RESUMO
We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr(175) (pThr(175)) in association with GSK3ß activation. We have now examined whether pThr(175) induces GSK3ß activation and whether this leads to pathologic fibril formation through Thr(231) phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr(175)Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3ß) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3ß inhibitors or small hairpin RNA knockdown of GSK3ß abolished fibril formation and prevented cell death. Inhibition of Thr(231) phosphorylation (Thr(231)Ala) prevented pathologic tau fibril formation, regardless of Thr(175) state, whereas Thr(231)Asp (pseudophosphorylated at Thr(231)) developed pathologic tau fibrils. Ser(235) mutations did not affect fibril formation, indicating an unprimed mechanism of Thr(231) phosphorylation. These findings suggest a mechanism of tau pathology by which pThr(175) induces GSK3ß phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Neurofibrilas/patologia , Proteínas tau/metabolismo , Motivos de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Morte Celular/genética , Células Cultivadas , Biologia Computacional , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Fosforilação , RNA Interferente Pequeno/genética , Tauopatias/genética , Tauopatias/patologia , Tauopatias/terapia , Treonina/metabolismo , Proteínas tau/químicaRESUMO
In NFL-/- mice, a model of motor neuron degeneration in ALS, degenerating spinal motor neurons express high levels of the receptor for the C5a anaphylatoxin (C5aR) early in the disease process. C5a is a potent in vitro neurotoxin for both Neuro2A and NGF-differentiated PC12 cells. While no interaction was observed between glutamate and C5a, both C5a and kainate upregulated the expression of activated C5aR. C5aR expression was increased in motor neurons in ALS. This data suggests that the early upregulation of C5aR may contribute to motor neuron damage that potentiates excitotoxicity in ALS.
Assuntos
Doença dos Neurônios Motores/imunologia , Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Proteínas de Neurofilamentos/genética , Receptor da Anafilatoxina C5a/metabolismo , Regulação para Cima/imunologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Complemento C5a/farmacologia , Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença dos Neurônios Motores/genética , Neurônios Motores/imunologia , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/imunologia , Degeneração Neural/metabolismo , Neurotoxinas/farmacologia , Receptor da Anafilatoxina C5a/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Although amyotrophic lateral sclerosis (ALS) can be associated with cognitive impairment (ALSci) as a reflection of frontotemporal lobar degeneration, the basis of this process is unknown. The observation of neuronal and extraneuronal tau deposition in ALSci in addition to a unique tau phosphorylation at Thr175 has suggested that ALSci can be associated with alterations in tau metabolism. We have examined the association between phosphorylation at Thr175 and tau fibril formation. Both soluble and insoluble tau was purified from control, patients with Alzheimer's disease (AD), ALS without cognitive impairment, and ALSci and the tendency to fibril formation assayed ex vivo using the thioflavin S fluorescence assay. The extent of fibril formation was significantly greater in tau derived from ALSci, with ALS-derived tau being intermediate between control and AD-derived tau. Using both Neuro2A and human embryonic kidney (HEK293T) cells, we expressed full-length tau constructs harboring either a pseudophosphorylation at Thr175 (Thr175-Asp-tau), inhibition of Thr175 phosphorylation (Thr175-Ala-tau) or intact tau (wild-type tau). Both tau fibril formation and cell death were significantly enhanced in the presence of Thr175-Asp-tau, regardless of the tau isoform, suggesting that phosphorylation of Thr175 is associated with tau fibril formation in ALSci.
