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.
Bibliographical noteFunding Information:
This work was supported by NINDS/NIH R01NS112503 (to D.W.C. and C.L.-T.); NINDS/NIH RF1NS124203 (D.W.C., C.L.-T., and C.L.); ALS Finding a Cure (C.L.-T.); The Massachusetts Center for Alzheimer Therapeutic Science (C.L.-T.); The Sean M. Healey & AMG Center for ALS at Mass General (C.L.-T.); U42 Mutant Mouse Resource Research Center OD010921 (C.L.); Ruth Kirschstein Institutional National Research Service Award T32 GM008666. (M.W.B. and M.S.B.), and T32 AG 66596-2 (M.S.B.); The Packard Center for ALS Research (D.W.C. and M.W.B.); The ALS association (D.W.C., M.W.B., and S.V.-S.); MDA development grants (Z.M. and J.L.-E.); The BrightFocus Foundation (A.R.A.d.A.Q.); and Cancer Center Support Grant CA034196 to The Jackson Laboratory.
For ultrastructural characterization of excitatory synaptic terminals, ASO treated human motor neurons were fixed for 1 hour with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and spun down to a cell pellet. Subsequently, the pellet was blocked with 1% osmium tetroxide in 0.15 M sodium cacodylate buffer (LADD Research), stained with 2% uranyl acetate in double distilled water (LADD Research), and dehydrated with ethanol (LADD Research). The cells were then embedded in Durcupan resin (Sigma) and sectioned on an Ultracut UC6 Ultramicrotome (Leica) by using a diamond knife (Diatome). The sections were next transferred onto copper mesh grids (LADD Research) and post-stained with uranyl acetate and lead citrate (Electron Microscopy Sciences). Images were acquired using a JEOL1400 (JEOL, Peabody, MA) transmission electron microscope (supported by NIH equipment grant 1S10OD023527-01) at 80kV using a Oneview 4KGatan digital camera (Gatan, Pleasanton, CA).
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