Fig 1: The number of VGLUT1-positive puncta was greater in CN-SC cocultures compared with CN slice controls. The number and size of VGLUT1 puncta in immunofluorescence images from CN-only controls (a) and CN-SC cocultures (b) at 14 DIV were analysed using ImageJ Analyze Particles function (c, d; ImageJ drawings of VGLUT1 puncta above a threshold). The number of VGLUT1-positive puncta (normalised to percentage of neurofilament-positive area in frame) was compared between CN-only controls and CN-SC cocultures. When independent two-sample t-tests were performed, the number of puncta was significantly higher compared with CN slice controls (e, p = 0.045, independent samples t-test with 95% confidence interval). Conversely, the average size of VGLUT1-positive puncta between the two groups did not differ significantly (ns) in these sample sizes (f, t-test: mean difference of -0.0015, 95% CI of -0.0035, 0.0005, p = 0.120). Scale bars in (b) and (d) = 20 µm, applies to panels (a) and (c), respectively.
Fig 2: CP13 antibody treatment does not alter neuronal markers(A and B) Quantification of protein levels and representative immunoblots detecting markers of axon damage (SMI32) in the cortex (A) and hippocampus (B). (C) Quantification of DARPP32 protein levels in striatal tissue homogenate by western blot. (D–F) Quantification of protein levels and representative immunoblots detecting the glutamatergic neuron marker VGlut1 in the cortex (D), hippocampus (E) and striatum (F). (G–I) Quantification of protein levels and representative immunoblots detecting the post-synaptic protein PSD95 in homogenates prepared from the cortex (G), hippocampus (H) and striatum (I). (G–I) Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 4–6; zQ175 (CP13) n = 4–6. For all graphs, statistics were performed using a two-way ANOVA with Sidak's post-hoc test. *p = 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. PSD95, postsynaptic density protein 95; VGLT1, vesicular glutamate transporter 1.
Fig 3: Corticospinal regeneration into neural progenitor cell graft.(A and B) Corticospinal axons robustly regenerate into the rostral half of a GFP-expressing neural progenitor cell graft (green). Horizontal section; rostral to left and caudal to right. Corticospinal axons were labeled with red fluorescent protein (RFP). (A) Neural progenitor cell (NPC) and (B) NPC and rehabilitation groups. (C and D) Higher-magnification images of images in A and B, and (E and F) single RFP channel of corticospinal axons. (G and H) Host corticospinal axons (labeled for RFP) regenerating into grafts form bouton-like structures that contain vesicular glutamate transporter 1 (vGlut1, blue) in close apposition to grafted (GFP-expressing, green) cell processes, suggesting synapse formation. (I and J) In host gray matter located 3 spinal cord segments below the lesion, GFP-labeled graft axons also form bouton-like structures that colocalize with the presynaptic marker synaptophysin (Syn, red). (K) Quantification of total corticospinal axon regeneration into NPC grafts normalized to intensity of corticospinal axon labeling in the dorsal columns at C3. There is significantly greater corticospinal regeneration into NPC graft among animals that underwent rehabilitation (P < 0.05, Student’s 1-tailed t test). NPC graft n = 8; NPC and rehabilitation groups, n = 9. Scale bars: 1 mm (A and B); 250 µm (C–F); 30 µm (G); 2 µm (H); 20 µm (I); 4.5 µm (J).
Fig 4: CP13 antibody treatment reduces tau hyperphosphorylation in human iPSC-derived cortical neurons(A) Graphical representation of the experimental procedure implemented to differentiate iPSCs into cortical neurons. (B) Representative confocal images of control and HD iPSC-derived neurons immunostained for MAP2 (red), VGlut1 (green)and nuclear stain DAPI (blue). (C) Quantification of the proportion of iPSC-derived neurons immunopositive for VGlut1 and MAP2 neuronal markers. Data are from three biological replicates and a total of 600 control and 572 HD neurons were counted. For both controls and HD, two independent iPSC lines each were analyzed (control lines, 17/18 CAG repeats; HD lines, 180 CAG repeats and 60 CAG repeats). (D) Representative confocal images of HD iPSC-derived neurons immunostained for MAP2 (green), anti-mouse secondary antibody (red) and nuclear stain DAPI (blue). (E) Quantification of pS202 tau normalized to total tau and beta actin protein levels as well as representative immunoblots showing pS202 tau, total tau and the loading control ß-actin in iPSC-derived cortical neurons. Data are from four biological replicates prepared using two independent control iPSC lines (17/18 CAG repeats) and one HD line (180 CAG repeats). (F) Quantification of neurite length after 14 days in culture in absence or presence of 15 µg/mL IgG or CP13. Data are from two biological replicates and more than 200 neurites were measured in each experimental condition (control lines, 17/18 CAG repeats; HD lines, 180 CAG repeats and 60 CAG repeats). Statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparisons test (E and F); *p = 0.05 and **p < 0.01. Outliers in (E) were identified using the Grubbs test (alpha = 0.05) and excluded from analysis. Scale bars: 25 µm (B) and 10 µm (D). IF, immunofluorescence; iPSCs, induced pluripotent stem cells; NPCs, neural progenitor cells; ns, not significant.
Fig 5: Stem cell-derived neurons innervated the centre of CN slices within 14 days. Cochlear nucleus (CN) slices were cultured with (right, a) or without (left, a) stem cell- (SC-) derived neurons for 14 days. After 14 days, both the CN-only (control) cultures and CN-SC cocultures were immunostained for neuronal (NFH, blue) (c, e, g), human neuronal (hNFM, red) (d, f, h), and the synaptic markers synapsin1 and VGLUT 1 (synapsin I: green (e, f) and VGLUT1: green (c, d, g, h)). In the CN-SC slice cocultures (b, red square), SC-derived neurites were observed innervating CN neurons within the centre of the slice (red, e–h). The stem cell-derived neurites were positive for both synapsin I (e, f) and VGLUT1 (g, h), similar to stem cell-derived neurites on the edge of CN slices. CN-only slice control (c, d) showed some VGLUT1 immunoreactivity but no hNFM immunoreactivity (g, h). Scale bar in (b) = 500 µm; scale bar in (h) = 20 µm, applies to all panels (c)–(h).
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