**3. Growth factors and signaling pathways involved in dendritic growth regulation**

Dendritogenesis in post-ganglionic sympathetic neurons begins around E14, with maturation of dendritic arbor continuing into postnatal development [13, 115]. Sympathetic neurons extend multiple dendrites with complex branching patterns. The size of the dendritic arbor is dependent on size of the target field and neuronal activity, suggesting that dendritic complexity is determined by the needs of the targets [116–120]. Similar to axonal growth, dendritogenesis can be divided into 3 stages – initiation of dendrites, elongation and branching of dendrites, and maturation coupled with pruning of the dendritic tree. In this section, we will explore the current understanding of the various growth factors, their signaling pathways and interactions between them to influence dendritic arborization in sympathetic neurons.

### **3.1 Bone morphogenetic proteins (BMPs)**

Members of the bone morphogenetic protein (BMP) family are important for dendritic growth initiation in sympathetic neurons *in vitro* and *in vivo.* BMPs bind and activate a heterotrimeric receptor complex of transmembrane serine/threonine kinase receptors made of type I receptor – BMP receptor type I A (BMPR1a), also known as activin receptor-like kinase-3 (ALK-3) or BMP receptor type IB (BMPR1b), also known as ALK-6, and one of the three type II receptors – BMPRII, Activin type II or IIB (Act II or ActRIIB). The activation of these kinases leads to phosphorylation of receptor Smads (Smads 1, 5 and/or 8), which complex with Smad 4 to translocate to the nucleus and regulate gene expression [121–123].

Sympathetic neurons and glial cells in the SCG from embryonic and postnatal ganglia express mRNA and protein for BMP-5, BMP-6 and BMP-7 [108, 124, 125]. Also, BMPR1a, BMPRIIB, ActRII and BMPRII are present in mouse SCG through later stages of embryonic development into postnatal life [126, 127], suggesting that BMP signaling pathway is functional in sympathetic neurons during periods of dendritogenesis. BMP-5, BMP-6, BMP-7 initiate dendritic growth in cultured perinatal sympathetic neurons by activation and translocation of the Smad complex and regulating gene expression [108, 128, 129]. Conditional knockouts of BMPR1a or both BMPR1a/1b show a decrease in dendritic length and branch complexity compared to congenic wildtype animals but do not show complete absence of

dendrites [130]. Also, BMP receptor knockouts showed a dramatic decrease in total dendritic length, branching and soma size later in postnatal development suggesting that BMP signaling may be important for maintenance of dendrites, rather than initiation of dendrites *in vivo* [130]. This difference in BMP function *in vitro* and *in vivo* may stem from the presence of other receptors such as the activin receptors to mediate BMP signaling [131]. Interestingly, although transfection of Smad1 dominant negative mutant blocks BMP-7-induced dendritic growth *in vitro*, the SCG neurons in conditional Smad 4 knockout mice show an increase in dendritic length and total dendritic arbor [130], suggesting that Smad 4 may play a limiting role *in vivo* and BMPs may be signaling through Smad-dependent and Smad-independent pathways for dendritic growth regulation.

Transcriptome and miRNome analyses have identified over 250 genes and over 40 microRNAs whose expression are altered in response to BMP-7 treatment in cultured sympathetic neurons during the period of dendritic growth initiation [132, 133]. Of the genes, p75NTR mRNA and protein are strongly upregulated by BMP-7 signaling in cultured SCG neurons. BMP-mediated effects on dendritic growth are not observed in p75NTR knockout mice, with p75NTR knockout mice showing stunted dendritic arbor compared to wildtype. Conversely, overexpression of p75NTR phenocopies the dendritic growth effects of BMP-7, suggesting that this is an important target of BMP-7 during dendritic growth regulation [132, 134]. However, p75NTR ligands, interplay between neurotrophins and BMP in activating p75NTR and downstream effectors of p75NTR signaling responsible for dendritogenesis in sympathetic neurons still need to be elucidated. Of the microRNAs identified, three miRNAs – miR-21, miR-23b and miR-664-1\* may regulate dendritic growth downstream of BMP-7 in sympathetic neurons *in vitro* [133]. Also, signaling pathways mediated by ubiquitin-proteasome system and by reactive oxygen species are suggested to be downstream of Smad signaling in sympathetic neurons *in vitro* with proteasome inhibitors and antioxidants inhibiting BMP-7 induced dendritic growth [135, 136].

### **3.2 Neuronal activity dependent dendritic growth**

Electric field stimulation or treatment of sympathetic neurons with potassium chloride can lead to neuronal depolarization and this neuronal activity triggers dendritic growth in postganglionic sympathetic neurons by the activation of calcium calmodulin dependent kinase II (CaMKII) [137]. Also, inhibition of integrinlinked kinase (ILK) using an siRNA prevents activity-dependent dendritic growth in sympathetic neurons *in vitro*, whereas pharmacological inhibition of glycogen synthase kinase-3β (GSK-3β) enhances activity-dependent dendritic growth in these neurons [138, 139].

### **3.3 Nerve growth factor and fibroblast growth factor**

NGF was one of the earliest growth factors recognized as important for dendritic growth with NGF injections leading to enhanced dendritic growth in sympathetic ganglia [116]. However, NGF, by itself, is unable to induce dendritic growth in cultured perinatal SCG neurons, but is required for BMP-7 induced dendritic growth [129, 140]. One of the downstream targets of NGF for dendritic growth appears to be Egr3 with Egr3−/− mice showing significant decrease in the number of primary dendrites, total dendritic length and maximum extent of dendritic arbor [74].

