In postganglionic sympathetic neurons, the size of the dendritic arbor determines presynaptic convergence, which correlates with tonic activity, and aberrant dendritic morphology is associated with disease. There is, therefore, great interest in understanding how dendritic morphology is regulated in these neurons. Early studies established a role for target-derived nerve growth factor (NGF) in regulating the size of the dendritic arbor of sympathetic neurons in vivo. However, in vitro studies revealed that even in the presence of optimal concentrations of NGF, rat sympathetic neurons cultured in the absence of serum or non-neuronal cells survive and elaborate extensive axonal arbors, but fail to form dendrites. Subsequently, it was discovered that bone morphogenetic proteins (BMPs) trigger cultured sympathetic neurons to extend a dendritic arbor comparable to that of their in vivo counterparts. The goals of this chapter are to: (i) summarize these early experiments; (ii) discuss evidence substantiating a role for BMPs in glial-induced dendritic growth in vitro and regulation of dendritic growth in vivo; (iii) review what is known about the molecular mechanisms by which NGF, BMPs and other factors influence dendritic arborization of sympathetic neurons; and (iv) identify key data gaps in understanding of how dendrites are regulated in sympathetic neurons.
- afferent input
- neuronal polarity
- reactive oxygen species (ROS)
- sympathetic neurons
- target-derived factors
Differences in dendritic morphology between neurons are a striking feature of the vertebrate nervous system with important functional implications. The shape of dendrites influences the propagation and integration of postsynaptic potentials , and determines presynaptic convergence [2, 3]. These observations coupled with evidence that aberrant dendritic structure is strongly associated with neurologic disease [4, 5] have generated significant interest in understanding how dendrites are regulated.
Postganglionic sympathetic neurons are a well-characterized model for studying dendrite development and plasticity . The dendritic arbor of these neurons is relatively complex with an average of two to six primary dendrites, depending on the animal species, and multiple orders of branching . In postganglionic sympathetic neurons, the size of the dendritic arbor correlates with not only the number and pattern of synaptic inputs [3, 8], but also tonic activity [8, 9]. As is true of central neurons, aberrant morphology of sympathetic neuron dendrites is associated with disease. For example, dendritic hypertropy of sympathetic neurons in stellate and superior cervical ganglia (SCG) is observed in the spontaneously hypertensive rat [10, 11], and is thought to contribute to the pathogenesis of hypertension in this model . Therapeutic intervention with statins not only decreases sympathetic activity and normalizes blood pressure in the spontaneously hypertensive rat , but also decreases dendritic arborization of both stellate and SCG neurons .
In this chapter, we will review what is known about the molecular and cellular mechanisms that regulate dendrites in postganglionic sympathetic neurons, and identify key data gaps.
2. Early studies of dendritic growth in sympathetic neurons
The majority of dendritic growth in postganglionic sympathetic neurons occurs during the postnatal period; however, dendrites continue to grow into adulthood [14, 15, 16], and
The effect of target tissues on dendritic growth in sympathetic neurons is mediated, at least in part, by nerve growth factor (NGF) [22, 23, 24, 25]. Separation of neurons from target tissues by axonal ligation causes dendritic atrophy in the few neurons that survive, and this effect is attenuated by systemic administration of NGF [26, 27]. However, exogenous NGF reverses axotomy-induced dendritic retraction by <50%, even though cell survival is completely rescued , indicating that additional target-derived factors are needed to fully account for the effects of target on dendritic growth. Consistent with this conclusion, the dendritic complexity of axotomized sympathetic neurons recovers to control levels upon ganglion cell reinnervation of the periphery .
The trophic actions of BMPs are specific to dendritic growth in that BMPs do not support cell survival, nor do they enhance axonal growth in cultured sympathetic neurons . Consistent with observations of dendritic growth in sympathetic neurons
These observations suggest that BMPs mediate the effects of ganglionic glia and target tissues on dendritic growth in sympathetic neurons. Immunocytochemical and
The question of whether BMPs also contribute to target effects on dendritic growth has yet to be addressed experimentally. Sympathetic targets, including the eye, heart, lung, kidney, and blood vessels, express significant levels of BMPs during embryonic development, throughout the postnatal period, and into adulthood [38, 39, 40]. Thus, target tissues may be a source of BMPs to sympathetic neurons not only during initial expansion of the dendritic arbor, but also in the maintenance and remodeling of dendritic arbors that continues throughout the life of the animal.
