Abstract
Because of the importance of the HNK-1 carbohydrate for preferential motor reinnervation after injury of the femoral nerve in mammals, we screened NIH Clinical Collection 1 and 2 Libraries and a Natural Product library comprising small organic compounds for identification of pharmacologically useful reagents. The reason for this attempt was to obviate the difficult chemical synthesis of the HNK-1 carbohydrate and its isolation from natural sources, with the hope to render such compounds clinically useful. We identified six compounds that enhanced neurite outgrowth from cultured spinal motor neurons at nM concentrations and increased their neurite diameter, but not their neurite branch points. Axons of dorsal root ganglion neurons did not respond to these compounds, a feature that is in agreement with their biological role after injury. We refer to the positive functions of some of these compounds in animal models of injury and delineate the intracellular signaling responses elicited by application of compounds to cultured murine central nervous system neurons. Altogether, these results point to the potential of the HNK-1 carbohydrate mimetics in clinically-oriented settings.
Highlights
Owing to the altogether favorable nature of the mammalian peripheral nervous system to allow regeneration after injury, the question arises how specificity for motor versus sensory reinnervation is achieved
These potential HNK1 mimicking compounds were thereafter validated with substrate-coated HNK1 antibody (1 μg/mL; 50 μL/well), incubated with each compound, and probed for binding using a biotinylated HNK1 peptide (1 μg/mL in PBS) that was targeted by horseradish peroxidase (HRP)-coupled streptavidin (1:1000 in PBS) (Figure 1)
The HNK1 peptide mimetic has been validated in several studies to be structurally and functionally equivalent to the native HNK1 glycan: it was obtained by screening a phage peptide display library with a commercial monoclonal HNK1 antibody and the monoclonal L1 antibody [9], which was thereafter termed HNK1 antibody because their similarity in epitope
Summary
Owing to the altogether favorable nature of the mammalian peripheral nervous system to allow regeneration after injury, the question arises how specificity for motor versus sensory reinnervation is achieved. After injury of the vertebrate femoral nerve before its bifurcation, motor axons regrow into the motor branch, largely ignoring the sensory branch [1,2,3]. The HNK1 glycan was discovered via a monoclonal antibody obtained by immunization with a human T cell line and found to be predominantly expressed by natural killer cells, the name [6]. Not all protein backbones of each molecule express the covalently attached HNK1 glycan but carry it differently at different stages during development and in different parts of the nervous system [12]. Interest in the HNK1 glycan pertains to its receptors, laminin [13,14], N-cadherin [15,16], and high mobility group box 1 (HMGB1, called amphoterin) [17,18]
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