Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

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.

Axon degeneration and regeneration: insights from Drosophila models of nerve injury.

Axon degeneration is the pivotal pathological event of acute traumatic neural injury as well as many chronic neurodegenerative diseases. It is an active cellular program and yet molecularly distinct from cell death. Much effort is devoted toward understanding the nature of axon degeneration and promoting axon regeneration. However, the fundamental mechanisms of self-destruction of damaged axons remain unclear, and there are still few treatments for traumatic brain injury (TBI) or spinal cord injury (SCI). Genetically approachable model organisms such as Drosophila melanogaster, the fruit fly, have proven exceptionally successful in modeling human neurodegenerative diseases. More recently, this success has been extended into the field of acute axon injury and regeneration. In this review, we discuss recent findings, focusing on how these models hold promise for accelerating mechanistic insight into axon injury and identifying potential therapeutic targets for TBI and SCI.

Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization.

In amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), cytoplasmic aggregates of hyperphosphorylated TDP-43 accumulate and colocalize with some stress granule components, but how pathological TDP-43 aggregation is nucleated remains unknown. In Drosophila, we establish that downregulation of tankyrase, a poly(ADP-ribose) (PAR) polymerase, reduces TDP-43 accumulation in the cytoplasm and potently mitigates neurodegeneration. We establish that TDP-43 non-covalently binds to PAR via PAR-binding motifs embedded within its nuclear localization sequence. PAR binding promotes liquid-liquid phase separation of TDP-43 in vitro and is required for TDP-43 accumulation in stress granules in mammalian cells and neurons. Stress granule localization initially protects TDP-43 from disease-associated phosphorylation, but upon long-term stress, stress granules resolve, leaving behind aggregates of phosphorylated TDP-43. Finally, small-molecule inhibition of Tankyrase-1/2 in mammalian cells inhibits formation of cytoplasmic TDP-43 foci without affecting stress granule assembly. Thus, Tankyrase inhibition antagonizes TDP-43-associated pathology and neurodegeneration and could have therapeutic utility for ALS and FTD.

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.

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.

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.

Poly(ADP-Ribosylation) in Age-Related Neurological Disease.

A central and causative feature of age-related neurodegenerative disease is the deposition of misfolded proteins in the brain. To devise novel approaches to treatment, regulatory pathways that modulate these aggregation-prone proteins must be defined. One such pathway is post-translational modification by the addition of poly(ADP-ribose) (PAR), which promotes protein recruitment and localization in several cellular contexts. Mounting evidence implicates PAR in seeding the abnormal localization and accumulation of proteins that are causative of neurodegenerative disease. Inhibitors of PAR polymerase (PARP) activity have been developed as cancer therapeutics, raising the possibility that they could be used to treat neurodegenerative disease. We focus on pathways regulated by PAR in neurodegenerative disease, with emphasis on amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).

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.

Impact of age-associated increase in 2'-O-methylation of miRNAs on aging and neurodegeneration in Drosophila.

MicroRNAs (miRNAs) are 20- to ∼24-nucleotide (nt) small RNAs that impact a variety of biological processes, from development to age-associated events. To study the role of miRNAs in aging, studies have profiled the levels of miRNAs with time. However, evidence suggests that miRNAs show heterogeneity in length and sequence in different biological contexts. Here, by examining the expression pattern of miRNAs by Northern blot analysis, we found that Drosophila miRNAs show distinct isoform pattern changes with age. Surprisingly, an increase of some miRNAs reflects increased 2'-O-methylation of select isoforms. Small RNA deep sequencing revealed a global increase of miRNAs loaded into Ago2, but not into Ago1, with age. Our data suggest increased loading of miRNAs into Ago2, but not Ago1, with age, indicating a mechanism for differential loading of miRNAs with age between Ago1 and Ago2. Mutations in Hen1 and Ago2, which lack 2'-O-methylation of miRNAs, result in accelerated neurodegeneration and shorter life span, suggesting a potential impact of the age-associated increase of 2'-O-methylation of small RNAs on age-associated processes. Our study highlights that miRNA 2'-O-methylation at the 3' end is modulated by differential partitioning of miRNAs between Ago1 and Ago2 with age and that this process, along with other functions of Ago2, might impact age-associated events in Drosophila.

Poly(ADP-ribose) Engages the TDP-43 Nuclear-Localization Sequence to Regulate Granulo-Filamentous Aggregation.

TAR DNA-binding protein of 43 kDa (TDP-43) forms granulo-filamentous aggregates in affected brain regions of >95% of patients with ALS and ∼50% of patients with frontotemporal degeneration (FTD). Furthermore, in disease, TDP-43 becomes N-terminally truncated resulting in protein deposits that are mainly composed of the C-terminal prion-like domain (PrLD). The PrLD is inherently aggregation-prone and is hypothesized to drive protein aggregation of TDP-43 in disease. Here, we establish that the N-terminal region of the protein is critical for rapid TDP-43 granulo-filamentous aggregation. We show that the biopolymer poly(ADP-ribose), or PAR, inhibits granulo-filamentous aggregation of TDP-43 by engaging PAR-binding motifs (PBMs) embedded in the TDP-43 nuclear-localization sequence. We demonstrate that progressive N-terminal truncation of TDP-43 can decelerate aggregation kinetics and promote formation of thread-like filaments. Thus, the N-terminal region and the PBMs of TDP-43 promote rapid granulo-filamentous aggregation and antagonize formation of thread-like fibrils. These findings illustrate the complexity of TDP-43 aggregation trajectories.

The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila.

Human neurodegenerative diseases have the temporal hallmark of afflicting the elderly population. Ageing is one of the most prominent factors to influence disease onset and progression, yet little is known about the molecular pathways that connect these processes. To understand this connection it is necessary to identify the pathways that functionally integrate ageing, chronic maintenance of the brain and modulation of neurodegenerative disease. MicroRNAs (miRNA) are emerging as critical factors in gene regulation during development; however, their role in adult-onset, age-associated processes is only beginning to be revealed. Here we report that the conserved miRNA miR-34 regulates age-associated events and long-term brain integrity in Drosophila, providing a molecular link between ageing and neurodegeneration. Fly mir-34 expression exhibits adult-onset, brain-enriched and age-modulated characteristics. Whereas mir-34 loss triggers a gene profile of accelerated brain ageing, late-onset brain degeneration and a catastrophic decline in survival, mir-34 upregulation extends median lifespan and mitigates neurodegeneration induced by human pathogenic polyglutamine disease protein. Some of the age-associated effects of miR-34 require adult-onset translational repression of Eip74EF, an essential ETS domain transcription factor involved in steroid hormone pathways. Our studies indicate that miRNA-dependent pathways may have an impact on adult-onset, age-associated events by silencing developmental genes that later have a deleterious influence on adult life cycle and disease, and highlight fly miR-34 as a key miRNA with a role in this process.

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans.

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

A fly model for the CCUG-repeat expansion of myotonic dystrophy type 2 reveals a novel interaction with MBNL1.

Expanded non-coding RNA repeats of CUG and CCUG are the underlying genetic causes for myotonic dystrophy type 1 (DM1) and type 2 (DM2), respectively. A gain-of-function of these pathogenic repeat expansions is mediated at least in part by their abnormal interactions with RNA-binding proteins such as MBNL1 and resultant loss of activity of these proteins. To study pathogenic mechanisms of CCUG-repeat expansions in an animal model, we created a fly model of DM2 that expresses pure, uninterrupted CCUG-repeat expansions ranging from 16 to 720 repeats in length. We show that this fly model for DM2 recapitulates key features of human DM2 including RNA repeat-induced toxicity, ribonuclear foci formation and changes in alternative splicing. Interestingly, expression of two isoforms of MBNL1, MBNL135 and MBNL140, leads to cleavage and concurrent upregulation of the levels of the RNA-repeat transcripts, with MBNL140 having more significant effects than MBNL135. This property is shared with a fly CUG-repeat expansion model. Our results suggest a novel mechanism for interaction between the pathogenic RNA repeat expansions of myotonic dystrophy and MBNL1.

Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.

The causes of amyotrophic lateral sclerosis (ALS), a devastating human neurodegenerative disease, are poorly understood, although the protein TDP-43 has been suggested to have a critical role in disease pathogenesis. Here we show that ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2, is a potent modifier of TDP-43 toxicity in animal and cellular models. ATXN2 and TDP-43 associate in a complex that depends on RNA. In spinal cord neurons of ALS patients, ATXN2 is abnormally localized; likewise, TDP-43 shows mislocalization in spinocerebellar ataxia type 2. To assess the involvement of ATXN2 in ALS, we analysed the length of the polyQ repeat in the ATXN2 gene in 915 ALS patients. We found that intermediate-length polyQ expansions (27-33 glutamines) in ATXN2 were significantly associated with ALS. These data establish ATXN2 as a relatively common ALS susceptibility gene. Furthermore, these findings indicate that the TDP-43-ATXN2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

Hsp104 suppresses polyglutamine-induced degeneration post onset in a drosophila MJD/SCA3 model.

There are no effective therapeutics that antagonize or reverse the protein-misfolding events underpinning polyglutamine (PolyQ) disorders, including Spinocerebellar Ataxia Type-3 (SCA3). Here, we augment the proteostasis network of Drosophila SCA3 models with Hsp104, a powerful protein disaggregase from yeast, which is bafflingly absent from metazoa. Hsp104 suppressed eye degeneration caused by a C-terminal ataxin-3 (MJD) fragment containing the pathogenic expanded PolyQ tract, but unexpectedly enhanced aggregation and toxicity of full-length pathogenic MJD. Hsp104 suppressed toxicity of MJD variants lacking a portion of the N-terminal deubiquitylase domain and full-length MJD variants unable to engage polyubiquitin, indicating that MJD-ubiquitin interactions hinder protective Hsp104 modalities. Importantly, in staging experiments, Hsp104 suppressed toxicity of a C-terminal MJD fragment when expressed after the onset of PolyQ-induced degeneration, whereas Hsp70 was ineffective. Thus, we establish the first disaggregase or chaperone treatment administered after the onset of pathogenic protein-induced degeneration that mitigates disease progression.

Maintaining the brain: insight into human neurodegeneration from Drosophila melanogaster mutants.

The fruitfly Drosophila melanogaster has enabled significant advances in neurodegenerative disease research, notably in the identification of genes that are required to maintain the structural integrity of the brain, defined by recessive mutations that cause adult onset neurodegeneration. Here, we survey these genes in the fly and classify them according to five key cell biological processes. Over half of these genes have counterparts in mice or humans that are also associated with neurodegeneration. Fly genetics continues to be instrumental in the analysis of degenerative disease, with notable recent advances in our understanding of several inherited disorders, Parkinson's disease, and the central role of mitochondria in neuronal maintenance.

Protein interacting with C kinase (PICK1) is a suppressor of spinocerebellar ataxia 3-associated neurodegeneration in Drosophila.

Spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant ataxia. The disease is caused by an expansion of a CAG-trinucelotide repeat region within the coding sequence of the ATXN3 gene, and this results in an expanded polyglutamine (polyQ) tract within the Ataxin-3 protein. The polyQ expansion leads to neuronal dysfunction and cell death. Here, we tested the ability of a number of proteins that interact with Ataxin-3 to modulate SCA3 pathogenicity using Drosophila. Of 10 candidates, we found four novel enhancers and one suppressor. The suppressor, PICK1 (Protein interacting with C kinase 1), is a transport protein that regulates the trafficking of ion channel subunits involved in calcium homeostasis to and from the plasma membrane. In line with calcium homeostasis being a potential pathway mis-regulated in SCA3, we also found that down-regulation of Nach, an acid sensing ion channel, mitigates SCA3 pathogenesis in flies. Modulation of PICK1 could be targeted in other neurodegenerative diseases, as the toxicity of SCA1 and tau was also suppressed when PICK1 was down-regulated. These findings indicate that interaction proteins may define a rich source of modifier pathways to target in disease situations.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.