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1.
Acta Neuropathol ; 130(1): 49-61, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25788357

RESUMO

The presence of lower molecular weight species comprising the C-terminal region of TAR DNA-binding protein 43 (TDP-43) is a characteristic of TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we have identified a novel splice variant of TDP-43 that is upregulated in ALS and generates a 35-kDa N-terminally truncated species through use of an alternate translation initiation codon (ATG(Met85)), denoted here as Met(85)-TDP-35. Met(85)-TDP-35 expressed ectopically in human neuroblastoma cells exhibited reduced solubility, cytoplasmic distribution, and aggregation. Furthermore, Met(85)-TDP-35 sequestered full-length TDP-43 from the nucleus to form cytoplasmic aggregates. Expression of Met(85)-TDP-35 in primary motor neurons resulted in the formation of Met(85)-TDP-35-positive cytoplasmic aggregates and motor neuron death. A neo-epitope antibody specific for Met(85)-TDP-35 labeled the 35-kDa lower molecular weight species on immunoblots of urea-soluble extracts from ALS-FTLD disease-affected tissues and co-labeled TDP-43-positive inclusions in ALS spinal cord sections, confirming the physiological relevance of this species. These results show that the 35-kDa low molecular weight species in ALS-FTLD can be generated from an abnormal splicing event and use of a downstream initiation codon and may represent a mechanism by which TDP-43 elicits its pathogenicity.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Morte Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Corpos de Inclusão/metabolismo , Neurônios Motores/metabolismo , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Proteínas de Ligação a DNA/genética , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Humanos , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia
2.
Mol Cell Neurosci ; 47(3): 167-80, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21421050

RESUMO

TDP-43 is a predominantly nuclear DNA/RNA binding protein involved in transcriptional regulation and RNA processing. TDP-43 is also a component of the cytoplasmic inclusion bodies characteristic of amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). We have investigated the premise that abnormalities of TDP-43 in disease would be reflected by changes in processing of its target RNAs. To this end, we have firstly identified RNA targets of TDP-43 using UV-Cross-Linking and Immunoprecipitation (UV-CLIP) of SHSY5Y cells, a human neuroblastoma cell line. We used conventional cloning strategies to identify, after quality control steps, 127 targets. Results show that TDP-43 binds mainly to introns at UG/TG repeat motifs (49%) and polypyrimidine rich sequences (17.65%). To determine if the identified RNA targets of TDP-43 were abnormally processed in ALS versus control lumbar spinal cord RNA, we performed RT-PCR using primers designed according to the location of TDP-43 binding within the gene, and prior evidence of alternative splicing of exons adjacent to this site. Of eight genes meeting these criteria, five were differentially spliced in ALS versus control. This supports the premise that abnormalities of TDP-43 in ALS are reflected in changes of RNA processing.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA/metabolismo , Medula Espinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Linhagem Celular Tumoral , Clonagem de Organismos , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Imunoprecipitação/métodos , Masculino , Pessoa de Meia-Idade , RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
3.
Ann Neurol ; 66(1): 75-80, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19670443

RESUMO

OBJECTIVE: In familial amyotrophic lateral sclerosis (fALS) harboring superoxide dismutase (SOD1) mutations (fALS1), SOD1 toxicity has been linked to its propensity to misfold and aggregate. It has recently been proposed that misfolded SOD1 may be causative of all types of ALS, including sporadic cases (sALS). In the present study, we have used a specific antibody to test for the presence of monomer/misfolded SOD1 in sALS. METHODS: Sections from lumbar spinal cords of 5 fALS1 cases, 13 sALS cases, and 1 non-SOD1 fALS case were labeled immunocytochemically using SOD1-exposed-dimer-interface (SEDI) antibody, which we have previously validated as being specific for pathological monomer/misfolded forms of SOD1. RESULTS: Monomer/misfolded SOD1 was detected with SEDI antibody in all 5 of the fALS1 cases, localizing predominantly to hyaline conglomerate inclusions, a specific pathological feature of fALS1. In contrast, monomer/misfolded SOD1 was not detected in any of the 13 sALS cases or in the non-SOD1 fALS cases. These results were confirmed by immunoprecipitation. INTERPRETATION: Although SEDI antibody does not necessarily label all misfolded forms of SOD1, these findings show a distinct difference between fALS1 and sALS, and do not support that monomer/misfolded SOD1 is a common disease entity linking all types of ALS. This is important to our understanding of ALS disease pathogenesis and to considerations of the applicability of using therapeutics that target misfolded SOD1 to non-SOD1-related cases. Ann Neurol 2009;66:75-80.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Dobramento de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase-1
4.
J Neurosci ; 28(8): 1833-40, 2008 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-18287500

