RESUMO
Following cerebral injuries such as stroke, a structural and functional reorganization of the impaired tissue occurs, which is often accompanied by a re-expression of developmental genes. During brain development, embryonic splice variants of the GABA-synthesizing GAD67 gene (collectively termed EGAD) participate in cell proliferation, migration, and neuronal differentiation. We thus hypothesized an involvement of EGAD in post-ischemic plasticity. EGAD transcripts were up-regulated at early reperfusion times in the injured area following transient middle cerebral artery occlusion (with a peak expression of 4.5-fold at 6h in C57BL/6 mice). Cell-specific analysis by a combination of radioactive in situ hybridization and immunolabeling revealed EGAD up-regulation in TUNEL-positive neurons. This unexpected cell death-associated expression of EGAD was confirmed in cell culture models of ischemia (combined oxygen-glucose deprivation) and apoptosis (staurosporine). Staurosporine-mediated cell death led to cleaved Caspase-3 activation, a key regulator of apoptosis following stroke. Blocking of staurosporine-associated EGAD expression via antisense RNA treatment reduced cleaved Caspase-3 activation by ~30%. In addition to the involvement of EGAD in proliferative processes during brain development, we found here that EGAD participates in cell death under pathophysiological conditions in the adult brain. Re-expression of EGAD in neurons following stroke may play a role in aberrant cell cycle activation, consequently being pro-apoptotic. Our observation of a new GABA related pro-apoptotic mechanism and its successful modification might be of significant clinical relevance.
Assuntos
Apoptose/fisiologia , Isquemia Encefálica/fisiopatologia , Encéfalo/fisiopatologia , Glutamato Descarboxilase/metabolismo , Neurônios/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Caspase 3/metabolismo , Hipóxia Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Glucose/deficiência , Glutamato Descarboxilase/genética , Infarto da Artéria Cerebral Média , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Ratos Wistar , Estaurosporina/toxicidadeRESUMO
BACKGROUND AND PURPOSE: The potassium chloride cotransporter 2 (KCC2) is the main neuronal chloride extruder in the adult nervous system. Therefore, KCC2 is responsible for an inwardly directed electrochemical gradient of chloride that leads to hyperpolarizing GABA-mediated responses. Under some pathophysiological conditions, GABA has been reported to be depolarizing because of a downregulation of KCC2. This is the first study to our knowledge analyzing the expression of KCC2 after a focal cerebral ischemia. METHODS: Mild and severe ischemia were induced in rats by a transient occlusion of the middle cerebral artery for 30 and 120 minutes, respectively. KCC2 mRNA and protein expression were studied in the ischemic hemisphere after different reperfusion times (2 hour, 1 day, 7 days, 30 days, 168 days) by using quantitative polymerase chain reaction, Western blotting, and immunohistological staining. RESULTS: We found a substantial decrease of KCC2 mRNA and protein levels in the ischemic hemisphere, with a stronger downregulation of KCC2 after severe vs mild ischemia. Long-term surviving cells expressing KCC2 could be detected in the infarct core. These cells were identified as GABAergic interneurons mainly expressing parvalbumin. CONCLUSIONS: Our study revealed a substantial neuron-specific downregulation of KCC2 after focal cerebral ischemia.
Assuntos
Cloretos/metabolismo , Regulação para Baixo/fisiologia , Ataque Isquêmico Transitório/metabolismo , Potássio/metabolismo , Simportadores/antagonistas & inibidores , Animais , Ataque Isquêmico Transitório/patologia , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Simportadores/metabolismo , Fatores de Tempo , Cotransportadores de K e Cl-RESUMO
Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4ß3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors.
Assuntos
Neurônios GABAérgicos/fisiologia , Atividade Motora , Neurônios Motores/fisiologia , Regeneração , Convulsões , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologia , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ácido Glutâmico/metabolismo , Camundongos , Proteoma/análise , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
BACKGROUND: The hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency. METHODS: We generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients. RESULTS: Homozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established. CONCLUSIONS: Our data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models.
