Proteomic screen of dorsal root ganglion neuron growth states reveals Glypican 1 as an autocrine and paracrine activator of axon growth

Following an injury in the adult central nervous system (CNS), axons fail to regenerate because of repressed intrinsic growth competence and a cellular environment that lacks growth support. Interestingly, injured axons of dorsal root ganglia neurons (DRG) can become growth competent and regenerate under specific conditions. The molecular program underlying their regenerative growth, however, has remained unclear. Thus far, studies of growth and regeneration in DRG axons have focused on candidate- or pathway-specific approaches or employed RNA sequencing for unbiased studies. However, transcriptomes only weakly predict the actual proteome. Especially non-transcriptional mechanisms, such as target degradation via autophagy or the proteasome or secretion of targets, can escape such transcriptomic analyses. Consequently, we set out to investigate the proteome of growing and regenerating DRGs. We used three previously published growth paradigms, in which DRG axons switch between low growth with high branching, to elongating growth with little branching. Proteomic analysis identified 39 proteins that inhabit the intersection of these growth paradigms. An overexpression screen of the identified candidates revealed multiple growth effectors. The largest growth effect was elicited by the cellsurface proteoglycan Glypican1 (Gpc1), which has previously been implicated in developmental axon guidance in both mammals and invertebrates. Overexpression of Gpc1 was sufficient to induce axon growth in cultured neurons, where it can overcome the growth inhibition conferred by chondroitin sulfate proteoglycans (CSPGs). Secretion of Gpc1 to the plasma membrane was necessary to bolster growth. Functional knock out of its heparan sulfate binding domain indicated that Gpc1 modulates cell-surface signalling receptors. We demonstrated that Gpc1 acts both autocrine and paracrine. Gpc1-overexpressing cells confer growth competence to WT neurons via extracellular vesicles which label positive for Gpc1. Further, we show that overexpression of Gpc1 changes the whole- and phospho proteome of the cell, to mirror a number of characteristics we found in the original growth paradigms. Together these results reveal Gpc1 as a promising candidate for growth activation, as it acts extracellularly to activate intracellular growth mechanisms, a challenge on which many other growth activators fail. <em>In vivo</em> studies will reveal the merit of Gpc1 in regenerating axons following CNS injury.