RESUMEN
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.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Neuroinmunomodulación , Recuperación de la Función , Traumatismos de la Médula Espinal/inmunología , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Formación de Anticuerpos/efectos de los fármacos , Astrocitos/patología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Peso Corporal/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunoglobulina G/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/patología , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/patología , Actividad Motora/efectos de los fármacos , Neuroinmunomodulación/efectos de los fármacos , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Piperidinas/uso terapéutico , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Ratas , Recuperación de la Función/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Bazo/patología , Sindecano-1/metabolismo , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. METHODS: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. RESULTS: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. INTERPRETATION: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.
Asunto(s)
Ácido 3-Hidroxibutírico/farmacología , Anticonvulsivantes/farmacología , Encéfalo/efectos de los fármacos , Dieta Cetogénica , Epilepsia del Lóbulo Temporal , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Ácido 3-Hidroxibutírico/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Electroencefalografía , Cuerpos Cetónicos/farmacología , Canal de Potasio Kv.1.1/genética , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Técnicas de Placa-ClampRESUMEN
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.
Asunto(s)
Calpaína/biosíntesis , Núcleo Celular/enzimología , Sistema Nervioso Central/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Cuerpos de Inclusión Intranucleares/enzimología , Señales de Localización Nuclear/metabolismo , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biosíntesis , Proteínas de Caenorhabditis elegans/genética , Calpaína/genética , Núcleo Celular/genética , Cuerpos de Inclusión Intranucleares/genética , Masculino , Ratones , Ratones Transgénicos , Señales de Localización Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The efficacy of the amphipathic ketoamide calpain inhibitor SNJ-1945 in attenuating calpain-mediated degradation of the neuronal cytoskeletal protein α-spectrin was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF-1 mice. Using a single early (15 min after CCI-TBI) i.p. bolus administration of SNJ-1945 (6.25, 12.5, 25, or 50-mg/kg), we identified the most effective dose on α-spectrin degradation in the cortical tissue of mice at its 24 h peak after severe CCI-TBI. We then investigated the effects of a pharmacokinetically optimized regimen by examining multiple treatment paradigms that varied in dose and duration of treatment. Finally, using the most effective treatment regimen, the therapeutic window of α-spectrin degradation attenuation was assessed by delaying treatment from 15 min to 1 or 3 h post-injury. The effect of SNJ-1945 on α-spectrin degradation exhibited a U-shaped dose-response curve when treatment was initiated 15 min post-TBI. The most effective 12.5 mg/kg dose of SNJ-1945 significantly reduced α-spectrin degradation by ~60% in cortical tissue. Repeated dosing of SNJ-1945 beginning with a 12.5 mg/kg dose did not achieve a more robust effect compared with a single bolus treatment, and the required treatment initiation was less than 1 h. Although calpain has been firmly established to play a major role in post-traumatic secondary neurodegeneration, these data suggest that even brain and cell-permeable calpain inhibitors, when administered alone, do not show sufficient cytoskeletal protective efficacy or a practical therapeutic window in a mouse model of severe TBI. Such conclusions need to be verified in the human clinical situation.
Asunto(s)
Lesiones Encefálicas/tratamiento farmacológico , Calpaína/antagonistas & inhibidores , Carbamatos/farmacología , Citoesqueleto/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Carbamatos/uso terapéutico , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Neuronas/metabolismo , Espectrina/metabolismoRESUMEN
Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on ß-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these ß-strands forming a ß-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the ß-strands, ß-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.
Asunto(s)
Neuroblastoma , Vitreorretinopatía Proliferativa , Humanos , Calpaína/genética , Calpaína/metabolismo , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/patología , MutaciónRESUMEN
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.
Asunto(s)
Calpaína , Cisteína , Acilación , Calcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Cisteína/genética , Cisteína/metabolismo , LipoilaciónRESUMEN
Blast-induced traumatic brain injury (bTBI) is the "signature wound" of the current wars in Iraq and Afghanistan. However, with no objective information of relative blast exposure, warfighters with bTBI may not receive appropriate medical care and are at risk of being returned to the battlefield. Accordingly, we have created a colorimetric blast injury dosimeter (BID) that exploits material failure of photonic crystals to detect blast exposure. Appearing like a colored sticker, the BID is fabricated in photosensitive polymers via multi-beam interference lithography. Although very stable in the presence of heat, cold or physical impact, sculpted micro- and nano-structures of the BID are physically altered in a precise manner by blast exposure, resulting in color changes that correspond with blast intensity. This approach offers a lightweight, power-free sensor that can be readily interpreted by the naked eye. Importantly, with future refinement this technology may be deployed to identify soldiers exposed to blast at levels suggested to be supra-threshold for non-impact blast-induced mild TBI.
