INVESTIGATING THE ROLE OF THE E3 UBIQUITIN LIGASE HECW IN AUTOPHAGY AND STRESS RESPONSE

As neurodegenerative diseases continue to rise in prevalence among the aging population, understanding the underlying molecular mechanisms becomes imperative. Autophagy plays a crucial role in maintaining cellular homeostasis by degrading aggregated proteins and damaged organelles. However, the mechanisms regulating autophagy during aging remain poorly understood. Ubiquitination has emerged as a critical mechanism in controlling autophagy. Hecw, the Drosophila melanogaster ortholog of HECW1/2, encodes a E3 ubiquitin implicated in ribonucleoparticles (RNPs) dynamics and neuroprotection. Hecw mutations in flies determine the onset of neurodegenerativelike phenotypes that resemble those of well-characterized autophagy mutants. Yet, the role of Hecw in autophagy remains unexplored. This study aimed to investigate whether Hecw is involved in the regulation of autophagy and how its loss contributes to the onset of neurodegenerative-like phenotypes and stress susceptibility. Our in vivo findings demonstrate that Hecw mutations cause age-related brain atrophy, emergence of locomotor deficits as well as reduction of the levels of ref(2)P, the Drosophila homolog of autophagy cargo receptor p62. Notably, we find that Hecw is essential in the aging brain for induction of autophagy, rather than for autophagosome maturation or cargo clearance. Additionally, investigations in the larval fat body and adult ovaries revealed that Hecw mutants show impaired autophagy induction in response to amino acid restriction, underscoring a disruption in autophagy regulation. Furthermore, we found that Hecw mutants are more susceptible to oxidative stress. Loss of Hecw in Atg7 mutants exacerbates their vulnerability to oxidative stress, indicating that Hecw and Atg7 operate in parallel pathways regulating autophagy. Using RNAi experiments, we showed that depletion of Atg1 or Atg7 in a Hecw mutant background exposed to thermal stress leads to accumulation of ref(2)P and of the autophagy protein Atg8a, suggesting that loss of Hecw further reduces the autophagic flux when initiation or maturation are compromised. To elucidate how Hecw controls ref(2)P levels, we examined its mRNA expression. We discovered that Hecw mutants display an early reduction in ref(2)P transcription in pre-symptomatic flies, indicating that Hecw supports ref(2)P expression from an early age. Consistent with this, we observed that restoring ref(2)P levels throughout development partially mitigated neurodegeneration in Hecw mutants. Additionally, we showed that Hecw influences the level of Mitf (fly TFEB, a transcription factor essential for autophagy induction), and observed that Hecw mutants experience a decline in Mitf protein levels that correlates with reduced mTORC1 activity. Notably, reduced Mitf in Hecw mutants correlates with reduced expression of its transcriptional targets during aging, a decline that can be reversed by increasing ref(2)P. Our findings indicate that Hecw plays a critical role in sustaining ref(2)P levels and modulating autophagy, particularly during aging. The decline in ref(2)P expression in Hecw mutants appears to be linked to lowered transcriptional regulation by Mitf and by the oxidative stress modulator CncC/NRF2, suggesting a complex interplay between these pathways. The relationship between ref(2)P regulation, the mTORC1-Mitf axis and CncC signaling remains unclear. However, recent studies on SMURF1, a E3 ligase of the NEDD4 subfamily, imply that such regulation may stem from the ability of NEDD4 family members to modulate phase separation. Our observations of ectopic formation of ref(2)P-positive bodies upon Hecw overexpression further support this idea, suggesting the involvement of Hecw in regulating protein aggregation during the autophagic response. Overall, this work furthers our understanding of Hecw, the least characterized member of the NEDD4 subfamily of E3 ligases, in supporting neuronal health during aging and upon stress response. Considering that HECW1 has been implicated in humans in the onset of familial Amyotrophic Lateral Sclerosis (ALS), our results related to p62/ref(2)P regulation may pave the way for new therapeutic strategies to treat neurodegenerative disorders associated with the accumulation of protein and RNA aggregates.