Bjarte Håvik

Identification of genes co‐upregulated with Arc during BDNF‐induced long‐term potentiation in adult rat dentate gyrus in vivo

Brain‐derived neurotrophic factor (BDNF) is a critical regulator of transcription‐dependent adaptive neuronal responses, such as long‐term potentiation (LTP). Brief infusion of BDNF into the dentate gyrus of adult anesthetized rats triggers stable LTP at medial perforant path‐granule synapses that is transcription‐dependent and requires induction of the immediate early gene Arc. Rather than acting alone, Arc is likely to be part of a larger BDNF‐induced transcriptional program. Here, we used cDNA microarray expression profiling to search for genes co‐upregulated with Arc 3 h after BDNF‐LTP induction. Of nine cDNAs encoding for known genes and up‐regulated more than four‐fold, we selected five genes, Narp, neuritin, ADP‐ribosylation factor‐like protein‐4 (ARL4L), TGF‐β‐induced immediate early gene‐1 (TIEG1) and CARP, for further validation. Real‐time PCR confirmed robust up‐regulation of these genes in an independent set of BDNF‐LTP experiments, whereas infusion of the control protein cytochrome C had no effect. In situ hybridization histochemistry further revealed up‐regulation of all five genes in somata of post‐synaptic granule cells following both BDNF‐LTP and high‐frequency stimulation‐induced LTP. While Arc synthesis is critical for local actin polymerization and stable LTP formation, several of the co‐upregulated genes have known functions in excitatory synaptogenesis, axon guidance and glutamate receptor clustering. These results provide novel insight into gene expression responses underlying BDNF‐induced synaptic consolidation in the adult brain in vivo.

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain.

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.

Familial diarrhea syndrome caused by an activating GUCY2C mutation.

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohns disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.).

The Complement Control-Related Genes CSMD1 and CSMD2 Associate to Schizophrenia

BACKGROUND Patients with schizophrenia often suffer from cognitive dysfunction, including impaired learning and memory. We recently demonstrated that long-term potentiation in rat hippocampus, a mechanistic model of learning and memory, is linked to gene expression changes in immunity-related processes involved in complement activity and antigen presentation. We therefore aimed to examine whether key regulators of these processes are genetic susceptibility factors in schizophrenia. METHODS Analysis of genetic association was based on data mining of genotypes from a German genome-wide association study and a multiplex GoldenGate tag single nucleotide polymorphism (SNP)-based assay of Norwegian and Danish case-control samples (Scandinavian Collaboration on Psychiatric Etiology), including 1133 patients with schizophrenia and 2444 healthy control subjects. RESULTS Allelic associations were found across all three samples for eight common SNPs in the complement control-related gene CSMD2 (CUB and Sushi Multiple Domains 2) on chromosome 1p35.1-34.3, of which rs911213 reached a statistical significance comparable to that of a genome wide threshold (p value = 4.0 × 10(-8); odd ratio = .73, 95% confidence interval = .65-.82). The second most significant gene was CSMD1 on chromosome 8p23.2, a homologue to CSMD2. In addition, we observed replicated associations in the complement surface receptor CD46 as well as the major histocompatibility complex genes HLA-DMB and HLA-DOA. CONCLUSIONS These data demonstrate a significant role of complement control-related genes in the etiology of schizophrenia and support disease mechanisms that involve the activity of immunity-related pathways in the brain.

Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo : evidence for compartment-specific translation control

Protein synthesis underlying activity‐dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor‐2 (eEF2), during long‐term potentiation (LTP) induced by local infusion of brain‐derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF‐induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal‐regulated kinase (ERK) inhibitor U0126 blocked BDNF‐LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho‐eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of α‐calcium/calmodulin‐dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK‐dependent regulation of the initiation and elongation steps of protein synthesis during BDNF‐LTP in vivo. Furthermore, the results suggest a compartment‐specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non‐synaptic sites.

Bursts of high-frequency stimulation trigger rapid delivery of pre-existing α-CaMKII mRNA to synapses: a mechanism in dendritic protein synthesis during long-term potentiation in adult awake rats

Messenger ribonucleic acid encoding the alpha‐subunit of calcium/calmodulin‐dependent protein kinase II (camkII) is abundantly and constitutively expressed in dendrites of pyramidal and granule cell neurons of the adult hippocampus. Recent evidence suggests that camkII messenger ribonucleic acid is stored in a translationally dormant state within ribonucleic acid storage granules. Delivery of camkII messenger ribonucleic acid from sites of storage to sites of translation may therefore be a key step in activity‐driven dendritic protein synthesis and synaptic plasticity. Here we explored possible camkII trafficking in the context of long‐term potentiation in the dentate gyrus of awake, adult rats. Long‐term potentiation was induced by patterned high‐frequency stimulation, synaptodendrosomes containing pinched‐off dendritic spines were obtained from microdissected dentate gyrus, and messenger ribonucleic acid levels were determined by real‐time polymerase chain reaction. High‐frequency stimulation triggered a rapid 2.5‐fold increase in camkII messenger ribonucleic acid levels in the synaptodendrosome fraction. This increase occurred in the absence of camkII upregulation in the homogenate fraction, indicating trafficking of pre‐existing messenger ribonucleic acid to synaptodendrosomes. The elevation in camkII messenger ribonucleic acid was paralleled by an increase in protein expression specific to the synaptodendrosome fraction, and followed by depletion of camkII message. Activity‐dependent regulation of camkII messenger ribonucleic acid and protein did not require N‐methyl‐d‐aspartate receptor activation. In contrast, N‐methyl‐d‐aspartate receptor activation was required for induction of the immediate early genes zif268 and activity‐regulated cytoskeleton‐associated protein in dentate gyrus homogenates. The results support a model in which locally stored camkII messenger ribonucleic acid is rapidly transported to dendritic spines and translated during long‐term potentiation in behaving rats.

