Myelin degeneration in multiple system atrophy detected by unique antibodies

Abstract

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.

Figure 1.

A: Specificity of anti-EP and QD-9 antibodies examined by Western blot. Lane a, anti-EP antibody; lane b, clone QD-9. The two antibodies detect a major 18.5-kd band (arrow) and a weaker 17.2-kd band (arrowhead) in extracts of MSA brain homogenates. B and C: Comparison between anti-EP and QD-9 immunohistochemistry in MSA brains. Anti-EP (B) and QD-9 (C) recognized identical structures in MSA brains. B and C are nearby sections from the putamen of an MSA patient (case M4). D: No immunoreactivity is detected in control brains by anti-EP or QD-9. D shows a control cerebellum stained by anti-EP (case C4). Bars, 50 μm (B to D).

Figure 2.

Double staining of specific markers and EP/QD-9 in MSA cases. The distribution of EP (A to C, purple) is different from those of leukocyte common antigen-labeled microglia (A, brown), SMI31-labeled neuronal axons (B, brown), and MAP-2B-labeled neuronal elements (C, brown). QD-9-positive structures (D, purple) do not overlap with glial fibrillary acidic peptide-positive astroglia (D, brown). All of these sections are from case M3. Bars, 50 μm (A to D).

Figure 3.

Various patterns of anti-EP/QD-9-positive oligodendroglial elements observed in MSA tissue. A: Long, thickened fibers (case M5). B: Densely stained cell bodies of irregular size and shape (arrow). Sometimes abnormally appearing fibers extend from them (case M3). C: Thickened or short, misshapen fibers extend from a positively staining oligodendrocyte (arrow) (case M5). Bars, 50 μm (A to C).

Figure 4.

Anti-EP/QD-9-positive oligodendroglial elements observed in various regions in the MSA brains. A: Cerebellar cortex (case M6). Long fibers extend from the Purkinje cell layer to the granular layer. Bar, 100 μm. B: Positive fibers in the middle cerebellar peduncle (case M4). C: Thick fibers around neurons of the dentate nucleus are stained (case M4). Bar, 25 μm. D: Both longitudinal and transverse bundles at the base of the pons contain positive structures (case M3). E: Myelinated fiber bundles are intensely stained in the posterior lateral part of the putamen (case M4). F: The intensity of staining in the caudate is less than that in the putamen (case M4). G: Globus pallidus, pars externa (case M4). H: Internal capsule (case M6). Bar in H, 50 μm (applies to B and D to H).

Figure 5.

Comparison between anti-EP staining (A) and the Klüver-Barrera method (B) in nearby sections from the temporal cortex of an MSA patient (case M4). Stars in A and B identify the same vessel. Notice that the anti-EP antibody stains abnormal oligodendroglial structures, whereas the Klüver-Barrera method shows little or no demyelination. Bar, 100 μm.

Figure 6.

Comparison between the Klüver-Barrera method (A, C, and E) and the anti-EP method (B, D, and F) in the cerebellar medulla of an MSA patient (case M4). A and B, C and D, and E and F are nearby sections. Stars and asterisks identify the same vessels. Anti-EP-positive structures are more intense in the moderately demyelinated area (C and D) than in the severely affected (A and B) or slightly affected (E and F) regions. Bar in F, 100 μm (applies to A to F).

Figure 7.

Comparison between intensity of Klüver-Barrera staining and EP/QD-9 density in MSA middle cerebellar peduncle. Anti-EP/QD-9 density is expressed as a percentage of each cross-sectional area. Grading was performed according to the average KBI measured in adjacent sections. *P < 0.05; **P < 0.01; ***P < 0.001. P values were calculated using the Mann-Whitney U test. See text for more details.

Figure 8.

Double staining with QD-9 and anti-ubiquitin in MSA cases. The distribution of QD-9 (purple) is different from that of ubiquinated GCIs (brown). A: Moderately affected cerebellar medulla (case M6). Ubiquinated GCIs and QD-9-positive fibers are detected. Note that QD-9 does not stain the GCIs. B: QD-9-positive fibers located in the globus pallidus in a case of MSA (case M4). GCIs are rarely observed. C: Plenty of GCIs are observed in the putamen in a case of MSA (case M5), whereas QD-9-positive fibers are relatively sparse in this case. Bar in C, 100 μm (applies to A to C).