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. 2024 Aug 21:17:1448215.
doi: 10.3389/fnmol.2024.1448215. eCollection 2024.

Proteomics analysis of periplaque and chronic inactive multiple sclerosis lesions

Jordan M Wilkins  1 , Kiran K Mangalaparthi  2 , Brian C Netzel  3 , William A Sherman  4 , Yong Guo  1 , Alicja Kalinowska-Lyszczarz  5 , Akhilesh Pandey  2 , Claudia F Lucchinetti  6
Affiliations

Proteomics analysis of periplaque and chronic inactive multiple sclerosis lesions

Jordan M Wilkins et al. Front Mol Neurosci. .

Abstract

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by increased inflammation and immune responses, oxidative injury, mitochondrial dysfunction, and iron dyshomeostasis leading to demyelination and axonal damage. In MS, incomplete remyelination results in chronically demyelinated axons and degeneration coinciding with disability. This suggests a failure in the ability to remyelinate in MS, however, the precise underlying mechanisms remain unclear. We aimed to identify proteins whose expression was altered in chronic inactive white matter lesions and periplaque white matter in MS tissue to reveal potential pathophysiological mechanisms.

Methods: Laser capture microdissection coupled to proteomics was used to interrogate spatially altered changes in formalin-fixed paraffin-embedded brain tissue from three chronic MS individuals and three controls with no apparent neurological complications. Histopathological maps guided the capture of inactive lesions, periplaque white matter, and cortex from chronic MS individuals along with corresponding white matter and cortex from control tissue. Label free quantitation by liquid chromatography tandem mass spectrometry was used to discover differentially expressed proteins between the various brain regions.

Results: In addition to confirming loss of several myelin-associated proteins known to be affected in MS, proteomics analysis of chronic inactive MS lesions revealed alterations in myelin assembly, metabolism, and cytoskeletal organization. The top altered proteins in MS inactive lesions compared to control white matter consisted of PPP1R14A, ERMN, SIRT2, CARNS1, and MBLAC2.

Conclusion: Our findings highlight proteome changes in chronic inactive MS white matter lesions and periplaque white matter, which may be crucial for proper myelinogenesis, bioenergetics, focal adhesions, and cellular function. This study highlights the importance and feasibility of spatial approaches such as laser capture microdissection-based proteomics analysis of pathologically distinct regions of MS brain tissue. Identification of spatially resolved changes in the proteome of MS brain tissue should aid in the understanding of pathophysiological mechanisms and the development of novel therapies.

Keywords: differentially expressed proteins; multiple sclerosis; pathology; protein networks; proteomics; spatial profiling.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Immunohistochemical myelin staining of brain tissue used in study. All sections were stained for myelin proteolipid protein 1 (PLP1) and for macrophages using CD68. Tissues from control individuals are seen in (A–C) and multiple sclerosis patients in (D–F). The green box indicated in (D-F) indicates the zoomed in regions of PLP1 (G–I) and CD68 (J–L) stains. Minimal cortex was present in MS case E and omitted from analysis. Brain tissue regions are indicated as follows: C, cortex; IA, inactive lesions; P, periplaque white matter; WM, white matter.
FIGURE 2
FIGURE 2
Proteome alterations are unique in MS lesions and periplaque white matter. Overview of proteome changes in multiple sclerosis brain tissue. (A) Principal component analysis of the top 500 variable proteins. (B) Bar plots showing significantly altered myelin-associated proteins in MS lesions compared to control and MS brain tissue regions. The data is centered around the log2-intensity. CTRL, control; CTX, cortex; IAL, inactive lesion; MS, multiple sclerosis; PPWM, periplaque white matter.
FIGURE 3
FIGURE 3
Cellular organization, biogenesis, and metabolism are altered in MS lesions. Proteome changes in MS inactive white matter lesions compared to control white matter. All proteins were significantly altered in MS white matter lesions when compared to control white matter tissue (p-value < 0.01 and fold-change ≥ 2). (A) A heatmap highlighting the top 100 differentially expressed proteins in MS inactive lesions when compared to control white matter tissue (ranked by p-value). All differentially expressed proteins can be seen in Supplementary Figure 1. (B) Protein-protein interaction network of differentially expressed proteins. The top 100 interconnected proteins are shown for clarity. A full interaction network can be seen in Supplementary Figure 2. (C) Enrichment of biological functions. (D) Enrichment of pathways. Gene count and Strength were determined by the STRING application in (C,D). CWM, control white matter; IAL, inactive lesion; MS, multiple sclerosis.
FIGURE 4
FIGURE 4
Network analysis reveals potentially unique alterations in periplaque white matter. Possible proteome changes that occur early in the white matter of MS brain tissue. (A) A Venn diagram showing the distribution of differentially expressed proteins amongst two comparisons in the white matter tissue (Inactive lesion versus control white matter, left; Inactive lesion versus periplaque white matter, right). (B) The top network perturbed within the 65 unique proteins likely affected in periplaque white matter. (C) The top network altered among the 267 unique proteins affected in chronic inactive lesions. (D) The top network altered in the 237 commonly affected proteins within MS inactive lesions. Green and red depict significantly decreased or increased proteins, respectively. Differentially expressed proteins were considered when the p-value < 0.01 and fold change ≥ 2. Abbreviations: CWM, control white matter; IAL, inactive lesion; PPWM, periplaque white matter.
FIGURE 5
FIGURE 5
Correlated proteome changes in spatially preserved MS brain tissue. Clustered heatmap reveals coordinated changes in MS tissue. The top spatially altered proteome changes in MS brain tissue when compared to control brains. Significantly altered proteins (adjusted p-value < 0.05 and fold change ≥ 2) with similar patterns were grouped into 8 clusters. Clusters with similar trends (e.g., Clusters 1/4 and 3/5) were evaluated for key altered functions, which is shown on the right side of the heatmap. Cluster 7 and Clusters 2/6 were considered to be white matter enriched and cortex enriched, respectively, which had no apparent perturbations in MS tissue. CTRL, control; CTX, cortex; IAL, inactive lesion; MS, multiple sclerosis; PPWM, periplaque white matter.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of the article. This study was supported by the Goldman Charitable Trust.

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