calpain and Wallerian-Degeneration

Topics: Animals; Axons; Calpain; Mice; Mitochondria; Nerve Regeneration; Trauma, Nervous System; Wallerian Degeneration

Traumatic brain injury (TBI) evokes widespread/diffuse axonal injury (TAI) significantly contributing to its morbidity and mortality. While classic theories suggest that traumatically injured axons are mechanically torn at the moment of injury, studies in the last two decades have not supported this premise in the majority of injured axons. Rather, current thought considers TAI a progressive process evoked by the tensile forces of injury, gradually evolving from focal axonal alteration to ultimate disconnection. Recent observations have demonstrated that traumatically induced focal axolemmal permeability leads to local influx of Ca2+ with the subsequent activation of the cysteine proteases, calpain and caspase, that then play a pivotal role in the ensuing pathogenesis of TAI via proteolytic digestion of brain spectrin, a major constituent of the subaxolemmal cytoskeletal network, the "membrane skeleton". In this pathological progression this local Ca2+ overloading with the activation of calpains also initiates mitochondrial injury that results in the release of cytochrome-c, with the activation of caspase. Both the activated calpain and caspases then participate in the degradation of the local axonal cytoskeleton causing local axonal failure and disconnection. In this review, we summarize contemporary thought on the pathogenesis of TAI, while discussing the potential diversity of pathological processes observed within various injured fiber types. The anterograde and retrograde consequences of TAI are also considered together with a discussion of various experimental therapeutic approaches capable of attenuating TAI.

Topics: Axons; Brain; Brain Injuries; Calcium; Calpain; Caspases; Cell Death; Cytoskeleton; Diffuse Axonal Injury; Humans; Wallerian Degeneration

Cisplatin is a commonly used chemotherapy agent with significant dose-limiting neurotoxicity resulting in peripheral neuropathy. Although it is postulated that formation of DNA-platinum adducts is responsible for both its cytotoxicity in cancer cells and side effects in neurons, downstream mechanisms that lead to distal axonal degeneration are unknown. Here we show that activation of calpains is required for both neurotoxicity and formation of DNA-platinum adduct formation in neurons but not in cancer cells. Furthermore, we show that neurotoxicity of cisplatin requires activation of Sarm1, a key regulator of Wallerian degeneration, as mice lacking the Sarm1 gene do not develop peripheral neuropathy as evaluated by both behavioral or pathological measures. These findings indicate that Sarm1 and/or specific calpain inhibitors could be developed to prevent cisplatin induced peripheral neuropathy.

Topics: Animals; Armadillo Domain Proteins; Calpain; Cells, Cultured; Cisplatin; Cytoskeletal Proteins; Enzyme Activation; Mice; Mice, Knockout; Neurotoxicity Syndromes; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Wallerian Degeneration

In both the central nervous system (CNS) and peripheral nervous system (PNS), transected axons undergo Wallerian degeneration. Even though Augustus Waller first described this process after transection of axons in 1850, the molecular mechanisms may be shared, at least in part, by many human diseases. Early pathology includes failure of synaptic transmission, target denervation, and granular disintegration of the axonal cytoskeleton (GDC). The Ca(2+)-dependent protease calpains have been implicated in GDC but causality has not been established. To test the hypothesis that calpains play a causal role in axonal and synaptic degeneration in vivo, we studied transgenic mice that express human calpastatin (hCAST), the endogenous calpain inhibitor, in optic and sciatic nerve axons. Five days after optic nerve transection and 48 h after sciatic nerve transection, robust neurofilament proteolysis observed in wild-type controls was reduced in hCAST transgenic mice. Protection of the axonal cytoskeleton in sciatic nerves of hCAST mice was nearly complete 48 h post-transection. In addition, hCAST expression preserved the morphological integrity of neuromuscular junctions. However, compound muscle action potential amplitudes after nerve transection were similar in wild-type and hCAST mice. These results, in total, provide direct evidence that calpains are responsible for the morphological degeneration of the axon and synapse during Wallerian degeneration.

