Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Transcriptome alterations of prefrontal cortical parvalbumin neurons in schizophrenia.

Schizophrenia (SZ) is associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC). This dysfunction is manifest as cognitive deficits that appear to arise from disturbances in gamma frequency oscillations. These oscillations are generated in DLPFC layer 3 (L3) via reciprocal connections between pyramidal cells (PCs) and parvalbumin (PV)-containing interneurons. The density of cortical PV neurons is not altered in SZ, but expression levels of several transcripts involved in PV cell function, including PV, are lower in the disease. However, the transcriptome of PV cells has not been comprehensively assessed in a large cohort of subjects with SZ. In this study, we combined an immunohistochemical approach, laser microdissection, and microarray profiling to analyze the transcriptome of DLPFC L3 PV cells in 36 matched pairs of SZ and unaffected comparison subjects. Over 800 transcripts in PV neurons were identified as differentially expressed in SZ subjects; most of these alterations have not previously been reported. The altered transcripts were enriched for pathways involved in mitochondrial function and tight junction signaling. Comparison with the transcriptome of L3 PCs from the same subjects revealed both shared and distinct disease-related effects on gene expression between cell types. Furthermore, network structures of gene pathways differed across cell types and subject groups. These findings provide new insights into cell type-specific molecular alterations in SZ which may point toward novel strategies for identifying therapeutic targets.

A New Provocative Test for Glaucoma.

PURPOSE: To compare the effects of the water-drinking test (WDT) with the 30° inverted body position test on intraocular pressure (IOP) in normal patients, suspected glaucoma patients and glaucoma patients. MATERIALS AND METHODS: Based on clinical evaluation of the optic disk, IOP, and standard achromatic perimetry (SAP) of 71 eyes, 18 were "normal" (normal SAP and optic disk evaluation, and IOP < 21 mm Hg), 30 were "glaucoma suspect" (GS; normal SAP, cup/disk (C/D) ratio > 0.5 or asymmetry > 0.2 and/or ocular hypertension), and 31 had "early glaucoma" (MD < -6 dB, glaucomatous optic neuropathy). Standard achromatic perimetry was performed with the Octopus 3.1.1 Dynamic 24-2 program. Patients fasted before the WDT, and four measurements were performed at basal, 15', 30, and 45' after drinking 1 liter of water (WDT) in 5 minutes. In the 30° inverted position, IOP measurement with Perkins applanation tonometer was taken after 5 minutes lying down. RESULTS: There was a statistical difference in all groups between the basal IOP and peak IOP during the WDT (p < 0.001) and in the inverted position IOP (p < 0.001). Controls (p = 0.50), suspects (p = 0.41) and glaucoma patients (p = 1.0) did not exhibit a difference between WDT-IOP and inverted position IOP. CONCLUSION: The 30° inverted position test was as efficient as WDT in detecting peak IOP. This new provocative test is easier, faster and more comfortable for both patients and doctors. How to cite this article: Kanadani FN, Moreira TCA, Campos LF, Vianello MP, Corradi J, Dorairaj SK, Freitas ALA, Ritch R. A New Provocative Test for Glaucoma. J Curr Glaucoma Pract 2016;10(1): 1-3.

Diurnal Curve of the Ocular Perfusion Pressure.

PURPOSE: To describe the diurnal variation of the ocular perfusion pressure (OPP) in normal, suspects and glaucoma patients. MATERIALS AND METHODS: Seventy-nine subjects were enrolled in a prospective study. The diurnal curve of intraocular pressure (IOP) was performed and blood pressure measurements were obtained. Each participant was grouped into one of the following based upon the clinical evaluation of the optic disk, IOP and standard achromatic perimetry (SAP): 18 eyes were classified as normal (normal SAP, normal optic disk evaluation and IOP < 21 mm Hg in two different measurements), 30 eyes as glaucoma suspect (GS) (normal SAP and mean deviation (MD), C/D ration > 0.5 or asymmetry > 0.2 and/or ocular hypertension), 31 eyes as early glaucoma (MD < -6 dB, glaucomatous optic neuropathy and SAP and MDs on SAP. Standard achromatic perimetry was performed with the Octopus 3.1.1 Dynamic 24-2 program. Intraocular pressure and blood pressure measurements were taken at 6 am, 9 am, 12, 3 and 6 pm. The patients stayed in the seated position for 5 minutes prior to blood pressure measurements. RESULTS: The mean IOP values in all groups did not follow any regular pattern. The peak IOP was found to be greater in suspect [18.70 ± 3.31 (mm Hg ± SD)] and glaucoma (18.77 ± 4.30 mm Hg) patients as compared to normal subjects (16.11 ± 2.27 mm Hg). In studying the diurnal variation of the OPP, we found lower values at 3 pm in normals (34.21 ± 2.07 mm Hg), at 9 am in suspects (54.35 ± 3.32 mm Hg) and at 12 pm in glaucoma patients (34.84 ± 1.44 mm Hg). CONCLUSION: Each group has a specific OPP variation during the day with the most homogeneous group being the suspect one. It is important to keep studying the IOP and OPP variation for increased comprehension of the pathophysiology of glaucomatous optic neuropathy. How to cite this article: Kanadani FN, Moreira TCA, Bezerra BSP, Vianello MP, Corradi J, Dorairaj SK, Prata TS. Diurnal Curve of the Ocular Perfusion Pressure. J Curr Glaucoma Pract 2016;10(1):4-6.

Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder.

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin-proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were <10(-7) and <10(-5), respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P<10(-6)) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular-cellular basis of differences in clinical phenotypes.

Alternative transcripts and evidence of imprinting of GNAL on 18p11.2.

Genetic studies implicating the region of human chromosome 18p11.2 in susceptibility to bipolar disorder and schizophrenia have observed parent-of-origin effects that may be explained by genomic imprinting. We have identified a transcriptional variant of the GNAL gene in this region, employing an alternative first exon that is 5' to the originally identified start site. This alternative GNAL transcript encodes a longer functional variant of the stimulatory G-protein alpha subunit, Golf. The isoforms of Golf display different expression patterns in the CNS and functionally couple to the dopamine D1 receptor when heterologously expressed in Sf9 cells. In addition, there are CpG islands in the vicinity of both first exons that are differentially methylated, a hallmark of genomic imprinting. These results suggest that GNAL, and possibly other genes in the region, is subject to epigenetic regulation and strengthen the case for a susceptibility gene in this region.

The Mouse Gene Expression Database (GXD).

The Gene Expression Database (GXD) is a community resource of gene expression information for the laboratory mouse. By combining the different types of expression data, GXD aims to provide increasingly complete information about the expression profiles of genes in different mouse strains and mutants, thus enabling valuable insights into the molecular networks that underlie normal development and disease. GXD is integrated with the Mouse Genome Database (MGD). Extensive interconnections with sequence databases and with databases from other species, and the development and use of shared controlled vocabularies extend GXD's utility for the analysis of gene expression information. GXD is accessible through the Mouse Genome Informatics web site at http://www.informatics.jax.org/ or directly at http://www.informatics.jax.org/menus/expression_menu. shtml.

The cytoplasmic Purkinje onconeural antigen cdr2 down-regulates c-Myc function: implications for neuronal and tumor cell survival.

Paraneoplastic cerebellar degeneration (PCD) is a disorder in which breast or ovarian tumors express an onconeural antigen termed cdr2, which normally is expressed in cerebellar Purkinje neurons. This leads to an immune response to cdr2 that is associated with tumor immunity and autoimmune cerebellar degeneration. We have found that cdr2, a cytoplasmic protein harboring a helix-leucine zipper (HLZ) motif, interacts specifically with the HLZ motif of c-Myc. Both proteins colocalize in the cytoplasm of adult cerebellar Purkinje neurons, and coimmunoprecipitate from tumor cell lines and cerebellar extracts. cdr2 down-regulates c-Myc-dependent transcription in cotransfection assays, and redistributes Myc protein in the cytoplasm. Disease antisera from six of six PCD patients specifically blocked the interaction between cdr2 and c-Myc in vitro. These data indicate that cdr2 normally sequesters c-Myc in the neuronal cytoplasm, thereby down-regulating c-Myc activity, and suggest a mechanism whereby inhibition of cdr2 function by autoantibodies in PCD may contribute to Purkinje neuronal death.

A post-transcriptional regulatory mechanism restricts expression of the paraneoplastic cerebellar degeneration antigen cdr2 to immune privileged tissues.

Paraneoplastic cerebellar degeneration (PCD) is believed to be an autoimmune disorder initiated by the ectopic expression of a neuron-specific protein in breast and ovarian tumors. PCD antisera was used previously to identify several cerebellar degeneration-related (cdr) genes encoding putative PCD antigens. We have found that the cdr2 gene, which encodes a cytoplasmic leucine zipper protein of unknown function, is expressed in PCD-associated tumors, whereas other cdr genes are not; thus, cdr2 encodes the PCD tumor antigen. To determine whether the expression pattern of cdr2 is consistent with its proposed role in PCD, we have isolated the mouse homolog and examined both the mRNA and protein distribution in adult tissues. We have found that cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis. Within the brain, both the cdr2 mRNA and immunoreactivity are confined primarily to neurons in the cerebellum and brainstem, the regions most affected in PCD. These results suggest first that the tissue-specific expression of cdr2 is regulated at a post-transcriptional level. Moreover, because the brain and testis are considered to be immune-privileged sites, the expression pattern of cdr2 is compatible with the autoimmune model of PCD pathogenesis.