Upregulation of calpain correlates with increased neurodegeneration in acute experimental auto-immune encephalomyelitis

Although calpain up-regulation is well established in experimental auto-immune encephalomyelitis (EAE), a link between increased calpain expression and activity and neurodegeneration has not been examined. Therefore, spinal cord tissue from Lewis rats with EAE was examined to test the hypothesis that increased calpain expression in neurons would correlate with increased cell death and axonal damage in a time-dependent manner following EAE induction. We found that increased calpain expression in EAE corresponded to increased TUNEL-positive neurons and to increased expression of dephosphorylated neurofilament protein, markers of cell death and axonal degeneration, respectively. An increase in internucleosomal DNA fragmentation in EAE spinal cord suggested that cell death was, at least partially, due to apoptosis. Axonal damage was further demonstrated in EAE spinal cord compared with control via morphological analysis, revealing granular degeneration of filament and microtubule integrity, loss of myelin, and mitochondrial damage. Calcium (Ca2+) influx, which is required for calpain activation, was also increased in EAE spinal cord. From these findings, we conclude that increases in Ca2+-induced calpain activity may play a crucial role in neurodegeneration in acute EAE.     

Activation of calpain and caspase pathways in demyelination and neurodegeneration in animal model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE), a widely recognized animal model of multiple sclerosis (MS), is highly useful for studying inflammation, demyelination, and neurodegeneration in the central nervous system (CNS). EAE exhibits many similarities with MS, which is a chronic inflammatory disease affecting CNS white matter in humans. Various studies have indicated that EAE is a particularly useful animal model for understanding both the mechanisms of immune-mediated CNS pathology and also the progressive clinical course of MS. Demyelination and axonal dysfunction have previously been shown in MS and EAE but current evidences indicate that axonal damage and neuron death also occur, demonstrating that these diseases harbor a neurodegenerative component. Recent studies also have shown that the activation of calpain and caspase pathways contribute to the apoptotic death of oligodendrocytes and neurons, promoting the pathological events leading to neurological deficits. Apoptosis is involved in the disease-regulating as well as in the disease-promoting processes in EAE. This review discusses the major involvement of calpain and caspase pathways in causing demyelination and neurodegeneration in EAE animals.     

Amyloid beta-induced neuronal death is bax-dependent but caspase-independent

Fibrillar amyloid beta (Abeta) peptides are major constituents of senile plaques in Alzheimer disease (AD) brain and cause neuronal apoptosis in vitro. Bax and caspase-3 have been implicated in the pathogenesis of AD and are components of a well-defined molecular pathway of neuronal apoptosis. To determine whether Abeta-induced neuronal apoptosis involves bax and/or caspase-3 activation, we examined the effect of Abeta on wild-type, bax-deficient, and caspase-3-deficient telencephalic neurons in vitro. In wild-type cultures, Abeta produced time- and concentration-dependent caspase-3 activation, apoptotic nuclear changes, and neuronal death. These neurotoxic effects of Abeta were not observed in bax-deficient cultures. Caspase-3 deficiency, or pharmacological inhibition of caspase activity, prevented caspase-3 activation and blocked the appearance of apoptotic nuclear features but not Abeta-induced neuronal death. Neither calpain inhibition nor microtubule stabilization with Taxol protected telencephalic neurons from Abeta-induced caspase activation or apoptosis. These results have potential implications regarding the underlying pathophysiology of AD and towards AD treatment strategies.     

Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.     

Caspase regulation of neuronal progenitor cell apoptosis

Programmed cell death (apoptosis) of both proliferating neuroblasts and postmitotic neurons is essential for normal nervous system development. To study the molecular regulation of apoptosis in neuronal progenitor cells, we developed a flow cytometric assay capable of distinguishing between viable, apoptotic, and necrotic cell populations. Incubation of freshly dissociated telencephalic cells from gestational day 12-13 mouse embryos with either cytosine arabinoside (AraC) or staurosporine caused a marked increase in the percentage of apoptotic cells. Both drugs induced caspase-3 activation, as determined by in vitro cleavage of a caspase-3 substrate and immunocytochemical detection of activated caspase-3. Treatment of telencephalic cells with the broad caspase inhibitor BAF, blocked caspase-3 activation and protected cells against both AraC and staurosporine-induced apoptotic death. These results indicate that neuronal progenitors possess a caspase-dependent apoptotic pathway, the activation of which may regulate neuronal progenitor cell numbers in vivo.     

Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures.

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen-glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of alpha-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3-mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen-glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.     

Calpain inhibitors delay injury-induced apoptosis in adult mouse spinal cord motor neurons

Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation.     

