Signalling Pathways Mediating the Effects of CD40-Activated CD40L Reverse Signalling on Inhibitory Medium Spiny Neuron Neurite Growth.

CD40-activated CD40L-mediated reverse signalling is a major physiological regulator of neurite growth from excitatory and inhibitory neurons in the developing central nervous system (CNS). Whereas in excitatory pyramidal neurons, CD40L reverse signalling promotes the growth and elaboration of dendrites and axons, in inhibitory GABAergic striatal medium spiny neurons (MSNs), it restricts neurite growth and branching. In pyramidal neurons, we previously reported that CD40L reverse signalling activates an interconnected and interdependent signalling network involving protein kinase C (PKC), extracellular regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK) signalling pathways that regulates dendrite and axon growth. Here, we have studied whether these signalling pathways also influence neurite growth from striatal inhibitory MSNs. To unequivocally activate CD40L reverse signalling, we treated MSN cultures from CD40-deficient mice with CD40-Fc. Here, we report that activation of CD40L reverse signalling in these cultures also increased the phosphorylation of PKC, ERK1/2, and JNK. Using pharmacological activators and inhibitors of these signalling pathways singularly and in combination, we have shown that, as in pyramidal neurons, these signalling pathways work in an interconnected and interdependent network to regulate the neurite growth, but their functions, relationships, and interdependencies are different from those observed in pyramidal neurons. Furthermore, immunoprecipitation studies showed that stimulation of CD40L reverse signalling recruits the catalytic fragment of Syk tyrosine kinase, but in contrast to pyramidal neurons, PKC does not participate in this recruitment. Our findings show that distinctive networks of three signalling pathways mediate the opposite effects of CD40L reverse signalling on neurite growth in excitatory and inhibitory neurons.

Neuregulin-4 Is Required for Maintaining Soma Size of Pyramidal Neurons in the Motor Cortex.

The regulation of neuronal soma size is essential for appropriate brain circuit function and its dysregulation is associated with several neurodevelopmental disorders. A defect in the dendritic growth and elaboration of motor neocortical pyramidal neurons in neonates lacking neuregulin-4 (NRG4) has previously been reported. In this study, we investigated whether the loss of NRG4 causes further morphologic defects that are specific to these neurons. We analyzed the soma size of pyramidal neurons of layer (L)2/3 and L5 of the motor cortex and a subpopulation of multipolar interneurons in this neocortical region in Nrg4+/+ and Nrg4-/- mice. There were significant decreases in pyramidal neuron soma size in Nrg4-/- mice compared with Nrg4+/+ littermates at all stages studied [postnatal day (P)10, P30, and P60]. The reduction was especially marked at P10 and in L5 pyramidal neurons. Soma size was not significantly different for multipolar interneurons at any age. This in vivo phenotype was replicated in pyramidal neurons cultured from Nrg4-/- mice and was rescued by NRG treatment. Analysis of a public single-cell RNA sequencing repository revealed discrete Nrg4 and Erbb4 expression in subpopulations of L5 pyramidal neurons, suggesting that the observed defects were due in part to loss of autocrine Nrg4/ErbB4 signaling. The pyramidal phenotype in the motor cortex of Nrg4-/- mice was associated with a lack of Rotarod test improvement in P60 mice, suggesting that absence of NRG4 causes alterations in motor performance.

Neuregulin-4 contributes to the establishment of cutaneous sensory innervation.

Recent work has shown that neuregulin-4 (NRG4) is a physiological regulator of the growth of sympathetic axons and CNS dendrites in the developing nervous system. Here, we have investigated whether NRG4 plays a role in sensory axon growth and the establishment of cutaneous sensory innervation. Imaging early nerve fibers in the well-characterized cutaneous trigeminal territory, the brachial plexus, and thorax revealed very marked and highly significant decreases in nerve fiber length and branching density in Nrg4-/- embryos compared with Nrg4+/+ littermates. NRG4 promoted neurotrophin-independent sensory axon growth from correspondingly early trigeminal ganglion and DRG neurons in culture but not from enteroceptive nodose ganglion neurons. High levels of Nrg4 mRNA were detected in cutaneous tissues but not in sensory ganglia. Our findings suggest that NRG4 is an important target-derived factor that participates in the establishment of early cutaneous sensory innervation.

