Fig 2: PTX-mediated regulation of local translationRepresentative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink signal in dendrite and soma, respectively. (Scale bars: in overview images 20 µm, in inserts of higher magnification 5 µm).Same as (A) with nascent Synapsin1-peptides.Quantification of Duolink-particle density of (A) and (B) assessing PTX treatment effect. n = 3 independent experiments with 10 images averaged per condition and experiment; Three-way ANOVA followed by Tukey's post hoc multiple comparison test; *P < 0.05; **P < 0.01).Quantification of Duolink-particle density of (A) and (B) assessing Puromycin-treatment effect. n = 3 independent experiments for Puromycin-Minus and n = 6 data points for Puromycin-Plus conditions (PTX and mock conditions combined together across n = 3 independent experiments, taken into account in ANOVA model) with 10 images averaged per condition and experiment; three-way ANOVA followed by Tukey's post hoc multiple comparison test; **P < 0.01, ***P < 0.001).Data information: Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5× interquartile range (IQR).

Fig 3: scRNAseq analysis reveals that Nng2-induced iNs are sensory neurons.Cell identity and heterogeneity were assessed using tSNE combined for all time points for ape (orange) and human (green) cells. (A) Filtered cell number for ape and human cells at different time points. (B) Identification of 8 different cell clusters by marker gene expression. NP, neural progenitor; SN, sensory neuron. (C) Marker gene expression (blue) for progenitor cells (SOX2), mature neurons (MAP2), and cortical cells (CUX1, BRN2, SATB2, and TRB1). Scale bars: uncorrected normalized expression. (D) Cell ratios of the three neuronal sub-classes of TAC1 (yellow), SSTR2 and GAL (green), and PIEZO and GAL (blue) expressing neurons for all cell lines and species. TAC1+, SSTR2+/GAL+, and PIEZO2+/GAL+ neurons on d5: 54%/36% (chimpanzees/humans), 16%/26%, and 31%/38; TAC1+, SSTR2+/GAL+, and PIEZO2+/GAL+ neurons on d14: 55%/57%, 9%/17%, and 36%/27%; TAC1+, SSTR2+/GAL+, and PIEZO2+/GAL+ neurons on d28: 71%/48%, 9%/20%, and 20%/32%; TAC1+, SSTR2+/GAL+, and PIEZO2+/GAL+ neurons on d35: 49%/31%, 12%/27%, and 40%/42%. (E) Heatmap showing scaled expression levels of top 10 significantly higher expressed genes in seven cell populations in iNs culture. The main populations we identified are fibroblasts (FB), neural progenitors, and neurons. Top annotation bars: ape (orange) and human (green). (F) The expression levels of sensory neurons markers are shown as a violin plot for neural progenitor’s clusters C1 and C2 and for neuron’s clusters C4, C5, C6, and C7. Note that the POU homeodomain transcription factors BRN3A (POU4F1) and Islet1 (ISL1) are highly expressed in a subset of early iNs. All iN clusters show high expression of growth factor FGF13 and voltage-gated sodium channel Nav1.7 (SCN9A) that regulate mechano-heat sensation in vivo.

Fig 4: Expression pattern of MAP2 in the CARMIL3(+) profiles. (A) Double labeling of CARMIL3 and MAP2 in the T2(+), mismatch, and control regions after pMCAO. The CARMIL3(+) profiles were colocalized with MAP2, indicating that CARMIL3(+) profiles were located on neurites. CARMIL3(+) neurites (arrows) were thicker than CARMIL3(-) neurites (arrowheads) in the mismatch region. (B) CARMIL3(+) neurite ratio [the number of CARMIL3(+) neurites divided by that of all identifiable neurites] in the three regions. There were significantly higher ratios of CARMIL3(+) neurites in the mismatch regions compared with control areas (p = 0.035). (C) Comparison of the neurite diameters of CARMIL3(+) and CARMIL3(-) neurites. CARMIL3(+) neurites had significantly larger neurite diameters than CARMIL3(-) ones in mismatch regions (p = 0.036). *p < 0.05. N = 4 rats in each group. Scale bar = 10 µm.

