The Perineuronal Net Protein Brevican Acts in Nucleus Accumbens Parvalbumin-Expressing Interneurons of Adult Mice to Regulate Excitatory Synaptic Inputs and Motivated Behaviors.

BACKGROUND: Experience-dependent functional adaptation of nucleus accumbens (NAc) circuitry underlies the development and expression of reward-motivated behaviors. Parvalbumin-expressing GABAergic (gamma-aminobutyric acidergic) interneurons (PVINs) within the NAc are required for this process. Perineuronal nets (PNNs) are extracellular matrix structures enriched around PVINs that arise during development and have been proposed to mediate brain circuit stability. However, their function in the adult NAc is largely unknown. Here, we studied the developmental emergence and adult regulation of PNNs in the NAc of male and female mice and examined the cellular and behavioral consequences of reducing the PNN component brevican in NAc PVINs. METHODS: We characterized the expression of PNN components in mouse NAc using immunofluorescence and RNA in situ hybridization. We lowered brevican in NAc PVINs of adult mice using an intersectional viral and genetic method and quantified the effects on synaptic inputs to NAc PVINs and reward-motivated learning. RESULTS: PNNs around NAc PVINs were developmentally regulated and appeared during adolescence. In the adult NAc, PVIN PNNs were also dynamically regulated by cocaine. Transcription of the gene that encodes brevican was regulated in a cell type- and isoform-specific manner in the NAc, with the membrane-tethered form of brevican being highly enriched in PVINs. Lowering brevican in NAc PVINs of adult mice decreased their excitatory inputs and enhanced both short-term novel object recognition and cocaine-induced conditioned place preference. CONCLUSIONS: Regulation of brevican in NAc PVINs of adult mice modulates their excitatory synaptic drive and sets experience thresholds for the development of motivated behaviors driven by rewarding stimuli.

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.

Differential Expression of miRNAs and Their Predicted Target Pathways in Cochlear Nucleus Following Chronic Noise Exposure in Rats.

Several recent preclinical studies have reported that dynamic changes in miRNA expression contribute to hearing function. This study aims to investigate miRNA expression changes in the cochlear nuclei (CN) of rats following chronic noise exposure. Eight-week-old rats (n = 14) were exposed to noise for 4 weeks. The control rats (n = 14) were raised under identical conditions without noise. Two months after noise exposure, the auditory brainstem response (ABR) was examined, and the cochlea and CN were harvested. In the CN, the expression levels of arc, neurocan, and brevican were measured (n = 6 per group). Furthermore, the expression levels of miRNAs and their predicted target genes were measured in the CN (n = 8 per group). ABR thresholds were elevated after 4 weeks of noise exposure, which were maintained for 3 months. In CN, the protein expression of arc and brevican was higher in the noise-exposed group than in the control group (0.95 [standard deviation (SD) = 0.53] vs. 3.19 [SD = 1.00], p < 0.001 for arc and 1.02 [SD = 0.10] vs. 1.66 [SD = 0.24], p < 0.001 for brevican). The noise-exposed rats exhibited lower expression levels of miR-758-5p, miR-15b-5p, miR-212-3p, miR-199a-5p, and miR-134-3p than the control rats (all p < 0.001). The AMPK signaling pathway was predicted to be regulated by these miRNAs. The predicted target genes AKT3, SIRT1, and PRKAA1 were highly expressed in noise-exposed rats. In CN of noise-exposed rats, the miRNAs of miR-758-5p, miR-15b-5p, miR-212-3p, miR-199a-5p, and miR-134-3p were reduced and related to AMPK signaling including AKT3 and SIRT1 expression. These modulation of signaling pathways could mediate the increased expression of brevican in the CN of noise-exposed rats.

Microglia Depletion-Induced Remodeling of Extracellular Matrix and Excitatory Synapses in the Hippocampus of Adult Mice.

