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Project

Drosophila models for TDP-43 and FUS proteinopathy, new therapeutic targets for neurodegeneration

TDP-43 is well known as a nuclear RNA/DNA binding protein involved in many aspects of

RNA metabolism. Accumulation of TDP-43 have been identified in the large majority of

patients within the amyotrophic lateral sclerosis-frontotemporal dementia spectrum of

disorders. TDP-43 proteinopathy is also found in more than 30% of Alzheimers disease

brains, suggesting a broad role for TDP-43 in neurodegeneration in the general population.

TDP-43 positive inclusions are the major pathological hallmark of the disease 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 mutations in hTDP-

43

, although in the majority of patients that suffer from TDP-43 proteinopathy 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 question 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 properties.

In the first part of this PhD thesis, we further clarified the normal function of dTDP-43 in

Drosophila

. We therefore combined a phenotypical analysis with next generation

transcriptome analysis (RNA-seq) of dTDP-43 gain and loss of function flies. We found in

Drosophila 

that dTDP-43 coordinates the switching of ecdysteroid receptor (EcR)-dependent

transcriptional programs from a pupal to an adult pattern and that a disturbance of this

function by gain or loss of dTDP-43 results in lethality and enhanced neuronal apoptosis.

dTDP-43 controls this switching by regulating the expression of a microtubule-binding

protein that is reponsible for the correct subcellular localization of EcR. Our results establish

disrupted EcR signaling as a cellular mechanism underlying dTDP-43 neurotoxicity in

Drosophila 

and identify steroid hormone receptor signaling as a potentially important

pathway in ALS and related TDP-43 proteinopathies. Since increasing and decreasing

expression of dTDP-43 lead to largely overlapping transcriptomic alterations, cytoplasmic

EcR accumulations and neurotoxic developmental phenotypes, our study suggests 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-rich region of the protein

cause ALS, but other atypical variants such as hTDP-43A90V (located in the nuclear

localization signal) and hTDP-43D169G (located in the first RNA-binding domain) are

described as well. In order to unravel the pathogenic nature of the different variants as well as

their gain- and/or loss-of-function properties, we have used site-specific transgene integration

to express hTDP-43WT, two typical ALS-causing mutations (hTDP-43G287S 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 rescue the neuronal loss, while hTDP-

43WT 

and hTDP-43D169G could rescue, suggesting that the hTDP-43A90VhTDP-43G287S and

hTDP-43A315T 

mutations have loss-of-function properties. We also reported 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 indeed pathogenic and might

increase the risk to develop Alzheimers disease (AD) in the French-Belgian population.

Together these in vivo data suggest that typical ALS-causing mutations (G287S, A315T) and

the rare variant A90V might be pathogenic through a loss-of-function mechanism and that

pathogenic mechanism in TDP-43 associated neurodegeneration is rather caused by a loss of

the normal molecular function of TDP-43 than a novel toxic gain of function.

In conclusion, we show that D. melanogaster is an excellent model to study TDP-43

proteinopathies and we provide substantial evidence that TDP-43-associated

neurodegeneration is rather caused by a loss of the normal molecular function of TDP-43 than
a novel toxic gain of function.

Date:1 Jan 2010 →  15 Apr 2014
Keywords:drosophila models
Disciplines:Genetics, Systems biology, Molecular and cell biology, Medical imaging and therapy, Other paramedical sciences
Project type:PhD project