TDP-43 dependent cryptic exons in neurodegenerative disorders: pathogenic mechanism and therapeutic strategy

About This Grant

PROJECT SUMMARY Tar DNA/RNA-binding protein 43kDa (TDP-43) is an RNA binding protein mainly localized in the nucleus, serving an essential role in RNA regulation. Our lab has shown that one of TDP- 43’s function is to repress splicing of non-conserved cryptic exons to ensure formation of a normal transcriptome. When TDP-43 is depleted from the nucleus, these cryptic exons get spliced into messenger RNAs. The inclusion of cryptic exons often introduces frameshifts or premature stop codons in mRNAs, disrupting their translation. To complement the loss of splicing repression by TDP-43, our lab has made a construct, termed CTR, that replaces the prion-like C-terminal domain of TDP-43 with a well-characterized splicing repressor domain derived from ribonucleoprotein, PTB-binding 1 (RAVER1). CTR has been shown to repress majority of cryptic exons in mouse embryonic stem cells lacking TDP-43. Cryptic exons of TDP- 43 in mice are distinct from those of primates even though TDP-43’s protein sequence is highly conserved among different organisms. These cryptic exons are diverse and cell type-specific in nature. Our preliminary analyses show that human astrocytes have a unique set of cryptic exons after TDP-43 loss that are not expressed in TDP-43 deficient neurons. Moreover, in preliminary data, we have demonstrated that CTR can rescue the disease phenotypes observed in TDP-43 deficient human ipsc-derived cortical i3 neurons (i3N). CTR can attenuate cell death, aid survival, and decrease cryptic HDGFL2 protein in TDP-43 deficient i3 neurons. The overarching goal of this proposal is that human cryptic exons are species and cell type specific and CTR can repress cryptic exon incorporation and rescue synaptic physiology and cell death in TDP- 43 deficient human cortical neurons. I will test this hypothesis by knocking down TDP-43 using lentivirus containing shRNA against TDP-43 in human cortical neurons, administering CTR by lentivirus transduction, using RNA sequencing, performing immunohistochemical analysis, and conducting mini electrode assay recording and whole-cell patch clamp. In Aim 1, I will determine whether there are cell-specific cryptic exons in human neurons, astrocytes, and oligodendrocyte progenitor cells. In Aim 2, I will determine whether CTR can prevent cryptic exon incorporation and restore normal synaptic function and rescue cell death in TDP-43 deficient human iPSC-derived neurons.