Activity-based anorexia drives persistent alterations of autophagic mechanisms in dorsal hippocampus of adolescent female rats

Anorexia nervosa (AN) is a life-threatening psychiatric disease marked by self-induced starvation and excessive exercise, mainly a>ecting young women. It often concurs with comorbid disorders and cognitive impairment. Though its cause remains unclear, recent study links body mass index with hippocampal volumes in AN, suggesting an impact of malnutrition on cognitive function. Additionally, autophagy, a self-degradative process, is involved in the regulation of energy balance during stressful circumstances such as starvation. Taking this into account, we investigated the potential involvement of autophagic mechanisms in regulating hippocampal neuroplastic processes in AN. Female rats were exposed to a combination of food restriction and free access to an activity wheel, to induce the activity-based anorexia (ABA) phenotype, during adolescence. Molecular analyses were conducted at two time points: post-natal-day 42 (PND42), at acute phase, and PND49, after body-weight recovery. Dorsal hippocampus (Dhip) tissues were collected, and both RNA and proteins were extracted and analyzed by means of Real-Time PCR and Western blot, respectively, using a two-way ANOVA followed by Tukey’s multiple comparisons test. Hippocampal functionality was assessed via the Spatial Order Object Recognition Test (SOR) and results were analyzed by an unpaired Student’s T test. We first evaluated transcription factor EB (TFEB), that is dephosphorylated during starvation and translocates into the nucleus where it binds target genes involved in autophagic and lysosomal functions. Indeed, in ABA rats at both time points pTFEB/TFEB ratio was decreased, whereas TFEB expression in the nucleus compared to the cytosol was increased, suggesting an increased translocation of the transcription factor into the nucleus. The analysis of the mRNA levels of Becn, encoding for Beclin-1, and Ctsb, encoding for Cathepsin B, two TFEB-responsive genes, showed an upregulation of both genes in ABA rats at PND42 and PND49. Moreover, the protein light chain 3 (LC3 II) levels, a marker of autophagosome formation, was incresed in ABA rats at both timepoints. Furthermore, the protein levels of caspase 3, a key apoptosis effector, was decreased in the ABA group at PND42 while increased at PND49, thus suggesting a switch from the autophagic pathway to a proapoptotic cell response. In parallel, ABA rats exhibited spatial cognitive impairment in the SOR test at both timepoints. Our findings indicate that food restriction and hyperactivity persistently increased autophagic mechanism, suggesting this process as a dysfunctional response potentially altering hippocampal-driven cognitive abilities. Autophagy, enrolled first to adapt the individual to starvation, switches toward persistent maladaptive alterations, that might trigger, at least in part, the reiteration of aberrant behaviors typical of AN patients and the vulnerability to develop other psychiatric comorbid symptoms. Sponsored by: MUR PRIN 2022CZTZ87