RESUMO

The neuronal intermediate filament protein peripherin is a component of ubiquitinated inclusions and of axonal spheroids in amyotrophic lateral sclerosis (ALS). Overexpression of peripherin causes motor neuron degeneration in transgenic mice and variations within the peripherin gene have been identified in ALS cases. We have shown previously the abnormal expression of a neurotoxic peripherin splice variant in transgenic mice expressing mutant superoxide dismutase-1. These findings indicated that abnormalities of peripherin splicing may occur in ALS. In the current study, peripherin splice variants were identified by reverse transcription-PCR of human neuronal RNA and comparisons in expression made between control and ALS spinal cord using Western blot analysis and immunocytochemistry. Using this approach we have identified a novel peripherin transcript retaining introns 3 and 4 that results in a 28 kDa splice isoform, designated Per 28. Using an antibody specific to Per 28, we show that this isoform is expressed at low stoichiometric levels from the peripherin gene, however causes peripherin aggregation when its expression is upregulated. Importantly we show an upregulation of Per 28 expression in ALS compared with controls, at both the mRNA and protein levels, and that Per 28 is associated with disease pathology, specifically round inclusions. These findings are the first to establish that peripherin splicing abnormalities occur in ALS, generating aggregation-prone splice isoforms.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/genética , Íntrons/fisiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Regulação para Cima/genética , Adolescente , Adulto , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Masculino , Glicoproteínas de Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Periferinas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia
5.
J Neuropathol Exp Neurol ; 66(12): 1147-53, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18090923

RESUMO

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the presence of various types of ubiquitinated inclusions in the cytoplasm of affected motor neurons. The identification of the ubiquitinated targets within these inclusions has represented a major challenge, as this may provide new gene candidates and/or clues to understanding the neurodegenerative mechanism(s) underlying the disease. As such, the nuclear factor TAR DNA-binding protein (TDP-43) was recently identified as a component of ubiquitinated skein-like inclusions and round inclusions in ALS. This identification combined with biochemical evidence led to the suggestion that TDP-43 is the key ubiquitinated target and major disease protein in ALS. Here, using 3-dimensional deconvolution imaging, we have obtained remarkable resolution of skein-like inclusions and round inclusions in ALS. Surprisingly we have found that in contrast to current thinking, TDP-43 is not the major ubiquitinated target within these types of inclusions. These findings raise the possibility that TDP-43 may not necessarily be the key disease protein in ALS and indicate that the major target(s) of ubiquitination remain to be identified.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Ubiquitina/metabolismo , Idoso , Feminino , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Periferinas
6.
Neurosci Lett ; 420(2): 128-32, 2007 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-17543992

RESUMO

Mislocalization of the TAR-DNA binding protein (TDP-43) from the nucleus to the cytoplasm of diseased motor neurons and association with intraneuronal ubiquitinated inclusions has recently been reported in amyotrophic lateral sclerosis (ALS). Here, we have investigated TDP-43 immunoreactivity in three lines of mutant SOD1 transgenic mice, G93A, G37R and G85R and compared with labeling in one sporadic ALS case and two familial ALS cases carrying mutations in SOD1, A4T and I113T. Our findings show that there is no mislocalization of TDP-43 to the cytoplasm in motor neurons of mutant SOD1 transgenic mice, nor association of TDP-43 with ubiquitinated inclusions. In contrast, mislocalization of TDP-43 to the cytoplasm and association with ubiquitinated inclusions was found in the ALS cases, including those carrying mutations in SOD1. Interestingly, there was no association of TDP-43 with ubiquitinated hyaline conglomerate inclusions, pathology closely associated with ALS cases carrying mutations in SOD1. Our findings indicate that the process of motor neuron degeneration in mutant SOD1 transgenic mice is unlikely to involve the abnormalities of TDP-43 described in the human disease.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença/genética , Mutação/genética , Superóxido Dismutase/genética , Transporte Ativo do Núcleo Celular/genética , Adulto , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Transporte Proteico/genética , Superóxido Dismutase-1
7.
J Neurol Sci ; 256(1-2): 39-51, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17368487

