathway and it has been implicated in AD (Roth, 2001). Thus, caspase-8 inhibition would at face value be therapeutically useful in the treatment of AD. Yet, a recent study indicates that caspase-8 inhibition leads directly to autophagic cell death (Yu et al., 2004). This observation led the authors to caution that "clinical therapies involving caspase inhibitors may arrest apoptosis but also have the unanticipated effect of promoting autophagic cell death" (page 1500, Yu et al., 2004). I feel that this warning is particularly relevant to AD, since anti-caspase therapy would necessarily be chronic and would not alleviate the primary stimulus or stimuli driving caspase activation. Under such circumstances, caspase inhibition therapy could convert dysfunctional neurons into dead neurons, eliminating all hope for clinical improvement. In total, a great deal has been learned over the last several years about the molecular regulation of caspase-dependent and -independent neuronal cell death and about Alzheimer's disease pathogenesis. However, my assessment of the field is similar to what it was three years ago when I wrote "an etiologic role for caspases in AD is far from proven" (page 829 Roth, 2001). Until such proof exists, treatment of AD patients with caspase inhibitors should proceed cautiously.

    References:

    Chan SL, Culmsee C, Haughey N, Klapper W, Mattson MP. Presenilin-1 mutations sensitize neurons to DNA damage-induced death by a mechanism involving perturbed calcium homeostasis and activation of calpains and caspase-12. Neurobiol.Dis. 11, 2-19. 2002. Abstract

    Leonard JR, Klocke BJ, D'Sa C, Flavell RA, Roth KA. Strain-dependent neurodevelopmental abnormalities in caspase-3-deficient mice. J.Neuropathol.Exp.Neurol 61[8], 673-677. 2002. Abstract

    Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner B A, Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-b. Nature 2000; (403): 98-103. Abstract

    MP, LaFerla FM, Rohn TT, Cotman CW. Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology. J.Clin.Invest. 114[1], 121-130. 2004. Abstract

    Roth K A. Caspases, apoptosis and Alzheimer's disease: causation, correlation and confusion. J Neuropathol Exp Neurol 2001; (60): 829-838. Abstract

    Roth K A, Kuan C, Haydar T F, D'Sa-Eipper C, Shindler K S, Zheng T S, Kuida K, Flavell R A, Rakic P. Epistatic and independent functions of caspase-3 and Bcl-XL in developmental programmed cell death. Proc Natl Acad Sci USA 2000; (97): 466-471. Abstract

    Saleh M, Vaillancourt JP, Graham RK, Huyck M, Srinivasula SM, Alnemri ES, Steinberg MH, Nolan V, Baldwin CT, Hotchkiss RS, Buchman TG, Zehnbauer BA, Hayden MR, Farrer LA, Roy S, Nicholson DW. Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 429, 75-79. 2004. Abstract

    Woo M, Hakem R, Furlonger C, Hakem A, Duncan GS, Sasaki T, Bouchard D, Lu L, Wu GE, Paige CJ, Mak TW. Caspase-3 regulates cell cycle in B cells: a consequence of substrate specificity. Nature Immunol 4[10], 1016-1022. 2003. Abstract

    Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S, Baehrecke EH, Lenardo MJ. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500-1502. 2004. Abstract

    Zaidi AU, McDonough JS, Klocke BJ, Latham CB, Korsmeyer SJ, Flavell RA, Schmidt RE, Roth KA. Chloroquine-induced neuronal cell death is p53 and Bcl-2 family-dependent but caspase-independent. J.Neuropathol.Exp.Neurol. 60[10], 937-945. 2001. Abstract

  9. I read Yong Shen's review on molecular events of neuron death in AD, and the comments, with great interest. As Paul mentioned, neuron death is a consequence of what happened in neurons in AD. I agree, but even after the horse is gone footprints and smells remain. One footprint on the AD brain was left by NFTs. The link between NFT formation and neuronal loss was initially posited because regions where NFTs were observed also exhibited neuronal loss. This link was strengthened when genetic studies of frontotemporal dementia parkinsonism-17 (FTDP-17) found that a mutation in the tau gene induced NFT formation and neuronal loss, implicating tau dysfunction in NFT formation and neuronal loss. Whatever forms of neuronal death mechanism, tau is directly involved. Tau-deficient neuronal culture did not show Aβ toxicity (Rapoport et al., 2002). This is a very important report showing that tau mediated Aβ neurotoxicity.

