Fig 1.
Global analysis of differential gene expression in striatum and cerebellum of Thap1+/- or Thap1C54Y/+ vs WT.
RNA-Seq was used to identify differentially expressed genes (DEGs) in the heterozygote Thap1+/- and Thap1C54Y P1 striatum and cerebellum as compared to WT. Diagrams show number of total DEGs as well as the number of up- or down-regulated genes in the (A) Thap1+/- striatum vs WT (B) Thap1C54Y striatum vs WT. (C) Venn diagrams show the number of overlapping DEGs (total, up-regulated or down-regulated) between Thap1+/- and Thap1C54Y/+ relative to WT striatum. Diagrams show number of total DEGs as well as the number of up- or down-regulated genes in the (D) Thap1+/- cerebellum vs WT (E) Thap1C54Y/+ cerebellum vs WT. (F) Venn diagrams show the number of overlapping DEGs (total, up-regulated or down-regulated) between Thap1+/- and Thap1C54Y/+ relative to WT cerebellum. Cogged gears in panels A, B, D and E represent the number and the direction of the differentially expressed genes for each genotype and brain region, as follows: up-regulated genes (turquoise color, upward right arrow); down-regulated genes (dark blue color, downward left arrow); total [number] of genes (purple color, downward right arrow).
Fig 2.
Top canonical pathways and gene ontology terms enriched in striatum and cerebellum of Thap1+/- and Thap1C54Y/+ relative to WT.
(A,B) Top canonical pathways as determined by IPA analysis, and (C,D) DAVID Gene Ontology (GO) terms show an enrichment of biological process based on the list of significant DEGs (DEseq p < 0.05) in the striatum of Thap1+/- and Thap1C54Y/+ relative to WT. (E,F) Top canonical pathways and (G,H) DAVID GO terms based on the list of significant DEGs in the cerebellum of Thap1+/- and Thap1C54Y/+ relative to WT.
Fig 3.
Key constituents of the eIF2α signaling pathway are down-regulated in the brains of Thap1+/- mice as compared to WT.
(A) mRNA expression gene profiles of key genes from the eIF2α signaling pathway were assayed with quantitative real-time PCR (RT-qPCR) using cerebellar samples. Data normalized relative to WT. Data are presented as means ± Standard Error of the Mean (SEM); n = 8 for each genotype and brain region with separate littermate WT controls, Student’s t test (*p<0.05; ** p<0.01; *** p<0.005). See also S9 Table. (B) Levels of protein expression in the striatum and cerebellum of P1 THAP1+/- mice relative to WT were assayed by western blot. Densitometry measurements (arbitrary units) are normalized to the housekeeping gene GAPDH, or for phosphoproteins, relative to GAPDH and their respective holoprotein level. Data are presented as means ± SEM; n = 4 for each genotype and region with separate, littermate WT controls, Student’s t test. *p<0.05. See also S9 Table.
Fig 4.
Tunicamycin challenge in P4 Thap1+/- and WT pups increases eiF2α signaling pathway proteins and reveals genotype-dependent differences in striatum and cerebellum.
Western blot analysis of (A) striatal and (B) cerebellar lysates from Thap1+/- and WT littermates for BiP, ATF4 and CHOP were performed 24 hrs after subcutaneous tunicamycin (TM) diluted in 150mM dextrose (or dextrose-only control; DEX). Protein expression levels represent normalization to the housekeeping gene GAPDH. Data are presented as means ± SEM; n = 5 for each genotype and region, data normalized to WT (dextrose-only) controls. Statistical differences were assessed by two-way ANOVAs with Tukey’s post hoc tests. *p < 0.05; **p < 0.01; ***p < 0.001. See also S9 Table.
Fig 5.
Cortico-striatal synaptic plasticity is altered in Thap1+/- and Thap1C54Y/+ derived slices.
(A) Thap1+/- mice are deficient in synaptically-induced LTD in dorsolateral striatum compared to wildtype controls (A1; p < .05), while LTP in the dorsomedial region is intact (A2). Representative excitatory postsynaptic potential (EPSP) traces in this and panel B were averaged over the baseline period (thin line) and over the final 5 min of recording (thick line), color coded to the graph. Calibration for these and all other traces: 1 mV / 5 ms. (B) In Thap1C54Y/+ mice, LTD was not significantly reduced (B1), but LTP was deficient (B2; p < .05). Note that wildtype data are the same as for panel A, and that all genotypes were analyzed together. (C) Paired-pulse ratio was not altered in Thap1+/- and Thap1C54Y/+ mice. The traces show averaged EPSPs recorded at inter-stimulus interval = 50 ms (thin and thick lines show responses to first and second stimuli, respectively). All graphs show group means ± SEM, and the number of slices/mice for each group are shown in parentheses. Data were analyzed by ANOVAs performed over the final 5 minutes of recording (panels A and B) or on averaged paired-pulse data for each interval (panel C), followed where appropriate by Newman-Keuls post-hoc tests. *p<0.05 See also S9 Table.
Fig 6.
Inhibition of eIF2α phosphatase rescues mGluR-LTD, but not synaptically-induced LTD.
The summary graphs in the right panels show mean ± SEM for the final 5 min of recording. (A) LTD after treatment with group 1 agonist DHPG (100 μM, applied during the gap in the graph) was reduced in Thap1+/- slices, and pretreatment with Sal003 (20 μM) (eIF2α phosphatase inhibitor) rescued LTD in Thap1+/- slices. (B) In high frequency stimulation (HFS)-induced LTD, Sal003 (10 μM) failed to reverse the deficit observed in Thap1+/- slices. Numbers in parentheses indicate number of slices/number of mice. Representative traces are shown during baseline period (solid lines) and at the end of the recording period (dashed lines). Calibrations: 1 mV / 5 ms. Asterisks indicate p < .05 (*) or p < .01 (**) by ANOVAs followed by Newman-Keuls post-hoc tests. See also S9 Table.
Fig 7.
Neurite length in Thap1+/- and Thap1C54Y/+ E16 primary striatal neurons in vitro is decreased relative to neurons from WT mice.
Traces and measures of neurite length using NeuriteTracer show that Thap1+/- striatal neurons have shorter processes as compared to WT as calculated after immunostaining with (A) MAP2, (B) TUJ1 or (C) Total Tau. Thap1C54Y/+ striatal neurons exhibit a milder phenotype and only have shorter axonal processes (Tau immunostaining) as compared to WT. The total number of nuclei (DAPI stain) was independent of genotype after plating equal numbers of neurons in all wells. Neurons were derived from 3 independent platings. Data are presented as means ± SEM, minimum of 25 neurons per well. Statistical differences were assessed by ANOVA with Student’s post hoc t-test. *p < 0.05; **p < 0.01; ***p < 0.001. See also S9 Table.