Differential effects of neuronal Cdkn2a over‐expression in mouse brain

Abstract

AbstractBackgroundAging is the primary risk factor for Alzheimer’s disease (AD). With aging, senescent cells accumulate in the body and the brain. Cells that become senescent cease contributing to homeostasis but do not typically undergo apoptosis. Prior studies have identified a broad association between increased cellular senescence and AD‐like pathology and have identified senescent cells in AD patient brain and mouse models of AD‐like pathology. However, contributions of senescent cells to disease progression remains uncertain.MethodThe two gene products of the senescence regulator Cdkn2a (p16‐Ink4a and p19‐Arf) were overexpressed using intracranial delivery of adeno‐associated viral vectors (AAV). Mice were injected with 2 µL of 1×1013, 5×1012, or 1×1012 vg/mL of AAV‐Arf and tissue was collected two months post‐injection. Control mice were injected with AAV empty capsid. Brain weights, NeuN IHC, and p19‐Arf mRNA were assessed. A second cohort of mice was injected with AAV‐Ink, AAV‐Arf, AAV‐Ink & AAV‐Arf (co‐injection), or AAV‐GFP and followed for four months post‐injection. We assessed body mass, brain mass, NeuN IHC, and expression of senescence‐induced genes by PCR‐array and qRT‐PCR. A third experiment over‐expressing p19‐Arf in PS19 mice is ongoing.ResultWe observed that animals over‐expressing p19‐Arf failed to gain body mass up to four months post‐injection. Post‐mortem, we found that over‐expression of p19‐Arf is toxic to neurons, reducing gross brain mass and NeuN area staining. Furthermore, a greater number of differentially regulated genes were observed in mice over‐expressing p19‐Arf than in mice over‐expressing p16‐Ink4a. We confirmed increased expression of the senescence‐regulator p21 in mice over‐expressing p19‐Arf, but not p16‐Ink4a, by qPCR. In a separate experiment, we replicated the finding of neurotoxicity induced by over‐expression of p19‐Arf at only two months post‐injection and determined the optimal viral titer to administer that induces significant over‐expression with minimal neurotoxicity for use in future studies.ConclusionAs observed in cultured cells, over‐expression of Cdkn2a seems to increase markers of senescence. Effects on tau pathology will be examined in the PS19 mouse model of tauopathy. Cell type‐specific expression in neurons, astroglia, and microglia will be used to elucidate cell type specific contributions to senescence‐induced disease progression.