3. Signaling pathways that regulate dendritic growth in sympathetic neurons
Research over the past few decades has provided insights into the signaling pathways and molecular mechanisms that control dendritic growth in sympathetic neurons. As discussed in the preceding section, BMPs and NGF play predominant roles in the initiation and maintenance of dendrites in these autonomic neurons. While the importance of these growth factors as regulators of dendritic growth in sympathetic neurons is well established, the downstream effectors that link BMP and NGF to increased dendritic growth are not fully understood. In this section, we will discuss the signaling pathways activated by these growth factors, the evidence implicating downstream effectors of BMPs and NGF in dendritic regulation, and the identification of factors that interact with these signaling pathways to alter their influence on the dendritic arborization of sympathetic neurons.
3.1. BMP signaling
BMPs mediate their cellular effects by binding to a heteromeric receptor complex of transmembrane serine/threonine kinas receptor subunits comprised of a type I receptor [BMP type I receptor A (BMPR1A), which is also known as activin receptor-like kinase
BMP signaling pathways are active in sympathetic ganglia during developmental periods corresponding to the initiation, extension and maintenance of dendrites. Quantitative PCR,
Several caveats of the
3.1.1. Smad-dependent transcriptional regulation of dendritic growth
Canonical BMP signaling involves the Smad family of transcription factors. Immunocyto-chemical analyses of primary rat SCG neurons have demonstrated that Smad 1/5/8 translocates to the nucleus within 20 minutes of exposure to BMP-7, with maximal nuclear translocation observed within 2 hours of adding BMP-7 to the culture medium. Transfection with a Smad1 dominant negative mutant, Smad1 (3SA), blocked BMP-7-induced dendritic growth in primary sympathetic neurons , indicating that Smad 1 activation is necessary for induction of dendritic growth by BMP-7. In contrast to the
A comprehensive analyses of Smad-dependent dendritic growth in sympathetic neurons provided evidence for early transcriptional regulation of dendritic growth downstream of BMP-7 . In neuronal cell cultures from embryonic rat SCG, BMP-7-induced dendritic growth could be blocked by pharmacologic inhibition of transcription with actinomycin-D when the inhibitor was added within the first 24 hours of BMP-7 exposure but not when it was added after 48 hours of BMP-7 exposure. Microarray analyses identified over 250 genes that were differentially regulated by BMP-7 within the first 24 hours after adding BMP-7 to the culture medium. Of these, 56 mRNAs were altered within the first 6 hours and 185 mRNAs were differentially regulated at 24 hours after BMP exposure . Many of the differentially regulated genes were linked to signaling pathways previously implicated in dendritogenesis in other neuronal cell types or neuronal morphogenesis and/or axonal guidance, such as BMP, Notch, integrin, Wnt, and NGF signaling molecules. However, the functional relevance of most of these genes to dendritic growth in sympathetic neurons has yet to be determined. Moreover, recent reports of limited correlation between transcriptome and proteome analysis in yeast, plants and mice [53, 54], suggest that in order to generate a more complete understanding of the molecular pathways that link BMPs to dendritic growth in sympathetic neurons, detailed proteome analyses are needed to complement the existing transcriptomic dataset.
