[Effect of Naotaifang on microglial polarization and glial scar following cerebral ischemia reperfusion injury].

This study aims to investigate the effect of Naotaifang(NTF) on the proteins associated with microglial polarization and glial scar in the rat model of cerebral ischemia reperfusion injury(CIRI). The CIRI model was established by middle cerebral artery occlusion/reperfusion. The 48 successfully modeled rats were randomized into model 7 d, model 14 d, NTF 7 d, and NTF 14 d groups(n=12). In addition, 12 SD rats were selected as the sham group. The NTF group was administrated with NTF suspension at 27 g·kg~(-1)·d~(-1) by gavage, and the sham, model 7 d, and model 14 d groups were administrated with the same volume of normal saline every day by gavage for 7 and 14 days, respectively. After the intervention, Longa score was evaluated. The infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) staining. Morris water maze and open field tests were carried out to evaluate the spatial learning, memory, cognitive function, and anxiety degree of rats. Hematoxylin-eosin(HE) staining was employed to observe the morphological structure and damage of the brain tissue. The immunofluorescence assay was employed to measure the expression of glial fibrillary acidic protein(GFAP) and glial scar. Western blot was employed to determine the protein levels of GFAP, neurocan, phosphacan, CD206, arginase-1(Arg-1), interleukin(IL)-1ß, IL-6, and IL-4. Compared with the sham, model 7 d and model 14 d groups showed cerebral infarction of different degrees, severe pathological injury of cerebral cortex and hippocampus, neurological impairment, reduced spatial learning and memory, cognitive dysfunction, severe anxiety, astrocyte hyperplasia, thickening penumbra glial scar, and up-regulated protein levels of IL-1ß, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P<0.01). Compared with the model group, NTF 7 d and NTF 14 d groups improved spatial learning, memory, and cognitive function, reduced anxiety, improved nerve function, reduced cerebral infarction volume, reduced astrocyte hyperplasia, thinned penumbra glial scar, down-regulated the protein levels of GFAP, neurocan, phosphacan, IL-6, and IL-1ß, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P<0.05 or P<0.01). NTF exerts a neuroprotective effect on CIRI by inducing the M2 polarization of microglia, inhibiting inflammatory response, and reducing the formation of glial scar.

Electroacupuncture promotes the repair of the damaged spinal cord in mice by mediating neurocan-perineuronal net.

AIMS: This study aimed to investigate the effect of perineuronal net (PNN) and neurocan (NCAN) on spinal inhibitory parvalbumin interneuron (PV-IN), and the mechanism of electroacupuncture (EA) in promoting spinal cord injury (SCI) repair through neurocan in PNN. METHODS: A mouse model of SCI was established. Sham-operated mice or SCI model mice were treated with chondroitin sulfate ABC (ChABC) enzyme or control vehicle for 2 weeks (i.e., sham+veh group, sham+ChABC group, SCI+veh group, and SCI+ChABC group, respectively), and then spinal cord tissues were taken from the T10 lesion epicenter for RNA sequencing (RNA-seq). MSigDB Hallmark and C5 databases for functional analysis, analysis strategies such as differential expression gene analysis (DEG), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI). According to the results of RNA-seq analysis, the expression of NCAN was knocked down or overexpressed by virus intervention, or/and EA intervention. Polymerase chain reaction (PCR), immunofluorescence, western blot, electrophysiological, and behavioral tests were performed. RESULTS: After the successful establishment of SCI model, the motor dysfunction of lower limbs, and the expression of PNN core glycan protein at the epicenter of SCI were reduced. RNA-seq and PCR showed that PNN core proteoglycans except NCAN showed the same expression trend in normal and injured spinal cord treated with ChABC. KEGG and GSEA showed that PNN is mainly associated with inhibitory GABA neuronal function in injured spinal cord tissue, and PPI showed that NCAN in PNN can be associated with inhibitory neuronal function through parvalbumin (PV). Calcium imaging showed that local parvalbumin interneuron (PV-IN) activity decreased after PNN destruction, whether due to ChABC treatment or surgical bruising of the spinal cord. Overexpression of neurocan in injured spinal cord can enhance local PV-IN activity. PCR and western blot suggested that overexpression or knockdown of neurocan could up-regulate or down-regulate the expression of GAD. At the same time, the activity of PV-IN in the primary motor cortex (M1) and the primary sensory cortex of lower (S1HL) extremity changed synchronously. In addition, overexpression of neurocan improved the electrical activity of the lower limb and promoted functional repair of the paralyzed hind limb. EA intervention reversed the down-regulation of neurocan, enhanced the expression of PNN in the lesioned area, M1 and S1HL. CONCLUSION: Neurocan in PNN can regulate the activity of PV-IN, and EA can promote functional recovery of mice with SCI by upregulating neurocan expression in PNN.

