UC San Diego

TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease (AD), commonly share features of nuclear clearance and cytoplasmic accumulation of the RNA-binding protein TDP-43 within affected neurons. TDP-43 suppression in an adult nervous system changes expression and splicing of RNAs encoded by hundreds of genes, however, the functional consequences and relevance to neurodegeneration of these changes have proven elusive. Our team recently discovered that TDP-43 suppression in human cells drives use of cryptic splice and polyadenylation sites in the pre-mRNA encoded by the STMN2 gene (aka SCG10), leading to loss of the stathmin-2 protein it encodes, preventing axonal recovery after injury. This discovery provides a new gene target in TDP-43 proteinopathy and links dysregulated RNA metabolism to impaired microtubule dynamics, two pathways suggested independently without recognized pathophysiologic commonality. I here demonstrate that concomitant loss of full length stathmin-2 mRNA and accumulation of its cryptically spliced and truncated form is a hallmark of TDP-43 proteinopathy. I show that removal of the proposed TDP-43 binding sites within intron 1 of the stathmin-2 pre-mRNA drives constitutive misprocessing. I determine TDP-43’s role at this site is to sterically block cryptic site utilization, as synthetically targeting other RNA binding proteins or RNA-targeted CRISPR effectors to this locus restores correct stathmin-2 pre-mRNA maturation. By eliminating either cryptic processing site, I determine that cryptic splicing, not cryptic polyadenylation, is the primary driver of stathmin-2 pre-mRNA misprocessing. Next, a therapeutic strategy for TDP-43 proteinopathies is identified using steric binding antisense oligonucleotides (ASOs) to maintain or restore stathmin-2 pre-mRNA maturation and rescue axonal regeneration in human motor neurons with TDP-43 deficiency. I developed AAV-based experimental approaches to modulate stathmin-2 in an adult mammalian central nervous system, including an AAV delivered RNAi against the stathmin-2 pre-mRNA that is used to determine the consequences of chronic reduction of murine stathmin-2, as well as gene therapy cargos to supplement stathmin-2 expression selectively in neurons with dysfunctional TDP-43. Finally, the STMN2 gene is humanized by knock-in of human exon 2a into the corresponding region of murine STMN2, with or without TDP-43 binding sites. I show humanized mice without the STMN2 TDP-43 binding sites constitutively misprocess stathmin-2 pre-mRNA, enabling therapy development in TDP-43 proteinopathies by identification of stathmin-2-restoring ASOs.