Fig 2: Increased CSF PVALB and Nf-L levels in MS patients. (A) PVALB protein concentration in CSF from controls (n = 32) and MS patients (n = 110). (B) Nf-L protein concentration in CSF from controls (n = 32) and MS patients (n = 110). (C) Correlation between CSF PVALB and Nf-L levels in MS patients (r = 0.036, P < 0.001, n = 110). Boxplots show the medians and the two hinges. Dots represent the samples analyzed. *P < 0.05, ***P < 0.001. MS: Multiple Sclerosis; Ctrl: controls; r: Pearson correlation coefficient; P: P-value.

Fig 3: Correlations between cognitive impairment and global cortical thickness and with CSF PVALB and NF-L levels in MS patients. (A) Boxplot representing the comparison of PVALB levels among the MS groups characterized by different levels of cognitive impairment. (B) Boxplot representing the comparison of Nf-L level among the MS groups characterized by different levels of cognitive impairment. Boxplots show the medians and the two hinges. Dots represent the individual samples analyzed. *P < 0.05. CTh: cortical thickness; CN: cognitively normal MS patients; mCI: MS patients with mild cognitive impairment; sCI: MS patients with severe cognitive impairment.

Fig 4: 3T MRI global outcomes correlations with CSF PVALB and Nf-L levels in MS patients. (A) Correlation plot showing different strength of correlation between CSF PVALB and Nf-L levels with MRI parameters. A false discovery rate (FDR) correction was applied. The Spearman rank index is proportional to the dimension/intensity of the bubbles. A color scale was used to determine the slope of the correlation. Blank squares indicate no significant correlation. (B) Scatter plot of relationship between CSF PVALB levels and global cortical thickness (CTh) in MS patients. (C) Scatter plot of relationship between CSF PVALB levels and number of cortical lesions (CLs) in MS patients. Dots represent the samples analyzed. CLs: cortical lesions; CTh: cortical thickness; R: Spearman correlation coefficient; P: P-value.

Fig 5: Molecular and neuropathology analysis of PVALB expression in post-mortem progressive MS. Gene ontology enrichment analysis for the top five biological process (A) and molecular function (B) clusters of differentially expressed genes in post-mortem precentral gyrus with grey matter (GM) lesions derived from SPMS cases characterized by the presence follicle-like structures (Fpos). A modified Fisher’s exact P-value (EASE) was used to determine the gene-enrichment analysis and Benjamini–Hochberg multiple test correction was applied to calculate the False Discovery Rate (FDR). Immunohistochemical assessment of PVALB+ cells in the precentral gyrus (motor cortex) of one post-mortem control (C) and one MS case (D). PVALB immunostaining detected a subpopulation of cortical neurons (E). (F) Boxplot reporting the percentage reduction of PVALB+ cells with respect to control in post-mortem precentral gyrus of the two independent MS cohorts (group I and II). (G) PVALB protein levels in available paired CSF post-mortem samples (n = 20) and age-matched healthy controls (n = 10). (H) Nf-L protein levels in available paired CSF post-mortem samples (n = 20) and age-matched healthy controls (n = 10). (I) Summary table of the correlation analyses between PVALB/Nf-L CSF levels and PVALB+ cell counts, % GM demyelination, MHC-II+ cell counts, age of disease onset (years), age at wheelchair (years; EDSS = 7), disease duration (years), and age at death (years). Means and SEM were used to represent the data. Dots represent the samples analyzed. *P < 0.05; **P < 0.01; ***P < 0.001. pm-MS: post-mortem Multiple Sclerosis cases; Ctrl: controls; R: correlation coefficient; P: P-value; GM: grey matter; MHC-II: major histocompatibility complex-II. Original magnifications: A, B 100×; B, D 400×.