Daniela C. Zarnescu

BACKGROUND: TAR DNA binding protein - 43 (TDP-43) nuclear loss is a pathological hallmark of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and related neurodegenerative disorders. While the consequences of TDP-43 dysfunction have been well-characterized, the mechanisms driving TDP-43 mislocalization remain poorly understood. Previous observations of altered localization and function of the adenosine-to-inosine (A-to-I) RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) in ALS/FTD tissue prompted us to investigate whether dysregulated RNA editing contributes to pathological TDP-43 nucleocytoplasmic trafficking. METHODS: TDP-43 cytoplasmic mislocalization was assessed following ADAR2 and TDP-43 co-overexpression in HEK293T cells and a Drosophila model co-overexpressing human TDP-43 and dADAR in motor neurons. We further evaluated TDP-43 mislocalization through both HeLa cell assays and interspecies heterokaryon assays. Next, we assessed TDP-43 binding to A-to-I edited RNA oligomers through electrophoretic mobility shift assays (EMSAs), and investigated inosine-containing RNAs in vivo via TDP-43 RNA immunoprecipitation followed by sequencing (RIP-seq) datasets from human TDP-43-expressing Drosophila. Finally, RNAseq and enhanced cross-linking and immunoprecipitation (eCLIP-seq) were performed in SH-SY5Y cells overexpressing three ADAR2 variants with differing editing activity to identify editing-related transcriptional alterations and RNAs differentially bound to TDP-43. RESULTS: ADAR2 overexpression reduced the nucleocytoplasmic (N:C) ratio of TDP-43 in HEK293T cells in a ADAR2 catalytic activity- and TDP-43 RNA-binding capacity-dependent manner. Drosophila motor neurons overexpressing dADAR also exhibited decreased nuclear TDP-43. Interspecies heterokaryons and permeabilized HeLa cell assays demonstrated that catalytically active ADAR2 and synthetic inosine-containing RNA oligomers, respectively, enhance nuclear export of endogenous TDP-43. EMSAs revealed preferential binding of TDP-43 to inosine-containing RNAs relative to unedited RNAs, and analysis of Drosophila RIP-seq datasets demonstrated enrichment of edited transcripts within TDP-43-bound RNAs. Finally, RNAseq and eCLIP-seq analyses identified editing-dependent alterations in gene expression and TDP-43 RNA-binding profiles in SH-SY5Y cells overexpressing active ADAR2 variants. CONCLUSIONS: Together, our findings identify A-to-I RNA editing as a previously unrecognized regulator of TDP-43 localization and RNA interactions. These results support a model where altered RNA editing modifies TDP-43-RNA interactions, promoting increased nuclear export of TDP-43. Broadly, our work highlights RNA editing dysregulation as a potential contributor to early pathogenic mechanisms underlying TDP-43 proteinopathies.

Hexanucleotide repeat expansions (HREs) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet the age-, sex-, repeat-length-, and circuit-specific influence on the pathology of neurons remains incompletely understood. Here, we established a Drosophila model of C9orf72 -associated dementia by expressing G4C2 repeats in mushroom body neurons (MBNs), a brain region critical for memory, locomotion, and sleep. Expression of 44X G4C2 repeats ((G4C2) _44X ) led to progressive axonal thinning, age-dependent accumulation of Repeat Associated Non-AUG (RAN) translated GR-GFP dipeptide repeat (DPR) puncta, premature nuclear-to-cytoplasmic mislocalization of endogenous TDP-43, increased caspase, reduced lifespan and a loss of presynaptic active zones. Behaviorally, (G4C2) _44X expression caused locomotor hyperactivity, altered spatial working memory, and fragmentation of sleep architecture in an age- and sex-dependent manner, recapitulating core features of FTD. Surprisingly, the shorter (G4C2) _12X repeat, traditionally considered a control, also produced detectable RAN translation and intermediate phenotypes in aging MBNs, suggesting that length- and tissue-associated factors modulate repeat toxicity. We further identified a repeat-length- and age-dependent reduction of the glypican Dally-like protein (Dlp) in (G4C2) _44X consistent with disrupted Wnt-related signaling linked to TDP-43 proteinopathies. Restoring Dlp expression in MBNs mitigated locomotor and working-memory alterations, and loss of presynaptic active zones. In contrast, axonal degeneration, TDP-43 mislocalization, and lifespan were not significantly improved by restoring Dlp, suggesting that multiple mechanisms contribute to G4C2-induced toxicity. Supporting our findings in Drosophila MBNs, a CRISPRi screen in TDP-43 knock-down iNeurons identified GPC6, a human ortholog of Dlp, as a significant contributor to TDP-43 dependent synaptic loss. Together, our findings reveal an aging-sensitive, circuit-specific model of C9orf72 -associated neurodegeneration and highlight roles for DPR accumulation and Dlp/GPC6 dependent synaptic loss in FTD pathomechanisms.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing our previous work in motor neurons, here we report a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset ofDrosophilaKenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions we show that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating our findings in the fly model, we find that the expression ofGPC6mRNA, a human ortholog ofdlp,is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specificDrosophilamodels provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies.

Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 puncta that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing our previous models of ALS, here we report a novel model of FTD based on overexpression of TDP-43 in the Drosophila mushroom body (MB) circuit. This model recapitulates several aspects of FTD pathology including age-dependent neuronal loss, and nuclear depletion and cytoplasmic accumulation of TDP-43, accompanied by behavioral deficits in working memory and sleep that occur before axonal degeneration ensues. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit while others are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), a modulator of Wg/Wnt signaling. Using genetic interactions we show that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits. These results highlight the utility of modelling TDP-43 proteinopathy in Drosophila and provide a novel platform for studying the molecular mechanisms underlying FTD, and potentially uncovering shared and circuit specific vulnerabilities in ALS/FTD.

Local control of gene expression provides critical mechanisms for regulating development, maintenance and plasticity in the nervous system. Among the strategies known to govern gene expression locally, mRNA transport and translation have emerged as essential for a neuron’s ability to navigate developmental cues, and to establish, strengthen and remove synaptic connections throughout lifespan. Substantiating the role of RNA processing in the nervous system, several RNA binding proteins have been implicated in both developmental and age dependent neurodegenerative disorders. Of these, TDP-43 is an RNA binding protein that has emerged as a common denominator in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and related disorders due to the identification of causative mutations altering its function and its accumulation in cytoplasmic aggregates observed in a significant fraction of ALS/FTD cases, regardless of etiology. TDP-43 is involved in multiple aspects of RNA processing including splicing, transport and translation. Given that one of the early events in disease pathogenesis is mislocalization from the nucleus to the cytoplasm, several studies have focused on elucidating the pathogenic role of TDP-43 in cytoplasmic translation. Here we review recent findings describing TDP-43 translational targets and potential mechanisms of translation dysregulation in TDP-43 proteinopathies across multiple experimental models including cultured cells, flies, mice and patient derived neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43^G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µδ6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µδ6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µδ6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.