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Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures (n = 5, p

Microglia/astrocyte and B cell neuroimmune responses are major contributors to the neurological deficits after traumatic spinal cord injury (SCI). Bruton tyrosine kinase (BTK) activation mechanistically links these neuroimmune mechanisms. Our objective is to use Ibrutinib, an FDA-approved BTK inhibitor, to inhibit the neuroimmune cascade thereby improving locomotor recovery after SCI. Rat models of contusive SCI, Western blot, immunofluorescence staining imaging, flow cytometry analysis, histological staining, and behavioral assessment were used to evaluate BTK activity, neuroimmune cascades, and functional outcomes. Both BTK expression and phosphorylation were increased at the lesion site at 2, 7, 14, and 28 days after SCI. Ibrutinib treatment (6 mg/kg/day, IP, starting 3 h post-injury for 7 or 14 days) reduced BTK activation and total BTK levels, attenuated the injury-induced elevations in Iba1, GFAP, CD138, and IgG at 7 or 14 days post-injury without reduction in CD45RA B cells, improved locomotor function (BBB scores), and resulted in a significant reduction in lesion volume and significant improvement in tissue-sparing 11 weeks post-injury. These results indicate that Ibrutinib exhibits neuroprotective effects by blocking excessive neuroimmune responses through BTK-mediated microglia/astroglial activation and B cell/antibody response in rat models of SCI. These data identify BTK as a potential therapeutic target for SCI.

Calpain-5 (CAPN5) is a member of the calpain family of calcium-activated neutral thiol proteases. CAPN5 is partly membrane associated, despite its lack of a transmembrane domain. Unlike classical calpains, CAPN5 contains a C-terminal C2 domain. C2 domains often have affinity to lipids, mediating membrane association. We recently reported that the C2 domain of CAPN5 was essential for its membrane association and the activation of its autolytic activity. However, despite the removal of the C2 domain by autolysis, the N-terminal fragment of CAPN5 remained membrane associated. S-acylation, also referred to as S-palmitoylation, is a reversible post-translational lipid modification of cysteine residues that promotes membrane association of soluble proteins. In the present study several S-acylated cysteine residues were identified in CAPN5 with the acyl-PEG exchange method. Data reported here demonstrate that CAPN5 is S-acylated on up to three cysteine residues including Cys-4 and Cys-512, and likely Cys-507. The D589N mutation in a potential calcium binding loop within the C2 domain interfered with the S-acylation of CAPN5, likely preventing initial membrane association. Mutating specific cysteine residues of CAPN5 interfered with both its membrane association and the activation of CAPN5 autolysis. Taken together, our results suggest that the S-acylation of CAPN5 is critical for its membrane localization which appears to favor its enzymatic activity.

The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca(2+) regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty μM Ca(2+) was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca(2+) binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca(2+)-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

We previously reported the serendipitous observation that fenbendazole, a benzimidazole anthelmintic, improved functional and pathological outcomes following thoracic spinal cord contusion injury in mice when administered pre-injury. Fenbendazole is widely used in veterinary medicine. However, it is not approved for human use and it was uncertain if only post-injury administration would offer similar benefits. In the present study we evaluated post-injury administration of a closely related, human anthelmintic drug, flubendazole, using a rat spinal cord contusion injury model. Flubendazole, administered i.p. 5 or 10 mg/kg day, beginning 3 h post-injury and daily thereafter for 2 or 4 weeks, resulted in improved locomotor function after contusion spinal cord injury (SCI) compared with vehicle-treated controls. Histological analysis of spinal cord sections showed that such treatment with flubendazole also reduced lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing. Flubendazole inhibited the activation of glial fibrillary acidic protein (GFAP); suppressed cyclin B1 expression and Bruton tyrosine kinase activation, markers of B cell activation/proliferation and inflammation; and reduced B cell autoimmune response. Together, these results suggest the use of the benzimidazole anthelmintic flubendazole as a potential therapeutic for SCI.

