Robert Siman

The role of calpain-mediated spectrin proteolysis in traumatically induced axonal injury.

In animals and man, traumatic brain injury (TBI) results in axonal injury (AI) that contributes to morbidity and mortality. Such injured axons show progressive change leading to axonal disconnection. Although several theories implicate calcium in the pathogenesis of AI, experimental studies have failed to confirm its pivotal role. To explore the contribution of Ca2+-induced proteolysis to axonal injury, this study was undertaken in an animal model of TBI employing antibodies targeting both calpain-mediated spectrin proteolysis (CMSP) and focal neurofilament compaction (NFC), a marker of intra-axonal cytoskeletal perturbation, at 15-120 minutes (min) postinjury. Light microscopy (LM) revealed that TBI consistently evoked focal, intra-axonal CMSP that was spatially and temporally correlated with NFC. These changes were seen at 15 min postinjury with significantly increasing number of axons demonstrating CMSP immunoreactivity over time postinjury. Electron microscopy (EM) demonstrated that at 15 min postinjury CMSP was confined primarily to the subaxolemmal network. With increasing survival (30-120 min) CMSP filled the axoplasm proper. These findings provide the first direct evidence for focal CMSP in the pathogenesis of generalized/diffuse AI. Importantly, they also reveal an initial subaxolemmal involvement prior to induction of a more widespread axoplasmic change indicating a spatial-temporal compartmentalization of the calcium-induced proteolytic process that may be amenable to rapid therapeutic intervention.

Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester.

Fibrillar amyloid deposits are defining pathological lesions in Alzheimer’s disease brain and are thought to mediate neuronal death. Amyloid is composed primarily of a 39–42 amino acid protein fragment of the amyloid precursor protein (APP), called amyloid β-protein (Aβ). Because deposition of fibrillar amyloid in vitrohas been shown to be highly dependent on Aβ concentration, reducing the proteolytic release of Aβ is an attractive, potentially therapeutic target. Here, the turnover rate of brain Aβ has been determined to define treatment intervals over which a change in steady-state concentration of Aβ could be measured. Mice producing elevated levels of human Aβ were used to determine approximate turnover rates for Aβ and two of its precursors, C99 and APP. Thet½ for brain Aβ was between 1.0 and 2.5 hr, whereas for C99, immature, and fully glycosylated forms of APP695 the approximate t½ values were 3, 3, and 7 hr, respectively. Given the rapid Aβ turnover rate, acute studies were designed using phorbol 12-myristate 13-acetate (PMA), which had been demonstrated previously to reduce Aβ secretion from cells in vitro via induction of protein kinase C (PKC) activity. Six hours after intracortical injection of PMA, Aβ levels were significantly reduced, as measured by both Aβ40- and Aβ42-selective ELISAs, returning to normal by 12 hr. An inactive structural analog of PMA, 4α-PMA, had no effect on brain Aβ levels. Among the secreted N-terminal APP fragments, APPβ levels were significantly reduced by PMA treatment, whereas APPα levels were unchanged, in contrast to most cell culture studies. These results indicate that Aβ is rapidly turned over under normal conditions and support the therapeutic potential of elevating PKC activity for reduction of brain Aβ.

Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat.

Calpain, a calcium-activated neutral protease family, has been implicated in the neuropathologic sequelae accompanying various neurological disorders. We have characterized the distribution and time course of calpain activation following brain injury in the rat, using a monoclonal antibody that recognizes calpain-generated breakdown products (BDPs) of spectrin. Adult male Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2–2.4 atm, n = 35) or served as controls (uninjured, n = 12). One group of animals (n = 21) were sacrificed at either 30 minutes (min), 1 day, or 3 days post-injury, and selected brain regions were prepared for Western blot analysis. The remaining animals (n = 26) were sacrificed at 90 min, 4 hours (h), 1 day, or 7 days post-injury, and immunohistochemistry was performed. Spectrin BDPs were found predominantly in the hemisphere ipsilateral to the injury site, located primarily in cortical and hippocampal regions which exhibit neuronal death. Calpain-mediated spectrin breakdown was detected at 90 min in dendrites and axons, and by 4 h in neuronal perikarya. By 1 day post-injury, cortical and hippocampal regions of calpain activation had increased in size. Delayed spectrin breakdown was observed in the thalamus, both at 3 days and 7 days after injury. These results suggest that calpain may play an role in the neurodegenerative process following brain injury.

Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury.

