In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3'UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.

Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.

We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

Neuronal RNA-binding protein HuD regulates addiction-related gene expression and behavior.

The neuronal RNA-binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD-mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuDOE ). When this set was further limited to genes in the knowledgebase of addiction-related genes database (KARG) that contains predicted HuD-binding sites in their 3' untranslated regions (3'UTRs), we found that HuD regulates networks that have been associated with addiction-like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction-related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild-type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuDOE and wild-type mice in CPP and found that HuDOE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction-related behaviors such as cocaine conditioning and seeking, through increased plasticity-related gene expression.

The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

Multimodal Neuroimaging in Schizophrenia: Description and Dissemination.

In this paper we describe an open-access collection of multimodal neuroimaging data in schizophrenia for release to the community. Data were acquired from approximately 100 patients with schizophrenia and 100 age-matched controls during rest as well as several task activation paradigms targeting a hierarchy of cognitive constructs. Neuroimaging data include structural MRI, functional MRI, diffusion MRI, MR spectroscopic imaging, and magnetoencephalography. For three of the hypothesis-driven projects, task activation paradigms were acquired on subsets of ~200 volunteers which examined a range of sensory and cognitive processes (e.g., auditory sensory gating, auditory/visual multisensory integration, visual transverse patterning). Neuropsychological data were also acquired and genetic material via saliva samples were collected from most of the participants and have been typed for both genome-wide polymorphism data as well as genome-wide methylation data. Some results are also presented from the individual studies as well as from our data-driven multimodal analyses (e.g., multimodal examinations of network structure and network dynamics and multitask fMRI data analysis across projects). All data will be released through the Mind Research Network's collaborative informatics and neuroimaging suite (COINS).

Polymorphisms in MIR137HG and microRNA-137-regulated genes influence gray matter structure in schizophrenia.

Evidence suggests that microRNA-137 (miR-137) is involved in the genetic basis of schizophrenia. Risk variants within the miR-137 host gene (MIR137HG) influence structural and functional brain-imaging measures, and miR-137 itself is predicted to regulate hundreds of genes. We evaluated the influence of a MIR137HG risk variant (rs1625579) in combination with variants in miR-137-regulated genes TCF4, PTGS2, MAPK1 and MAPK3 on gray matter concentration (GMC). These genes were selected based on our previous work assessing schizophrenia risk within possible miR-137-regulated gene sets using the same cohort of subjects. A genetic risk score (GRS) was determined based on genotypes of these four schizophrenia risk-associated genes in 221 Caucasian subjects (89 schizophrenia patients and 132 controls). The effects of the rs1625579 genotype with the GRS of miR-137-regulated genes in a three-way interaction with diagnosis on GMC patterns were assessed using a multivariate analysis. We found that schizophrenia subjects homozygous for the MIR137HG risk allele show significant decreases in occipital, parietal and temporal lobe GMC with increasing miR-137-regulated GRS, whereas those carrying the protective minor allele show significant increases in GMC with GRS. No correlations of GMC and GRS were found in control subjects. Variants within or upstream of genes regulated by miR-137 in combination with the MIR137HG risk variant may influence GMC in schizophrenia-related regions in patients. Given that the genes evaluated here are involved in protein kinase A signaling, dysregulation of this pathway through alterations in miR-137 biogenesis may underlie the gray matter loss seen in the disease.

A pilot study on commonality and specificity of copy number variants in schizophrenia and bipolar disorder.

