**2.2 Artemin**

Artemin, a member of the glial derived neurotrophic factor (GDNF) family ligands (GFLs) plays an important role in the axonal elongation and guidance of the postganglionic axons to their targets [21–24]. In addition to Artemin, other members of the GDNF family, including GDNF and Nerturin have been shown to enhance neurite growth in subpopulations of sympathetic neurons [21, 22, 24]. Artemin mRNA is expressed at high levels near the dorsal aorta around E12.5 and then in the smooth muscles of many of the blood vessels along which the sympathetic axons migrate to their targets [25, 26]. The receptors for Artemin – Ret and GFRa3 are both expressed in the sympathetic ganglia as early as E11.5 and then expression gets restricted to subsets of cells later in embryonic development [26–29]. Treatment of nascent sympathetic ganglia (E13.5) with artemin induces axonal growth with axons showing branching and radial outgrowth. Also, axonal growth from explant cultures of the ganglia are directed towards beads coated with artemin, suggesting artemin has the ability to guide axons to their targets [30]. In addition, Artemin knockout mice show decreased axonal growth postnatally, [31] and mice lacking either GFRa3 or Ret show reduced, depleted or abnormal neuronal projections and abnormal branching indicating that Artemin signaling mediated by Ret:GFRa3 receptor complex is necessary for proper migration of sympathetic neurons during development [26, 28]. Although early studies suggest a role for Artemin in sympathetic neuron survival with the superior cervical ganglia being smaller in Artemin, Ret and GFRα3 knockout animals compared to wild type animals [29, 32], more recent studies suggest that the decreased neuronal cell numbers in the absence of Artemin signaling are an indirect effect of aberrant axonal migration and target innervation [28]. Taken together, the data suggest that the members of the GDNF family act as early guidance molecules to promote axon elongation and target innervation.

## **2.3 Neurotrophins**

Neurotrophin family of growth factors – nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) have been implicated in many aspects of neuronal function, including differentiation, survival, axonal growth and dendritic growth [33–37]. These neurotrophins are synthesized and secreted as proneurotrophins, which are the proteolytically cleaved to generate mature neurotrophins that activate different isoforms of Trk tyrosine kinase receptors (Trk) and p75 neurotrophin receptors (p75NTR) and activate a variety of downstream signaling pathways [33–36, 38].

### *2.3.1 Nerve growth factor*

NGF synthesis begins in targets of sympathetic neurons in concert with the arrival of the sympathetic axons and is correlated with increased expression of TrkA [39–43]. Although NGF is necessary for survival for sympathetic neurons in the early stages, the neurons lose their dependence on NGF for survival in the later stages *in vivo* and *in vitro* [42, 44, 45]. Exposure of sympathetic neurons to exogenous NGF, overexpression of NGF in the target tissues or adding NGF to compartments containing the distal axons leads to increased axonal growth and hyperinnervation of the target tissues [46–51]. Conversely, mice lacking NGF or TrkA (NGF receptor) show decreased survival of sympathetic neurons and decreased target innervation [52–54]. In addition to NGF, proNGF promotes axonal elongation and branching in postnatal sympathetic neurons through activation of the p75NTR receptor rather than the TrkA receptor [55].

NGF's axonal growth effects are independent of its effects on neuronal survival. Mice lacking both NGF and Bax, a pro-apoptotic gene necessary for apoptosis in sympathetic neurons [56, 57], show normal early axonal growth and guidance along the vasculature but show differential loss of innervations in the different target tissues with sympathetic innervation being completely absent in salivary glands and cardiac ventricles, reduced in the liver and unaffected in the trachea [58]. These evidence supports the argument that NGF is important for axon growth of the distal axons and target innervation. It is interesting to note that this requirement for target-derived NGF in the terminal axonal growth varies between the different targets, suggesting that other growth factors are important for target innervation in some of these tissue [58].

