mitochondrial function. Animal models based on genetically modified mice with null mutation, an extra gene copy, or point mutations of these genes have helped us to obtain insights into the mechanism of several symptoms of PD. However, none of the genetic models based on PD-linked genes recapitulate the key symptom of the disease, such as the loss of dopamine neurons, but more subtle effects on the DA system have been detected. α- synuclein transgenic mice and DJ-1 knockout mice were reported to be more susceptible to MPTP toxin, suggesting that the progression of PD might be due to a combination of genetic factors and environmental exposures.

It is noteworthy that targeting apoptosis upstream of its execution phase results in a marked attenuation of neurodegeneration in PD animal models, whereas interfering at a more downstream level, such as caspase activation, produces variable results. Inhibition of caspase activation by gene transfer of X-linked inhibitor of apoptosis or peptide inhibitors prevents the loss of DA neuron cell body but not nerve fibers. Because the symptoms of PD are caused by the loss of DA terminals in the striatum, preventing the death of DA cell bodies without preventing the degeneration of their axons is unlikely to be a good therapeutic strategy. A combinatorial strategy might be required to prevent both the loss of cell body and axon degeneration to obtain clinical benefits.

2.1.3. Huntington’s disease

HD is an autosomal-dominant neurodegenerative disease characterized by involuntary movements and dementia that result from selective neuronal loss in the striatum and cerebral cortex. HD is caused by expansions of CAG in the huntingtin gene, producing a protein containing elongated poly-glutamine (poly-Q) repeats. The length of poly-Q repeats shows a rough inverse correlation with the ages of disease onset, suggesting the length of poly-Q is an important factor in the pathogenesis of this disease. Proteins with poly-Q repeats can aggregate in vitro and in vivo. Transgenic mice that overexpress an N-terminal fragment of human huntingtin with expanded poly-Q region show reduced survival, intraneuronal aggregates, and behavior deficits similar to HD. The oligomerization of expanded poly-Q repeats play a pivotal role in the neurodegeneration of HD.

Caspase activation has been proposed to participate in the mutant huntingtin-mediated cell death. TUNEL-positive cells and caspase-1 and caspase-8 activation have been detected in the HD patient brains. In mouse models of HD, expression of a dominantnegative form of caspas-1 or the intracerebroventricular

administration of a pan-caspase inhibitor zVAD-fmk delays the progression and mortality of the disease.

How mutant huntingtin triggers apoptosis remains unclear. Expression of expanded poly-Q repeats in vitro can directly activate caspase-3, -8, and -9. Caspase-3 and caspase-6 can also cleave wild-type and mutant huntingtin proteins, generating truncated fragments. Truncated fragments that contain expanded poly-Q repeats show increased toxicity and propensity to aggregate compared with full-length protein. Caspase- 8 has been found to be recruited into the intracellular aggregates and is activated in neuronal cells that express expanded poly-Q repeats. Caspase-8 activation in HD is mediated by the formation of heterodimers between Hip1 (huntingtin-interacting protein 1) and Hippi (Hip1 protein interactor), which is favored by the disease-associated poly-Q expansion. Despite the insights offered by these studies, we need to be cautious when considering caspases as therapeutic targets for HD because the physiologic relevance of caspase-mediated cleavage of huntingtin in vivo remains to be established.

A central issue is the relative contribution of neuronal apoptosis to neurological deficits in HD and other agerelated neurodegenerative disorders. Early-stage HD patients develop characteristic motor deficits without evidence of striatal atrophy; striatal atrophy becomes prominent only in later stages of the disease. Furthermore, in a conditional huntingtin transgenic mouse, neurological deficits could be reversed by turning off expression of the mutant transgene. Thus neural dysfunction, rather than irreversible cell death, might be responsible for early neurological deficits. This conclusion suggests that the primary target for the therapy of HD should be the mechanism of neural dysfunction, rather than that of cell death.

2.1.4. Amyotrophic lateral sclerosis

ALS is a fatal disorder characterized by the loss of motor neurons in the cerebral cortex and spinal cord, leading to progressive and ultimately fatal paralysis. A major advance in the understanding of the disease mechanism came from the identification of mutations in the gene encoding superoxide dismutase (SOD1) that is responsible for a significant portion of familial ALS cases. Mice deficient in SOD1 do not develop any motor neuron disease. Transgenic expression of different human ALS-linked SOD1 mutations in mice and rats replicates the clinical and pathological characteristics of ALS, without a correlation on the free radical scavenging activity of SOD1. These indicate that the cytotoxicity of mutant SOD1 is a gain of function. It has been

shown that mutant SOD1 can form intraneuronal aggregates and induce oxidative stress. The mechanism by which mutant SOD1 induces ALS appears to be complex because it may involve cell-autonomous effects in motor neurons that may determine the onset of the disease, as well as effects in nonmotor neurons, such as microglia, astrocytes, and oligodendrocytes, which affect the disease progression.