How CD40L reverse signaling regulates axon and dendrite growth.

CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.

CD40L Reverse Signaling Influences Dendrite Spine Morphology and Expression of PSD-95 and Rho Small GTPases.

CD40-activated CD40L reverse signaling is a major physiological regulator of neural process growth from many kinds of developing neurons. Here we have investigated whether CD40L-reverse signaling also influences dendrite spine number and morphology in striatal medium spiny neurons (MSNs). Golgi preparations revealed no differences in the spine density, but because the dendrite arbors of MSNs were larger and branched in Cd40 -/- mice, the total number of spines was greater in Cd40 -/- mice. We also detected more mature spines compared with wild-type littermates. Western blot revealed that MSN cultures from Cd40 -/- mice had significantly less PSD-95 and there were changes in RhoA/B/C and Cdc42. Immunocytochemistry revealed that PSD-95 was clustered in spines in Cd40 -/- neurons compared with more diffuse labeling in Cd40 +/+ neurons. Activation of CD40L-reverse signaling with CD40-Fc prevented the changes observed in Cd40 -/- cultures. Our findings suggest that CD40L-reverse signaling influences dendrite spine morphology and related protein expression and distribution.

Initial axon growth rate from embryonic sensory neurons is correlated with birth date.

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later-born neurons within each population have further to extend their axons to reach their targets than early-born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.

CD40 forward signalling is a physiological regulator of early sensory axon growth.

Multiple members of the tumour necrosis factor superfamily (TNFSF) regulate the growth and branching of neural processes late in development, when neurons are establishing and refining connections. Here, we present the first evidence that a TNFSF member acts much earlier in development, when axons are growing to their targets. CD40L transiently enhanced axon growth from embryonic mouse DRG neurons cultured at this early stage. Early spinal nerves of embryos lacking the CD40L receptor (Cd40-/- mice) were significantly shorter in vivo than those of Cd40+/+ littermates. CD40L was synthesized in early DRG targets and was co-expressed with CD40 in early DRG neurons. Whereas CD40L enhanced early axon growth independently of neurotrophins, disruption of a CD40L/CD40 autocrine loop impaired early neurotrophin-promoted axon growth. In marked contrast to the widespread regulation of axon and dendrite growth by CD40L reverse signalling later in development, CD40-Fc, which activates reverse signalling, had no effect on early sensory axon growth. These results suggest that CD40 forward signalling is a novel physiological regulator of early axon growth that acts by target-derived and autocrine mechanisms.

Neuregulin-4 Is Required for the Growth and Elaboration of Striatal Medium Spiny Neuron Dendrites.

Medium spiny neurons (MSNs) comprise the vast majority of neurons in the striatum. Changes in the exuberant dendrites of these widely connected neurons are associated with a multitude of neurological conditions and are caused by a variety of recreational and medicinal drugs. However, we have a poor understanding of the physiological regulators of dendrite growth and elaboration of this clinically important population of neurons. Here, we show that MSN dendrites are markedly smaller and less branched in neonatal mice that possess a homozygous null mutation in the neuregulin-4 gene (Nrg4-/-) compared with wild type (Nrg4+/+) littermates. Nrg4-/- mice also had a highly significant reduction in MSN dendrite spine number in neonates and adults. The striking stunted dendrite arbor phenotype of MSNs observed in Nrg4-/- neonates was replicated in MSNs cultured from Nrg4-/- embryos and was completely rescued by soluble recombinant neuregulin-4. MSNs cultured from wild type mice coexpressed NRG4 and its receptor ErbB4. Our findings show that NRG4 is a major novel regulator of dendritic growth and arborization and spine formation in the striatum and suggest that it exerts its effects by an autocrine/paracrine mechanism.

Regional Differences in the Contributions of TNF Reverse and Forward Signaling to the Establishment of Sympathetic Innervation.

