Brevican and Neurocan Cleavage Products in the Cerebrospinal Fluid - Differential Occurrence in ALS, Epilepsy and Small Vessel Disease

Abstract

The neural extracellular matrix (ECM) composition shapes the neuronal microenvironment and undergoes substantial changes upon development and aging, but also due to cerebral pathologies. In search for potential biomarkers, cerebrospinal fluid (CSF) and serum concentrations of brain ECM molecules have been determined recently to assess ECM changes during neurological conditions including Alzheimer’s disease or vascular dementia. Here, we measured the levels of two signature proteoglycans of brain ECM, neurocan and brevican, in the CSF and serum of 96 neurological patients currently understudied regarding ECM alterations: 16 cases with amyotrophic lateral sclerosis (ALS), 26 epilepsy cases, 23 cerebral small vessel disease (CSVD) patients and 31 controls. Analysis of total brevican and neurocan was performed via sandwich Enzyme-linked immunosorbent assays (ELISAs). Major brevican and neurocan cleavage products were measured in the CSF using semiquantitative immunoblotting. Total brevican and neurocan concentrations in serum and CSF did not differ between groups. The 60 kDa brevican fragment resulting from cleavage by the protease ADAMTS-4 was also found unchanged among groups. The presumably intracellularly generated 150 kDa C-terminal neurocan fragment, however, was significantly increased in ALS as compared to all other groups. This group also shows the highest correlation between cleaved and total neurocan in the CSF. Brevican and neurocan levels strongly correlated with each other across all groups, arguing for a joint but yet unknown transport mechanism from the brain parenchyma into CSF. Conclusively our findings suggest an ALS-specific pattern of brain ECM remodeling and may thus contribute to new diagnostic approaches for this disorder.

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

“…As the biosynthesis of brevican and neurocan occurs largely in neural tissue and fragments of these proteins have been detected in CSF ( ; accessed on 1 September 2022) [ 31 ] it is of interest to analyze their concentration in the CSF of patients as a potential proxy of brain levels of these signature ECM components. In agreement with our recent study [ 32 ], we show that CSF concentrations of these proteoglycans and their major cleavage products positively correlate with the age of the patients and with Aβ1-40, an indicator for the glymphatic flux from the parenchyma to the CSF [ 33 ]. Although we did not see a group-wise difference between control and AD patients, which is in line with earlier studies from other labs [ 18 , 19 , 34 ], we found clear correlations with tau measures, NF-L, and the Aβ ratio, and—at least for brevican—also with the MMSE score.…”

“…Commercial ELISA kits (RayBiotech Norcross, GA, USA) (ELH-BCAN-1 and ELH-NCAN-1) were used for the quantitative measurement of total brevican and neurocan in the CSF samples (dilution 1:300 for anti-brevican ELISAs and 1:10 for anti-neurocan ELISAs). ELISA measurements were essentially conducted according to the manufacturer’s instructions and to [ 32 ], with slight modifications: To compensate for sensitivity differences in ELISA batches the concentration of the recombinant calibrator protein for the standard curve was adjusted according to OD values obtained (highest standard concentrations were set to 3 ng/mL in brevican ELISAs and 25 ng/mL in neurocan ELISAs; standard dilution series: 6 × 1:2 dilution steps). HRP-streptavidin was used at concentrations of 1:200 for brevican and 1:500 for neurocan.…”

“…Before immuno-blotting protein gels were activated under UV light for 5 min. Protein transfer onto PVDF membranes (Merck Millipore, Burlington, MA, USA), blocking and immunodetection were performed according to standard protocols, see also [ 32 ]. Immunodetection was performed using an ECL Chemocam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany).…”

Proteomic and Bioinformatic Tools to Identify Potential Hub Proteins in the Audiogenic Seizure-Prone Hamster GASH/Sal

“…Alterations in the expression levels of BCAN (brevican core protein isoform X2; overexpressed DEP with 2.08 FC) and NCAN (neurocan core protein isoform X2; overexpressed DEP with 4.16 FC) have been reported in epileptic patients, as in the case of focal cortical dysplasia [ 103 , 104 ]. Epilepsies with a genetic basis can manifest early in life, and both BCAN and NCAN are believed to play crucial roles in terminal differentiation during development, as well as in the adult nervous system during postnatal development.…”

CCR5 deficiency normalizes TIMP levels, working memory, and gamma oscillation power in APOE4 targeted replacement mice

“…Both forms may be cleaved. As shown in Figure 2e, our blots show high molecular weight soluble brevican as well as a previously described approximately 50-60 kDa metalloprotease generated cleavage fragment (Seidenbecher et al ., 1995; Hussler et al ., 2022). This proteolytic fragment is significantly increased in APOE3/3 as compared to APOE4/4 CSF samples ( p = 0.0039, Student’s t test).…”