Background The extent and severity of traumatic brain injuries (TBIs) can be difficult to determine with current diagnostic methods. To address this, there has been increased interest in developing biomarkers to assist in the diagnosis, determination of injury severity, evaluation of recovery and therapeutic efficacy, and prediction of outcomes. Several promising serum TBI biomarkers have been identified using hypothesis-driven approaches, largely examining proteins that are abundant in neurons and non-neural cells in the CNS. New method An unbiased approach, phage display, was used to identify serum TBI biomarkers. In this proof-of-concept study, mice received a TBI using the controlled cortical impact model of TBI (1 mm injury depth, 3.5 m/s velocity) and phage display was utilized to identify putative serum biomarkers at 6 h postinjury. Results An engineered phage which preferentially bound to injured serum was sequenced to identify the 12-mer ‘recognizer’ peptide expressed on the coat protein. Following synthesis of the recognizer peptide, pull down, and mass spectrometry analysis, the target protein was identified as glial fibrillary acidic protein (GFAP). Comparison with existing methods and conclusions GFAP has previously been identified as a promising TBI biomarker. The results provide proof of concept regarding the ability of phage display to identify TBI serum biomarkers. This methodology is currently being applied to serum biomarkers of mild TBI.

Neuronal damage, glial inflammation, and astrogliosis/astroglial scar formation are major secondary injury mechanisms that are significant contributors to functional deficits after spinal cord injury (SCI). The objectives of the study were to evaluate the distinct roles of ERK2 vs. ERK1/2 and ERK1/2-calpain 1–NF-κB signal transduction in the tissue damage and astrogliosis/astroglial scar formation following SCI in rats. RNAi approaches, pharmacological intervention (U0126), Western blot analysis, immunofluorescence analysis, and histological assessment were used to target ERK1/2-calpain 1-NF-κB signal transduction pathway for neuroprotection. Histological staining analysis demonstrated that selectively reducing pERK2 using ERK2 siRNA, but not inhibition of pERK1/2 with U0126, significantly reduced lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing in spinal cord two weeks after contusive SCI. An ERK1/2-calpain 1-NF-κB signal transduction pathway was involved in the astroglial scar formation after SCI. Blockade of ERK1/2 by U0126 decreased calpain 1 expression 4 h following SCI. Selective calpain 1 reduction by lentiviral shRNA attenuated astroglial NF-κB activity and astroglial scar formation after SCI in rats. Taken together, these results demonstrate the involvement of individual ERK2 and calpain 1 signaling pathways in tissue damage and astrogliosis/astroglial scar formation in animal models of SCI. Therefore, targeting individual ERK and its downstream signal transduction of calpain 1-NF-κB may provide greater potential as novel therapeutics for minimizing tissue damage and astroglial scar formation following SCI.

PURPOSE We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Calpains and caspases are cysteine endopeptidases which share many similar substrates. Caspases are essential for caspase-dependent apoptotic death where calpains may play an augmentive role, while calpains are strongly implicated in necrotic cell death morphologies. Previous studies have demonstrated a down-regulation in the expression of many components of the caspase-dependent cell death pathway during CNS development. We therefore sought to determine if there is a corresponding upregulation of calpains. The major CNS calpains are the μ- and m-isoforms, composed of the unique 80 kDa calpain 1 and 2 subunits, respectively, and the shared 28 kDa small subunit. In rat brain, relative protein and mRNA levels of calpain 1, calpain 2, caspase 3, and the endogenous calpain inhibitor calpastatin, were evaluated using western blot and real-time RT-PCR. The developmental time points examined ranged from embryonic day 18 until postnatal day 90. Calpain 1 and calpastatin protein and mRNA levels were low at early developmental time points and increased dramatically by P30. Conversely, Caspase-3 expression was greatest at E18, and was rapidly downregulated rapidly down-regulated between by P30. Calpain 2 protein and mRNA levels were relatively constant throughout the E18-P90 age range examined. The inverse relationship of calpain 1 and caspase 3 levels during CNS development is consistent with the shift from caspase-dependent to caspase-independent cell death mechanisms following CNS injury in neonatal vs. adult rat brain.