Neurocan expression associates with better survival and viral positivity in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.

Identification of Neurocan and Phosphacan as Early Biomarkers for Open Neural Tube Defects.

Open neural tube defects (NTDs) such as myelomeningocele (MMC) are debilitating and the most common congenital defects of the central nervous system. Despite their apparent clinical importance, the existing early prenatal diagnostic options for these defects remain limited. Using a well-accepted retinoic-acid-induced model of MMC established in fetal rats, we discovered that neurocan and phosphacan, the secreted chondroitin sulfate proteoglycans of the developing nervous system, are released into the amniotic fluid (AF) of fetal rats displaying spinal cord defects. In contrast to normal controls, elevated AF levels of neurocan and phosphacan were detected in MMC fetuses early in gestation and continued to increase during MMC progression, reaching the highest level in near-term fetuses. The molecular forms of neurocan and phosphacan identified in the AF of MMC fetuses and those found in MMC spinal cords were qualitatively similar. In summary, this is the first report demonstrating the presence of neurocan and phosphacan in the AF of MMC fetuses. The identification of elevated levels of neurocan and phosphacan in the AF of MMC fetuses provides two prospective biomarkers with the potential for early prenatal diagnosis of open NTDs.

Extracellular Matrix Changes in Subcellular Brain Fractions and Cerebrospinal Fluid of Alzheimer's Disease Patients.

The brain's extracellular matrix (ECM) is assumed to undergo rearrangements in Alzheimer's disease (AD). Here, we investigated changes of key components of the hyaluronan-based ECM in independent samples of post-mortem brains (N = 19), cerebrospinal fluids (CSF; N = 70), and RNAseq data (N = 107; from The Aging, Dementia and TBI Study) of AD patients and non-demented controls. Group comparisons and correlation analyses of major ECM components in soluble and synaptosomal fractions from frontal, temporal cortex, and hippocampus of control, low-grade, and high-grade AD brains revealed a reduction in brevican in temporal cortex soluble and frontal cortex synaptosomal fractions in AD. In contrast, neurocan, aggrecan and the link protein HAPLN1 were up-regulated in soluble cortical fractions. In comparison, RNAseq data showed no correlation between aggrecan and brevican expression levels and Braak or CERAD stages, but for hippocampal expression of HAPLN1, neurocan and the brevican-interaction partner tenascin-R negative correlations with Braak stages were detected. CSF levels of brevican and neurocan in patients positively correlated with age, total tau, p-Tau, neurofilament-L and Aß1-40. Negative correlations were detected with the Aß ratio and the IgG index. Altogether, our study reveals spatially segregated molecular rearrangements of the ECM in AD brains at RNA or protein levels, which may contribute to the pathogenic process.

Reorganization of the Brain Extracellular Matrix in Hippocampal Sclerosis.

The extracellular matrix (ECM) is an important regulator of excitability and synaptic plasticity, especially in its highly condensed form, the perineuronal nets (PNN). In patients with drug-resistant mesial temporal lobe epilepsy (MTLE), hippocampal sclerosis type 1 (HS1) is the most common histopathological finding. This study aimed to evaluate the ECM profile of HS1 in surgically treated drug-resistant patients with MTLE in correlation to clinical findings. Hippocampal sections were immunohistochemically stained for aggrecan, neurocan, versican, chondroitin-sulfate (CS56), fibronectin, Wisteria floribunda agglutinin (WFA), a nuclear neuronal marker (NeuN), parvalbumin (PV), and glial-fibrillary-acidic-protein (GFAP). In HS1, besides the reduced number of neurons and astrogliosis, we found a significantly changed expression pattern of versican, neurocan, aggrecan, WFA-specific glycosylation, and a reduced number of PNNs. Patients with a lower number of epileptic episodes had a less intense diffuse WFA staining in Cornu Ammonis (CA) fields. Our findings suggest that PNN reduction, changed ECM protein, and glycosylation expression pattern in HS1 might be involved in the pathogenesis and persistence of drug-resistant MTLE by contributing to the increase of CA pyramidal neurons' excitability. This research corroborates the validity of ECM molecules and their modulators as a potential target for the development of new therapeutic approaches to drug-resistant epilepsy.