Topics: Animals; Axons; Calcium-Binding Proteins; Calpain; Cerebral Cortex; Cytoskeleton; Electromyography; Electrophysiological Phenomena; Female; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Neurofilament Proteins; Neuromuscular Junction; Optic Nerve; Retina; Sciatic Nerve; Wallerian Degeneration

Axon degeneration is widespread both in neurodegenerative disease and in normal neural development, but the molecular pathways regulating these degenerative processes and the extent to which they are distinct or overlapping remain incompletely understood. We report that calpastatin, an inhibitor of calcium-activated proteases of the calpain family, functions as a key endogenous regulator of axon degeneration. Calpastatin depletion was observed in degenerating axons after physical injury, and maintaining calpastatin inhibited degeneration of transected axons in vitro and in the optic nerve in vivo. Calpastatin depletion also occurred in a caspase-dependent manner in trophic factor-deprived sensory axons and was required for this in vitro model of developmental degeneration. In vivo, calpastatin regulated the normal pruning of retinal ganglion cell axons in their target field. These findings identify calpastatin as a key checkpoint for axonal survival after injury and during development, and demonstrate downstream convergence of these distinct pathways of axon degeneration.

Topics: Animals; Animals, Newborn; Armadillo Domain Proteins; Axotomy; Brain; Calcium-Binding Proteins; Calpain; Cell Survival; Cells, Cultured; Cytoskeletal Proteins; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Green Fluorescent Proteins; HEK293 Cells; Humans; In Vitro Techniques; Mice; Microscopy, Electron, Transmission; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Neurons; Nicotinamide-Nucleotide Adenylyltransferase; RNA, Messenger; RNA, Small Interfering; Sciatic Neuropathy; Time Factors; Transduction, Genetic; Wallerian Degeneration

Axonal injury in white matter is an important consequence of many acute neurological diseases including ischemia. A role for glutamate-mediated excitotoxicity is suggested by observations from in vitro and in situ models that AMPA/kainate blockers can reduce axonal injury. We assessed axonal vulnerability in primary murine neuronal cultures, with axons isolated from their cell bodies using a compartmented chamber design. Transient removal of oxygen and glucose in the axon compartment resulted in irreversible loss of axon length and neurofilament labeling. This injury was not prevented by addition of ionotropic glutamate receptor blockers and could not be reproduced by glutamate receptor agonists. However, hypoxic injury was prevented by blockade of voltage-gated sodium channels, inhibition of calpain and removal of extracellular calcium. These results suggest that isolated, unmyelinated axons are vulnerable to hypoxic injury which is mediated by influx of sodium and calcium but is independent of glutamate receptor activation.

Topics: Animals; Axons; Calcium; Calcium Signaling; Calpain; Cell Culture Techniques; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; Hypoxia-Ischemia, Brain; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurotoxins; Receptors, Glutamate; Sodium; Sodium Channel Blockers; Sodium Channels; Wallerian Degeneration

Nerve injury initiates Wallerian degeneration with subsequent alterations of cytokine expression contributing to neuropathic pain. To investigate the very early temporal pattern of cytokine regulation we studied 140 mice of C57Bl/6J background after chronic constriction injury (CCI) of the right sciatic nerve and measured the relative mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1beta) and of the anti-inflammatory cytokines IL-4 and IL-10 with quantitative real-time polymerase chain reaction (qRT-PCR). The measurements were performed in ipsi- and contralateral sciatic nerves and dorsal root ganglia (DRG) 1, 3, 6, 9, 12, 24 h, and 3 and 7 days after CCI. We found an ipsilateral upregulation of TNF, IL-1beta and IL-10 mRNA levels as early as one hour after CCI. To investigate upstream regulatory mechanisms, we used inhibitors to the N-methyl-d-aspartate (NMDA) receptor ((+)-MK-801) and to calpain (MDL-28170). MDL-28170, but not (+)-MK-801 inhibited TNF and IL-1beta upregulation one hour after CCI. This leads us to suggest that calpain is one of the earliest mediators of cytokine upregulation in injured peripheral nerves.