NGF's effects on axonal growth are primarily mediated through activation of the TrkA receptors. NGF and phosphorylated TrkA are retrogradely transported in endosomes from the axon terminals [59–62] and regulate axonal growth through changes to cytoskeletal proteins and transcription factors such as cyclic AMP response element binding protein (CREB) and early growth regulator 3 (Egr3) [49, 63–67]. Also, local reintroduction of NGF to NGF-deprived neurons in culture results in profuse axonal growth, suggesting that NGF promotes axonal growth both locally and through retrograde signaling [68]. NGF also upregulates the expression of its receptor TrkA in sympathetic neurons [41] and activates downstream effectors such as PI-3Kinase-Akt pathways and MAPK pathways leading to cytoskeletal changes resulting in axonal growth [69]. In addition, NGF, through its binding to TrkA receptors, activates glycogen synthase kinase-3 (GSK-3), which results in the phosphorylation of microtubuleassociated protein 1B (MAP1B) and decrease in MAP1B phosphorylation is correlated with decreased axonal growth [70]. NGF signaling during axonal elongation and termination is dependent on activation SHP-2, a protein tyrosine phosphatase. Inhibition of SHP-2 *in vitro* leads to decreased axonal growth by inhibiting extracellular signalregulated kinase (ERK) signaling, however interfering with SHP-2 signaling results in increased axonal density within the targets [71]. Also, studies suggest that Wnt 5a is upregulated in sympathetic targets in response to NGF, and blocking Wnt5a activation using an antibody suppresses NGF-induced axonal growth [72]. Early growth response (Egr) proteins – Egr1 and Egr3 are induced by NGF signaling in sympathetic neurons with inhibition of Egr1 *in vitro* using a dominant negative and Egr3 knockout *in vivo* show decreased neurite outgrowth and target innervation [49, 73, 74]. Recent studies have suggest a role of non-coding RNAs and post-translational modifications downstream of NGF signaling during axonal growth [75, 76]. Untranslated axonal mRNA Tp53inp2 upregulates NGF-TrkA signaling during axonal growth [75] and NGF-dependent prenylation of proteins such as Rac GTPase appears to be important for receptor trafficking to promote axonal growth [76].

Once the axons reach the target, NGF-TrkA signaling increases the expression of Coronin-1, a protein that interacts with the actin cytoskeleton [77]. Coronin-1

acts as a molecular switch to convert downstream effectors of NGF-TrkA from the PI-3 K pathway to calcium signaling, leading to the suppression of axonal growth and branching [77, 78].

### *2.3.2 Neurotrophin 3 (NT-3)*

In addition to NGF, neurotrophin-3 is expressed in sympathetic neurons, although its main receptor TrkC is expressed at low levels in neonatal sympathetic neurons [44, 79–81]. NT-3 mutant mice show severe defects in their sympathetic nervous system with 50% fewer neurons, and defects in axonal branching and axonal innervation of target tissues such as the pineal gland and cardiac myocytes [82–84]. In addition, neurotrophin-3 (NT-3) promotes axonal growth and branching in sympathetic neurons *in vitro* [41, 84]*.* Overexpression of NT-3 in adipose tissue leads to increased sympathetic innervation through its activation of TrkC receptors [85]. However, NT-3's effects on axonal growth are mediated by activation of TrkA receptors as opposed to TrkC, with NT-3 selectively promoting neurite outgrowth rather than for survival in neonatal sympathetic neurons [41]. Although both NGF and NT-3 signal using the same receptor, unlike the NGF-TrkA complex, NT-3-TrkA complex does not mediate retrograde signaling [61]. Recent studies also suggest that NT3-TrkA complex prevents axons from branching into intermediate targets and enables larger growth cones in the absence of Coronin-1, through activation of Ras-MAPK and PI3K-Akt pathways [86].

### *2.3.3 Brain-derived neurotrophic factor (BDNF)*

Similar to other neurotrophins, BDNF is expressed in sympathetic neurons and sympathetic neuron targets [79, 87], and serves as target-derived growth factor for pre-ganglionic sympathetic neurons [88]. Unlike NGF and NT-3, BDNF null mutants show a slight increase in the number of sympathetic neurons compared to wildtype animals, indicating that BDNF is not important for survival of sympathetic neurons [89]. Addition of exogenous BDNF inhibits axonal growth and inhibiting BDNF activity using antibodies against BDNF promotes axonal growth in sympathetic neurons *in vitro* [79]. Also, BDNF +/− and BDNF −/− mice show hyperinnervation of the target tissues [87]. Although Trk B (the main BDNF receptor) is not present in sympathetic neurons [41], the sympathetic axons express p75NTR during target innervation [87] and BDNF's effects on axonal growth are mediated through its interaction with this receptor. BDNF-p75NTR signaling inhibits the activity of NGF-TrkA complex leading to axonal growth inhibition *in vitro* and axon pruning *in vivo* [87, 90].

### **2.4 Tumor necrosis factor superfamily**

Multiple members of the tumor necrosis factor superfamily (TNFSF) are known to regulate axonal growth in sympathetic neurons. Members of the TNFSF act as either as membrane-bound ligands or soluble ligands once cleaved from the membrane and bind to receptors belonging to the TNF superfamily (TNFRSF) [91, 92]. These molecules can also serve as reverse signaling molecules with TNFRSF acting as ligands and membrane-bound TNFSF functioning as receptors [93].

