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1.  A Human-Specific De Novo Protein-Coding Gene Associated with Human Brain Functions 
PLoS Computational Biology  2010;6(3):e1000734.
To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203). Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.
Author Summary
For decades, gene duplication, retrotranspositions and gene fusions were believed to be major ways to increase gene number. All involve “mother” genes as the “building blocks” for new genes. However, several recently identified “motherless” genes challenged the idea in that some proteins might have emerged de novo from ancestral non-coding DNAs. Did any such genes emerge in human after the divergence from chimpanzee? If yes, such genes might help understand what makes us human. Here we report the first experimentally verified case of a human-specific protein-coding gene, FLJ33706 (alternative gene symbol C20orf203), that originated de novo since the divergence of human and chimpanzee. FLJ33706 was formed by the insertion of repeat elements, especially Alu sequences, that contributed to the formation of the first coding exon and six standard splice junctions, followed by two human-specific substitutions that escaped stop codons. The functional protein-coding features of the FLJ33706 gene are supported by population genetics, transcriptome profiling, Western-blot and immunohistochemistry assays. Data suggest that FLJ33706 may be involved in nicotine addiction and Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and be involved in human brain functions.
doi:10.1371/journal.pcbi.1000734
PMCID: PMC2845654  PMID: 20376170
2.  RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention 
Neuron  2013;80(2):415-428.
SUMMARY
A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.
doi:10.1016/j.neuron.2013.10.015
PMCID: PMC4098943  PMID: 24139042
3.  A Prolyl-isomerase Mediates Dopamine-dependent Plasticity and Cocaine Motor Sensitization 
Cell  2013;154(3):637-650.
Summary
Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1 dopamine receptor signaling that is induced by drug administration, the glutamate receptor protein mGluR5 is phosphorylated by MAP kinase, which we show potentiates Pin1-mediated prolyl isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate NMDA receptor-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses, and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction.
doi:10.1016/j.cell.2013.07.001
PMCID: PMC3785238  PMID: 23911326
4.  Nucleus accumbens mGluR5-associated signaling regulates binge alcohol drinking under Drinking-in-the-Dark procedures 
Background
Alcohol increases the expression of Group 1 metabotropic glutamate receptors (mGluRs), their associated scaffolding protein Homer2, and stimulates phosphatidylinositol 3-kinase (PI3K) within the nucleus accumbens (NAC). Moreover, functional studies suggest that NAC Group 1 mGluR/Homer2/PI3K signaling may be a potential target for pharmacotherapeutic intervention in alcoholism.
Methods
Immunoblotting was conducted to examine the effects of alcohol consumption under Drinking-in-the-Dark (DID) procedures on Group 1 mGluR-associated proteins in C57BL/6J (B6) mice. Follow-up behavioral studies examined the importance of Group 1 mGluR/Homer2/PI3K signaling within the NAC shell for limited access alcohol drinking. Finally, immunoblotting examined whether the NAC expression of Group 1 mGluR-associated proteins is a genetic correlate of high alcohol drinking using a selectively bred high DID (HDID-1) mouse line.
Results
Limited access alcohol drinking under DID procedures up-regulated NAC shell Homer2 levels, concomitant with increases in mGluR5 and NR2B. Intra-NAC shell blockade of mGluR5, Homer2, or PI3K signaling, as well as transgenic disruption of the Homer binding site on mGluR5 decreased alcohol consumption in B6 mice. Moreover, transgenic disruption of the Homer binding site on mGluR5 and Homer2 deletion both prevented the attenuating effect of mGluR5 and PI3K blockade upon intake. Finally, the basal NAC shell protein expression of mGluR1 and Homer2 was increased in offspring of HDID-1 animals.
Conclusions
Taken together, these data further implicate Group1 mGluR signaling through Homer2 within the NAC in excessive alcohol consumption.
doi:10.1111/j.1530-0277.2012.01776.x
PMCID: PMC3382009  PMID: 22432643
mGluR5 receptors; PI3 kinase; Homer; mGluR1 receptors; HDID-1
5.  Homers at the Interface between Reward and Pain 
Pain alters opioid reinforcement, presumably via neuroadaptations within ascending pain pathways interacting with the limbic system. Nerve injury increases expression of glutamate receptors and their associated Homer scaffolding proteins throughout the pain processing pathway. Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs. Thus, we investigated a potential role for Homers in the interactions between pain and drug reward in mice. Chronic constriction injury (CCI) of the sciatic nerve elevated Homer1b/c and/or Homer2a/b expression within all mesolimbic structures examined and for the most part, the Homer increases coincided with elevated mGluR5, GluN2A/B, and the activational state of various down-stream kinases. Behaviorally, CCI mice showed pain hypersensitivity and a conditioned place-aversion (CPA) at a low heroin dose that supported conditioned place-preference (CPP) in naïve controls. Null mutations of Homer1a, Homer1, and Homer2, as well as transgenic disruption of mGluR5-Homer interactions, either attenuated or completely blocked low-dose heroin CPP, and none of the CCI mutant strains exhibited heroin-induced CPA. However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI’s effects) were sufficient to blunt heroin CPP in uninjured mice. However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA. Together, these data implicate increases in glutamate receptor/Homer/kinase activity within limbic structures, perhaps outside the NAC, as possibly critical for switching the incentive motivational properties of heroin following nerve injury, which has relevance for opioid psychopharmacology in individuals suffering from neuropathic pain.
