Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation, and can occur selectively for substrates including aggregated proteins. It remains unclear, however, if starvation-induced and selective macroautophagy share identical adapter molecules to capture their cargo. Here we report that Alfy, a phosphatidylinositol 3-phosphate binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner, and likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy, by bridging cargo to the molecular machinery that builds autophagosomes.

A common feature of many human neurodegenerative diseases is the accumulation of insoluble ubiquitin-containing protein aggregates in the CNS. Although Drosophila has been helpful in understanding several human neurodegenerative disorders, a loss-of-function mutation has not been identified that leads to insoluble CNS protein aggregates. The study of Drosophila mutations may identify unique components that are associated with human degenerative diseases. The Drosophila blue cheese (bchs) gene defines such a novel degenerative pathway. bchs mutants have a reduced adult life span with the age-dependent formation of protein aggregates throughout the neuropil of the CNS. These inclusions contain insoluble ubiquitinated proteins and amyloid precursor-like protein. Progressive loss of CNS size and morphology along with extensive neuronal apoptosis occurs in aged bchs mutants. BCHS protein is widely expressed in the cytoplasm of CNS neurons and is present over the entire length of axonal projections. BCHS is nearly 3500 amino acids in size, with the last 1000 amino acids consisting of three functional protein motifs implicated in vesicle transport and protein processing. This region along with previously unidentified proteins encoded in the human, mouse, and nematode genomes shows striking homology along the full length of the BCHS protein. The high degree of conservation between Drosophila and human bchs suggests that study of the functional pathway of BCHS and associated mutant phenotype may provide useful insights into human neurodegenerative disorders.

Summary

Mutations in the Drosophila retained/dead ringer (retn) gene lead to female behavioral defects and alter a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body β-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. We posit that some of these retn-expressing cells function to repress a male behavioral pathway activated by fruM.