Members of the TNF and TNF receptor superfamilies acting by both forward and reverse signaling are increasingly recognized as major physiological regulators of axon growth and tissue innervation in development. Studies of the experimentally tractable superior cervical ganglion (SCG) neurons and their targets have shown that only TNF reverse signaling, not forward signaling, is a physiological regulator of sympathetic innervation. Here, we compared SCG neurons and their targets with prevertebral ganglion (PVG) neurons and their targets. Whereas all SCG targets were markedly hypoinnervated in both TNF-deficient and TNFR1-deficient mice, PVG targets were not hypoinnervated in these mice and one PVG target, the spleen, was significantly hyperinnervated. These in vivo regional differences in innervation density were related to in vitro differences in the responses of SCG and PVG neurons to TNF reverse and forward signaling. Though TNF reverse signaling enhanced SCG axon growth, it did not affect PVG axon growth. Whereas activation of TNF forward signaling in PVG axons inhibited growth, TNF forward signaling could not be activated in SCG axons. These latter differences in the response of SCG and PVG axons to TNF forward signaling were related to TNFR1 expression, whereas PVG axons expressed TNFR1, SCG axons did not. These results show that both TNF reverse and forward signaling are physiological regulators of sympathetic innervation in different tissues.

Adipocyte-secreted BMP8b mediates adrenergic-induced remodeling of the neuro-vascular network in adipose tissue.

Activation of brown adipose tissue-mediated thermogenesis is a strategy for tackling obesity and promoting metabolic health. BMP8b is secreted by brown/beige adipocytes and enhances energy dissipation. Here we show that adipocyte-secreted BMP8b contributes to adrenergic-induced remodeling of the neuro-vascular network in adipose tissue (AT). Overexpression of bmp8b in AT enhances browning of the subcutaneous depot and maximal thermogenic capacity. Moreover, BMP8b-induced browning, increased sympathetic innervation and vascularization of AT were maintained at 28 °C, a condition of low adrenergic output. This reinforces the local trophic effect of BMP8b. Innervation and vascular remodeling effects required BMP8b signaling through the adipocytes to 1) secrete neuregulin-4 (NRG4), which promotes sympathetic axon growth and branching in vitro, and 2) induce a pro-angiogenic transcriptional and secretory profile that promotes vascular sprouting. Thus, BMP8b and NRG4 can be considered as interconnected regulators of neuro-vascular remodeling in AT and are potential therapeutic targets in obesity.

TWE-PRIL reverse signalling suppresses sympathetic axon growth and tissue innervation.

TWE-PRIL is a naturally occurring fusion protein of components of two TNF superfamily members: the extracellular domain of APRIL; and the intracellular and transmembrane domains of TWEAK with no known function. Here, we show that April-/- mice (which lack APRIL and TWE-PRIL) exhibited overgrowth of sympathetic fibres in vivo, and sympathetic neurons cultured from these mice had significantly longer axons than neurons cultured from wild-type littermates. Enhanced axon growth from sympathetic neurons cultured from April-/- mice was prevented by expressing full-length TWE-PRIL in these neurons but not by treating them with soluble APRIL. Soluble APRIL, however, enhanced axon growth from the sympathetic neurons of wild-type mice. siRNA knockdown of TWE-PRIL but not siRNA knockdown of APRIL alone also enhanced axon growth from wild-type sympathetic neurons. Our work reveals the first and physiologically relevant role for TWE-PRIL and suggests that it mediates reverse signalling.

An essential role for neuregulin-4 in the growth and elaboration of developing neocortical pyramidal dendrites.