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80 kDa μ-calpain large subunit and 28 kDa small subunit. In rat cortex, antibodies against domains II and III of the large μ-calpain subunit also detected a 40 kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte μ-calpain with Ca2+. Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of μ-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca2+ released from mitochondrial stores.

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.

Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased axonal damage and neurofilament proteolysis with postinjury intravenous administration of relatively low concentrations of the calpain inhibitors leupeptin, E-64-D, and calpeptin. We sought to determine if conditions under which calpain inhibitors were administered in previous studies resulted in effective calpain inhibition, and to identify conditions that result in significant calpain inhibition following spinal cord injury. Contusive spinal cord injury was produced in female Long-Evans rats using the NYU impactor at the 12.5-25-mm height setting. The results demonstrate that intravenous administration of 1 mg/kg E-64-D or 250 micro g/kg calpeptin does not inhibit total calpain activity in the rat spinal cord, measured using a BODIPY-FL labeled casein assay. Intravenous administration of MDL28170 (20 mg/kg) resulted in mild calpain inhibition and a modest decrease in the proteolysis of calpain substrates alpha-spectrin and MAP2. Intraspinal microinjection of 50 nmoles/19 micro g MDL28170, either 30 min prior to or 20 min following contusion injury, resulted in a more robust inhibition of total calpain activity and greater attenuation of alpha-spectrin breakdown and MAP2 proteolysis. The decreased proteolysis persisted 24 h postinjury. Together, the results demonstrate that direct microinjection of the calpain inhibitor MDL28170 is more effective than intravenous infusion in reducing calpain activity and decreasing the injury-induced proteolysis of calpain substrates.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.

Prostate apoptosis response-4 (Par-4) is a protein containing both a leucine zipper and a death domain that was isolated by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis. Par-4 is expressed in the nervous system, where its function is unknown. In Alzheimer disease (AD), neurons may die by apoptosis, and amyloid beta-protein (A beta) may play a role in this. We report here that Par-4 expression is increased in vulnerable neurons in AD brain and is induced in cultured neurons undergoing apoptosis. Blockade of Par-4 expression or function prevented neuronal apoptosis induced by Ab and trophic factor withdrawal. Par-4 expression was enhanced, and mitochondrial dysfunction and apoptosis exacerbated, in cells expressing presenilin-1 mutations associated with early-onset inherited AD.

Excess glutamatergic neurotransmission may contribute to excitotoxic loss of nigrostriatal neurons in Parkinson’s disease (PD). Here, we determined if increasing glutamate uptake could reduce the extent of tyrosine hydroxylase (TH) loss in PD progression. The beta-lactam antibiotic, ceftriaxone, increases the expression of glutamate transporter 1 (GLT-1), a glutamate transporter that plays a major role in glutamate clearance in central nervous system and may attenuate adverse behavioral or neurobiological function in other neurodegenerative disease models. In association with >80 % TH loss, we observed a significant decrease in glutamate uptake in the established 6-hydroxydopamine (6-OHDA) PD model. Ceftriaxone (200 mg/kg, i.p.) increased striatal glutamate uptake with ≥ 5 consecutive days of injection in nonlesioned rats and lasted out to 14 days postinjection, a time beyond that required for 6-OHDA to produce >70 % TH loss (~9 days). When ceftriaxone was given at the time of 6-OHDA, TH loss was ~57 % compared to ~85 % in temporally matched vehicle-injected controls and amphetamine-induced rotation was reduced about 2-fold. This attenuation of TH loss was associated with increased glutamate uptake, increased GLT-1 expression, and reduced Serine 19 TH phosphorylation, a calcium-dependent target specific for nigrostriatal neurons. These results reveal that glutamate uptake can be targeted in a PD model, decrease the rate of TH loss in a calcium-dependent manner, and attenuate locomotor behavior associated with 6-OHDA lesion. Given that detection of reliable PD markers will eventually be employed in susceptible populations, our results give credence to the possibility that increasing glutamate uptake may prolong the time period before locomotor impairment occurs.

