Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most com- mon chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA 21 that directly contribute to cognitive de fi cits remain incompletely understood. Here, we found that the HSA21- encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued de fi cits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes

Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimers pathology.

Conventional genetic approaches and computational strategies have converged on immune-inflammatory pathways as key events in the pathogenesis of late onset sporadic Alzheimers disease (LOAD). Mutations and/or differential expression of microglial specific receptors such as TREM2, CD33, and CR3 have been associated with strong increased risk for developing Alzheimers disease (AD). DAP12 (DNAX-activating protein 12)/TYROBP, a molecule localized to microglia, is a direct partner/adapter for TREM2, CD33, and CR3. We and others have previously shown that TYROBP expression is increased in AD patients and in mouse models. Moreover, missense mutations in the coding region of TYROBP have recently been identified in some AD patients. These lines of evidence, along with computational analysis of LOAD brain gene expression, point to DAP12/TYROBP as a potential hub or driver protein in the pathogenesis of AD. Using a comprehensive panel of biochemical, physiological, behavioral, and transcriptomic assays, we evaluated in a mouse model the role of TYROBP in early stage AD. We crossed an Alzheimers model mutant APP KM670/671NL /PSEN1 Δexon9 (APP/PSEN1) mouse model with Tyrobp -/- mice to generate AD model mice deficient or null for TYROBP (APP/PSEN1; Tyrobp +/- or APP/PSEN1; Tyrobp -/-). While we observed relatively minor effects of TYROBP deficiency on steady-state levels of amyloid-β peptides, there was an effect of Tyrobp deficiency on the morphology of amyloid deposits resembling that reported by others for Trem2 -/- mice. We identified modulatory effects of TYROBP deficiency on the level of phosphorylation of TAU that was accompanied by a reduction in the severity of neuritic dystrophy. TYROBP deficiency also altered the expression of several AD related genes, including Cd33. Electrophysiological abnormalities and learning behavior deficits associated with APP/PSEN1 transgenes were greatly attenuated on a Tyrobp-null background. Some modulatory effects of TYROBP on Alzheimers-related genes were only apparent on a background of mice with cerebral amyloidosis due to overexpression of mutant APP/PSEN1. These results suggest that reduction of TYROBP gene expression and/or protein levels could represent an immune-inflammatory therapeutic opportunity for modulating early stage LOAD, potentially leading to slowing or arresting the progression to full-blown clinical and pathological LOAD

Mutations in THAP1/DYT6 reveal that diverse dystonia genes disrupt similar neuronal pathways and functions

<div><p>Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in <i>THAP1</i> [THAP (Thanatos-associated protein) domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, <i>THAP1</i> downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the <i>in vivo</i> effect of a heterozygote <i>Thap1</i> C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia.</p></div

Cortico-striatal synaptic plasticity is altered in <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} derived slices.

<p><b>(A)</b><i>Thap1</i>^<i>+/-</i> mice are deficient in synaptically-induced LTD in dorsolateral striatum compared to wildtype controls (A₁; <i>p</i> < .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>(B)</b> In <i>Thap1</i>^{<i>C54Y/+</i>} 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. <b>(C)</b> Paired-pulse ratio was not altered in <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} 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 <i>post-hoc</i> tests. *p<0.05 See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p

Top canonical pathways and gene ontology terms enriched in striatum and cerebellum of <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} relative to WT.

<p><b>(A,B)</b> Top canonical pathways as determined by IPA analysis, and <b>(C,D)</b> 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 <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} relative to WT. <b>(E,F)</b> Top canonical pathways and <b>(G,H)</b> DAVID GO terms based on the list of significant DEGs in the cerebellum of <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} relative to WT.</p

Global analysis of differential gene expression in striatum and cerebellum of <i>Thap1</i>^<i>+/-</i> or <i>Thap1</i>^{<i>C54Y/+</i>} vs WT.

<p>RNA-Seq was used to identify differentially expressed genes (DEGs) in the heterozygote <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^<i>C54Y</i> 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 <b>(A)</b> <i>Thap1</i>^<i>+/-</i> striatum vs WT <b>(B)</b> <i>Thap1</i>^<i>C54Y</i> striatum vs WT. <b>(C)</b> Venn diagrams show the number of overlapping DEGs (total, up-regulated or down-regulated) between <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} relative to WT striatum. Diagrams show number of total DEGs as well as the number of up- or down-regulated genes in the <b>(D)</b> <i>Thap1</i>^<i>+/-</i> cerebellum vs WT <b>(E)</b> <i>Thap1</i>^{<i>C54Y/+</i>} cerebellum vs WT. <b>(F)</b> Venn diagrams show the number of overlapping DEGs (total, up-regulated or down-regulated) between <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} 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).</p

Tunicamycin challenge in P4 <i>Thap1</i>^<i>+/-</i> and WT pups increases eiF2α signaling pathway proteins and reveals genotype-dependent differences in striatum and cerebellum.

<p>Western blot analysis of <b>(A)</b> striatal and <b>(B)</b> cerebellar lysates from <i>Thap1</i>^<i>+/-</i> 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 <i>post hoc</i> tests. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p

Neurite length in <i>Thap1</i>^<i>+/-</i> and <i>Thap1</i>^{<i>C54Y/+</i>} E16 primary striatal neurons <i>in vitro</i> is decreased relative to neurons from WT mice.

<p>Traces and measures of neurite length using NeuriteTracer show that <i>Thap1</i>^<i>+/-</i> striatal neurons have shorter processes as compared to WT as calculated after immunostaining with <b>(A)</b> MAP2, <b>(B)</b> TUJ1 or <b>(C)</b> Total Tau. <i>Thap1</i>^{<i>C54Y/+</i>} 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 <i>post hoc</i> t-test. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p

Key constituents of the eIF2α signaling pathway are down-regulated in the brains of <i>Thap1</i>^<i>+/-</i> mice as compared to WT.

<p><b>(A)</b> 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, <i>Student’s t test</i> (*p<0.05; ** p<0.01; *** p<0.005). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>. <b>(B)</b> Levels of protein expression in the striatum and cerebellum of P1 <i>THAP1</i>^<i>+/-</i> 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, <i>Student’s t test</i>. *p<0.05. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p

Inhibition of eIF2α phosphatase rescues mGluR-LTD, but not synaptically-induced LTD.

<p>The summary graphs in the right panels show mean ± SEM for the final 5 min of recording. <b>(A)</b> LTD after treatment with group 1 agonist DHPG (100 μM, applied during the gap in the graph) was reduced in <i>Thap1</i>^<i>+/-</i> slices, and pretreatment with Sal003 (20 μM) (eIF2α phosphatase inhibitor) rescued LTD in <i>Thap1</i>^<i>+/-</i> slices. <b>(B)</b> In high frequency stimulation (HFS)-induced LTD, Sal003 (10 μM) failed to reverse the deficit observed in <i>Thap1</i>^<i>+/-</i> 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 <i>p</i> < .05 (*) or p < .01 (**) by ANOVAs followed by Newman-Keuls <i>post-hoc</i> tests. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007169#pgen.1007169.s012" target="_blank">S9 Table</a>.</p