Amyloid beta peptide toxicity in differentiated PC12 cells: calpain-calpastatin, caspase, and membrane damage

Amyloid beta peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease (AD). The peptide is toxic to neurons, possibly by causing initial synaptic dysfunction and neuronal membrane dystrophy, promoted by increased cellular Ca(2+). Calpain (Ca(2+)-dependent protease) and caspase have also been implicated in AD. There is little information on communication between the two proteases or on the involvement of calpastatin (the specific calpain inhibitor) in Abeta toxicity. We studied the effects of Abeta25-35 (sAbeta) on calpain, calpastatin, and caspase in neuronal-like differentiated PC12 cells. sAbeta-treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide, without apparent nuclear changes of apoptosis, and little poly (ADP-ribose) polymerase [PARP] degradation). The sAbeta-induced membrane damage is in contrast with staurosporine-induced damage (nuclear apoptotic changes, PARP degradation, without membrane propidium iodide permeability). sAbeta led to activation of caspase-8 and calpain, promotion of calpastatin degradation (by caspase-8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). The results support the idea that Abeta causes primarily neuronal membrane dysfunction, and point to cross-talk between calpain and caspase (protease activation and degradation of calpastatin) in Abeta toxicity. Increased expression of calpastatin and/or decrease in calpain and caspase-8 may serve as means for ameliorating early symptoms of AD.     

Calpain facilitates the neuron death induced by 3-nitropropionic acid and contributes to the necrotic morphology

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.     

BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia

Hypoxic-ischemic (H-I) injury to the brain in the perinatal period often leads to significant long-term neurological deficits. In a model of neonatal H-I injury in postnatal day 7 rats, our previous data have shown that cell death with features of apoptosis is prominent between 6 and 24 h after H-I and that neurotrophins, particularly BDNF, can markedly protect against tissue loss. During brain development, caspase-3 is required for normal levels of programmed cell death. Utilizing an antibody specific for the activated form of caspase-3, CM1, we now show that caspase-3 is specifically activated in neuronal cell bodies and their processes beginning at 6 h and peaking 24 h following unilateral carotid ligation and exposure to hypoxia in postnatal day 7 rats. Caspase-3 activation began to occur in cortex at 6 h and in striatum and hippocampus at 12-18 h. Caspase-3 activation was also observed in developing oligodendrocytes. Intracerebroventricular injection of BDNF prior to H-I injury almost completely abolished evidence of H-I-induced caspase-3 activation in vivo. Utilizing a specific molecular marker of an apoptotic pathway, these findings demonstrate that H-I injury to the developing brain is a strong apoptotic stimulus leading to caspase-3 activation, that BDNF can block this process in vivo, and that the ability of BDNF to inhibit caspase activation and subsequent apoptosis likely accounts in large part for its protection against neuronal injury in this model.     

The calpain inhibitor VI prevents apoptosis of adult motor neurons

Motor neuron cell death was studied in organotypic cultures of adult spinal cord slices from the mouse. Six hours after excision, many motor neuron nuclei displayed apoptotic features including nuclear and chromatin condensation. At this time point, many motor neurons also exhibited immunoreactivity to calpain II. Both the calpain inhibitor VI and ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the appearance of apoptotic nuclei whereas the pan caspase inhibitor Z-VAD.fmk had no effect. The results suggest that calpain is required for apoptosis of motor neurons and that this type of apoptosis is independent of caspase activation.     

Contribution of caspase-mediated apoptosis to the cell death caused by oxygen-glucose deprivation in cortical cell cultures

Several evidences suggest that cell death after cerebral ischemia involves both necrosis and apoptosis. However, it is still unknown which is the relative contribution of both types of cell death. Exposing rat cortical cultures to oxygen-glucose deprivation (OGD), we show the simultaneous presence of necrotic and apoptotic cells. The relative contribution of necrosis and apoptosis was dependent on the duration of the OGD. OGD-mediated apoptotic cell death is caspase-dependent because the addition of a pan-caspase inhibitor specifically blocked the apoptotic component of the OGD-mediated cell death. Moreover, we observed the activation of caspase-3, -7, and -9 after OGD in neurons and microglial cells. No activation of these caspases was observed in GFAP positive cells. Our results also show that calpain is related to OGD-mediated proteolysis of caspase-3 and -9 but not of caspase-7. These data suggest that different pathways could be involved in OGD-mediated caspase activation.     

Altered apoptotic responses in neurons lacking RhoB GTPase

Caspase 3 activation has been linked to the acute neurotoxic effects of central nervous system damage, as in traumatic brain injury or cerebral ischaemia, and also to the early events leading to long-term neurodegeneration, as in Alzheimer's disease. However, the precise mechanisms activating caspase 3 in neuronal injury are unclear. RhoB is a member of the Rho GTPase family that is dramatically induced by cerebral ischaemia or neurotrauma, both in preclinical models and clinically. In the current study, we tested the hypothesis that RhoB might directly modulate caspase 3 activity and apoptotic or necrotic responses in neurons. Over-expression of RhoB in the NG108-15 neuronal cell line or in cultured corticohippocampal neurons elevated caspase 3 activity without inducing overt toxicity. Cultured corticohippocampal neurons from RhoB knockout mice did not show any differences in sensitivity to a necrotic stimulus - acute calcium ionophore exposure - compared with neurons from wild-type mice. However, corticohippocampal neurons lacking RhoB exhibited a reduction in the degree of DNA fragmentation and caspase 3 activation induced by the apoptotic agent staurosporine, in parallel with increased neuronal survival. Staurosporine induction of caspase 9 activity was also suppressed. RhoB knockout mice showed reduced basal levels of caspase 3 activity in the adult brain. These data directly implicate neuronal RhoB in caspase 3 activation and the initial stages of programmed cell death, and suggest that RhoB may represent an attractive target for therapeutic intervention in conditions involving elevated caspase 3 activity in the central nervous system.     

