Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth

Abstract

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 + inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS-and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Doublehomozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.neurodegeneration | zinc finger nuclease | proteomics A myotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin and TDP-43 + inclusions (FTLD-TDP) are incurable fatal neurodegenerative diseases. ALS is characterized by the loss of upper and lower motor neurons and FTLD patients suffer from degeneration of the frontal and temporal lobes. Both diseases belong to an ALS-FTLD disease spectrum (1, 2). TDP-43 is a RNA binding protein, which modulates RNA splicing, and systematic cross-linking and immunoprecipitation studies identified numerous RNA targets (3-5). About 90% of ALS cases are sporadic and the majority is pathologically characterized by insoluble TDP-43 inclusions (1, 6). Mutations in TDP-43 can lead to familial forms of ALS and FTLD-TDP (7,8). Most of the patientassociated mutations cluster in the C-terminal glycine-rich domain of TDP-43 (9), which mediates protein-protein interactions and is required for splicing-associated activities as well as autoregulation (10)(11)(12)(13)(14). Pathologically, ALS and FTLD-TDP are characterized by nuclear clearance and deposition of insoluble . Whether neurotoxicity of the TDP-43 inclusions or reduced TDP-43 function upon nuclear clearance is responsible for ALS and FTLD is under debate. However, little is known about the physiological function of TDP-43. To obtain insights into the in vivo function of TDP-43, we investigated the morphological, developmental, and molecular consequences of a loss of TDP-43 in zebrafish.

Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis

“…Loss-of-function models in invertebrates and zebrafish have shown a wide range of defects, ranging from embryonic lethality to morphological and functional defects of the nervous system, vascular system, and muscle degeneration (27,31,(34)(35)(36)(37). In mammalian species, TDP-43 gene deletion leads to early embryonic lethality (38)(39)(40)(41).…”

“…Because both nuclear depletion and cytoplasmic aggregation are expected to reduce the amount of functional TDP-43, dysfunction of TDP-43 is probable in these cells. Assays of TDP-43 function derived from human CNS tissue implicate the existence of TDP-43 dysfunction in both ALS and FTLD (30,31). The consequences of TDP-43 dysfunction may be serious.…”

Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function

“…Under specific cellular stress conditions, such as oxidative stress, TDP-43 localizes to cytoplasmic stress granules (11), which may contribute to pathological aggregate formation (12). TDP-43 is essential for development and survival in animal models and cultured cells (13)(14)(15)(16)(17). In human cells and mouse brain, TDP-43 binds thousands of transcripts with a strong preference for GU-rich sequences and regulates Ͼ600 protein-coding genes.…”

Between new genetic discoveries and large randomized trials—neurological research in the era of systems medicine

“…Due to a partial genome duplication, zebrafish have two TDP-43-related genes, both of which the Schmid and Haass groups …the most remarkable… revision of the classical amyloid hypothesis is the rapidly spreading view of amyloid 'infectivity' … …neurodegeneration research needs to integrate more closely with research into neuronal cell biology and physiology… upfront meeting p oint deleted by zinc-finger-based genome editing. Remarkably, the double-deletion results in a profound vascular phenotype with vessels mis-patterning and accompanying muscle and motor axon pathology [19]. Comprehensive proteomics analysis in collaboration with Lichtenthaler-in this form a novelty in zebrafish-subsequently revealed mis-regulation of certain muscle proteins that could also be detected in the brains of a subpopulation of human FTLD patients, demonstrating the power of zebrafish-based 'omics' analysis to discover molecular phenotypes of potential disease relevance.…”