Oxygen Levels Regulate the Development of Human Cortical Radial Glia Cells.

The oxygen (O2) concentration is a vital parameter for controlling the survival, proliferation, and differentiation of neural stem cells. A prenatal reduction of O2 levels (hypoxia) often leads to cognitive and behavioral defects, attributable to altered neural development. In this study, we analyzed the effects of O2 levels on human cortical progenitors, the radial glia cells (RGCs), during active neurogenesis, corresponding to the second trimester of gestation. Small changes in O2 levels profoundly affected RGC survival, proliferation, and differentiation. Physiological hypoxia (3% O2) promoted neurogenesis, whereas anoxia (<1% O2) and severe hypoxia (1% O2) arrested the differentiation of human RGCs, mainly by altering the generation of glutamatergic neurons. The in vitro activation of Wnt-ß-catenin signaling rescued the proliferation and neuronal differentiation of RGCs subjected to anoxia. Pathologic hypoxia (≤1% O2) also exerted negative effects on gliogenesis, by decreasing the number of O4+ preoligodendrocytes and increasing the number of reactive astrocytes derived from cortical RGCs. O2-dependent alterations in glutamatergic neurogenesis and oligodendrogenesis can lead to significant changes in cortical circuitry formation. A better understanding of the cellular effects caused by changes in O2 levels during human cortical development is essential to elucidating the etiology of numerous neurodevelopmental disorders.

The Role of Sonic Hedgehog in the Specification of Human Cortical Progenitors In Vitro.

Impaired sonic hedgehog (Shh) signaling is involved in the pathology of cortical formation found in neuropsychiatric disorders. However, its role in the specification of human cortical progenitors is not known. Here, we report that Shh is expressed in the human developing cortex at mid-gestation by radial glia cells (RGCs) and cortical neurons. We used RGC cultures, established from the dorsal (cortical) telencephalon of human brain at mid-gestation to study the effect of Shh signaling. Cortical RGCs in vitro maintained their regional characteristics, expressed components of Shh signaling, and differentiated into Nkx2.1, Lhx6, and calretinin-positive (CalR(+)) cells, potential cortical interneuron progenitors. Treatment with exogenous Shh increased the pool of Nkx2.1(+) progenitors, decreased Lhx6 expression, and suppressed the generation of CalR(+) cells. The blockade of endogenous Shh signaling increased the number of CalR(+) cells, but did not affect Nkx2.1 expression, implying the existence of parallel Shh-independent pathways for cortical Nkx2.1 regulation. These results support the idea that, during human brain development, Shh plays an important role in the specification of cortical progenitors. Since direct functional studies in humans are limited, the in vitro system that we established here could be of great interest for modeling the development of human cortical progenitors.

A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3. We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients.

COUP-TFII expressing interneurons in human fetal forebrain.

Transcription factor COUP-TFII in rodents is important for migration of cortical interneurons from caudal ganglionic eminence (CGE) to the neocortex. Since in human, unlike in rodents, cortical interneurons have both ganglionic eminence (GE) and dorsal cortical origin, we studied the distribution of COUP-TFII in the human developing neocortex from 9 to 22 gestational weeks. COUP-TFII is expressed at all stages studied in the GE and in various cortical zones, from the proliferative ventricular/subventricular zone (VZ/SVZ) to layer I. Gradients of COUP-TFII expression are present in the GE, with peak expression in the CGE, and in the neocortex, from high expression in the temporal and occipital cortex to moderate in the frontal and dorsal cortex. Double immunofluorescence with γ-aminobutyric acid (GABA), calretinin, or calbindin, established that subpopulations of interneurons express COUP-TFII. A small fraction of COUP-TFII(+) cells are progenitor cells that proliferate in the CGE (3.4 ± 0.3%) and in the cortical VZ/SVZ (1.7 ± 0.1%). In summary, COUP-TFII is expressed in the human fetal forebrain in GABAergic cells, according to its possible role in migration of cortical interneurons. The source of these cells seems to be the CGE and, to a smaller extent, the cortical VZ/SVZ.

Characterizing clinical features and location-specific gene expression profiles associated with pain burden in children with functional dyspepsia.

