Corticocortical disconnection in Alzheimer’s disease occurs by the progressive impairment and eventual loss of a small subset of pyramidal neurons in layers III and V of association areas of the neocortex. These neurons exhibit large somatic size, extensive dendritic arborization and high levels of nonphosphorylated neurofilaments of medium and high molecular weight that can be identified using a monoclonal SMI-32 antibody. It is thought that the accumulation of amyloid Aβ and neurofibrillary tangles may provoke metabolic disturbances that result in the loss of these SMI-32 immunoreactive neurons. The recent detection of increased levels of caspase-3 cleaved fodrin in frontal, temporal and parietal association areas in Alzheimer’s disease brains suggests that programmed cell death may contribute to the destruction of SMI-32 positive neurons. In the present study, we utilized an antibody that selectively recognizes the 120 kDa breakdown product of αIIspectrin (fodrin) generated by caspase-3 to determine whether this protease is activated in vulnerable pyramidal neurons located in layers III and V of Alzheimer’s disease brains. Neurons immunoreactive for caspase-3 cleaved αIIspectrin were located predominantly in layers III and V of the inferior frontal and superior temporal cortices of patients with Alzheimer’s disease but not age-matched controls. Pyramidal neurons immunoreactive for caspase-3 cleaved αIIspectrin invariably displayed SMI-32 immunoreactivity suggesting that caspase-3 activation is a pathological event that may be responsible for the loss of a subset of pyramidal neurons that comprise corticocortical projections.

The ability of fimbria-fornix bilateral axotomy to elicit calpain and caspase-3 activation in the rat septohippocampal pathway was determined using antibodies that selectively recognize either calpain- or caspase-cleaved products of the cytoskeletal protein αII-spectrin. Radioenzymatically determined choline acetyl transferase (ChAT) activity was elevated in the septum at day 5, but reduced in the dorsal hippocampus at days 3, 5 and 7, after axotomy. Prominent accumulation of calpain-, but not caspase-3-, cleaved spectrin proteolytic fragments was observed in both the septum and dorsal hippocampus 1–7 days after axotomy. ChAT-positive neuronal cell bodies in the septum also displayed calpain-cleaved spectrin indicating that calpain activation occurred in cholinergic septal neurons as a consequence of transection of the septohippocampal pathway. Calpain-cleaved αII-spectrin immunoreactivity was observed in cholinergic fibers coursing through the fimbria-fornix, but not in pyramidal neurons of the dorsal hippocampus, suggesting that degenerating cholinergic nerve terminals were the source of calpain activity in the dorsal hippocampus following axotomy. Accumulation of calpain-cleaved spectrin proteolytic fragments in the dorsal hippocampus and septum at day 5 after axotomy was reduced by i.c.v. administration of two calpain inhibitors. Calpain inhibition partially reduced the elevation of ChAT activity in the septum produced by transection but failed to decrease the loss of ChAT activity in the dorsal hippocampus following axotomy. These findings suggest that calpain activation contributes to the cholinergic cell body response and hippocampal axonal cytoskeletal degradation produced by transection of the septohippocampal pathway.

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model.

Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD.

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp “safety-catch” regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this “safety catch” results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.

Parkinson's disease (PD) is a progressive neurodegenerative disorder of the basal ganglia, associated with the inappropriate death of dopaminergic neurons of the substantia nigra pars compacta (SNc). Here, we show that adenovirally mediated expression of neuronal apoptosis inhibitor protein (NAIP) ameliorates the loss of nigrostriatal function following intrastriatal 6‐OHDA administration by attenuating the death of dopamine neurons and dopaminergic fibres in the striatum. In addition, we also addressed the role of the cysteine protease caspase‐3 activity in this adult 6‐OHDA model, because a role for caspases has been implicated in the loss of dopamine neurons in PD, and because NAIP is also a reputed inhibitor of caspase‐3. Although caspase‐3‐like proteolysis was induced in the SNc dopamine neurons of juvenile rats lesioned with 6‐OHDA and in adult rats following axotomy of the medial forebrain bundle, caspase‐3 is not induced in the dopamine neurons of adult 6‐OHDA‐lesioned animals. Taken together, these results suggest that therapeutic strategies based on NAIP may have potential value for the treatment of PD.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.

Sepsis induces lymphocyte apoptosis and prevention of lymphocyte death may improve the chances of surviving this disorder. We compared the efficacy of a selective caspase-3 inhibitor to a polycaspase inhibitor and to caspase-3 −/− mice. Both inhibitors prevented lymphocyte apoptosis and improved survival. Caspase-3 −/− mice shared a decreased, but not total, block of apoptosis. The polycaspase inhibitor caused a very substantial decrease in bacteremia. Caspase inhibitors did not benefit RAG-1 −/− mice, which had a >tenfold increase in bacteremia compared to controls. Adoptive transfer of T cells that overexpressed the anti-apoptotic protein Bcl-2 increased survival. T cells stimulated with anti-CD3 and anti-CD28 produced increased interleukin 2 and interferon γ by 6 h. Thus, caspase inhibitors enhance immunity by preventing lymphocyte apoptosis and lymphocytes act rapidly, within 24 h, to control infection.

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death‐inducing stimulus. We demonstrate that the activation of caspase‐3, in Jurkat cells stimulated to undergo apoptosis by a Fas‐independent pathway, is catalyzed by caspase‐6. Caspase‐6 was found to co‐purify with caspase‐3 as part of a multiprotein activation complex from extracts of camptothecin‐treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase‐3 could be demonstrated by co‐immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase‐3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP‐dependent ‘foldase’ activity of Hsp60 improves the vulnerability of pro‐caspase‐3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.

Caspase-3–mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

Expression of the 243-residue form of the adenovirus E1A protein in the absence of other viral proteins triggers apoptosis by a pathway that requires p53. This pathway includes processing and activation of initiator procaspase-8, redistribution of cytochrome c, and activation of procaspase-3. Bcl-2 functions at or upstream of procaspase-8 processing to inhibit all of these events and prevent cell death. This contrasts with the anti-apoptotic influence of Bcl-2 family proteins in the cell death pathway induced by Fas ligand or tumor necrosis factor (TNF), in which Bcl-2 typically acts downstream of Fas/TNFR1-mediated activation of caspase-8. Moreover, E1A induces procaspase-8 processing and cell death in cells deleted of FADD, an adaptor protein critical for Fas/TNFR1 activation of caspase-8. The results indicate that E1A is capable of activating caspase-8 by a Bcl-2-inhibitable pathway that does not involve autocrine stimulation of FADD-dependent death receptor pathways.

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.

Caspase-mediated proteolysis of downstream substrates is a critical element of the execution pathway common to all forms of apoptosis studied to date. While this caspase-dependent pathway is activated during cytotoxic lymphocyte granule-induced cell death, recent studies have also provided evidence for caspase-independent pathways. However, the mechanisms mediating these additional pathways have not been defined. The current study demonstrates that DNA-PKcs, and NuMA are directly and efficiently cleaved by granzyme B in vitro and in vivo, generating unique substrate fragments not observed during other forms of apoptosis. This direct, caspase-independent ability of granzyme B to cleave downstream death substrates constitutes an apoptotic effector mechanism that is insensitive to inhibitors of the signaling or execution components of the endogenous apoptotic cascade.