Investigating the role of ATG9a-mediated autophagy in shaping stress resilience

Dysregulation of autophagy is implicated in many stress-related diseases, including metabolic syndrome and neuropsychiatric conditions [1]. Among core autophagy proteins, ATG9a, the only transmembrane protein in the pathway, plays a pivotal role in autophagosome formation. Despite this, its role in stress-related behavioural regulation is poorly understood. This project investigates whether stabilising ATG9a-driven autophagy in the ventral hippocampus promotes stress resilience, with potential relevance for novel nanobody-based therapeutic strategies.

To address this, we first examined the effects of viral-mediated manipulation of ATG9a in the glutamatergic neurons of the ventral hippocampus (vHipp) in male and female mice. Behavioural phenotyping was performed by combining conventional behavioural tests for anxiety and cognition with supervised and unsupervised deep phenotyping approaches using DeepLabCut and DeepOF to quantify individual and social behavioural features in semi-naturalistic settings. We investigated the impact of chronic stress using a validated chronic social defeat stress (CSDS) model on ATG9a mRNA expression levels via RNAScope. To dissect how acute stress affects Atg9a dynamics, we quantified Atg9a mRNA levels at three time points following a 1-hour restraint stress. In addition, metabolic stress was induced using a short-term high-fat diet (HFD). We then investigated whether AAV-mediated Atg9a overexpression rescues high-fat diet-induced stress phenotypes. Statistical analyses included analysis of variance with post hoc Tukey tests and Welch’s t-tests where appropriate.

We observed that ATG9a did not alter cognitive performance in either sex. However, male mice overexpressing ATG9a exhibited reduced anxiety-like behaviour as reflected by increased time spent in the centre of the open field compared to mice wth knockdown of ATG9a in the vHipp (KD vs OE: p = 0.0402, Cohen’s d = 0.969, 95% CI [0.28,1.91]), whereas no significant effect was observed in females. Chronic social defeat stress did not significantly alter ATG9a transcript levels in the vHipp of stress and non-stressed mice of either sex. Following acute restraint stress, male mice showed a trend in increased ATG9a mRNA levels post-stress, although this did not reach statistical significance. Furthermore, we observed that high-fat diet exposure reduced the ATG9a levels in the CA1 region of the ventral hippocampus in male mice (Welch’s t test, p = 0.0268), with no signficant effect in females.

Together, our current work suggests that ATG9a-dependent autophagy in the ventral hippocampus contributes to a sex-specific modulation of anxiety phenotype and metabolic sensitivity. The absence of significant cognitive effects and the presence of non-significant trends in acute stress conditions highlight the need for further investigation at the protein and circuit level. Further investigation on ATG9a protein levels and deep phenotyping of anxiety and social behaviour will be performed to underpin ATG9a as a linking autophagy and stress-related behavioural outcomes. This work bridges behavioural, molecular and metabolic approaches to highlight the therapeutic potential of ATG9a in stress resilience.

References

1. Pierone, B.C., Pereira, C.A., Garcez, M.L., Kaster, M.P., 2020. Stress and signaling pathways regulating autophagy: From behavioral models to psychiatric disorders. Experimental Neurology 334, 113485.