TDP-43 is a splicing factor and regulates various aspects of RNA metabolism, and its levels are tightly regulated through a regulatory feedback loop (Avendano-Vazquez et al. 2012). Thousands of RNA targets of TDP-43 have been identified by cross-linking and immunoprecipitation (Polymenidou et al. 2011; Tollervey et al. 2011; Xiao et al. 2011); however, the physiological function of TDP-43 is still unclear. FTLD- and ALS-associated mutations in TDP-43 cluster in the C-terminal glycine-rich domain (Fig. 2a). It has been suggested that they increase the propensity to aggregate as nuclear TDP-43 is mainly found in cytosolic aggregates (Johnson et al. 2009). Alternatively, mutations may cause a partial loss of function as the nucleus becomes cleared of TDP-43 upon cytosolic aggregation (Neumann et al. 2006). Interestingly, the zebrafish genome harbors two TDP-43 ortholgues, Tardbp and Tardbp-like (Tardbpl). The second orthologue, Tardbpl, lacks a glycin-rich domain (Fig. 2a). Potential gain and loss of function of TDP-43 has been investigated in zebrafish. Injection of wildtype and mutant human TDP-43 mRNA causes shorter and aberrantly branched primary spinal motor neuron axons in zebrafish (Kabashi et al. 2010; Laird et al. 2010). The motor neuron axon phenotype is more pronounced upon injection of mutant compared with wildtype human TDP-43 in zebrafish (Kabashi et al. 2010). Interestingly, MO-mediated KD of zebrafish Tardbp has been reported to cause a similar motor neuron axon phenotype, leading the authors to speculate that ALS is generated by a combined loss and gain of function mechanism (Kabashi et al. 2010). However, two independent studies have subsequently demonstrated that loss of Tardbp in zebrafish is fully compensated by alternative splicing of Tardbpl (Fig. 2b) and therefore Tardbp mutants do not show any spinal motor neuron axon phenotype. Under wildtype conditions, Tardbpl is mainly expressed as a protein, lacking a C-terminal glycine-rich domain. Upon loss of Tardbp, a novel Tardbpl splice variant (Tardbpl_tv1) with a glycine-rich domain, highly homologous to that of Tardbp, is generated. (Hewamadduma et al. 2013; Schmid et al. 2013) (Fig. 2b). Tardbpl_tv1 can fully compensate for the loss of Tardbp because of the presence of the glycine-rich domain, which is known to be important for interaction with other hnRNPs and splicing (Buratti et al. 2005). Consistent with this finding, the reported Tardbp KD-induced shorter and hyperbranched motor neurons have not been observed in several lines of stable Tardbp mutants generated by genome editing (Schmid et al. 2013). Thus, MO KD phenotypes of Tardbp have to be interpreted with caution, as they may be the result of unspecific off-target effects or the MO injection procedure itself. In contrast to the single mutants, the double homozygous Tardbp and Tardbpl mutants are lethal, characterized by muscle degeneration and shorter spinal motor neuron axon phenotype (Schmid et al. 2013) (Fig. 2c). In addition, they entail mis-patterning of the vasculature and strongly reduced blood circulation (Fig. 2c). A quantitative proteomics analysis identified the muscle-specific actin-binding protein Filamin Ca as a twofold up-regulated protein in double homozygous Tardbp/Tardbpl mutant embryos, whereas all the other identified muscle proteins were down-regulated because of muscle degeneration. This alteration in Filamin C expression could be confirmed in frontal cortex of FTLD patients with TDP-43 pathology. Interestingly, in brain tissues, Filamin C is expressed in vascular smooth muscle cells. This correlation suggests a loss of the function component in FTLD-TDP cases, which may result in vascular symptoms (Schmid et al. 2013) such as disturbed blood flow. Vascular dysfunction might therefore precede neuronal loss in ALS and FTLD. Interestingly, in mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), vascular malfunction is observed prior to neurodegeneration (Zhong et al. 2008). However, whether vascular dysfunction indeed contributes to the human disease remains to be proved. If this turns out to be the case, the zebrafish would provide an ideal model to study the disturbed signaling pathways responsible for reduced blood flow and identify potential therapeutic targets.
Figure 2. The Tar DNA-binding protein (TARDBP) orthologues in zebrafish, Tardbp and Tardbpl, and their double homozygous loss of function phenotype. (a) Schematic representation of human TARDBP with the RNA recognition motif 1 (RRM1), the RNA recognition motif 2 (RRM2) and the glycine-rich domain where frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) associated mutation cluster. (b) Schematic representation of the exon/intron structure of Tardbpl. Upon loss of Tardbp, the tardbpl exon 5 splice donor site is not recognized, and intron 5–6 is transcribed. Translation of this novel transcript variant 1 (Tardbpl_tv1) generates a protein with a glycine rich domain, which is functionally redundant to Tardbpl. (c) Combined loss of Tardbp and Tardbpl leads to vascular mispatterning, shorter spinal motor neuron axons, and muscle degeneration (data from (Schmid et al. 2013)).
Download figure to PowerPoint