Fig 2: PS deficiency causes abnormal NF levels and pathological ß-sheet protein structures. a Representative immunohistological images of NF-L in the hippocampus of 6 month-old WT, PS1 cKO, PS cKO, Tau, PS1 cKO;Tau and PS cKO;Tau mice. Insets: magnified images indicated by squares showing prominent NF-L somatic staining in hippocampal neurons of PS cKO;Tau mice. Scale bar = 100 µm. b Left: Biochemical analysis of hippocampal lysates using SMI312 (NF-M/H) and NF-L antibodies. Right: Quantification of NF-H (~ 200 kDa), NF-M (~ 150 kDa), SMI-labeled (~ 60–70 kDa) and NF-L (~ 70 kDa) bands in independent membranes. Protein levels were normalized to GAPDH. Values represent mean ± s.e.m. (n = 4 mice/group). Statistical analysis was determined by two-way ANOVA followed by Tukey’s post hoc tests. ***P < 0.001. c Synchrotron-based µFTIR analysis of the retrosplenial cortex (RSC) and corpus callosum (CC) of WT and PS cKO;Tau mice at 6 months of age. Second derivative absorbances (d2A) of ß-sheet/a-helix (d2A1635/d2A1656), and ß-intermolecular/Amide I (d2A1625/d2A1635+1656) and ß-antiparallel/Amide I (d2A1695/d2A1635+1656) protein structures. Values represent the minimum, the maximum and the median of the average of 100 spectra/mouse (n = 4 mice/group). Statistical analysis was determined by one-way ANOVA followed by Sidak’s post hoc tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. d Representative infrared heat maps of ß-intermolecular/Amide I (left images; scale bar = 50 µm) and consecutive immunohistological sections of aggregated tau detected by MC1 staining (middle images) and Congo red staining (right images; scale bar = 25 µm) in the RSC and CC of WT and PS cKO;Tau mice.

Fig 3: Expression of neuronal proteins in FtMt clones. (A) Composite picture of western blots showing the differences in levels of NF-M, PSD-95, SNAP-25, and FtMt between undifferentiated (-) and RA differentiated (+) of untransfected (Un) and FtMt high expressing clone (High B). It was noticed in all experiments that FtMt expression was increased with differentiation RA treatment, even though the expression vector was using the CMV immediate-early promoter, and not the native FtMt promoter. (B) Relative levels of NF-M (B) in different FtMt clones. Results shown in B represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Relative levels of PSD-95 (C) in different FtMt clones. Results shown in C represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Relative levels of SNAP-25 (D) expression in different FtMt clones. Results shown in -D represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05. (E) Composite Western blot showing pattern of expression for NF-M, PSD-95, and SNAP-25. Representative western blots of one experiment showing patterns of expression in untransfected and different FtMt clones. Experiment represents the sequential probing of single membrane for NF-M, PSD-95, and SNAP-25 followed by normalization for ß-actin.

Fig 4: Immunocytochemistry of FtMt expressing clones. (A–C). Untransfected (A), Medium (B), and High B (C) clones differentiated for 7 days stained with antibody to FtMt to identify positive expressing cells. Positive staining is observed for Medium and High B clone, but not untransfected cells. Antibody reacted cells were identified by reaction with Alexa568 labeled-anti rabbit immunoglobulin G. All images were recorded using an Olympus FV1000 laser confocal microscope. (D-F). Higher magnification images of FtMt antibody reacted cells. Images from areas indicated with white lines on (A–C). (G–I) Untransfected (G), Medium (H), and High B (I) clones differentiated for 7 days stained with antibody SMI-312 that stains neurofilament proteins, but predominantly NF-M. All cells showed SMI-312 positive neuronal processes consistent with differentiated morphology. Undifferentiated cells show no staining for SMI312 (not shown). Antibody-reacted cells were identified by reaction with Alexa568 labeled-anti mouse immunoglobulin (G). (J–L). Untransfected (J), Medium (K), and High B (L) clones differentiated for 7 days stained with antibody MtCox1 that stains mitochondrial cytochrome oxidase-1. Morphology of MtCox1 immunoreactive mitochondria in FtMt expressing clones (K-Medium and L- High B) compared to untransfected cells (J). Antibody reacted cells were identified by reaction with Alexa568-labeled anti mouse immunoglobulin (G). (M) Quantitative estimation of aggregated/abnormal mitochondria. Estimation of percentage of aggregated mitochondria in untransfected (Un), Med, and High B expressing clones. The numbers were estimated by visual examination of 3 fields/image and 3 images for each clone at magnification shown in (A–C). Results show mean ± SEM, with standard significance levels indicated from data analyzed by one way ANOVA with Tukey post-hoc test. ***p < 0.001, ****p < 0.0001.

Fig 5: Let-7 functions by preferentially destabilizing its target mRNAs in soma of PCNs.a, Let-7 is the most abundant miRNA in PCNs. Pie charts showing the percentage of different miRNAs in neurites and soma of PCNs, determined by small RNA-seq. b, Mutations of let-7 binding sites lead to a stronger increase of N-zip mRNA reporter levels in soma. Box plots showing changes in the normalized expression levels between tiles that contain intact let-7 seed sites (wt let-7) and tiles in which let-7 seeds were mutated (mutated let-7). In the mutated tiles, at least one let-7 seed site was lost. The data are shown separately for each tile (as indicated on x) and each subcellular compartment (green: neurite, blue: soma). P values, computed with two-sided Wilcoxon rank-sum test, are shown above the plots (n = 3 independent biological replicates). Box plot elements, here and further, unless otherwise specified: center line, medium; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. c, Proteomic analysis of isolated soma and neurites indicates somatic enrichment of the core components of the miRNA repression complex (AGO1, 2, 3 and 4; TNRC6A, B and C, orange). Volcano plot showing -log10 P values (y) plotted against log2FC of iBAQ values, normalized to GAPDH in each compartment. P values were computed with limma’s eBayes function (Methods) and adjusted for multiple testing using the Benjamini–Hochberg method. Significant values (FDR < 5%) are shown in dark gray and non-significant in light gray. Neurofilaments (NEFL and NEFM) and synapsin (SYN1) were used as neurite markers (green) and histones as soma markers (blue) to confirm the efficiency of the separation of soma and neurites.