Multiple sclerosis patient-derived CSF induces transcriptional changes in proliferating oligodendrocyte progenitors

Abstract

Background: Cerebrospinal fluid (CSF) is in contact with brain parenchyma and ventricles, and its composition might influence the cellular physiology of oligodendrocyte progenitor cells (OPCs) thereby contributing to multiple sclerosis (MS) disease pathogenesis.

Objective: To identify the transcriptional changes that distinguish the transcriptional response induced in proliferating rat OPCs upon exposure to CSF from primary progressive multiple sclerosis (PPMS) or relapsing remitting multiple sclerosis (RRMS) patients and other neurological controls.

Methods: We performed gene microarray analysis of OPCs exposed to CSF from neurological controls, or definitive RRMS or PPMS disease course. Results were confirmed by quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry and western blot of cultured cells, and validated in human brain specimens.

Results: We identified common and unique oligodendrocyte genes for each treatment group. Exposure to CSF from PPMS uniquely induced branching of cultured progenitors and related transcriptional changes, including upregulation (P<0.05) of the adhesion molecule GALECTIN-3/Lgals3, which was also detected at the protein level in brain specimens from PPMS patients. This pattern of gene expression was distinct from the transcriptional programme of oligodendrocyte differentiation during development.

Conclusions: Despite evidence of morphological differentiation induced by exposure to CSF of PPMS patients, the overall transcriptional response elicited in cultured OPCs was consistent with the activation of an aberrant transcriptional programme.

Keywords: Cerebrospinal fluid; differentiation; gene expression; oligodendrocyte progenitor cells.

Figure 1. Multiple sclerosis and control patient cohorts and experimental plan

(A) Table describing patient cohort including the neurological controls and PPMS and RRMS patients. (B) Schematic diagram of treatment protocol for rat OPCs exposed to CSF. (C) MTT assay reduction of rat OPCs exposed to different patient group CSFs for 24 h. Statistical differences were determined using one-way ANOVA with Dunnett’s correction (NS = p > 0.05).

Figure 2. Cerebrospinal fluid from patients with a PPMS disease course promotes changes in OPC which are consistent with differentiation

(A) Phase contrast micrographs showing the morphology of OPCs exposed to CSF for 24 h. Scale bar = 20 µm. (B) Quantification of primary processes in OPCs exposed to CSF from neurological controls, PPMS or RRMS patients. (C) qPCR analysis of differentiation markers in OPCs treated with CSF from different patient groups. Statistical differences in (B), and (C) were determined using one-way ANOVA with Dunnett’s correction (*p<0.05; **p<0.01; ***p<0.001 vs. control).

Figure 3. Exposure of rat OPC to human CSF form patients with distinct disease course results in distinctive transcriptional responses

(A) Flowchart of the microarray analysis performed in OPCs incubated with CSF from patients with PPMS, RRMS or neurological controls for 24 h. (B) Principal component analysis of samples revealed a tight clustering of the samples within each treatment group. (C) Venn diagrams depicting uniquely and commonly up-regulated genes amongst PPMS and RRMS groups compared to control group. (D) Venn diagrams depicting uniquely and commonly down-regulated genes amongst PPMS and RRMS groups compared to control group. (E) Gene ontology analysis of up-regulated genes shared between PPMS and RRMS. (F) Gene ontology analysis of down-regulated genes shared between PPMS and RRMS.

Figure 4. Common and unique gene expression changes associated with exposure of rat OPCs to CSF from patients with distinct disease course

(A) qPCR analysis of genes commonly regulated by exposure of rat OPCs to CSF from RRMS and PPMS patients. (B) qPCR analysis of genes uniquely regulated by exposure of rat OPCs to CSF from RRMS patients. Different colors are used to indicate triplicates for each sample. Values were normalized to Gapdh and statistical differences using one-way ANOVA with Tukey’s multiple comparison correction (*p<0.05; **p<0.01; ***p<0.001).

Figure 5. Cerebrospinal fluid from PPMS patients induces aberrant transcriptional changes in oligodendrocyte progenitors

(A) Schematic diagram showing the analysis between microarray gene changes induced by PPMS-CSF on proliferating OPCs and RNAseq data from Zhang Y, et al 2014 during oligodendrocyte differentiation. (B) Pie chart showing the overlap between the developmentally regulated genes and the genes down-regulated by exposure to the CSF of PPMS patients. Note that genes were labeled as “common” (present in both data sets) or “unique” (only present in the OPC treated with CSF); the table depicts developmentally regulated genes also modulated by CSF exposure. (C) qPCR analysis of down-regulated gene targets. Different colors are used to indicate triplicates for each culture exposed t CSF. Values were normalized to Gapdh and statistical differences using one-way ANOVA with Tukey’s multiple comparison correction (*p<0.05; **p<0.01). (D) qPCR analysis of genes down-regulated by CSF exposure but up-regulated during oligodendrocyte differentiation. (E) Pie chart showing the overlap between the developmentally regulated genes and those up-regulated by CSF exposure. The table identifies some of the genes regulated by the development al process and by exposure to CSF.

Figure 6. Exposure of rat OPCs to the CSF from PPMS patients results in high Galectin-3 transcript and protein levels

(A) qPCR analysis of Lgals3 in OPCs treated with CSF from different patient groups. Values were normalized to Gapdh and statistical differences evaluated using one-way ANOVA with Tukey’s multiple comparison correction (**p<0.01). (B) GALECTIN-3 staining in rat OPCs treated with CSF from control, PPMS and RRMS patients. Scale bar = 20 µm. (C) GALECTIN-3 signal intensity, quantified from (B). (D) Western blot analysis of GALECTIN-3 levels in OPCs treated with CSF from different patient groups. (E) GALECTIN-3 signal intensity, quantified from (D) relative to ACTIN levels. Statistical differences in (C) and (E) were determined using one-way ANOVA with Dunnett’s correction (*p<0.05; ***p<0.001 vs. control).

Figure 7. LGALS3, the gene encoding for Galectin 3 is up-regulated in human normal-appearing white matter (NAWM) from secondary progressive and PPMS patients

(A) Quantitative PCR analysis of LGALS3 in human NAWM, normalized to the geometric average of GAPDH, DHX32 and RPLP0. Different colors are used to indicate triplicates for each sample. Statistical differences were determined using one-way ANOVA with Dunnett’s correction (**p<0.01; ***p< 0.001 vs. control). (B) Immunostaining of NAWM slides with antibodies against GALECTIN-3 and counterstained with hematoxylin to identify cellular nuclei. Scale bar = 50 µm.