Schizophrenia (SZ) and bipolar disorder (BD) are known to share genetic risks. In this work, we conducted whole-genome scanning to identify cross-disorder and disorder-specific copy number variants (CNVs) for these two disorders. The Database of Genotypes and Phenotypes (dbGaP) data were used for discovery, deriving from 2416 SZ patients, 592 BD patients and 2393 controls of European Ancestry, as well as 998 SZ patients, 121 BD patients and 822 controls of African Ancestry. PennCNV and Birdsuite detected high-confidence CNVs that were aggregated into CNV regions (CNVRs) and compared with the database of genomic variants for confirmation. Then, large (size⩾500 kb) and small common CNVRs (size <500 kb, frequency⩾1%) were examined for their associations with SZ and BD. Particularly for the European Ancestry samples, the dbGaP findings were further evaluated in the Wellcome Trust Case Control Consortium (WTCCC) data set for replication. Previously implicated variants (1q21.1, 15q13.3, 16p11.2 and 22q11.21) were replicated. Some cross-disorder variants were noted to differentially affect SZ and BD, including CNVRs in chromosomal regions encoding immunoglobulins and T-cell receptors that were associated more with SZ, and the 10q11.21 small CNVR (GPRIN2) associated more with BD. Disorder-specific CNVRs were also found. The 22q11.21 CNVR (COMT) and small CNVRs in 11p15.4 (TRIM5) and 15q13.2 (ARHGAP11B and FAN1) appeared to be SZ-specific. CNVRs in 17q21.2, 9p21.3 and 9q21.13 might be BD-specific. Overall, our primary findings in individual disorders largely echo previous reports. In addition, the comparison between SZ and BD reveals both specific and common risk CNVs. Particularly for the latter, differential involvement is noted, motivating further comparative studies and quantitative models.

Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia.

BACKGROUND: Identifying brain changes in schizophrenia has been a major research focus for many years. Although impressive gains have been made in neuroimaging and brain electrophysiology, molecular and cellular markers of schizophrenia have lagged. There are no consistent biochemical markers for schizophrenia pathophysiology and none that reflect treatment course. METHODS: Samples were obtained from 25 postmortem schizophrenic brains and 31 nonschizophrenic controls. These samples were processed, and the synaptosomal fraction was isolated. Ten micrograms of protein from each of these samples was solubilized in a sodium dodecylsulfate sample buffer and separated on 10% (wt/vol) polyacrylamide gels. Monoclonal antibody (SMI-81) was incubated with the blots and, using quantitative Western blotting, we measured the relative amounts of SNAP-25 in these samples. RESULTS: We report altered levels of SNAP-25 in both the inferior temporal cortex (Brodmann area 20) and prefrontal association cortex (Brodmann areas 9 and 10) in postmortem brains of patients with schizophrenia relative to nonschizophrenic controls. Normal levels of SNAP-25 are noted in schizophrenics in area 17, decreased levels in areas 10 and 20, and an elevated level in area 9. CONCLUSIONS: These data support cytoarchitectural observations that the cerebral cortex of schizophrenic patients has extensive pathology. The data presented here, along with data on other brain-specific proteins, indicate a complicated molecular adaptation to the causative factors of schizophrenia.

Role of highly conserved pyrimidine-rich sequences in the 3' untranslated region of the GAP-43 mRNA in mRNA stability and RNA-protein interactions.

We have shown previously that the mRNA for the growth-associated protein GAP-43 is selectively stabilized during neuronal differentiation. In this study, we explored the role of its highly conserved 3' untranslated region (3'UTR) in mRNA stability and RNA-protein interactions. The 3'UTRs of the rat and chicken GAP-43 mRNAs show 78% sequence identity, which is equivalent to the conservation of their coding regions. In rat PC12 cells stably transfected with the full-length rat or chicken GAP-43 cDNAs, the transgene mRNAs decayed with same half-life of about 3 h. The GAP-43 3'UTR also caused the rabbit beta-globin mRNA to decay with a half-life of 4 h, indicating that the major determinants for GAP-43 mRNA stability are localized in its highly conserved 3'UTR. Three brain cytosolic RNA-binding proteins (molecular mass 40, 65 and 95 kDa) were found to interact with both the rat and chicken GAP-43 mRNAs. These RNA-protein interactions were specific and involved pyrimidine-rich sequences in the 3'UTR. Like the GAP-43 mRNA, the activity of these proteins was enriched in brain and increased during development. We propose that highly conserved pyrimidine-rich sequences in the 3'UTR of this mRNA regulate GAP-43 gene expression via interactions with specific RNA-binding proteins.

cis-acting regulatory elements in the GAP-43 mRNA 3'-untranslated region can function in trans to suppress endogenous GAP-43 gene expression.

