TDP-43 impairs sleep in Drosophila through Ataxin-2-dependent metabolic disturbance.

Neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia are associated with substantial sleep disruption, which may accelerate cognitive decline and brain degeneration. Here, we define a role for trans-activation response element (TAR) DNA binding protein 43 (TDP-43), a protein associated with human neurodegenerative disease, in regulating sleep using Drosophila. Expression of TDP-43 severely disrupts sleep, and the sleep deficit is rescued by Atx2 knockdown. Brain RNA sequencing revealed that Atx2 RNA interference regulates transcripts enriched for small-molecule metabolic signaling in TDP-43 brains. Focusing on these Atx2-regulated genes, we identified suppressors of the TDP-43 sleep phenotype enriched for metabolism pathways. Knockdown of Atx2 or treatment with rapamycin attenuated the sleep phenotype and mitigated the disruption of small-molecule glycogen metabolism caused by TDP-43. Our findings provide a connection between toxicity of TDP-43 and sleep disturbances and highlight key aspects of metabolism that interplay with TDP-43 toxicity upon Atx2 rescue.

Cell type-specific regulation of m6 A modified RNAs in the aging Drosophila brain.

The aging brain is highly vulnerable to cellular stress, and neurons employ numerous mechanisms to combat neurotoxic proteins and promote healthy brain aging. The RNA modification m6 A is highly enriched in the Drosophila brain and is critical for the acute heat stress response of the brain. Here we examine m6 A in the fly brain with the chronic stresses of aging and degenerative disease. m6 A levels dynamically increased with both age and disease in the brain, marking integral neuronal identity and signaling pathway transcripts that decline in level with age and disease. Unexpectedly, there is opposing impact of m6 A transcripts in neurons versus glia, which conferred different outcomes on animal health span upon Mettl3 knockdown to reduce m6 A: whereas Mettl3 function is normally beneficial to neurons, it is deleterious to glia. Moreover, knockdown of Mettl3 in glial tauopathy reduced tau pathology and increased animal survival. These findings provide mechanistic insight into regulation of m6 A modified transcripts with age and disease, highlighting an overall beneficial function of Mettl3 in neurons in response to chronic stresses, versus a deleterious impact in glia.

dTrmt10A impacts Hsp70 chaperone m6A levels and the stress response in the Drosophila brain.

Chronic cellular stress has a profound impact on the brain, leading to degeneration and accelerated aging. Recent work has revealed the vital role of RNA modifications, and the proteins responsible for regulating them, in the stress response. In our study, we defined the role of CG14618/dTrmt10A, the Drosophila counterpart of human TRMT10A a N1-methylguanosine methyltransferase, on m6A regulation and heat stress resilience in the Drosophila brain. By m6A-IP RNA sequencing on Drosophila head tissue, we demonstrated that manipulating dTrmt10A levels indirectly regulates m6A levels on polyA + RNA. dTrmt10A exerted its influence on m6A levels on transcripts enriched for neuronal signaling and heat stress pathways, similar to the m6A methyltransferase Mettl3. Intriguingly, its impact primarily targeted 3' UTR m6A, setting it apart from the majority of Drosophila m6A-modified transcripts which display 5' UTR enrichment. Upregulation of dTrmt10A led to increased resilience to acute heat stress, decreased m6A modification on heat shock chaperones, and coincided with decreased decay of chaperone transcripts and increased translation of chaperone proteins. Overall, these findings establish a potential mechanism by which dTrmt10A regulates the acute brain stress response through m6A modification.

m1A in CAG repeat RNA binds to TDP-43 and induces neurodegeneration.

Microsatellite repeat expansions within genes contribute to a number of neurological diseases1,2. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology3-9. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N1-methyladenosine (m1A) by TRMT61A, and that m1A can be demethylated by ALKBH3. We also observed that the m1A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m1A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m1A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m1A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.

A perspective on Drosophila genetics and its insight into human neurodegenerative disease.

