RESUMO
Alzheimer's disease (AD) is the leading cause of dementia and has no cure. Genetic, cell biological, and biochemical studies suggest that reducing amyloid-ß (Aß) production may serve as a rational therapeutic avenue to delay or prevent AD progression. Inhibition of RhoA, a Rho GTPase family member, is proposed to curb Aß production. However, a barrier to this hypothesis has been the limited understanding of how the principal downstream effectors of RhoA, Rho-associated, coiled-coil containing protein kinase (ROCK) 1 and ROCK2, modulate Aß generation. Here, we report that ROCK1 knockdown increased endogenous human Aß production, whereas ROCK2 knockdown decreased Aß levels. Inhibition of ROCK2 kinase activity, using an isoform-selective small molecule (SR3677), suppressed ß-site APP cleaving enzyme 1 (BACE1) enzymatic action and diminished production of Aß in AD mouse brain. Immunofluorescence and confocal microscopy analyses revealed that SR3677 alters BACE1 endocytic distribution and promotes amyloid precursor protein (APP) traffic to lysosomes. Moreover, SR3677 blocked ROCK2 phosphorylation of APP at threonine 654 (T654); in neurons, T654 was critical for APP processing to Aß. These observations suggest that ROCK2 inhibition reduces Aß levels through independent mechanisms. Finally, ROCK2 protein levels were increased in asymptomatic AD, mild cognitive impairment, and AD brains, demonstrating that ROCK2 levels change in the earliest stages of AD and remain elevated throughout disease progression. Collectively, these findings highlight ROCK2 as a mechanism-based therapeutic target to combat Aß production in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/biossíntese , Quinases Associadas a rho/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , DNA/genética , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Imuno-Histoquímica , Lentivirus/genética , Camundongos , Microscopia Confocal , Plasmídeos/genética , Técnicas Estereotáxicas , Espectrometria de Massas em TandemRESUMO
Our understanding of Alzheimer's disease (AD) pathophysiology remains incomplete. Here we used quantitative mass spectrometry and coexpression network analysis to conduct the largest proteomic study thus far on AD. A protein network module linked to sugar metabolism emerged as one of the modules most significantly associated with AD pathology and cognitive impairment. This module was enriched in AD genetic risk factors and in microglia and astrocyte protein markers associated with an anti-inflammatory state, suggesting that the biological functions it represents serve a protective role in AD. Proteins from this module were elevated in cerebrospinal fluid in early stages of the disease. In this study of >2,000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brains that may serve as therapeutic targets and fluid biomarkers for the disease.