Neuregulins, with the exception of neuregulin-4 (NRG4), have been shown to be extensively involved in many aspects of neural development and function and are implicated in several neurological disorders, including schizophrenia, depression and bipolar disorder. Here we provide the first evidence that NRG4 has a crucial function in the developing brain. We show that both the apical and basal dendrites of neocortical pyramidal neurons are markedly stunted in Nrg4-/- neonates in vivo compared with Nrg4+/+ littermates. Neocortical pyramidal neurons cultured from Nrg4-/- embryos had significantly shorter and less branched neurites than those cultured from Nrg4+/+ littermates. Recombinant NRG4 rescued the stunted phenotype of embryonic neocortical pyramidal neurons cultured from Nrg4-/- mice. The majority of cultured wild type embryonic cortical pyramidal neurons co-expressed NRG4 and its receptor ErbB4. The difference between neocortical pyramidal dendrites of Nrg4-/- and Nrg4+/+ mice was less pronounced, though still significant, in juvenile mice. However, by adult stages, the pyramidal dendrite arbors of Nrg4-/- and Nrg4+/+ mice were similar, suggesting that compensatory changes in Nrg4-/- mice occur with age. Our findings show that NRG4 is a major novel regulator of dendritic arborisation in the developing cerebral cortex and suggest that it exerts its effects by an autocrine/paracrine mechanism.

CD40 is a major regulator of dendrite growth from developing excitatory and inhibitory neurons.

Dendrite size and morphology are key determinants of the functional properties of neurons and neural circuits. Here we show that CD40, a member of the TNF receptor superfamily, is a major regulator of dendrite growth and elaboration in the developing brain. The dendrites of hippocampal excitatory neurons were markedly stunted in Cd40-/- mice, whereas those of striatal inhibitory neurons were much more exuberant. These striking and opposite phenotypic changes were also observed in excitatory and inhibitory neurons cultured from Cd40-/- mice and were rescued by soluble CD40. The changes in excitatory and inhibitory neurons cultured from Cd40-/- mice were mimicked in neurons of Cd40+/+ mice by treatment with soluble CD40L and were dependent on PKC-ß and PKC-γ, respectively. These results suggest that CD40-activated CD40L reverse signalling has striking and opposite effects on the growth and elaboration of dendrites among major classes of brain neurons by PKC-dependent mechanisms.

TNF superfamily member APRIL enhances midbrain dopaminergic axon growth and contributes to the nigrostriatal projection in vivo.

We have studied the role of the tumor necrosis factor superfamily member APRIL in the development of embryonic mouse midbrain dopaminergic neurons in vitro and in vivo. In culture, soluble APRIL enhanced axon growth during a window of development between E12 and E14 when nigrostriatal axons are growing to their targets in the striatum in vivo. April transcripts were detected in both the striatum and midbrain during this period and at later stages. The axon growth-enhancing effect of APRIL was similar to that of glial cell-derived neurotrophic factor (GDNF), but in contrast to GDNF, APRIL did not promote the survival of midbrain dopaminergic neurons. The effect of APRIL on axon growth was prevented by function-blocking antibodies to one of its receptors, BCMA (TNFRSF13A), but not by function-blocking antibodies to the other APRIL receptor, TACI (TNFRSF13B), suggesting that the effects of APRIL on axon growth are mediated by BCMA. In vivo, there was a significant reduction in the density of midbrain dopaminergic projections to the striatum in April-/- embryos compared with wild type littermates at E14. These findings demonstrate that APRIL is a physiologically relevant factor for the nigrostriatal projection. Given the importance of the degeneration of dopaminergic nigrostriatal connections in the pathogenesis and progression of Parkinson's disease, our findings contribute to our understanding of the factors that establish nigrostriatal integrity.

T-type Ca2+ channels are required for enhanced sympathetic axon growth by TNFα reverse signalling.

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth.

Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation.

BACKGROUND: Nerve growth factor (NGF) is the prototypical target-derived neurotrophic factor required for sympathetic neuron survival and for the growth and ramification of sympathetic axons within most but not all sympathetic targets. This implies the operation of additional target-derived factors for regulating terminal sympathetic axon growth and branching. RESULTS: Here report that growth differentiation factor 5 (GDF5), a widely expressed member of the transforming growth factor beta (TGFß) superfamily required for limb development, promoted axon growth from mouse superior cervical ganglion (SCG) neurons independently of NGF and enhanced axon growth in combination with NGF. GDF5 had no effect on neuronal survival and influenced axon growth during a narrow window of postnatal development when sympathetic axons are ramifying extensively in their targets in vivo. SCG neurons expressed all receptors capable of participating in GDF5 signaling at this stage of development. Using compartment cultures, we demonstrated that GDF5 exerted its growth promoting effect by acting directly on axons and by initiating retrograde canonical Smad signalling to the nucleus. GDF5 is synthesized in sympathetic targets, and examination of several anatomically circumscribed tissues in Gdf5 null mice revealed regional deficits in sympathetic innervation. There was a marked, highly significant reduction in the sympathetic innervation density of the iris, a smaller though significant reduction in the trachea, but no reduction in the submandibular salivary gland. There was no reduction in the number of neurons in the SCG. CONCLUSIONS: These findings show that GDF5 is a novel target-derived factor that promotes sympathetic axon growth and branching and makes a distinctive regional contribution to the establishment of sympathetic innervation, but unlike NGF, plays no role in regulating sympathetic neuron survival.