Fig 5: miR-329 and miR-495 are required for Prr7 down-regulation by PTX.(A) Predicted miRNA-binding sites for human Prr7 3' UTR and seed matches for miR-329 and miR-495 from TargetScan (http://www.targetscan.org/). (B, C, D) Rat hippocampal neurons were co-transfected at DIV13 with either pmiRGLO Prr7 WT or Mut 3' UTR plasmids (50 ng) and control pLNA, 329 pLNA, 495 pLNA (20 pmol), or miR-329/495 pLNA mix (10 pmol each) and treated with either EtOH or 100 µM PTX at DIV18. Cells were lysed at DIV20 and firefly/renilla luciferase activity ratios measured. Data = mean normalized to EtOH condition ± SD, n = 3–4, two-way ANOVA with Tukey’s post hoc homeostatic synaptic depression test. 329/495 pLNA data: Ctr WT versus 329/495 WT: *P = 0.0194; Ctr WT versus Ctr Mut: ns P = 0.4724; Ctr Mut versus 329/495 Mut: ns P = 0.9452. 495 pLNA data: Ctr WT versus 495 WT: *P = 0.0397; Ctr Mut versus 495 Mut: ns P = 0.8484. 329 pLNA data: Ctr WT versus 329 WT: ns P = 0.2275; Ctr Mut versus 329 Mut: ns P = 0.9841. (E) Representative dendrite images showing Prr7 expression (gray scale, top panels) and merged Prr7 (red), GFP (green), and Map2 (blue) signals (bottom panels) in Ctr or 329/495 pLNA-transfected hippocampal neurons treated with 48 h EtOH or PTX. Scale bars = 5 µm. On the right, average Prr7 punctum intensity in hippocampal cells transfected with GFP (150 ng) and control, miR-329, miR-495 pLNA (20 pmol), or miR-329/495 pLNA mix (10 pmol each) at DIV13 and treated with PTX or EtOH on DIV19 for 48 h. Data = mean normalized to EtOH condition ± SD, n = 3, one-way ANOVA with Tukey’s post hoc homeostatic synaptic depression test. Ctr versus 329: ns P = 0.9946; Ctr versus 495: ns P = 0.1739; Ctr versus 329/495: *P = 0.0023. (F, G) Schematic of single-cell dual-fluorescence miRNA sensor assay and measurement of endogenous miR-329 and miR-495 activity upon PTX treatment in hippocampal neurons. Cells were transfected with miR-329, miR-495, or control sensor (125 ng), with or without Ctr, miR-329, or miR-495 pLNA (5 pmol) at DIV13, treated with 100 µM PTX or EtOH at DIV19, and fixed at DIV21. The number of neurons expressing GFP only without dsRed versus those expressing dsRed was counted for three coverslips per condition. Data = proportion of GFP+ cells/total cell count for three coverslips counted per independent experiment ± SD, n = 4, binomial generalized mixed effects model (GLMM) with post hoc tests conducted using the lme4 R and multcomp packages, with P-values adjusted by Bonferroni’s method. Ctr sensor data: PTX versus mock: *P = 0.041206; Ctr pLNA PTX versus Ctr pLNA mock: ns P = 0.457119. 329 sensor data: PTX versus mock: ***P = 0.000316; Ctr pLNA PTX versus Ctr pLNA mock: **P = 0.001658; 329 pLNA PTX versus 329 pLNA mock: ns P = 0.05349. 495 sensor data: PTX versus mock: ***P = 0.000145; Ctr pLNA PTX versus Ctr pLNA mock: ****P = 3.78 × 10-6; 495 pLNA PTX versus 495 pLNA mock: ns P = 0.23609. (H, I) Mature miR-329 and miR-495 levels in cell body and dendrite compartments of hippocampal neurons treated with either EtOH or PTX for 48 h. Data = mean normalized to EtOH condition ± SD, n = 3, one-sample t test with hypothetical mean set to 1. Cell body: miR-329 ns P = 0.2923, miR-495 ns P = 0.2742: dendrites: miR-329 ns P = 0.8777, miR-495 ns P = 0.1641.Source data are available for this figure.