Electrophysiological evaluation of extracellular spermine and alkaline pH on synaptic human GABAA receptors.

Polyamines have fundamental roles in brain homeostasis as key modulators of cellular excitability. Several studies have suggested alterations in polyamine metabolism in stress related disorders, suicide, depression, and neurodegeneration, making the pharmacological modulation of polyamines a highly appealing therapeutic strategy. Polyamines are small aliphatic molecules that can modulate cationic channels involved in neuronal excitability. Previous indirect evidence has suggested that polyamines can modulate anionic GABAA receptors (GABAARs), which mediate inhibitory signaling and provide a direct route to reduce hyperexcitability. Here, we attempted to characterize the effect that spermine, the polyamine with the strongest reported effect on GABAARs, has on human postmortem native GABAARs. We microtransplanted human synaptic membranes from the dorsolateral prefrontal cortex of four cases with no history of mental or neurological disorders, and directly recorded spermine effects on ionic GABAARs responses on microtransplanted oocytes. We show that in human synapses, inhibition of GABAARs by spermine was better explained by alkalization of the extracellular solution. Additionally, spermine had no effect on the potentiation of GABA-currents by diazepam, indicating that even if diazepam binding is enhanced by spermine, it does not translate to changes in functional activity. Our results clearly demonstrate that while extracellular spermine does not have direct effects on human native synaptic GABAARs, spermine-mediated shifts of pH inhibit GABAARs. Potential spermine-mediated increase of pH in synapses in vivo may therefore participate in increased neuronal activity observed during physiological and pathological states, and during metabolic alterations that increase the release of spermine to the extracellular milieu.

Systematic reanalysis of genomic data improves quality of variant interpretation.

As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize the benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants that were not initially reported in 23 individuals, 6 described here, comprising a 16% increase in P/LP yield. We also downgraded 3 LP and 6 variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after 3 years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.

Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations.

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

Variable telomere length across post-mortem human brain regions and specific reduction in the hippocampus of major depressive disorder.

Stress can be a predisposing factor to psychiatric disorders and has been associated with decreased neurogenesis and reduced hippocampal volume especially in depression. Similarly, in white blood cells chronic psychological stress has been associated with telomere shortening and with mood disorders and schizophrenia (SZ). However, in previous post-mortem brain studies from occipital cortex and cerebellum, no difference in telomere length was observed in depression. We hypothesized that in psychiatric disorders, stress-driven accelerated cellular aging can be observed in brain regions particularly sensitive to stress. Telomere length was measured by quantitative-PCR in five brain regions (dorsolateral prefrontal cortex, hippocampus (HIPP), amygdala, nucleus accumbens and substantia nigra (SN)) in major depressive disorder (MDD), bipolar disorder, SZ and normal control subjects (N = 40, 10 subjects per group). We observed significant differences in telomere length across brain regions suggesting variable levels of cell aging, with SN and HIPP having the longest telomeres and the dorsolateral prefrontal cortex the shortest. A significant decrease (P < 0.02) in telomere length was observed specifically in the HIPP of MDD subjects even after controlling for age. In the HIPP of MDD subjects, several genes involved in neuroprotection and in stress response (FKBP5, CRH) showed altered levels of mRNA. Our results suggest the presence of hippocampal stress-mediated accelerated cellular aging in depression. Further studies are needed to investigate the cellular specificity of these findings.

Circadian dysregulation of clock genes: clues to rapid treatments in major depressive disorder.

