De novo variants of NR4A2 are associated with neurodevelopmental disorder and epilepsy

Purpose This study characterizes the clinical and genetic features of nine unrelated patients with de novo variants in the NR4A2 gene. Methods Variants were identified and de novo origins were confirmed through trio exome sequencing in all but one patient. Targeted RNA sequencing was performed for one variant to confirm its splicing effect. Independent discoveries were shared through GeneMatcher. Results Missense and loss-of-function variants in NR4A2 were identified in patients from eight unrelated families. One patient carried a larger deletion including adjacent genes. The cases presented with developmental delay, hypotonia (six cases), and epilepsy (six cases). De novo status was confirmed for eight patients. One variant was demonstrated to affect splicing and result in expression of abnormal transcripts likely subject to nonsense-mediated decay. Conclusion Our study underscores the importance of NR4A2 as a disease gene for neurodevelopmental disorders and epilepsy. The identified variants are likely causative of the seizures and additional developmental phenotypes in these patients

De novo variants of NR4A2 are associated with neurodevelopmental disorder and epilepsy

PURPOSE: This study characterizes the clinical and genetic features of nine unrelated patients with de novo variants in the NR4A2 gene. METHODS: Variants were identified and de novo origins were confirmed through trio exome sequencing in all but one patient. Targeted RNA sequencing was performed for one variant to confirm its splicing effect. Independent discoveries were shared through GeneMatcher. RESULTS: Missense and loss-of-function variants in NR4A2 were identified in patients from eight unrelated families. One patient carried a larger deletion including adjacent genes. The cases presented with developmental delay, hypotonia (six cases), and epilepsy (six cases). De novo status was confirmed for eight patients. One variant was demonstrated to affect splicing and result in expression of abnormal transcripts likely subject to nonsense-mediated decay. CONCLUSION: Our study underscores the importance of NR4A2 as a disease gene for neurodevelopmental disorders and epilepsy. The identified variants are likely causative of the seizures and additional developmental phenotypes in these patients

Genome engineering with TALE and CRISPR systems in neuroscience

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience

The endocannabinoid gene <i>faah2a</i> modulates stress-associated behavior in zebrafish

<div><p>The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (<i>abhd4</i>, <i>gde1</i>, <i>napepld)</i>, enzymes that degrade (<i>faah</i>, <i>faah2a</i>, <i>faah2b</i>), and receptors that bind (<i>cnr1</i>, <i>cnr2</i>, <i>gpr55-like</i>) endocannabinoids are expressed in zebrafish (<i>Danio rerio</i>). These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in <i>cnr1</i> and <i>faah2a</i> to test the role of these genes in modulating stress-associated behavior. We showed that disruption of <i>cnr1</i> potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of <i>faah2a</i> attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.</p></div

<i>faah2a</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>faah2a</i> (HET) mutant, and homozygous <i>faah2a</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p

Temporal expression patterns of eCB receptor genes.

<p>The time points on all graphs are represented as means ± 95% CI (0.25–1 dpf: 20 larvae/n, n = 3; 2–7 dpf: 10 larvae/n, n = 3). * Indicates that a group is significantly different from the 5 dpf group (Sidak's multiple comparisons test, <i>P <</i> 0.05). (A) The fold change of <i>cnr1</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (B) The fold change of <i>cnr2</i> transcript levels relative to 5 dpf as determined by qRT-PCR. (C) The fold change of <i>loc793909</i> transcript levels relative to 5 dpf as determined by qRT-PCR.</p

TALEN-mediated mutagenesis of <i>faah2a</i>.

<p>(A) The <i>faah2a</i> TALEN target site was designed in exon 3 so that mutagenesis would disrupt all predicted splice variants. NCBI Accessions: Gene ID, 436973; DNA, NC_007112.6; mRNA (i), NM_001002700.2; Protein (i), NP_001002700.1. The target site is just upstream of the sequence encoding a lysine in the predicted serine 228 (S) / serine 204 (S) / lysine 129 (K) catalytic triad. (B) An alignment of wild type and mutant <i>faah2a</i> sequences reveals the TALEN-induced indel in the target BsrI restriction enzyme site.</p

<i>cnr1</i> modulates stress-associated behavior.

<p>(A) The rolling means of distances travelled by wild type (WT), heterozygous <i>cnr1</i> (HET) mutant, and homozygous <i>cnr1</i> (HOM) mutant zebrafish. The pre-treatment baseline locomotor activity was recorded from −15–0 min, and the post-treatment locomotor activity was recorded from 0–31 min. At time 0 the zebrafish were treated with either E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity at each second is represented as a mean of the distance travelled during the preceding 60 s. (B) The means of distances travelled after the zebrafish were treated with E2 media (Control) or E2 media + NaCl (Stress). The locomotor activity of each group is represented as a mean of the distance travelled per min during the 5–25 min time bin ± 95% CI. Groups with all different letters above the columns are statistically different from each other, while groups with a conserved letter above the columns are not statistically different from each other (Tukey's honest significant difference test, <i>P <</i> 0.05).</p

GLI1 interaction with p300 modulates SDF1 expression in cancer-associated fibroblasts to promote pancreatic cancer cells migration

Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300–GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.Fil: Vera, Renzo Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lamberti, María Julia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Almada, Luciana L.. No especifíca;Fil: Tolosa, Ezequiel Julian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vrabel, Anne M.. No especifíca;Fil: Sigafoos, Ashley N.. No especifíca;Fil: Toruner, Merih D.. No especifíca;Fil: Flores, Luis F.. No especifíca;Fil: Rivarola, Viviana Alicia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Rumie Vittar, Natalia Belen. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Fernández Zapico, Martín E.. No especifíca

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1

International audienceUpon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process