Amyotrophic Lateral Sclerosis Pathogenesis Converges on Defects in Protein Homeostasis Associated with TDP-43 Mislocalization and Proteasome-Mediated Degradation Overload.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that affects upper and/or lower motor neurons. It usually affects people between the ages of 40-70. The average life expectancy is about 3-5 years after diagnosis and there is no effective cure available. Identification of variants in more than 20 different loci has provided insight into the pathogenic molecular mechanisms mediating disease pathogenesis. In this review, we focus on seven ALS-causing genes: TDP-43, FUS, C9orf72, VCP, UBQLN2, VAPB and SOD-1, which encompass about 90% of the variants causing familial ALS. We examine the biological functions of these genes to assess how these pathogenic variants contribute to ALS pathogenesis by integrating findings from studies in Drosophila melanogaster and mammals. Additionally, we highlight the functional and genetic connections between these loci. Altogether, this review reveals that the majority of biological studies converge on defects in proteostasis due to the mislocalization of TDP-43 and/or altering the function of specific proteins mediating or modulating proteasomal degradation.

Pri sORF peptides induce selective proteasome-mediated protein processing.

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

Probing mechanisms that underlie human neurodegenerative diseases in Drosophila.

The fruit fly, Drosophila melanogaster, is an excellent organism for the study of the genetic and molecular basis of metazoan development. Drosophila provides numerous tools and reagents to unravel the molecular and cellular functions of genes that cause human disease, and the past decade has witnessed a significant expansion of the study of neurodegenerative disease mechanisms in flies. Here we review the interplay between oxidative stress and neuronal toxicity. We cover some of the studies that show how proteasome degradation of protein aggregates, autophagy, mitophagy, and lysosomal function affect the quality control mechanisms required for neuronal survival. We discuss how forward genetic screens in flies have led to the isolation of a few loci that cause neurodegeneration, paving the way for large-scale systematic screens to identify such loci in flies as well as promoting gene discovery in humans.

Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation.

Bonus, a Drosophila TIF1 homolog, is a nuclear receptor cofactor required for viability, molting, and numerous morphological events. Here we establish a role for Bonus in the modulation of chromatin structure. We show that weak loss-of-function alleles of bonus have a more deleterious effect on males than on females. This male-enhanced lethality is not due to a defect in dosage compensation or somatic sex differentiation, but to the presence of the Y chromosome. Additionally, we show that bonus acts as both an enhancer and a suppressor of position-effect variegation. By immunostaining, we demonstrate that Bonus is associated with both interphase and prophase chromosomes and through chromatin immunoprecipitation show that two of these sites correspond to the histone gene cluster and the Stellate locus.

senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye.

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. We show that in senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. We identify senseless repression of rough in R8 as an essential mechanism of R8 cell fate determination and demonstrate that misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment. A genetic model of R8 differentiation is presented.

Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission.

The cytoplasmic H3 helical domain of syntaxin is implicated in numerous protein-protein interactions required for the assembly and stability of the SNARE complex mediating vesicular fusion at the synapse. Two specific hydrophobic residues (Ala-240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin1A have been suggested to be involved in SNARE complex stability and required for the inhibitory effects of syntaxin on N-type calcium channels. We have generated the equivalent double point mutations in Drosophila syntaxin1A (A243V, V247A; syx(4) mutant) to examine their significance in synaptic transmission in vivo. The syx(4) mutant animals are embryonic lethal and display severely impaired neuronal secretion, although non-neuronal secretion appears normal. Synaptic transmission is nearly abolished, with residual transmission delayed, highly variable, and nonsynchronous, strongly reminiscent of transmission in null synaptotagmin I mutants. However, the syx(4) mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx(4) mutant animals have severely impaired hypertonic saline response in vivo, an assay indicating loss of fusion-competent synaptic vesicles, and in vitro SNARE complexes containing Syx(4) protein have significantly compromised stability. These data suggest that the same residues required for syntaxin-mediated calcium channel inhibition are required for the generation of fusion-competent vesicles in a neuronal-specific mechanism acting at synapses.

Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.

Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction.

Proprioceptor pathway development is dependent on Math1.