Conventional antidepressants require 2-8 weeks for a full clinical response. In contrast, two rapidly acting antidepressant interventions, low-dose ketamine and sleep deprivation (SD) therapy, act within hours to robustly decrease depressive symptoms in a subgroup of major depressive disorder (MDD) patients. Evidence that MDD may be a circadian-related illness is based, in part, on a large set of clinical data showing that diurnal rhythmicity (sleep, temperature, mood and hormone secretion) is altered during depressive episodes. In a microarray study, we observed widespread changes in cyclic gene expression in six regions of postmortem brain tissue of depressed patients matched with controls for time-of-death (TOD). We screened 12 000 transcripts and observed that the core clock genes, essential for controlling virtually all rhythms in the body, showed robust 24-h sinusoidal expression patterns in six brain regions in control subjects. In MDD patients matched for TOD with controls, the expression patterns of the clock genes in brain were significantly dysregulated. Some of the most robust changes were seen in anterior cingulate (ACC). These findings suggest that in addition to structural abnormalities, lesion studies, and the large body of functional brain imaging studies reporting increased activation in the ACC of depressed patients who respond to a wide range of therapies, there may be a circadian dysregulation in clock gene expression in a subgroup of MDDs. Here, we review human, animal and neuronal cell culture data suggesting that both low-dose ketamine and SD can modulate circadian rhythms. We hypothesize that the rapid antidepressant actions of ketamine and SD may act, in part, to reset abnormal clock genes in MDD to restore and stabilize circadian rhythmicity. Conversely, clinical relapse may reflect a desynchronization of the clock, indicative of a reactivation of abnormal clock gene function. Future work could involve identifying specific small molecules capable of resetting and stabilizing clock genes to evaluate if they can rapidly relieve symptoms and sustain improvement.

Preclinical efficacy of the oncolytic measles virus expressing the sodium iodide symporter in iodine non-avid anaplastic thyroid cancer: a novel therapeutic agent allowing noninvasive imaging and radioiodine therapy.

Anaplastic thyroid cancer is an extremely aggressive disease resistant to radioiodine treatment because of loss of sodium iodide symporter (NIS) expression. To enhance prognosis of this fatal cancer, we validated the preclinical efficacy of measles virus (MV)-NIS, the vaccine strain of the oncolytic MV (MV-Edm), modified to include the NIS gene. Western blotting analysis confirmed that a panel of eight anaplastic thyroid cancer (ATC)-derived cell lines do not express NIS protein, but do express CD46, the MV receptor. In vitro cell death assays and in vivo xenograft studies demonstrate the oncolytic efficacy of MV-NIS in BHT-101 and KTC-3, ATC-derived cell lines. Radioactive iodine uptake along with single-photon emission computed tomography (SPECT)-computed tomography imaging of KTC-3 xenografts after (99)Tc(m) administration confirmed NIS expression in vitro and in vivo, respectively, after virus treatment. Adjuvant administration of RAI, to MV-NIS-treated KTC-3 tumors showed a trend for increased tumor cell killing. As current treatment for ATC is only palliative, and MV-NIS is currently Food and Drug Administration approved for human clinical trials in myeloma, our data indicate that targeting ATC with MV-NIS could prove to be a novel therapeutic strategy for effective treatment of iodine-resistant ATC and will expedite its testing in clinical trials for this aggressive disease.

Cancer cell proliferation is inhibited by specific modulation frequencies.

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology.

Association of DRD2 and DRD3 polymorphisms with Parkinson's disease in a multiethnic consortium.

OBJECTIVE: To examine genetic associations of polymorphisms in the dopamine receptor D2 (DRD2) and D3 (DRD3) genes with risk of Parkinson's disease (PD). METHODS: The study included 1325 newly diagnosed patients with PD and 1735 controls from a consortium of five North American case-control studies. We collected risk factor information by in-person or telephone interview. Six DRD2 and two DRD3 polymorphisms were genotyped using a common laboratory. Odds ratios were estimated using logistic regression. RESULTS: Among non-Hispanic whites, homozygous carriers of Taq1A DRD2 (rs1800497) polymorphism had an increased risk of PD compared to homozygous wildtype carriers (OR=1.5, 95% CI 1.0-2.3). In contrast, the direction of association for Taq1A polymorphism was opposite for African-Americans, showing an inverse association with PD risk (OR=0.10, 95% CI 0.2-0.7). Among white Hispanics who carried two alleles, the Ser9Gly DRD3 (rs6280) polymorphism was associated with a decreased risk of PD (OR=0.4, 95% CI 0.2-0.8). The inverse association of smoking with PD risk was not modified by any of the DRD2 or DRD3 polymorphisms. CONCLUSIONS: DRD2 polymorphisms are unlikely to be true disease-causing variants; however, three DRD2 polymorphisms (including Taq1A) may be in linkage disequilibrium with possible disease associated variants in the DRD2-ANKK1-NCAM1-TTC12 gene cluster.

