The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy

Abstract

Drosophila blue cheese ( bchs ) encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. bchs mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on bchs -related degeneration, nor bchs’ placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in bchs mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation. Further, pharmacological rescue requires at least one intact BEACH-containing isoform of the two identified in this study. Genetically augmenting a late step in autophagy, however, rescues even a strong mutation which retains only a third, non-BEACH containing isoform. Using living primary larval brain neurons, we elucidate the primary defect in bchs to be an excess of early autophagic compartments and a deficit in mature compartments. Conversely, rescuing the mutants by full-length Bchs over-expression induces mature compartment proliferation and rescues neuronal death. Surprisingly, only the longest Bchs isoform colocalizes well with autophagosomes, and shuttles between different vesicular locations depending on the type of autophagic impetus applied. Our results are consistent with Bchs promoting autophagic maturation, and the BEACH domain being required for this function. HIGHLIGHTS The autophagic adaptor blue cheese is placed in an epistatic hierarchy, using pharmacological and genetic modulation of bchs - motor neuron degeneration. An intact BEACH isoform can promote autophagic proliferation, and in primary larval brain neurons Bchs shuttles to different components of the autophagy machinery, dependent on the stimulus.

Short and long sleeping mutants reveal links between sleep and macroautophagy

“…Overall autophagy is impaired in both aus and bchs mutants, and yet they have opposing effects on sleep. However, we noticed that while the aus mutant increases the accumulation of autophagosomes, likely by blocking their clearance ( Figure 5 ), the bchs mutant has been shown to decrease the accumulation of autophagosomes, by blocking the maturation of immature Atg5(+) autophagosomes and recruitment of Atg8 ( Sim et al, 2019 ). We hypothesized that the opposite changes in the level of Atg8(+) autophagosomes in aus and bchs mutants drive their respective sleep phenotypes.…”

“…To better understand the effect of autophagy disruptions on sleep, we exploited the vast existing mutant and RNAi resources available in Drosophila to conduct directed screening. This demonstrated sleep gain in multiple scenarios that impede the production of autophagosomes, including homozygotes for bchs58 ( Figure 6A ), a loss-of-function mutant known to impair autophagosome maturation ( Sim et al, 2019 ), and RNAi knockdown of a number of genes involved in autophagy initiation, autophagosome nucleation, and autophagosome maturation, in particular atg1 and atg8a/b ( Figure 7 and Figure 7—figure supplement 1 , Figure 7—figure supplement 2 ). Using a subset of these tools that were too weak to drive sleep gain in wild-type flies, we find complete suppression of aus/+ sleep loss by bchs/+ , and rescue of aus/+ and aus/aus sleep loss by pan-neuronal RNAi for either atg5 or atg7 ( Figure 6B–D ).…”

A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing

“…WDFY3 is a cargo adaptor for selective degradation of ubiquitinated protein aggregates and physically interacts with Atg5 and p62 ( Clausen et al, 2010 ; Filimonenko et al, 2010 ). Mutants in the Drosophila Wdfy3 homolog Bchs show shortened lifespan and neurodegeneration ( Finley et al, 2003 ; Sim et al, 2019 ). Given that the expression of Bchs was increased in response to rapamycin treatment or over-expression of H3/H4 in ECs, we examined if Bchs was required for the effects of these treatments on the intestine and lifespan.…”

“…Instead Atg1 protein functioned downstream of increased histones in ECs. Increased transcription of Bchs, which was sufficient to mediate autophagy ( Sim et al, 2019 ), was a key downstream effector of histone-induced intestinal health and longevity.…”

The role of lipids in autophagy and its implication in neurodegeneration

“…The role of PI3P is not restricted to bulk macroautophagy, as for instance PI3P is also necessary to localize Alfy, the mammalian orthologue of the Drosophila Blue Cheese, and TECPR1 in selective autophagy to eliminate aggregates and bacteria [92]. Interestingly, another phosphoinositide that may impact initiation of the autophagosome is PI4P, a lipid that recruits ATG13, a subunit of the ULK1 complex, to the nascent autophagosomes.…”