A comprehensive atlas of Aggrecan, Versican, Neurocan and Phosphacan expression across time in wildtype retina and in retinal degeneration.

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1-/- and Pde6brd1/rd1, while Versican showed regional increases in the periphery of Rho-/- mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair.

Neurocan regulates vulnerability to stress and the anti-depressant effect of ketamine in adolescent rats.

Depression is more prevalent among adolescents than adults, but the underlying mechanisms remain largely unknown. Using a subthreshold chronic stress model, here we show that developmentally regulated expressions of the perineuronal nets (PNNs), and one of the components, Neurocan in the prelimbic cortex (PrL) are important for the vulnerability to stress and depressive-like behaviors in both adolescent and adult rats. Reduction of PNNs or Neurocan with pharmacological or viral methods to mimic the expression of PNNs in the PrL during adolescence compromised resilience to stress in adult rats, while virally mediated overexpression of Neurocan reversed vulnerability to stress in adolescent rats. Ketamine, a recent-approved drug for treatment-resistant depression rescued impaired function of Parvalbumin-positive neurons function, increased expression of PNNs in the PrL, and reversed depressive-like behaviors in adolescent rats. Furthermore, we show that Neurocan mediates the anti-depressant effect of ketamine, virally mediated reduction of Neurocan in the PrL abolished the anti-depressant effect of ketamine in adolescent rats. Our findings show an important role of Neurocan in depression in adolescence, and suggest a novel mechanism for the anti-depressant effect of ketamine.

Valproic Acid Inhibits Glial Scar Formation after Ischemic Stroke.

INTRODUCTION: Cerebral ischemia induces reactive proliferation of astrocytes (astrogliosis) and glial scar formation. As a physical and biochemical barrier, the glial scar not only hinders spontaneous axonal regeneration and neuronal repair but also deteriorates the neuroinflammation in the recovery phase of ischemic stroke. OBJECTIVES: Previous studies have shown the neuroprotective effects of the valproic acid (2-n-propylpentanoic acid, VPA) against ischemic stroke, but its effects on the ischemia-induced formation of astrogliosis and glial scar are still unknown. As targeting astrogliosis has become a therapeutic strategy for ischemic stroke, this study was designed to determine whether VPA can inhibit the ischemic stroke-induced glial scar formation and to explore its molecular mechanisms. METHODS: Glial scar formation was induced by an ischemia-reperfusion (I/R) model in vivo and an oxygen and glucose deprivation (OGD)-reoxygenation (OGD/Re) model in vitro. Animals were treated with an intraperitoneal injection of VPA (250 mg/kg/day) for 28 days, and the ischemic stroke-related behaviors were assessed. RESULTS: Four weeks of VPA treatment could markedly reduce the brain atrophy volume and improve the behavioral deficits in rats' I/R injury model. The results showed that VPA administrated upon reperfusion or 1 day post-reperfusion could also decrease the expression of the glial scar makers such as glial fibrillary acidic protein, neurocan, and phosphacan in the peri-infarct region after I/R. Consistent with the in vivo data, VPA treatment showed a protective effect against OGD/Re-induced astrocytic cell death in the in vitro model and also decreased the expression of GFAP, neurocan, and phosphacan. Further studies revealed that VPA significantly upregulated the expression of acetylated histone 3, acetylated histone 4, and heat-shock protein 70.1B in the OGD/Re-induced glial scar formation model. CONCLUSION: VPA produces neuroprotective effects and inhibits the glial scar formation during the recovery period of ischemic stroke via inhibition of histone deacetylase and induction of Hsp70.1B.

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.

Inhibition of Autophagy Flux Promotes Secretion of Chondroitin Sulfate Proteoglycans in Primary Rat Astrocytes.

Following spinal cord injury (SCI), reactive astrocytes in the glial scar produce high levels of chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal regeneration. Transforming growth factor beta (TGFß) is a well-known factor that induces the production of CSPGs, and in this study, we report a novel mechanism underlying TGFß's effects on CSPG secretion in primary rat astrocytes. We observed increased TGFß-induced secretion of the CSPGs neurocan and brevican, and this occurred simultaneously with inhibition of autophagy flux. In addition, we show that neurocan and brevican levels are further increased when TGFß is administered in the presence of an autophagy inhibitor, Bafilomycin-A1, while they are reduced when cells are treated with a concentration of rapamycin that is not sufficient to induce autophagy. These findings suggest that TGFß mediates its effects on CSPG secretion through autophagy pathways. They also represent a potential new approach to reduce CSPG secretion in vivo by targeting autophagy pathways, which could improve axonal regeneration after SCI.