A novel g.-1258G>A mutation in a conserved putative regulatory element of PAX9 is associated with autosomal dominant molar hypodontia.

Mutations in the transcription factor PAX9 which plays a critical role in the switching of odontogenic potential from the epithelium to the mesenchyme during tooth development cause autosomal dominant non-syndromic hypodontia primarily affecting molars. Linkage analysis on a family segregating autosomal dominant molar hypodontia with markers flanking and within PAX9 yielded a maximum multipoint LOD score of 3.6. No sequence variants were detected in the coding or 5'- and 3'-untranslated regions (UTRs) of PAX9. However, we identified a novel g.-1258G>A sequence variant in all affected individuals of the family but not in the unaffected family members or in 3088 control chromosomes. This mutation is within a putative 5'-regulatory sequence upstream of PAX9 highly conserved in primates, somewhat conserved in ungulates and carnivores but not conserved in rodents. Bioinformatics analysis of the sequence determined that there was no abolition or creation of a putative binding site for known transcription factors. Based on our previous findings that haploinsufficiency for PAX9 leads to hypodontia, we postulate that the g.-1258G>A variant reduces the expression of PAX9 which underlies the hypodontia phenotype in this family.

The genetic basis of divergent pigment patterns in juvenile threespine sticklebacks.

Animal pigment patterns are important for a range of functions, including camouflage and communication. Repeating pigment patterns, such as stripes, bars and spots have been of particular interest to developmental and theoretical biologists, but the genetic basis of natural variation in such patterns is largely unexplored. In this study, we identify a difference in a periodic pigment pattern among juvenile threespine sticklebacks (Gasterosteus aculeatus) from different environments. Freshwater sticklebacks exhibit prominent vertical bars that visually break up the body shape, but sticklebacks from marine populations do not. We hypothesize that these distinct pigment patterns are tuned to provide crypsis in different habitats. This phenotypic difference is widespread and appears in most of the freshwater populations that we sampled. We used quantitative trait locus (QTL) mapping in freshwater-marine F2 hybrids to elucidate the genetic architecture underlying divergence in this pigmentation pattern. We identified two QTL that were significantly associated with variation in barring. Interestingly, these QTL were associated with two distinct aspects of the pigment pattern: melanophore number and overall pigment level. We compared the QTL locations with positions of known pigment candidate genes in the stickleback genome. We also identified two major QTL for juvenile body size, providing new insights into the genetic basis of juvenile growth rates in natural populations. In summary, although there is a growing literature describing simple genetic bases for adaptive coloration differences, this study emphasizes that pigment patterns can also possess a more complex genetic architecture.

Coffee, ADORA2A, and CYP1A2: the caffeine connection in Parkinson's disease.

BACKGROUND AND PURPOSE: In 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine animal models of Parkinson's disease (PD), caffeine protects neurons by blocking the adenosine receptor A2A (ADORA2A). Caffeine is primarily metabolized by cytochrome P450 1A2 (CYP1A2). Our objective was to examine whether ADORA2A and CYP1A2 polymorphisms are associated with PD risk or modify the caffeine-PD association. METHODS: Parkinson's Epidemiology and Genetic Associations Studies in the United States (PEGASUS) included five population-based case-control studies. One laboratory genotyped four ADORA2A and three CYP1A2 polymorphisms in 1325 PD cases and 1735 age- and sex-matched controls. Information regarding caffeine (coffee) consumption and other lifestyle factors came from structured in-person or telephone interviews. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. RESULTS: Two ADORA2A polymorphisms were inversely associated with PD risk - rs71651683, a 5' variant (adjusted allelic OR = 0.51, 95% CI 0.33-0.80, permutation-adjusted P = 0.015) and rs5996696, a promoter region variant (adjusted OR for AC and CC genotypes compared with the AA wild-type genotype were 0.76 (95% CI 0.57-1.02) and 0.37 (95% CI 0.13-1.01), respectively (permutation-adjusted P for trend = 0.04). CYP1A2 polymorphisms were not associated with PD risk; however, the coffee-PD association was strongest among subjects homozygous for either variant allele rs762551 (P(interaction) = 0.05) or rs2470890 (P(interaction) = 0.04). CONCLUSION: In this consortium study, two ADORA2A polymorphisms were inversely associated with PD risk, but there was weak evidence of interaction with coffee consumption. In contrast, the coffee-PD association was strongest among slow metabolizers of caffeine who were homozygous carriers of the CYP1A2 polymorphisms.

