Neurexophilin 3 is highly localized in cortical and cerebellar regions and is functionally important for sensorimotor gating and motor coordination

Neurexophilin 3 (Nxph3) is a specific ligand of synaptic alpha-neurexins that are essential for efficient neurotransmitter release. Previous biochemical work demonstrated that Nxph3 interacts with an extracellular domain of alpha-neurexins in a tight complex; however, no information is available on the localization or functional role of Nxph3 in the brain. Here, we generated lacZ reporter gene knock-in mice to investigate the distribution of Nxph3 at the single-cell level and Nxph3 knockout mice to examine its functional importance. Nxph3 expression was restricted mostly to subplate-derived neurons in cortical layer 6b, granule cells in the vestibulocerebellum, and Cajal-Retzius cells during development. Colabeling experiments demonstrated that neurons expressing Nxph3 do not belong to a uniform cell type. Morphological analyses and systematic behavioral testing of knockout mice revealed no anatomical defects but uncovered remarkable functional abnormalities in sensory information processing and motor coordination, evident by increased startle response, reduced prepulse inhibition, and poor rotarod performance. Since Nxph3-deficient mice behaved normally while performing a number of other tasks, our data suggest an important role for Nxph3 as a locally and temporally regulated neuropeptide-like molecule, presumably acting in a complex with alpha-neurexins in select neuronal circuits.     

Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 microg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.     

Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis

In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1(-/-)) and heterozygous (Egr-1(+/-)) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1(+/-) cells (22.8%; p < 0.0001) when compared with Egr-1(-/-) cells (3.5%). Radiation elevated p53 protein in Egr-1(+/-) cells in 3-6 h. However, in Egr-1(-/-) cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1(+/-) cells but not in Egr-1(-/-) cells. Interestingly, transient overexpression of EGR-1 in p53(-/-) MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53(+/+) MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed in Egr-1(-/-) MEF cells before and after radiation when compared with the levels observed in Egr-1(+/-) cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1(-/-) cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1(-/-) MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53.     

Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)

The fathead minnow (Pimephales promelas) has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals (EDCs) on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows (n=70) were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean (+/-SE) spawning intervals for the fish in this study were 3.0 and 3.7+/-0.1 days, respectively. The mean number of eggs produced per spawn was 85+/-2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition (gonadosomatic index [GSI], histopathology) and plasma concentrations of vitellogenin and sex steroids (beta-estradiol, testosterone, 11-ketotestosterone). The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma beta-estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of beta-estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations.     

DNA mismatch repair MSH2 gene-based SNP associated with different populations

We screened for the major essential single-nucleotide polymorphism (SNP) variant that might be associated with the MSH2 gene based on the data available from three types of human tissue samples [156 lymphoblastoid cell variations (LCL), 160 epidermis, 166 fat]. An association analysis confirmed that the KCNK12 SNP variant (rs748780) was highly associated (p value 9 × 10^?4) with the MSH2 gene for all three samples. Using SNP identification, we further found that the recognized SNP was also relevant among Hapmap populations. Techniques that display specific SNPs associated with the gene of interest or nearby genes provide more reliable genetic associations than techniques that rely on data from individual SNPs. We investigated the MSH2 gene regional linkage association with the determined SNP (rs748780), KCNK12 variant (Allele T>C) in the intronic region, in HapMap3 full dataset populations, Yoruba in Ibadan, Nigeria (YRI), Utah residents with ancestry from northern Europe (CEU), Han Chinese in Beijing, China (CHB), and a population of Mexican ancestry in Los Angeles, California (MEX). A gene-based SNP association analysis analyzes the combined impact of every variant within the gene while creating referrals to linkage disequilibrium or connections between markers. Our results indicated that among the four populations studied, this association was highest in the MEX population based on the r ² value; a similar pattern was also observed in the other three populations. The relevant SNP rs748780 in KCNK12 is related to a superfamily of potassium channel pore-forming P-domain proteins as well as to other non-pore-forming proteins and has been shown to be relevant to neurological disorder predisposition in MEX as well as in other populations.     

Loss-of-function mutations in RAB18 cause Warburg micro syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.     

Rod Photoreceptors Express GPR55 in the Adult Vervet Monkey Retina

Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R) and cannabinoid CB2 receptor (CB2R). In recent years, the G-protein coupled receptor 55 (GPR55) was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its neural (cone photoreceptor, horizontal, bipolar, amacrine and ganglion cells) and CB2R in glial components (Müller cells). The aim of this study was to determine the expression pattern of GPR55 in the monkey retina by using confocal microscopy. Our results show that GPR55 is strictly localized in the photoreceptor layer of the extrafoveal portion of the retina. Co-immunolabeling of GPR55 with rhodopsin, the photosensitive pigment in rods, revealed a clear overlap of expression throughout the rod structure with most prominent staining in the inner segments. Additionally, double-label of GPR55 with calbindin, a specific marker for cone photoreceptors in the primate retina, allowed us to exclude expression of GPR55 in cones. The labeling of GPR55 in rods was further assessed with a 3D visualization in the XZ and YZ planes thus confirming its exclusive expression in rods. These results provide data on the distribution of GPR55 in the monkey retina, different than CB1R and CB2R. The presence of GPR55 in rods suggests a function of this receptor in scotopic vision that needs to be demonstrated.     

Cardiomyocyte Protection by GATA-4 Gene Engineered Mesenchymal Stem Cells Is Partially Mediated by Translocation of miR-221 in Microvesicles

Introduction

microRNAs (miRs), a novel class of small non-coding RNAs, are involved in cell proliferation, differentiation, development, and death. In this study, we found that miR-221 translocation by microvesicles (MVs) plays an important role in cardioprotection mediated by GATA-4 overexpressed mesenchymal stem cells (MSC).

Methods and Results

Adult rat bone marrow MSC and neonatal rat ventricle cardiomyocytes (CM) were harvested as primary cultures. MSC were transduced with GATA-4 (MSC^GATA-4) using the murine stem cell virus (pMSCV) retroviral expression system. Empty vector transfection was used as a control (MSC^Null). The expression of miRs was assessed by real-time PCR and localized using in situ hybridization (ISH). MVs collected from MSC cultures were characterized by expression of CD9, CD63, and HSP70, and photographed with electron microscopy. Cardioprotection during hypoxia afforded by conditioned medium (CdM) from MSC cultures was evaluated by lactate dehydrogenase (LDH) release, MTS uptake by CM, and caspase 3/7 activity. Expression of miR-221/222 was significantly higher in MSC than in CM and miR-221 was upregulated in MSC^GATA-4. MSC overexpression of miR-221 significantly enhanced cardioprotection by reducing the expression of p53 upregulated modulator of apoptosis (PUMA). Moreover, expression of PUMA was significantly decreased in CM co-cultured with MSC. MVs derived from MSC expressed high levels of miR-221, and were internalized quickly by CM as documented in images obtained from a Time-Lapse Imaging System.

Conclusions

Our results demonstrate that cardioprotection by MSC^GATA-4 may be regulated in part by a transfer of anti-apoptotic miRs contained within MVs.