Paresh D. Patel

Variant Brain-Derived Neurotrophic Factor (BDNF) (Met66) Alters the Intracellular Trafficking and Activity-Dependent Secretion of Wild-Type BDNF in Neurosecretory Cells and Cortical Neurons

Brain-derived neurotrophic factor (BDNF) plays a critical role in nervous system and cardiovascular development and function. Recently, a common single nucleotide polymorphism in the bdnf gene, resulting in a valine to methionine substitution in the prodomain (BDNFMet), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders in humans heterozygous for the variant BDNF. When expressed by itself in hippocampal neurons, less BDNFMet is secreted in an activity-dependent manner. The nature of the cellular defect when both BDNFMet and wild-type BDNF (BDNFVal) are present in the same cell is not known. Given that this is the predominant expression profile in humans, we examined the effect of coexpressed BDNFMet on BDNFVal intracellular trafficking and processing. Our data indicate that abnormal trafficking of BDNFMet occurred only in neuronal and neurosecretory cells and that BDNFMet could alter the intracellular distribution and activity-dependent secretion of BDNFVal. We determined that, when coexpressed in the same cell, ∼70% of the variant BDNF forms BDNFVal·BDNFMet heterodimers, which are inefficiently sorted into secretory granules resulting in a quantitative decreased secretion. Finally, we determined the form of BDNF secreted in an activity-dependent manner and observed no differences in the forms of BDNFMet or the BDNFVal·BDNFMet heterodimer compared with BDNFVal. Together, these findings indicate that components of the regulated secretory machinery interacts specifically with a signal in the BDNF prodomain and that perturbations in BDNF trafficking may lead to selective impairment in CNS function.

Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Mineralocorticoid Receptor Antagonism in Experimental Atherosclerosis

Background—Aldosterone has been implicated in the effects of angiotensin II in the vasculature. We hypothesized that there is local expression of the mineralocorticoid receptor (MR) in the vasculature and that the use of a selective aldosterone receptor antagonist (SARA) improves endothelial function in early atherosclerosis. Methods and Results—New Zealand rabbits were placed on normal chow or 1% cholesterol diets, randomized to placebo or SARA (eplerenone, 50 mg/kg twice daily), and killed at the end of 6 weeks for various studies. In the hyperlipidemic (HL) chow group, there was a 2.3-fold increase in superoxide (O2·−) generation. SARA normalized O2· − generation in intact aortas and reduced NADH and NADPH oxidase activity to basal levels (0.31±0.04 and 0.27±0.02 in HL versus 0.16±0.05 and 0.07±0.02 in HL-SARA, respectively;P <0.01 by ANOVA). This was associated with improvements in peak relaxations to the endothelial-dependent agonist acetylcholine (82±6% in HL-SARA versus 61±4 in HL;P <0.01 by ANOVA; ED50 6.8×10−8 mol/L in HL-SARA and 1.2×10−7 mol/L in HL;P =NS) to near-normal levels. Vessels from the HL group demonstrated hyperreactivity to angiotensin II that could not be corrected with SARA. Plasma aldosterone levels by radioimmunoassay demonstrated a 4- to 5-fold increase in response to SARA but no differences with lipid feeding. Real-time reverse transcriptase–polymerase chain reaction studies revealed expression of MR in the aorta of HL rabbits and those of controls. Conclusions—MR antagonism improves endothelial function and reduces O2·− generation in diet-induced atherosclerosis. Targeting aldosterone by blocking its receptor has potential antiatherosclerotic effects.

Glucocorticoid and mineralocorticoid receptor mRNA expression in squirrel monkey brain.

Corticosteroids have been implicated in hippocampal atrophy in patients with severe psychiatric disorders, but little is known about receptor expression for corticosteroids in human or nonhuman primate brain. Both the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were surveyed in this study of squirrel monkey brain using in situ hybridization histochemistry. Regions of high GR mRNA levels included CA1 and CA2 of hippocampus, dentate gyrus, paraventricular hypothalamus, lateral geniculate, lateral>medial amygdala, and cerebellum. Western analysis confirmed that GR immunoreactivity in squirrel monkey brain tissue most likely reflects the alpha isoform. Regions of high MR mRNA levels included all hippocampal pyramidal cell fields, dentate gyrus granule cell layer, lateral septum, medial>lateral amygdala, and to a lesser extent, cerebellum. Low levels of MR were also expressed in caudate and putamen. Receptor expression for corticosteroids in deep brain structures and the hippocampal formation was similar to that previously reported in rodents, but GR and MR mRNA were expressed at higher levels in squirrel monkey cerebral cortex. GR expression was evident in all cortical layers, particularly the pyramidal cell-rich layers II/III and V. MR expression was restricted to the more superficial cortical layers, and was only moderately represented in layer V. Laminar patterns were apparent in all regions of cortex for GR expression in squirrel monkeys, but low MR mRNA levels were found in dorsomedial prefrontal cortex (PFC). Different subregional distributions and distinctive laminar patterns suggest specialized functions or coordinated interactions between GR and MR mediated functions in primate PFC.

Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland

BACKGROUND Regulation of raphe serotonergic cells is fundamental to the prevailing hypothesis of major depression pathophysiology. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis, but brainstem TPH mRNA expression has been difficult to measure and study. Recently, a novel paralog of TPH, TPH2 (or neuronal TPH), was described, but its anatomic expression is unknown. METHODS In situ hybridization histochemical survey was conducted across Sprague-Dawley rat brain for TPH1 and TPH2 mRNA. Semiquantitative techniques were used to estimate relative mRNA levels in individual cells. RESULTS Almost exclusively, TPH2 mRNA is expressed in raphe, in a pattern overlapping the histologically defined raphe nuclei. In sharp contrast, TPH1 (the previously known TPH) is expressed predominantly in pineal gland. There is no appreciable overlap in the expression of these paralogs. The level of TPH2 mRNA expression in individual raphe cells is approximately 2.5-fold greater than the level of TPH1 expression in pinealocytes. CONCLUSIONS TPH2 mRNA has an anatomic expression pattern consistent with brainstem raphe nuclei and is likely to be the gene giving rise to the majority of TPH activity in these cells. The robust expression of TPH2 in brainstem should facilitate studies on the transcriptional regulation of raphe serotonin biosynthesis.

Mutations in a novel gene encoding a CRAL-TRIO domain cause human Cayman ataxia and ataxia/dystonia in the jittery mouse.

Cayman ataxia is a recessive congenital ataxia restricted to one area of Grand Cayman Island. Comparative mapping suggested that the locus on 19p13.3 associated with Cayman ataxia might be homologous to the locus on mouse chromosome 10 associated with the recessive ataxic mouse mutant jittery. Screening genes in the region of overlap identified mutations in a novel predicted gene in three mouse jittery alleles, including the first mouse mutation caused by an Alu-related (B1 element) insertion. We found two mutations exclusively in all individuals with Cayman ataxia. The gene ATCAY or Atcay encodes a neuron-restricted protein called caytaxin. Caytaxin contains a CRAL-TRIO motif common to proteins that bind small lipophilic molecules. Mutations in another protein containing a CRAL-TRIO domain, alpha-tocopherol transfer protein (TTPA), cause a vitamin E–responsive ataxia. Three-dimensional protein structural modeling predicts that the caytaxin ligand is more polar than vitamin E. Identification of the caytaxin ligand may help develop a therapy for Cayman ataxia.

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.

Stress coping stimulates hippocampal neurogenesis in adult monkeys

Coping with intermittent social stress is an essential aspect of living in complex social environments. Coping tends to counteract the deleterious effects of stress and is thought to induce neuroadaptations in corticolimbic brain systems. Here we test this hypothesis in adult squirrel monkey males exposed to intermittent social separations and new pair formations. These manipulations simulate conditions that typically occur in male social associations because of competition for limited access to residency in mixed-sex groups. As evidence of coping, we previously confirmed that cortisol levels initially increase and then are restored to prestress levels within several days of each separation and new pair formation. Follow-up studies with exogenous cortisol further established that feedback regulation of the hypothalamic-pituitary-adrenal axis is not impaired. Now we report that exposure to intermittent social separations and new pair formations increased hippocampal neurogenesis in squirrel monkey males. Hippocampal neurogenesis in rodents contributes to spatial learning performance, and in monkeys we found that spatial learning was enhanced in conditions that increased hippocampal neurogenesis. Corresponding changes were discerned in the expression of genes involved in survival and integration of adult-born granule cells into hippocampal neural circuits. These findings support recent indications that stress coping stimulates hippocampal neurogenesis in adult rodents. Psychotherapies designed to promote stress coping potentially have similar effects in humans with major depression.

Regulation of tryptophan hydroxylase-2 gene expression by a bipartite RE-1 silencer of transcription/neuron restrictive silencing factor (REST/NRSF) binding motif.

Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in raphe serotonin biosynthesis, and polymorphisms of TPH2 are implicated in vulnerability to psychiatric disorders. Dynamic transcription regulation of TPH2 may underlie differences in vulnerability. We identified a transcription element in the TPH2 promoter that resembles the binding motif for RE-1 silencer of transcription (REST; also known as NRSF) transcription factor. REST limits tissue expression of non-neuronal genes through a canonical 21-bp motif called the NRSE (neuron-restrictive silencing element). The NRSE in TPH2 is a novel bipartite variant interrupted by a 6-base insertion. We confirmed that this bipartite NRSE permits transcriptional repression by REST identical to canonical NRSE in rat C6-glioma cells. Synthetic permutations of the motif revealed considerable flexibility in the juxtaposition of the two halves of bipartite NRSE. Computational analysis revealed many bipartite NRSE variants conserved between mouse and human genomes. A subgroup of these was found to bind REST by chromatin immunoprecipitation. Messenger RNAs for TPH2 and potassium channel H6, another gene with a bipartite NRSE, were up-regulated by dominant-negative REST in C6-glioma cells. These findings, which indicate that TPH2 expression is part of the developmental program regulated by REST and suggest that many previously unrecognized genes may be regulated by REST through the novel motif, have implications for the mechanism of REST action.

Maternal behavior and offspring resiliency to maternal separation in c57bl/6 mice ☆

Adverse early life experience, such as childhood abuse, neglect, and trauma, increases lifetime risk for mental illness. To investigate underlying mechanisms, the maternal separation (MS) paradigm was developed and validated as an animal model of early adversity in rats, reliably effecting long-term changes to anxiety, gene expression, and stress response. However, across-species validation of core findings in mice has met with limited success. To re-visit parameters governing the effectiveness of MS in mice, this study investigated the effect of MS on maternal care, offspring behavior, and offspring stress-induced corticosterone response in the c57bl/6 mouse strain. The results from this study suggest that: (i) levels of maternal care increase as a function of separation duration immediately after daily MS, but long-term care remains unchanged; and (ii) c57bl/6 mice are resilient to MS, exhibiting subtle decreases in anxiety and unchanged stress-induced corticosterone response as adults, irrespective of separation duration.