Biology subjects

Dubnau, J.

Publications and source records attributed to Dubnau, J..

8 recordsLinked to original sources

bioRxiv2025

Glial cell-intrinsic and non-cell autonomous toxicity in a Drosophila C9orf72 neurodegeneration model

The most common genetic cause of both familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G4C2 repeat in the first intron of the gene C9orf72. The C9orf72 repeat expansion is bidirectionally transcribed into sense and anti-sense RNA foci, and also produces dipeptide repeats (DPRs) via a non-canonical translation mechanism known as repeat-associated (RAN) translation. Each of these components of the G4C2 repeat expansion cause neurodegenerative effects in animal models when expressed in neurons, but impacts from glial expression are more poorly understood. Here, we use glial cell type-specific expression of individual DPRs, of RNA repeat-only, or of the G4C2 repeat that is capable of producing both DPRs and RNA repeats to systematically investigate both the glial cell-intrinsic and non-cell autonomous toxicity of each of these components. Our results show that as with neurons, the GR and G4C2 transgenes, produce the highest degree of cell-intrinsic toxicity when expressed in glia. Both of these transgenes are capable of producing the GR DPR, which is also typically found to be the most toxic factor in neurons. We demonstrate that both the GR and G4C2 transgenes cause activation of mdg4, an endogenous retrovirus (ERV). Such ERV expression is a hallmark of TDP-43 dysfunction that is commonly observed in C9orf72 patients and contributes to both cell intrinsic and non-cell autonomous toxicity. We find that only the G4C2 transgene produces measurable non-cell autonomous effects that result in loss of nearby neurons. But manipulations of apoptosis reveal non-cell autonomous or systemic effects from either GR or G4C2 expressing glia. Blocking apoptotic cell death of either GR or G4C2 expressing glia via the p35 caspase inhibitor further exacerbates effects on lifespan and ablating such glia via expression of the proapoptotic reaper gene partially ameliorates these effects.

neuroscience↗

bioRxiv2024

Targeted single cell expression profiling identifies integrators of sleep and metabolic state

Animals modulate sleep in accordance with their internal and external environments. Metabolic cues are particularly potent regulators of sleep, allowing animals to alter their sleep timing and amount depending on food availability and foraging duration. The fruit fly, Drosophila melanogaster, suppresses sleep in response to acute food deprivation, presumably to forage for food. This process is dependent on a single pair of Lateral Horn Leucokinin (LHLK) neurons, that secrete the neuropeptide Leucokinin. These neurons signal to insulin producing cells and suppress sleep under periods of starvation. The identification of individual neurons that modulate sleep-metabolism interactions provides the opportunity to examine the cellular changes associated with sleep modulation. Here, we use single-cell sequencing of LHLK neurons to examine the transcriptional responses to starvation. We validate that a Patch-seq approach selectively isolates RNA from individual LHLK neurons. Single-cell CEL-Seq comparisons of LHLK neurons between fed and 24-hr starved flies identified 24 genes that are differentially expressed in accordance with starvation state. In total, 12 upregulated genes and 12 downregulated genes were identified. Gene-ontology analysis showed an enrichment for Attacins, a family of anti-microbial peptides, along with several transcripts with diverse roles in regulating cellular function. Targeted knockdown of differentially expressed genes identified multiple genes that function within LHLK neurons to regulate sleep-metabolism interactions. Functionally validated genes include an essential role for the E3 ubiquitin Ligase insomniac, the sorbitol dehydrogenase Sodh1, as well as AttacinC and AttacinB in starvation-induced sleep suppression. Taken together, these findings provide a pipeline for identifying novel regulators of sleep-metabolism interactions within individual neurons.

neuroscience↗

bioRxiv2022

Endogenous Retroviruses and TDP-43 Proteinopathy Form a Sustaining Feedback to Drive the Intercellular Spread of Neurodegeneration

