Glycosylation in Axonal Guidance

Abstract

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.

Keywords: attraction; axonal guidance; chemotaxis; chondroitin sulfate; glycosaminoglycan; glycosylation; haptotaxis; heparan sulfate proteoglycan; hyaluronan; repulsion.

Figure 1

Overview of axonal guidance. (A) Growth cone (i) extends and turns toward the attractive cues like netrin, shh (ii) and repelled by repulsive cues like slits, semaphorins (iii). Attractive and repulsive cues are shown with + (green) and –(red), respectively. (B) Short-range and long-range guidance within the spinal cord. (i) Axons are repelled by BMP7 and draxin from the roof plate. Shh and netrin-1 attract axons toward the floor plate by long-range attraction. Netrin-1 produced by neural progenitor cells has also been proposed to guide commissural axons locally (not shown here) while whether floor plate netrin is dispensable or not is debated [9]. (ii) Axons enter the floor plate through short-range guidance, for example, through Nr-CAM. (iii) Slit allows axons to exit the floor plate. (iv) Axons move rostrally through the repulsive gradient of Shh (acts as an attractive cue pre-crossing and repulsive cue post-crossing) and attractive gradient of Wnt. Examples of glycosylated proteins regulating adhesion at the midline are shown (dystroglycan, and endoglycan). Receptors and multiple other guidance cues e.g., semaphorins involved in regulating midline crossing that are beyond the scope of this review are not shown here.

Figure 2

Functions of glycosylation in axon guidance. (A) PSA (shown in red) acts as negative regulator of axon fasciculation. (B) L1 on neurons binds to α 2,3-sialic acid carried by CD24. Neuronal contactin and TAG1 bind to Lewis^x carried by CD24. This CD24, L1, TAG1 and contactin complex causes signal transduction to induce neurite outgrowth in cerebellar neurons (right). In DRG neurons, TAG1 of the TAG1-CD24 complex interacts with Cspr2 and contactin of the contacin-CD24 complex interacts with Cspr1 (left). These two independent complexes cause signal transduction to inhibit neurite outgrowth (box in the right corner shows all the complexes, top: outgrowth inhibiting complex, bottom: outgrowth promoting complex. (C) HSPGs are major components of ECM and can modulate neuronal microenvironment by organizing ECM and guidance molecules. (D) CSPGs form an inhibitory zone around neurons (left), enzymatic removal CSPGs causes the reversal of inhibition and promotes the growth. (E) Role of glycosylation in receptor trafficking and surface representation. On right, (i) ER-Golgi transition (ii) vesicle containing glycosylated receptor and (iii) surface representation of glycosylated receptor are shown. Multiple proteins bind and form a complex with PSA to regulate their functions. Cell surface-exposed PSA interacts with morphogenic factors such as BDNF, NGF, and NT3, and activate their receptors. PSA-bound BDNF leads to activation of BDNF receptors TrkB and p75NTR, resulting in increased survival and growth of neuroblastoma cells [37]. Apart from the NCAM-mediated adhesion by PSA discussed above, PSA has multiple binding partners in the nervous system that regulate processes from neurogenesis to synapse formation in the developing nervous system [24]. However, detailed molecular mechanisms of how PSA modulates these functions need to be investigated to understand higher-order functions of PSA in the nervous system.

Figure 3

Glycosylation sites prediction of netrin-1 (A) Crystal structure of mouse netrin-1 generated using PyMOL using PDB files obtained from UniProt. Glycosylation sites predicted from GlycoMine ^struct [92] are presented with black and yellow spheres in crystal structure. Yellow and black spheresindicate N-linked (probability more than 0.8) and l O-linked (probability more than 0.95) glycosylation sitesrespectively. (B,C) Domain organization of mouse (B) and chicken (C) netrin-1 with predicted glycosylations sites. N-linked glycosylation sites known from the crystal structure are marked with asterisks (N91, N116, and N131). Color code of domains is consistent with structure shown in A (NTR domain is not represented in A).