Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development

The inflammatory chemokine Cxcl18b exerts neutrophil-specific chemotaxis via the promiscuous chemokine receptor Cxcr2 in zebrafish

Cxcl18b is a chemokine found in zebrafish and in other piscine and amphibian species. Cxcl18b is a reliable inflammatory marker; however, its function is yet to be elucidated. Here, we found that Cxcl18b is chemotactic towards neutrophils, similarly to Cxcl8a/Interleukin-8, the best characterised neutrophil chemoattractant in humans and teleosts. Like Cxcl8a, Cxcl18b-dependent recruitment required the chemokine receptor Cxcr2, while it was unaffected by depletion of the other two neutrophil receptors cxcr1 and cxcr4b. To visualise cxcl18b induction, we generated a Tg(cxcl18b:eGFP) reporter line. The transgene is induced locally upon bacterial infection with the fish pathogen Mycobacterium marinum, but strikingly is not directly expressed by infected cells. Instead, cxcl18b is induced by non-phagocytic uninfected cells that compose the stroma of the granulomas, typical inflammatory lesions formed upon mycobacterial infections. Together, these results suggest that Cxcl18b might be an important contributor to neutrophil chemotaxis in the inflammatory microenvironment and indicate that the zebrafish model could be explored to further investigate in vivo the biological relevance of different Cxcl8-like chemokine lineages

Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo

Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholino-mediated knockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammation resolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation

Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import

The majority of familial ALS (fALS)-associated mutations occurs within the nuclear localization signal (NLS) and impairs nuclear import. Nuclear import of FUS depends on Transportin and interference with this pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules

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

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. Double-homozygous 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

Table1_Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1.XLSX

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development.</p