CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content.

Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.SIGNIFICANCE STATEMENT Menkes and Wilson disease affect copper homeostasis and characteristically afflict the nervous system. However, their molecular neuropathology mechanisms remain mostly unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors that localize to the Golgi apparatus, ATP7 and the conserved oligomeric Golgi (COG) complex. Disruption of these mechanisms affect mitochondrial function and localization to synapses as well as neurotransmission and synaptic plasticity. These findings suggest communication between the Golgi apparatus and mitochondria through homeostatically controlled cellular copper levels and copper-dependent enzymatic activities in both organelles.

Basal autophagy is required for promoting dendritic terminal branching in Drosophila sensory neurons.

Dendrites function as the primary sites for synaptic input and integration with impairments in dendritic arborization being associated with dysfunctional neuronal circuitry. Post-mitotic neurons require high levels of basal autophagy to clear cytotoxic materials and autophagic dysfunction under native or cellular stress conditions has been linked to neuronal cell death as well as axo-dendritic degeneration. However, relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization or the mechanisms by which the autophagic machinery may be transcriptionally regulated to promote dendritic diversification. We demonstrate that autophagy-related (Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. We demonstrate that the Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.

Drosophila caspase activity is required independently of apoptosis to produce active TNF/Eiger during nociceptive sensitization.

Tumor necrosis factor (TNF) signaling is required for inflammatory nociceptive (pain) sensitization in Drosophila and vertebrates. Nociceptive sensitization in Drosophila larvae following UV-induced tissue damage is accompanied by epidermal apoptosis and requires epidermal-derived TNF/Eiger and the initiator caspase, Dronc. Major gaps remain regarding TNF function in sensitization, including the relationship between apoptosis/tissue damage and TNF production, the downstream signaling in this context, and the target genes that modulate nociceptive behaviors. Here, apoptotic cell death and thermal nociceptive sensitization are genetically and procedurally separable in a Drosophila model of UV-induced nociceptive sensitization. Activation of epidermal Dronc induces TNF-dependent but effector caspase-independent nociceptive sensitization in the absence of UV. In addition, knockdown of Dronc attenuated nociceptive sensitization induced by full-length TNF/Eiger but not by a constitutively soluble form. UV irradiation induced TNF production in both in vitro and in vivo, but TNF secretion into hemolymph was not sufficient to induce thermal nociceptive sensitization. Downstream mediators of TNF-induced sensitization included two TNF receptor-associated factors, a p38 kinase, and the transcription factor nuclear factor kappa B. Finally, sensory neuron-specific microarray analysis revealed downstream TNF target genes induced during thermal nociceptive sensitization. One of these, enhancer of zeste (E(z)), functions downstream of TNF during thermal nociceptive sensitization. Our findings suggest that an initiator caspase is involved in TNF processing/secretion during nociceptive sensitization, and that TNF activation leads to a specific downstream signaling cascade and gene transcription required for sensitization. These findings have implications for both the evolution of inflammatory caspase function following tissue damage signals and the action of TNF during sensitization in vertebrates.

The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse.

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT: The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization complex Arp2/3 is reduced in dysbindin-deficient cells, thus affecting actin-dependent phenotypes in two cellular compartments where dysbindin resides, endosomes and presynapses. Our studies indicate that a central cellular structure affected by schizophrenia susceptibility loci is the actin cytoskeleton, an organelle necessary for synaptic function in the presynaptic and postsynaptic compartment.

Human ApoE ε4 alters circadian rhythm activity, IL-1ß, and GFAP in CRND8 mice.

Disruptions to daily living, inflammation, and astrogliosis are characteristics of Alzheimer's disease. Thus, circadian rhythms, nest construction, IL-1ß and TNF-α, and glial fibrillary acidic protein (GFAP) were examined in a mouse model developed to model late-onset Alzheimer's disease-the most common form of the disease. Mice carrying both the mutated human AßPP transgene found in the CRND8 mouse and the human apolipoprotein E ε4 allele (CRND8/E4) were compared with CRND8 mice and wildtype (WT) mice. Circadian rhythms were evaluated by wheel-running behavior. Activity of daily living was measured by nest construction. This study then examined mRNA levels of the inflammatory cytokines IL-1ß and TNF-α as well as protein levels of GFAP. Behavioral outcomes were then correlated with cytokines and GFAP. Compared to WT controls, both CRND8 and CRND8/E4 mice showed significantly more frequent, but shorter, bouts of activity. In the three groups, the CRND8/E4 mice had intermediate disruptions in circadian rhythms. Both CRND8/E4 mice and CRND8 mice showed significant impairments in nesting behavior compared to WTs. While CRND8 mice expressed significantly increased IL-1ß and GFAP expression compared to WT controls, CRND8/E4 mice expressed intermediate IL-1ß and GFAP levels. Significant correlations between IL-1ß, GFAP, and behavior were observed. These data are congruent with other studies showing that human ApoE ε4 is protective early in life in transgenic mice modeling Alzheimer's disease.

Genomic phenotype of non-cultured pulmonary fibroblasts in idiopathic pulmonary fibrosis.

Activated fibroblasts are the central effector cells of the progressive fibrotic process in idiopathic pulmonary fibrosis (IPF). Characterizing the genomic phenotype of isolated fibroblasts is essential to understanding IPF pathogenesis. Comparing the genomic phenotype of non-cultured pulmonary fibroblasts from advanced IPF patients' and normal lungs revealed novel genes, biological processes and concomitant pathways previously unreported in IPF fibroblasts. We demonstrate altered expression in proteasomal constituents, ubiquitination-mediators, Wnt, apoptosis and vitamin metabolic pathways and cell cycle regulators, suggestive of loss of cellular homeostasis. Specifically, FBXO32, CXCL14, BDKRB1 and NMNAT1 were up-regulated, while RARA and CDKN2D were down-regulated. Paradoxically, pro-apoptotic inducers TNFSF10, BAX and CASP6 were also found to be increased. This comprehensive description of altered gene expression in isolated IPF fibroblasts underscores the complex biological processes characteristic of IPF and may provide a foundation for future research into this devastating disease.

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation.

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. We demonstrate that Piwi and Bam proteins are expressed independently of each other in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam- ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam- ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. We propose that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division.

Bazooka is a permissive factor for the invasive behavior of discs large tumor cells in Drosophila ovarian follicular epithelia.

Drosophila Bazooka and atypical protein kinase C are essential for epithelial polarity and adhesion. We show here that wild-type bazooka function is required during cell invasion of epithelial follicle cells mutant for the tumor suppressor discs large. Clonal studies indicate that follicle cell Bazooka acts as a permissive factor during cell invasion, possibly by stabilizing adhesion between the invading somatic cells and their substratum, the germline cells. Genetic epistasis experiments demonstrate that bazooka acts downstream of discs large in tumor cell invasion. In contrast, during the migration of border cells, Bazooka function is dispensable for cell invasion and motility, but rather is required cell-autonomously in mediating cell adhesion within the migrating border cell cluster. Taken together, these studies reveal Bazooka functions distinctly in different types of invasive behaviors of epithelial follicle cells, potentially by regulating adhesion between follicle cells or between follicle cells and their germline substratum.