Neurons cultured from heterozygous preCGG KI mice possess elevated heterochromatin at 14 and 21 DIV. Hippocampal neurons from littermate heterozygous preCGG and WT mice were stained with DAPI (4',6-diamidino-2-phenylindole) to label the nucleus. (A) Representative Dapi nuclear staining at three time points for each genotype; (B) the average (mean ± SE) nuclear area; (C) the number; and (D) the area of heterochromatin in each individual nucleus. Heterozygous preCGG neurons have similar nuclear area, but more abundant and smaller heterochromatin compared with WT at 14 and 21 DIV. Data are from N = 3 separate cultures with 90 randomly selected nuclei analyzed from each experimental group. Scale bar: 10 µm.

Heterozygous preCGG KI neurons show decreased viability from 21 DIV. Separate cultures of hippocampal neurons from littermate heterozygous preCGG and WT mice were fixed weekly after plating, and Map2B and Dapi immunofluorescent staining were quantified. Map2B and Dapi fluorescent staining are shown in (A) (WT) and (B) (preCGG). Images are taken using low magnification (×20). (C) Quantification of the number of Map2B-positive neurons divided by the number of Dapi-positive cells as a measure of viability. preCGG neurons have a higher fractions of Dapi-positive nuclei that lack Map2B staining on 21 and 28 DIV (P < 0.0001). N = 3 separate cultures and approximately 30 randomly selected images for each experimental group. Scale bar: 100 µm.

Heterozygous preCGG KI neurons show decreased survival independent of glia cell composition. Separate cultures of hippocampal neurons from littermate heterozygous preCGG and WT mice were fixed weekly, and Map2B and GFAP in-cell western analysis performed. MTS assay was performed weekly to study the survival rate of the neurons. (A and B) Results of Map2B and GFAP in-cell western analyses, respectively, demonstrating decreased Map2B protein on 14, 21 and 28 DIV, whereas GFAP protein from the glia cell population remained comparable between genotype. Data are from N = 3 separate cultures with two to three wells for each experimental group. (C) The relative survival rate of the heterozygous preCGG and WT neurons using MTS assay. Data from N = 3 separate cultures with 16 wells from each experimental group.

Heterozygous preCGG KI neurons show significantly reduced dendritic complexity throughout in vitro development. Hippocampal neuronal cultures from heterozygous preCGG mice and WT littermates were fixed weekly after plating. Immunofluorescent labeling was performed using Map2B antibody. (A) Representative images from the two different genotypes at three time points. (B and C) Quantification of dendrite length and the number of branch points of individual neurons, respectively, calculated with Imaris FilamentTracer software as described in Materials and Methods. The heterozygous preCGG neurons show delayed dendritic growth from an early developmental stage (7 DIV) throughout their maturation (21 DIV). N = 3 separate cultures with 30–38 randomly selected neurons for each time points for each genotype. Scale bar: 20 µm.

Neurons from heterozygous preCGG KI mice have increased presynaptic synapsin and postsynaptic phalloidin puncta volume. Mature 14 and 21 DIV neurons from heterozygous preCGG and WT littermate mice were analyzed for patterns of synapsin (A and C) and phalloidin (B and D) labeling. (A and B) Representative images from 21 DIV neurons labeled with synapsin and phalloidin (scale bar: 5 µm). Boxed regions are shown in higher magnification below each lower magnification image. The synapsin and phalloidin puncta were quantified, and summary data shown in (C) and (D) reveal that heterozygous preCGG neurons have significantly bigger synapsin and phalloidin puncta compared with the WT (P < 0.0001). The puncta average intensity and total counts did not differ between genotype at either 14 or 18 DIV (data not shown). N = 3 individual cultures with a total of 30 randomly selected neurons measured per genotype for each synapsin and phalloidin puncta quantification. (E) Results from in-cell western experiments showing that, at 14 DIV, heterozygous preCGG hippocampal neurons have higher synapsin levels than WT neurons cultured from littermates (P = 0.0041).

Heterozygous preCGG KI neurons express elevated stress protein and mRNAs. Hippocampal neurons from littermate heterozygous preCGG and WT mice were collected 14 DIV after plating. Upper panels: Levels of mRNA of the stress proteins alpha B-crystallin (Cryab), Hsp27 and Hsp70 were measured by RT–PCR and normalized to expression of GUS. mRNA signals were normalized to expression of the GUS gene. Significantly higher mRNA levels for alpha B-crystallin (Cryab) (4.5 ± 0.9-fold), Hsp27 (9.9 ± 0.3), and Hsp70 (2.8 ± 0.7) were detected in 14 DIV preCGG KI neurons compared with WT control neurons. P-values are calculated on the basis of RT–PCR results obtained from two independent cultures derived from WT and preCGG littermates (N = 2), each performed in duplicate with three starting RNA concentrations (see Materials and Methods for details). Lower panels: In-cell western analysis of the corresponding proteins in the upper panels. Signals were normalized to cell number using DraQ5 staining of nuclei as described in Materials and Methods. Data from N = 2 separate cultures with six wells from each experimental group.

Heterozygous preCGG neurons express more Fmr1 mRNAs and less FMRP proteins compared with WT littermate neurons. Fourteen DIV hippocampal neurons from heterozygous preCGG and WT littermate mice were fixed in a 96-well plate, and in-cell western analysis performed using chicken anti-FMRP antibody. (A) The 800 nm infrared wavelength signal detected by the LI-COR Odyssey scanner. The first row was stained with a secondary antibody (goat anti-chicken 800) without a primary antibody (chicken FMRP antibody). Rows 2 and 3 show FMRP protein levels expressed by the heterozygous preCGG (left six columns) and WT (right six columns) neuron-enriched low-density cultures. (B) DraQ5 nuclear staining at 700 nm, which serves to normalize FMRP signals to cell density. (C) Quantification of the signal ratio of the two channels (800:700 nm) using Odyssey 2.1 software, and the heterozygous preCGG results are presented as percentage of WT. Heterozygous preCGG KI neurons show 72.6 ± 5.4% of total FMRP level compared with WT littermate neurons 100.0 ± 2.4% (N = 3 separate cultures, four determinations per culture) (P = 0.0002). (D) Fmr1 mRNA-level comparison between heterozygous preCGG KI neurons and WT at 14 DIV after plating. Quantitative RT–PCR was performed and Fmr1 mRNA level was measured; heterozygous preCGG neurons show 2.6 ± 0.3-fold higher Fmr1 mRNA level than the WT littermate control (P < 0.0001). The P-values are calculated using two-way ANOVA based on RT–PCR results obtained from two independent cultures derived from WT and preCGG littermates (N = 2), each performed in duplicate with three starting RNA concentrations (see Materials and Methods for details).