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram-centimeter force (g-cm) injury was induced in rats by a weight-drop method and allowed to progress for 4 hr. One-centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2-AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca(2+)) levels following injury, compared with corresponding tissue sections from sham-operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double-immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl-2 ratio, indicating the induction of the mitochondria-mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double-immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca(2+) levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.     

Calpain activation and apoptosis in motor neurons of cultured adult mouse spinal cord

Calpain, a Ca2+-dependent cysteine protease, has been implicated in neuronal apoptosis following spinal cord injury. In this study, activation of calpain was investigated in motor neurons of adult spinal cord slices from the mouse, using a cell-permeable fluorogenic calpain substrate and Western blotting. Calpain was rapidly activated in the motor neurons of excised spinal cord slices and calpain activity was observed both in the cytoplasm and the nuclei. In these neurons, nuclear and chromatin condensation were pronounced. Both calpain inhibitor VI and EGTA (ethyleneglycol-bis(beta-aminoethyl ether) N' ,N' ,N' ,N' -tetraacetic acid) inhibited calpain activation and subsequent appearance of apoptotic nuclei. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved alpha-fodrin fragment. These results show that calpain is rapidly activated in injured motor neurons and imply that this activation could be responsible for execution of caspase-independent apoptosis in injured adult motor neurons.     

Caspase inhibitors protect against neuronal apoptosis induced by cerebrospinal fluid from multiple sclerosis patients

Neuronal apoptosis has recently been implicated in multiple sclerosis (MS). Apoptotic cell death of neurons is induced in cultures exposed to cerebrospinal fluid (CSF) from MS patients. Since caspases are essential in the regulation of apoptosis, direct evidence was sought linking caspases to CSF-induced neuronal death. Caspase activity was measured in cell extracts from MS CSF-treated cultured neurons by the cleavage of caspase-1 and caspase-3 substrates. Caspase-3 activity, but not caspase-1, was induced in neuronal cultures in response to MS CSF treatment. This caspase-3 activity was inhibited in vitro by Ac-YVAD-cmk and Ac-DEVD-cmk caspase inhibitors. Treatment of MS CSF-incubated neuronal cells with these caspase inhibitors completely preserved neuronal survival and largely attenuated DNA fragmentation detected in situ. These findings show that neuronal cells are rescued from MS CSF-induced death by caspase inhibitors and suggest ways to treat MS.     

Caspases that are activated during generation of nuclear polyglutamine aggregates are necessary for DNA fragmentation but not sufficient for cell death

Truncated polypeptides containing expanded polyglutamine (polyQ) stretches tend to form cytoplasmic or nuclear aggregates in cultured cells, leading to cell death. Although it has been shown recently that caspase-8 coaggregates with polyQ and is activated during polyQ-mediated cell death, little is known of the location and timing of caspase-8 activation by nuclear polyQ aggregates. Also, the relationship between nuclear polyQ aggregate-mediated cell death and activation of other caspases is unclear. In P19 embryonal carcinoma (EC) cells, which can be made to differentiate into neuronal cells, polyQ72 repeats preferentially aggregate in the nucleus. Nuclear aggregates of polyQ72 induced P19 EC cell death, with a high frequency of cells exhibiting morphology characteristic of apoptosis (i.e., roundness, cell shrinkage, chromatin condensation) and DNA fragmentation. In the present study, we used antisera that specifically recognized the active forms of caspase-8, -3, and -9 but not their proforms, and showed that only caspase-8 and -3 were activated during the generation of polyQ72 aggregates in P19 EC cell nuclei. Furthermore, we showed that the caspase inhibitor z-VAD-fmk inhibited DNA fragmentation, but only partially inhibited the appearance of apoptotic morphology. Thus, caspase activation, including caspase-8 and -3, is necessary for polyQ-mediated DNA fragmentation but not sufficient for polyQ-mediated cell death in P19 EC cells.     

Role of protein conformational dynamics and DNA integrity in relevance to neuronal cell death in neurodegeneration

Apoptosis has been implicated in the pathogenesis of various neurodegenerative disorders, although the extent to which it is responsible for the neurodegeneration along with other kind of cell death events is not known. Eventhough much information is available today on the apoptotic cascades in general, the precise mechanism and the exact sequence of events leading to neuronal degeneration in Alzheimer's disease (AD) and other neurodegenerative disorders is not understood till now. Amyloid beta (Abeta) proteins are the hallmark toxic proteins known to cause the activation of apoptotic cascades via caspase dependent and caspase-independent pathways. Abeta can cause neuronal apoptosis through multiple mechanisms involving mitochondria and endoplasmic reticulum as the key organelles. In this review, we have discussed the role of apoptosis in neurodegeneration and provided new thoughts on the role of protein conformational dynamics and DNA integrity associated with neurodegenerative disorders. An insight on whether the apoptosis observed in the neurodegenerative disorders is of any functional advantage has been discussed.