Pairwise effects between lipid GWAS genes modulate lipid plasma levels and cellular uptake.

Complex traits are characterized by multiple genes and variants acting simultaneously on a phenotype. However, studying the contribution of individual pairs of genes to complex traits has been challenging since human genetics necessitates very large population sizes, while findings from model systems do not always translate to humans. Here, we combine genetics with combinatorial RNAi (coRNAi) to systematically test for pairwise additive effects (AEs) and genetic interactions (GIs) between 30 lipid genome-wide association studies (GWAS) genes. Gene-based burden tests from 240,970 exomes show that in carriers with truncating mutations in both, APOB and either PCSK9 or LPL ("human double knock-outs") plasma lipid levels change additively. Genetics and coRNAi identify overlapping AEs for 12 additional gene pairs. Overlapping GIs are observed for TOMM40/APOE with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate plasma and cellular lipid levels primarily via AEs and nominates putative drug target pairs for improved lipid-lowering combination therapies.

Neurocan genome-wide psychiatric risk variant affects explicit memory performance and hippocampal function in healthy humans.

Alterations of the brain extracellular matrix (ECM) can perturb the structure and function of brain networks like the hippocampus, a key region in human memory that is commonly affected in psychiatric disorders. Here, we investigated the potential effects of a genome-wide psychiatric risk variant in the NCAN gene encoding the ECM proteoglycan neurocan (rs1064395) on memory performance, hippocampal function and cortical morphology in young, healthy volunteers. We assessed verbal memory performance in two cohorts (N = 572, 302) and found reduced recall performance in risk allele (A) carriers across both cohorts. In 117 participants, we performed functional magnetic resonance imaging using a novelty-encoding task with visual scenes. Risk allele carriers showed higher false alarm rates during recognition, accompanied by inefficiently increased left hippocampal activation. To assess effects of rs1064395 on brain morphology, we performed voxel-based morphometry in 420 participants from four independent cohorts and found lower grey matter density in the ventrolateral and rostral prefrontal cortex of risk allele carriers. In silico eQTL analysis revealed that rs1064395 SNP is linked not only to increased prefrontal expression of the NCAN gene itself, but also of the neighbouring HAPLN4 gene, suggesting a more complex effect of the SNP on ECM composition. Our results suggest that the NCAN rs1064395 A allele is associated with lower hippocampus-dependent memory function, variation of prefrontal cortex structure and ECM composition. Considering the well-documented hippocampal and prefrontal dysfunction in bipolar disorder and schizophrenia, our results may reflect an intermediate phenotype by which NCAN rs1064395 contributes to disease risk.

Variants of Lipid-Related Genes in Adult Japanese Patients with Severe Hypertriglyceridemia.

AIM: Hypertriglyceridemia is a type of dyslipidemia that contributes to atherosclerosis and coronary heart disease. Variants in lipoprotein lipase (LPL), apolipoprotein CII (APOC2), apolipoprotein AV (APOA5), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), lipase maturation factor 1 (LMF1), and glucokinase regulator (GCKR) are responsible for hypertriglyceridemia. We investigated the molecular basis of severe hypertriglyceridemia in adult patients referred to the Clinical Laboratory at Fukuoka University Hospital. METHODS: Twenty-three adult patients with severe hypertriglyceridemia (＞1,000 mg/dL, 11.29 mmol/L) were selected. The coding regions of candidate genes were sequenced by next-generation sequencing. Forty-nine genes reportedly associated with hypertriglyceridemia were analyzed. RESULTS: In the 23 patients, we detected 70 variants: 28 rare and 42 common ones. Among the 28 rare variants with ＜1% allele frequency, p.I4533L in APOB, p.M490I in MLXIPL, p.L152M in NCAN, and p.S264T in TIMD4 were novel. We did not observe single gene homozygous or compound heterozygous disease-causing rare variants in any of the 23 hypertriglyceridemia cases. However, in silico algorithms and previous reports indicated that five rare variants, APOA5 (p.T184S), GCKR (c.354＋1G＞A), LMF1 (p.G410R), and LRP1 (p.G813R; p.R2173Q), and seven common variants, APOA5 (pG185C), APOE (p.C130R; p.E262K/p.E263K), GCKR (p.V103M), GPIHBP1 (p.C14F), LRP1 (p.Y4054F), and MLXIPL (p.Q241H), can cause hypertriglyceridemia. However, all five disease-causing rare variants detected in this study were heterozygous. CONCLUSIONS: The prevalence of disease-causing rare variants in candidate genes in severe hypertriglyceridemia patients was low. The major causes of severe hypertriglyceridemia were not single gene abnormalities, but involved multiple gene variations and environmental factors.

