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.

Hyalectanase Activities by the ADAMTS Metalloproteases.

The hyalectan family is composed of the proteoglycans aggrecan, versican, brevican and neurocan. Hyalectans, also known as lecticans, are components of the extracellular matrix of different tissues and play essential roles in key biological processes including skeletal development, and they are related to the correct maintenance of the vascular and central nervous system. For instance, hyalectans participate in the organization of structures such as perineural nets and in the regulation of neurite outgrowth or brain recovery following a traumatic injury. The ADAMTS (A Disintegrin and Metalloprotease domains, with thrombospondin motifs) family consists of 19 secreted metalloproteases. These enzymes also perform important roles in the structural organization and function of the extracellular matrix through interactions with other matrix components or as a consequence of their catalytic activity. In this regard, some of their preferred substrates are the hyalectans. In fact, ADAMTSs cleave hyalectans not only as a mechanism for clearance or turnover of proteoglycans but also to generate bioactive fragments which display specific functions. In this article we review some of the physiological and pathological effects derived from cleavages of hyalectans mediated by ADAMTSs.

Neurocan is a New Substrate for the ADAMTS12 Metalloprotease: Potential Implications in Neuropathies.

BACKGROUND/AIMS: The composition of the extracellular matrix (ECM) in the central nervous system (CNS) has several features that make it unique. For instance, it is remarkable for the presence of proteoglycans such as versican, brevican, and neurocan, some of which have been identified as substrates of different members of the ADAMTS family of secreted metalloproteases. Previous studies have associated ADAMTSs with the repair of the CNS, including recovery following degradation of glial scar tissue and the stimulation of axonal growth after brain injury. However, the involvement of ADAMTSs in diseases of the CNS is complex and not understood fully, and a current challenge is unraveling the precise roles of these metalloproteases in the brain. METHODS: ADAMTS12 and neurocan gene expression was examined by quantitative PCR. Western blot analysis was employed to detect ADAMTS12 and neurocan protein expression in cell lines, and immunostaining techniques were used to detect neurocan in mouse brain tissues. Neurocan cleavage using recombinant ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS12 metalloproteases was evaluated by western blotting. Cell adhesion and migration were assessed using uncoated culture dishes or dishes coated with Matrigel or ECM components. RESULTS: We identified neurocan as a novel component of brain ECM that can be cleaved by ADAMTS12. In addition, we showed that neurocan cleavage by ADAMTS12 altered the adhesive properties of the human neuroglioma H4 cell line. Moreover, immunohistochemical analysis of Adamts12-deficient mice revealed the significant accumulation of neurocan in the brain of neonatal mice. CONCLUSION: Overall, our results suggest that ADAMTS12 could be involved in the repair of the CNS through its ability to degrade neurocan. Moreover, it can be inferred that alterations in neurocan degradation processes could be associated with the pathogenesis of neurological disorders.

Dynamics of extracellular matrix proteins in cerebrospinal fluid and serum and their relation to clinical outcome in human traumatic brain injury.

Background Brevican, neurocan, tenascin-C and tenascin-R are extracellular matrix proteins present in brain that show increased expression in experimental animal models of brain injury. However, little is known about the dynamics of these proteins in human body fluids, such as cerebrospinal fluid (CSF) and serum, after traumatic brain injury (TBI). The aims of this study were to investigate if matrix proteins in CSF and serum are associated with functional outcome following traumatic brain injury, if their concentrations change over time and to compare their levels between brain injured patients to controls. Methods In total, 42 traumatic brain injury patients, nine healthy controls and a contrast group consisting of 38 idiopathic normal pressure hydrocephalus patients were included. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of proteins. Results Increased concentrations of brevican, tenascin-C and tenascin-R in CSF correlated with unfavourable outcome, with stronger outcome prediction ability compared to other biomarkers of brain tissue injury. CSF brevican, tenascin-R and serum neurocan gradually decreased with time (p = 0.04, p = 0.008, p = 0.005, respectively), while serum tenascin-C (p = 0.01) increased. CSF concentrations of brevican, neurocan and tenascin-R (only in time point 3) after TBI were lower than in the idiopathic normal pressure hydrocephalus group (p < 0.0001, p < 0.0001, and p = 0.0008, respectively). In serum, tenascin-C concentration was higher and neurocan lower compared to healthy controls (p = 0.02 and p = 0.0009). Conclusions These findings indicate that levels of extracellular matrix proteins are associated with clinical outcome following TBI and may act as markers for different pathophysiology than currently used protein biomarkers.

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.

Antihypoxic effect of miR-24 in SH-SY5Y cells under hypoxia via downregulating expression of neurocan.