Drosophila has been long appreciated as a classic genetic system for its ability to define gene function in vivo. Within the last several decades, the fly has also emerged as a premiere system for modeling and defining mechanisms of human disease by expressing dominant human disease genes and analyzing the effects. Here I discuss key aspects of this latter approach that first intrigued me to focus my laboratory research on this idea. Differences between the loss-of-function vs. the gain-of-function approach are raised-and the insight of these approaches for appreciating mechanisms that contribute to human neurodegenerative disease. The application of modifier genetics, which is a prominent goal of models of human disease, has implications for how specific genes or pathways intersect with the dominant disease-associated mechanisms. Models of human disease will continue to reveal unanticipated insight into fundamental cellular processes-insight that might be harder to glean from classical genetic methodologies vs modifier genetics of disease.

Mettl3-dependent m6A modification attenuates the brain stress response in Drosophila.

N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain's susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5'UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain's biological response to stress.

Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain.

Aging is a risk factor for neurodegenerative disease, but precise mechanisms that influence this relationship are still under investigation. Work in Drosophila melanogaster identified the microRNA miR-34 as a modifier of aging and neurodegeneration in the brain. MiR-34 mutants present aspects of early aging, including reduced lifespan, neurodegeneration, and a buildup of the repressive histone mark H3K27me3. To better understand how miR-34 regulated pathways contribute to age-associated phenotypes in the brain, here we transcriptionally profiled the miR-34 mutant brain. This identified that genes associated with translation are dysregulated in the miR-34 mutant. The brains of these animals show increased translation activity, accumulation of protein aggregation markers, and altered autophagy activity. To determine if altered H3K27me3 was responsible for this proteostasis dysregulation, we studied the effects of increased H3K27me3 by mutating the histone demethylase Utx. Reduced Utx activity enhanced neurodegeneration and mimicked the protein accumulation seen in miR-34 mutant brains. However, unlike the miR-34 mutant, Utx mutant brains did not show similar altered autophagy or translation activity, suggesting that additional miR-34-targeted pathways are involved. Transcriptional analysis of predicted miR-34 targets identified Lst8, a subunit of Tor Complex 1 (TORC1), as a potential target. We confirmed that miR-34 regulates the 3' UTR of Lst8 and identified several additional predicted miR-34 targets that may be critical for maintaining proteostasis and brain health. Together, these results present novel understanding of the brain and the role of the conserved miRNA miR-34 in impacting proteostasis in the brain with age.

Glial AP1 is activated with aging and accelerated by traumatic brain injury.

The emergence of degenerative disease after traumatic brain injury is often described as an acceleration of normal age-related processes. Whether similar molecular processes occur after injury and in age is unclear. Here we identify a functionally dynamic and lasting transcriptional response in glia, mediated by the conserved transcription factor AP1. In the early post-TBI period, glial AP1 is essential for recovery, ensuring brain integrity and animal survival. In sharp contrast, chronic AP1 activation promotes human tau pathology, tissue loss, and mortality. We show a similar process activates in healthy fly brains with age. In humans, AP1 activity is detected after moderate TBI and correlates with microglial activation and tau pathology. Our data provide key molecular insight into glia, highlighting that the same molecular process drives dynamic and contradictory glia behavior in TBI, and possibly age, first acting to protect but chronically promoting disease.

Synergistic effects of brain injury and aging: common mechanisms of proteostatic dysfunction.

The aftermath of TBI is associated with an acute stress response and the accumulation of insoluble protein aggregates. Even after the symptoms of TBI are resolved, insidious molecular processes continue to develop, which often ultimately result in the development of age-associated neurodegenerative disorders. The precise molecular cascades that drive unhealthy brain aging are still largely unknown. In this review, we discuss proteostatic dysfunction as a converging mechanism contributing to accelerated brain aging after TBI. We examine evidence from human tissue and in vivo animal models, spanning both the aging and injury contexts. We conclude that TBI has a sustained debilitating effect on the proteostatic machinery, which may contribute to the accelerated pathological and cognitive hallmarks of aging that are observed following injury.

Toxicity of pathogenic ataxin-2 in Drosophila shows dependence on a pure CAG repeat sequence.