    How does tau induce toxicity in neurons? Tau is highly hydrophilic, and normally binds to and stabilizes microtubules. In the disease state, tau is highly phosphorylated and turns into insoluble protein aggregates, resulting in some neurons dying. The activation of the tau kinases (GSK-3, JNK, and CDK5) generates hyperphosphorylated tau, which possibly triggers the cell death signal. This activation is triggered even when Aβ induces neuronal death, because cell death did not occur in Aβ-treated, tau-deficient neurons. Eliminating all other possibilities, the accumulation of tau in cytoplasm, formation of insoluble tau aggregate and hyperphosphorylated tau may either individually or in combination be required for Aβ to induce neuronal death. For example, the accumulation of tau in cytoplasm was reported to affect anterograde vesicle trafficking (Ebneth et al., 1998; Stamer et al., 2002; Trinczek et al., 1999). This may lead to a loss of synaptic function, and consequently induce neuronal death at a later stage.

    Our recent study may be able to add yet another possibility for tau toxicity. We prepared N2a cells stably over-expressing P301L tau, and N2a cells over-expressing both P301L tau and CHIP, a ubiquitin ligase that induces ubiquitination of tau. When cells are treated with MG132, a protease inhibitor, they are marked for death. More tau over-expressing cells died than mock or CHIP-overexpressing cells. Thus, tau overexpression increases cell vulnerability to stress while CHIP overexpression protected cells from this vulnerability. Then we examined the SDS-insoluble tau in each of the cells. Tau overexpressing cells showed larger amounts of SDS-insoluble tau, and MG132 treatment enhanced the formation of SDS-insoluble tau, while CHIP over-expression dramatically reduced the formation of SDS-insoluble tau. SDS-insoluble tau increases cell vulnerability under stress, and tau accumulation in cytoplasm induces detergent-insoluble tau formation and vulnerability to insult.

    References:
    Ebneth, A., Godemann, R., Stamer, K., Illenberger, S., Trinczek, B., and Mandelkow, E. (1998). Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J Cell Biol 143, 777-794. Abstract

    Rapoport, M., Dawson, H. N., Binder, L. I., Vitek, M. P., and Ferreira, A. (2002). Tau is essential to beta -amyloid-induced neurotoxicity. Proc Natl Acad Sci U S A 99, 6364-6369. Abstract

    Hatakeyama, S., Matsumoto, M., Kamura, T., Murayama, M., Chui, D-H, Planel, E, Takahashi, R., Nakayama, K.I., and Takashima, A. (2004). U-box protein CHIP mediates poly-ubiquitilation on preferentially 4-repeat Tau and is involved in neurodegeneration of tauopathy. J. Neurochem (in press).

    Stamer, K., Vogel, R., Thies, E., Mandelkow, E., and Mandelkow, E. M. (2002). Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol 156, 1051-1063. Abstract

    Trinczek, B., Ebneth, A., Mandelkow, E. M., and Mandelkow, E. (1999). Tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles. J Cell Sci 112 ( Pt 14), 2355-2367. Abstract

  10. Reply to Paul Coleman by Yong Shen
    Paul Coleman raised a good point in arguing against neuronal death as the major contributor to clinical symptoms. My understanding is that the "clinical symptoms" mainly means "cognition, or learning and memory". Yes, I also think that synaptic transmission is one event that affects learning and memory. Paul indicated in the comments that "synapses are the means by which the nervous system transmits, processes, and stores information". However, as Paul also said, without their parent neurons, synapses do not even exist. Moreover, although we discussed neuron death, more importantly, we would like to find out why the neurons in AD brains die and what factors, including synapse deficit, cause that. From this point, I agree with Paul that as an indicator neuron death is the latecomer. But all of the cellular cascades, death-signal transduction pathways, and destructive factors that cause synapse deficits also contribute to neuron death. From this view, neuron death is an important and significant event to study.

    Then we discuss who is the "murderer" of cognitive decline, synapse or neuron, or both? While we definitely appreciate the studies that show synapse loss and dysfunction are the critical events in the deficits in AD, there is also much solid evidence to prove that neuronal death or loss contribute to the cognitive deficits of AD (there are 557 references found by searching "neuron death and Alzheimer brains"). Recently, one study reported that early Alzheimer patients still can learn and memorize (reference). The interesting to me is that although synapse deficits occur early in AD brains, these patients still can learn and memorize. This can be interpreted that synapses have strong flexibility (or plasticity) even after injury. Alternatively, it also could be explained by the brain still functioning even after synapses are reduced. However, when neurons are lost (actually neurons are gradually lost, perhaps parallel to synapse deficits, not all lost at one time in the end stages of Alzheimer's disease), brain cognition starts to decline. It is not the other way around-that clinical symptoms start then neurons start to be lost. Thus, it is valuable and important to study mechanisms of neuron death and identify therapeutic target molecules that cause cell death. We may then have an opportunity to slow down the neurodegenerative process of Alzheimer's disease.

References

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  1. Dying to Bind: DENN/MADD Promotes Neuron Death in AD Brain
  2. Polyglutamine Disease Therapy—Bypass the Glutamine?

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Paper Citations

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  1. . A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Nature. 1998 Aug 13;394(6694):694-7. PubMed.