Topics: Animals; Calpain; Disease Models, Animal; Female; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Interleukins; Mice; Mice, Inbred C57BL; Nerve Crush; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Sciatic Neuropathy; Time Factors; Tumor Necrosis Factor-alpha; Wallerian Degeneration

It is known that calpain activation is involved in human traumatic brain injury (TBI) and that calpain inhibition can have neuroprotective effects on both gray matter and white matter injury of TBI models. However, the role of calpain activation in the corpus callosum remains unclear and requires elucidation given its potential clinical relevance. We evaluated the neuroprotective effects of calpain inhibitor MDL-28170 on corpus callosum function and structural destruction using a fluid percussion injury (FPI) model. The therapeutic time window for a single administration of MDL-28170 was up to 4 h post injury in protecting the corpus callosum structural integrity, and up to 30 min in protecting the axonal function evaluated 1 day following injury. When given 30 min prior injury, MDL-28170 showed neuroprotective effects that lasted up to 7 days. However, 30 min post injury administration of the drug afforded neuroprotection only up to 3 days. In contrast, two additional reinforcement injections at 24 and 48 h in addition to 30 min post FPI significantly protected both axonal function and structural integrity that lasted 14 days following FPI. Our data indicated that calpain inhibitor MDL-28170 is an effective neuroprotectant for axonal injury in corpus callosum following FPI with a therapeutic time window up to 4 hours. Although delayed treatment (2 or 4 h post FPI) was effective in protecting the axonal structure, the axons saved may not be as functional as normal fibers. Multiple drug administrations may be necessary for achieving a persisting effectiveness of this compound.

Topics: Action Potentials; Animals; Axons; Brain Injuries; Calpain; Corpus Callosum; Cysteine Proteinase Inhibitors; Diffuse Axonal Injury; Dipeptides; Disease Models, Animal; Drug Administration Schedule; Male; Neural Conduction; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Time Factors; Treatment Outcome; Wallerian Degeneration

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.

Topics: Animals; Calpain; Cells, Cultured; Cerebellum; Electrophoresis, Gel, Two-Dimensional; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Neurites; Peptide Fragments; Superior Cervical Ganglion; Wallerian Degeneration

Local axon degeneration is a common pathological feature of many neurodegenerative diseases and peripheral neuropathies. While it is believed to operate with an apoptosis-independent molecular program, the underlying molecular mechanisms are largely unknown. In this study, we used the degeneration of transected axons, termed "Wallerian degeneration," as a model to examine the possible involvement of the ubiquitin proteasome system (UPS). Inhibiting UPS activity by both pharmacological and genetic means profoundly delays axon degeneration both in vitro and in vivo. In addition, we found that the fragmentation of microtubules is the earliest detectable change in axons undergoing Wallerian degeneration, which among other degenerative events, can be delayed by proteasome inhibitors. Interestingly, similar to transected axons, degeneration of axons from nerve growth factor (NGF)-deprived sympathetic neurons could also be suppressed by proteasome inhibitors. Our findings suggest a possibility that inhibiting UPS activity may serve to retard axon degeneration in pathological conditions.

Topics: Amino Acids; Animals; Animals, Newborn; Axons; Benzimidazoles; Blotting, Western; Calpain; Cells, Cultured; Chelating Agents; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeleton; Disease Models, Animal; Drug Interactions; Egtazic Acid; Endopeptidases; Ganglia, Sympathetic; Immunohistochemistry; Leupeptins; Microtubules; Multienzyme Complexes; Nerve Growth Factor; Optic Nerve; Optic Nerve Injuries; Peptide Fragments; Proteasome Endopeptidase Complex; Rats; Time Factors; Tubulin; Ubiquitin; Wallerian Degeneration

Peripheral nerve injury results in a series of events culminating in degradation of the axonal cytoskeleton (Wallerian degeneration). In the time period between axotomy and cytoskeletal degradation (24-48 h in rodents), there is calcium entry and activation of calpains within the axon. The precise timing of these events during this period is unknown. In the present study, antibodies were generated to three distinct peptide epitopes of m-calpain, and a fusion protein antibody was generated to the intrinsic calpain inhibitor calpastatin. These antibodies were used to measure changes in these proteins in mouse sciatic nerves during Wallerian degeneration. In sciatic nerve homogenates and cultured dorsal root ganglion (DRG) neurites, m-calpain protein was significantly reduced in transected nerves very early after nerve injury, long before axonal degeneration occurred. Levels of m-calpain protein remained low as compared to control nerves for the remainder of the 72-h time course. No changes in calpastatin protein were evident. Systemic treatment of animals with the protease inhibitor leupeptin partially prevented the rapid loss of calpain protein. Removal of calcium in DRG cultures had the same effect. These data indicate that m-calpain protein is lost very early after axonal injury, and likely reflect activation and degradation of this protein long before the cytoskeleton is degraded. Calpain activation may be an early event in a proteolytic cascade that is initiated by axonal injury and culminates with axonal degeneration.