One gene identified as being strongly upregulated by BMP-7 in primary sympathetic neurons, the gene encoding the p75 neurotrophin receptor (p75NTR) , has been evaluated for a role in BMP-induced dendritic growth. A member of the tumor necrosis factor (TNF) receptor family, p75NTR regulates diverse neurobiological processes, including axonal growth, synaptic plasticity, dendritic growth in central neurons, and neuronal cell death [55, 56, 57, 58, 59, 60, 61]. p75NTR binds diverse ligands to mediate its effects, including NGF, other neurotrophins, myelin-derived polypeptides, such as myelin-associated glycoprotein (MAG) or Nogo, and β-amyloid peptide [60, 62, 63]. In cultured embryonic rat SCG neurons, p75NTR mRNA and protein expression are significantly upregulated within 24 hours of exposure to BMP-7 [52, 64], and pharmacologic inhibition of signaling via BMPRI prevents induction of p75NTR protein expression in primary sympathetic neurons exposed to BMP-7 . Functional studies revealed that BMP7 does not trigger dendritic growth in primary sympathetic neurons derived from SCG of p75NTR knockout mice; conversely, ligand-independent activation of p75NTR via overexpression of a p75NTR cDNA construct in p75NTR−/− neurons , phenocopies the dendrite-promoting effects of BMP-7 . Morphometric analyses of SCG from wildtype
An outstanding question regarding p75NTR effects on dendritic growth in sympathetic neurons is the identity of ligand(s) and co-receptor(s) that p75NTR interacts with to mediate BMP-induced dendritic growth. Several lines of evidence argue against a direct interaction between p75NTR and the BMP receptor complex: (
Similarly, the downstream effector molecule(s) that link p75NTR to increased dendritic arborization remain to be determined. Key candidates include the Rho GTPases. Rho GTPases function as central regulators of dendritic morphology, linking extracellular signals to changes in the dendritic actin cytoskeleton [68, 69]. p75NTR has been shown to interact with RhoA in the yeast two-hybrid system . In cultured rat sympathetic neurons, exposure to BMP-7 increases the levels of the GTP-bound form of RhoA, but not GTP-Rac1 or GTP-Cdc42, as determined by a GTP pull down assay, and triggers RhoA translocation from the cytoplasm to the membrane . The observation that BMP-7-induced dendritic growth in primary sympathetic neurons requires RhoA activation , suggests a model in which BMP-7 sequentially activates BMPRIA, Smad 1/5/8, p75NTR, and then RhoA to induce dendritic growth in sympathetic neurons.
3.2. Signaling pathways that interact with Smad signaling to modulate BMP-induced dendritic growth
The shape of the dendritic arbor of developing sympathetic neurons is determined by interactions between positive and negative regulators of dendritic growth. A number of signaling pathways have been shown to interact with BMP signaling to modulate the number of dendrites, total dendritic length and dendritic branching in sympathetic neurons. In this section, we will review known positive and negative regulators of Smad signaling that impact BMP-induced dendritogenesis in sympathetic neurons.
3.2.1. Positive regulators of Smad signaling that enhance dendritic growth
Biochemical studies have shown that R-Smads interact with components of the proteasome complex, as well as enzymes and proteins involved in the ubiquitination-deubiquitination of proteins, in many systems, and the ability of Smads to regulate transcription is dependent on association with the proteasome complex in various cell types [77, 78]. Interactions between BMP signaling molecules and the proteasome pathway have been reported in perinatal rat sympathetic neuronal cultures prior to dendritic growth induction by BMP-7 . In this study, interactions between Smad1 and multiple proteasome components were confirmed using a yeast two-hybrid assay, and pharmacologic inhibition of proteasome activity by lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth in primary sympathetic neurons in the absence of any effect on axonal growth . These proteasome inhibitors also suppressed Smad-mediated transcriptional regulation in a biochemical assay using a Tlx -luciferase construct transfected into P19 cells . One caveat of this study is that although there was clearly a functional interaction between BMP signaling and the proteasome pathway in the context of dendritic growth, a biochemical interaction between Smads and proteasomes were not demonstrated in primary sympathetic neurons. Further studies are necessary to fully understand the genetic and biochemical interactions between Smads and ubiquitin-proteasome pathway during dendritogenesis in sympathetic neurons.
Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth, and suggest that ROS-mediated signaling positively modulates dendritic complexity in sympathetic neurons. One caveat of this study, however, is that while BMP-7 was observed to increase NOX2 levels and oxygen consumption in sympathetic neurons, increased ROS levels were not detected in sympathetic neurons exposed to BMP-7. Likely, this reflects the fact that physiologic BMP signaling generates levels of ROS that are below the detection threshold for standard ROS detection assays. Further work is needed to determine whether these
3.2.2. Negative regulators of SMAD signaling that influence dendritic growth
In addition to modulating BMP effects on dendritic arborization via activation of MAPK signaling, FGFs regulate neuronal differentiation via the integrative nuclear FGFR1 signaling (INFS) pathway . FGFR1 is expressed in the nucleus of adult rat SCG neurons following axotomy . Nuclear localization of FGFR1 is also increased in perinatal rat sympathetic neurons following exposure to BMP-7, and transfection of a mutant FGFR1 receptor inhibits FGFR1 nuclear localization and decreases the dendritic response to BMP-7 . These data suggest that the INFS-mediated FGF signaling pathway functions downstream of BMP signaling to limit BMP-induced dendritogenesis in sympathetic neurons.
