Fig 2: Control immunolocalization of plasma membrane AMPA receptor subunits (external epitopes) in hippocampal neuronal cultures.(A) Beta-tubulin (green, anti-TuJ1) labeling of dendrite, after anti-GluA1 external epitope [66] labeling and subsequent plasma membrane permeabilization. Note punctate GluA1 [66] labeling along the dendrite. (B) Synaptophysin (P38, green) and GluA1 [66]. (C) Synaptophysin (green) and GluA2 [66]. (D) Labeling with anti-GluA1. (E) Labeling with anti-GluA1, but the antibody was preincubated with the peptide antigen before staining. (F) Labeling with anti-GluA2. (G) Labeling with anti-GluA2, but the antibody was preincubated with the peptide antigen before staining. (H) Immunolabeling of non-permeabilized neuronal cultures with ani-GluA1 (green). (I) Immunolabeling with the postsynaptic marker anti-PSD95 [66] after permeabilization of neuronal cultures. (J) Merging of (H) and (I) showing postsynaptic labeling of GluA1. Scale bar (in A, valid for all images): 10 µm.

Fig 3: HSF1 binding to regulatory elements of PSD-95 and its regulation is impaired in HD. (A–D) HSF1-ChIP on PSD-95 HSE #3 in (A) STHdhQ7 and STHdhQ111 cells (n = 6), (B) the striatum of WT and zQ175 mice at 3 (n = 4 mice/genotype) and (C) 6 months (n = 4 mice/genotype), and (D) in the striatum of patients with HD and sex and age-matched controls (n = 6 individuals/group). (E) Luciferase activity in STHdhQ7 and STHdhQ111 cells. Data is normalized to Renilla luciferase and relativized to Q7 cells (n = 5). (F) Luciferase activity in WT and Hsf1(+/-) STHdhQ7 and STHdhQ111 cells. Data is normalized to Renilla luciferase and relativized to Q7. Error bars denote mean ± SEM. Unpaired Student’s t-test in (A–E), and One-way ANOVA with Tukey’s post-hoc test in (F). * p < 0.05, **** p < 0.0001.

Fig 4: CS10 is effective for the cryostorage of primary hippocampal neurons. A, Percentage of recovered and lost hippocampal cells after cryostorage in CS10. Percentage of “recovered” cells was calculated with ((# cells alive + # cells dead) ÷ (# cells in initial aliquot)) × 100. Percentage of “lost” cells was calculated with 100% recovered. Mean ± SD, N = 7 source dissections. B, Post-thaw viability of CS10-cryostored hippocampal cells compared to the viability of freshly dissected cells as evaluated by Trypan blue exclusion. Mean ± SD, N = 7 source dissections. C, RT-qPCR of RNA samples from fresh or CS10-cryostored hippocampal cultures collected at DIV12. CTs were normalized to housekeeping genes and relative copy numbers were generated using 2-?CT × 106. Floating bar graph spans the minimum and maximum data points, vertical line denotes mean. N = 3 source dissections. Statistical comparisons made by unpaired parametric t test with Holm–Sidak correction for multiple comparisons. Multiplicity adjusted p value for each comparison listed in table (right). D, Immunofluorescence labeling of freshly dissected or CS10-cryostored hippocampal neurons at DIV4, using antibodies against the dendrite marker MAP2 (magenta) and axon marker Tau (green). Scale bar = 100 µm. E–G, Quantification of number of primary neurites (E), primary neurite length (F), and axon length (G) of freshly dissected or CS10-cryostored hippocampal neurons at DIV4. Box-and-whisker plots show data pooled from four independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (????) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. N = 4 source dissections, n > 115 neurons per condition. H, Immunofluorescence labeling of freshly dissected or CS10-cryostored hippocampal neurons expressing Lifeact-mRuby2 (inverted white) at DIV16, using antibodies against excitatory postsynaptic marker PSD95 (Novus; green), inhibitory postsynaptic marker gephyrin (GPHN; cyan), and presynaptic marker synaptophysin (SYP; magenta). Scale bar = 5 µm. I, Density of actin-rich protrusions from secondary dendrites ay DIV16 from indicated conditions. J, Percentage of PSD95-positive excitatory synapses and GPHN-positive inhibitory synapses at DIV16 from indicated conditions. Mean ± SD, N = 4 source dissections, n > 16 neurons per condition. All statistical comparisons between biological replicate means made by unpaired parametric t test (E–G, I, J). n.s. = not significant.

Fig 5: Synaptogenesis occurs normally in neurons following cryostorage and recovery. A–C, Neurons expressing Lifeact-mRuby2 (inverted white) from indicated conditions were fixed at DIV12 (A), DIV14 (B), and DIV16 (C), and immunolabeled using antibodies against excitatory postsynaptic marker PSD95 (NeuroMab; green), inhibitory postsynaptic marker gephyrin (GPHN; cyan), and presynaptic marker synaptophysin (SYP; magenta). Bottom panel is magnified segment of boxed dendrite region. Scale bar = 100 µm (top) and 5 µm (bottom panel). D, Density of all actin-rich protrusions from secondary dendrites at indicated timepoints and conditions. E, Ratio of dendritic spines to dendritic filopodia as determined by presence or absence of PSD95 on actin-rich protrusions at indicated timepoints and conditions. Box-and-whisker plots (D, E) show data pooled from three independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (???) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. F, Percentage of PSD95-positive excitatory synapses and GPHN-positive inhibitory synapses at DIV16 from indicated conditions. Mean ± SD, N = 3 source dissections, n = 6–7 neurons per condition. All statistical comparisons between biological replicate means made by unpaired parametric t test. n.s. = not significant.