The proprioceptive system provides continuous positional information on the limbs and body to the thalamus, cortex, pontine nucleus, and cerebellum. We showed previously that the basic helix-loop-helix transcription factor Math1 is essential for the development of certain components of the proprioceptive pathway, including inner-ear hair cells, cerebellar granule neurons, and the pontine nuclei. Here, we demonstrate that Math1 null embryos lack the D1 interneurons and that these interneurons give rise to a subset of proprioceptor interneurons and the spinocerebellar and cuneocerebellar tracts. We also identify three downstream genes of Math1 (Lh2A, Lh2B, and Barhl1) and establish that Math1 governs the development of multiple components of the proprioceptive pathway.

Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit betaFTZ-F1-dependent transcription.

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the AF-2 activation domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo.

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis.

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Drosophila Lyra mutations are gain-of-function mutations of senseless.

The Lyra mutation was first described by Jerry Coyne in 1935. Lyra causes recessive pupal lethality and adult heterozygous Lyra mutants exhibit a dominant loss of the anterior and posterior wing margins. Unlike many mutations that cause loss of wing tissue (e.g., scalloped, Beadex, cut, and apterous-Xasta), Lyra wing discs do not exhibit increased necrotic or apoptotic cell death, nor do they show altered BrdU incorporation. However, during wing disc eversion, loss of the anterior and posterior wing margins is apparent. We have previously shown that senseless, a gene that is necessary and sufficient for peripheral nervous system (PNS) development, is allelic to Lyra. Here we show by several genetic criteria that Lyra alleles are neomorphic alleles of senseless that cause ectopic expression of SENSELESS in the wing pouch. Similarly, overexpression of SENSELESS in the wing disc causes loss of wing margin tissue, thereby mimicking the Lyra phenotype. Lyra mutants display aberrant expression of DELTA, VESTIGIAL, WINGLESS, and CUT. As in Lyra mutants, overexpression of SENSELESS in some areas of the wing pouch also leads to loss of WINGLESS and CUT. In summary, our data indicate that overexpression of SENSELESS causes a severe reduction in NOTCH signaling that in turn may lead to decreased transcription of several key genes required for wing development, leading to a failure in cell proliferation and loss of wing margin tissue.

Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins.

In our quest for novel genes required for the development of the embryonic peripheral nervous system (PNS), we have performed three genetic screens using MAb 22C10 as a marker of terminally differentiated neurons. A total of 66 essential genes required for normal PNS development were identified, including 49 novel genes. To obtain information about the molecular nature of these genes, we decided to complement our genetic screens with a molecular screen. From transposon-tagged mutations identified on the basis of their phenotype in the PNS we selected 31 P-element strains representing 26 complementation groups on the second and third chromosomes to clone and sequence the corresponding genes. We used plasmid rescue to isolate and sequence 51 genomic fragments flanking the sites of these P-element insertions. Database searches using sequences derived from the ends of plasmid rescues allowed us to assign genes to one of four classes: (1) previously characterized genes (11), (2) first mutations in cloned genes (1), (3) P-element insertions in genes that were identified, but not characterized molecularly (1), and (4) novel genes (13). Here, we report the cloning, sequence, Northern analysis, and the embryonic expression pattern of candidate cDNAs for 10 genes: astray, chrowded, dalmatian, gluon, hoi-polloi, melted, pebble, skittles, sticky ch1, and vegetable. This study allows us to draw conclusions about the identity of proteins required for the development of the nervous system in Drosophila and provides an example of a molecular approach to characterize en masse transposon-tagged mutations identified in genetic screens.

Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila.

The senseless (sens) gene is required for proper development of most cell types of the embryonic and adult peripheral nervous system (PNS) of Drosophila. Sens is a nuclear protein with four Zn fingers that is expressed and required in the sensory organ precursors (SOP) for proper proneural gene expression. Ectopic expression of Sens in many ectodermal cells causes induction of PNS external sensory organ formation and is able to recreate an ectopic proneural field. Hence, sens is both necessary and sufficient for PNS development. Our data indicate that proneural genes activate sens expression. Sens is then in turn required to further activate and maintain proneural gene expression. This feedback mechanism is essential for selective enhancement and maintenance of proneural gene expression in the SOPs.

A genome-wide search for synaptic vesicle cycle proteins in Drosophila.

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain.

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.