Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies

Abstract

Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre–messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3′ splice site in STMN2 pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2–dependent lysosome trafficking in TDP-43–deficient human motor neurons. In mice that were gene-edited to contain human STMN2 cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected Stmn2 pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding.

WITHDRAWN: (CA)noligonucleotides correct RNA mis-splicing in TDP-43 pathology

“…TDP-43 deficiency has been linked to widespread cryptic splicing events, including but not limited to the splicing of STMN2, UNC13A, KALRN, RANBP1, and HDGFL2, eventually leading to neuronal dysfunction and death [1][2][3][4][5][6][7][8][9] . CLIP-seq data have revealed that TDP-43 predominantly binds to intronic GU repeats within pre-mRNAs 10 , where it sterically blocks the access of splicing factors to cryptic sites, resulting in the exclusion of cryptic exons from mature transcripts 11 .…”

“…Recently, attention has been drawn to this mechanism of action for treating aberrant RNA splicing caused by TDP-43 deficiency. To date, research has primarily focused on correcting individual misspliced targets 11 . Although genes such as STMN2 and UNC13A have been shown to be important for neuronal health [1][2][3] , the downstream targets responsible for neuronal degeneration in TDP-43 pathology remain unknown, and it is possible that several targets act in concert.…”

Highly variable molecular signatures of TDP-43 loss of function are associated with nuclear pore complex injury in a population study of sporadic ALS patient iPSNs

“…similar to that reported for UNC13A 20,22 ) or co-regulation of mRNA targets by multiple RNA binding proteins (as has been previously demonstrated for TDP-43 and FMRP in Drosophila models 51,52 ) may be contributing factors. Nonetheless, this variability holds implications for future sALS focused clinical trials either targeting individual mRNA targets of TDP-43 themselves 19,23,27,28 or those seeking to utilize TDP-43 loss of function associated “cryptic” peptides or RNAs as biomarkers or readouts of therapeutic efficacy 21,25 .…”

“…Nuclear depletion, and in some cells complete nuclear clearance, is thought to precede cytoplasmic mislocalization 17 and has been demonstrated to lead to a loss of nuclear function 18–27 . Although early studies evaluated alterations in gene expression and mRNA splicing that occur in mice following TDP-43 depletion 24,26 , there is great dissimilarity between mouse and human TDP-43 targets due to a lack of conservation in the consensus sequence for TDP-43 binding in TDP-43 targets in mice 19,23,28 . To more accurately evaluate the role of TDP-43 nuclear depletion in human disease, recent studies utilized induced pluripotent stem cell (iPSC) technologies to identify alterations in gene expression and mRNA splicing that occur upon artificial depletion of TDP-43 in human neurons 19,20,25 .…”

Induced pluripotent stem cells‐based disease modeling, drug screening, clinical trials, and reverse translational research for amyotrophic lateral sclerosis

“…Among them, cryptic splicing-polyadenylation of the stathmin-2 (STMN2) gene, encoding a protein required for axonal regeneration, is caused by TDP-43 mislocalization and is likely to be involved for axonal phenotypes and vulnerability of ALS MNs (Klim et al, 2019;Melamed et al, 2019). It is remarkable that ASOs targeted for TDP-43 binding sites within human STMN2 gene suppressed cryptic splicing, which restored axonal regeneration and STMN2-dependent lysosome trafficking in TDP-43-deficient human MNs (Baughn et al, 2023). This suggests the merit of ASO targeting STMN2 cryptic splicing for clinical application.…”