RESUMO

We have previously shown that the co-localization of neuronal nitric oxide synthase (nNOS) with neurofilament (NF) aggregates in motor neurons derived from transgenic mice over-expressing the human low molecular weight NF protein (hNFL+/+) is associated with a deregulation of calcium influx via the N-methyl-d-aspartate (NMDA) receptor, resulting in apoptosis. Because the absence of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor confers calcium permeability and has been implicated in the process of excitotoxicity in ALS, we have examined the role of the AMPA receptor in this model. GluR2 protein expression and mRNA were examined in hNFL+/+ and wild-type motor neurons (wt). Live cell calcium imaging was performed using Oregon-Green Bapta and Fura-2 calcium dyes. For apoptotic studies, neurons were treated with glutamate, with or without glutamate receptor antagonists [6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) or (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801)] and examined for active caspase-3 or phospholipid inversion. We observed that although both GluR2 mRNA and protein levels were decreased in hNFL+/+ motor neurons compared to wt, there was no appreciable calcium influx via the AMPA receptor. These studies demonstrate that calcium mediated excitotoxicity in NF aggregate-bearing neurons is NMDA receptor dependant.


Assuntos
Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios Motores/metabolismo , Proteínas de Neurofilamentos/metabolismo , Receptores de AMPA/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Proteínas de Neurofilamentos/genética , Receptores de AMPA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Medula Espinal/citologia , Fatores de Tempo
8.
Free Radic Biol Med ; 42(1): 143-51, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17157201

RESUMO

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which excitotoxicity has been implicated as a cause for cell death. To examine neurofilament (NF) aggregate-mediated sensitization of motor neurons to NMDA excitotoxicity, we examined NMDA receptor expression and the impact of NO donors (NOC12 or NOC5) or sodium cyanide (NaCN) on calcium influx and viability in dissociated motor neurons derived from wt and hNFL+/+ (NF aggregate-forming) mice. Alterations in intracellular calcium were assayed using Oregon Green calcium dye and the extent of apoptosis using active caspase-3 immunoreactivity. Although NF aggregate-bearing neurons demonstrated increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation, this was not associated with increased NMDA receptor expression. The down-regulation of the NMDA receptor using NO donors decreased calcium influx and caspase-3 activation in aggregate-bearing neurons, but had no effect on wt cultures. The converse was observed with NaCN in which intracellular calcium levels increased significantly in wt cultures in association with increased cell death. No effect was observed in aggregate-bearing neurons. These findings suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity, and that this can be reversed by NO.


Assuntos
Agregação Celular/fisiologia , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Óxido Nítrico/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Caspase 3/metabolismo , Agregação Celular/efeitos dos fármacos , Morte Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença dos Neurônios Motores/patologia , Neurônios Motores/patologia , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/fisiologia , Compostos Nitrosos/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Cianeto de Sódio/farmacologia
9.
Neurobiol Dis ; 23(2): 342-50, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16730180

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3' Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.


