Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish

Abstract

Neurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in the differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit (NEFL), whose expression is reduced in motor neurons in amyotrophic lateral sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via a splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects the NF assembly and motor axon growth, with resultant motor deficits. This equilibrium is also disrupted upon the partial depletion of TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the gene TARDBP that is mislocalized into cytoplasmic inclusions in ALS. The study supports the interaction of the NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

Diving deep: zebrafish models in motor neuron degeneration research

“…Interestingly, TDP-43 binds with NEFL mRNA at its 3'UTR (MJ, 2007 ). A recent study in zebrafish revealed that MO-mediated knockdown of TDP-43 disrupts the splicing of NEFL zebrafish ortholog neflb , as the overexpression of one isoform of neflb rescued the reduction in the motor neuron axon length of TDP-43 knockdown embryos, while the other isoform exacerbated the phenotypes (Demy et al, 2020 ).…”

Posttranscriptional regulation of neurofilament proteins and tau in health and disease

“…Two isoforms of the latter gene, neflE3 and neflE4, are generated by alternative splicing of neflb mRNA ( Fig. 1B ) ( Demy et al, 2020 ). Alternative use of two polyadenylation sites in exon 4 in the mouse NfL gene also generates 2.3-and 3.5-kb mRNAs ( Nakahira et al, 1990 ).…”

“…The functional significance of this observation is not yet understood. Recent studies showed that disruption of the balance of the two isoforms of NfL mRNAs through partial deletion of TDP43 affects Nf assembly and motor axon growth with resultant motor deficits in zebrafish ( Demy et al, 2020 ). More recent studies demonstrated that TDP43 proteinopathy can cause translational suppression of Nf mRNAs resulting in 3-4-fold decrease in the levels of NfPs in mouse brain ( Kumar et al, 2021 ).…”

Exploring the alternative: Fish, flies and worms as preclinical models for ALS

“…Moreover, depleting both tardbp and tardbpl simultaneously caused a harsher zebrafish phenotype, comprising weight loss, reduced survival and muscle atrophy on top of the swimming and motor deficits, which were already present upon a tardbp-only KO [188,190,191]. Recently, zebrafish experiments showed that neurofilament splicing, essential for proper axonal transport and overall motor neuron health, was regulated by TDP-43, suggesting that both factors might be biologically and pathologically associated [192]. Further substantiation of zebrafish transgenic lines as assets for the investigation of TDP-43 ALS was found by performing comparative sequencing analyses.…”