We recently reported that perineuronal nets (PNNs), which are chondroitin sulfate (CS) containing matrices that function to maintain synaptic integrity of key neurocircuits, are present in the hypothalamus of mice, rats and humans (Mizadeh et al, Nat Metabolism, 2019). AgRP neurons are among those enmeshed by PNNs in this brain area and excessive activation of these neurons is suggested to play a role in the pathogenesis of T2D. Our finding that PNNs are abnormal in the hypothalamus of T2D rodents offers a plausible link between brain mediated glucose-sensing dysfunction and T2D pathogenesis. PNN matrix stability correlates closely with the abundance of specific CS variants that constitute the PNN matrix, with an increase in 6S-CS promoting matrix destabilization. Traditional methods for CS-based ECM analysis in fixed brain tissues depend primarily upon IHC detection of CS-specific disaccharides, but this method is limited by cross-reactivity of CS-specific antibodies. Here we report the development and validation of two novel and quantitative biochemical methods for the in-depth compositional analysis of four biologically relevant CS-derived disaccharides present in rodent and human fixed brain tissue: 1) electrospray ionization-tandem mass spectrometry (MS/MS) to determine the percentages of 4S- and 6S-CS within the monosulfated population, and 2) liquid chromatography (LC)-MS/MS for resolving and determining percentages of 0S- 4S- 6S- and 4S6S-CS. These methods can detect CS from fixed coronal brain slices with nanomolar sensitivity and high reproducibility. After optimization using synthetic standards, both methods were validated using a developmental time course series in fixed rodent brain tissue and then applied to the analysis of formalin-fixed human brain tissue. These methods offer a useful approach with which to investigate the role of aberrant CS composition in the pathogenesis of diabetes and related metabolic disorders.


K.M. Alonge: Research Support; Self; Novo Nordisk Inc. A. Logsdon: Research Support; Spouse/Partner; Novo Nordisk Inc. W.A. Banks: None. C. Keene: None. M.W. Schwartz: Consultant; Self; Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S. M. Guttman: None.


National Institutes of Health; Novo Nordisk

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