Neurodegenerative disorders are thought to initiate focally and then spread over time through neural circuits. A mechanism that has been proposed to explain this is the inter-cellular movement of misfolded "prion-like" proteins that then recruit normally folded proteins in recipient cells to also adopt pathological conformations. Such a mechanism has been proposed, for example, to explain the propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) during the progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases such as mad cow disease, which can be caused by ingestion or by injection into animal brains of misfolded PrP, ALS and FTD are not infectious and the injection of phosphorylated and aggregated TDP-43 protein into rodent brains is not sufficient to cause disease phenotypes unless those animals also express high levels of transgenic TDP-43 protein. This suggests a missing component of a positive feedback that is necessary to sustain disease progression. We provide evidence that endogenous retroviruses (ERVs) are that missing component. We demonstrate that ERV expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either the Drosophila ERV, gypsy (mdg4) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Importantly. We also demonstrate that viral ERV transmission causes propagation of such TDP-43 pathology to cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism underlies the toxicity of glial cells to neurons and other glia in a Drosophila in vivo model.

neuroscience↗

bioRxiv2019

Postmortem Cortex Samples Identify Distinct Molecular Subtypes of ALS: Retrotransposon Activation, Oxidative Stress, and Activated Glia

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several inherited pathogenic mutations have been identified as causative, the vast majority of cases are sporadic with no family history of disease. Thus, for the majority of ALS cases, a specific causal abnormality is not known and the disease may be a product of multiple inter-related pathways contributing to varying degrees in different ALS patients. Using unsupervised machine learning algorithms, we stratified the transcriptomes of 148 ALS decedent cortex tissue samples into three distinct and robust molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 20% of the ALS patient samples exhibited high levels of retrotransposon expression and other signatures of TDP-43 dysfunction. Finally, a third group showed predominant signatures of glial activation (19%). Together these results demonstrate that at least three distinct molecular signatures contribute to ALS disease. While multiple dysregulated components and pathways comprising these clusters have previously been implicated in ALS pathogenesis, unbiased analysis of this large survey demonstrated that sporadic ALS patient tissues can be segregated into distinct molecular subsets.

genomics↗

bioRxiv2018

Cellular labeling of endogenous virus replication (CLEVR) reveals de novo insertions of the gypsy endogenous retrovirus in cell culture and in both neurons and glial cells of aging fruit flies

Evidence is rapidly mounting that transposable element expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, successful reporters of LINE element mobilization have been in use for some time, but similar strategies have not been developed to report Long Terminal Repeat (LTR) retrotransposons and endogenous retroviruses. We describe Cellular Labeling of Endogenous Virus Replication (CLEVR), which reports replication of the gypsy element in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR-retrotransposons, endogenous retroviruses and exogenous retroviruses.

genetics↗

bioRxiv2018

Nuclear transcriptomes of the seven neuronal cell types that constitute the Drosophila mushroom bodies.

The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The Drosophila melanogaster MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes ({gamma}, /{beta} and /{beta}) and 7 cell subtypes ({gamma}d, {gamma}m, /{beta}ap, /{beta}m, /{beta}p, /{beta}s and /{beta}c) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used /{beta} class marker Fas2 and the /{beta} class marker trio. Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the Drosophila MB provides a valuable resource for the fly neuroscience community.

neuroscience↗

bioRxiv2018

A computational framework for converting high-throughput DNA sequencing data into neural circuit connectivity.

There is growing interest in determining the connectivity of neural circuits-the "connectome"-at single neuron resolution. Most approaches to circuit mapping rely on either microscopy or physiology, but these approaches have very limited throughput. We have recently proposed BOINC (Barcoding of Individual Neuronal Connectivity), a radically different approach to connectivity mapping based on high-throughput DNA sequencing. Here we describe the set of computational algorithms that serve to convert sequencing data into neural connectivity. We apply our computational pipeline to the results of proof-of-principle experiments illustrating an implementation of BOINC based on pseudorabies virus (PRV). PRV is capable of traversing individual synapses and carry DNA barcodes from one cell to another. Using this high-throughput sequencing data, we obtain 456-by-486 connectivity matrix between putative neurons. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution would represent a major advance in neuroscience.

neuroscience↗

bioRxiv2016

Retrotransposon Activation Contributes to Neurodegeneration in a DrosophilaTDP-43 Model of ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in [~]40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.\n\nAUTHOR SUMMARYFunctional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in [~]40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.

neuroscience↗