Asunto(s)
Traumatismos por Explosión/diagnóstico , Lesiones Encefálicas/diagnóstico , Colorimetría/instrumentación , Colorimetría/métodos , Compuestos Epoxi , Polímeros , Cristalización , Humanos , Microscopía Electrónica de Rastreo , Nanoestructuras/análisis , Nanoestructuras/ultraestructura , FotonesRESUMEN
Cyclophilin D (CypD) has been shown to play a critical role in mitochondrial permeability transition pore (mPTP) opening and the subsequent cell death cascade. Studies consistently demonstrate that mitochondrial dysfunction, including mitochondrial calcium overload and mPTP opening, is essential to the pathobiology of cell death after a traumatic brain injury (TBI). CypD inhibitors, such as cyclosporin A (CsA) or NIM811, administered following TBI, are neuroprotective and quell neurological deficits. However, some pharmacological inhibitors of CypD have multiple biological targets and, as such, do not directly implicate a role for CypD in arbitrating cell death after TBI. Here, we reviewed the current understanding of the role CypD plays in TBI pathobiology. Further, we directly assessed the role of CypD in mediating cell death following TBI by utilizing mice lacking the CypD encoding gene Ppif. Following controlled cortical impact (CCI), the genetic knockout of CypD protected acute mitochondrial bioenergetics at 6 h post-injury and reduced subacute cortical tissue and hippocampal cell loss at 18 d post-injury. The administration of CsA following experimental TBI in Ppif-/- mice improved cortical tissue sparing, highlighting the multiple cellular targets of CsA in the mitigation of TBI pathology. The loss of CypD appeared to desensitize the mitochondrial response to calcium burden induced by TBI; this maintenance of mitochondrial function underlies the observed neuroprotective effect of the CypD knockout. These studies highlight the importance of maintaining mitochondrial homeostasis after injury and validate CypD as a therapeutic target for TBI. Further, these results solidify the beneficial effects of CsA treatment following TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Peptidil-Prolil Isomerasa F/genética , Animales , Lesiones Traumáticas del Encéfalo/fisiopatología , Región CA3 Hipocampal/patología , Cognición/efectos de los fármacos , Peptidil-Prolil Isomerasa F/deficiencia , Peptidil-Prolil Isomerasa F/metabolismo , Ciclosporina/farmacología , Metabolismo Energético/efectos de los fármacos , Memoria/efectos de los fármacos , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuroprotección/efectos de los fármacosRESUMEN
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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.
Asunto(s)
Dominios C2/fisiología , Calpaína/genética , Calpaína/metabolismo , Secuencia de Aminoácidos/genética , Dominios C2/genética , Movimiento Celular , Activación Enzimática/genética , Humanos , Modelos Moleculares , Mutación/genética , Conformación Proteica , Dominios Proteicos/fisiologíaRESUMEN
Activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) are implicated in the pathophysiology of spinal cord injury (SCI). However, the specific functions of individual ERK isoforms in neurodegeneration are largely unknown. We investigated the hypothesis that ERK2 activation may contribute to pathological and functional deficits following SCI and that ERK2 knockdown using RNA interference may provide a novel therapeutic strategy for SCI. Lentiviral ERK2 shRNA and siRNA were utilized to knockdown ERK2 expression in the spinal cord following SCI. Pre-injury intrathecal administration of ERK2 siRNA significantly reduced excitotoxic injury-induced activation of ERK2 (p < 0.001) and caspase 3 (p < 0.01) in spinal cord. Intraspinal administration of lentiviral ERK2 shRNA significantly reduced ERK2 expression in the spinal cord (p < 0.05), but did not alter ERK1 expression. Administration of the lentiviral ERK2 shRNA vector 1 week prior to severe spinal cord contusion injury resulted in a significant improvement in locomotor function (p < 0.05), total tissue sparing (p < 0.05), white matter sparing (p < 0.05), and gray matter sparing (p < 0.05) 6 weeks following severe contusive SCI. Our results suggest that ERK2 signaling is a novel target associated with the deleterious consequences of spinal injury.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Traumatismos de la Médula Espinal/enzimología , Animales , Modelos Animales de Enfermedad , Femenino , Inyecciones Espinales/métodos , Laminectomía/métodos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Células PC12 , Ácido Quiscuálico , ARN Interferente Pequeño/farmacología , Ratas , Ratas Long-Evans , Traumatismos de la Médula Espinal/etiología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Transfección/métodosRESUMEN
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 < 0.05). The PrP-hCAST mice (n = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls (n = 9, p < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume (p < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls (p < 0.05, n = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Hipocampo/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Animales , Hipocampo/patología , Humanos , Locomoción/fisiología , Ratones , Ratones TransgénicosRESUMEN
There is accumulating evidence that excitotoxicity and oxidative stress resulting from excessive activation of glutamate (N-methyl-D-aspartate) NMDA receptors are major participants in striatal degeneration associated with 3-nitropropionic acid (3NP) administration. Although excitotoxic and oxidative mechanisms are implicated in 3NP toxicity, there are conflicting reports as to whether NMDA receptor antagonists attenuate or exacerbate the 3NP-induced neurodegeneration. In the present study, we investigated the involvement of NMDA receptors in striatal degeneration, protein oxidation and motor impairment following systemic 3NP administration. We examined whether NMDA receptor antagonists, memantine and ifenprodil, influence the neurotoxicity of 3NP. The development of striatal lesion and protein oxidation following 3NP administration is delayed by memantine but not affected by ifenprodil. However, in behavioral experiments, memantine failed to improve and ifenprodil exacerbated the motor deficits associated with 3NP toxicity. Together, these findings suggest caution in the application of NMDA receptor antagonists as a neuroprotective agent in neurodegenerative disorders associated with metabolic impairment.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nitrocompuestos/toxicidad , Propionatos/toxicidad , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Adenosina Difosfato/metabolismo , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Maleato de Dizocilpina/farmacología , Interacciones Farmacológicas , Masculino , Memantina/farmacología , Actividad Motora/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Piperidinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
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.