Spinocerebellar ataxia type 2 is a polyglutamine (polyQ) disease associated with an expanded polyQ domain within the protein product of the ATXN2 gene. Interestingly, polyQ repeat expansions in ATXN2 are also associated with amyotrophic lateral sclerosis (ALS) and parkinsonism depending upon the length of the polyQ repeat expansion. The sequence encoding the polyQ repeat also varies with disease presentation: a pure CAG repeat is associated with SCA2, whereas the CAG repeat in ALS and parkinsonism is typically interrupted with the glutamine encoding CAA codon. Here, we asked if the purity of the CAG sequence encoding the polyQ repeat in ATXN2 could impact the toxicity of the ataxin-2 protein in vivo in Drosophila. We found that ataxin-2 encoded by a pure CAG repeat conferred toxicity in the retina and nervous system, whereas ataxin-2 encoded by a CAA-interrupted repeat or CAA-only repeat failed to confer toxicity, despite expression of the protein at similar levels. Furthermore, the CAG-encoded ataxin-2 protein aggregated in the fly eye, while ataxin-2 encoded by either a CAA/G or CAA repeat remained diffuse. The toxicity of the CAG-encoded ataxin-2 protein was also sensitive to the translation factor eIF4H, a known modifier of the toxic GGGGCC repeat in flies. These data indicate that ataxin-2 encoded by a pure CAG versus interrupted CAA/G polyQ repeat domain is associated with differential toxicity, indicating that mechanisms associated with the purity of the sequence of the polyQ domain contribute to disease.

An Integrated Multi-omics Approach Identifies Therapeutic Potential for ATP6V1A in Late Onset Alzheimer's Disease.

In this issue of Neuron, Wang et al. employ an impressive multi-omics approach, including gene co-expression network analyses and drug repositioning, to normalize disease-disrupted gene modules and identify a therapeutically relevant driver of Alzheimer's disease.

Inducing different severities of traumatic brain injury in Drosophila using a piezoelectric actuator.

Drosophila models have been instrumental in providing insights into molecular mechanisms of neurodegeneration, with wide application to human disease. The brain degeneration associated with traumatic brain injury (TBI) has been modeled in Drosophila using devices that inflict trauma on multiple parts of the fly body, including the head. However, the injuries produced by these models are not specific in location and are inconsistent between individual animals. We have recently developed a device that can be used to inflict controlled head injury to flies, resulting in physiological responses that are remarkably similar to those observed in humans with TBI. This protocol describes the construction, calibration and use of the Drosophila TBI (dTBI) device, a platform that employs a piezoelectric actuator to reproducibly deliver a force in order to briefly compress the fly head against a metal surface. The extent of head compression can be controlled through an electrical circuit, allowing the operator to set different levels of injury. The entire device can be assembled and calibrated in under a week. The device components and the necessary electrical tools are readily available and cost ~$800. The dTBI device can be used to harness the power of Drosophila genetics and perform large-scale genetic or pharmacological screens, using a 7-d post-injury survival curve to identify modifiers of injury.

Author Correction: An integrated multi-omics approach identifies epigenetic alterations associated with Alzheimer's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An integrated multi-omics approach identifies epigenetic alterations associated with Alzheimer's disease.

Protein aggregation is the hallmark of neurodegeneration, but the molecular mechanisms underlying late-onset Alzheimer's disease (AD) are unclear. Here we integrated transcriptomic, proteomic and epigenomic analyses of postmortem human brains to identify molecular pathways involved in AD. RNA sequencing analysis revealed upregulation of transcription- and chromatin-related genes, including the histone acetyltransferases for H3K27ac and H3K9ac. An unbiased proteomic screening singled out H3K27ac and H3K9ac as the main enrichments specific to AD. In turn, epigenomic profiling revealed gains in the histone H3 modifications H3K27ac and H3K9ac linked to transcription, chromatin and disease pathways in AD. Increasing genome-wide H3K27ac and H3K9ac in a fly model of AD exacerbated amyloid-ß42-driven neurodegeneration. Together, these findings suggest that AD involves a reconfiguration of the epigenome, wherein H3K27ac and H3K9ac affect disease pathways by dysregulating transcription- and chromatin-gene feedback loops. The identification of this process highlights potential epigenetic strategies for early-stage disease treatment.

Dynamic neural and glial responses of a head-specific model for traumatic brain injury in Drosophila.

