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<p>Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1A cause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila, loss of the only ortholog of Cc2d1a, lgd, is embryonically lethal, while in vertebrates, Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have a redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b LOF to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID.</p

Primer sequences used for quantitative PCR.

<p>Primer sequences used for quantitative PCR.</p

Scheme showing the three major ER-stress sensors: PERK, ATF6 and IRE1 and the link to the targets assayed in this report.

<p>Activated PERK phosphorylates eIF2α to attenuate protein translation but allowing the expression of ATF4-dependent genes, CHOP and ASNS, involved in redox- and apoptosis-related pathways. Cleaved -active- ATF6 leads to induction of molecular chaperones (GPR78) and of the transcription factor XBP1. IRE1 activation leads to XBP1 splicing, transcriptional activation of chaperones and stimulation of protein degradation through ErdJ4, which is a component of the ER-associated degradation (ERAD) system.</p

Induction of CHOP and ASNS expression in SHSY-5Y tunicamycin ER stressed cells is reversed by PBA or PVA treatment.

<p>Cells were treated for 24 h with tunicamycin and with PBA or PVA for the times indicated. The expression of CHOP and ASNS was determined by RT-PCR and normalized to that of the corresponding 36b4 internal control. Bars represent the mean ± SEM of the relative change with respect to tunicamycin treated cells: **p<0.005 <i>vs</i> tunicamycin treated cells.</p

PBA and PVA protect SH-SY5Y cells against tunicamycin induced ER stress.

<p>SH-SY5Y cells were exposed to tunicamycin (500 nm) for 24 h in the absence (medium) or in the presence of PBA (panel A) or PVA (panel B). Cell viability was determined using a LDH release assay and the results are expressed as the means ± SEM. Bars represent the percentage of LDH release over that obtained in untreated cells. **p<0.005; ***p<0.0001 <i>vs</i> tunicamycin treated cells (Medium). Panel C. Immunoblot of procaspase 3 and cleaved caspase 3. A representative image is shown and the bar diagram represents the ratio of cleaved versus total protein (mean ± SEM) in the different conditions and normalizad to the ratio in absence of tunicamycin. ^##p<0.005 <i>vs</i> untreated cells; **p<0.005 <i>vs</i> tunicamycin treated cells.</p

PBA and PVA neutralize the ER stress sensors induced by tunicamycin.

<p>SH-SY5Y cells were treated as described in the Materials and Methods and the expression of GPR78 (A), spliced XBP1 (B) and ERdJ4 (C) was determined by RT-PCR. The bars represent the expression (mean ± SEM) normalized to that of the corresponding 36b4 internal control: *p<0.05; **p<0.005 <i>vs</i> tunicamycin treated cells.</p

Tunicamycin-induced ER stress in SH-SY5Y cells.

<p>A) SH-SY5Y cells were treated for 48 h with the indicated concentrations of tunicamycin and cell viability was determined using a LDH release assay. Bars represent the percentage of LDH release over that obtained in untreated cells: *p<0.05; ***p<0.0001 <i>vs</i> untreated cells. B–C) The transcriptional activity of different sensors was used to monitor the induction of ER stress by tunicamycin in SHSY-5Y (B) and HEK-293T (C) cells. Bars represent the fold change (mean ± SEM) in gene expression normalized to the control untreated cells: *p<0.05; **p<0.005; <i>vs</i> control untreated cells.</p