Fig 2: Characterization of gene-edited iPSC and neurons. (A) Sanger sequencing was used to confirm the generation of a PSEN1 knockout (i), a corrected wild-type PSEN1 line (ii), an unedited heterozygous mutant line (iii) and a homozygous PSEN1 int4del mutation line (iv). (B) Immunocytochemistry was performed on all iPSC lines following CRISPR/Cas9 editing to confirm pluripotency with the pluripotency markers OCT4 (red) and SSEA4 (green). Scale bar 100 µm. (C) Successful differentiation of iPSC into neurons was characterized 50 days post-induction by immunocytochemistry for the neuronal marker TUJ1 (red) and deep-layer cortical neuronal marker TBR1 (green). Scale bar 25µm. (D) Further characterization of cortical differentiation was performed using qPCR analysis 100 days post-neural induction to assess expression of neuronal marker TUBB3 and cortical layer markers TBR1 and CTIP2. Numbers within histogram represent the number of independent neural inductions. (E) PCR analysis of PSEN1 splicing in cDNA from day 100 neurons using primers recognizing exons 3 and 5 of PSEN1 mRNA. The full-length transcript is depicted at 374 bp and one short transcript caused by aberrant splicing is evident at 193 bp in mutation-bearing neurons (we do not see evidence of a second aberrantly spliced band at 111 bp).

Fig 3: AAV8-mediated deletion of Tsc2 in adult mice induces a pro-regenerative environment in DRG. A, Immunohistochemistry of uninjured L4 DRG stained for Tsc2 and Tubb3. Mice had intrathecal injections three weeks before a sciatic nerve crush. Uninjured DRG were isolated 3 d post-injury. B, Quantification of percentage of Tsc2-positive neurons from A. C, Immunohistochemistry of uninjured L4 DRG stained for cJun and Isl1. D, Quantification of percentage of cJun-positive neurons from C as well as linear regression of cJun expression and infection efficiency for Tsc2 KO-AAV8 mice. E, Immunohistochemistry of uninjured L4 DRG stained for Atf3 and Isl1. F, Quantification of percentage of Atf3-positive neurons from E as well as linear regression of Atf3 expression and infection efficiency for Tsc2 KO-AAV8 mice. G, Immunohistochemistry of uninjured L4 DRG stained for Iba1 and Tubb3. Image from DRG with high infection efficiency. H, Quantification of macrophage density by Iba1-positive area per neuron from G as well as linear regression of macrophage density and infection efficiency for Tsc2 KO-AAV8 mice. N.S., not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars: 50 µm. Extended Data Figure 8-1 shows data values of mean and SEM, number of replicates, statistical tests, and values for all comparisons.

Fig 4: FL2 depletion results in a more dynamic MT array near the growth cone in regenerating neurites.(A and B) Immuno-micrographs of DRG neurites of GFP AV– and Cre AV–treated neurons 2 days after replating, dual-stained for tubulin isoform ß3 (TUBB3) (green) and tyrosinated tubulin (Tyr tub) (red) (A) or for TUBB3 (green) and acetylated tubulin (Act tub) (red). Brightness/contrast adjusted linearly to more clearly see acetylated tubulin stain (B). Scale bars: 50 µm. (C) Tyr/TUBB3 ratios in GFP AV– and Cre AV–treated neurons from 1 representative experiment, starting at the tip of the growth cone and moving toward the soma (dark line is the mean intensity ratio; shaded area represents the SEM). (D) Tyr/TUBB3 fluorescence intensity ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1.00 ± 0.03, Cre AV: 1.26 ± 0.05, n = 86 GFP, n = 101 Cre). (E) Distribution of Act/TUBB3 ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1 ± 0.06, Cre AV: 0.74 ± 0.04. n = 87 GFP; n = 116 Cre). (F) Act/TUBB3 ratios in the proximal to mid region of the axonal shaft (GFP AV: 1.00 ± 0.13; Cre AV: 0.94 ± 0.07, P = 0.68, n = 43 GFP, n = 51 Cre). Experiment performed in duplicate. (G) Inverted images of MTs in the growth cones (GCs) of GFP AV– and CRE AV–treated neurons, immunostained for TUBB3. Brightness/contrast adjusted to better visualize individual MTs. Scale bar: 10 µm. (H) Mean TUBB3 fluorescence intensities in the distalmost 50 µm of neurites (GFP: 1.00 ± 0.06, Cre: 1.18 ± 0.09, Welch’s t test, P = 0.12, n = 91 GFP, n = 101 Cre). Experiments performed in triplicate unless otherwise indicated. Data were analyzed using unpaired 2-tailed Welch’s t test. Bars represent mean ± SEM. ****P < 0.0001.

Fig 5: Representative immunostaining of the relevant stages is presenting the basic morphology and expression of main neuronal markers of the cell cultures: Nestin (magenta, or green)—NPC marker; TUBB3 (red) and MAP2 (red) are neuronal markers, and GFAP (green) as an intermediate filament astrocyte marker; nuclei are counterstained with DAPI in all cases (blue). (A) RT-qPCR detection of main neuronal (TUBB3 and MAP2) and astrocytic (GFAP) markers on neuronal cultures from TD7 until TD63 stage at weekly intervals. Bars represent the relative expression data (mean ± SEM). Data were normalized with the positive control GAPDH. *p < 0.05; **p < 0.01. The presented analyses are representative of three independent experiments. (B,C) Representative Western blot results show the expression of TUBB3, MAP2, and GFAP in the neuronal cultures. 10 µg of protein per lane was used. (D,E) Representative Western blots confirm the presence of Alzheimer’s disease-related proteins in our culture system. The presence of the C-terminal fragment of APP (APP-CTF) in the fAD cell cultures but not in the control cell line cultures signifies the ongoing Alzheimer’s disease phenotype. Five micrograms of protein per lane was used. As a positive control, GAPDH was used in all Western blots. (F,G) Quantification and comparison of APP and APP-CTF protein expressions in con control and fAD cell lines. Error bars represent the mean ± SD of three measurements. ***p < 0.001.