A role for apoptosis in ALS is suggested by the proapoptotic activity of mutant SOD1 in cultured neuronal cell lines and the neuroprotective effect of overexpressing Bcl-2 in mutant SOD1 transgenic mice. In the lumbar cord of patients with ALS and mutant SOD1 transgenic mice, the mRNA content and protein level of Bcl-2 are decreased, whereas those of Bax mRNA are increased as compared with that of control. Activated caspase-1 and caspase-3 can be detected in the spinal cords of ALS patients and mutant SOD1 transgenic mice. Inhibition of caspase-1 activity delays disease progression and prolongs the life span in SOD1 transgenic mice. Evidence for a prominent recruitment of the mitochondrial pathway has been found in the spinal cord of patients and transgenic SOD1 mice, and the treatment with minocycline, which has an inhibitory effect on mitochondrial dysfunction, delays disease onset and extends survival of the transgenic SOD1 mice. Bcl-2 and mutant SOD1 protein physically interact in spinal cord mitochondria. Motor neurons isolated from mutant SOD1 transgenic mice show increased susceptibility to the activation of the Fas-triggered cell death pathway, suggesting the extrinsic pathway might also make a contribution to the neurodegenerative process.

Although the studies just discussed support the involvement of apoptosis in the death of motor neurons involved in ALS, they are by no means conclusive. As with other chronic neurodegenerative diseases, neural dysfunction, which occurs considerably earlier than that of neuronal cell death, may be responsible for the onset of ALS, whereas neuronal cell death is only manifested in late stage of the disease. Although Bax deletion prolongs survival and completely blocks mutant SOD1-mediated motor neuron cell death, it only delays the timing of neuromuscular denervation, which began long before the activation of cell death proteins in SOD mutants. Expression of wild-type or mutant human SOD1 in the motor neurons of Drosophila induced progressive climbing deficits associated with defective neural electrophysiology, focal accumulation of SOD1, and a stress response in surrounding glia without a loss of neurons. Thus maintaining normal neural connectivity and function should be an important goal for the treatment of chronic neurodegeneration such as ALS.

2.2. Necrotic cell death in neurodegenerative diseases

Necrosis typically occurs after acute neurological injuries, such as ischemia, hypoxia, stroke, or trauma, as well as in chronic neurodegenerative diseases, including AD, PD, HD, and ALS. Although the molecular mechanisms of necrotic cell death are mostly not understood, elevated intracellular calcium, cytosolic calpains, and spilled lysosomal cathepsins are the major players in necrotic neuronal death.

2.2.1. Calpains

Calpains are a family of calcium-dependent cysteine proteases that cleave a variety of cellular substrates. The cross-talk between caspases and calpains has been reported in a numbers of in vivo and cell culture models of apoptosis. Calpain-mediated cleavage of caspases results in both caspase inhibition and activation. Conversely, caspases regulate calpain activity by mediating degradation of calpastatin, the endogenous inhibitor of calpain.

The importance of calpain activation in acute cell injury and necrotic cell death triggered by calcium influx has been established. One of the mechanisms by which calpain activation contributes to cell death is the cleavage of several cytoskeletal proteins of neuronal axons, such as neurofilaments, cain/cabin 1, and fodrin. Degradation of neurofilaments induced by oxygen/glucose deprivation could be attenuated by calcium removal, blockade of voltage-gated sodium channels, or inhibition of calpains. Another mechanism by which calpain contributes to cell demise is the cleavage of membrane channels (e.g., the subunit NR2B of the N-methyl-D- aspartate receptor and the plasma membrane Na/Ca exchanger [NCX]), during excitotoxicity. NCX operates in cellular calcium extrusion, and its proteolytic inactivation by calpain is responsible for the secondary phase of excitotoxic calcium upregulation and the death of the neurons.

The role of calpains in neuronal cell death has also been examined in chronic neurodegenerative diseases. Inhibition of calpains prevents neuronal and behavioral deficits in a mouse model of PD, and calpain activation was evident in postmortem midbrain tissues from PD patients. In the case of AD, calpain activation occurs before abnormalities in the microtubuleassociated protein tau. Activated calpain associates with neurofibrillary tangles, which are abnormal aggregates of hyperphosphorylated tau and a major pathological feature of AD. Calpain activation has been detected in human HD caudate but not in age-matched controls.