Biology subjects

Gera, J.

Publications and source records attributed to Gera, J..

7 recordsLinked to original sources

bioRxiv2024

Synaptic connectome of a neurosecretory network in the Drosophila brain

Hormones mediate inter-organ signaling which is crucial in orchestrating diverse behaviors and physiological processes including sleep and activity, feeding, growth, metabolism and reproduction. The pars intercerebralis and pars lateralis in insects represent major hubs which contain neurosecretory cells (NSC) that produce various peptide hormones. To obtain insight into how hormonal signaling is regulated, we have characterized the synaptic connectome of NSC in the adult Drosophila brain. Identification of neurons providing inputs to multiple NSC classes implicate diuretic hormone 44-expressing NSC as a major coordinator of physiology and behavior. Surprisingly, despite most NSC having dendrites in the subesophageal zone (primary taste processing center), inputs from peripheral gustatory neurons to NSC are largely indirect. We also deciphered pathways via which diverse olfactory inputs are relayed to NSC. Linear dynamical modeling of signal propagation through the connectome identifies enteric neurons as the strongest influencers of NSC activity compared to other sensory modalities. Further, our analyses revealed substantial inputs from brain descending neurons to NSC, suggesting that descending neurons regulate both endocrine and motor output to synchronize physiological changes with appropriate behaviors. In contrast to NSC inputs, synaptic output from NSC is sparse and mostly mediated by corazonin NSC. We show that both corazonin-expressing NSC and their downstream synaptic partner DNg27 influence egg-laying. We additionally explore putative paracrine interconnectivity between NSC classes and peptide hormone pathways from NSC to peripheral tissues by analyzing single-cell transcriptomic datasets. Our comprehensive characterization of the Drosophila neurosecretory network connectome provides a platform to understand complex hormonal networks and how they orchestrate animal behaviors and physiology.

neuroscience↗

bioRxiv2024

Anti-diuretic hormone ITP signals via a guanylate cyclase receptor to modulate systemic homeostasis in Drosophila

Insects have evolved a variety of neurohormones that enable them to maintain their nutrient and osmotic homeostasis. While the identities and functions of various insect metabolic and diuretic hormones have been well-established, the characterization of an anti-diuretic signaling system that is conserved across most insects is still lacking. To address this, here we characterized the ion transport peptide (ITP) signaling system in Drosophila. The Drosophila ITP gene encodes five transcript variants which generate three different peptide isoforms: ITP amidated (ITPa) and two ITP-like (ITPL1 and ITPL2) isoforms. Using a combination of anatomical mapping and single-cell transcriptome analyses, we comprehensively characterized the expression of all three ITP isoforms in the nervous system and peripheral tissues. Our analyses reveal wide-spread expression of ITP isoforms. Moreover, we show that ITPa-producing neurons are activated and release ITPa during dehydration. Further, recombinant Drosophila ITPa inhibits diuretic peptide-induced renal tubule secretion ex vivo, thus confirming its role as an anti-diuretic hormone. Using a phylogenetic-driven approach, an ex vivo secretion assay and a heterologous mammalian cell-based assay, we identified and functionally characterized Gyc76C, a membrane guanylate cyclase, as a bona fide Drosophila ITPa receptor. Thus, recombinant ITPa application leads to increased cGMP production in HEK293T cells expressing Drosophila Gyc76C. Moreover, knockdown of Gyc76C in renal tubules abolishes the inhibitory effect of ITPa on diuretic hormone stimulated secretion. Extensive anatomical mapping of Gyc76C reveals that it is highly expressed in larval and adult tissues associated with osmoregulation (renal tubules and rectum) and metabolic homeostasis (fat body). Consistent with this expression, knockdown of Gyc76C in renal tubules impacts tolerance to osmotic and ionic stresses, whereas knockdown specifically in the fat body impacts feeding, nutrient homeostasis and associated behaviors. We also complement receptor knockdown experiments with ITP knockdown and ITPa overexpression in ITPa-producing neurons. Interestingly, the ITPa-Gyc76C pathway examined here is reminiscent of the atrial natriuretic peptide signaling in mammals. Lastly, we utilized connectomics and single-cell transcriptomics to identify synaptic and paracrine pathways upstream and downstream of ITPa-expressing neurons. Our analysis identifies pathways via which ITP neurons integrate hygrosensory inputs and interact with other homeostatic hormonal pathways. Taken together, our systematic characterization of ITP signaling establishes a tractable system to decipher how a small set of neurons integrates diverse inputs to orchestrate systemic homeostasis in Drosophila.

physiology↗

bioRxiv2023

Cellular protection from H2O2 toxicity by Fv-Hsp70 and mutants of Fv-Hsp70. Protection via catalase and gamma-glutamyl cysteine synthase.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain, and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 uM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection. Cellular protection was protective via the antioxidant proteins catalase and GGCS.

