Abstract
We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.
Highlights
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS), typically beginning as a relapsing-remitting disease (RRMS) [1]
Immunocytochemistry (IC) of fixed human oligodendroglioma (HOG) cells showed that most cells expressed myelin basic protein (MBP) (Figure 1C) and 2,3 -cyclic nucleotide-3 -phosphodiesterase (CNPase) (Figure 1D)
Data for a population of HOG cells incubated with no aptamer are shown in Figure 2A, compared to 50 nM 3060-BS (Figure 2B), and Myaptavin-3064 (Figure 2C)
Summary
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS), typically beginning as a relapsing-remitting disease (RRMS) [1]. MS is thought to initiate with inflammation of unknown etiology, which leads to focal demyelination in the CNS, and is treated with a wide range of non-curative immunosuppressive agents [2]. Even in actively-treated patients, 36% of cases evolve into a progressive phase [3]. In pathologically inactive progressive cases, inflammation and axonal injury diminish to levels seen in age-matched controls, but neurodegeneration continues [4]. The remyelinating capacity of the CNS is apparently exhausted, leading to further progression and disability. Anti-inflammatory drugs are usually not beneficial in this stage
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