No abstract

Activation of brain melanocortin 4 receptors (MC4Rs) leads to reduced food intake, increased energy expenditure, increased insulin sensitivity, and reduced linear growth. MC4R effects on energy expenditure and glucose metabolism are primarily mediated by the G protein G(s)α in brain regions outside of the paraventricular nucleus of the hypothalamus (PVN). However, the G protein(s) that is involved in MC4R-mediated suppression of food intake and linear growth, which are believed to be regulated primarily though action in the PVN, is unknown. Here, we show that PVN-specific loss of G(q)α and G11α, which stimulate PLC, leads to severe hyperphagic obesity, increased linear growth, and inactivation of the hypothalamic-pituitary-adrenal axis, without affecting energy expenditure or glucose metabolism. Moreover, we demonstrate that the ability of an MC4R agonist delivered to PVN to inhibit food intake is lost in mice lacking G(q/11)α in the PVN but not in animals deficient for G(s)α. The blood pressure response to the same MC4R agonist was only lost in animals lacking G(s)α specifically in the PVN. Together, our results exemplify how different physiological effects of GPCRs may be mediated by different G proteins and identify a pathway for appetite regulation that could be selectively targeted by G(q/11)α-biased MC4R agonists as a potential treatment for obesity.

CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

Among Escherichia coli strains isolated from stool specimens from patients with acute diarrhea, 1.4% were found to harbor cdtB by use of enrichment cytolethal distending toxin (CDT) PCR. These isolates were identified as being enteropathogenic E. coli (EPEC). In a retrospective study using a probe hybridization assay, 6 of 138 EPEC strains were found to harbor the cdtB locus. cdtB-positive isolates mostly belong to the O86a and O127a serogroups, with the former being associated with higher expression of CDT. Pulsed-field gel electrophoresis profiles showed that the EPEC strains harboring cdtB strains are genetically diverse.Cytolethal distending toxin (CDT) is a novel class of bacterial genotoxin that induces characteristic elongation of eukaryotic cells followed by progressive cellular distention and death (12,14,23). CDT is considered to be an important factor in intestinal pathogenesis (3), as this toxin is able to induce tissue damage and fluid accumulation in the descending colon of orally infected suckling mice (21). Three genes, cdtA, cdtB, and cdtC, arranged in an apparent operon are required for the production of active CDT (25). The deduced amino acid sequences of these genes from Escherichia coli strains E6468-62 (serogroup O86) and 9142-88 (serogroup O128) are 38, 56, and 37% homologous, respectively (24, 25), and the corresponding toxins are called . The amino acid sequence of Cdt-III from strain S5 (serogroup O15) has Ͼ90% homology to Cdt-II and 55 to 69% homology to . The presence of cdt in different bacterial species (8,20,24,28) and the results of analysis of its flanking regions suggest that this gene has been acquired from heterologous species by horizontal gene transfer (7,18,22) or through a phage (13). Even though the data on the structural and functional aspects of CDT are expanding, knowledge of the epidemiological association of E. coli harboring cdt remains scanty (1,15,17,19).To investigate the incidence of cdt-harboring E. coli, a total of 284 stool specimens collected from acute-diarrhea patients of all age groups admitted to the Infectious Diseases Hospital and B. C. Roy Memorial Hospital for Children (Calcutta, India) from May to July 2002 were examined. Relevant clinical information such as presence of fever, vomiting, dehydration status, and type and duration of diarrhea was recorded for each patient. For enrichment CDT PCR, overnight stool cultures in Luria-Bertani broth (Difco, Detroit, Mich.) were directly tested for the presence of the cdtB gene in a standard PCR assay. The primer pair used in this study was based on the cdt nucleotide sequence of E. coli (25) and had the sequences 5Ј-GATTTTGCCGGGTATTTCT-3Ј and 5Ј-CCCTCAACAG AGGAAGAA-3Ј. These primers are specific for Cdt-I. After a hot start at 94°C for 5 min, the DNA was subjected to 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min 30 s. The expected size of the PCR amplicon was 707 bp. The sensitivity of the CDT PCR assay was 10 3 CFU. For confirmation, a PCR amplicon...

