RESUMO
The early-stage pathologies of frontotemporal lobal degeneration (FTLD) remain largely unknown. In VCPT262A-KI mice carrying VCP gene mutation linked to FTLD, insufficient DNA damage repair in neural stem/progenitor cells (NSCs) activated DNA-PK and CDK1 that disabled MCM3 essential for the G1/S cell cycle transition. Abnormal neural exit produced neurons carrying over unrepaired DNA damage and induced early-stage transcriptional repression-induced atypical cell death (TRIAD) necrosis accompanied by the specific markers pSer46-MARCKS and YAP. In utero gene therapy expressing normal VCP or non-phosphorylated mutant MCM3 rescued DNA damage, neuronal necrosis, cognitive function, and TDP43 aggregation in adult neurons of VCPT262A-KI mice, whereas similar therapy in adulthood was less effective. The similar early-stage neuronal necrosis was detected in PGRNR504X-KI, CHMP2BQ165X-KI, and TDPN267S-KI mice, and blocked by embryonic treatment with AAV-non-phospho-MCM3. Moreover, YAP-dependent necrosis occurred in neurons of human FTLD patients, and consistently pSer46-MARCKS was increased in cerebrospinal fluid (CSF) and serum of these patients. Collectively, developmental stress followed by early-stage neuronal necrosis is a potential target for therapeutics and one of the earliest general biomarkers for FTLD.
Assuntos
Degeneração Lobar Frontotemporal/patologia , Células-Tronco Neurais/metabolismo , Proteína com Valosina/metabolismo , Animais , Ciclo Celular , Linhagem da Célula/genética , Células Cultivadas , Dano ao DNA/genética , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/líquido cefalorraquidiano , Degeneração Lobar Frontotemporal/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Necrose/metabolismo , Necrose/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Proteína com Valosina/genéticaRESUMO
ß-Galactosidase encoded by the Escherichia coli lacZ gene, is widely used as a reporter molecule in molecular biology in a wide variety of animals. ß-Galactosidase retains its enzymatic activity in cells or tissues even after fixation and can degrade X-Gal, a frequently used colormetric substrate, producing a blue color. Therefore, it can be used for the activity staining of fixed tissues. However, the enzymatic activity of the ß-galactosidase that is ectopically expressed in the non-fixed tissues of animals has not been extensively studied. Here, we report the characterization of ß-galactosidase activity in Drosophila tissues with and without fixation in various experimental conditions comparing the activity of two evolutionarily orthologous ß-galactosidases derived from the E. coli lacZ and Drosophila melanogaster DmelGal genes. We performed quantitative analysis of the activity staining of larval imaginal discs and an in vitro assay using larval lysates. Our data showed that both E. coli and Drosophila ß-galactosidase can be used for cell-type-specific activity staining, but they have their own preferences in regard to conditions. E. coli ß-galactosidase showed a preference for neutral pH but not for acidic pH compared with Drosophila ß-galactosidase. Our data suggested that both E. coli and Drosophila ß-galactosidase show enzymatic activity in the physiological conditions of living animals when they are ectopically expressed in a desired specific spatial and temporal pattern. This may enable their future application to studies of chemical biology using model animals.