Neuronal and perineuronal changes of cerebral cortex after exposure to inhaled particulate matter.

The inhalation of particulate matter (PM) increases the perineuronal nets (PNNs) in the cerebral cortex; however, little is known about the related molecular changes. We explored how PM exposure impacted cognitive function and the levels of PNN-related genes. BALB/c mice (6-week-old females, n = 32) were exposed to 1-5-µm diesel-extracted particles (DEPs) (100 µg/m3, 5 hours per day, 5 days per week) and categorized into the following four groups: 1) 4-week DEP exposure (n = 8); 2) 4-week control (n = 8); 3) 8-week DEP exposure (n = 8); and 4) 8-week control (n = 8). The Y-maze test and olfactory function test were conducted after 4 and 8 weeks of DEP exposure. The prefrontal cortex, olfactory bulb and temporal cortex were harvested from the animals in each group. The expression of genes related to PNNs (Tenascin C, matrix metalloproteinase [MMP]14, MMP9) and synaptic vesicular transporters of vesicular glutamergic transporter 1 (VGLUT1), VGLUT2, vesicular GABAergic transporter (VGAT) were measured. The temporal cortex was immunostained for neurocan, VGLUT1, and VGAT. The 4-week DEP group had lower total arm entry in the Y-maze test and olfactory sensitivity. These impaired behavioral functions recovered in the 8-week DEP group. Expression of tenascin C and MMP9 were increased in the cerebral cortex in the 8-week DEP group compared with the control group. The levels of VGLUT1, VGLUT2, and VGAT were elevated in the cerebral cortex of the 8-week DEP group compared with the control group. In immunostaining of the temporal cortex, the expression of neurocan, VGLUT1, and GAD67 were increased in the 8-week DEP group compared with the control group. The 4-week DEP inhalation impaired spatial activities and olfactory sensitivities. After 8 weeks of DEP exposure, the PNN components and their proteolytic enzymes and the vesicular transporters increased in the cerebral cortex.

Intrinsic cellular and molecular properties of in vivo hippocampal synaptic plasticity are altered in the absence of key synaptic matrix molecules.

Hippocampal synaptic plasticity comprises a key cellular mechanism for information storage. In the hippocampus, both long-term potentiation (LTP) and long-term depression (LTD) are triggered by synaptic Ca2+ -elevations that are typically mediated by the opening of voltage-gated cation channels, such as N-methyl-d-aspartate receptors (NMDAR), in the postsynaptic density. The integrity of the post-synaptic density is ensured by the extracellular matrix (ECM). Here, we explored whether synaptic plasticity is affected in adult behaving mice that lack the ECM proteins brevican, neurocan, tenascin-C, and tenascin-R (KO). We observed that the profiles of synaptic potentiation and depression in the dentate gyrus (DG) were profoundly altered compared to plasticity profiles in wild-type littermates (WT). Specifically, synaptic depression was amplified in a frequency-dependent manner and although late-LTP (>24 hr) was expressed following strong afferent tetanization, the early component of LTP (<75 min post-tetanization) was absent. LTP (>4 hr) elicited by weaker tetanization was equivalent in WT and KO animals. Furthermore, this latter form of LTP was NMDAR-dependent in WT but not KO mice. Scrutiny of DG receptor expression revealed significantly lower levels of both the GluN2A and GluN2B subunits of the N-methyl-d-aspartate receptor, of the metabotropic glutamate receptor, mGlu5 and of the L-type calcium channel, Cav 1.3 in KO compared to WT animals. Homer 1a and of the P/Q-type calcium channel, Cav 1.2 were unchanged in KO mice. Taken together, findings suggest that in mice that lack multiple ECM proteins, synaptic plasticity is intact, but is fundamentally different.

Beneficial effects of local profound hypothermia and the possible mechanism after experimental spinal cord injury in rats.