Assuntos
Variação Genética/genética , Mutação/genética , Proteínas/genética , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Paraplegia/diagnóstico , Paraplegia/genética , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/genéticaRESUMO
Age-induced neuroinflammation could be a contributing factor to the restricted neurogenesis in aged mice. Indomethacin, a common non-steroidal anti-inflammatory drug, has been demonstrated to partially restore neurogenesis under pathophysiological inflammation-associated conditions in adult C57BL/6 mice. This study investigated whether indomethacin is able to decrease age-related neuroinflammation in the hippocampus (24-month-old mice) and thereby stimulate neurogenesis. During hippocampal aging, the transcript expression of pro-inflammatory cytokines (Tnfα, Il-1α, Il-1ß), the chemokine Mip-1α, and markers for activated astrocytes (Gfap, Lcn2, but not Vim and Serpina3n) and microglia (Iba1, F4/80, Cd68, Cd86) significantly increased. Treatment with indomethacin significantly decreased COX-1 and COX-2 transcript expression. Of the age-related inflammatory mediators, only Gfap and Iba1 were affected by indomethacin treatment in the hippocampus, with a significantly reduced transcript expression being detected for both markers. Neurogenesis was unaffected by indomethacin. Thus, our data reveal that administration of indomethacin to aged mice is not able to effectively decrease neuroinflammation and promote neurogenesis.
Assuntos
Envelhecimento/patologia , Anti-Inflamatórios não Esteroides/farmacologia , Hipocampo/efeitos dos fármacos , Indometacina/farmacologia , Inflamação/patologia , Neurogênese/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Contagem de Células , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Microglia/metabolismo , Microglia/patologiaRESUMO
Increased age is a major risk factor for stroke incidence and post-ischemic mortality. To develop age-adjusted therapeutic interventions, a clear understanding of the complexity of age-related post-ischemic mechanisms is essential. Transient occlusion of the middle cerebral artery--a model that closely resembles human stroke--was used to induce cerebral infarction in mice of 4 different ages (2, 9, 15, 24 months). By using Illumina cDNA microarrays and quantitative PCR we detected a distinct age-dependent response to stroke involving 350 differentially expressed genes. Our analyses also identified 327 differentially expressed genes that responded to stroke in an age-independent manner. These genes are involved in different aspects of the inflammatory and immune response, oxidative stress, cell cycle activation and/or DNA repair, apoptosis, cytoskeleton reorganization and/or astrogliosis, synaptic plasticity and/or neurotransmission, and depressive disorders and/or dopamine-, serotonin-, GABA-signaling. In agreement with our earlier work, aged brains displayed an attenuated inflammatory and immune response (Sieber et al., 2011) and a reduced impairment of post-stroke synaptic plasticity. Our data also revealed a distinct age-related susceptibility for post-ischemic depression, the most common neuropsychiatric consequence of stroke, which has a major influence on functional outcome.
Assuntos
Envelhecimento/genética , Acidente Vascular Cerebral/genética , Transcrição Gênica/genética , Envelhecimento/imunologia , Animais , Encéfalo/imunologia , Encéfalo/fisiopatologia , Ciclo Celular/genética , Reparo do DNA/genética , Transtorno Depressivo/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Predisposição Genética para Doença , Humanos , Inflamação/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Regeneração Nervosa , Plasticidade Neuronal/genética , Estresse Oxidativo/genética , Fatores de Risco , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/fisiopatologia , Sinapses/fisiologiaRESUMO
BACKGROUND: Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear. METHODS AND RESULTS: As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1ß, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed. CONCLUSIONS: Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.
Assuntos
Apoptose , Inflamação/metabolismo , Glicoproteínas de Membrana/fisiologia , Células Mieloides/citologia , Receptores Imunológicos/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Encéfalo/patologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Homeostase , Imuno-Histoquímica , Inflamação/patologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Artéria Cerebral Média/patologia , Células Mieloides/metabolismo , Fagocitose , Receptores Imunológicos/metabolismo , ReperfusãoRESUMO
BACKGROUND: Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia. METHODOLOGY/PRINCIPAL FINDINGS: Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed. CONCLUSIONS/SIGNIFICANCE: TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study.