αA-Crystallin inhibits optic nerve astrocyte activation induced by oxygen-glucose deprivation in vitro.

AIMS: A previous study reported that intravitreal injection of αA-crystallin inhibits glial scar formation after optic nerve traumatic injury. The purpose of this study was to investigate the effect of αA-crystallin on optic nerve astrocytes induced by oxygen glucose deprivation (OGD) in vitro. MATERIALS AND METHODS: Optic nerve astrocytes from newborn Long Evans rats were cultured with αA-crystallin (10-4 g/l) to detect the effects of αA-crystallin on astrocytes. Using a scratch assay, the effect of αA-crystallin treatment on astrocyte migration was assessed. Astrocytes were exposed to OGD and glucose reintroduction/reoxygenation culture for 24 h and 48 h. The expression of glial fibrillary acidic protein (GFAP) and neurocan were subsequently evaluated via immunocytochemistry and western blot. BMP2/4, BMPRIa/Ib and Smad1/5/8 mRNA expression levels were detected by RT-PCR. KEY FINDINGS: The results showed that αA-crystallin slowed the migration of astrocytes in filling the scratch gaps. GFAP and neurocan expression in astrocytes was increased after OGD. However, after treatment with αA-crystallin, GFAP and neurocan expression levels clearly decreased. Furthermore, RT-PCR showed that BMP2 and BMP4 mRNA expression levels decreased significantly. SIGNIFICANCE: These results suggest that αA-crystallin inhibits the activation of astrocytes after OGD injury in vitro. Inhibition of the BMP/Smad signaling pathway might be the mechanism underlying this effect.

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

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.

Cerebrospinal fluid brevican and neurocan fragment patterns in human traumatic brain injury.

BACKGROUND: Altered levels of two extracellular matrix (ECM) proteoglycans, brevican and neurocan, have been found in brain injury models; however, their proteolytic processing in traumatic brain injury (TBI) remains unexplored. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a possible contributor to ECM remodelling following TBI. The aims of this study were to evaluate proteolytic brevican/neurocan patterns and ADAMTS-like activity in cerebrospinal fluid (CSF) in the context of TBI. MATERIALS AND METHODS: Forty-two acute TBI patients and 37 idiopathic normal pressure hydrocephalus (iNPH) patients were included in the analysis of tryptic brevican and neurocan peptides in CSF using parallel reaction monitoring mass spectrometry. Twenty-nine TBI and 36 iNPH patients were analysed for ADAMTS-like activity in CSF using a quenched fluorescent substrate. RESULTS: The majority of CSF concentrations of brevican peptides significantly decreased in TBI patients compared with the iNPH group (p ≤ 0.002), while ADAMTS-like activity increased (p < 0.0001). Two C-terminal brevican peptides strongly correlated with unfavourable outcome of TBI patients (rho = 0.85-0.93, p ≤ 0.001). CONCLUSIONS: The decreased CSF concentrations of brevican peptides in TBI are associated with their increased degradation by ADAMTS enzymes. Furthermore, the N- and C-terminal parts of brevican are differentially regulated following TBI and may serve as outcome markers.

Brevican and Neurocan Peptides as Potential Cerebrospinal Fluid Biomarkers for Differentiation Between Vascular Dementia and Alzheimer's Disease.

BACKGROUND: Brevican and neurocan are central nervous system-specific extracellular matrix proteoglycans. They are degraded by extracellular enzymes, such as metalloproteinases. However, their degradation profile is largely unexplored in cerebrospinal fluid (CSF). OBJECTIVE: The study aim was to quantify proteolytic peptides derived from brevican and neurocan in human CSF of patients with Alzheimer's disease (AD) and vascular dementia (VaD) compared with controls. METHODS: The first cohort consisted of 75 individuals including 25 patients with AD, 7 with mild cognitive impairment (MCI) diagnosed with AD upon follow-up, 10 patients with VaD or MCI diagnosed with VaD upon follow-up, and 33 healthy controls and cognitively stable MCI patients. In the second cohort, 31 individuals were included (5 AD patients, 14 VaD patients and 12 healthy controls). Twenty proteolytic peptides derived from brevican (n = 9) and neurocan (n = 11) were quantified using high-resolution parallel reaction monitoring mass spectrometry. RESULTS: In the first cohort, the majority of CSF concentrations of brevican and neurocan peptides were significantly decreased inVaDas compared withADpatients (AUC = 0.83.0.93, p≤0.05) and as compared with the control group (AUC = 0.79.0.87, p ≤ 0.05). In the second cohort, CSF concentrations of two brevican peptides (B87, B156) were significantly decreased in VaD compared with AD (AUC = 0.86.0.91, p ≤ 0.05) and to controls (AUC = 0.80.0.82, p ≤ 0.05), while other brevican and neurocan peptides showed a clear trend to be decreased in VaD compared with AD (AUC = 0.64.80, p > 0.05). No peptides differed between AD and controls. CONCLUSION: Brevican and neurocan peptides are potential diagnostic biomarkers for VaD, with ability to separate VaD from AD.