Rit GTPase, a member of the small GTPase family, has also been shown to activate ERK1/2 in primary sympathetic neurons, and the transfection of dominant negative (dn) Rit or constitutively active (ca) Rit were observed to increase or decrease BMP-induced dendritic growth, respectively . Rit GTPase also negatively modulates dendritic growth as a downstream target of IFNγ signaling as demonstrated by inhibition of IFNγ-mediated dendritic retraction in primary sympathetic neurons transfected with dnRit constructs . Addition of IFNγ to cultures of pheochromocytoma cells, which are often used as a model for sympathetic neurons, increased levels of GTP-Rit, and transfection of dnRit inhibited IFNγ-induced activation of p38 MAPK . These observations suggest that a novel Rit-p38 MAP kinase signaling pathway functions in parallel with the canonical JAK–STAT signaling pathway to mediate IFNγ-induced dendritic retraction. Collectively, these studies provide evidence for crosstalk between BMP signaling and MAPK signaling during dendritogenesis in sympathetic neurons, and suggest that in contrast to its effects in central neurons, MAPK signaling functions as a negative regulator of BMP-induced dendritic growth in sympathetic neurons.
In summary, signaling by cytokines, growth factors, small molecules, and peptides, such as retinoic acid, PACAP and VIP, antagonize BMP signaling during dendritogenesis in sympathetic neurons. Most of the relevant data were collected from studies of primary perinatal sympathetic neurons cultured from rodent SCG. While the findings from this model have provided a glimpse into the complexity of the interactions that influence dendritic arborization of these neurons, further studies are required to understand the mechanisms by which these factors interact to regulate dendritic growth, how these pathways are spatially and temporally coordinated to influence dendritic arborization of sympathetic neurons
3.3. Other pathways that regulate dendritic growth in sympathetic neurons
As described earlier, NGF is an important regulator of dendritic growth in sympathetic neurons. However, the molecular mechanisms by which NGF regulates dendritic growth are not well characterized. Early growth response-3 (Egr3), a transcriptional regulator known to be induced by NGF via MAPK signaling, has been identified as a potential downstream regulator of NGF-induced dendritic growth . Sympathetic neurons from a conditional
Neuronal depolarization induced by electric field stimulation or the addition of potassium chloride to primary postnatal sympathetic neurons cultured was shown to trigger the formation of dendrites in the presence of NGF that retracted in the absence of neuronal activity . Neuronal depolarization enhanced stability of microtubules and activated calcium calmodulin dependent kinase II (CaMKII) in dendrites. The latter was shown to be causally related to the effects of neuronal depolarization on dendritic growth: pharmacologic inhibition of CaMKII activity using KN62 or mAIP completely blocks activity-dependent dendritic growth in cultured sympathetic neurons .
Signaling by integrin-linked kinase (ILK) and glycogen synthase kinase-3β (GSK-3β) have also been shown to be downstream effectors of activity-dependent dendritic growth in postnatal sympathetic neurons . ILK and GSK-3β are serine threonine kinases that are downstream effectors of integrin and neurotrophin signaling . ILK has been shown to phosphorylate and inactivate GSK-3β to regulate NGF-mediated axonal growth . Increased phosphorylation of GSK-3β protein was observed in cultured postnatal rat SCG neurons in response to increased neuronal activity, and inhibition of ILK activity by QLT0254, as well as transfection of dominant negative ILK or siRNA for ILK, blocked activity-dependent dendritic growth in these neurons. Similarly, inhibition of GSK-3β activity using kenpaullone or genetic knockdown of GSK-3β expression increased the number of primary dendrites formed in response to potassium chloride, suggesting that GSK-3β inhibition is necessary for early stages of activity-dependent dendritic growth in sympathetic neurons.
Interestingly, unlike BMP-induced dendritic growth, inhibition of ERK activity inhibited activity-dependent dendritic growth in postnatal sympathetic neurons
4. The path forward
The experimental evidence clearly implicate NGF, BMP and neuronal activity as positive regulators of dendritic growth in perinatal sympathetic neurons
The authors acknowledge Dennis Higgins, PhD (University of Buffalo) who dedicated his research career to understanding the regulation of dendritic growth in sympathetic neurons, and whose enthusiasm for this topic, and scientific research in general, was the inspiration and motivation for much of the authors’ own work in this field. This work was supported by the National Institutes of Health (grants R01 NS097808, R01 ES014901 and R21 NS45037).
Conflict of interest
The authors declare no conflict of interest.