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved alpha-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

Colchicine, a microtubule disrupting agent, has been used to model several aspects of Alzheimer's disease-related neuropathology. The formation of neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, involves the loss of tau (a low mol. wt. microtubule-associated protein) from axons and accumulation of abnormally phosphorylated tau in somatodendritic compartments. Other cytoskeletal proteins, such as microtubule-associated protein 2 (MAP2), disappear as tau accumulates. The present study was directed at evaluating the effects of colchicine on tau and MAP2, to determine if changes in their levels or distribution might be similar to those which precede the formation of neurofibrillary tangles in Alzheimer's disease. Six hours following intrahippocampal colchicine injection (3.5 micrograms injected into two rostro-caudal locations) tau-1 immunostaining was enhanced in CA1 s. radiatum and decreased in the outer molecular layer of the dentate gyrus. In addition, a shift in the relative abundance of tau isoforms was observed in Western blots. Both the immunocytochemical and immunoblot results are consistent with a dephosphorylation of tau. Loss of MAP2 was evident 3 days postinjection which coincided with a loss of Cresyl violet staining in granule cell, CA3, subicular and entorhinal neurons. Accumulation of tau or MAP2 in neuronal perikarya was not observed at any postinjection time points. Thus, intrahippocampal colchicine administration does not model the shift in tau localization, excessive tau phosphorylation, or other cytoskeletal alterations that are suggested to precede or accompany the formation of neurofibrillary pathology in Alzheimer's disease.

Caspase-independent cell death, an important death pathway in many cells including neurons, is executed via apoptosis-inducing factor (AIF), an oxidoreductase, localized to the mitochondrial intermembrane space. AIF is processed and released from mitochondria following mitochondrial permeability transition pore (mPTP) formation, and translocates to the nucleus to induce DNA fragmentation and cell death. The release of AIF requires cleavage of its N-terminus anchored in the inner mitochondrial membrane. The protease responsible for this AIF truncation has not been established, although there is considerable evidence suggesting a role for micro-calpain. We previously found that a pool of micro-calpain is localized to the mitochondrial intermembrane space, the submitochondrial compartment in which AIF truncation occurs. The close submitochondrial proximity of mitochondrial micro-calpain and AIF gives support to the hypothesis that mitochondrial micro-calpain may be the protease responsible for processing AIF prior to its release. In the present study, AIF was released from rat liver mitochondria following mPTP induction by atractyloside. This release was inhibited by the cysteine protease inhibitor MDL28170, but not by more specific calpain inhibitors PD150606 and calpastatin. Atractyloside caused swelling in rat brain mitochondria, but did not induce AIF release. In a mitochondrial fraction from SH-SY5Y neuroblastoma cells, incubation with 5 mM Ca(2+) resulted in the activation of micro-calpain but not in AIF truncation. In summary, the localization of micro-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive.

Excessive activation of calpains (calcium-activated neutral proteases) is observed following spinal cord contusion injury, traumatic brain injury, stroke, and in neurodegenerative disorders including Alzheimer's disease. Calpain inhibition represents an attractive therapeutic target, but current calpain inhibitors possess relatively weak potency, poor specificity, and in many cases, limited cellular and blood–brain barrier permeability. We developed novel calpain inhibitors consisting of the endogenous inhibitor, calpastatin or its inhibitory domain I, fused to the protein transduction domain of the HIV trans-activator (Tat) protein (Tat 47–57 ). The Tat-calpastatin fusion proteins were potent calpain inhibitors in a cell-free activity assay, but did not inhibit cellular calpain activity in primary rat cortical neurons when applied exogenously at concentrations up to 5 μM. The fusion proteins were able to transduce neurons, but were localized within endosome-like structures. A similar endosomal uptake was observed for Tat-GFP. Together, the results suggest that endosomal uptake of the Tat-calpastatin prevents its interaction with calpain in other cellular compartments. Endosomal uptake of proteins fused to the Tat protein transduction domain severely limits the applications of this methodology.

