Developmental expression of 4-repeat-Tau induces neuronal aneuploidy in Drosophila tauopathy models

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development

Functional analysis of TDP-43 neurotoxicity in Drosophila

TDP-43 is well known as a nuclear RNA/DNA binding protein involved in ma ny aspects of RNA metabolism. Accumulation of TDP-43 have been identified in the large majority of patients within the amyotrophic lateral sclerosis-frontotemporal dementi a spectrum of disorders. TDP-43 proteinopathy is also found in more than 30% of Alzhei mer s disease brains, suggesting a broad role for TDP-43 in neurodegeneration in the g eneral population. TDP-43 positive inclusions are the major pathological hallmark of the di sease and are typically located in the neuronal cytoplasm and accompanied by a loss of nuclear TDP-43 expression. ALS (and sometimes FTD) can be caused by heterozygous mutati ons in hTDP- 43, although in the majority of patients that suffer from TDP-43 prot einopathy no hTDP-43 mutations are present. Although this proteinopathy has been discovered ~ 8 years ago, its pathogenetic mechanism is still not clear and a major unresolved questio n is whether TDP-43- mediated neurotoxicity is caused by a gain or loss-of-function mechanism . TDP-43 is evolutionary well-conserved and in Drosophila, there is a homolog called TBPH or dTDP-43 which, similar to its human counterpart, has two RNA recognition motifs, a glycine rich region and displays similar nucleic acid binding and mRNA splicing prope rties. In the first part of this PhD thesis, we further clarified the normal fu nction of dTDP-43 in Drosophila. We therefore combined a phenotypical analysis with next g eneration transcriptome analysis (RNA-seq) of dTDP-43 gain and loss of function fl ies. We found in Drosophila that dTDP-43 coordinates the switching of ecdysteroid rece ptor (EcR)-dependent transcriptional programs from a pupal to an adult pattern and that a dis turbance of this function by gain or loss of dTDP-43 results in lethality and enhanced ne uronal apoptosis. dTDP-43 controls this switching by regulating the expression of a microt ubule-binding protein that is reponsible for the correct subcellular localization of E cR. Our results establish disrupted EcR signaling as a cellular mechanism underlying dTDP-43 neuro toxicity in Drosophila and identify steroid hormone receptor signaling as a poten tially important pathway in ALS and related TDP-43 proteinopathies. Since increasing and decreasing expression of dTDP-43 lead to largely overlapping transcriptomic alterat ions, cytoplasmic EcR accumulations and neurotoxic developmental phenotypes, our study sug gests that TDP- 43 aggregation results in its loss-of-function. Secondly, we wanted to clarify the pathogenic character of mutations in TDP-43. Most heterozygous hTDP-43 mutations located in the C-terminal glycine-r ich region of the protein cause ALS, but other atypical variants such as hTDP-43A90V (locate d in the nuclear localization signal) and hTDP-43D169G (located in the first RNA-bi nding domain) are described as well. In order to unravel the pathogenic nature of the diff erent variants as well as their gain- and/or loss-of-function properties, we have used site-specif ic transgene integration to express hTDP-43WT, two typical ALS-causing mutations (hTDP-4 3G287S and hTDP-43A315T) and two atypical variants (hTDP-43A90V and hTDP-43D169G) in dTDP-43 loss-of-function flies and checked their ability to rescue the loss-of-function phenotypes. We discovered that hTDP-43A90V, hTDP-43G287S and hTDP-43A315T failed to resc ue the neuronal loss, while hTDP- 43WT and hTDP-43D169G could rescue, suggesting that the hTDP -43A90V, hTDP-43G287S and hTDP-43A315T mutations have loss-of-function properties. We also repo rted a shift of hTDP- 43 from the nucleus to the cytoplasm in ~10% of the bursicon neurons in hTDP-43A90V, hTDP-43G287S and hTDP-43A315T but not hTDP-43D169G and hTDP-43WT flies. Next, we provide additional evidence that the rare variant hTDP-43A90V is i ndeed pathogenic and might increase the risk to develop Alzheimer s disease (AD) in the French-Belg ian population. Together these in vivo data suggest that typical ALS-causing mutat ions (G287S, A315T) and the rare variant A90V might be pathogenic through a loss-of-function mec hanism and that pathogenic mechanism in TDP-43 associated neurodegeneration is rather ca used by a loss of the normal molecular function of TDP-43 than a novel toxic gain of funct ion. In conclusion, we show that D. melanogaster is an excellent model to study TDP-43 proteinopathies and we provide substantial evidence that TDP-43-associat ed neurodegeneration is rather caused by a loss of the normal molecular fun ction of TDP-43 thana novel toxic gain of function.status: publishe

TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis?

