CSMD1 regulates brain complement activity and circuit development.

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

Neuropsychological deficits in mice depleted of the schizophrenia susceptibility gene CSMD1.

Recent meta-analyses of schizophrenia genome-wide association studies (GWASs) have identified the CUB and SUSHI multiple domains 1 (CSMD1) gene as a statistically strong risk factor. CSMD1 is a complement control-related protein suggested to inhibit the classical complement pathway, being expressed in developing neurons. However, expression of CSMD1 is largely uncharacterized and relevance for behavioral phenotypes is not previously demonstrated. Here, we assess neuropsychological behaviors of a Csmd1 knockout (KO) mouse in a selection of standard behavioral tests. Deregulation of neuropsychological responses were observed in both the open field and the elevated plus maze tests, in which KO mice spent 55% and 33% less time than WT littermate mice in open areas, respectively. Altered behaviors were also observed in tail suspension and to higher acoustic stimuli, for which Csmd1 KO mice showed helplessness and moderate increase in startle amplitude, respectively. Furthermore, Csmd1 KO mice also displayed increased weight-gain and glucose tolerance, similar to a major phenotype of the metabolic syndrome that also has been associated to the human CSMD1 locus. Consistent with a role in the control of behaviors, Csmd1 was found highly expressed in the central nervous system (CNS), and with some expression in visceral fat and ovary, under tissue-specific control by a novel promoter-associated lncRNA. In summary, disruption of Csmd1 induces behaviors reminiscent of blunted emotional responses, anxiety and depression. These observations suggest an influence of the CSMD1 schizophrenia susceptibility gene on psychopathology and endophenotypes of the negative symptom spectra.

Silencing of doublecortin-like (DCL) results in decreased mitochondrial activity and delayed neuroblastoma tumor growth.

Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy.

LOC689986, a unique gene showing specific expression in restricted areas of the rodent neocortex.

BACKGROUND: The neocortex is a highly specialised and complex brain structure, involved in numerous tasks, ranging from processing and interpretation of somatosensory information, to control of motor functions. The normal function linked to distinct neocortical areas might involve control of highly specific gene expression, and in order to identify such regionally enriched genes, we previously analysed the global gene expression in three different cortical regions (frontomedial, temporal and occipital cortex) from the adult rat brain. We identified distinct sets of differentially expressed genes. One of these genes, namely the hypothetical protein LOC689986 (LOC689986), was of particular interest, due to an almost exclusive expression in the temporal cortex. RESULTS: Detailed analysis of LOC689986 in the adult rat brain confirmed the expression in confined areas of parieto-temporal cortex, and revealed highly specific expression in layer 4 of the somatosensory cortex, with sharp borders towards the neighbouring motor cortex. In addition, LOC689986 was found to be translated in vivo, and was detected in the somatosensory cortex and in the Purkinje cells of the cerebellar cortex. The protein was present in neuronal dendrites and also in astrocyte cells. Finally, this unique gene is apparently specific for, and highly conserved in, the vertebrate lineage. CONCLUSIONS: In this study, we have partially characterised the highly conserved LOC689986 gene, which is specific to the vertebrate linage. The gene displays a distinct pattern of expression in layer 4 of the somatosensory cortex, and areas of the parieto-temporal cortex in rodents.

Doublecortin and doublecortin-like are expressed in overlapping and non-overlapping neuronal cell population: implications for neurogenesis.

We have characterized the expression of doublecortin-like (DCL), a microtubule-associated protein involved in embryonic neurogenesis that is highly homologous to doublecortin (DCX), in the adult mouse brain. To this end, we developed a DCL-specific antibody and used this to compare DCL expression with DCX. In the neurogenic regions of the adult brain like the subventricular zone (SVZ), the rostral migratory stream (RMS), the olfactory bulb (OB), and the hippocampus, DCL colocalizes with DCX in immature neuronal cell populations. In contrast to DCX, we also found high DCL expression in three other brain regions with suspected neurogenesis or neuronal plasticity. First, the radial glia-like, hypothalamic tanycytes show high DCL expression that partly colocalizes with the neural stem cell marker vimentin. Second, DCL expression is found in cells of the suprachiasmatic nucleus (SCN), which lacks expression of the adult neuron marker NeuN. Third, a novel region exhibiting DCL expression is part of the olfactory tubercle where DCL is found in the neuropil of the islands of Calleja (ICj). Our findings define DCL as a novel marker for specific aspects of adult neurogenesis, which partly overlap with DCX. In addition, we propose unique roles for DCL in adult neurogenesis and we suggest high levels of neuronal plasticity in tanycytes, SCN, and ICj.

DCLK1 variants are associated across schizophrenia and attention deficit/hyperactivity disorder.

