Abstract DP349: Optogenetic Neuronal Stimulation Modulates Cholesterol Dynamics in Post-Stroke Recovery

Background: Post-stroke brain stimulation is a promising neurorestorative strategy, yet the underlying molecular mechanisms driving recovery remain unclear. Our prior work demonstrated that post-stroke optogenetic stimulation of the ipsilesional motor cortex (iM1) enhances functional recovery, and our RNA sequencing suggested cholesterol metabolism as a key pathway modulated by stimulation. Here, we examined the temporal dynamics of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), a key cholesterol enzyme, after stroke and assessed how optogenetic iM1 stimulation influences this expression. Methods: Male C57BL/6 mice (6-7 weeks) underwent stereotaxic surgery to express Channelrhodopsin in iM1 excitatory neurons and optical fiber implantation. After 5-6 weeks, mice received transient middle cerebral artery occlusion (30 min). Optogenetic stimulations were delivered from post-stroke days (PD) 5–14. Motor performance was assessed using the rotating beam test at pre-stroke baseline, PD4, 7 and 14. Brains were collected from stroke (PD1, 7&15) and sham control mice (n=4-5/group), and processed for immunohistochemistry using antibodies against HMGCS1, NeuN and CD68. Results: iM1 stimulation enhanced recovery at PD14, improving beam performance in both travel distance and speed (p<0.05). Previous RNA sequencing revealed involvement of multiple cholesterol biosynthesis and metabolism pathways at PD15 in iM1-stimulated mice. Immunostaining demonstrated that HMGCS1 was primarily expressed in primary motor cortex neurons and in peri-infarct glia. Compared with sham, iM1 showed a transient HMGCS1 increase at PD1, followed by reduced levels at PD7 and PD14, while contralateral M1 displayed reduced expression at all time points. At PD15, neuronal HMGCS1 in both stimulated and non-stimulated iM1 was significantly lower than in their respective contralateral M1 (p<0.05). In both regions, stimulated mice showed a trend toward higher HMGCS1 than non-stimulated mice. Stimulated mice also showed increased glial HMGCS1 in the peri-infarct region. Conclusions: Our findings link optogenetic stimulation-induced recovery to modulation of cholesterol metabolism, highlighting HMGCS1 as a potential molecular contributor. Future studies will validate cholesterol-related gene changes and clarify cell-type-specific contributions, with implications for targeting metabolic pathways to enhance neurorestoration.