Topics: Animals; Axons; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Membrane; Cytoskeleton; Down-Regulation; Leupeptins; Male; Mice; Mice, Inbred C57BL; Peptide Hydrolases; Peripheral Nerves; Peripheral Nervous System Diseases; Protease Inhibitors; Rabbits; Signal Transduction; Time Factors; Tubulin; Wallerian Degeneration

Peripheral neuropathies and Wallerian degeneration share a number of pathological features; the most prominent of which is axonal degeneration. We asked whether common pathophysiologic mechanisms are involved in these 2 disorders by directly comparing in vitro models of axonal degeneration after axotomy or exposure to the neurotoxin vincristine. Embryonic rat dorsal root ganglia (DRG) were allowed to extend neurites for 5 days in culture, and then were either axotomized or exposed to 0.01 microM vincristine. Neurites universally degenerated by 3 days after axotomy or after 6 days of vincristine exposure. The neuroprotective effects of a low calcium environment or pharmacologic inhibition of the cysteine protease calpain were compared in these 2 models of axonal degeneration. Addition of EGTA or growth in zero-calcium media provided significant protection against axonal degeneration after either axotomy or vincristine exposure. Treatment with the experimental calpain inhibitor AK295 was equally protective in both models. Chronic exposure to AK295 was not toxic to the cultures. These data suggest that common mechanisms involving calcium and calpains are involved in both axotomy-induced and vincristine-induced axonal degeneration. In addition, calpain inhibition may provide a strategy for preventing axonal degeneration and preserving neurologic function in a variety of PNS and CNS disorders.

Topics: Animals; Antineoplastic Agents, Phytogenic; Axons; Axotomy; Calpain; Cells, Cultured; Chelating Agents; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Female; Ganglia, Spinal; Pregnancy; Rats; Rats, Sprague-Dawley; Vincristine; Wallerian Degeneration

The biological basis for the phenotype of delayed Wallerian degeneration in the WLDs mouse has yet to be elucidated, although it is known that the characteristic is intrinsic to the axon. Previous data suggested that nerves from the WLD(S) are relatively resistant to proteolytic degradation. We investigated the time-course of neurofilament degradation in response to addition of the calcium-activated protease m-calpain, comparing nerves from WLD(S) and wild-type mice. During 10 min of in vitro proteolysis, neurofilaments from the WLD(S) were consistently slower to degrade than were neurofilaments from wild-type mice. Direct comparisons were performed on Western blots, with statistically significant differences in neurofilament immunoreactivity at 2, 4, and 6 min of reaction time (p < 0.01). These findings suggest that the mutation leading to the WLD(S) phenotype may affect the proteolytic interaction between calpain and neurofilaments.

Topics: Animals; Axons; Calpain; Cloning, Molecular; Male; Mice; Mice, Neurologic Mutants; Neurofilament Proteins; Wallerian Degeneration

Biopsy specimens of human and monkey peripheral nerves, when incubated in calcium containing media, showed a loss of neurofilaments and microtubules with replacement by granular debris. Cytoskeletal structures remained intact when incubated in calcium-free media. Disruption of neurofilaments and microtubules in calcium containing media was inhibited by the thiol protease inhibitor, leupeptin. Similar incubations of excised Pacinian corpuscles revealed evidence of early terminal axon degeneration in the presence of calcium. These data substantiate the hypothesis that neural cytoskeletal degradation in primates and in man is calcium-mediated.

Topics: Animals; Calcium; Calpain; Cebus; Culture Media; Cytoskeleton; Humans; Intermediate Filaments; Leupeptins; Microtubules; Nerve Degeneration; Peripheral Nerves; Wallerian Degeneration