SUMMARY Emerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S tau mice. Reducing TIA1 decreases the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibits the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles (NFTs). Despite the increase in NFTs, TIA1 reduction increases neuronal survival and rescues behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity, and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a paradigm in which dysfunction of the translational stress response leads to tau-mediated pathology.

Background Eating disorders are lethal and heritable; however, the underlying genetic factors are unknown. Binge eating is a highly heritable trait associated with eating disorders that is comorbid with mood and substance use disorders. Therefore, understanding its genetic basis will inform therapeutic development that could improve several comorbid neuropsychiatric conditions. Methods We assessed binge eating in closely related C57BL/6 mouse substrains and in an F2 cross to identify quantitative trait loci (QTL) associated with binge eating. We used gene targeting to validated candidate genetic factors. Finally, we used transcriptome analysis of the striatum via mRNA sequencing (RNA-seq) to identify the premorbid transcriptome and the binge-induced transcriptome to inform molecular mechanisms mediating binge eating susceptibility and establishment. Results C57BL/6NJ but not C57BL/6J mice showed rapid and robust escalation in palatable food consumption. We mapped a single genome-wide significant QTL on chromosome 11 (LOD=7.4) to a missense mutation in cytoplasmic FMR1-interacting protein 2 (Cyfip2). We validated Cyfip2 as a major genetic factor underlying binge eating in heterozygous knockout mice on a C57BL/6N background that showed reduced binge eating toward a wild-type C57BL/6J-like level. Transcriptome analysis of premorbid genetic risk identified the enrichment terms “morphine addiction” and “retrograde endocannabinoid signaling” whereas binge eating resulted in the downregulation of a gene set enriched for decreased myelination, oligodendrocyte differentiation, and expression. Conclusions We identified Cyfip2 as a major significant genetic factor underlying binge eating and provide a behavioral paradigm for future genome-wide association studies in populations with increased genetic complexity.

Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512–50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)—a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes—heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10−15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1 ϩ/Ϫ) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1 ϩ/Ϫ mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1 ϩ/Ϫ mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1 ϩ/Ϫ mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1 ϩ/Ϫ decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

Transcriptional and post-transcriptional regulation of gene expression defines the neurobiological mechanisms that bridge genetic and environmental risk factors with neurobehavioral dysfunction underlying the addictions. More than 1000 genes in the eukaryotic genome code for multifunctional RNA binding proteins (RBPs) that can regulate all levels of RNA biogenesis. More than 50% of these RBPs are expressed in the brain where they regulate alternative splicing, transport, localization, stability, and translation of RNAs during development and adulthood. RBP dysfunction can exert global effects on their targetomes that underlie neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease as well as neurodevelopmental disorders, including autism and schizophrenia. Here, we consider the evidence that RBPs influence key molecular targets, neurodevelopment, synaptic plasticity, and neurobehavioral dysfunction underlying the addictions. Increasingly well-powered genome-wide association studies in humans and mammalian model organisms combined with ever more precise transcriptomic and proteomic approaches will continue to uncover novel and possibly selective roles for RBPs in the addictions. Key challenges include identifying the biological functions of the dynamic RBP targetomes from specific cell types throughout subcellular space (e.g., the nuclear spliceome versus the synaptic translatome) and time and manipulating RBP programs through post-transcriptional modifications to prevent or reverse aberrant neurodevelopment and plasticity underlying the addictions.

Fifteen of the 21 innexin (Inx) genes (Hve-inx) found in the genome of the medicinal leech, Hirudo verbana, are expressed in the CNS (Kandarian et al., 2012). Two are expressed pan-neuronally, while the others are restricted in their expression to small numbers of cells, in some cases reflecting the membership of known networks of electrically coupled and dye-coupled neurons or glial cells. We report here that when Hve-inx genes characteristic of discrete coupled networks were expressed ectopically in neurons known not to express them, the experimental cells were found to become dye coupled with the other cells in that network. Hve-inx6 is normally expressed by only three neurons in each ganglion, which form strongly dye-coupled electrical connections with each other [Shortening-Coupling interneuron (S-CI) network] (Muller and Scott, 1981; Dykes and Macagno, 2006). But when Hve-inx6 was ectopically expressed in a variety of central embryonic neurons, those cells became dye coupled with the S-CI network. Similarly, Hve-inx2 is normally uniquely expressed by the ganglion’s large glial cells, but when it was ectopically expressed in different central neurons, they became dye coupled to the glial cells. In contrast, overexpression of the pan-neuronal Inx genes Hve-inx1 and Hve-inx14 did not yield any novel instances of dye coupling to pre-existent neuronal networks. These results reveal that expression of certain innexins is sufficient to couple individual neurons to pre-existing networks in the CNS. We propose that a primary determinant of selective neuronal connectivity and circuit formation in the leech is the surface expression of unique subsets of gap junctional proteins.

Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. We report here that the punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). We then demonstrate that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction-coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild-type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. We show in cell culture with dye-uptake and plasma membrane labeling experiments that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cell's perinuclear region. Lastly, we use these mutant innexin transgenes to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 571-586, 2013.