Elevated nuclear TDP-43 induces constitutive exon skipping.

BACKGROUND: Cytoplasmic inclusions and loss of nuclear TDP-43 are key pathological features found in several neurodegenerative disorders, suggesting both gain- and loss-of-function mechanisms of disease. To study gain-of-function, TDP-43 overexpression has been used to generate in vitro and in vivo model systems. METHODS: We analyzed RNA-seq datasets from mouse and human neurons overexpressing TDP-43 to explore species specific splicing patterns. We explored the dynamics between TDP-43 levels and exon repression in vitro. Furthermore we analyzed human brain samples and publicly available RNA datasets to explore the relationship between exon repression and disease. RESULTS: Our study shows that excessive levels of nuclear TDP-43 protein lead to constitutive exon skipping that is largely species-specific. Furthermore, while aberrant exon skipping is detected in some human brains, it is not correlated with disease, unlike the incorporation of cryptic exons that occurs after loss of TDP-43. CONCLUSIONS: Our findings emphasize the need for caution in interpreting TDP-43 overexpression data and stress the importance of controlling for exon skipping when generating models of TDP-43 proteinopathy.

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.