Altered expression of glutamate signaling, growth factor, and glia genes in the locus coeruleus of patients with major depression.

Several studies have proposed that brain glutamate signaling abnormalities and glial pathology have a role in the etiology of major depressive disorder (MDD). These conclusions were primarily drawn from post-mortem studies in which forebrain brain regions were examined. The locus coeruleus (LC) is the primary source of extensive noradrenergic innervation of the forebrain and as such exerts a powerful regulatory role over cognitive and affective functions, which are dysregulated in MDD. Furthermore, altered noradrenergic neurotransmission is associated with depressive symptoms and is thought to have a role in the pathophysiology of MDD. In the present study we used laser-capture microdissection (LCM) to selectively harvest LC tissue from post-mortem brains of MDD patients, patients with bipolar disorder (BPD) and from psychiatrically normal subjects. Using microarray technology we examined global patterns of gene expression. Differential mRNA expression of select candidate genes was then interrogated using quantitative real-time PCR (qPCR) and in situ hybridization (ISH). Our findings reveal multiple signaling pathway alterations in the LC of MDD but not BPD subjects. These include glutamate signaling genes, SLC1A2, SLC1A3 and GLUL, growth factor genes FGFR3 and TrkB, and several genes exclusively expressed in astroglia. Our data extend previous findings of altered glutamate, astroglial and growth factor functions in MDD for the first time to the brainstem. These findings indicate that such alterations: (1) are unique to MDD and distinguishable from BPD, and (2) affect multiple brain regions, suggesting a whole-brain dysregulation of such functions.

The fibroblast growth factor family and mood disorders.

While there has been a great deal of interest in the role of brain-derived neurotrophic factor (BDNF) in mood disorders and/or the mode of action of antidepressants, less is known about the role of other growth factors. This paper is focused on a group of growth factors, the fibroblast growth factor (FGF) family and their potential role in mood disorders.

Preclinical pharmacology and toxicology of intravenous MV-NIS, an oncolytic measles virus administered with or without cyclophosphamide.

MV-NIS is an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS). Here, we report the results of preclinical pharmacology and toxicology studies conducted in support of our clinical protocol "Phase I Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or Refractory Multiple Myeloma." Dose-response studies in the KAS-6/1 myeloma xenograft model demonstrated a minimum effective dose of 4 x 10(6) TCID50 (tissue culture infectious dose 50)/kg. Toxicity studies in measles-naive squirrel monkeys and measles-susceptible transgenic mice were negative at intravenous doses up to 10(8) and 4 x 10(8) TCID50/kg, respectively. Abundant viral mRNA, maximal on day 8, was detected in cheek swabs of squirrel monkeys, more so after pretreatment with cyclophosphamide. On the basis of these data, the safe starting dose of MV-NIS for our clinical protocol was set at 1-2 x 10(4) TCID50/kg (10(6) TCID50 per patient).

Stress-induced changes in primate prefrontal profiles of gene expression.

