Complete spatial characterisation of N-glycosylation upon striatal neuroinflammation in the rodent brain

Abstract

Background: Neuroinflammation is an underlying pathology of all neurological conditions, the understanding of which is still being comprehended. A specific molecular pathway that has been overlooked in neuroinflammation is glycosylation (i.e., post-translational addition of glycans to the protein structure). N-glycosylation is a specific type of glycosylation with a cardinal role in the central nervous system (CNS), which is highlighted by congenital glycosylation diseases that result in neuropathological symptoms such as epilepsy and mental retardation. Changes in N-glycosylation can ultimately affect glycoproteins' functions, which will have an impact on cell machinery. Therefore, characterisation of N-glycosylation alterations in a neuroinflammatory scenario can provide a potential target for future therapies.

Methods: With that aim, the unilateral intrastriatal injection of lipopolysaccharide (LPS) in the adult rat brain was used as a model of neuroinflammation. In vivo and post-mortem, quantitative and spatial characterisation of both neuroinflammation and N-glycome was performed at 1-week post-injection of LPS. These aspects were investigated through a multifaceted approach based on positron emission tomography (PET), quantitative histology, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), liquid chromatography and matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI).

Results: In the brain region showing LPS-induced neuroinflammation, a significant decrease in the abundance of sialylated and core fucosylated structures was seen (approximately 7.5% and 8.5%, respectively), whereas oligomannose N-glycans were significantly increased (13.5%). This was confirmed by MALDI-MSI, which provided a high-resolution spatial distribution of N-glycans, allowing precise comparison between normal and diseased brain hemispheres.

Conclusions: Together, our data show for the first time the complete profiling of N-glycomic changes in a well-characterised animal model of neuroinflammation. These data represent a pioneering step to identify critical targets that may modulate neuroinflammation in neurodegenerative diseases.

Keywords: Glycomics; LPS model; Liquid chromatography; MALDI-MSI; N-glycosylation; Neuroinflammation; Protein glycosylation; Striatum.

Fig. 1

Schematic representation of the experimental design and procedures. a Establishment of a neuroinflammation model upon LPS injection in the rat striatum. The striatum was collected 7 days post-injection and further analysed for neuroinflammatory markers and N-glycomic profile. b Experimental plan of N-glycan characterisation. Regions of interest were either sectioned for MALDI-TOF-MSI or punched for N-glycan isolation and characterisation using HILIC-UPLC. The combination of both techniques allowed for the spatial and quantitative N-glycome profiling in the rat striatum

Fig. 2

Pathological validation of the model of striatal neuroinflammation where LPS was injected into the striatum, in comparison to the contralateral (non-injected (NI)) striatum at seven days post-injection (dpi). a In vivo analysis of translocator protein (TSPO) expression as a marker of neuroinflammation in the striatum post-LPS injection. Average quantified TSPO-PET image in non-displaceable binding potential (BP_ND) in a coronal section. b Quantification of TSPO-PET imaging in the striatum. Results are expressed as means ± the standard error of the mean (SEM). n = 9; paired Student t test and statistically significant difference was set at **p < 0.01. c Striatal mRNA expression of different genes related to the inflammatory response–Glial fibrillary protein (GFAP), Iba1, vimentin (Vim), TSPO and tumour necrosis factor α (TNFα). Results are expressed as means ± SEM. n = 11–12; paired Student t test and statistically significant difference was set at ****p < 0.0001. d Spearman correlation between in vivo individual PET BPnd data and post-mortem expression data. Spearman correlation coefficients are marked in the corresponding case; blue signifies a positive correlation. Statistical significance was set at *p < 0.01. e, f, g Histological evaluation of the expression of Iba1, GFAP and Vim (respectively) in LPS-injected vs non-injected striata at seven dpi. Scale bar = 50 μm. h, i, j Striatal optical density of Iba+, GFAP+ or Vim+ (respectively) in the LPS-injected and NI striata. Results are expressed as means ± SEM. n = 10–12; paired Student t test and statistically significant difference was set at ***p < 0.001, and ****p < 0.0001

Fig. 3

N-glycome changes between LPS-injected and NI striata using HILIC-UPLC. a HILIC-UPLC chromatograms for N-glycans isolated from rat striata (LPS-injected vs NI) during a 30-min run, separated into 26 main chromatographic glycan peaks (GP) following the characterisation performed by Samal et al. [41]. Detailed composition of each of these peaks is described in Supplementary table S1. b Relative abundances represented as percentages of total N-glycans divided into the three main biosynthetic classes: oligomannose, complex and hybrid. c Summary table of the GP that are statistically significantly different between LPS-injected and NI striatum at seven dpi. Red indicates significantly increased peak area (abundance) in the LPS-injected striatum, whereas green represents significantly decreased peak area in the LPS-injected striatum, compared to NI striatum. The abundance of these was Log transformed for statistical analysis. n = 5; paired Student’s t test was used to compare groups in each GP. All other GPs (not mentioned) did not show any significant difference in abundance between LPS-injected and NI striata

Fig. 4

Changes in the N-glycosylation traits between LPS-injected striatum and contralateral (non-injected (NI)) striatum at seven days post-injection. The common glycosylation features amongst the main structures in each glycan peak were grouped in the main glycosylation traits, according to Supplementary table S3. Only the most abundant glycan in each GP was considered. Data presented as the mean ± SD, n = 5. Paired Student’s t test was used and statistically significant difference was set at *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Changes in the percentage fraction of fucosylated and sialylated N-glycans between LPS-injected striatum and contralateral (non-injected (NI)) striatum at seven days post-injection. a Proportion of total N-glycans decorated with either core or outer arm fucose (or both), considering the main glycan structure in each GP. b Percentage fraction of fucosylated N-glycans according to the degree of fucosylation (n = 1, 2, 3, 4), which includes both sialylated and unsialylated fucosylated glycans. Data presented as the mean ± SD, n = 5. Paired Student’s t test was used, and statistically significant difference was set at ****p < 0.0001. c Percentage fraction of sialylated N-glycans according to the degree of sialylation, considering the main glycan structure in each GP. d Percentage fraction of sialylated N-glycans according to the linkage of the sialic acid to galactose residue indicates the significantly high abundance of α(2-3)-linkage to galactose compared to that of α(2-6)-linkage. Data presented as the mean ± SD, n = 5. Paired Student’s t test was used, and statistical significant difference was set at ****p < 0.0001

Fig. 6

N-glycan imaging of the brain of LPS-injected rats. Main N-glycan structures that are visualised in the lesion core belong to specific groups: oligomannose, bisected structures and galactosylated glycans, mainly with α-galactose. Each image is accompanied by the putative structures determined by combinations of accurate m/z, CID fragmentation patterns and glycan database structure