Hypoxia-induced apoptosis-related mechanisms involved in the brain damage following cerebral ischemia injury. A subset of the small noncoding microRNA (miRNAs) is regulated by tissue oxygen levels, and miR-24 was found to be activated by hypoxic conditions. However, the roles of miR-24 and its target gene in neuron are not well understood. Here, we validated miRNA-24 is down-regulated in patients with cerebral infarction. Hypoxia suppressed the expression of miR-24, but increased the expression of neurocan in both mRNA and protein levels in SH-SY5Y cells. MiR-24 mimics reduced the expression of neurocan, suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. By luciferase reporter assay, neurocan is validated a direct target gene of miR-24. Furthermore, knockdown of neurocan suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. Taken together, miR-24 overexpression or silencing of neurocan shows an antihypoxic effect in SH-SY5Y cells. Therefore, miR-24 and neurocan play critical roles in neuron cell apoptosis and are potential therapeutic targets for ischemic brain disease.

Decline in arylsulfatase B and Increase in chondroitin 4-sulfotransferase combine to increase chondroitin 4-sulfate in traumatic brain injury.

In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-ß1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-ß1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-ß1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the glial scar.

Arylsulfatase B modulates neurite outgrowth via astrocyte chondroitin-4-sulfate: dysregulation by ethanol.

In utero ethanol exposure causes fetal alcohol spectrum disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement. Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the levels of inhibitory chondroitin sulfate proteoglycans (CSPGs) in astrocytes. Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons cocultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB. Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure. ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in cocultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression. In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in cocultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth. An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.

tPA promotes ADAMTS-4-induced CSPG degradation, thereby enhancing neuroplasticity following spinal cord injury.

Although tissue plasminogen activator (tPA) is known to promote neuronal remodeling in the CNS, no mechanism of how this plastic function takes place has been reported so far. We provide here in vitro and in vivo demonstrations that this serine protease neutralizes inhibitory chondroitin sulfate proteoglycans (CSPGs) by promoting their degradation via the direct activation of endogenous type 4 disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4). Accordingly, in a model of compression-induced spinal cord injury (SCI) in rats, we found that administration of either tPA or its downstream effector ADAMTS-4 restores the tPA-dependent activity lost after the SCI and thereby, reduces content of CSPGs in the spinal cord, a cascade of events leading to an improved axonal regeneration/sprouting and eventually long term functional recovery. This is the first study to reveal a tPA-ADAMTS-4 axis and its function in the CNS. It also raises the prospect of exploiting such cooperation as a therapeutic tool for enhancing recovery after acute CNS injuries.

A common polymorphism in the NCAN gene is associated with hepatocellular carcinoma in alcoholic liver disease.

BACKGROUND & AIMS: The genetic background of alcoholic liver diseases and their complications are increasingly recognized. A common polymorphism in the neurocan (NCAN) gene, which is known to be expressed in neuronal tissue, has been identified as a risk factor for non-alcoholic fatty liver disease (NAFLD). We investigated if this polymorphism may also be related to alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). METHODS: We analysed the distribution of the NCAN rs2228603 genotypes in 356 patients with alcoholic liver cirrhosis, 126 patients with alcoholic HCC, 382 persons with alcohol abuse without liver damage, 362 healthy controls and in 171 patients with hepatitis C virus (HCV) associated HCC. Furthermore, a validation cohort of 229 patients with alcoholic cirrhosis (83 with HCC) was analysed. The genotypes were determined by LightSNiP assays. The expression of NCAN was studied by RT-PCR and immunofluorescence microscopy. RESULTS: The frequency of the NCAN rs2228603 T allele was significantly increased in patients with HCC due to ALD (15.1%) compared to alcoholic cirrhosis without HCC (9.3%), alcoholic controls (7.2%), healthy controls (7.9%), and HCV associated HCC (9.1%). This finding was confirmed in the validation cohort (15.7% vs. 6.8%, OR=2.53; 95%CI: 1.36-4.68; p=0.0025) and by multivariate analysis (OR=1.840; 95%CI: 1.22-2.78; p=0.004 for carriage of the rs2228603 T allele). In addition, we identified and localised NCAN expression in human liver. CONCLUSIONS: NCAN is not only expressed in neuronal tissue, but also in the liver. Its rs2228603 polymorphism is a risk factor for HCC in ALD, but not in HCV infection.

Morphological and Molecular Characteristics of Perineuronal Nets in the Human Prefrontal Cortex-A Possible Link to Microcircuitry Specialization.

