Effects of heterozygous deletion of autism-related gene Cullin-3 in mice.

Autism Spectrum Disorder (ASD) is a developmental disorder in which children display repetitive behavior, restricted range of interests, and atypical social interaction and communication. CUL3, coding for a Cullin family scaffold protein mediating assembly of ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors, has been identified as a high-risk gene for autism. Although complete knockout of Cul3 results in embryonic lethality, Cul3 heterozygous mice have reduced CUL3 protein, demonstrate comparable body weight, and display minimal behavioral differences including decreased spatial object recognition memory. In measures of reciprocal social interaction, Cul3 heterozygous mice behaved similarly to their wild-type littermates. In area CA1 of hippocampus, reduction of Cul3 significantly increased mEPSC frequency but not amplitude nor baseline evoked synaptic transmission or paired-pulse ratio. Sholl and spine analysis data suggest there is a small yet significant difference in CA1 pyramidal neuron dendritic branching and stubby spine density. Unbiased proteomic analysis of Cul3 heterozygous brain tissue revealed dysregulation of various cytoskeletal organization proteins, among others. Overall, our results suggest that Cul3 heterozygous deletion impairs spatial object recognition memory, alters cytoskeletal organization proteins, but does not cause major hippocampal neuronal morphology, functional, or behavioral abnormalities in adult global Cul3 heterozygous mice.

Purine metabolism regulates DNA repair and therapy resistance in glioblastoma.

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.

The Role of First Ray Insufficiency in the Development of Metatarsalgia.

Two theories exist in the development of central or transfer metatarsalgia. First, as the severity of hallux valgus increases, there is mechanical overload of the second metatarsal. Second, increased relative lesser metatarsal length is thought to contribute to metatarsalgia. It is imperative, in the treatment of first ray disorders (hallux valgus or hallux rigidus), to not overshorten the first ray when addressing the first ray pathologic condition. Treatment of metatarsalgia in the setting of failed hallux valgus correction can be treated with both conservative and surgical options.

Kctd13 deletion reduces synaptic transmission via increased RhoA.

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.

Whole-Body Exposure to 28Si-Radiation Dose-Dependently Disrupts Dentate Gyrus Neurogenesis and Proliferation in the Short Term and New Neuron Survival and Contextual Fear Conditioning in the Long Term.

Astronauts traveling to Mars will be exposed to chronic low doses of galactic cosmic space radiation, which contains highly charged, high-energy (HZE) particles. 56Fe-HZE-particle exposure decreases hippocampal dentate gyrus (DG) neurogenesis and disrupts hippocampal function in young adult rodents, raising the possibility of impaired astronaut cognition and risk of mission failure. However, far less is known about how exposure to other HZE particles, such as 28Si, influences hippocampal neurogenesis and function. To compare the influence of 28Si exposure on indices of neurogenesis and hippocampal function with previous studies on 56Fe exposure, 9-week-old C57BL/6J and Nestin-GFP mice (NGFP; made and maintained for 10 or more generations on a C57BL/6J background) received whole-body 28Si-particle-radiation exposure (0, 0.2 and 1 Gy, 300 MeV/n, LET 67 KeV/µ, dose rate 1 Gy/min). For neurogenesis assessment, the NGFP mice were injected with the mitotic marker BrdU at 22 h postirradiation and brains were examined for indices of hippocampal proliferation and neurogenesis, including Ki67+, BrdU+, BrdU+NeuN+ and DCX+ cell numbers at short- and long-term time points (24 h and 3 months postirradiation, respectively). In the short-term group, stereology revealed fewer Ki67+, BrdU+ and DCX+ cells in 1-Gy-irradiated group relative to nonirradiated control mice, fewer Ki67+ and DCX+ cells in 0.2 Gy group relative to control group and fewer BrdU+ and DCX+ cells in 1 Gy group relative to 0.2 Gy group. In contrast to the clearly observed radiation-induced, dose-dependent reductions in the short-term group across all markers, only a few neurogenesis indices were changed in the long-term irradiated groups. Notably, there were fewer surviving BrdU+ cells in the 1 Gy group relative to 0- and 0.2-Gy-irradiated mice in the long-term group. When the short- and long-term groups were analyzed by sex, exposure to radiation had a similar effect on neurogenesis indices in male and female mice, although only male mice showed fewer surviving BrdU+ cells in the long-term group. Fluorescent immunolabeling and confocal phenotypic analysis revealed that most surviving BrdU+ cells in the long-term group expressed the neuronal marker NeuN, definitively confirming that exposure to 1 Gy 28Si radiation decreased the number of surviving adult-generated neurons in male mice relative to both 0- and 0.2-Gy-irradiated mice. For hippocampal function assessment, 9-week-old male C57BL/6J mice received whole-body 28Si-particle exposure and were then assessed long-term for performance on contextual and cued fear conditioning. In the context test the animals that received 0.2 Gy froze less relative to control animals, suggesting decreased hippocampal-dependent function. However, in the cued fear conditioning test, animals that received 1 Gy froze more during the pretone portion of the test, relative to controls and 0.2-Gy-irradiated mice, suggesting enhanced anxiety. Compared to previously reported studies, these data suggest that 28Si-radiation exposure damages neurogenesis, but to a lesser extent than 56Fe radiation and that low-dose 28Si exposure induces abnormalities in hippocampal function, disrupting fear memory but also inducing anxiety-like behavior. Furthermore, exposure to 28Si radiation decreased new neuron survival in long-term male groups but not females suggests that sex may be an important factor when performing brain health risk assessment for astronauts traveling in space.

