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.HIGHLIGHTSThe 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.
Highlights
A common pathological hallmark in many neurodegenerative disorders is the accumulation of intracellular toxic aggregates
We generated bchs17(M) and bchs58(M) by precise excision of the EP insertion. bchs17(M) introduces a stop codon at Trp2640, and is expected to disrupt isoforms 1 and 2 (Figure 1B). bchsLL03462 is a strong allele resulting from an insertion into bchs of a splice acceptor site followed by stop codons into the 7th coding exon at aa 1229 preceding the BEACH domain (Flybase allele report FBti0124589)
Physical interactions of the proposed aggrephagy adaptor and p62-binding BEACH domain protein (Nezis et al, 2008; Clausen et al, 2010) blue cheese (Bchs) with other parts of the autophagic machinery have been demonstrated in biochemical studies
Summary
A common pathological hallmark in many neurodegenerative disorders is the accumulation of intracellular toxic aggregates. Degradation of misfolded proteins and protein aggregates is mediated by two main intracellular systems, the ubiquitinproteasome system (UPS) and autophagy (Glickman and Ciechanover, 2002; Levine and Klionsky, 2004). Elongation of the isolation membrane requires the E1-like activating enzyme Atg and E2-like conjugating enzyme Atg to bring Atg to Atg (an E3-like ligase) which binds Atg to form the Atg12Atg5-Atg multimeric complex (Ohsumi, 2001; Kuma et al, 2002) This E3-like ligase complex aids in the lipidation of Atg protein and dissociates from the membrane upon formation of the autophagosome (Kabeya et al, 2000; Hanada et al, 2007). In mammals one of these adaptors, ALFY (autophagylinked FYVE protein), scaffolds the machinery associated with isolation membrane elongation around sequestered protein aggregates by binding to the autophagy receptor, p62, through its PH-BEACH (Beige and Chediak–Higashi) domain, Atg through its WD40 repeats, and phosphatidylinositol 3-phosphate (PI3P)-containing autophagic membrane via its FYVE domain (Clausen et al, 2010; Filimonenko et al, 2010)
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