Fig 2: TMP promotes cell viability and suppresses cell apoptosis in H2O2-treated PRGCs. (A) NF-L expression was detected by immunofluorescence (magnification, ×200). (B-D) The mRNA expression levels of NF-L, Thy-1 and Brn3a were measured by reverse transcription-quantitative PCR. (E) Protein expression of NF-L, Thy-1 and Brn3a as determined by western blotting. ß-actin was used as a loading control. (F) PRGCs were treated with 10, 100, 200, 400 and 800 µM of H2O2 for 24 h, and then cell viability was determined by a CCK-8 assay. PRGCs were pre-treated with 25, 50 and 100 µM TMP for 24 h prior to exposure with 400 µM of H2O2. (G) Cell viability was determined by a CCK-8 assay. (H) The activity of caspase-3 was determined using a caspase-3 assay kit. (I) Apoptosis was detected via flow cytometric analysis. Data are presented as mean of three replicates ± standard deviation. *P<0.05, **P<0.01 vs. Control group, #P<0.05, ##P<0.01 vs. Vehicle + H2O2 group. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; NF-L, neurofilament-L; PI, propidium iodide; PRGCs, primary retinal ganglion cells; Thy-1, thymus cell antigen 1; TMP, tetramethylpyrazine.

Fig 3: Comparison of NF-L and Vps11 Immunolocalization in Wild-Type, Plp1 Null Mutants and Plp1 Mice With Duplications. A to D: Confocal images from the ventral Basal Ganglia of adult wild-type (WT) B6 mice immunostained with NF-L (green) and Vps11 (red). Longitudinal sections through the axon of WT-mice (A to C) show Vps11 show Vps11 expression alternates with NF-L expression. Confocal, deconvolved, cross-sections through the axon (D) confirmed that Vps11 expression was immediately outside of the axon. E-G. Confocal images from 35DPN Plp1 null mutant mice immunostained with NF-L (green) and Vps11 (red). Longitudinal and cross sections through the axon (E to G) show strong Vps11 expression was largely distinct from NF-L, and in agreement with quantitative Vps11 immunostaining visualized with DAB. H. Confocal deconvolved, cross-sections through the axon confirmed Vps11 expression was immediately outside the axon. I to L. Confocal images from Plp1 mice with duplications immunostained with NF-L (green) and Vps11 (red). Images suggest degeneration of axon cylinder. Longitudinal sections through the axon (I to K) show very weak Vps11 expression overlapping with a subset of NF-L-positive puncta. Again, cross-sections through the axon (L) confirmed that Vps11 expression was immediately outside of the axon and NF-L staining within the axon proper.

Fig 4: Systemic AAV dissemination, eGFP expression in Schwann cells and inflammation at the site of AAV delivery using SciNDi(A) Dual-IF detection of eGFP and neurofilament light chain (NF-L) in the sciatic nerve trunk following bilateral IM application of PBS (vehicle). Representative images showing eGFP alone (green), NF-L (red), the merge images of eGFP and NF-L, and nuclear marker DAPI (blue). IF procedures and image acquisition were performed as described under Figures 2E and 2F (nerve trunk and 10× magnified views).(B) Immunohistochemical (IHC) detection of eGFP in the right lumbar L4/L5 spinal ventral horn (VH-R) or both dorsal horns (DH-R and DH-L) after AAV delivery using SciNDi. IHC procedures and image acquisition were performed as described under Figures 2G–2I.(C) qRT-PCR determination of vector genome copy numbers (AAV2-ITR/2N*ß- tubulin) comparing AAV delivery via IM route or SciNDi (Mean ± s.e.m.; SC: lumbar spinal cord, n = 6; B: whole brain, n = 4–6; and L: liver, n = 6; One- way ANOVA; ns, not significant).(D) qRT-PCR determination of eGFP (transgene) expression (??CT, relative to PBS injected cohorts). Statistics in A-B (Mean ± s.e.m.; SC: lumbar spinal cord, n = 6; B: whole brain, n = 4–6; and L: liver, n = 6; Mann-Whitney test: *= p = 0.05, ns = not significant).(E and F) Dual-IF co-detection of eGFP in sciatic nerve along with either the Schwann cell marker (S100, in E), or monocyte/macrophage marker (CD68, in F) following bilateral IM application of PBS, and AAV delivery using IM injection or SciNDi. Representative images (20× views) showing eGFP alone (green), S100 alone (red) (in E) or CD68 alone (red) (in F), and the respective merge images along with the nuclear marker DAPI (blue). Antibodies: eGFP (abcam #13970ab, Polyclonal chicken, 1:200), S100 (DAKO #Z0311, rabbit polyclonal, 1:200), CD68 (Novus Biologicals #NBP2-33337, rat mAb FA-11, 1:200). High resolution IF images were acquired using a Zeiss observer inverted microscope equipped with colibri 7 LED illumination. (G–I) Dual-IF co-detection of eGFP and and monocyte/macrophage marker (CD68) in hindlimb femoris muscle following PBS application using SciNDi (in G), and AAV delivery using IM injection (in H) or SciNDi (in I). Representative images (20× view, merge with DAPI, IF procedures and image acquisition were as described under Figure 3F).

Fig 5: Application I. Demonstrating trans-synaptic spreading as a route for spreading of a-syn-aggregate pathology from DG via CA3 to the CA1 region using surgical and viral transgene methods. a Illustration of PFF injection in CA1 in WT OHSCs to test the efficiency of the retrograde route of spreading. 1 Composite image 14 dpi of S129A PFFs at CA1. Scale bar: 200 µm. MJF-14-positive aggregates are seen at the CA1 region (i, ii), but there is no spreading to DG (iii). Scale bar i & iii: 50 µm, ii: 20 µm. b Diagram showing transection of axonal projections between DG and CA3, which blocks spreading of a-syn aggregate pathology from DG to CA1. The surgical destruction of the tissue is demonstrated by the absence of nuclei (1), axonal marker NF-L (2), and MJF-14 staining (3). Scale bars: 200 µm. Magnified images from 3 show aggregates at DG (i) and proximal to the cut (ii), but not distal to the lesion (ii, iii). Scale bars: 50 µm. c Diagram showing expression of WT-a-syn in a-syn KO slices by AAV vectors injected in DG, CA3, and CA1. 1 a-syn expression in DG, CA3, and CA1 supports spreading of aggregated pS129 a-syn (11A5) to CA1 7 dpi of PFFs in DG, as seen from the magnified panels i-iiii. Scale bar: 200 µm, i & iiii: 20 µm, ii & iii: 10 µm. Note the strong AAV-dependent expression of pS129 in some neuronal nuclei. d Illustration of WT-a-syn expression in DG and CA1 only of a-syn KO slices. 1 Absence of a-syn expression in the CA3 abolishes spreading of aggregation to CA1 at 7 dpi. Scale bar: 200 µm. i, ii pS129-positive aggregates are detectable at DG. iii No pS129-positive aggregates are found at the CA1 region. Iiii A few neurons show nuclear expression of pS129-a-syn at CA3. Scale bars: i: 20 µm, ii, iii & iiii: 10 µm. Data in a are illustrative of 12 slices divided over 3 experiments. Images in b are representative of 4 experiments with 18 slices in total, while c & d representative of 3 separate experiments/18–21 slices in total per condition