Investigating neuronal senescence marker, p19INK4d, in mouse and human brains

Abstract

AbstractBackgroundThe cellular senescence stress response allows damaged, toxic cells to survive. Clearing senescent cells is a promising therapeutic approach and has moved to clinical trials for Alzheimer’s disease and related dementias (ADRD). We previously identified a link between the cell cycle inhibitor CDKN2D/p19INK4d and neuronal senescence in human postmortem brains with various levels of AD neuropathology. We hypothesized that senescence could be induced by overexpressing this gene in mouse neurons in vivo.MethodAdeno‐associated virus 9 (AAV9) with the neuron specific human Synapsin I (hSyn1) promoter was used to deliver experimental Cdkn2d‐GFP, or control GFP gene expression. Precise delivery of 0.5µl of 1013 IU virus into four neuronal populations per mouse, bilateral hippocampal CA1 and cortical layer 5 neurons was accomplished with the assistance of a stereotaxic robot. Male and female wild type, human tau transgenic and mouse tau knockout mice were used (12‐13 months‐old, n = 47). Mice were euthanized at either two weeks or eight weeks post‐surgery to determine the short‐ and long‐term effects of Cdkn2d expression. GeoMx digital spatial profiler was utilized to quantify protein expression between Cdkn2d infected and uninfected neighboring neurons. ROI selection is displayed in Figure 1.ResultHistological analyses confirmed p19INK4d protein expression in AAV9‐infected cells. GeoMx DSP analyses performed on hippocampus, cortex, and thalamus revealed a protein expression pattern consistent with senescence (i.e., resistance to apoptosis), in Cdkn2d‐expressing neurons of the hippocampus and cortex compared to the un‐infected thalamus (Figure 2). Moreover, proteins associated with apoptosis were higher in the thalamus compared to the cortex and hippocampus.ConclusionsThe data indicates that AAV9‐hSyn1‐Cdkn2d‐GFP is an appropriate method to increase p19INK4d expression in mouse neurons in vivo. Over‐expressing p19INK4d caused neurons to acquire a protein expression signature consistent with senescence. The concomitant expression of apoptosis proteins in the thalamus leads me to hypothesize that the senescent cells in the cortex may cause cell death in the thalamus through the corticothalamic tract. Further studies with increased sample size are needed to confirm the results and continue exploring the effects of Cdkn2d on neuronal senescence, and how tau protein may influence this stress response.