Danielle Diaper

Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes.

Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.

TDP-43 Loss-of-Function Causes Neuronal Loss Due to Defective Steroid Receptor-Mediated Gene Program Switching in Drosophila

TDP-43 proteinopathy is strongly implicated in the pathogenesis of amyotrophic lateral sclerosis and related neurodegenerative disorders. Whether TDP-43 neurotoxicity is caused by a novel toxic gain-of-function mechanism of the aggregates or by a loss of its normal function is unknown. We increased and decreased expression of TDP-43 (dTDP-43) in Drosophila. Although upregulation of dTDP-43 induced neuronal ubiquitin and dTDP-43-positive inclusions, both up- and downregulated dTDP-43 resulted in selective apoptosis of bursicon neurons and highly similar transcriptome alterations at the pupal-adult transition. Gene network analysis and genetic validation showed that both up- and downregulated dTDP-43 directly and dramatically increased the expression of the neuronal microtubule-associated protein Map205, resulting in cytoplasmic accumulations of the ecdysteroid receptor (EcR) and a failure to switch EcR-dependent gene programs from a pupal to adult pattern. We propose that dTDP-43 neurotoxicity is caused by a loss of its normal function.

Drosophila TDP-43 dysfunction in glia and muscle cells cause cytological and behavioural phenotypes that characterize ALS and FTLD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that are characterized by cytoplasmic aggregates and nuclear clearance of TAR DNA-binding protein 43 (TDP-43). Studies in Drosophila, zebrafish and mouse demonstrate that the neuronal dysfunction of TDP-43 is causally related to disease formation. However, TDP-43 aggregates are also observed in glia and muscle cells, which are equally affected in ALS and FTLD; yet, it is unclear whether glia- or muscle-specific dysfunction of TDP-43 contributes to pathogenesis. Here, we show that similar to its human homologue, Drosophila TDP-43, Tar DNA-binding protein homologue (TBPH), is expressed in glia and muscle cells. Muscle-specific knockdown of TBPH causes age-related motor abnormalities, whereas muscle-specific gain of function leads to sarcoplasmic aggregates and nuclear TBPH depletion, which is accompanied by behavioural deficits and premature lethality. TBPH dysfunction in glia cells causes age-related motor deficits and premature lethality. In addition, both loss and gain of Drosophila TDP-43 alter mRNA expression levels of the glutamate transporters Excitatory amino acid transporter 1 (EAAT1) and EAAT2. Taken together, our results demonstrate that both loss and gain of TDP-43 function in muscle and glial cells can lead to cytological and behavioural phenotypes in Drosophila that also characterize ALS and FTLD and identify the glutamate transporters EAAT1/2 as potential direct targets of TDP-43 function. These findings suggest that together with neuronal pathology, glial- and muscle-specific TDP-43 dysfunction may directly contribute to the aetiology and progression of TDP-43-related ALS and FTLD.

A lineage-related reciprocal inhibition circuitry for sensory-motor action selection

The insect central complex and vertebrate basal ganglia are forebrain centres involved in selection and maintenance of behavioural actions. However, little is known about the formation of the underlying circuits, or how they integrate sensory information for motor actions. Here, we show that paired embryonic neuroblasts generate central complex ring neurons that mediate sensory-motor transformation and action selection in Drosophila. Lineage analysis resolves four ring neuron subtypes, R1-R4, that form GABAergic inhibition circuitry among inhibitory sister cells. Genetic manipulations, together with functional imaging, demonstrate subtype-specific R neurons mediate the selection and maintenance of behavioural activity. A computational model substantiates genetic and behavioural observations suggesting that R neuron circuitry functions as salience detector using competitive inhibition to amplify, maintain or switch between activity states. The resultant gating mechanism translates facilitation, inhibition and disinhibition of behavioural activity as R neuron functions into selection of motor actions and their organisation into action sequences.

A feedback loop between dipeptide-repeat protein, TDP-43 and karyopherin-α mediates C9orf72-related neurodegeneration

TDP-43 accumulation is a major pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia, including the most common genetic cause, G4C2 hexanucleotide repeat expansion in C9ORF72 (C9ALS/FTD). Solomon et al. report that G4C2-derived dipeptide repeat protein but not G4C2-RNA accumulation causes TDP-43 proteinopathy that triggers onset and progression of disease.

Immunostaining of the Developing Embryonic and Larval Drosophila Brain

Immunostaining is used to visualize the spatiotemporal expression pattern of developmental control genes that regulate the genesis and specification of the embryonic and larval brain of Drosophila. Immunostaining uses specific antibodies to mark expressed proteins and allows their localization to be traced throughout development. This method reveals insights into gene regulation, cell-type specification, neuron and glial differentiation, and posttranslational protein modifications underlying the patterning and specification of the maturing brain. Depending on the targeted protein, it is possible to visualize a multitude of regions of the Drosophila brain, such as small groups of neurons or glia, defined subcomponents of the brains axon scaffold, or pre- and postsynaptic structures of neurons. Thus, antibody probes that recognize defined tissues, cells, or subcellular structures like axons or synaptic terminals can be used as markers to identify and analyze phenotypes in mutant embryos and larvae. Several antibodies, combined with different labels, can be used concurrently to examine protein co-localization. This protocol spans over 3-4 days.

TDP-43 neurotoxicity by failed steroid receptor-dependent transcriptional program switching

S FOR TALKS (IN CHRONOLOGICAL ORDER)

Both loss and gain of TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration in Drosophilia

Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. The genetics of FTD has been one of the success stories in genetics over the past 15 years. Classic family based linkage studies have identified genes that explain a large part of the families with a Mendelian inheritance of the disease. This group of familial FTD patients has now been linked to mutations in several genes, including the microtubule-associated protein tau (MAPT), progranulin (GRN), valosin-containing protein (VCP), charged multivescicular body protein 2B (CHMP2B), TAR DNA-binding protein 43 (TDP43) and Fused in Sarcoma (FUS) and most recently C9Orf72. Over the years the identified genes have triggered many studies that increased our understanding of the disease process. Neuropathologically the disease can be divided in two major groups that have a clear correlation with their genetic background; hose with tau-positive inclusions and those with ubiquitin-positive and TDP43 positive inclusions. The field of genetics keeps changing rapidly thanks to technological developments, first with the development of Genome Wide Association Studies (GWAS) studies but now also with the use of next generation sequencing, as was already demonstrated with the identification of the expanded repeat in C9Orf72, and we can also expect many whole exome or whole genome sequencing studies. This review provides an overview of the genetics of FTD, with an update of recent discoveries.

TDP-43 neurotoxicity is caused by a failure of steroid receptor–dependent transcriptional program switching in Drosophila

CAUSED BYA FAILURE OF STEROID RECEPTOR–DEPENDENT TRANSCRIPTIONAL PROGRAM SWITCHING IN DROSOPHILA Bart Dermaut, Lies Vanden Broeck, Marina Naval Sanchez, Yoshitsugu Adachi, Danielle Diaper, Gernot Kleinberger, Marc Gistelinck, Christine Van Broeckhoven, Frank Hirth, Stein Aerts, Patrick Callaerts, Pasteur Institute of Lille, Lille, France; 2 VIB University of Leuven, Leuven, Belgium; 3 University of Leuven, Leuven, Belgium; 4 King’s College London, London, United Kingdom; University of Antwerp, Antwerpen, Belgium; King’s College London, London, Belgium.