BACKGROUND: In children with functional dyspepsia (FD), genes involved in pain modulation may be differentially expressed contributing to chronic pain. METHODS: Children with suspected FD (cases) and known eosinophilic esophagitis (controls) undergoing esophagogastroduodenoscopy completed the Rome IV Diagnostic, Pain Burden and Frequency Severity-Duration questionnaires. Two antral and two duodenal biopsies were collected and relative fold differences in gene expression for 84 pain-associated genes compared to pain-free controls were calculated. RESULTS: Sixty-six subjects with FD (postprandial distress syndrome = 34, epigastric pain syndrome = 7, both = 25; 65% female; mean age 13.7 years) and 13 pain-free controls (8% female; mean age 12.7) were studied. There were no significant differences in antral and duodenal eosinophilic counts or distribution between the pain and pain-free groups. Pain severity and burden did not differ significantly between FD subgroups and neither measure significantly correlated with eosinophil counts in the antrum or duodenum. Analysis of 47 antral and 39 duodenal biospecimens revealed 5 candidate genes significantly associated with pain burden: antral EDN1, PTGES3 and duodenal HTR1A, P2Y1, SCN3A (p < 0.01). Subsequent stringent statistical analysis comparing those with significant pain versus no pain revealed antral PTGES3 and duodenal SCN3A were the highest priority candidate genes (p < 0.001). CONCLUSIONS: Pain burden in pediatric FD may be linked to antral EDN1, PTGES3 and duodenal HTR1A, P2Y1, SCN3A differential expression. These genes are known to be involved in pain conduction, modulation, and neurotransmission, suggesting potential therapeutic targets for managing pain in FD.

Identification of a Pain-Specific Gene Expression Profile for Pediatric Recurrent Abdominal Pain.

Objectives: Functional Abdominal Pain (FAP) and Irritable Bowel Syndrome (IBS) are common recurrent abdominal pain diagnoses with the hallmark, lack of inflammation. To identify a biological signature for IBS/FAP in the colon, this study used genetic profiling to uncover gene expression changes associated with IBS/FAP and abdominal pain. Methods: Patients (8 to 17 years) newly diagnosed with IBS or FAP were enrolled in the study. At diagnostic colonoscopy, three rectal biopsies were collected, and gene expression analysis was performed using a Qiagen PCR Array. Relative fold difference in gene expression for 84 pain-associated genes was calculated using the 2-ΔΔ Cq method compared with pain-free controls. Factors affecting pain burden (Pain Burden Interview; PBI) were analyzed, including age, sex, rectal inflammation, and gene expression. Data were analyzed using multiple stepwise linear regression and 2-tailed t tests (P ≤ 0.05). Results: Of the 22 total patients in the study, 19 were diagnosed with either IBS-Constipation (frequency of 5.26%), IBS-Diarrhea (47.37%), IBS-Mixed (10.53%), or FAP (36.84%). IBS/FAP patients reported significantly higher pain burden at the time of diagnosis compared to pain-free controls (p < 0.001), as well as significantly higher abdominal pain (p = 0.01). Of the 84 genes, expression of GRIN1 (p = 0.02), MAPK3 (p = 0.04), P2X4 (p = 0.04), and PTGES3 (p = 0.02) were all significantly associated with PBI score. Discussion: Abdominal pain associated with IBS/FAP in pediatric patients may be linked to the expression of GRIN1, MAPK3, P2X4, and PTGES3, pointing to potential novel therapeutic targets for management of recurring abdominal pain.

A Role for Global DNA Methylation Level and IL2 Expression in the Transition From Acute to Chronic Low Back Pain.

Objectives: The transition from acute low back pain (aLBP) to chronic LBP (cLBP) results from a variety of factors, including epigenetic modifications of DNA. The aim of this study was to (1) compare global DNA (gDNA) methylation and histone acetylation at LBP onset between the aLBP and cLBP participants, (2) compare mRNA expression of genes with known roles in the transduction, maintenance, and/or modulation of pain between the aLBP and cLBP participants, (3) compare somatosensory function and pain ratings in our participants, and (4) determine if the aforementioned measurements were associated. Methods: A total of 220 participants were recruited for this prospective observational study following recent onset of an episode of LBP. We retained 45 individuals whose gDNA was of sufficient quality for analysis. The final sample included 14 participants whose pain resolved within 6 weeks of onset (aLBP),15 participants that reported pain for 6 months (cLBP), and 16 healthy controls. Participants were subjected to quantitative sensory testing (QST), blood was drawn via venipuncture, gDNA isolated, and global DNA methylation and histone acetylation, as well as mRNA expression of 84 candidate genes, were measured. Results: Individuals that develop cLBP display multimodal somatosensory hypersensitivity relative to aLBP participants. cLBP participants also had significantly lower global DNA methylation, which was negatively correlated with interleukin-2 (IL2) mRNA expression. Discussion: cLBP is characterized by somatosensory hypersensitivity, lower global DNA methylation, and higher IL2 expression level compared to those whose pain will resolve quickly (aLBP). These results suggest potential diagnostic and therapeutic relevance for global DNA methylation and IL2 expression in the pathology underlying the transition from acute to chronic LBP.

Characterizing Clinical Features and Creating a Gene Expression Profile Associated With Pain Burden in Children With Inflammatory Bowel Disease.

