Beyond development: LINX/Islr2 expression and TrkA interaction in the adult nervous system under neuroinflammatory challenge.

Abstract

Introduction: LINX, also known as immunoglobulin superfamily containing leucine-rich repeat 2 (Islr2), was identified as a TrkA pathway component and demonstrated to modulate the NGF/TrkA-mediated axonal growth, guidance, and branching during development. However, there is limited information on LINX expression in the adult nervous system, and the possible functions of LINX in mature neurons remain unexplored. These two questions are addressed by the present study, which first investigates LINX expression in the adult spinal cord (SC), dorsal root ganglia (DRG), and cerebral cortex (Cx), and subsequently explores LINX/TrkA functions using the DRG organ culture model.

Methods: Different commercially available anti-LINX antibodies were tested, and the most selective antibody was used to investigate LINX expression in the SC, Cx, and DRG of healthy rats, and to explore the LINX/TrkA action in DRG organ cultures using a model of neuroinflammation/injury induced by lipopolysaccharides (LPS). The LINX/TrkA interaction was demonstrated by immunoprecipitation in the Cx of healthy and LPS-treated rats. The DRG from healthy and LPS-treated rats were dissected and cultured in the presence of anti-LINX, with or without stimulation using NGF, and a TrkA selective inhibitor. TrkA phosphorylation levels and the expression of the axonal growth marker GAP43 were analyzed by bio-molecular assays and confocal immunofluorescence microscopy.

Results: The WB analysis shows that LINX is expressed in the adult central and peripheral nervous systems, and the immunoprecipitation analysis demonstrates LINX and TrkA interaction in the Cx of both adult healthy and LPS-treated rats. In vitro experiments using an LPS-DRG model confirm the association between LINX and TrkA. However, it was found that while anti-LINX administration abolishes the effects of NGF and TrkA activation in LPS-DRG, it results in GAP43 increase in the presence of a TrkA inhibitor.

Discussion: Our study confirms that LINX is expressed in the adult central nervous system and demonstrates that the LINX/TrkA interaction is maintained following LPS-induced systemic inflammation. Moreover, our findings that antibody-mediated perturbation of endogenous LINX is associated with the modulation of axonal growth markers in adult LP-DRG cultures, in serum-free conditions and/or in the presence of a TrkA inhibitor, suggest a potential role for LINX in regulating neurotrophin-dependent responses in injured adult neurons.