Drug-induced activation of SREBP-controlled lipogenic gene expression in CNS-related cell lines: Marked differences between various antipsychotic drugs

BackgroundThe etiology of schizophrenia is unknown, but neurodevelopmental disturbances, myelin- and oligodendrocyte abnormalities and synaptic dysfunction have been suggested as pathophysiological factors in this severe psychiatric disorder. Cholesterol is an essential component of myelin and has proved important for synapse formation. Recently, we demonstrated that the antipsychotic drugs clozapine and haloperidol stimulate lipogenic gene expression in cultured glioma cells through activation of the sterol regulatory element-binding protein (SREBP) transcription factors. We here compare the action of chlorpromazine, haloperidol, clozapine, olanzapine, risperidone and ziprasidone on SREBP activation and SREBP-controlled gene expression (ACAT2, HMGCR, HMGCS1, FDPS, SC5DL, DHCR7, LDLR, FASN and SCD1) in four CNS-relevant human cell lines.ResultsThere were marked differences in the ability of the antipsychotic drugs to activate the expression of SREBP target genes, with clozapine and chlorpromazine as the most potent stimulators in a context of therapeutically relevant concentrations. Glial-like cells (GaMg glioma and CCF-STTG1 astrocytoma cell lines) displayed more pronounced drug-induced SREBP activation compared to the response in HCN2 human cortical neurons and SH-SY5Y neuroblastoma cells, indicating that antipsychotic-induced activation of lipogenesis is most prominent in glial cells.ConclusionOur present data show a marked variation in the ability of different antipsychotics to induce SREBP-controlled transcriptional activation of lipogenesis in cultured human CNS-relevant cells. We propose that this effect could be relevant for the therapeutic efficacy of some antipsychotic drugs.

Synaptic activity-induced global gene expression patterns in the dentate gyrus of adult behaving rats: Induction of immunity-linked genes

Gene expression in adult neuronal circuits is dynamically modulated in response to synaptic activity. Persistent changes in synaptic strength, as seen during high-frequency stimulation (HFS)-induced long-term potentiation (LTP), require new gene expression. While modulation of many individual genes has been shown, an understanding of LTP as a complex dynamical response requires elucidation of the global gene expression signature and its impact on biologically meaningful gene sets. In this study, we demonstrate that LTP induction in the dentate gyrus of awake freely moving rats was associated with changes in the expression of genes linked to signal transduction, protein trafficking, cell structure and motility, and other processes consistent with the induction of mechanisms of synaptic reorganization and growth. Interestingly, the most significantly over-represented gene sets were related to immunity and defense, including T-cell-mediated immunity and major histocompatibility complex (MHC) class I-mediated immunity. Real-time PCR confirmed the upregulation of a panel of immune-linked genes including the rt1-a/ce family, and the MHC class II members cd74, rt1-Ba and rt1-Da. These genes were N-methyl-d-aspartate receptor-independent and not induced following HFS-LTP induction in anesthetized rats, indicating a gene response specific to behaving rats. Our data support recent assumptions that immunity-associated processes are functionally linked to adaptive neuronal responses in the brain, although the differential expression of immunity-linked genes could also be related to the HFS per se.

Lithium differentially affects clock gene expression in serum-shocked NIH-3T3 cells

Bipolar disorder has been associated with disturbances in circadian rhythms. Lithium is frequently used in the long-term treatment of bipolar disorder, and has been shown to prolong such rhythms in animals and humans. To examine whether lithium affects the expression of genes regulating the circadian clock, cultured NIH-3T3 cells were synchronized by serum-shocking, and the relative expression of the clock genes Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), Cryptochrome2 (Cry2), Brain and muscle aryl hydrocarbon nuclear translocator-like 1 (Bmal1), Circadian locomotor output cycles kaput (Clock), Rev-Erb-α (Nr1d1), RAR-related orphan receptor α (Ror-α), Glycogen synthase kinase-3β (Gsk-3β), Casein kinase 1−ε (CK1-ε; Csnk1ε), E4 binding protein 4 (E4BP4; Nfil-3) and albumin D-binding protein (Dbp) was examined for three consecutive days in the presence of lithium (20 mM) or vehicle (20 mM NaCl). We found that lithium significantly increased the expression of Per2 and Cry1, whereas Per3, Cry2, Bmal1, E4BP4 and Rev-Erb-α expression was reduced. We also found that lithium prolonged the period of Per2. Taken together, these effects on clock gene expression may be relevant for the effects of lithium on biological rhythms and could also give new leads to further explore its mood-stabilizing actions in the treatment of bipolar disorder.

Variants in doublecortin- and calmodulin kinase like 1, a gene up-regulated by BDNF, are associated with memory and general cognitive abilities

Background Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF) plays an important role in mammalian memory formation. Methodology / Principal Finding Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1) gene, are significantly associated with general cognition (IQ scores) and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus). We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. Conclusion These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.