In traumatic axonal injury, Ca2+ influx across a focally damaged axolemma precipitates local mitochondrial failure, degradation of the subaxolemmal spectrin network and compaction of neurofilaments, which collectively contribute to axonal failure. In previous studies, cyclosporin A pretreatment preserved mitochondrial integrity and attenuated axonal failure following trauma. Here we investigate whether this CsA-linked protection was related to the concomitant blunting of intra-axonal, Ca2+-induced cytoskeletal changes in traumatic axonal injury, assessed with antibodies targeting spectrin proteolysis and neurofilament compaction. CsA pretreatment dramatically reduced Ca2+-induced cytoskeletal damage following injury; CsA-treated rats, compared with vehicle-treated rats, displayed a 70% decrease in immunoreactive/damaged profiles. We suggest that CsA-mediated preservation of mitochondrial integrity enables the restoration of ionic and metabolic homeostasis thereby short-circuiting Ca2+-induced proteolysis in injured axons.

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Diffuse plaques contain C-terminal Aβ42 and not Aβ40: Evidence from cats and dogs ☆

Abstract Recent reports have suggested that β-amyloid (Aβ) species of variable length C-termini are differentially deposited within early and late-stage plaques and the cerebrovasculature. Specifically, longer C-terminal length A β 42 3 fragments (i.e., Aβ forms extending to residues 42 and/or 43) are thought to be predominant within diffuse plaques while both A β 42 3 and Aβ40 (Aβ forms terminating at residue 40) are present within a subset of neuritic plaques and cerebrovascular deposits. We sought to clarify the issue of differential Aβ deposition using aged canines, a partial animal model of Alzheimers disease that exhibits extensive diffuse plaques and frequent vascular amyloid, but does not contain neuritic plaques or neurofibrillary tangles. We examined the brains of 20 aged canines, 3 aged felines, and 17 humans for the presence of Aβ immunoreactive plaques, using antibodies to Aβ1–17, Aβ17–24, Aβ1–28, Aβ40, and Aβ42. We report that plaques within the canine and feline brain are immunopositive for Aβ42 but not Aβ40. This is the first observation of nascent AD pathology in the aged feline brain. Canine plaques also contained epitopes within Aβ1–17, Aβ17–24, and Aβ1–28. In all species examined, vascular deposits were immunopositive for both Aβ40 and Aβ42. In the human brain, diffuse plaques were preferentially Aβ42 immunopositive, while neuritic plaques and vascular deposits were both Aβ40 and Aβ42 immunopositive. However, not all neuritic plaques contain Aβ40 epitopes.

FAD mutant PS-1 gene-targeted mice: increased Aβ42 and Aβ deposition without APP overproduction

To investigate the consequences of mutant presenilin-1 (PS-1) expression under the control of the normal PS-1 gene, a gene-targeted mouse bearing the FAD mutation P264L was made. Gene-targeted models are distinct from transgenic models because the mutant gene is expressed at normal levels, in the absence of the wild-type protein. PS-1(P264L/P264L) mice had normal expression of PS-1 mRNA, but levels of the N- and C-terminal protein fragments of PS-1 were reduced while levels of the holoprotein were increased. When crossed into Tg(HuAPP695.K670N/M671L)2576 mice, the PS-1(P264L) mutation accelerated the onset of amyloid (Abeta) deposition in a gene-dosage dependent manner. Tg2576/PS-1(P264L/P264L) mice also had Abeta deposition that was widely distributed throughout the brain and spinal cord. APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mice had elevated levels of Abeta42, sufficient to cause Abeta deposition beginning at 6 months of age. Abeta deposition increased linearly over time in APP(NLh/NLh)/PS-1(P264L/P264L) mice, whereas the increase in Tg2576 mice was exponential. The APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mouse represents an animal model that exhibits Abeta deposition without overexpression of APP.

Proteasome inhibition enhances the stability of mouse Cu Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis

Point mutations occurring within the Cu/Zn superoxide dismutase (SOD1) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SOD1 gene and studied the expression of the mutant templates in stably transformed cell lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu/Zn SOD isoforms indicate that the FALS mutant Cu/Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu/Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu/Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins.

Distribution of calpain I, an enzyme associated with degenerative activity, in rat brain

The calcium-activated protease calpain I was localized in rat brain by immunocytochemistry. Calpain I-like immunoreactivity (CLI) was prominent in several structures in which degeneration is an ongoing feature, e.g. spinal motoneurons, olfactory nerve. Also noteworthy was the presence of CLI in regions susceptible to age-related pathologies, e.g. cerebellar Purkinje cells, substantia nigra and subiculum. This distribution suggests that calpain I may be involved with both normal and pathological neuronal degeneration.

Aurintricarboxylic acid protects hippocampal neurons from NMDA- and ischemia-induced toxicity in vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium‐dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia‐or excitotoxin‐induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA‐and ischemia‐induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.