Drosophila melanogaster as a model to study autophagy in neurodegenerative diseases induced by proteinopathies.

Proteinopathies are a large group of neurodegenerative diseases caused by both genetic and sporadic mutations in particular genes which can lead to alterations of the protein structure and to the formation of aggregates, especially toxic for neurons. Autophagy is a key mechanism for clearing those aggregates and its function has been strongly associated with the ubiquitin-proteasome system (UPS), hence mutations in both pathways have been associated with the onset of neurodegenerative diseases, particularly those induced by protein misfolding and accumulation of aggregates. Many crucial discoveries regarding the molecular and cellular events underlying the role of autophagy in these diseases have come from studies using Drosophila models. Indeed, despite the physiological and morphological differences between the fly and the human brain, most of the biochemical and molecular aspects regulating protein homeostasis, including autophagy, are conserved between the two species.In this review, we will provide an overview of the most common neurodegenerative proteinopathies, which include PolyQ diseases (Huntington's disease, Spinocerebellar ataxia 1, 2, and 3), Amyotrophic Lateral Sclerosis (C9orf72, SOD1, TDP-43, FUS), Alzheimer's disease (APP, Tau) Parkinson's disease (a-syn, parkin and PINK1, LRRK2) and prion diseases, highlighting the studies using Drosophila that have contributed to understanding the conserved mechanisms and elucidating the role of autophagy in these diseases.

Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis.

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.

Dispersive forces and resisting spot welds by alternative homolog conjunction govern chromosome shape in Drosophila spermatocytes during prophase I.

The bivalent chromosomes that are generated during prophase of meiosis I comprise a pair of homologous chromosomes. Homolog pairing during prophase I must include mechanisms that avoid or eliminate entanglements between non-homologous chromosomes. In Drosophila spermatocytes, non-homologous associations are disrupted by chromosome territory formation, while linkages between homologous chromosomes are maintained by special conjunction proteins. These proteins function as alternative for crossovers that link homologs during canonical meiosis but are absent during the achiasmate Drosophila male meiosis. How and where within bivalents the alternative homolog conjunction proteins function is still poorly understood. To clarify the rules that govern territory formation and alternative homolog conjunction, we have analyzed spermatocytes with chromosomal aberrations. We examined territory formation after acute chromosome cleavage by Cas9, targeted to the dodeca satellite adjacent to the centromere of chromosome 3 specifically in spermatocytes. Moreover, we studied territory organization, as well as the eventual orientation of chromosomes during meiosis I, in spermatocytes with stable structural aberrations, including heterozygous reciprocal autosomal translocations. Our observations indicate that alternative homolog conjunction is applied in a spatially confined manner. Comparable to crossovers, only a single conjunction spot per chromosome arm appears to be applied usually. These conjunction spots resist separation by the dispersing forces that drive apart homologous pericentromeric heterochromatin and embedded centromeres within territories, as well as the distinct chromosomal entities into peripheral, maximally separated territories within the spermatocyte nucleus.

Bivalent individualization during chromosome territory formation in Drosophila spermatocytes by controlled condensin II protein activity and additional force generators.

Reduction of genome ploidy from diploid to haploid necessitates stable pairing of homologous chromosomes into bivalents before the start of the first meiotic division. Importantly, this chromosome pairing must avoid interlocking of non-homologous chromosomes. In spermatocytes of Drosophila melanogaster, where homolog pairing does not involve synaptonemal complex formation and crossovers, associations between non-homologous chromosomes are broken up by chromosome territory formation in early spermatocytes. Extensive non-homologous associations arise from the coalescence of the large blocks of pericentromeric heterochromatin into a chromocenter and from centromere clustering. Nevertheless, during territory formation, bivalents are moved apart into spatially separate subnuclear regions. The condensin II subunits, Cap-D3 and Cap-H2, have been implicated, but the remarkable separation of bivalents during interphase might require more than just condensin II. For further characterization of this process, we have applied time-lapse imaging using fluorescent markers of centromeres, telomeres and DNA satellites in pericentromeric heterochromatin. We describe the dynamics of the disruption of centromere clusters and the chromocenter in normal spermatocytes. Mutations in Cap-D3 and Cap-H2 abolish chromocenter disruption, resulting in excessive chromosome missegregation during M I. Chromocenter persistence in the mutants is not mediated by the special system, which conjoins homologs in compensation for the absence of crossovers in Drosophila spermatocytes. However, overexpression of Cap-H2 precluded conjunction between autosomal homologs, resulting in random segregation of univalents. Interestingly, Cap-D3 and Cap-H2 mutant spermatocytes displayed conspicuous stretching of the chromocenter, as well as occasional chromocenter disruption, suggesting that territory formation might involve forces unrelated to condensin II. While the molecular basis of these forces remains to be clarified, they are not destroyed by inhibitors of F actin and microtubules. Our results indicate that condensin II activity promotes chromosome territory formation in co-operation with additional force generators and that careful co-ordination with alternative homolog conjunction is crucial.

Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease.

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.