Perineuronal nets (PNNs) are a type of extracellular matrix (ECM) that play a significant role in synaptic activity and plasticity of interneurons in health and disease. We researched PNNs' regional and laminar representation and molecular composition using immunohistochemistry and transcriptome analysis of Brodmann areas (BA) 9, 14r, and 24 in 25 human postmortem brains aged 13-82 years. The numbers of VCAN- and NCAN-expressing PNNs, relative to the total number of neurons, were highest in cortical layers I and VI while WFA-binding (WFA+) PNNs were most abundant in layers III-V. The ECM glycosylation pattern was the most pronounced regional difference, shown by a significantly lower proportion of WFA+ PNNs in BA24 (3.27 ± 0.69%) compared to BA9 (6.32 ± 1.73%; P = 0.0449) and BA14 (5.64 ± 0.71%; P = 0.0278). The transcriptome of late developmental and mature stages revealed a relatively stable expression of PNN-related transcripts (log2-transformed expression values: 6.5-8.5 for VCAN and 8.0-9.5 for NCAN). Finally, we propose a classification of PNNs that envelop GABAergic neurons in the human cortex. The significant differences in PNNs' morphology, distribution, and molecular composition strongly suggest an involvement of PNNs in specifying distinct microcircuits in particular cortical regions and layers.

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.

Astrocyte-secreted neurocan controls inhibitory synapse formation and function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, in vivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

Association between variants in or near PNPLA3, GCKR, and PPP1R3B with ultrasound-defined steatosis based on data from the third National Health and Nutrition Examination Survey.

BACKGROUND & AIMS: A genome-wide association study associated 5 genetic variants with hepatic steatosis (identified by computerized tomography) in individuals of European ancestry. We investigated whether these variants were associated with measures of hepatic steatosis (HS) in non-Hispanic white (NHW), non-Hispanic black, and Mexican American (MA) participants in the US population-based National Health and Nutrition Examination Survey III, phase 2. METHODS: We analyzed data from 4804 adults (1825 NHW, 1442 non-Hispanic black, and 1537 MA; 51.7% women; mean age at examination, 42.5 y); the weighted prevalence of HS was 37.3%. We investigated whether ultrasound-measured HS, with and without increased levels of alanine aminotransferase (ALT), or level of ALT alone, was associated with rs738409 (patatin-like phospholipase domain-containing protein 3 [PNPLA3]), rs2228603 (neurocan [NCAN]), rs12137855 (lysophospholipase-like 1), rs780094 (glucokinase regulatory protein [GCKR]), and rs4240624 (protein phosphatase 1, regulatory subunit 3b [PPP1R3B]) using regression modeling in an additive genetic model, controlling for age, age-squared, sex, and alcohol consumption. RESULTS: The G allele of rs738409 (PNPLA3) and the T allele of rs780094 (GCKR) were associated with HS with a high level of ALT (odds ratio [OR], 1.36; P = .01; and OR, 1.30; P = .03, respectively). The A allele of rs4240624 (PPP1R3B) and the T allele of rs2228603 (NCAN) were associated with HS (OR, 1.28; P = .03; and OR, 1.40; P = .04, respectively). Variants of PNPLA3 and NCAN were associated with ALT level among all 3 ancestries. Some single-nucleotide polymorphisms were associated with particular races or ethnicities: variants in PNPLA3, NCAN, GCKR, and PPP1R3B were associated with NHW and variants in PNPLA3 were associated with MA. No variants were associated with NHB. CONCLUSIONS: We used data from the National Health and Nutrition Examination Survey III to validate the association between rs738409 (PNPLA3), rs780094 (GCKR), and rs4240624 (PPP1R3B) with HS, with or without increased levels of ALT, among 3 different ancestries. Some, but not all, associations between variants in NCAN, lysophospholipase-like 1, GCKR, and PPP1R3B with HS (with and without increased ALT level) were significant within subpopulations.

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.

Brevican, Neurocan, Tenascin-C, and Tenascin-R Act as Important Regulators of the Interplay Between Perineuronal Nets, Synaptic Integrity, Inhibitory Interneurons, and Otx2.

Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix (ECM) structure called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons, and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C, and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. In addition, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Furthermore, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs, did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin interneuron and PNN maturation, and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C, and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution, and Otx2 in the V1.

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.

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.

The emerging role of the chondroitin sulfate proteoglycan family in neurodegenerative diseases.

Chondroitin sulfate (CS) is a kind of linear polysaccharide that is covalently linked to proteins to form proteoglycans. Chondroitin sulfate proteoglycans (CSPGs) consist of a core protein, with one or more CS chains covalently attached. CSPGs are precisely regulated and they exert a variety of physiological functions by binding to adhesion molecules and growth factors. Widely distributed in the nervous system in human body, CSPGs contribute to the major component of extracellular matrix (ECM), where they play an important role in the development and maturation of the nervous system, as well as in the pathophysiological response to damage to the central nervous system (CNS). While there are more than 30 types of CSPGs, this review covers the roles of the most important ones, including versican, aggrecan, neurocan and NG2 in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. The updated reports of the treatment of neurodegenerative diseases are involving CSPGs.

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery. METHODS: Paired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays. RESULTS: Brevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = - 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84-0.94) compared with those without. CONCLUSION: Levels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics.