RESUMO
Disease-associated variants of TUBA4A (alpha-tubulin 4A) have recently been identified in familial ALS. Interestingly, a downregulation of TUBA4A protein expression was observed in familial as well as sporadic ALS brain tissue. To investigate whether a decreased TUBA4A expression could be a driving factor in ALS pathogenesis, we assessed whether TUBA4A knockdown in zebrafish could recapitulate an ALS-like phenotype. For this, we injected an antisense oligonucleotide morpholino in zebrafish embryos targeting the zebrafish TUBA4A orthologue. An antibody against synaptic vesicle 2 was used to visualize motor axons in the spinal cord, allowing the analysis of embryonic ventral root projections. Motor behavior was assessed using the touch-evoked escape response. In post-mortem ALS motor cortex, we observed reduced TUBA4A levels. The knockdown of the zebrafish TUBA4A orthologue induced a motor axonopathy and a significantly disturbed motor behavior. Both phenotypes were dose-dependent and could be rescued by the addition of human wild-type TUBA4A mRNA. Thus, TUBA4A downregulation as observed in ALS post-mortem motor cortex could be modeled in zebrafish and induced a motor axonopathy and motor behavior defects reflecting a motor neuron disease phenotype, as previously described in embryonic zebrafish models of ALS. The rescue with human wild-type TUBA4A mRNA suggests functional conservation and strengthens the causal relation between TUBA4A protein levels and phenotype severity. Furthermore, the loss of TUBA4A induces significant changes in post-translational modifications of tubulin, such as acetylation, detyrosination and polyglutamylation. Our data unveil an important role for TUBA4A in ALS pathogenesis, and extend the relevance of TUBA4A to the majority of ALS patients, in addition to cases bearing TUBA4A mutations.
RESUMO
Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.