Doublecortin and calmodulin like kinase 1 (DCLK1) is implicated in synaptic plasticity and neurodevelopment. Genetic variants in DCLK1 are associated with cognitive traits, specifically verbal memory and general cognition. We investigated the role of DCLK1 variants in three psychiatric disorders that have neuro-cognitive dysfunctions: schizophrenia (SCZ), bipolar affective disorder (BP) and attention deficit/hyperactivity disorder (ADHD). We mined six genome wide association studies (GWASs) that were available publically or through collaboration; three for BP, two for SCZ and one for ADHD. We also genotyped the DCLK1 region in additional samples of cases with SCZ, BP or ADHD and controls that had not been whole-genome typed. In total, 9895 subjects were analysed, including 5308 normal controls and 4,587 patients (1,125 with SCZ, 2,496 with BP and 966 with ADHD). Several DCLK1 variants were associated with disease phenotypes in the different samples. The main effect was observed for rs7989807 in intron 3, which was strongly associated with SCZ alone and even more so when cases with SCZ and ADHD were combined (P-value = 4 × 10(-5) and 4 × 10(-6), respectively). Associations were also observed with additional markers in intron 3 (combination of SCZ, ADHD and BP), intron 19 (SCZ+BP) and the 3'UTR (SCZ+BP). Our results suggest that genetic variants in DCLK1 are associated with SCZ and, to a lesser extent, with ADHD and BP. Interestingly the association is strongest when SCZ and ADHD are considered together, suggesting common genetic susceptibility. Given that DCLK1 variants were previously found to be associated with cognitive traits, these results are consistent with the role of DCLK1 in neurodevelopment and synaptic plasticity.

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 Crohn's 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.

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-3ß (Gsk-3ß), 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.

Acute clozapine exposure in vivo induces lipid accumulation and marked sequential changes in the expression of SREBP, PPAR, and LXR target genes in rat liver.

BACKGROUND: Several antipsychotic drugs (APDs) have high propensity to induce weight gain and dyslipidemia in patients, with clozapine and olanzapine as the most potent drugs. These lipid-related effects have been attributed to drug-mediated blockade or antagonism of histamine H1 and serotonin 5-HT2 receptors as well as activation of hypothalamic AMP-activated protein kinase. We recently showed that APDs activate lipid biosynthesis in cultured liver cells through stimulation of the sterol regulatory element-binding protein (SREBP) transcription factors. OBJECTIVE: The objective of the study was to search for clozapine-related lipogenic effects in peripheral tissues in vivo using rat liver as target organ. MATERIALS AND METHODS: Adult female Sprague-Dawley rats were administered single intraperitoneal injections of clozapine (25 and 50 mg/kg). Hepatic lipid levels were measured during a 48-h time course. Real-time quantitative PCR was used to analyze expression of genes involved in lipid biosynthesis, oxidation, efflux, and lipolysis. RESULTS: We identified an initial up-regulation of central lipogenic SREBP target genes, followed by a marked and sustained down-regulation. We also observed a sequential transcriptional response for fatty acid beta-oxidation and cholesterol efflux genes, normally controlled by the peroxisome proliferator activated receptor alpha and liver X receptor alpha transcription factors, and also down-regulation of genes encoding major lipases. The transcriptional responses were associated with a significant accumulation of triacylglycerol, phospholipids, and cholesterol in the liver. CONCLUSION: These results demonstrate that acute clozapine exposure affects SREBP-regulated lipid biosynthesis as well as other lipid homeostasis pathways. We suggest that such drug-induced effects on lipid metabolism in peripheral tissues are relevant for the metabolic adverse effects associated with clozapine and possibly other APDs.

Additive viability-loss following hsp70/hsc70 double interference and Hsp90 inhibition in two breast cancer cell lines.

Hsp70 is an anti-apoptotic protein over-expressed in breast, lung, rectum, endometrial and bladder cancers. In this study, we investigated the impact of Hsp70 protein expression, and its close family member, Hsc70, on breast cancer cell viability and on the activity of the Hsp90/Hsp70 chaperone complex, a complex whose activity is important for the survival of cancer cells and tumours. The simultaneous blockade of Hsp70, Hsc70 and Hsp90 was most efficient in reducing breast cancer cell viability, as compared to their respective separate blockades. However, while the Hsp90 inhibition alone correlates with lost cell viability and reduced Hsp90/Hsp70 chaperone complex activity, the single or mixed reduction in Hsp70 and Hsc70 expression displayed no ability to reduce the activity of the Hsp90/Hsp70 chaperone complex. The results suggest that targeting both the Hsp70 family, as well as the Hsp90 protein will have an additive negative effect on cancer cell survival, even though their pathways of action are separate.

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 alpha-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.

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

BACKGROUND: The 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. RESULTS: There 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. CONCLUSION: Our 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.

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-beta-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.

Bursts of high-frequency stimulation trigger rapid delivery of pre-existing alpha-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.