Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients

Abstract

Studies of the properties of soluble oligomer species of amyloidogenic proteins, derived from different proteins with little sequence homology, have indicated that they share a common structure and may share similar pathogenic mechanisms. Amyloid β, tau protein, as well as amyloid precursor protein normally associated with Alzheimer's disease and Parkinson's disease were found in lesions and plaques of multiple sclerosis patients. The objective of the study is to investigate whether brain and cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients demonstrate the presence of soluble oligomers normally associated with protein-misfolding diseases such as Alzheimer's disease. We have used anti-oligomer monoclonal antibodies to immunodetect soluble oligomers in CSF and brain tissues derived from multiple sclerosis patients. In this report, we describe the presence of soluble oligomers in the brain tissue and cerebral spinal fluid of multiple sclerosis patients detected with our monoclonal anti-oligomer antibodies with Western blot and Sandwich enzyme-linked immunosorbent assay (sELISA). These results might suggest that protein aggregation plays a role in multiple sclerosis pathogenesis although further and more refined studies are needed to confirm the role of soluble aggregates in multiple sclerosis.

Keywords: monoclonal antibodies; multiple sclerosis; protein-misfolding disease; soluble oligomers.

Figure 1

Immunodetection of Aβ and tau oligomers in brains of Alzheimer’s disease patients with the PRIOC monoclonal antibodies. Brain sections (7 μm thickness) derived from Alzheimer’s disease patients (patient case number 51486; 60649; 60980) and from a control brain derived from a patient with cancer (patient case number 58695) were stained with PRIOC, 4G8 (stains Aβ plaques), or AT8 mAb (stains phosphorylated tau). The control 58695 case displayed no staining for oligomers, Aβ plaques, and pathological tau. The 51486 case showed a diffuse pattern of medium-sized aggregates with PRIOC mAb and large plaques with 4G8. Confocal studies displayed co-staining of both the plaques and the aggregates. Cases 60649 and 60980 displayed extensive intracellular staining (green fluorescence) and smaller plaques.

Figure 2

Presence of oligoclonal bands in CSF samples from MS patients. CSF samples from MS (primary progressive: MD01 and secondary progressive: MD02) were electrophoresed then probed with autologous serum (1, 2, 3, and 4). Secondary anti-IgM (1 and 2) or anti-IgG (3 and 4) HRP-conjugate was used for detection of the autologous sera. Sera were omitted from lanes 5, 6, 7, and 8 to confirm their binding specificity after addition of secondary anti-IgM (5 and 6) or anti-IgG (7 and 8) HRP-conjugate.

Figure 3

(A) Detection of soluble oligomers in brain homogenate and CSF of patients with multiple sclerosis. Brain homogenates and CSF samples MS094; MS074; MS076; MS079) from MS patients were probed with anti-oligomer mAbs. Several bands (90–200 kDa range) consistent with the oligomeric profile recognized on Western blotting have been detected. Positive control Parkinson’s disease (PD) brain homogenate and CSF displayed similar pattern as opposed to the human control (Ct, CO41) brain, which failed to show oligomer detection. (B) Brain homogenates and/or CSF derived from MS, PD, AD, and lung cancer were used to assess specific binding of PRIOC mAb to soluble oligomers in a Sandwich ELISA assay. PRIOC was used to coat the ELISA plate in coating buffer. Brain homogenate and/or CSF were added to the wells followed by a biotinylated PRIOC mAb. Error bars represent the mean antibody level derived from n = 4 wells.

Figure 4

(A) Proteinase K sensitive oligomers or prions in brain homogenate of patients with multiple sclerosis. Brain homogenates from MS patients (MS079) were probed with anti-oligomer mAbs. The bands (90–200 kDa range) demonstrated with the anti-oligomer antibody in both MS and the positive control Parkinson’s disease (PD) brain homogenates via western blotting, were no longer seen following proteinase K digestion. (B) Brain homogenates (MS074; MS076) from MS patients and the human control (Ct; CO41) brain were probed with anti-PrP 3F4 mAb to immunodetect the typical PrP bands. RML (PrP^Sc)-infected brain homogenate was used to probe for the PK sensitivity and was shown to display the 27–30 kDa band.