RESUMO
TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desacetilase 6 de Histona , Humanos , Neurônios/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/metabolismoRESUMO
The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.
