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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Nat Neurosci. 2023 Nov 23;27(1):34–47. doi: 10.1038/s41593-023-01496-0

Figure 2: Focal, chronic, and selective suppression of stathmin-2 by subpial delivery of AAV9 shStmn2 in lumbar spinal cord results in motor deficits.

Figure 2:

(a) Schematic of experiments evaluating sustained stathmin-2 depletion by AAV9-delivered shRNA subpially delivered into 12-month-old adult wildtype mice. Figure created using Biorender. (b) Measurement of Stmn2 mRNA expression by qRT-PCR in mice after subpial delivery of AAV9-shControl or AAV9-shStmn2 across cervical, thoracic, and lumbar segments at 1- and 8-months post-administration. Statistics by two-sided, one-way ANOVA with Tukey’s multiple comparisons correction. N=4 animals/condition at 1 month. N=18 animals with AAV9-shControl and n=17 animals with AAV9-shStmn2 at 8 months. P < 0.0001. (c) Measurement of mRNA expression for a panel of genes by qRT-PCR in lumbar spinal cord 8 months post-injection of AAV9-shRNAs. Statistics by two-sided, unpaired t-tests. P < 0.0001. (b,c) Gapdh used as an endogenous control gene. Each data point represents an individual mouse. (d) Stmn2 mRNA levels detected by single molecule FISH in mice lumbar spinal cord. (e) Immunoblots of stathmin-2 protein in mice lumbar spinal cord 8 months post-administration of AAV9-shControl (n= 4 animals) or Stmn2 shRNA (n=4 animals). HSP-90 was used as a loading control. *Indicates non-specific band. Each lane was loaded with a different animal. (f-h) Longitudinal analysis of hindlimb clasping (f), grip strength (g), and forelimb grip strength (h) of AAV9-shControl (n=18) or AAV9-shStmn2 (n=24); Statistics by two-sided, two-way ANOVA and Sidak’s multiple comparisons test. p values range from * P = 0.0141 to P < 0.0001. (i) Representative confocal images of gastrocnemius muscle immunostained with synaptophysin and neurofilament-H antibodies for axon terminals (green) and α-bungarotoxin for muscle endplates (magenta), 8 months post-injection of AAV9-shControl or AAV9-shStmn2. (j) Quantification of the neuromuscular innervation status. Statistics by two-sided, two-way ANOVA and Sidak’s multiple comparisons test. N=5 animals/condition. P <0.0001. (k,l) Representative images (k) and quantification (l) of ChAT positive motor neurons at the lumbar spinal cord of mice 8 months post-administration of AAV9-shControl (n=3 animals) or AAV9-shStmn2 (n=5 animals). Statistics by Mann-Whitney’s test. All panels: Error bars plotted as SEM. ****, p <0.0001; ***, p < 0.001; **, p < 0.01; *, p <0.05; ns, p>0.05.