ERK signaling mediates CaSR-promoted axon growth.

The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor that monitors the systemic extracellular free ionized calcium level ([Ca(2+)]o) in organs involved in systemic [Ca(2+)]o homeostasis. CaSR is widely expressed in the nervous system and its activation promotes axon and dendrite growth during development, but the mechanism by which it does this is not known. Here we show that enhanced axon growth and branching from cultured embryonic sympathetic neurons by activation of the endogenous CaSR depends on the presence of nerve growth factor (NGF). Our observation that activation of overexpressed CaSR promotes axon growth in NGF-free medium has enabled us to investigate CaSR downstream signaling contributing to axon growth in the absence of NGF signaling. We show that activation of overexpressed CaSR leads to activation of ERK1 and ERK2, and pharmacological inhibition of CaSR-dependent ERK1/ERK2 activation prevents CaSR-dependent axon growth. Analysis of axon growth from cultured neurons expressing deletion mutants of the CaSR cytoplasmic tail revealed that the region between alanine 877 and glycine 907 is required for promoting axon growth that is distinct from the high-affinity filamin-A binding site that has previously been implicated in ERK1/ERK2 activation.

Regulation of Autocrine Signaling in Subsets of Sympathetic Neurons Has Regional Effects on Tissue Innervation.

The regulation of innervation by target-derived factors like nerve growth factor (NGF) is the cornerstone of neurotrophic theory. Whereas autocrine signaling in neurons affecting survival and axon growth has been described, it is difficult to reconcile autocrine signaling with the idea that targets control their innervation. Here, we report that an autocrine signaling loop in developing mouse sympathetic neurons involving CD40L (TNFSF5) and CD40 (TNFRSF5) selectively enhances NGF-promoted axon growth and branching, but not survival, via CD40L reverse signaling. Because NGF negatively regulates CD40L and CD40 expression, this signaling loop operates only in neurons exposed to low levels of NGF. Consequently, the sympathetic innervation density of tissues expressing low NGF is significantly reduced in CD40-deficient mice, whereas the innervation density of tissues expressing high levels of NGF is unaffected. Our findings reveal how differential regulation of autocrine signaling in neurons has region-specific effects on axon growth and tissue innervation.

Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL.

APRIL (A Proliferation-Inducing Ligand, TNFSF13) is a member of the tumor necrosis factor superfamily that regulates lymphocyte survival and activation and has been implicated in tumorigenesis and autoimmune diseases. Here we report the expression and first known activity of APRIL in the nervous system. APRIL and one of its receptors, BCMA (B-Cell Maturation Antigen, TNFRSF17), are expressed by hippocampal pyramidal cells of fetal and postnatal mice. In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation. Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation. The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA. APRIL promoted phosphorylation and activation of ERK1, ERK2 and Akt and serine phosphorylation and inactivation of GSK-3ß in cultured hippocampal pyramidal cells. Inhibition of MEK1/MEK2 (activators of ERK1/ERK2), PI3-kinase (activator of Akt) or Akt inhibited the axon growth-promoting action of APRIL, as did pharmacological activation of GSK-3ß and the expression of a constitutively active form of GSK-3ß. These findings suggest that APRIL promotes axon elongation by a mechanism that depends both on ERK signaling and PI3-kinase/Akt/GSK-3ß signaling.