Arid1b haploinsufficient mice reveal neuropsychiatric phenotypes and reversible causes of growth impairment.

Sequencing studies have implicated haploinsufficiency of ARID1B, a SWI/SNF chromatin-remodeling subunit, in short stature (Yu et al., 2015), autism spectrum disorder (O'Roak et al., 2012), intellectual disability (Deciphering Developmental Disorders Study, 2015), and corpus callosum agenesis (Halgren et al., 2012). In addition, ARID1B is the most common cause of Coffin-Siris syndrome, a developmental delay syndrome characterized by some of the above abnormalities (Santen et al., 2012; Tsurusaki et al., 2012; Wieczorek et al., 2013). We generated Arid1b heterozygous mice, which showed social behavior impairment, altered vocalization, anxiety-like behavior, neuroanatomical abnormalities, and growth impairment. In the brain, Arid1b haploinsufficiency resulted in changes in the expression of SWI/SNF-regulated genes implicated in neuropsychiatric disorders. A focus on reversible mechanisms identified Insulin-like growth factor (IGF1) deficiency with inadequate compensation by Growth hormone-releasing hormone (GHRH) and Growth hormone (GH), underappreciated findings in ARID1B patients. Therapeutically, GH supplementation was able to correct growth retardation and muscle weakness. This model functionally validates the involvement of ARID1B in human disorders, and allows mechanistic dissection of neurodevelopmental diseases linked to chromatin-remodeling.

Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse.

Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight, and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously by several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP immunoreactivity was detected in several areas, including the prefrontal cortex, insular cortex, olfactory bulb, amygdala, and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei, particularly the arcuate nucleus, where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of colocalization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool for studying GHSR function, particularly within the brainstem and hippocampus; however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.

Role of calcium and EPAC in norepinephrine-induced ghrelin secretion.

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to ß1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells.

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the ß1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

Expression of Serum Retinol Binding Protein and Transthyretin within Mouse Gastric Ghrelin Cells.

Ghrelin is an orexigenic peptide hormone produced mainly by a distinct group of dispersed endocrine cells located within the gastric oxyntic mucosa. Besides secreted gene products derived from the preproghrelin gene, which include acyl-ghrelin, desacyl-ghrelin and obestatin, ghrelin cells also synthesize the secreted protein nesfatin-1. The main goal of the current study was to identify other proteins secreted from ghrelin cells. An initial gene chip screen using mRNAs derived from highly enriched pools of mouse gastric ghrelin cells demonstrated high levels of serum retinol-binding protein (RBP4) and transthyretin (TTR), both of which are known to circulate in the bloodstream bound to each other. This high expression was confirmed by quantitative RT-PCR using as template mRNA derived from the enriched gastric ghrelin cell pools and from two ghrelin-producing cell lines (SG-1 and PG-1). RBP4 protein also was shown to be secreted into the culture medium of ghrelin cell lines. Neither acute nor chronic caloric restriction had a significant effect on RBP4 mRNA levels within stomachs of C57BL/6J mice, although both manipulations significantly decreased stomach TTR mRNA levels. In vitro studies using PG-1 cells showed no effect on RBP4 release of octanoic acid, epinephrine or norepinephrine, all of which are known to act directly on ghrelin cells to stimulate ghrelin secretion. These data provide new insights into ghrelin cell physiology, and given the known functions of RBP4 and TTR, support an emerging role for the ghrelin cell in blood glucose handling and metabolism.