Stressful experiences that consistently increase cortisol levels appear to alter the expression of hundreds of genes in prefrontal limbic brain regions. Here, we investigate this hypothesis in monkeys exposed to intermittent social stress-induced episodes of hypercortisolism or a no-stress control condition. Prefrontal profiles of gene expression compiled from Affymetrix microarray data for monkeys randomized to the no-stress condition were consistent with microarray results published for healthy humans. In monkeys exposed to intermittent social stress, more genes than expected by chance appeared to be differentially expressed in ventromedial prefrontal cortex compared to monkeys not exposed to adult social stress. Most of these stress responsive candidate genes were modestly downregulated, including ubiquitin conjugation enzymes and ligases involved in synaptic plasticity, cell cycle progression and nuclear receptor signaling. Social stress did not affect gene expression beyond that expected by chance in dorsolateral prefrontal cortex or prefrontal white matter. Thirty four of 48 comparisons chosen for verification by quantitative real-time polymerase chain reaction (qPCR) were consistent with the microarray-predicted result. Furthermore, qPCR and microarray data were highly correlated. These results provide new insights on the regulation of gene expression in a prefrontal corticolimbic region involved in the pathophysiology of stress and major depression. Comparisons between these data from monkeys and those for ventromedial prefrontal cortex in humans with a history of major depression may help to distinguish the molecular signature of stress from other confounding factors in human postmortem brain research.

Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression.

Abnormalities in L-glutamic acid (glutamate) and GABA signal transmission have been postulated to play a role in depression, but little is known about the underlying molecular determinants and neural mechanisms. Microarray analysis of specific areas of cerebral cortex from individuals who had suffered from major depressive disorder demonstrated significant down-regulation of SLC1A2 and SLC1A3, two key members of the glutamate/neutral amino acid transporter protein family, SLC1. Similarly, expression of L-glutamate-ammonia ligase, the enzyme that converts glutamate to nontoxic glutamine was significantly decreased. Together, these changes could elevate levels of extracellular glutamate considerably, which is potentially neurotoxic and can affect the efficiency of glutamate signaling. The astroglial distribution of the two glutamate transporters and L-glutamate-ammonia ligase strongly links glia to the pathophysiology of depression and challenges the conventional notion that depression is solely a neuronal disorder. The same cortical areas displayed concomitant up-regulation of several glutamate and GABA(A) receptor subunits, of which GABA(A)alpha1 and GABA(A)beta3 showed selectivity for individuals who had died by suicide, indicating their potential utility as biomarkers of suicidality. These findings point to previously undiscovered molecular underpinnings of the pathophysiology of major depression and offer potentially new pharmacological targets for treating depression.

Dysregulation of the fibroblast growth factor system in major depression.

In this report we describe findings that imply dysregulation of several fibroblast growth factor (FGF) system transcripts in frontal cortical regions of brains from human subjects with major depressive disorder (MDD). This altered gene expression was discovered by microarray analysis of frontal cortical tissue from MDD, bipolar, and nonpsychiatric control subjects and was verified by quantitative real-time PCR analysis and, importantly, in a separate cohort of MDD subjects. Furthermore, we show, through a separate analysis of specific serotonin reuptake inhibitor (SSRI)-treated and non-SSRI-treated MDD subjects that the observed changes in expression of FGF transcripts are not secondary to drug treatment. Rather, changes in specific FGF transcripts are attenuated by SSRIs and may thus be partially responsible for the mechanism of action of these drugs. We also make available the gene-expression profile of all of the other growth factors and growth factor receptors detected in these postmortem samples.

DNA microarray analysis of functionally discrete human brain regions reveals divergent transcriptional profiles.

Transcriptional profiles within discrete human brain regions are likely to reflect structural and functional specialization. Using DNA microarray technology, this study investigates differences in transcriptional profiles of highly divergent brain regions (the cerebellar cortex and the cerebral cortex) as well as differences between two closely related brain structures (the anterior cingulate cortex and the dorsolateral prefrontal cortex). Replication of this study across three independent laboratories, to address false-positive and false-negative results using microarray technology, is also discussed. We find greater than a thousand transcripts to be differentially expressed between cerebellum and cerebral cortex and very few transcripts to be differentially expressed between the two neocortical regions. We further characterized transcripts that were found to be specifically expressed within brain regions being compared and found that ontological classes representing signal transduction machinery, neurogenesis, synaptic transmission, and transcription factors were most highly represented.