*Signaling Pathways Regulating Axogenesis and Dendritogenesis in Sympathetic Neurons DOI: http://dx.doi.org/10.5772/intechopen.102442*

Fibroblast growth factor receptor 1 (FGFR1) is expressed in adult SCG neurons and its nuclear localization increases in perinatal sympathetic neurons upon BMP-7 exposure [141, 142]. Also, expression of mutant FGFR1 decreases the dendritic growth induced by BMP-7 in sympathetic neurons, through the activation of the integrative nuclear FGFR1 signaling pathway [142].

Interestingly, stimulation of the MAPK signaling pathways has differential effects on activity-dependent dendritic growth and BMP-7 induced dendritic growth. While pharmacological inhibition of ERK activity using PD98059 inhibits activitydependent dendritic growth, the treatment with the same inhibitor enhances BMP-7 induced dendritic growth [137, 139, 143]. Stimulation of the MAPK signaling through overexpression of MEK1 leads to inhibition of BMP-7 induced dendritic growth and the inhibition of MAPK signaling pathway with dominant negative MEK1 or ERK2 mutant increases the number of dendrites and total dendritic arbor in BMP-7 treated [143]. Further studies are needed to understand the opposing roles of ERK in BMP-induced vs. activity-dependent dendritic growth.

### **3.4 Cytokines**

Several members of the cytokine family have been shown to regulate dendritic growth in sympathetic neurons. These growth factors function through the activation of the Janus kinase (JAK), leading to the nuclear translocation of proteins known as signal transducers and activators of transcription (STAT) [144]. In perinatal sympathetic neurons, interferons gamma (IFNg), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) decrease the number of primary dendrites and total dendritic arbor, without affecting axonal growth and neuronal survival. In addition, these cytokines can lead to retraction of pre-existing dendrites through the activation of STAT proteins [145–147]. In addition to activating STATs, IFNg activates Rit, (a small GTPase related to Ras GTPase) and p38-MAPK pathway to effect the dendritic retraction observed in these neurons [148]. Rit is expressed in sympathetic neurons and has opposite effects on axonal and dendritic growth. Dominant negative Rit transgenes decrease axonal elongation but enhance BMP-7 induced dendritic growth in an ERK-signaling dependent manner and constitutively active Rit enhances number of axons and axonal branching in sympathetic neurons while inhibiting dendritic growth [149].

### **3.5 Cytoskeletal proteins**

Dendritic growth and remodeling requires changes to the actin and microtubule cytoskeleton [150, 151]. Signaling pathways downstream of Rho GTPases act as intermediates to connect extracellular signals and actin cytoskeletal remodeling during dendritic growth [152]. In cultured sympathetic neurons, BMP-7 treatment increases the GTP bound RhoA [153] and decreases GTP-bound Rit [149], with no effects on other small GTPases. In cultured SCG neurons, BMP-7 induced dendritic growth requires the activation of RhoA [153], suggesting that activation of this GTPase may be the link to actin cytoskeleton remodeling necessary for dendritic growth.

The microtubule polarity in axons is different from that in dendrites. Unlike microtubules in axons, which have a uniform polarity, microtubules in dendrites have a mixed orientation that is driven by the different motor proteins [154, 155]. A kinesin related motor protein kinesin 6 (also known as CHO/MKLP1) mRNA and protein are

expressed in cultured embryonic sympathetic neuron, with CHO/MKLP1 protein extending from the cell body to the newly formed dendrites [156, 157]. Two other kinesin related motors – Kinesin 5 (also known as Eg5 or Kif11) and kinesin 12 (also known as Kif15) – are also expressed in embryonic sympathetic neurons [158, 159], with kinesin 5 associating only with the microtubule cytoskeleton and kinesin 12 being enriched in the dendrites and associating with both actin and microtubule cytoskeleton [158–160]. Treatment with antisense oligonucleotides against kinesin 6 lead to an increase in axonal length but a decrease in dendritic width and inhibition of BMP-7- induced dendritic growth in these neurons [156, 157]. Knockdown of kinesin 12 in cultured embryonic SCG neurons using an siRNA lead to longer axons that are less branched than control neurons and decrease in dendritic width [157, 160]. Both kinesin 6 and 12 appear to be important for the mixed polarity of microtubules in the dendrites with a decrease in these kinesins leading t0 fewer minus-end directed microtubules in the dendrites and increased frequency of microtubule transport. Similar to the others kinesins, inhibition of kinesin 5 leads to increase in axonal length, however a decrease in kinesin5 also leads to axons being non-responsive to navigational cues [161, 162]. In addition to a decrease in dendritic width like other kinesins, a reduction in kinesin 5 causes a decrease in dendritic length, a small decrease in number of dendrites and a significant effect on dendritic morphology especially during dendritic maturation stages [163]. In contrast to other kinesin mutants, a decrease in kinesin 5 leads to more minus-end microtubules in the dendrites [163]. Interestingly, kinesin5 appears to be regulated by phosphorylation with more phosphorylated kinesin5 being localized to the dendrites, suggesting that kinesin5 could be a potential link between signaling pathways and the cytoskeletal remodeling during dendritogenesis [163].