OBJECTIVE: The primary focus of this study was to investigate the effects of local profound hypothermia and to explore the possible mechanism in adult rats with spinal cord injury. STUDY DESIGN AND METHODS: Spinal cord injury models were established by placing aneurysm clips on T10. An epidural perfusion device was applied to maintain a steady temperature (18 °C) for 120 min with gradual rewarming to 37 °C Total hypothermic duration lasted up to about 170 min. The expression of axon regeneration inhibitors was tested by Western blot and real-time PCR. Luxol Fast Blue (LFB) stain and Bielschowsky silver stain were used to observe spinal cord morphology. Motor function of the hind limbs (BBB score) was monitored for 21 days. RESULTS: The expressions of RhoA, ROCK-II, NG2, Neurocan, Brevican, and Nogo-A were downregulated by regional hypothermia (RH) after spinal cord injury. Subsequent observation showed that rats that had received RH had an alleviated demyelinating condition and a greater number of nerve fibers. Furthermore, the RH group achieved higher BBB scores than the spinal cord injury (SCI) group. CONCLUSIONS: Recovery of hind limb function in rats can be promoted by local profound hypothermia; this may be caused by the suppression of axon regeneration inhibitors.

The Role of bFGF in the Excessive Activation of Astrocytes Is Related to the Inhibition of TLR4/NFκB Signals.

Astrocytes have critical roles in immune defense, homeostasis, metabolism, and synaptic remodeling and function in the central nervous system (CNS); however, excessive activation of astrocytes with increased intermediate filaments following neuronal trauma, infection, ischemia, stroke, and neurodegenerative diseases results in a pro-inflammatory environment and promotes neuronal death. As an important neurotrophic factor, the secretion of endogenous basic fibroblast growth factor (bFGF) contributes to the protective effect of neuronal cells, but the mechanism of bFGF in reactive astrogliosis is still unclear. In this study, we demonstrated that exogenous bFGF attenuated astrocyte activation by reducing the expression of glial fibrillary acidic protein (GFAP) and other markers, including neurocan and vimentin, but not nestin and decreased the levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), via the regulation of the upstream toll-like receptor 4/nuclear factor κB (TLR4/NFκB) signaling pathway. Our study suggests that the function of bFGF is not only related to the neuroprotective and neurotrophic effect but also involved in the inhibition of excessive astrogliosis and glial scarring after neuronal injury.

Neuronal and astroglial TGFß-Smad3 signaling pathways differentially regulate dendrite growth and synaptogenesis.

To address the role of the transforming growth factor beta (TGFß)-Smad3 signaling pathway in dendrite growth and associated synaptogenesis, we used small inhibitory RNA to knockdown the Smad3 gene in either cultured neurons and or primary astrocytes. We found that TGFß1 treatment of primary neurons increased dendrite extensions and the number of synapsin-1-positive synapses. When Smad3 was knockdown in primary neurons, dendrite growth was inhibited and the number of synapsin-1-positive synapses reduced even with TGFß1 treatment. When astrocyte-conditioned medium (ACM), collected from TGFß1-treated astrocytes (TGFß1-stimulated ACM), was added to cultured neurons, dendritic growth was inhibited and the number of synapsin-1-positive puncta reduced. When TGFß1-stimulated ACM was collected from astrocytes with Smad3 knocked down, this conditioned media promoted the growth of dendrites and the number of synapsin-1-positive puncta in cultured neurons. We further found that TGFß1 signaling through Smad3 increased the expression of chondroitin sulfate proteoglycans, neurocan, and phosphacan in ACM. Application of chondroitinase ABC to the TGFß1-stimulated ACM reversed its inhibitory effects on the dendrite growth and the number of synapsin-1-positive puncta. On the other hand, we found that TGFß1 treatment caused a facilitation of Smad3 phosphorylation and translocation to the nucleus induced by status epilepticus (SE) in wild-type (Smad3(+/+)) mice, and this treatment also caused a promotion of γ-aminobutyric acid-ergic synaptogenesis impaired by SE in Smad3(+/+) as well as in Smad3(-/-) mice, but more dramatic promotion in Smad3(+/+) mice. Thus, we provide evidence for the first time that TGFß-Smad3 signaling pathways within neuron and astrocyte differentially regulate dendrite growth and synaptogenesis, and this pathway may be involved in the pathogenesis of some central nervous system diseases, such as epilepsy.

Selective expression of hyaluronan and receptor for hyaluronan mediated motility (Rhamm) in the adult mouse subventricular zone and rostral migratory stream and in ischemic cortex.