Gβ5 is a divergent member of the signal-transducing G protein β subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gβ isoforms, Gβ5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gβ5 is an essential stabilizer of such RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gβ5 in the brain outside the visual system. We show here that mice lacking Gβ5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gβ5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gβ5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression.

Mitochondrial complex‐I dysfunction has been observed in patients of Huntington’s disease (HD). We assessed whether such a defect is present in the 3‐nitropropionic acid (3‐NP) model of HD. Rats treated with 3‐NP (10–20 mg/kg i.p., for 4 days) exhibited weight loss, gait abnormalities, and striatal lesions with increased glial fibrillary acidic protein immunostaining on fifth and ninth days, while increase in striatal dopamine and loss of tyrosine hydroxylase immunoreactivity were observed on fifth day following treatment. We report for the first time a dose‐dependent reduction in complex‐I activity in the cerebral cortex when analyzed spectrophotometrically and by blue native‐polyacrylamide gel electrophoresis following 3‐NP treatment. The citrate synthase normalized activities of mitochondrial complex‐I, ‐II, ‐(I + III) and ‐IV were decreased in the cortex of 3‐NP treated rats. In addition, succinate driven State 3 respiration was also significantly inhibited in vivo and in the isolated mitochondria. These findings taken together with the observation of a significant decrease in vivo but not in vitro of State 3 respiration with NAD+‐linked substrates, suggest complex‐I dysfunction in addition to irreversible inhibition of complex‐II and succinate dehydrogenase activity as a contributing factor in 3‐NP‐induced cortico‐striatal lesion.

Thyroid hormone serves many functions throughout brain development, but the mechanisms that control the timing of its actions in specific brain regions are poorly understood. In the cerebellum, thyroid hormone controls formation of the transient external germinal layer, which contains proliferative granule cell precursors, subsequent granule cell migration, and cerebellar foliation. We report that the thyroid hormone-inactivating type 3 deiodinase (encoded by Dio3) is expressed in the mouse cerebellum at embryonic and neonatal stages, suggesting a need to protect cerebellar tissues from premature stimulation by thyroid hormone. Dio3(-/-) mice displayed reduced foliation, accelerated disappearance of the external germinal layer, and premature expansion of the molecular layer at juvenile ages. Furthermore, Dio3(-/-) mice exhibited locomotor behavioral abnormalities and impaired ability in descending a vertical pole. To ascertain that these phenotypes resulted from inappropriate exposure to thyroid hormone, thyroid hormone receptor α1 (TRα1) was removed from Dio3(-/-) mice, which substantially corrected the cerebellar and behavioral phenotypes. Deletion of TRα1 did not correct the previously reported small thyroid gland or deafness in Dio3(-/-) mice, indicating that Dio3 controls the activation of specific receptor isoforms in different tissues. These findings suggest that type 3 deiodinase constrains the timing of thyroid hormone action during cerebellar development.

Huntington's disease (HD) is caused due to an abnormal expansion of polyglutamine repeats in the first exon of huntingtin gene. The mutation in huntingtin causes abnormalities in the functioning of protein, leading to deleterious effects ultimately to the demise of specific neuronal cells.The disease is inherited in an autosomal dominant manner and leads to a plethora of neuropsychiatric behaviour and neuronal cell death mainly in striatal and cortical regions of the brain, eventually leading to death of the individual. The discovery of the mutant gene led to a surge in molecular diagnostics of the disease and in making different transgenic models in different organisms to understand the function of wild-type and mutant proteins. Despite difficult challenges, there has been a significant increase in understanding the functioning of the protein in normal and other gain-of-function interactions in mutant form. However, there have been no significant improvements in treatments of the patients suffering from this ailment and most of the treatment is still symptomatic. HD warrants more attention towards better understanding and treatment as more advancement in molecular diagnostics and therapeutic interventions are available. Several different transgenic models are available in different organisms, ranging from fruit flies to primate monkeys, for studies on understanding the pathogenicity of the mutant gene. It is the right time to assess the advancement in the field and try new strategies for neuroprotection using key pathways as target. The present review highlights the key ingredients of pathology in the HD and discusses important studies for drug trials and future goals for therapeutic interventions.