Guidance molecules in axon regeneration

Abstract

The regenerative capacity of injured adult mammalian central nervous system (CNS) tissue is very limited. Disease or injury that causes destruction or damage to neuronal networks typically results in permanent neurological deficits. Injury to the spinal cord, for example, interrupts vital ascending and descending fiber tracts of spinally projecting neurons. Because neuronal structures located proximal or distal to the injury site remain largely intact, a major goal of spinal cord injury research is to develop strategies to reestablish innervation lost as a consequence of injury. The growth inhibitory nature of injured adult CNS tissue is a major barrier to regenerative axonal growth and sprouting. An increasing complexity of molecular players is being recognized. CNS inhibitors fall into three general classes: members of canonical axon guidance molecules (e.g., semaphorins, ephrins, netrins), prototypic myelin inhibitors (Nogo, MAG, and OMgp) and chondroitin sulfate proteoglycans (lecticans, NG2). On the other end of the spectrum are molecules that promote neuronal growth and sprouting. These include growth promoting extracellular matrix molecules, cell adhesion molecules, and neurotrophic factors. In addition to environmental (extrinsic) growth regulatory cues, cell intrinsic regulatory mechanisms exist that greatly influence injury-induced neuronal growth. Various degrees of growth and sprouting of injured CNS neurons have been achieved by lowering extrinsic inhibitory cues, increasing extrinsic growth promoting cues, or by activation of cell intrinsic growth programs. More recently, combination therapies that activate growth promoting programs and at the same time attenuate growth inhibitory pathways have met with some success. In experimental animal models of spinal cord injury (SCI), mono and combination therapies have been shown to promote neuronal growth and sprouting. Anatomical growth often correlates with improved behavioral outcomes. Challenges ahead include testing whether some of the most promising treatment strategies in animal models are also beneficial for human patients suffering from SCI.

Figure 1.

Strategies to reestablish neuronal innervation following injury. (A) Long-distance axonal regeneration. Following axon transection, the distal segment of the nerve undergoes Wallerian degeneration (dotted black lines). New axons (red) sprout from the proximal axon segment (blue) and reestablish synaptic contact with preinjury targets. (B) short-distance growth of injured axons. Collateral sprouts (red) form synaptic contact with neighboring neuronal elements to by-pass the injury site. (C) Sprouting of spared axons to maintain connectivity beyond the injury site. The strategy shown in A is typically observed following compression injury in the PNS, whereas neuronal responses shown in B and C, have been observed in the injured adult mammalian CNS.

Figure 2.

Schematic of adult rat spinal cord with dorsal lesion cavity as a result of injury. Transected axons of ascending sensory fibers (green) display dystrophic end-bulbs (inset) at the edge of the lesion cavity. The lesion cavity is surrounded by a glial scar (red) composed of reactive astrocytes, microglia and meningeal fibroblasts that migrate into the lesion site. Growth inhibitory molecules, including semaphorins and chondroitin sulfate proteoglycans (CSPGs) are enriched in the glial scar contributing to the growth inhibitory nature of injured adult mammalian CNS tissue.

Figure 3.

The prototypic myelin inhibitors MAG, Nogo-A, and OMgp and their receptors. MAG (Siglec 4a) is a type-1 transmembrane protein with an ectodomain composed of five Ig-like domains. Nogo-A (RTN4A) has two inhibitory domains: Amino-Nogo and Nogo-66. The membrane topology of Nogo-A appears to be variable. Although Amino-Nogo can be detected extracellulary, a significant portion of Amino-Nogo is thought to have a cytoplasmic orientation (dotted line). OMgp is a member of the large family of leucine-rich repeat (LRR) proteins and linked to the cell surface by a GPI anchor. Several receptors for prototypic myelin inhibitors have been identified: PirB (and its human homolog LILRB2) binds to MAG, Nogo-66, and OMgp and signals neuronal growth cone collapse and neurite outgrowth inhibition in vitro. The ectodomain of PirB is composed of Ig-like domains and the cytoplamsic portion contains four immunoreceptor tyrosine-based inhibitory motifs (ITIM). The Nogo-66 receptor 1 (NgR1) binds directly to Nogo-66, MAG, and OMgp and is important for growth cone collapse responses toward acutely presented inhibitors. In some neurons, NgR1 is thought to form a tripartite receptor complex with Lingo-1 and the death domain containing TNFR superfamily members p75 or its functional substitute TROY (TNFRSF19). NgR2 is a receptor selective for MAG, functionally redundant with NgR1. The lectin-activity of MAG forms a complex with select gangliosides, including GT1b. Integrins, a family of heterodimeric cell surface receptors composed of α- and β-subunits, have been found to participate in Nogo-A and MAG inhibition in vitro.

Figure 4.

Mechanisms for CSPG inhibition. Proteoglycans are a heterogenous class of extracellular proteins with distinct protein core structures bearing covalently attached sulfated glycosaminoglycan (GAG) side chains. Prominent members of neural proteoglycans bearing heparan sulfate GAG chains (HSPGs) include the syndecans, glycpicans and agrin. Chondroitin sulfate GAG bearing proteoglycans (CSPGs) include members of the lectican family (neurocan, aggrecan, versican, and brevican). The leukocyte antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs) includes the mammalian members LAR, RPTPσ and RPTPδ. LAR type RPTPs are transmembrane proteins with cell adhesion molecule-like ectodomains and a large cytoplasmic region with two conserved phosphatase domains. The Drosophila homolog of LAR-RPTPσ-RPTPδ is called DLAR and is a functional receptor for the fly HSPGs syndecan and glypican (dally-like). Avian RPTPσ binds directly to the HSPGs agrin and collagen XVIII. In addition, CSPGs belonging to the lectican family (including neurocan and aggrecan) bind to the first Ig-like domain of mouse RPTPσ to signal neuronal growth inhibition. The interaction between lecticans and RPTPσ is direct, depends on the presence of CS-GAG chains, and is sensitive to chondroitinase ABC treatment.

Figure 5.

Corticospinal regeneration induced by over-expression of the high-affinity BDNF receptor trkB. (A) Corticospinal motor neurons are retrogradely infected with adeno-associated virus expressing GFP (green), whereas lentivirus encoding trkB (red) is delivered directly to the motor cortex. Some cells will coexpress both GFP and trkB (yellow). (B) Traced GFP-immunoreactive corticospinal axons demonstrate regeneration into a subcortical lesion grafted with BDNF-secreting syngeneic fibroblast substrate (red outlines). (C) Regenerated corticospinal axon within the subcortical graft. (D–F) Other examples of regenerated corticospinal axons within subcortical BDNF-secreting grats after trkB over-expression demonstrate growth cone-like morphology (D, E) and associations with the vasculature; axons indicated with arrowheads in (F) wrap around a blood vessel (asterisk). Scale bars, 500um (A), 25um (C–F). Adapted from Hollis et al. PNAS (2009).