0029 The Effect of BMAL1 Expression on Autophagy Activity in Astrocytes

Abstract

Abstract Introduction Circadian rhythm disruption is an established symptom of neurodegenerative disease, however, there is mounting evidence that circadian systems drive early pathogenesis. The core circadian clock gene, BMAL1, mediates daily oscillations in transcription for homeostatic processes. Furthermore, the deletion of BMAL1 renders tissue transcriptionally arrhythmic and influences non-circadian genes, which causes reactive astrogliosis, oxidative stress, and neuronal injury in the brain. We have recently observed in-vitro that BMAL1 knockout astrocytes exhibit increases in the pH-sensitive lysosomal dye lysotracker. This suggests that the clock potentially modulates an even broader array of cellular processes, like autophagy, which maintains cellular homeostasis by delivering proteins to lysosomes for degradation. Given that deficits in the clearance of stable protein aggregates is characteristic of many neurodegenerative disorders, the manipulation of autophagic machinery displays therapeutic potential. Therefore, the purpose of this project is to evaluate whether autophagy activity in astrocytes is regulated rhythmically by circadian clocks or through non-rhythmic genes linked to BMAL1 expression. Methods Through employing the fluorescent (RFP)-EGFP-LC3 reporter, we can detect differential organelle pH values to reveal the autophagy dynamics. Specifically, the RFP:EGFP signal ratio represents the progression from the autophagosome to the acidic autolysosome, thereby quantitatively illustrating the protein degradation arising from autophagy machinery. Results When comparing astrocyte-specific BMAL1 knockout mice to the wildtype littermates, there are significant increases for EGFP and RFP channels within the white matter. Furthermore, the greater RFP:EGFP value exhibits upregulated protein degradation, which validates that BMAL1 deletion can modulate autophagy activity. Upon establishing the baseline fluorescence for wildtypes across the 24-hour cycle, the EGFP and RFP signals suggested rhythmic activity for the light-stimulated groups. Nevertheless, the insignificant sinusoidal patterns among the dark-only counterparts reduces the probability that circadian clocks regulate autophagy activity. Conclusion The effect of BMAL1 deletion on autophagosomes in white matter tissue likely originates from other glial cell types that are uniquely susceptible to dysfunction in non-rhythmic pathways related to BMAL1 expression in astrocytes. Ultimately, investigating how BMAL1 protects against neurodegeneration will reveal therapeutic approaches for manipulating the autophagy machinery to promote pathology clearance, along with emphasizing the importance for maintaining sleeping patterns. Support (if any)