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Mebendazol/análogos & derivados , Fármacos Neuroprotectores/farmacología , Recuperación de la Función/efectos de los fármacos , Traumatismos de la Médula Espinal/patología , Médula Espinal/efectos de los fármacos , Animales , Antinematodos/farmacología , Reposicionamiento de Medicamentos , Femenino , Mebendazol/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Mitochondria isolated from synaptosomes are more sensitive to Ca2+ overload and the resultant opening of the mitochondrial permeability transition pore (mPTP) than nonsynaptic mitochondria. To identify the mechanisms underlying these differences in Ca2+ dynamics, we examined relative levels of mPTP components in synaptic versus nonsynaptic mitochondria. Synaptic mitochondria had higher levels of cyclophilin D when compared with nonsynaptic mitochondria, whereas levels of the voltage-dependent anion channel and the adenine nucleotide translocase were similar in the two mitochondrial fractions. These differences in Ca2+ handling between synaptic and nonsynaptic mitochondria were greatly reduced in cyclophilin D null [Ppif-/- (peptidylprolyl isomerase F)] mice. Higher concentrations of cyclosporine A, which interacts with cyclophilin D to delay mPTP opening, were necessary to increase the Ca2+ uptake capacity of synaptic versus nonsynaptic mitochondria. To determine whether the differences in Ca2+ handling might reflect the relative abundance of neuronal and glial mitochondria in the two mitochondrial fractions, we compared cyclophilin D levels in primary cortical neurons and astrocytes. Primary rat cortical neurons possess higher cyclophilin D levels than do primary astrocytes. In the adult rat brain, cyclophilin D immunoreactivity was abundant in neurons but sparse in astrocytes. Together, these results demonstrate that the Ca2+ handling differences observed in synaptic versus nonsynaptic mitochondria are primarily the result of the high levels of cyclophilin D in synaptic mitochondria, reflecting the greater proportion of neuronal mitochondria in this fraction. The high levels of cyclophilin D in neuronal mitochondria result in their greater vulnerability to mPT and in higher levels of cyclosporine A being required to inhibit mPTP opening.
Asunto(s)
Ciclofilinas/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Astrocitos/metabolismo , Tampones (Química) , Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Peptidil-Prolil Isomerasa F , Ciclosporina/administración & dosificación , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Concentración Osmolar , Ratas , Ratas Sprague-DawleyRESUMEN
Although calpain (calcium-activated cysteine protease) inhibition represents a rational therapeutic target for spinal cord injury (SCI), few studies have reported improved functional outcomes with post-injury administration of calpain inhibitors. This reflects the weak potency and limited aqueous solubility of current calpain inhibitors. Previously, we demonstrated that intraspinal microinjection of the calpain inhibitor MDL28170 resulted in greater inhibition of calpain activity as compared to systemic administration of the same compound. In the present study, we evaluated the ability of intraspinal MDL28170 microinjection to spare spinal tissue and locomotor dysfunction following SCI. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon impactor at the 200-kdyn force setting. Open-field locomotion was evaluated until 6 weeks post-injury. Histological assessment of tissue sparing was performed at 6 weeks after SCI. The results demonstrate that MDL28170, administered with a single post-injury intraspinal microinjection (50 nmoles), significantly improves both locomotor function and pathological outcome measures following SCI.