The expression of the GAP-43 gene is controlled partly by changes in the stability of its mRNA, a process that is mediated by the interaction of specific sequences in the 3'-untranslated region (3'UTR) with neuronal-specific RNA-binding proteins. Limiting amounts of these trans-acting factors are available in the cell, thus we proposed that overexpression of the GAP-43 3'UTR could affect the levels of the endogenous mRNA via competitive binding to specific RNA-binding proteins. In this study, we show that chronic expression of GAP-43 3'UTR sequences in PC12 cells causes the depletion of the endogenous mRNA and consequent reduction of GAP-43 protein levels. The levels of the mRNAs for c-fos, the amyloid precursor protein (APP) and the microtubule associated protein tau, all three containing similar 3'UTR sequences, were not affected by the treatment. These results thus suggest that the effect of excess GAP-43 3'UTR is specific for its corresponding mRNA. We also used an HSV (herpes simplex virus)-1 vector and a mammalian expression vector with an inducible promoter to acutely express a 10 to 50 fold excess of 3'UTR sequences. Under these conditions, we found that transient expression of the GAP-43 3'UTR was effective in inhibiting both GAP-43 gene expression and neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells and in primary neuronal cultures. These results underscore the role of 3'UTR sequences in the control of GAP-43 gene expression and suggest that overexpression of specific 3'UTR sequences could be used as a potential tool for probing the function of other post-transcriptionally-regulated proteins during neuronal differentiation.

Partial purification and characterization of a neurite-promoting factor from the injured goldfish optic nerve.

We have partially purified and characterized a neurite-promoting factor derived from the injured goldfish optic nerve (ON). This factor is secreted into conditioned media (CM) by the injured, but not intact goldfish ON, and has potent outgrowth-promoting effects on neurons of the embryonic mammalian brain. Based on its elution properties on ion-exchange and gel-filtration chromatography, this factor appears to be an acidic protein of Mr ca. 26 kilodaltons (kDa) that is distinct from previously characterized growth factors with described effects on mammalian CNS neurons.

Measuring synaptosomal associated protein-25 kDa in human cerebral spinal fluid.

Changes in the quantity and distribution of neuronal proteins have been reported in psychiatric and neurological illnesses. The majority of this work has been performed in post-mortem samples and the results are difficult to apply to clinical care. The objective of this study is to develop a methodology that can identify trace amounts of brain proteins in cerebral spinal fluid (CSF). Human cerebral spinal fluid was processed to remove albumin and immunoglobulins. CSF samples were analyzed on Western blots using a monoclonal antibody against SNAP-25. These samples were compared to SNAP-25 immunoprecipitated from CSF, rat and human brain homogenates. The monoclonal antibody Mab 331 identified a single band of 25 kDa in all samples. These results demonstrate that the presynaptic protein SNAP-25 can be identified and measured in CSF.

The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs.

The protein that has been designated as GAP-43, B-50, F1 or pp46 is associated with the growth and modulation of neuronal connections. cDNA clones for the rat and human genes were isolated and used to demonstrate that the messenger RNA for the protein is expressed only in neurons, that its overall level is highest in the developing brain, and that in the adult human brain levels of the mRNA are highest in the associative neocortex.

Conditioned media from the injured lower vertebrate CNS promote neurite outgrowth from mammalian brain neurons in vitro.

Unlike most pathways of the mature mammalian central nervous system (CNS), the CNS of lower vertebrates can regenerate after jury, a capacity that may be due to the secretion of neurite-promoting factors from the injured CNS. We report that conditioned media (CM) from the injured optic nerve of the mature goldfish promoted marked neurite outgrowth from dissociated embryonic rat cortical and hindbrain neurons in serum-free, neuron-enriched culture. This property was not shared by CM from intact goldfish optic nerve, or from intact or injured optic nerve of mature rats. Neurite-promoting activity was obtained at concentrations as low as 100 ng total protein/ml of CM from injured goldfish optic nerve, and was associated with a distinctive morphology of neurite outgrowth. Due its properties of non-dialyzability, heat lability, and trypsin sensitivity, the neurite-promoting factor(s) appeared to be one or more protein species of MW greater than 12,000. Factors secreted by the regenerating CNS of lower vertebrates can directly promote outgrowth of mammalian CNS neurons.

A factor from the injured lower vertebrate CNS promotes outgrowth from human fetal brain neurons.

Unlike mammals, lower vertebrates retain the capacity to regenerate damaged central nervous system (CNS) pathways throughout life. In previous studies, we have used the goldfish optic nerve (ON) as a model for CNS regeneration, and found that the injured goldfish ON selectively secretes a factor that promotes process outgrowth of cultured neurons, including neurons of the developing rodent CNS. In the current study, we found that a factor similarly obtained from the injured goldfish ON also has potent outgrowth-promoting effects on cerebrocortical neurons of the fetal human brain, and that these effects are dependent on the age of fetal neurons. This factor appeared to be a protein of mol. wt. greater than 12,000, and was associated with a distinctive morphology of neurite outgrowth. The neurite-promoting factor from the injured goldfish ON may be homologous to factors within the developing human brain.

Alterations in GAP-43-immunoreactive innervation in the aging rat pituitary.

The levels and distribution of the growth-associated protein, GAP-43, were examined in the pituitary glands of young and aging Sprague-Dawley rats, using immunohistochemical techniques on tissue sections and Western blot analyses. GAP-43-immunoreactive innervation was observed in sections in the intermediate and neural lobes of animals aged 8-15 months, while in the oldest rats studied (17 months), stained fibers were observed mainly in the neural, but not the intermediate lobe. Western blots revealed reduced levels of GAP-43 in samples from 15 month old animals, as compared to 12 month old rats, in the neurointermediate lobes. There was no immunoreactivity for GAP-43 in the anterior lobes in the tissue sections or in the blots in any of the glands examined. A diminished level of GAP-43 in pituitary innervation in aged animals suggests a reduced ability for nerve terminals to undergo 'plastic' changes in their relationship to target endocrine cells. Since GAP-43 has also been suggested to modulate neurotransmitter release, a reduction in the protein in aging nerve terminals may diminish availability of transmitters at presynaptic sites.

Opposite effects of acute ethanol exposure on GAP-43 and BDNF expression in the hippocampus versus the cerebellum of juvenile rats.

The adolescent brain is particularly vulnerable to the effects of alcohol, with intoxications at this developmental age often producing long-lasting effects. The present study addresses the effects of a single acute ethanol exposure on growth-associated protein-43 (GAP-43) and brain-derived neurotrophic factor (BDNF) gene expression in neurons in the cerebellum and hippocampus of adolescent rats. Male postnatal day 23 (P23) Sprague-Dawley rats were exposed to ethanol vapors for 2h and after a recovery period of 2h, the cerebellum and hippocampus were harvested and samples were taken for blood alcohol concentration (BAC) determinations. We found that this exposure resulted in a mean BAC of 174 mg/dL, which resembles levels in human adolescents after binge drinking. Analyses of total RNA and protein by quantitative reverse transcription PCR and western blotting, respectively, revealed that this single ethanol exposure significantly decreased the levels of GAP-43 mRNA and protein in the cerebellum but increased the levels of mRNA and protein in the hippocampus. BDNF mRNA and protein levels were also increased in the hippocampus but not in the cerebellum of these animals. In situ hybridizations revealed that GAP-43 and BDNF mRNA levels were primarily increased by alcohol exposure in hippocampal dentate granule cells and CA3 neurons. Overall, the reported alterations in the expression of the plasticity-associated genes GAP-43 and BDNF in juvenile rats are consistent with the known deleterious effects of binge drinking on motor coordination and cognitive function.