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.

A neural circuit for vocal production responds to viscerosensory input in the songbird.

Motor performance is monitored continuously by specialized brain circuits and used adaptively to modify behavior on a moment-to-moment basis and over longer time periods. During vocal behaviors, such as singing in songbirds, internal evaluation of motor performance relies on sensory input from the auditory and vocal-respiratory systems. Sensory input from the auditory system to the motor system, often referred to as auditory feedback, has been well studied in singing zebra finches (Taeniopygia guttata), but little is known about how and where nonauditory sensory feedback is evaluated. Here we show that brief perturbations in air sac pressure cause short-latency neural responses in the higher-order song control nucleus HVC (used as proper name), an area necessary for song learning and song production. Air sacs were briefly pressurized through a cannula in anesthetized or sedated adult male zebra finches, and neural responses were recorded in both nucleus parambigualis (PAm), a brainstem inspiratory center, and HVC, a cortical premotor nucleus. These findings show that song control nuclei in the avian song system are sensitive to perturbations directly targeted to vocal-respiratory, or viscerosensory, afferents and support a role for multimodal sensory feedback integration in modifying and controlling vocal control circuits.NEW & NOTEWORTHY This study presents the first evidence of sensory input from the vocal-respiratory periphery directly activating neurons in a motor circuit for vocal production in songbirds. It was previously thought that this circuit relies exclusively on sensory input from the auditory system, but we provide groundbreaking evidence for nonauditory sensory input reaching the higher-order premotor nucleus HVC, expanding our understanding of what sensory feedback may be available for vocal control.

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.

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.

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.