Asunto(s)
Calpaína/antagonistas & inhibidores , Dipéptidos/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Animales , Calpaína/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Femenino , Microinyecciones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/enzimología , Trastornos del Movimiento/fisiopatología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/enzimología , Fibras Nerviosas Mielínicas/patología , Ratas , Ratas Long-Evans , Médula Espinal/enzimología , Médula Espinal/patología , Traumatismos de la Médula Espinal/enzimología , Traumatismos de la Médula Espinal/patología , Resultado del TratamientoRESUMEN
Extensive dendritic beading of MAP2 (microtubule-associated protein 2) immunoreactivity has previously been observed in the contused rat spinal cord. However, we have also observed dendritic beading in occasional uninjured animals. The purpose of this study was to examine the possibility that perfusion conditions contributed to the dendritic beading. Under deep anesthesia, uninjured rats (adult female Long-Evans, 200-225 g) were transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde at cold (4 degrees C) or warm (20 degrees C) temperature, and at a low (20 ml/min) or high (50 ml/min) flow rate. Dendrites were visualized by MAP2 immunoreactivity. The results demonstrate that perfusion with cold solutions at a high flow rate induces pronounced dendritic beading, and when perfused at a low flow rate, results in moderate dendritic beading. Warm perfusates did not induce dendritic beading when administered at a low flow rate, but occasional beading was observed with a high flow rate. Western blots revealed spectrin breakdown, but not MAP2 loss, in rats perfused with cold saline solution at a high flow rate, conditions that also resulted in dendritic beading. These findings demonstrate that dendritic morphology is sensitive to both temperature and flow rate of the perfusate. Warm fixative and a low perfusion flow rate minimized the perfusion-induced dendritic beading.
Asunto(s)
Frío , Dendritas/efectos de los fármacos , Fijadores/farmacología , Formaldehído/farmacología , Polímeros/farmacología , Cloruro de Sodio/farmacología , Traumatismos de la Médula Espinal/patología , Análisis de Varianza , Animales , Frío/efectos adversos , Dendritas/metabolismo , Dendritas/patología , Femenino , Proteínas Asociadas a Microtúbulos/metabolismo , Perfusión/efectos adversos , Perfusión/métodos , Ratas , Ratas Long-Evans , Espectrina/metabolismoRESUMEN
Mild traumatic brain injury (mTBI) affects millions of people annually and is difficult to diagnose. Mild injury is insensitive to conventional imaging techniques and diagnoses are often made using subjective criteria such as self-reported symptoms. Many people who sustain a mTBI develop persistent post-concussive symptoms. Athletes and military personnel are at great risk for repeat injury which can result in second impact syndrome or chronic traumatic encephalopathy. An objective and quantifiable measure, such as a serum biomarker, is needed to aid in mTBI diagnosis, prognosis, return to play/duty assessments, and would further elucidate mTBI pathophysiology. The majority of TBI biomarker research focuses on severe TBI with few studies specific to mild injury. Most studies use a hypothesis-driven approach, screening biofluids for markers known to be associated with TBI pathophysiology. This approach has yielded limited success in identifying markers that can be used clinically, additional candidate biomarkers are needed. Innovative and unbiased methods such as proteomics, microRNA arrays, urinary screens, autoantibody identification and phage display would complement more traditional approaches to aid in the discovery of novel mTBI biomarkers.
Asunto(s)
Lesiones Encefálicas/sangre , Lesiones Encefálicas/inducido químicamente , Animales , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Lesiones Encefálicas/diagnóstico , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/líquido cefalorraquídeo , MicroARNs/sangre , MicroARNs/líquido cefalorraquídeo , Personal Militar , Síndrome Posconmocional/sangre , Síndrome Posconmocional/líquido cefalorraquídeo , Síndrome Posconmocional/diagnósticoRESUMEN
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 (1mm injury depth, 3.5m/s velocity) and phage display was utilized to identify putative serum biomarkers at 6h 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.
Asunto(s)
Bacteriófagos , Análisis Químico de la Sangre/métodos , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/diagnóstico , Técnicas de Visualización de Superficie Celular , Proteína Ácida Fibrilar de la Glía/sangre , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Bacteriófagos/metabolismo , Biomarcadores/sangre , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones Endogámicos C57BL , Lóbulo Parietal , Biblioteca de PéptidosRESUMEN
Selenium is an essential element required for activity of several antioxidant enzymes, including glutathione peroxidase. Because of the critical role of the antioxidant system in responding to traumatic events, we hypothesized that dietary selenium supplementation would enhance neuroprotection in a rodent model of spinal cord injury. Rats were maintained on either a control or selenium-enriched diet prior to, and following, injury. Dietary selenium supplementation, provided as selenized yeast added to normal rat chow, resulted in a doubling of selenium levels in the spinal cord. Dietary selenium reduced the time required for recovery of bladder function following thoracic spinal cord injury. However, this was not accompanied by improvement in locomotor function or tissue sparing.
Asunto(s)
Suplementos Dietéticos , Locomoción/efectos de los fármacos , Compuestos de Organoselenio/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Animales , Suplementos Dietéticos/análisis , Femenino , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Vejiga Urinaria/fisiopatologíaRESUMEN
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.