The extracellular matrix (ECM) plays a key role in synaptogenesis and the regulation of synaptic functions in the central nervous system. Recent studies revealed that in addition to dopaminergic and serotoninergic neuromodulatory systems, microglia also contribute to the regulation of ECM remodeling. In the present work, we investigated the physiological role of microglia in the remodeling of perineuronal nets (PNNs), predominantly associated with parvalbumin-immunopositive (PV+) interneurons, and the perisynaptic ECM around pyramidal neurons in the hippocampus. Adult mice were treated with PLX3397 (pexidartinib), as the inhibitor of colony-stimulating factor 1 receptor (CSF1-R), to deplete microglia. Then, confocal analysis of the ECM and synapses was performed. Although the elimination of microglia did not alter the overall number or intensity of PNNs in the CA1 region of the hippocampus, it decreased the size of PNN holes and elevated the expression of the surrounding ECM. In the neuropil area in the CA1 str. radiatum, the depletion of microglia increased the expression of perisynaptic ECM proteoglycan brevican, which was accompanied by the elevated expression of presynaptic marker vGluT1 and the increased density of dendritic spines. Thus, microglia regulate the homeostasis of pre- and postsynaptic excitatory terminals and the surrounding perisynaptic ECM as well as the fine structure of PNNs enveloping perisomatic-predominantly GABAergic-synapses.

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.

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.

The Role of BEHAB/Brevican in the Tumor Microenvironment: Mediating Glioma Cell Invasion and Motility.

Malignant gliomas are the most common tumors in the central nervous system (CNS) and, unfortunately, are also the most deadly. The lethal nature of malignant gliomas is due in large part to their unique and distinctive ability to invade the surrounding neural tissue. The invasive and dispersive nature of these tumors makes them particularly challenging to treat, and currently there are no effective therapies for malignant gliomas. The brain tumor microenvironment plays a particularly important role in mediating the invasiveness of gliomas, and, therefore, understanding its function is key to developing novel therapies to treat these deadly tumors. A defining aspect of the tumor microenvironment of gliomas is the unique composition of the extracellular matrix that enables tumors to overcome the typically inhibitory environment found in the CNS. One conspicuous component of the glioma tumor microenvironment is the neural-specific ECM molecule, brain-enriched hyaluronan binding (BEHAB)/brevican (B/b). B/b is highly overexpressed in gliomas, and its expression in these tumors contributes importantly to the tumor invasiveness and aggressiveness. However, B/b is a complicated protein with multiple splice variants, cleavage products, and glycoforms that contribute to its complex functions in these tumors and provide unique targets for tumor therapy. Here we review the role of B/b in glioma tumor microenvironment and explore targeting of this protein for glioma therapy.

The CNS-specific proteoglycan, brevican, and its ADAMTS4-cleaved fragment show differential serological levels in Alzheimer's disease, other types of dementia and non-demented controls: A cross-sectional study.

Evidence indicate that the brain-specific protein, brevican, is proteolytically cleaved during neurodegeneration, hence positioning fragments of brevican as potential blood biomarkers of neurodegenerative diseases, such as dementia. We aimed to develop two assays capable of detecting the brevican N-terminal (N-Brev) and the ADAMTS4-generated fragment (Brev-A), cleaved at Ser401, in serum and to perform a preliminary assessment of their diagnostic potential in dementias. Monoclonal antibodies against N-Brev and Brev-A were used to develop two ELISAs detecting each epitope. A comparison of brevican fragments in serum from individuals with AD (n = 28), other dementia (OD) (n = 41), and non-dementia-related memory complaints (NDCs) (n = 48) was conducted. Anti-N-Brev and anti-Brev-A antibodies selectively recognized their targets and dilution and spike recoveries were within limits of ±20%. Intra- and inter-assay CVs were below limits of 10% and 15%, respectively. For the N-Brev biomarker, serum from patients with OD showed significantly lower levels than those with AD (p = 0.05) and NDCs (p < 0.01). The opposite pattern was evident for Brev-A: serum levels in patients with OD were significantly higher than for AD (p = 0.04) and NDCs (p = 0.01). For both N-Brev and Brev-A, levels did not differ between AD and NDCs. The ratio of N-Brev/Brev-A resulted in increased significant differences between OD and AD (p < 0.01) and between OD and NDCs (p < 0.0001). The ratio discriminated between NDCs and OD (AUC: 0.75, 95% CI: 0.65-0.85, p < 0.0001) and between OD and AD (AUC: 0.72, 95% CI: 0.59-0.85, p < 0.01). In conclusion, we developed the first assays detecting the N-terminal of brevican as well as an ADAMTS4-cleaved fragment of brevican in blood. Differential levels of N-Brev and Brev-A between AD and OD allow for these biomarkers to possibly distinguish between different forms of dementias.

