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.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily impacting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and Ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are striking modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster, eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component polyA binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α-phosphorylation in ALS models. This treatment mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that dysfunction induced by prolonged stress granule formation may contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

With the increase in the ageing population, neurodegenerative disease is devastating to families and poses a huge burden on society. The brain and spinal cord are extraordinarily complex: they consist of a highly organized network of neuronal and support cells that communicate in a highly specialized manner. One approach to tackling problems of such complexity is to address the scientific questions in simpler, yet analogous, systems. The fruit fly, Drosophila melanogaster, has been proven tremendously valuable as a model organism, enabling many major discoveries in neuroscientific disease research. The plethora of genetic tools available in Drosophila allows for exquisite targeted manipulation of the genome. Due to its relatively short lifespan, complex questions of brain function can be addressed more rapidly than in other model organisms, such as the mouse. Here we discuss features of the fly as a model for human neurodegenerative disease. There are many distinct fly models for a range of neurodegenerative diseases; we focus on select studies from models of polyglutamine disease and amyotrophic lateral sclerosis that illustrate the type and range of insights that can be gleaned. In discussion of these models, we underscore strengths of the fly in providing understanding into mechanisms and pathways, as a foundation for translational and therapeutic research.

Flies with mutations in the single Drosophila Adar gene encoding an RNA editing enzyme involved in editing 4% of all transcripts have severe locomotion defects and develop age-dependent neurodegeneration. Vertebrates have two ADAR-editing enzymes that are catalytically active; ADAR1 and ADAR2. We show that human ADAR2 rescues Drosophila Adar mutant phenotypes. Neither the short nuclear ADAR1p110 isoform nor the longer interferon-inducible cytoplasmic ADAR1p150 isoform rescue walking defects efficiently, nor do they correctly edit specific sites in Drosophila transcripts. Surprisingly, human ADAR1p110 does suppress age-dependent neurodegeneration in Drosophila Adar mutants whereas ADAR1p150 does not. The single Drosophila Adar gene was previously assumed to represent an evolutionary ancestor of the multiple vertebrate ADARs. The strong functional similarity of human ADAR2 and Drosophila Adar suggests rather that these are true orthologs. By a combination of direct cloning and searching new invertebrate genome sequences we show that distinct ADAR1 and ADAR2 genes were present very early in the Metazoan lineage, both occurring before the split between the Bilateria and Cnidarians. The ADAR1 gene has been lost several times, including during the evolution of insects and crustacea. These data complement our rescue results, supporting the idea that ADAR1 and ADAR2 have evolved highly conserved, distinct functions.

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2–5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease in which the loss of spinal cord motor neurons leads to paralysis and death within a few years of clinical disease onset. In almost all cases of ALS, TAR DNA binding protein of 43 kDa (TDP-43) forms cytoplasmic neuronal inclusions. A second causative gene for a subset of ALS is fused in sarcoma (FUS), an RNA binding protein that also forms cytoplasmic inclusions in spinal cord motor neurons. Poly A binding protein 1 (PABP-1) is a marker of stress granules, i.e. accumulations of proteins and RNA indicative of translational arrest in cells under stress. We report on the colocalization PABP-1 to both TDP-43 and FUS inclusions in 4 patient cohorts: ALS without a mutation, ALS with an intermediate poly glutamine repeat expansion in ATXN2, ALS with a GGGGCC-hexanucleotide repeat expansion in C9orf72, and ALS with basophilic inclusion body disease. Notably, PABP-1 colocalization to TDP-43 was twice as frequent in ALS with C9orf72 expansions compared to ALS with no mutation. This study highlights PABP-1 as a protein important to the pathology of ALS and indicates that the proteomic profile of TDP-43 inclusions in ALS may be different depending on the causative genetic mutation.

BackgroundThe major hindrance to imaging the intact adult Drosophila is that the dark exoskeleton makes it impossible to image through the cuticle. We have overcome this obstacle and describe a method whereby the internal organs of adult Drosophila can be imaged in 3D by bleaching and clearing the adult and then imaging using a technique called optical projection tomography (OPT). The data is displayed as 2D optical sections and also in 3D to provide detail on the shape and structure of the adult anatomy.MethodologyWe have used OPT to visualize in 2D and 3D the detailed internal anatomy of the intact adult Drosophila. In addition this clearing method used for OPT was tested for imaging with confocal microscopy. Using OPT we have visualized the size and shape of neurodegenerative vacuoles from within the head capsule of flies that suffer from age-related neurodegeneration due to a lack of ADAR mediated RNA-editing. In addition we have visualized tau-lacZ expression in 2D and 3D. This shows that the wholemount adult can be stained without any manipulation and that this stain penetrates well as we have mapped the localization pattern with respect to the internal anatomy.ConclusionWe show for the first time that the intact adult Drosophila can be imaged in 3D using OPT, also we show that this method of clearing is also suitable for confocal microscopy to image the brain from within the intact head. The major advantage of this is that organs can be represented in 3D in their natural surroundings. Furthermore optical sections are generated in each of the three planes and are not prone to the technical limitations that are associated with manual sectioning. OPT can be used to dissect mutant phenotypes and to globally map gene expression in both 2D and 3D.