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.

3-Nitropropionic acid (3-NA) was originally identified as a component of moldy sugarcane, produced by a fungus, Arthrinium sp. Ingestion of the mildewed sugarcane results in motor impairment and muscle weakness that is neither progressive nor reversible, and is accompanied by bilateral damage to the striatum (1,2) (see also Chapters 1, 2, 5, 10, and 12). In rats, repeated systemic administration of 3-NPA results in motor impairment and consistent morphologic injury to striatum (Fig. 1). Hippocampal damage is less consistent, neocortical damage is infrequent, and the cerebellum is spared (3–5). The striatal damage induced by repeated systemic 3-NPA resembles many of the neuropathologic aspects of Huntington’s disease (6,7),(see also Chapters 2, 5, 10, and 12), particularly when low doses of 3-NPA are used (8). In addition, the regional damage caused by chronic 3-NPA resembles that produced by severe global ischemia (9) and hypoglycemia (10). Thus, 3-NPA is a useful tool for understanding neuronal damage resulting from late-onset neurodegenerative disorders such as Huntington’s disease, as well as acute insults such as ischemia and hypoglycemia.

Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.

The neuronal cytoskeleton is disrupted in neurodegenerative disorders such as Alzheimer's disease. Due to the lack of suitable animal models, studies examining the events involved in the neurodegeneration have relied on postmortem human brain tissue obtained from individuals with the disease and from normal controls. However, it is uncertain if the neuronal cytoskeleton is stable during the postmortem interval. Immunohistochemistry and immunoblots were used to examine the microtubule-associated proteins tau, MAP2, and MAP1B in the rat hippocampus at various times after death. Shortly after death, tau immunoreactivity was lost from axons and accumulated in somatodendritic compartments. MAP2 and MAP1B also accumulated in neuronal cell bodies prior to a loss of immunostaining in some regions, notably subiculum. Immunoblots confirmed a loss of MAP2 and MAP1B within a few hours after death. Tau levels remained constant during the 8-hour postmortem interval examined, although the electrophoretic mobility of some tau bands was altered. Human brain tissue obtained at autopsy and at surgery demonstrated similar cytoskeletal alterations in postmortem tissue. These results demonstrate that microtubules and associated proteins are not stable postmortem.

Nucleolar antigens p145 and p120 are associated with proliferating cells (Freeman, J.W.; McRorie, D.K.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Spohn, W.H.; Busch, H. Cancer Res. 46:3593; 1986 and Freeman, J.W.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Busch, H. Cancer Res. 48:1244; 1988) and are not detectable in normal resting cells. Recent immunoelectron microscopic studies (Ochs, R.L.; Reilly, M.T.; Freeman, J.W.; Busch, H. Cancer Res. 48:6523; 1988) suggest that the two antigens have overlapping nucleolar localizations. In this study the nucleolus was physicochemically and biochemically studied to determine whether p145 and p120 were associated with a common nucleolar component. Antigen p145 was associated with 40-80 S ribonucleoprotein particles (RNPs), and the p145 antigen was not detected in HeLa cells following in situ RNAse digestion. P120 was found in a 40-80 S, RNAse resistant complex. Sequential extraction of HeLa nucleoli showed that most of antigen p145 was extractable in 10 mM Tris with 0.2% deoxycholate, whereas p120 was found in a nucleolar residue fraction requiring DNAse and high salt treatment for optimal extraction. Neither antigen p145 nor p120 was detectable in normal resting tissues. Antigen p145 was detected in all proliferating tissues examined, including a variety of malignant tumors (ten of ten), benign tissues including adenomas and hyperplasias (eight of eight), and in normal proliferating cells such as colonic epithelium and spermatogonia of the testes. Antigen p120 was not detected in all tumors, being absent in three of seven lymphomas and in one melanoma examined.(ABSTRACT TRUNCATED AT 250 WORDS)