Growth associated protein (GAP-43): Cloning and the development of a sensitive ELISA for neurological disorders Sharmilee Gnanapavan a, ⁎, Nasim Yousaf b , Wendy Heywood c , Donna Grant a , Kevin Mills c , Yuti Chernajovsky b , Geoff Keir a , Gavin Giovannoni d a Department of Neuroimmunology, Institute of Neurology, Queen Square, London, UK b Bone and Joint Research Unit, William Harvey Research Unit, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK c Biochemistry Group, Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, London, UK d Blizzard Institute, Queen Mary University London, London, UK abstract article info Article history: Received 2 March 2014 Received in revised form 2 July 2014 Accepted 15 July 2014 Keywords: GAP-43 Neuroplasticity CSF Biomarker ELISA GAP-43 has been studied in the rodent and mammalian brain and shown to be present specifically in areas undergoing axonal elongation and synapse formation. GAP-43 was cloned using the baculovirus expression system and purified. A sandwich ELISA was developed using the recombinant GAP-43 as standard and validated. CSF GAP-43 levels were analysed in benign intracranial hypertension, movement disorders, multiple sclerosis, neuropathy, CNS infections, motor neuron disease, and headache (neurological controls). GAP-43 levels were low in all disorders analysed (in particular motor neuron disease; p = 0.001, and movement disorders and multiple sclerosis; p b 0.0001) compared to controls, aside from CNS infections. GAP-43 is preferentially reduced in the CSF of neurological disorders associated with neurodegeneration. © 2014 Published by Elsevier B.V. 1. Introduction The membrane associated protein GAP-43 (growth-associated protein of 43 kDa, MIM: 162060), neuromodulin or B-50, was originally isolated in 1980 (Zwiers et al., 1980) and appeared to be a major protein present in synaptic plasma membranes. It is found in axons specifically associated with growth cones and immature synaptic terminals (Skene et al., 1986). While GAP-43's precise function is not known, there are several lines of evidence to support for a role in path finding in axons. GAP-43 expression has been found to be greatly increased in growing (Jacobson et al., 1986) and regenerating axons, in some mature axons capable of regeneration (i.e. those of fish and amphibians), axotomy resulted in a gross upregulation of the protein that coincided with, or preceded the initiation of axonal outgrowth (Skene and Willard, 1981). Knockout mice lacking GAP-43 die early in the postnatal period, as well as having axons stall and then take random courses when they reach decision points such as the optic chiasm, suggesting that GAP-43 serves to amplify the response of growth cones to external cues (Strittmatter et al., 1995; Fishman, 1996). When GAP-43 is overexpressed in mice, lesion-induced nerve sprouting and terminal arborisation during re-innervation were greatly potentiated (Aigner et al., 1995). GAP-43 is found exclusively in particulate fractions of brain tissue and absent from cytosolic subcellular fractions of peripheral organs (Kristjansson et al., 1982). Throughout life GAP-43 is found in high levels in neocortical association areas and the limbic system, presum- ably localised to areas rich in synaptic contacts (Oestreicher et al., 1981; Oestreicher et al., 1986; Benowitz and Routtenberg, 1997). While in certain brain areas, in particular those involved in ascending somatosensory information (e.g. cochlear nuclei, the vestibular complex or the sensory cortex) and motor control (e.g. red nucleus, the motor nuclei of cranial nerves or the motor cortex), GAP-43 is essentially absent (Benowitz et al., 1988; Benowitz et al., 1989). In disease, GAP-43 at both a tissue level and within the CSF is reduced unless there is recovery in terms of remyelination or nerve growth. For instance, it has been found to be reduced in a majority of white matter plaques in multiple sclerosis (MS), with the exception of remyelinated lesions where the expression is increased (Teunissen et al., 2006). Low CSF GAP-43 levels have been associated with greater brain atrophy. While, in a study looking at the early stages of inflamma- tory lesions axons were demonstrated to remodel by recruiting local in- terneurons and collaterals in close association with the temporal expression of GAP-43 (Kerschensteiner et al., 2004). This trend is also evident in other neurodegenerative disorders, with reduced GAP-43 levels mainly in the late stage of Alzheimer's disease (AD) Journal of Neuroimmunology 276 (2014) 18–23 ⁎ Corresponding author at: Institute of Neurology, Queen Square, London WC1N 3BG, UK. Tel.: +44 0203 448 4488; fax: +44 0203 448 3797. E-mail address: s.gnanapavan@ucl.ac.uk (S. Gnanapavan). http://dx.doi.org/10.1016/j.jneuroim.2014.07.008 0165-5728/© 2014 Published by Elsevier B.V. Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim