Deregulation of mTORC1-TFEB axis in human iPSC model of GBA1-associated Parkinson's disease.

Mutations in the GBA1 gene are the single most frequent genetic risk factor for Parkinson's disease (PD). Neurodegenerative changes in GBA1-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 GBA1 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 GBA1 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 GBA1 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 GBA1 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 GBA1-associated neurodegeneration.

C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages.

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme ß-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.

Cytokine exocytosis and JAK/STAT activation in the Drosophila ovary requires the vesicle trafficking regulator α-Snap.

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 Drosophila 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 α-Snap or the SNARE family member Syntaxin1A in epithelia blocks polar cells maintenance and prevents specification of motile border cells. Blocking apoptosis rescues polar cell maintenance in α-Snap-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 Ca2+ 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.

A hormonal cue promotes timely follicle cell migration by modulating transcription profiles.

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.

Circuitous Genetic Regulation Governs a Straightforward Cell Migration.

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.

Identification of Novel Regulators of the JAK/STAT Signaling Pathway that Control Border Cell Migration in the Drosophila Ovary.

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.