(M1330-03-17) Harnessing Mass Spectrometry Approaches to Quantify Glycan Composition at the Blood-Brain Barrier

Purpose: Chondroitin sulfate glycosaminoglycans (CS-GAGs) are major constituents of the brain extracellular matrix that enmesh neural cells and regulate various neurological functions. Maladaptive changes to the composition of CS-GAGs have emerged as important mediators of cognitive function, and as possibly contributing to blood-brain barrier (BBB) function. Management of BBB function is a promising target for improving patient outcomes after repetitive mild traumatic brain injury (TBI), yet it remains unknown whether TBI-related changes in CS-GAG composition at the BBB can affect vascular and neurobehavioral function.

Methods: A lack of quantitative methods to assess CS-GAG composition at the BBB has severely limited our ability to understand how glycan changes contribute to cerebrovascular function. Addressing this limitation, we developed innovative liquid chromatography tandem mass spectrometry (LC-MSMS) techniques capable of quantifying CS-GAG sulfation patterns in fixed brain tissue, and in fixed brain microvessels (BMVs). In this study, a Neuropactor^TM device will be used to deliver five daily bouts of TBI to the dorsal midline of adult male and female CD-1 mice placed on a Marmarou foam pad. At one week, and at one month after the final TBI exposure, we will quantify sucrose permeability across the BBB in the right hemisphere using LC-MSMS. In the left hemisphere we will identify domains of fluorescent dextran leakage using immunohistochemistry. Between one week and one month after the final TBI exposure, we will measure sensorimotor and cognitive function in mice using the Rotarod and T-maze, respectively.

Results: Our innovative LC-MSMS approach uses chondroitinase digestion of fixed brain tissue to measure the levels of each individual CS-GAG isomer¹. Using this method, we recently discovered a significant increase in total CS-GAG sulfation in cortical brain tissue from humans with dementia, compared to non-demented controls². Here, we have applied our LC-MSMS approach to measure a significant increase in total CS-GAG sulfation in fixed brain tissue from mice after TBI (p < 0.001; Figure 1). Whereas successful isolation of BMVs from mouse cortical tissue revealed a unique CS-GAG sulfation pattern compared to the brain parenchyma, TBI exposure increased total CS-GAG sulfation in isolated BMVs (p < 0.01; Figure 2). Our central hypothesis is that TBI-induced vascular and neurobehavioral dysfunctions are mediated, in part, through mechanisms of CS-GAG sulfation at the BBB.

Conclusion: Currently, no drugs are FDA-approved for the treatment of TBI; however, Arylsulfatase B (ARSB) is an enzymatic inhibitor of CS-GAG sulfation that is FDA-approved for Maroteaux-Lamy syndrome. We seek to repurpose ARSB as a viable treatment option for TBI by targeting the blood-facing glycocalyx of the BBB. ARSB has a reported half-life of 36 hours, and so we propose to treat mice with ARSB (IV; 0.1 mg/kg) at 3, 7, 10, 14, 17 and 21 days after the final TBI exposure. We will confirm steady-state pharmacokinetics of ARSB using LC-MSMS in terminal plasma collections at one week, and at one month after the final TBI exposure. We will determine whether ARSB attenuates TBI-induced CS-GAG sulfation in both brain tissue and in BMVs. We will also determine whether ARSB ameliorates vascular and neurobehavioral dysfunctions associated with TBI. Successful results will provide substantial evidence in support of repurposing ARSB for managing BBB function and for possibly treating the clinical sequelae associated with TBI.

References: 1. Alonge KM, Logsdon AF, Murphree TA, Banks WA, Keene CD, Edgar JS, Whittington D, Schwartz MW, Guttman M. Quantitative analysis of chondroitin sulfate disaccharides from human and rodent fixed brain tissue by electrospray ionization-tandem mass spectrometry. Glycobiology. 2019. 29(12):847-60. PMID: 31361007
2. Logsdon AF, Francis KL, Richardson NE, Hu S, Faber CL, Phan BA, Nguyen V, Setthavongsack N, Banks WA, Woltjer RL, Keene CD, Latimer CS, Schwartz MW, Scarlett JM, Alonge KM. Decoding perineuronal net glycan sulfation patterns in the Alzheimer’s disease brain. Alzheimer's & Dementia: The Journal of the Alzheimer's Association. 2022. 18(5):942-954. PMID: 34482642

Acknowledgements:

Funding: AFL, NIH-NIA K22AG081264; Start-up funding from the Texas Tech University Health Sciences Center’s Office of Research and Department of Pharmacology & Neuroscience


Figure 1 | Liquid chromatography tandem mass spectrometry (LC-MSMS) to measure chondroitin sulfate glycosaminoglycan (CS-GAG) sulfation. (A) Definition of CS isomers and sulfate positions on Glucuronic acid (GlcA) or N-acetylgalactosamine (GalNAc). (B) Retention time for each CS isomer standard during LC-MSMS run with multiple reaction monitoring (MRM). (C) Increased total CS-GAG sulfation in fixed brain tissue from mice after TBI (***p < 0.001). Values: mean ± SEM (t test, n=6).


Figure 2 | Purity of mouse brain microvessel (BMV) isolation and LC-MSMS analysis of CS-GAG composition in BMV after TBI. (A) Brightfield image of freshly isolated BMVs, scale bar = 20 μm. (B) Immunofluorescent image of endothelial marker (GLUT1; red) with DAPI nuclear stain (blue), scale bar = 20 μm. (C) Immunoblots of the BMV partition were positive for endothelial marker (CLD5) and immunoblots of the parenchyma partition were positive for neuronal marker (NeuN), each lane represents a single mouse brain. (D) LC-MSMS Chromatogram showing the relative abundance of CS-GAG isomers (CSA and CSC) from isolated BMV. (E) The mouse brain parenchyma exhibits more CSA, and less CSC, compared to the mouse BMV. (F) TBI increased the total CS-GAG sulfation in BMVs isolated after 7 days. (**p < 0.01; ***p < 0.001); n=5; two-way ANOVA, Bonferroni; t-test; Values: mean ± SEM.