Glutamate receptor expression in multiple sclerosis lesions

Abstract

Blockade of receptors for the excitatory neurotransmitter glutamate ameliorates neurological clinical signs in models of the CNS inflammatory demyelinating disease multiple sclerosis (MS). To investigate whether glutamate excitoxicity may play a role in MS pathogenesis, the cellular localization of glutamate and its receptors, transporters and enzymes was examined. Expression of glutamate receptor (GluR) 1, a Ca(++)-permeable ionotropic AMPA receptor subunit, was up-regulated on oligodendrocytes in active MS lesion borders, but Ca(++)-impermeable AMPA GluR2 subunit levels were not increased. Reactive astrocytes in active plaques expressed AMPA GluR3 and metabotropic mGluR1, 2/3 and 5 receptors and the GLT-1 transporter, and a subpopulation was immunostained with glutamate antibodies. Activated microglia and macrophages were immunopositive for GluR2, GluR4 and NMDA receptor subunit 1. Kainate receptor GluR5-7 immunostaining showed endothelial cells and dystrophic axons. Astrocyte and macrophage populations expressed glutamate metabolizing enzymes and unexpectedly the EAAC1 transporter, which may play a role in glutamate uptake in lesions. Thus, reactive astrocytes in MS white matter lesions are equipped for a protective role in sequestering and metabolizing extracellular glutamate. However, they may be unable to maintain glutamate at levels low enough to protect oligodendrocytes rendered vulnerable to excitotoxic damage because of GluR1 up-regulation.

Figure 1

Distribution of GluR1 and GluR2 immunostaining. A–C. Anti‐GluR1 immunostaining of (A) weakly immunolabeled oligodendrocytes in normal control brain white matter, (B) strongly stained oligodendrocytes in the border of an active plaque; (C) same area as (B) on an adjacent section; the antibody was antigen‐blocked by pre‐incubation with the peptide sequence used to raise the antibody; no oligodendrocytes were stained and only weakly immunolabeled macrophages seen. D. 14E immunostaining of oligodendrocytes in same area as B and C. E,F. Anti‐GluR2 staining of (E) oligodendrocytes in normal control brain white matter and (F) weak staining of oligodendrocytes and macrophages in a section serial to those shown in B–D. All acetone fixed, no hematoxylin counterstaining; scale bar 25 µm.

Figure 2

GluR5–7 immunostaining. A. Normal control white matter, longitudinal section of spinal cord; only very weak GluR5–7 staining of a blood vessel is seen. B. Punctate GluR5–7 immunostaining of endothelial cells in a spinal cord active plaque blood vessel. C. High power view of a GluR5–7‐immunopositive axon subpopulation around a blood vessel in a brain active plaque. D. Lower power view of GluR5–7‐immunopositive axons around another blood vessel in the same active plaque as in C. Scale bars 25 µm.

Figure 3

Expression of mGluRs. A. Normal control white matter. B–F. Adjacent sections from the same active plaque. A–C. Anti‐mGluR1. A. Weakly immunolabeled oligodendrocytes. B. Active plaque, possible glial progenitor cells and small reactive astrocytes. C. Positive axons in another area of the same plaque. D. Anti‐mGR2/3: possible glial progenitor cells and immature bipolar astrocytes. E. Anti‐mGR5. F. Monoclonal anti‐GFAP antibody immunostaining of dense astrocyte processes. Scale bar 25 µm.

Figure 4

Anti‐GLT‐1, EAAC1 and GDH immunostaining. A,B. Anti‐GLT‐1 immunostaining of only occasional astrocytes which have complex branching processes in normal control white matter. However, in (B) dense astrocyte process staining is seen throughout an active multiple sclerosis lesion. C,D. Adjacent sections of a brain active plaque. C. EAAC1 expression in a reactive astrocyte subpopulation and granules in macrophages. D. GDH immunostaining of hypertrophic astrocytes. Scale bar 25 µm.

Figure 5

Anti‐glutamate antibody immunostaining. A–C. Anti‐glutamate monoclonal antibody, 1:2000. A. Weakly immunolabeled glia in normal control brain white matter. B. Hypertrophic astrocytes surrounding a small blood vessel in an active MS plaque border. C. Top: white matter; bottom: active MS plaque. D. Anti‐GFAP, astrocyte cell bodies and processes in the same area as (B) on an adjacent section. All sections carbodiimide fixed and no hematoxylin staining. Scale bars 25 µm in A, B and D; 250 µm in C.