Assuntos
Doença dos Neurônios Motores/genética , Mutação , RNA Mensageiro/genética , Superóxido Dismutase/genética , Humanos , Doença dos Neurônios Motores/enzimologia , Neurônios , RNA/genética , RNA/isolamento & purificação , Sondas RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteínas/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Transcrição Gênica
10.
Neurosci Res ; 55(1): 87-95, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16529832

RESUMO

We have previously reported that supernatant derived from LPS-activated BV-2 cells, an immortalized microglial cell line, induces death of NSC-34 cells (a motor neuron hybridoma) through a TNFalpha and nitric oxide synthase (NOS) dependant mechanism. In this study, we have observed that LPS-activated BV-2 supernatant induces NSC-34 cell death in association with an upregulation of the TNF receptor 1 (TNFR1) expression on NSC-34 cells, both at the transcription level and at the cell surface protein level. The upregulation of TNFR1 receptor was independent of TNFalpha, and could be partly inhibited by the inhibition of iNOS activation in the BV-2 cells. The TNFR2 receptor was not involved. These observations have important implications in understanding the mechanism by which microglial activation contributes to the motor neuron degeneration.


Assuntos
Meios de Cultivo Condicionados/toxicidade , Microglia/metabolismo , Neurônios Motores/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Imunofluorescência/métodos , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , RNA Mensageiro/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sais de Tetrazólio , Tiazóis , Fator de Necrose Tumoral alfa/imunologia
11.
Glia ; 52(1): 59-69, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15920739

RESUMO

Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low-molecular-weight NF subunit [NFL (-/-)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild-type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein-70 (HSP-70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (-/-) mice demonstrated significant astrogliosis and HSP-70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non-neuronal cells may differ significantly depending on the etiology of the NF aggregate itself.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Gliose/fisiopatologia , Degeneração Neural/fisiopatologia , Proteínas de Neurofilamentos/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Caspase 3 , Caspases/metabolismo , Morte Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Gliose/genética , Gliose/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Fatores de Tempo
12.
Brain Res ; 1004(1-2): 8-17, 2004 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-15033415

RESUMO

The significance of copper/zinc superoxide dismutase (SOD1) and neuronal nitric oxide synthase (nNOS) co-localization to neurofilamentous (NF) aggregates in amyotrophic lateral sclerosis (ALS) is unknown. In this study, we have used dissociated motor neurons from either C57BL/6 or mice that over-express the human low molecular weight neurofilament protein (hNFL+/+) to examine the relationship between NF aggregate formation, SOD1 and nNOS co-localization, and the regulation of NMDA-mediated calcium influx in vitro. The intracellular distribution of NF aggregates, SOD1 and nNOS was examined by confocal microscopy and NMDA-induced alterations in intracellular calcium levels using either Oregon green fluorescence or FURA-2 photometric imaging. Cell death was assessed using an antibody to activated caspase-3. C57 Bl/6 motor neurons expressed nNOS in a punctate manner, whereas SOD1 was distributed homogeneously throughout the cytosol. In contrast, hNFL+/+ motor neurons demonstrated co-localization of SOD1 and nNOS by day 9 post-plating, preceding the formation of NF aggregates. Both proteins co-localized to NF aggregates once formed. With NMDA stimulation, aggregate-bearing hNFL+/+ motor neurons demonstrated significant increases in intracellular calcium, whereas only a minimal alteration in intracellular calcium was observed in C57 Bl/6 neurons. Following stimulation with 100 microM NMDA, 75.5+/-5.5% of hNFL+/+ neurons became apoptotic, whereas only 16.3+/-5.3% of C57 Bl/6 were. These observations suggest that the presence of NF aggregates results in a failure of regulation of NMDA-mediated calcium influx, and that this occurs due to the sequestration of nNOS to the NF aggregate, preventing its down-regulation of the NMDA receptor.


Assuntos
Cálcio/metabolismo , N-Metilaspartato/metabolismo , Proteínas de Neurofilamentos/biossíntese , Neurônios/metabolismo , Óxido Nítrico Sintase/biossíntese , Animais , Agregação Celular/efeitos dos fármacos , Agregação Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Proteínas de Neurofilamentos/análise , Neurônios/química , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo I
13.
Brain Res ; 976(2): 149-58, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12763249

RESUMO

Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.


Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , N-Metilaspartato/farmacologia , Neurônios/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Diferenciação Celular , Membrana Celular/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I , Células PC12 , Ratos , Frações Subcelulares/enzimologia
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