doi:10.3389/fpsyt.2013.00039
PMCID: PMC3675508  PMID: 23761764
Homer proteins; Group1 metabotropic glutamate receptors; NMDA receptors; neuropathic pain; heroin; nucleus accumbens; conditioned place-preference; conditioned place-aversion
6.  Preso1 dynamically regulates group I metabotropic glutamate receptors 
Nature neuroscience  2012;15(6):836-844.
Group I metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, are G protein–coupled receptors (GPCRs) that are expressed at excitatory synapses in brain and spinal cord. GPCRs are often negatively regulated by specific G protein–coupled receptor kinases and subsequent binding of arrestin-like molecules. Here we demonstrate an alternative mechanism in which group I mGluRs are negatively regulated by proline-directed kinases that phosphorylate the binding site for the adaptor protein Homer, and thereby enhance mGluR–Homer binding to reduce signaling. This mechanism is dependent on a multidomain scaffolding protein, Preso1, that binds mGluR, Homer and proline-directed kinases and that is required for their phosphorylation of mGluR at the Homer binding site. Genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, and Preso1−/− mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. Preso1 creates a microdomain for proline-directed kinases with broad substrate specificity to phosphorylate mGluR and to mediate negative regulation.
doi:10.1038/nn.3103
PMCID: PMC3434267  PMID: 22561452
7.  Oligodendroglia metabolically support axons and contribute to neurodegeneration 
Nature  2012;487(7408):443-448.
Summary
Oligodendroglia support axon survival and function through mechanisms independent of myelination and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been hypothesized. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and utilization. We show the most abundant lactate transporter in the CNS, monocarboxylate transporter 1 (MCT1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and mouse models of, amyotrophic lateral sclerosis (ALS), suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.
doi:10.1038/nature11314
PMCID: PMC3408792  PMID: 22801498
8.  Harmine, A Natural Beta-Carboline Alkaloid, Upregulates Astroglial Glutamate Transporter Expression 
Neuropharmacology  2010;60(7-8):1168-1175.
Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2 /GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2 /GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation.
doi:10.1016/j.neuropharm.2010.10.016
PMCID: PMC3220934  PMID: 21034752
harmine; GLT-1; EAAT2; glutamate transporter; astroglia; ALS
9.  Meta-analysis and genome-wide interpretation of genetic susceptibility to drug addiction 
BMC Genomics  2011;12:508.
Background
Classical genetic studies provide strong evidence for heritable contributions to susceptibility to developing dependence on addictive substances. Candidate gene and genome-wide association studies (GWAS) have sought genes, chromosomal regions and allelic variants likely to contribute to susceptibility to drug addiction.
Results
Here, we performed a meta-analysis of addiction candidate gene association studies and GWAS to investigate possible functional mechanisms associated with addiction susceptibility. From meta-data retrieved from 212 publications on candidate gene association studies and 5 GWAS reports, we linked a total of 843 haplotypes to addiction susceptibility. We mapped the SNPs in these haplotypes to functional and regulatory elements in the genome and estimated the magnitude of the contributions of different molecular mechanisms to their effects on addiction susceptibility. In addition to SNPs in coding regions, these data suggest that haplotypes in gene regulatory regions may also contribute to addiction susceptibility. When we compared the lists of genes identified by association studies and those identified by molecular biological studies of drug-regulated genes, we observed significantly higher participation in the same gene interaction networks than expected by chance, despite little overlap between the two gene lists.
Conclusions
These results appear to offer new insights into the genetic factors underlying drug addiction.
doi:10.1186/1471-2164-12-508
PMCID: PMC3215751  PMID: 21999673
10.  Genome Wide Association for Addiction: Replicated Results and Comparisons of Two Analytic Approaches 
PLoS ONE  2010;5(1):e8832.
Background
Vulnerabilities to dependence on addictive substances are substantially heritable complex disorders whose underlying genetic architecture is likely to be polygenic, with modest contributions from variants in many individual genes. “Nontemplate” genome wide association (GWA) approaches can identity groups of chromosomal regions and genes that, taken together, are much more likely to contain allelic variants that alter vulnerability to substance dependence than expected by chance.