BACKGROUND: There is often dissociation between inflammatory activity and abdominal pain in children with inflammatory bowel disease (IBD), suggesting other factors may play a role in the pain experience. METHODS: Patients (8 to 17 years) newly diagnosed with IBD were enrolled in the ALLAY Study: Assessing Risk Factors for Abdominal Pain in Children with Inflammatory Bowel Disease (NCT02984059). At diagnostic colonoscopy, 3 rectal biopsies were collected, and gene expression analysis was performed using Qiagen RT2 Profiler Neuropathic and Inflammatory Pain PCR Array. Relative fold difference in gene expression for 84 pain-associated genes was calculated using the 2-ΔΔ Cq method compared with pain-free controls. Factors affecting pain burden (Pain Burden Interview; PBI) were analyzed, including age, sex, rectal inflammation, and gene expression. Data were analyzed using multiple stepwise linear regression and 2-tailed t tests (P ≤ 0.05). RESULTS: Thirty-nine newly diagnosed IBD patients were included (65% male, mean age 12.75 years [SD 2.63], 23 Crohn's disease, 16 ulcerative colitis), along with 3 controls. Mean PBI score was 7.73 (SD 6.4, range 0 to 23) for all patients. Age and sex were not predictive of pain burden, but disease activity score was (P = 0.03). Expression of TRPV3, OPRM1, P2X3, SCN9A, PTGS2, and MAPK14 were associated with PBI score. Subsequent 2-tailed t tests comparing patients with no pain (PBI score ≦ 2, N = 11) to those with pain (PBI > 2, N = 28) confirmed differential expression of TRPV3, PTGS2, and MAPK14 was in patients with pain (all P < 0.05). CONCLUSION: Pain burden in newly diagnosed IBD patients may be linked to TRPV3, PTGS2, and MAPK14 expression, suggesting potential therapeutic targets for managing pain in IBD.

Contribution of COMT and BDNF Genotype and Expression to the Risk of Transition From Acute to Chronic Low Back Pain.

OBJECTIVES: A number of factors, including heritability and the environment, contribute to risk of transition from acute low back pain to chronic low back pain (CLBP). The aim of this study was to (1) compare somatosensory function and pain ratings at low back pain (LBP) onset between the acute low back pain and CLBP conditions and (2) evaluate associations between BDNF and COMT polymorphisms and expression levels at LBP onset to acute and chronic pain burden and risk for transition to the chronic pain state. METHODS: In this longitudinal study, 220 participants were enrolled following recent onset of LBP and data were collected until the LBP resolved or until the end of the study at 6 months. Forty-two participants' pain resolved before 6 weeks from onset and 42 participants continued to have pain at 6 months. Patient-reported pain burden, somatosensory function (quantitative sensory testing), and blood samples were collected at each study visit. RESULTS: CLBP is associated with greater pain burden and somatosensory hypersensitivity at the time of LBP onset. COMT rs4680 genotype (GG) was associated with acute cold pain sensitivity and with the risk for transition to CLBP while COMT expression was independently associated with risk for transition. DISCUSSION: CLBP was characterized by higher reported pain burden and augmented hypersensitivity at LBP onset. COMT expression and genotype were associated with acute pain burden and likelihood of transition to CLBP.

Genetic mapping in Diversity Outbred mice identifies a Trpa1 variant influencing late-phase formalin response.

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

Myocyte enhancer factor 2 (MEF2) tethering to muscle selective A-kinase anchoring protein (mAKAP) is necessary for myogenic differentiation.

Differentiation of skeletal myoblast cells to functional myotubes involves highly regulated transcriptional dynamics. The myocyte enhancer factor 2 (MEF2) transcription factors are critical to this process, synergizing with the master regulator MyoD to promote muscle specific gene transcription. MEF2 is extensively regulated by myogenic stimuli, both transcriptionally and post-translationally, but to date there has been little progress in understanding how signals upstream of MEF2 are coordinated to produce a coherent response. In this study, we define a novel interaction between the muscle A-kinase anchoring protein (mAKAP) and MEF2 in skeletal muscle. Discrete domains of MEF2 and mAKAP bind directly. Their interaction was exploited to probe the function of mAKAP-tethered MEF2 during myogenic differentiation. Dominant interference of MEF2/mAKAP binding was sufficient to block MEF2 activation during the early stages of differentiation. Furthermore, extended expression of this disrupting domain effectively blocked myogenic differentiation, halting the formation of myotubes and decreasing expression of several differentiation markers. This study expands our understanding of the regulation of MEF2 in skeletal muscle and identifies the mAKAP scaffold as a facilitator of MEF2 transcription and myogenic differentiation.