TNFa protein is present in postnatal SCG neurons throughout the cell body and neurites with strong immunoreactivity for TNF receptors R1 (TNFR1) in the cell body and in target tissues [94]. *tnfa*−/− and *tnfr*−/− mice show decreased innervation of sympathetic targets, with no effect on neuronal numbers [94]. While soluble TNFa inhibits NGF-induced axonal growth *in vitro* through activation of NF-kB [95], the reverse signaling mediated by TNFR1 at the axon terminal enhances axonal growth and target innervation through elevation of opening of T-type calcium channels leading to rapid activation of protein kinase C, ERK1 and ERK2 [94, 96]. Another TNF superfamily member – receptor-activator of NF-κB (RANK, also known as TNFRSF11A)) is expressed in embryonic and early postnatal sympathetic neurons, while its ligand RANKL is expressed in target tissues [97]. Similar to TNFa, local activation of RANKL-RANK signaling is necessary for axonal growth effects, and addition of soluble RANKL or activation of RANK signaling inhibits NGF-induced axonal extension and branching, through activation of NF-κB signaling [97]. The glucocorticoid induced tumor necrosis factor receptor related protein (GITR) and its ligand GITRL are also expressed in sympathetic neurons [98]. The activation of GITR by its ligand GITRL leads to activation of ERK signaling and the downregulation of NF-kB signaling pathways and regulation of both of these pathways are necessary for NGF-induced axonal growth [98, 99]. A recent study showed that *TWE-PRIL*, an alternative spliced form that combines extracellular domain of one TNFSF member APRIL (TNFSF13) and the transmembrane and cytoplasmic domains of another member TWEAK (TNFSF12), is expressed in developing SCG neurons [100]. *April* −/− mice show increased axonal growth in the presence of NGF, that can be rescued by overexpression of TWE-PRIL. TWE-PRIL reverse signaling leads to axonal growth inhibition by preventing NGF-dependent activation of ERK [100]. Similarly, CD40 (TNFRSF5) and its ligand CD40L are expressed in embryonic and early postnatal SCG neurons [101]. While CD40 by itself does not affect axonal growth, the reverse autocrine signaling mediated by CD40-CD40L enhances NGF induced axonal growth in these neurons, especially when there is low NGF with high levels of NGF inhibiting CD40 and CD40L expression [102].

Interestingly, two TNF family members have differential effects on paravertebral and prevertebral ganglia. Unlike SCG targets which showed hypoinnervation in *tnfa*−/− and *tnfr*−/− mice, the targets of the prevertebral sympathetic ganglia showed no change in innervation and reverse signaling mediated by TNFR1 did not alter axonal growth from prevertebral ganglia neurons [103]. Similarly, CD40 null mutants show hyperinnervation in targets of prevertebral ganglia and CD40-CD40L reverse signaling inhibits axonal growth in prevertebral ganglia neurons [101].

## **2.5 Extracellular matrix proteins**

As axons extend from the sympathetic ganglia to the target, they are exposed to a complex environment composed of extracellular matrix molecules such as laminin, collagen, fibronectin and thrombospondin. Laminin, collagen IV and thrombospondin promote axonal growth in perinatal superior cervical ganglia neurons *in vitro* and mediate their effects on axonal growth through activation of specific classes of integrin receptors [104–107]. Exposure of SCG neurons to laminin leads to formation of multiple axons, whereas neurons exposed to collagen IV extend only a single axon suggesting distinct signaling pathways downstream of integrin activation [104, 105, 108]. In addition, exposure to laminin causes bundling of microtubules, leading to rapidly growing axons [109]. Conversely, chondroitin sulfate proteoglycans inhibit axonal growth in cultured neonatal SCG neurons and may be responsible for lack of sympathetic reinnervation of the heart during ischemia-reperfusion injury [110].

### **2.6 Other signaling pathways involved in axonal growth**

Interleukin 1b (IL-1b) and Interleukin 1 receptor (IL-1R) are expressed in neonatal sympathetic neurons with IL-1R1 being present in the cell body and axons, and IL-1b being expressed in the sympathetic neurons and target tissues [111, 112]. IL-1b inhibits axonal growth in cultured sympathetic neurons by promoting the nuclear translocation of NF-kB [112].

Ceramide, a lipid second messenger, generated from glycosphingolipid metabolism or sphingomyelin metabolism is known to be important for cell proliferation or cell death downstream of extracellular agents such as TNF, interleukins and other molecules [113, 114]. Although newly synthesized glycosphingolipids are not important for axonal growth, when added to the distal axons ceramide inhibits neuronal outgrowth, possibly by decreasing the uptake of NGF by the distal axons [113, 114].