Hyaluronan is a large glycosaminoglycan, which is abundant in the extracellular matrix of the developing rodent brain. In the adult brain however, levels of hyaluronan are significantly reduced. In this study, we used neurocan-GFP as a histochemical probe to analyze the distribution of hyaluronan in the adult mouse subventricular zone (SVZ), as well as in the rostral migratory stream (RMS). Interestingly, we observed that hyaluronan is generally downregulated in the adult brain, but notably remains at high levels in the SVZ and RMS; areas in which neural stem/progenitor cells (NSPCs) persist, proliferate and migrate throughout life. In addition, we found that the receptor for hyaluronan-mediated motility (Rhamm) was expressed in migrating neuroblasts in these areas, indicating that Rhamm could be involved in regulating hyaluronan-mediated cell migration. Hyaluronan levels are balanced by synthesis through hyaluronan synthases (Has) and degradation by hyaluronidases (Hyal). We found that Has1 and Has2, as well as Hyal1 and Hyal2 were expressed in GFAP positive cells in the adult rodent SVZ and RMS, indicating that astrocytes could be regulating hyaluronan-mediated functions in these areas. We also demonstrate that hyaluronan levels are substantially increased at six weeks following a photothrombotic stroke lesion to the adult mouse cortex. Furthermore, GFAP positive cells in the peri-infarct area express Rhamm. Thus, hyaluronan may be involved in regulating cell migration in the normal SVZ and RMS and could also be responsible for priming the peri-infarct area following an ischemic lesion for cell migration.

Studies in humans and mice implicate neurocan in the etiology of mania.

OBJECTIVE: Genome-wide association has been reported between the NCAN gene and bipolar disorder. The aims of this study were to characterize the clinical symptomatology most strongly influenced by NCAN and to explore the behavioral phenotype of Ncan knockout (Ncan(-/-)) mice. METHOD: Genotype/phenotype correlations were investigated in patients with bipolar disorder (N=641) and the genetically related disorders major depression (N=597) and schizophrenia (N=480). Principal components and genotype association analyses were used to derive main clinical factors from 69 lifetime symptoms and to determine which of these factors were associated with the NCAN risk allele. These analyses were then repeated using the associated factor(s) only in order to identify the more specific clinical subdimensions that drive the association. Ncan(-/-) mice were tested using diverse paradigms, assessing a range of behavioral traits, including paradigms corresponding to bipolar symptoms in humans. RESULTS: In the combined patient sample, the NCAN risk allele was significantly associated with the "mania" factor, in particular the subdimension "overactivity." Ncan(-/-) mice were hyperactive and showed more frequent risk-taking and repetitive behaviors, less depression-like conduct, impaired prepulse inhibition, amphetamine hypersensitivity, and increased saccharin preference. These aberrant behavioral responses normalized after the administration of lithium. CONCLUSIONS: NCAN preferentially affected mania symptoms in humans. Ncan(-/-) mice showed behavioral abnormalities that were strikingly similar to those of the human mania phenotype and may thus serve as a valid mouse model.

Genome-wide association study identifies genetic variation in neurocan as a susceptibility factor for bipolar disorder.

We conducted a genome-wide association study (GWAS) and a follow-up study of bipolar disorder (BD), a common neuropsychiatric disorder. In the GWAS, we investigated 499,494 autosomal and 12,484 X-chromosomal SNPs in 682 patients with BD and in 1300 controls. In the first follow-up step, we tested the most significant 48 SNPs in 1729 patients with BD and in 2313 controls. Eight SNPs showed nominally significant association with BD and were introduced to a meta-analysis of the GWAS and the first follow-up samples. Genetic variation in the neurocan gene (NCAN) showed genome-wide significant association with BD in 2411 patients and 3613 controls (rs1064395, p = 3.02 × 10(-8); odds ratio = 1.31). In a second follow-up step, we replicated this finding in independent samples of BD, totaling 6030 patients and 31,749 controls (p = 2.74 × 10(-4); odds ratio = 1.12). The combined analysis of all study samples yielded a p value of 2.14 × 10(-9) (odds ratio = 1.17). Our results provide evidence that rs1064395 is a common risk factor for BD. NCAN encodes neurocan, an extracellular matrix glycoprotein, which is thought to be involved in cell adhesion and migration. We found that expression in mice is localized within cortical and hippocampal areas. These areas are involved in cognition and emotion regulation and have previously been implicated in BD by neuropsychological, neuroimaging, and postmortem studies.