Noise exposure alters MMP9 and brevican expression in the rat primary auditory cortex.

BACKGROUND: This study aimed to investigate the changes in molecules related to perineuronal nets (PNNs) and synaptic transporters in the primary auditory cortices of rats with noise-induced hearing loss. Female Sprague-Dawley rats at postnatal day 7 were divided into the noise and control groups. Four hours of 115 dB SPL white noise was delivered for 10 days to the noise group. Thirty days after noise exposure, the primary auditory cortex and the inferior colliculus were harvested. The expression levels of vesicular glutamatergic transporter (VGLUT)1, VGLUT2, vesicular GABA transporter (VGAT), glutamate decarboxylase (GAD)67, brevican, aggrecan, MMP9, and MMP14 were evaluated using real-time reverse transcription polymerase chain reaction or western blot. An immunofluorescence assay was conducted to assess parvalbumin (PV), Wisteria floribunda agglutinin (WFA), and brevican. The immune-positive cells were counted in the primary auditory cortex. RESULTS: The expression level of VGLUT1 in the primary auditory cortex was decreased in the noise group. The expression level of VGLUT2 in the inferior colliculus was elevated in the noise group. The expression levels of brevican and PV + WFA in the primary auditory cortex were decreased in the noise group. The expression level of MMP9 in the primary auditory cortex was increased in the noise group. CONCLUSION: Noise-induced hearing loss during the precritical period impacted PNN expression in the primary auditory cortex. Increased MMP9 expression may have contributed to the decrease in brevican expression. These changes were accompanied by the attenuation of glutamatergic synaptic transporters.

Establishment and Evaluation of a 6-Gene Survival Risk Assessment Model Related to Lung Adenocarcinoma Microenvironment.

OBJECTIVE: A survival risk assessment model associated with a lung adenocarcinoma (LUAD) microenvironment was established and evaluated to identify effective independent prognostic factors for LUAD. METHODS: The public data were downloaded from the TCGA database, and ESTIMATE prediction software was used to score immune cells and stromal cells for tumor purity prediction. The samples were divided into the high-score group and the low-score group by the median value of the immune score (or stromal score). The Wilcoxon test was used for differential analysis. GO and KEGG enrichment analysis of differentially expressed genes (DEGs) was performed using "clusterProfiler" of R package. Meanwhile, univariate and multivariate regression analysis was performed on DEGs to construct a multivariate Cox risk regression model with variable gene expression levels as independent prognostic factors affecting a tumor microenvironment (TME) and tumor immunity. RESULTS: This study found that LUAD patients with high immune cell (stromal cell) infiltration had better prognosis and were in earlier staging. Functional enrichment analysis revealed that most DEGs were related to the proliferation and activation of immune cells or stromal cells. A survival prediction model composed of 6 TME-related genes (CLEC17A, TAGAP, ABCC8, BCAN, FLT3, and CCR2) was established, and finally, the 6 feature genes closely related to the prognosis of LUAD were proved. The AUC value of the ROC curve in this model was 0.7, indicating that the model was reliable. CONCLUSION: Six genes related to the LUAD microenvironment have a predictive prognostic value in LUAD.