Fig 2: Dual C9ORF72 expanded repeat and Stmn2 loss of function model recapitulates C9-associated pathology. (A–H) DPR accumulation, pTdp-43 pathology, and gliosis were analyzed in the brains of 6-month-old mice. (A,B) Immunoassay measuring soluble poly-GP (A) and poly-GA (B) show a significant accumulation of DPRs in sgCtrl-149R and sgStmn2-149R mice. (C) Representative images of immunohistochemical analysis of poly(GP) with quantitative analysis of the number of GP-positive inclusions in the cortex (D). (E) Representative images of pTdp-43 immunoreactivity and quantitative analysis of the number of pTdp-43 inclusions observed in the cortex (F). (G) Representative images of immunohistochemical analysis of Gfap levels with quantitative analysis of percent Gfap immunoreactivity (H). Data presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, non-significant (ns), as analyzed using two-way ANOVA, followed by Tukey’s post hoc analysis, n = 4–6 animals per group (A–H). Scale bar is 100 uM (C) and 60 uM (E,G). Solid arrows indicate pTdp-43 inclusions (E).

Fig 3: Generation of Stmn2 knockdown mouse model using CRISPRi. (A) Schematic of CRISPRi machinery used to create CRISPRi knockin mice. (B) Schematic of CRISPRi mice, AAV sgRNA construct, and delivery of virus by ICV injection to create an in vivo model of Stmn2 loss of function. (C–E) Stmn2 RNA and protein levels were assessed in the brain tissue of 2-month-old CRISPRi mice and/or non-transgenic littermate controls injected with AAV encoding eGFP, or four guides targeting Stmn2 (sgStmn2#1-4). (C) Stmn2 RNA levels, normalized to endogenous controls Rplp0 and Gapdh, as measured by qRT-PCR, are significantly reduced in CRISPRi mice injected with AAV sgStmn2. Immunoblot (D) and quantification of Stmn2 protein normalized to Gapdh levels (E) reveal a reduction of Stmn2 protein in CRISPRi mice injected with sgStmn2. Data presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, non-significant (ns), as analyzed using one-way ANOVA, followed by Dunnett’s post hoc analysis, n = 2–4 animals per group (C,E). Schematic (B) created with BioRender.com.

Fig 4: Soluble STMN2 and TDP-43 protein are reduced in the frontal cortex of familial FTLD-TDP cases. (A,B) Soluble STMN2 (A), TDP-43 (B) protein levels were measured by Western blot in the frontal cortex of FTLD-TDP cases (n = 90) and non-neurological disease controls (n = 31). Following quantification of STMN2 (A) or TDP-43 (B) immunoreactivity, levels were normalized to GAPDH to control for protein loading. (C) pTDP-43 protein levels, as measured by immunoassay, are significantly elevated in cases with a C9ORF72 repeat expansion or GRN mutations compared to those without mutations. Horizontal bars represent median ± 95% confidence intervals. p-values from the linear regression model adjusting for age and sex are shown. Values for both unadjusted and adjusted linear regression models are reported in Supplementary Tables S2, S4.