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are clinically distinct fatal neurodegenerative disorders. Increasing molecular evidence indicates that both disorders are linked in a continuous spectrum (ALS-FTD spectrum). Neuronal cytoplasmic inclusions consisting of the nuclear TAR DNA-binding protein 43 (TDP-43) are found in the large majority of patients in the ALS-FTD spectrum and dominant mutations in the TDP-43 gene cause ALS. A major unresolved question is whether TDP-43-mediated neuronal loss is caused by toxic gain of function of cytoplasmic aggregates, or by a loss of its normal function in the nucleus. Here we argue that based on recent genetic studies in worms, flies, fish, and rodents, loss of function of TDP-43, rather than toxic aggregates, is the key factor in TDP-43-related proteinopathies.publisher: Elsevier articletitle: TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis? journaltitle: Trends in Molecular Medicine articlelink: http://dx.doi.org/10.1016/j.molmed.2013.11.003 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved.status: publishe

TDP-43-mediated neurodegeneration : towards a loss-of-function hypothesis?

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are clinically distinct fatal neurodegenerative disorders. Increasing molecular evidence indicates that both disorders are linked in a continuous spectrum (ALS-FTD spectrum). Neuronal cytoplasmic inclusions consisting of the nuclear TAR DNA-binding protein 43 (TDP-43) are found in the large majority of patients in the ALS-FTD spectrum and dominant mutations in the TDP-43 gene cause ALS. A major unresolved question is whether TDP-43-mediated neuronal loss is caused by toxic gain of function of cytoplasmic aggregates, or by a loss of its normal function in the nucleus. Here we argue that based on recent genetic studies in worms, flies, fish, and rodents, loss of function of TDP-43, rather than toxic aggregates, is the key factor in TDP-43-related proteinopathies

Hearing regulates Drosophila aggression

Aggression is a universal social behavior important for the acquisition of food, mates, territory, and social status. Aggression in Drosophila is context-dependent and can thus be expected to involve inputs from multiple sensory modalities. Here, we use mechanical disruption and genetic approaches in Drosophila melanogaster to identify hearing as an important sensory modality in the context of intermale aggressive behavior. We demonstrate that neuronal silencing and targeted knockdown of hearing genes in the fly's auditory organ elicit abnormal aggression. Further, we show that exposure to courtship or aggression song has opposite effects on aggression. Our data define the importance of hearing in the control of Drosophila intermale aggression and open perspectives to decipher how hearing and other sensory modalities are integrated at the neural circuit level.status: publishe

Functional complementation in Drosophila to predict the pathogenicity of TARDBP variants: evidence for a loss-of-function mechanism

The human TAR DNA binding protein 43 (TDP-43), encoded by the gene TARDBP, plays a central role in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 inclusions are also found in up to approximately 60% of Alzheimer's disease (AD) brains. Although ALS-causing TARDBP mutations cluster in the C-terminal glycine-rich region of the protein, the pathogenic nature of the atypical missense variants p.A90V (located between the bipartite nuclear localization signal) and p.D169G (located in the first RNA-binding domain) is unclear. In addition, whether causal ALS mutations represent gain or loss-of-function alleles remains unknown. We recently reported that loss-of-function of the highly conserved TARDBP ortholog in Drosophila (called TBPH) leads to death of bursicon neurons resulting in adult maturation and wing expansion defects. Here, we compared wild-type TARDBP, 2 typical ALS-causing mutations (p.G287S and p.A315T) and 2 atypical variants (p.A90V and p.D169G), for their ability to complement neuronal TBPH loss-of-function. Although p.D169G rescued organismal pupal lethality and neuronal loss to a similar extent as wild-type TARDBP, p.A90V, p.G287S, and p.A315T were less efficient. Accordingly, p.A90V, p.G287S, and p.A315T but not p.D169G or wild-type protein promoted a shift of TDP-43 from the nucleus to the cytoplasm in approximately 12%-14% of bursicon neurons. Finally, we found that the carrier frequency of rare variant p.A90V was higher in French-Belgian AD cases (5/1714, 0.29%) than in controls of European descent (5/9436, 0.05%) (odds ratio = 5.5; 95% confidence interval, 1.6-19.0; p = 0.009). We propose that pathogenic TARDBP mutations have partial loss-of-function properties and that TARDBP p.A90V may increase AD risk by the same mechanism.publisher: Elsevier articletitle: Functional complementation in Drosophila to predict the pathogenicity of TARDBP variants: evidence for a loss-of-function mechanism journaltitle: Neurobiology of Aging articlelink: http://dx.doi.org/10.1016/j.neurobiolaging.2014.09.001 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe