Insulin-mediated changes in tau hyperphosphorylation and autophagy in a drosophila model of tauopathy and neuroblastoma cells

Almost 50 million people in the world are affected by dementia; the most prevalent form of which is Alzheimer’s disease (AD). Although aging is considered to be the main risk factor for AD, growing evidence from epidemiological studies suggests that type 2 diabetes mellitus (T2DM) increases the risk of dementia including AD. Defective brain insulin signaling has been suggested as an early event in AD and other tauopathies but the mechanisms that link these diseases are largely unknown. Tau hyperphosphorylation is a hallmark of neurofibrillary pathology and insulin resistance increases the number of neuritic plaques particularly in AD. Utilizing a combination of our Drosophila models of tauopathy (expressing the 2N4R-Tau) and neuroblastoma cells, we have attempted to decipher the pathways downstream of the insulin signaling cascade that lead to tau hyperphosphorylation, aggregation and autophagic defects. Using cell-based, genetic, and biochemical approaches we have demonstrated that tau phosphorylation at AT8 and PHF1 residues is enhanced in an insulin-resistant environment. We also show that insulin-induced changes in total and phospho-tau are mediated by the crosstalk of AKT, glycogen synthase kinase-3β, and extracellular regulating kinase located downstream of the insulin receptor pathway. Finally, we demonstrate a significant change in the levels of the key proteins in the mammalian target of rapamycin/autophagy pathway, implying an increased impairment of aggregated protein clearance in our transgenic Drosophila models and cultured neuroblastoma cells

Holographic Josephson Junctions and Berry holonomy from D-branes

We construct a holographic model for Josephson junctions with a defect system of a Dp brane intersecting a D(p+2) brane. In addition to providing a geometrical picture for the holographic dual, this leads us very naturally to suggest the possibility of non-Abelian Josephson junctions characterized in terms of the topological properties of the branes. The difference between the locations of the endpoints of the Dp brane on either side of the defect translates into the phase difference of the condensate in the Josephson junction. We also add a magnetic flux on the D(p+2) brane and allow it evolve adiabatically along a closed curve in the space of the magnetic flux, while generating a non-trivial Berry holonomy.Comment: 20 pages, 2 figure

Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes

Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.National Institutes of Health (U.S.) (grant R01 EB007042)National Institutes of Health (U.S.

Dysregulation of autophagy and stress granule-related proteins in stress-driven Tau pathology

Imbalance of neuronal proteostasis associated with misfolding and aggregation of Tau protein is a common neurodegenerative feature in Alzheimer's disease (AD) and other Tauopathies. Consistent with suggestions that lifetime stress may be an important AD precipitating factor, we previously reported that environmental stress and high glucocorticoid (GC) levels induce accumulation of aggregated Tau; however, the molecular mechanisms for such process remain unclear. Herein, we monitor a novel interplay between RNA-binding proteins (RBPs) and autophagic machinery in the underlying mechanisms through which chronic stress and high GC levels impact on Tau proteostasis precipitating Tau aggregation. Using molecular, pharmacological and behavioral analysis, we demonstrate that chronic stress and high GC trigger mTOR-dependent inhibition of autophagy, leading to accumulation of Tau aggregates and cell death in P301L-Tau expressing mice and cells. In parallel, we found that environmental stress and GC disturb cellular homeostasis and trigger the insoluble accumulation of different RBPs, such as PABP, G3BP1, TIA-1, and FUS, shown to form stress granules (SGs) and Tau aggregation. Interestingly, an mTOR-driven pharmacological stimulation of autophagy attenuates the GC-driven accumulation of Tau and SG-related proteins as well as the related cell death, suggesting a critical interface between autophagy and the response of the SG-related protein in the neurodegenerative potential of chronic stress and GC. These studies provide novel insights into the RNA-protein intracellular signaling regulating the precipitating role of environmental stress and GC on Tau-driven brain pathology.We would like to thank Professor Juergen Gotz, (University of Queensland, Australia) for the kind offer of eGFP-P301LTau SH-SY5Y cells and Dr. Bruno Almeida for his technical assistance. J.M.S. was granted with a PhD fellowship (SRFH/BD/88932/2012) by Portuguese Foundation for Science & Technology (FCT); I.S. is holder of FCT Investigator grants (IF/01799/2013), C.D. is a recipient of PhD fellowship of PHDoc program and co-tutelle PhD student of UMinho-UPMC universities. This work was funded by FCT research grants "PTDC/SAU-NMC/113934/2009" (I.S.), the Portuguese North Regional Operational Program (ON. 2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER) as well as the Project Estrategico co-funded by FCT (PEst-C/SAU/LA0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01-0124-FEDER-037298) as well as the project NORTE-01-0145-FEDER000013, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). In addition, this work was partly funded by Canon Foundation in Europe. This work has been also funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145FEDER-007038. This study was also supported to BW by grants from NIH (AG050471, NS089544, and ES020395), the BrightFocus Foundation, the Alzheimer Association and the Cure Alzeimer Foundation. Human brain tissue was generously provided by the National Institute of Aging Boston University AD Center (P30AG13846).info:eu-repo/semantics/publishedVersio

Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease

Common neurodegenerative proteinopathies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid beta (Aß) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila; however, potential limitations of prior studies include poor discrimination between effects on the adult versus developing nervous system and neuronal versus glial cell types. In addition, variable expression paradigms and outcomes hinder systematic comparison of toxicity profiles. Using standardized conditions and medium-throughput assays, we express human Tau, Aß or αSyn selectively in neurons of the adult Drosophila retina and monitor age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. We find that each protein causes a unique profile of neurodegenerative pathology, demonstrating distinct and separable impacts on neuronal death and dysfunction. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aß induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression, using a codon-optimized transgene, is characterized by marked retinal vacuolar change, progressive photoreceptor cell death, and delayed-onset but modest ERG changes. Lastly, to address potential mechanisms, we perform transmission electron microscopy (TEM) to reveal potential degenerative changes at the ultrastructural level. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. Our findings highlight variable and dynamic properties of neurodegeneration triggered by these disease-relevant proteins in vivo, and suggest that Drosophila may be useful for revealing determinants of neuronal dysfunction that precede cell loss, including synaptic changes, in the adult nervous system

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43.

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations and these facilitate a number of cellular functions. However, the mechanisms by which regions of the ER become tethered to mitochondria are not properly known. Understanding these mechanisms is not just important for comprehending fundamental physiological processes but also for understanding pathogenic processes in some disease states. In particular, disruption to ER-mitochondria associations is linked to some neurodegenerative diseases. Here we show that the ER-resident protein VAPB interacts with the mitochondrial protein tyrosine phosphatase-interacting protein-51 (PTPIP51) to regulate ER-mitochondria associations. Moreover, we demonstrate that TDP-43, a protein pathologically linked to amyotrophic lateral sclerosis and fronto-temporal dementia perturbs ER-mitochondria interactions and that this is associated with disruption to the VAPB-PTPIP51 interaction and cellular Ca(2+) homeostasis. Finally, we show that overexpression of TDP-43 leads to activation of glycogen synthase kinase-3β (GSK-3β) and that GSK-3β regulates the VAPB-PTPIP51 interaction. Our results describe a new pathogenic mechanism for TDP-43