Traumatic brain injury (TBI) is the strongest environmental risk factor for the accelerated development of neurodegenerative diseases. There are currently no therapeutics to address this due to lack of insight into mechanisms of injury progression, which are challenging to study in mammalian models. Here, we have developed and extensively characterized a head-specific approach to TBI in Drosophila, a powerful genetic system that shares many conserved genes and pathways with humans. The Drosophila TBI (dTBI) device inflicts mild, moderate, or severe brain trauma by precise compression of the head using a piezoelectric actuator. Head-injured animals display features characteristic of mammalian TBI, including severity-dependent ataxia, life span reduction, and brain degeneration. Severe dTBI is associated with cognitive decline and transient glial dysfunction, and stimulates antioxidant, proteasome, and chaperone activity. Moreover, genetic or environmental augmentation of the stress response protects from severe dTBI-induced brain degeneration and life span deficits. Together, these findings present a tunable, head-specific approach for TBI in Drosophila that recapitulates mammalian injury phenotypes and underscores the ability of the stress response to mitigate TBI-induced brain degeneration.

TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2.

In >95% of cases of amyotrophic lateral sclerosis (ALS) and ∼45% of frontotemporal degeneration (FTD), the RNA/DNA-binding protein TDP-43 is cleared from the nucleus and abnormally accumulates in the cytoplasm of affected brain cells. Although the cellular triggers of disease pathology remain enigmatic, mounting evidence implicates the poly(ADP-ribose) polymerases (PARPs) in TDP-43 neurotoxicity. Here we show that inhibition of the PARP enzymes tankyrase 1 and tankyrase 2 (referred to as Tnks-1/2) protect primary rodent neurons from TDP-43-associated neurotoxicity. We demonstrate that Tnks-1/2 interacts with TDP-43 via a newly defined tankyrase-binding domain. Upon investigating the functional effect, we find that interaction with Tnks-1/2 inhibits the ubiquitination and proteasomal turnover of TDP-43, leading to its stabilization. We further show that proteasomal turnover of TDP-43 occurs preferentially in the nucleus; our data indicate that Tnks-1/2 stabilizes TDP-43 by promoting cytoplasmic accumulation, which sequesters the protein from nuclear proteasome degradation. Thus, Tnks-1/2 activity modulates TDP-43 and is a potential therapeutic target in diseases associated with TDP-43, such as ALS and FTD.This article has an associated First Person interview with the first author of the paper.

New Roles for Canonical Transcription Factors in Repeat Expansion Diseases.

The presence of microsatellite repeat expansions within genes is associated with >30 neurological diseases. Of interest, (GGGGCC)>30-repeats within C9orf72 are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). These expansions can be 100s to 1000s of units long. Thus, it is perplexing how RNA-polymerase II (RNAPII) can successfully transcribe them. Recent investigations focusing on GGGGCC-transcription have identified specific, canonical complexes that may promote RNAPII-transcription at these GC-rich microsatellites: the DSIF complex and PAF1C. These complexes may be important for resolving the unique secondary structures formed by GGGGCC-DNA during transcription. Importantly, this process can produce potentially toxic repeat-containing RNA that can encode potentially toxic peptides, impacting neuron function and health. Understanding how transcription of these repeats occurs has implications for therapeutics in multiple diseases.

Repeat-associated non-AUG (RAN) translation mechanisms are running into focus for GGGGCC-repeat associated ALS/FTD.

Many human diseases are associated with the expansion of repeat sequences within the genes. It has become clear that expressed disease transcripts bearing such long repeats can undergo translation, even in the absence of a canonical AUG start codon. Termed "RAN translation" for repeat associated non-AUG translation, this process is becoming increasingly prominent as a contributor to these disorders. Here we discuss mechanisms and variables that impact translation of the repeat sequences associated with the C9orf72 gene. Expansions of a G4C2 repeat within intron 1 of this gene are associated with the motor neuron disease ALS and dementia FTD, which comprise a clinical and pathological spectrum. RAN translation of G4C2 repeat expansions has been studied in cells in culture (ex vivo) and in the fly in vivo. Cellular states that lead to RAN translation, like stress, may be critical contributors to disease progression. Greater elucidation of the mechanisms that impact this process and the factors contributing will lead to greater understanding of the repeat expansion diseases, to the potential development of novel approaches to therapeutics, and to a greater understanding of how these players impact biological processes in the absence of disease.