PU.1 Is A Key Regulator For Amyloid β Protein Induced TREM2 Gene Expression, And Cellular Senescence In A Transgenic Model Of Alzheimer’s Disease

Object Amyloid β(Aβ) which may stimuli and determine the changes of microglial phenotypes has been established as a key factor for the pathological changes in Alzheimer’s disease(AD). Triggering receptor expressed on myeloid cells-2(TREM2)is a newly identified receptor which mutation significantly increases the risk of late-onset Alzheimer’ disease(LOAD). Although the up-regulation of TREM2 have been reported in AD mice models and patient, the precise regulated mechanism of TREM2 in AD remains unclear. Transcription factor PU.1/Spi1 plays a pivotal role in microglia activation and immune response. In this study, we aimed to elucidate the age-related gene expression profile of TREM2, DAP12, PU.1, and the transcriptional regulation relationship between TREM2 and PU.1 in 5XFAD mice and then explore the potential mechanisms. In addition, cellular senescence is vital component of brain aging and closely associated with cognitive impairment. However, it remains blurred whether Aβ accelerates the neuronal senescence which may have some potential role in AD.Therefore, this study also explore preliminarily the relationship between senescence-associated genes and cognitive deficit in 5XFAD mice.Methods The brain slices or.hippocampal tissues from 5XFAD transgenic mice and age match wild type controls of different months old were used to detect the expression of TREM2 and PU.1, followed by q PCR, western blotting, immunohistochemistry or immunofluorescence staining. The situmulation of o Aβ and f Aβ to BV2 microglial cell and primary microglia in vtro were probe for comfirm the the phenomenon in vivo.Whereafter, Cell models of knockdown and overexpression of PU.1 were established by PU.1 silencing RNA and PU.1 overexpression vector which were mediated by lentiviral vectors. Ch IP-PCR, EMSA and promoter luciferase reporter gene activity were performed to explore the transcription regulation mechanisms between TREM2 and PU.1. Also, the effects of lentiviral particles(TREM2 sh RNA virus or TREM2 OE virus)on PU.1 gene expression levels in BV2 cells were confirmed by western immunoblot.Phagocytosis of f Aβ and nile red microspheres were used to evaluate the phagocytosis function of microglia. The senescence-associated genes in the hippocampus were analyzeda nd screened by q PCR. Cognitive performance of the mice was evaluated by Y maze and Morris water maze tests. OAβ was applied to culture neurons to simulate the in vivo manifestation. Aging-related proteins were detected by western blot and immunofluorescence.Results1. TREM2 gene expression levels are significantly elevated in activated microglia in the hippocampus of 5XFAD mice during disease progression, as well as PU.12. TREM2 gene expression is positively correlated with PU.1 and coexpress in activated microglia.3. Aβ induced TREM2 and PU.1 upregulation and loss of PU.1 abrogated Aβ induced TREM2 upregulation in microglia in vitro.4. Overexpression of PU.1 increased TREM2 gene expression, while inhibition of PU.1significantly reduced TREM2 gene expression.5. PU.1 contributed to the transcription of TREM2 by directly regulate TREM2 promoter activity.6. TREM2 regulate PU.1 and microglial viability associated gene expression7. Knockdown TREM2 or PU.1 impared microglia phagocytosis in vitro.8. The m RNA levels of inflammatory were significantly upregulated in early to medium stage of AD and significantly negative correlation was found between the m RNA levels of inflammatory mediators and protein expression of TREM2 with age from7-month-old to oldest age in 5XFAD mice brain.9. Age-dependent cognitive impairment was exacerbated in 5XFAD mice by cognitive behavioristics test10. Of all the differentially-expressed genes, the senescence-associated marker p16 was most significantly increased, even at the early age.11. Senescence-associated marker p16 is up-regulated under AD context and mainly localized on neuron, sparingly expressed in astrocyte labeled with GS, but not in activated microglia.12. The treatment of o Aβ increased the expression of p16 in cultured neuron in a concentration- and time-dependent manner.13. The treatment of oAβ did not increase SA-β-gal staining in primary neuron in vitro.Conclusion Aβ contributed to the upregulation TREM2 of activated microglial cells in5 XFAD mice, which was depand on transcription factor PU.1. Both PU.1 and TREM2 play a key role in regulating specialized genes expression, which are assoiated with cell survival, and mediated microglial phenotype shifting, which may facilitates phagocytosis and inhibits inflammation induced by Aβ. Importantly, the upregulated TREM2 further promotes the expression of PU.1 by type of feedback, which may form a circulation in microglia under AD milieu. This compensatory effect is insufficient to defent the redundant Aβ or inflammation in progression of early to medium stage of AD and then develops cognitive impairment. Aβ-accelerated senescence of neurons may be associated with the cognitive impairment in 5XFAD mice. Senescence-associated marker p16 can serve as an indicator to estimate the cognitive prognosis for AD population. AD can be a form of accelerated brain aging. Anti-aging molecules may present promising as disease-modified treatment in Alzheimer’s disease in the future.