TDP-43 AND NOVA-1 RNA BINDING PROTEINS AS SPLICING REGULATORS OF TNIK GENE, A SCHIZOPHRENIA GENETIC RISK FACTOR

TDP-43, a RNA-binding protein (RBP) involved in different steps of RNA metabolism, forms pathological aggregates in affected tissues of the majority of amyotrophic lateral sclerosis (ALS) patients and of a subset of frontotemporal dementia (FTLD) cases. Our group has recently demonstrated that TDP-43 regulates the alternative splicing of several pre-mRNAs related to neuronal metabolism, including TNIK, encoding for a Ser/Thr kinase highly expressed in the brain. TDP-43 promotes the skipping of TNIK alternative exon 15, which encodes for a 29-amino-acid sequence in a region of the protein with unknown function. The TNIK gene was found to be genetically associated to psychiatric disorders, such as schizophrenia, as a risk factor, and to intellectual disability for null-mutations. In neurons, TNIK is involved in regulating different processes, including synapse formation, dendrite arborization and neurogenesis. In this study, our first aim was to investigate the regulation of TNIK exon 15 alternative splicing during neuronal differentiation in order to better characterize the role played by the ubiquitously expressed and ALS/FTLD-associated TDP-43 splicing factor in a neuronal context. Our second aim was to investigate if the specific expression of TNIK exon15-cointaining (TNIKex15) protein isoforms may be important to maintain TNIK physiological function in neurons, mainly focusing on its activity in neurite development. We first observed that TNIK exon 15 alternative splicing was differently regulated in human adult tissues and TNIKex15 isoforms were exclusively expressed in brain, spinal cord and skeletal muscle. Given the prevalent expression of TNIKex15 isoforms in the central nervous system, we further investigated this alternative splicing event in in vitro models to evaluate its regulation during the neuronal differentiation process. We found a significant increase of TNIKex15 transcripts in both SK-N-BE cells treated with retinoic acid and in human iPSCs differentiated into neurons. Moreover, TNIKex15 protein isoforms were specifically expressed in neuron-differentiated cells showing a prevalent perinuclear distribution in immunofluorescence analyses. Since we previously showed that TNIK exon 15 inclusion increases upon TDP-43 knock-down, we measured TDP-43 protein content during neuronal differentiation in vitro but we found no changes. We therefore investigated the possible involvement of the neuron-specific splicing factor NOVA-1, specifically expressed in our neuron-differentiated cells, in regulating TNIK processing. In HEK293T cells, NOVA-1 over-expression increased TNIK exon 15 inclusion without negatively affecting TDP-43 protein content compared to mock-transfected cells. Furthermore, by minigene splicing assays we evaluated the NOVA-1 interplay with TDP-43 in regulating TNIK exon 15 splicing. In this analysis, we also included hnRNPA2/B1, an ubiquitous RBP that co-operates with TDP-43 in regulating its splicing activity and that we found to promote TNIK exon 15 skipping, similarly to TDP-43. In competition assay with TDP-43 and hnRNPA2/B1, NOVA-1 completely abrogated their exon skipping activity on TNIK gene interacting with TDP-43 and hnRNPA2/B1 proteins in a RNA-dependent manner. As our results suggested a neuronal relevance for TNIKex15 protein isoforms, we further investigated the specific function of these isoforms in neurite development in murine primary cortical neurons. TNIKex15 over-expression negatively affected neurite development, reducing neurite number, as already describe in literature. When we analyzed TNIKex15-expressing neurons, we observed reduced filopodia number at growth cones, reduced soma area and filamentous actin (F-actin) levels compared to control cells. In contrast, upon TNIK exon 15-deleted (TNIKΔ15) over-expression, we found similar neurite development, soma and growth cones morphology and F-actin levels to control cells and also to TNIK KM, a TNIK mutant with a defective kinase activity. Moreover, TNIKex15 over-expression affected cell spreading in HEK293T cells that showed a prevalent round morphology compared to control cells. In contrast, TNIKΔ15-expressing cells presented an intermediate phenotype. In conclusion, our study has demonstrated that TNIKex15 isoforms are specifically expressed in neuronal tissues and during neuronal differentiation in vitro and that these isoforms show a specific function in regulating neurite development, influencing F-actin organization. Moreover, the neuron-specific splicing factor NOVA-1, probably promoting the formation of new ribonucleoprotein complex(es) with the ubiquitous splicing factors TDP-43 and hnRNPA2B1, acts as “silencer” of their splicing inhibitory activity on TNIK pre-mRNA. These results suggest that investigating NOVA-1/TDP-43 competitive mechanism also for other TDP-43 splicing targets may further help the understanding of TDP-43 splicing activity in a neuronal environment and in ALS/FTD diseases.