Neurocan Contributes to Perineuronal Net Development.

Perineuronal nets (PNs) are matrix molecule assemblies surrounding neuronal somata, dendrites and axon initial segments in a lattice-like appearance. PN molecules are involved in many structural and physiological processes during development and in adulthood, suggesting a crucial role in normal brain function. Neurocan, as one of the main PN proteoglycans, is suggested to control important developmental processes of neuronal tissue. This statement relies on thorough and excellent experimental work mainly conducted in reduced systems, such as cell cultures. However, previous data collected in neurocan-deficient mice do not seem to support neurocan's role in development since brain development in general and the formation of PNs especially in the hippocampus were reported to be undisturbed in neurocan-deficient mice. Here, we aim to re-address the role of neurocan in developmental processes by investigating the influence of neurocan on PN formation in the medial nucleus of the trapezoid body, a PN-enriched nucleus in the auditory brainstem, using neurocan-deficient mice. Immunohistochemical and biochemical analyses demonstrate that neurocan controls the regulation of PN development by influencing mRNA and protein quantity of various PN molecules. Resulting alterations in PN fine structure are critical for PN function as estimated by reduced amount of GAD65/67 and prolongation of synaptic transmission delay of calyx of Held synapses. Thus, neurocan contributes to proper PN formation and synapse physiology in the MNTB.

Transcriptome analysis and functional characterization of cerebral organoids in bipolar disorder.

BACKGROUND: Reprogramming human induced pluripotent stem cells (iPSCs) from somatic cells and generating three-dimensional brain organoids from these iPSCs provide access to live human neuronal tissue with disease-specific genetic backgrounds. METHODS: Cerebral organoids were generated from iPSCs of eight bipolar disorder (BPI) patients and eight healthy control individuals. RNA-seq experiments were undertaken using RNA isolated from the cerebral organoids. Functional activity in the cerebral organoids was studied using microelectrode arrays. RESULTS: RNA-seq data comparing gene expression profiles in the cerebral organoids showed downregulation of pathways involved in cell adhesion, neurodevelopment, and synaptic biology in bipolar disorder along with upregulation of genes involved in immune signaling. The central hub in the network analysis was neurocan (NCAN), which is located in a locus with evidence for genome-wide significant association in BPI. Gene ontology analyses suggested deficits related to endoplasmic reticulum biology in BPI, which was supported by cellular characterization of ER-mitochondria interactions. Functional studies with microelectrode arrays revealed specific deficits in response to stimulation and depolarization in BPI cerebral organoids. CONCLUSIONS: Our studies in cerebral organoids from bipolar disorder showed dysregulation in genes involved in cell adhesion, immune signaling, and endoplasmic reticulum biology; implicated a central role for the GWAS hit NCAN in the biology of BPI; and showed evidence of deficits in neurotransmission.

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.

Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents.

The accumulation of chondroitin sulfate proteoglycans (CSPGs) in the glial scar following acute damage to the central nervous system (CNS) limits the regeneration of injured axons. Given the rich diversity of CSPG core proteins and patterns of GAG sulfation, identifying the composition of these CSPGs is essential for understanding their roles in injury and repair. Differential expression of core proteins and sulfation patterns have been characterized in the brain and spinal cord of mice and rats, but a comprehensive study of these changes following optic nerve injury has not yet been performed. Here, we show that the composition of CSPGs in the optic nerve and retina following optic nerve crush (ONC) in mice and rats exhibits an increase in aggrecan, brevican, phosphacan, neurocan and versican, similar to changes following spinal cord injury. We also observe an increase in inhibitory 4-sulfated (4S) GAG chains, which suggests that the persistence of CSPGs in the glial scar opposes the growth of CNS axons, thereby contributing to the failure of regeneration and recovery of function.