Mutations in the
gene are the single most frequent genetic risk factor for Parkinson's disease (PD). Neurodegenerative changes in
-associated PD have been linked to the defective lysosomal clearance ...of autophagic substrates and aggregate-prone proteins. To elucidate novel mechanisms contributing to proteinopathy in PD, we investigated the effect of
mutations on the transcription factor EB (TFEB), the master regulator of the autophagy-lysosomal pathway (ALP). Using PD patients' induced-pluripotent stem cells (iPSCs), we examined TFEB activity and regulation of the ALP in dopaminergic neuronal cultures generated from iPSC lines harboring heterozygous
mutations and the CRISPR/Cas9-corrected isogenic controls. Our data showed a significant decrease in TFEB transcriptional activity and attenuated expression of many genes in the CLEAR network in
mutant neurons, but not in the isogenic gene-corrected cells. In PD neurons, we also detected increased activity of the mammalian target of rapamycin complex1 (mTORC1), the main upstream negative regulator of TFEB. Increased mTORC1 activity resulted in excess TFEB phosphorylation and decreased nuclear translocation. Pharmacological mTOR inhibition restored TFEB activity, decreased ER stress and reduced α-synuclein accumulation, indicating improvement of neuronal protiostasis. Moreover, treatment with the lipid substrate reducing compound Genz-123346, decreased mTORC1 activity and increased TFEB expression in the mutant neurons, suggesting that mTORC1-TFEB alterations are linked to the lipid substrate accumulation. Our study unveils a new mechanism contributing to PD susceptibility by
mutations in which deregulation of the mTORC1-TFEB axis mediates ALP dysfunction and subsequent proteinopathy. It also indicates that pharmacological restoration of TFEB activity could be a promising therapeutic approach in
-associated neurodegeneration.
The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway is an essential regulator of cell migration both in mammals and fruit flies. Cell migration is required ...for normal embryonic development and immune response but can also lead to detrimental outcomes, such as tumor metastasis. A cluster of cells termed "border cells" in the Drosophila ovary provides an excellent example of a collective cell migration, in which two different cell types coordinate their movements. Border cells arise within the follicular epithelium and are required to invade the neighboring cells and migrate to the oocyte to contribute to a fertilizable egg. Multiple components of the STAT signaling pathway are required during border cell specification and migration; however, the functions and identities of other potential regulators of the pathway during these processes are not yet known. To find new components of the pathway that govern cell invasiveness, we knocked down 48 predicted STAT modulators using RNAi expression in follicle cells, and assayed defective cell movement. We have shown that seven of these regulators are involved in either border cell specification or migration. Examination of the epistatic relationship between candidate genes and Stat92E reveals that the products of two genes, Protein tyrosine phosphatase 61F (Ptp61F) and brahma (brm), interact with Stat92E during both border cell specification and migration.
Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic
mutations that reduce the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). GCase catalyzes the conversion of ...glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8-10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.
How vesicle trafficking components actively contribute to regulation of paracrine signaling is unclear. We genetically uncovered a requirement for α-soluble NSF attachment protein (α-Snap) in the ...activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway during
egg development. α-Snap, a well-conserved vesicle trafficking regulator, mediates association of N-ethylmaleimide-sensitive factor (NSF) and SNAREs to promote vesicle fusion. Depletion of
or the SNARE family member
in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in
-depleted egg chambers, indicating that the lack of border cells in mutants is due to impaired signaling. Genetic experiments implicate α-Snap and NSF in secretion of a STAT-activating cytokine. Live imaging suggests that changes in intracellular Ca
are linked to this event. Our data suggest a cell-type specific requirement for particular vesicle trafficking components in regulated exocytosis during development. Given the central role for STAT signaling in immunity, this work may shed light on regulation of cytokine release in humans.
Drosophila border cells undergo a straightforward and stereotypical collective migration during egg development. However, a complex genetic program underlies this process. A variety of approaches, ...including biochemical, genetic, and imaging strategies have identified many regulatory components, revealing layers of control. This complexity suggests that the active processes of evaluating the environment, remodeling the cytoskeleton, and coordinating movements among cells, demand rapid systems for modulating cell behaviors. Multiple signaling inputs, nodes of integration, and feedback loops act as molecular rheostats to fine-tune gene expression levels and physical responses. Since key genetic regulators of border cell migration have been shown to be required in other types of cell migration, this model system continues to provide an important avenue for genetic discovery.
Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) is an evolutionarily conserved signaling cascade that converts extracellular signals to regulation of gene expression, which ...ultimately leads to regulation of various biological processes. Some biological events that rely on the JAK/STAT signaling pathway include hematopoiesis, immune function, stem cell maintenance and some types of cell migration. Insufficient or excessive activity of the pathway leads to developmental defects and/or pathological conditions such as autoimmune disorders and some types of cancer. A cluster of migratory cells termed border cells within the Drosophila melanogaster ovary has provided an excellent system for studying the JAK/STAT pathway and its regulatory effect on cell migration. Drosophila is advantageous as a model genetic organism because of minimized gene redundancy compared to mammals and the amenability for a wide variety of genetic manipulations. The border cell cluster is composed of a pair of non-migratory cells termed polar cells that secrete the JAK/STAT activating signals, and four to six signal-receiving cells that are specified as migratory as a result of pathway activity. Previous studies have elucidated some genetic regulatory networks underlying border cell specification and migration, but these studies did not determine all regulators of STAT signaling activity. Through in vivo RNA interference of 48 candidate genes in the border cell precursors, we identified multiple novel regulators of border cell migration. Through various genetic analyses, we further characterized three candidate genes for their effects on border cell specification and migration. We showed that Protein Tyrosine Phosphatase 61F and Brahma, homolog to yeast SWI2/SNF2 chromatin remodeler, function as negative regulators of STAT activity during both border cell specification and migration. We also showed that α-Soluble NSF Attachment Protein, an evolutionarily conserved regulator of vesicle fusion, is required for border cell specification. We demonstrated that α-Snap is specifically required in polar cells for release of the JAK/STAT pathway activator. Interestingly, our findings suggest that despite the inevitable requirement for exocytosis in border cells and their precursors, α-Snap is dispensable in those cells for their specification. Overall, this research has identified novel molecular and cellular regulators of the JAK/STAT signaling pathway that are required for motile cell specification and migration.
Cell migration is essential during animal development. In the Drosophila ovary, the steroid hormone ecdysone coordinates nutrient sensing, growth, and the timing of morphogenesis events including ...border cell migration. To identify downstream effectors of ecdysone signaling, we profiled gene expression in wild-type follicle cells compared to cells expressing a dominant negative Ecdysone receptor or its coactivator Taiman. Of approximately 400 genes that showed differences in expression, we validated 16 candidate genes for expression in border and centripetal cells, and demonstrated that seven responded to ectopic ecdysone activation by changing their transcriptional levels. We found a requirement for seven putative targets in effective cell migration, including two other nuclear hormone receptors, a calcyphosine-encoding gene, and a prolyl hydroxylase. Thus, we identified multiple new genetic regulators modulated at the level of transcription that allow cells to interpret information from the environment and coordinate cell migration in vivo.
•Varied categories of genes transcriptionally respond to EcR and Tai signaling in ovarian follicle cells.•Proper border cell migration requires signaling from additional nuclear hormone receptors.•Prolyl-4-hydroxylase alpha EFB (PH4αEFB) acts to synchronize cell migration and egg development.
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