Functional analysis of TDP-43 neurotoxicity in Drosophila

Abstract

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