Methodology/Principal Findings
We report pooled “nontemplate” genome-wide association studies of two independent samples of substance dependent vs control research volunteers (n = 1620), one European-American and the other African-American using 1 million SNP (single nucleotide polymorphism) Affymetrix genotyping arrays. We assess convergence between results from these two samples using two related methods that seek clustering of nominally-positive results and assess significance levels with Monte Carlo and permutation approaches. Both “converge then cluster” and “cluster then converge” analyses document convergence between the results obtained from these two independent datasets in ways that are virtually never found by chance. The genes identified in this fashion are also identified by individually-genotyped dbGAP data that compare allele frequencies in cocaine dependent vs control individuals.
Conclusions/Significance
These overlapping results identify small chromosomal regions that are also identified by genome wide data from studies of other relevant samples to extents much greater than chance. These chromosomal regions contain more genes related to “cell adhesion” processes than expected by chance. They also contain a number of genes that encode potential targets for anti-addiction pharmacotherapeutics. “Nontemplate” GWA approaches that seek chromosomal regions in which nominally-positive associations are found in multiple independent samples are likely to complement classical, “template” GWA approaches in which “genome wide” levels of significance are sought for SNP data from single case vs control comparisons.
doi:10.1371/journal.pone.0008832
PMCID: PMC2809089  PMID: 20098672
11.  Binge drinking up-regulates accumbens mGluR5-Homer2-PI3K signaling: Functional implications for alcoholism 
The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC mGluR-Homer2-PI3K signaling in regulating excessive alcohol consumption within the context of the Scheduled High Alcohol Consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking (∼1.5 g/kg/30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knock-down of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP (0.1-1 μg/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared to wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5-Homer-PI3K signaling may be a mechanism governing excessive alcohol intake, the “anti-binge” effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5F1128R transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2 and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5-Homer2-PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5-Homer2-PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.
doi:10.1523/JNEUROSCI.5900-08.2009
PMCID: PMC2761716  PMID: 19587272
Homer2; nucleus accumbens; alcoholism; binge drinking; mGluR5; PI3K
12.  Genome wide association for substance dependence: convergent results from epidemiologic and research volunteer samples 
BMC Medical Genetics  2008;9:113.
Background
Dependences on addictive substances are substantially-heritable complex disorders whose molecular genetic bases have been partially elucidated by studies that have largely focused on research volunteers, including those recruited in Baltimore. Maryland. Subjects recruited from the Baltimore site of the Epidemiological Catchment Area (ECA) study provide a potentially-useful comparison group for possible confounding features that might arise from selecting research volunteer samples of substance dependent and control individuals. We now report novel SNP (single nucleotide polymorphism) genome wide association (GWA) results for vulnerability to substance dependence in ECA participants, who were initially ascertained as members of a probability sample from Baltimore, and compare the results to those from ethnically-matched Baltimore research volunteers.
Results
We identify substantial overlap between the home address zip codes reported by members of these two samples. We find overlapping clusters of SNPs whose allele frequencies differ with nominal significance between substance dependent vs control individuals in both samples. These overlapping clusters of nominally-positive SNPs identify 172 genes in ways that are never found by chance in Monte Carlo simulation studies. Comparison with data from human expressed sequence tags suggests that these genes are expressed in brain, especially in hippocampus and amygdala, to extents that are greater than chance.
Conclusion
The convergent results from these probability sample and research volunteer sample datasets support prior genome wide association results. They fail to support the idea that large portions of the molecular genetic results for vulnerability to substance dependence derive from factors that are limited to research volunteers.
doi:10.1186/1471-2350-9-113
PMCID: PMC2637238  PMID: 19094236
13.  OKCAM: an ontology-based, human-centered knowledgebase for cell adhesion molecules 
Nucleic Acids Research  2008;37(Database issue):D251-D260.
‘Cell adhesion molecules’ (CAMs) are essential elements of cell/cell communication that are important for proper development and plasticity of a variety of organs and tissues. In the brain, appropriate assembly and tuning of neuronal connections is likely to require appropriate function of many cell adhesion processes. Genetic studies have linked and/or associated CAM variants with psychiatric, neurologic, neoplastic, immunologic and developmental phenotypes. However, despite increasing recognition of their functional and pathological significance, no systematic study has enumerated CAMs or documented their global features. We now report compilation of 496 human CAM genes in six gene families based on manual curation of protein domain structures, Gene Ontology annotations, and 1487 NCBI Entrez annotations. We map these genes onto a cell adhesion molecule ontology that contains 850 terms, up to seven levels of depth and provides a hierarchical description of these molecules and their functions. We develop OKCAM, a CAM knowledgebase that provides ready access to these data and ontologic system at http://okcam.cbi.pku.edu.cn. We identify global CAM properties that include: (i) functional enrichment, (ii) over-represented regulation modes and expression patterns and (iii) relationships to human Mendelian and complex diseases, and discuss the strengths and limitations of these data.
doi:10.1093/nar/gkn568
PMCID: PMC2686464  PMID: 18790807

Results 1-13 (13)