CLK inhibition alters tau splicing and has a neuroprotective effect in vitro

Abstract

Background Microtubule‐associated protein tau (MAPT) undergoes alternative splicing at exon 10 which encodes one of its four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau isoforms with all four repeats (4R) whilst exon 10 exclusion leads to isoforms with three repeats (3R). The 1:1 ratio between 3R and 4R isoforms is tightly regulated by splicing machinery and crucial to normal function. Kinases such as CLKs regulate the activity of splicing factors and their interaction with target transcripts, including MAPT. Disruption to this balance leads to the formation of neurofibrillary tangles (NFTs) and neurodegeneration but the mechanism of this is not yet wholly understood. Methods Differentiated Neuro2a and SH‐SY5Y cells were used as neuronal models as these cell lines show an imbalance of MAPT isoforms with bias towards 4R and 3R respectively. Splicing factor SRSF1 was transiently knocked down to confirm its role in MAPT splicing. Novel kinase inhibitors were used to reduce the activity of CLKs and determine the effect on MAPT splicing. The expression ratio of 4R and 3R isoforms was measured by RT‐PCR. Okadaic acid (OA) was used to produce a neurotoxic model and investigate the use of kinase inhibitors for neuronal protection. As a functional measure β3‐tubulin fluorescent marker was used to quantify OA dependent reduction in neurite outgrowth. Results 4R:3R tau expression significantly decreased by 65% in Neuro2a cells with knockdown of SRSF1 (students t‐test, p<0.001). CLK inhibition increased 4R:3R by 50% in SH‐SY5Y cells compared to control (one‐way ANOVA, p<0.05). SHSY5Y cells neurite outgrowth was significantly reduced by 60% and 85% with 1nM and 5nM OA treatment respectively, demonstrating a concentration dependent effect (one‐way ANOVA, p<0.001). At 1nM OA, neurite outgrowth was rescued by co‐treatment with a novel CLK inhibitor (two‐way ANOVA, p<0.0001). Conclusions Novel CLK inhibitors can modulate the expression of MAPT splicing isoforms and exert a functional effect on neurite outgrowth. This suggests these compounds could have a therapeutic effect by rectifying the balance of 4R:3R tau expression.