cell biology↗

bioRxiv2023

cGAS-activating lupus autoantibody for cancer immunotherapy

Cytoplasmic DNA triggers a cGAS-mediated signaling cascade that promotes an innate immune response and is potentially actionable in cancer immunotherapy. Here we show that a cytoplasmic-localizing lupus anti-DNA autoantibody activates cGAS and facilitates an immune-mediated prolongation of survival in orthotopic models of glioblastoma (GBM). Mechanistically, cellular penetration and blood-brain barrier crossing by the anti-DNA autoantibody is linked to nucleoside transport. Pulldown, knockdown, signaling, and cytotoxicity assays demonstrate autoantibody association with and activation of cGAS. In orthotopic GBM models, the autoantibody localizes to brain tumor, increases tumor CD8+ T cell content, and prolongs survival in immunocompetent but not immunodeficient mice. This work introduces the new concept of a cGAS-activating anti-DNA autoantibody, which impacts theories on mechanisms of autoimmunity and has translational applications in cancer immunotherapy.

immunology↗

bioRxiv2019

Targeting the mTORC2 signaling complex in B cell malignancies.

Hyperactive PI3 kinase-Akt (PI3K-Akt) signaling has an important role in cell growth and resistance to apoptosis in B cell malignancies. Inhibition of this pathway by blocking PI3K activity, and or inhibiting mTORC1/2 signaling complexes is an active area of research in B cell leukemia/lymphoma such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). With a tissue-scan array, the expression of Rictor is a component of the mTORC2 complex was determined by quantitative PCR in a number of B cell malignancies. Rictor was found to be over-expressed in CLL and MCL cells as compared to normal B cells with no over-expression in Hodgkins and non-Hodgkins lymphomas. Inactivation of Rictor was performed by shRNA in two Mantle cell lines and these stable Rictor knockdown cell lines demonstrated a slower growth of cells as compared to scrambled shRNA control. In addition, there was a decrease of mTORC2 signaling and B cell receptor (BCR) cross-linking mediated Akt (Ser473) and NDRG1 (Thr 346) phosphorylation. To specifically disrupt the mTORC2 signaling complex and target Rictor overexpression, previously identified inhibitors that block Rictor and MTOR interaction in a yeast two-hybrid system were analyzed. Treatment of primary CLL specimens with these inhibitors followed by immunoprecipitation experiments confirmed the disruption of the mTORC2 complex. These inhibitors also induced apoptosis in CLL specimens and were more effective than rapamycin, an MTOR inhibitor and pp242, an mTORC1 and 2 inhibitors, at equimolar concentrations. Treatment of CLL specimens with the lead inhibitor, compound#6, resulted in inhibition of p-Akt, p-GSK 3 beta, p-PKC alpha, p-Foxo1, and p-Foxo3, with minimal effect on the phosphorylation of an mTORC1 target gene, S6 kinase. In comparison with Idelalisib (CAL-101), a clinically approved PI3Kinase p110 delta inhibitor in CLL, comp#6 is more effective in inducing apoptosis in primary CLL specimens at equimolar concentrations (mean 51.2, SD 21.7 as compared to mean 26.9, SD 17.2). The data support the effectiveness of these novel inhibitors that specifically disrupt the mTORC2 complex in primary CLL specimens.

cancer biology↗

bioRxiv2017

Synthetic single domain antibodies for the conformational trapping of membrane proteins

Single domain antibodies called nanobodies are excellent affinity reagents for membrane proteins. However, their generation relies on immunizations, which is only amenable to robust proteins and impedes selections in the presence of non-covalent or toxic ligands. To overcome these key limitations, we developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. Using ribosome display, exceptionally large libraries were pre-enriched against membrane protein targets and subsequently funneled into a robust phage display process, thereby reducing selection bias. We successfully generated conformation-selective, high affinity sybodies against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. Our platform builds exclusively on commercially available reagents and enables non-specialized labs to generate conformation-specific binders against previously intractable protein targets.

bioengineering↗

bioRxiv2017

mTORC2/AKT/HSF1/HuR Constitute A Feed-Forward Loop Regulating Rictor Expression And Tumor Growth In Glioblastoma

Overexpression of Rictor has been demonstrated to result in increased mTORC2 nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via HSF1-induced HuR activity. HuR is shown to directly bind the 3' UTR of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppresses xenograft growth in mice. We further demonstrate that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. In an additional level of regulation, miR-218, a known Rictor targeting miRNA is shown to be subject to mTORC2/STAT3-mediated repression. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency and suppresses miR-218 resulting in enhanced mTORC2 activity in these tumors.

cancer biology↗