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.

Age-dependent maintenance of motor control and corticostriatal innervation by death receptor 3.

Death receptor 3 is a proinflammatory member of the immunomodulatory tumor necrosis factor receptor superfamily, which has been implicated in several inflammatory diseases such as arthritis and inflammatory bowel disease. Intriguingly however, constitutive DR3 expression has been detected in the brains of mice, rats, and humans, although its neurological function remains unknown. By mapping the normal brain expression pattern of DR3, we found that DR3 is expressed specifically by cells of the neuron lineage in a developmentally regulated and region-specific pattern. Behavioral studies on DR3-deficient (DR3(ko)) mice showed that constitutive neuronal DR3 expression was required for stable motor control function in the aging adult. DR3(ko) mice progressively developed behavioral defects characterized by altered gait, dyskinesia, and hyperactivity, which were associated with elevated dopamine and lower serotonin levels in the striatum. Importantly, retrograde tracing showed that absence of DR3 expression led to the loss of corticostriatal innervation without significant neuronal loss in aged DR3(ko) mice. These studies indicate that DR3 plays a key nonredundant role in the retention of normal motor control function during aging in mice and implicate DR3 in progressive neurological disease.

Developmental switch in NF-kappaB signalling required for neurite growth.

For a given cell type, particular extracellular signals generate characteristic patterns of activity in intracellular signalling networks that lead to distinctive cell-type specific responses. Here, we report the first known occurrence of a developmental switch in the intracellular signalling network required for an identical cellular response to the same extracellular signal in the same cell type. We show that although NF-kappaB signalling is required for BDNF-promoted neurite growth from both foetal and postnatal mouse sensory neurons, there is a developmental switch between these stages in the NF-kappaB activation mechanism and the phosphorylation status of the p65 NF-kappaB subunit required for neurite growth. Shortly before birth, BDNF activates NF-kappaB by an atypical mechanism that involves tyrosine phosphorylation of IkappaBalpha by Src family kinases, and dephosphorylates p65 at serine 536. Immediately after birth, BDNF-independent constitutive activation of NF-kappaB signalling by serine phosphorylation of IkappaBalpha and constitutive dephosphorylation of p65 at serine 536 are required for BDNF-promoted neurite growth. This abrupt developmental switch in NF-kappaB signalling in a highly differentiated cell type illustrates an unsuspected plasticity in signalling networks in the generation of identical cellular responses to the same extracellular signal.

Developmental regulation of sensory neurite growth by the tumor necrosis factor superfamily member LIGHT.

In a PCR screen to identify novel cytokine candidates involved in neuronal development, we identified transcripts for the tumor necrosis factor superfamily member 14 (TNFSF14), generally known as LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells), together with its receptors, lymphotoxin-beta receptor (LTbetaR) and TNF family receptor herpesvirus entry mediator (HVEM), in the experimentally tractable sensory neurons of the mouse nodose ganglion. Immunocytochemistry revealed coexpression of LIGHT and its receptors in all nodose ganglion neurons in neonates. Enhancing LIGHT signaling in these neurons by overexpressing LIGHT inhibited BDNF-promoted neurite growth during a narrow window of development in the immediate perinatal period without affecting neuronal survival. Overexpressing a LIGHT mutant that selectively activates HVEM, but not one that selectively activates LTbetaR, also inhibited BDNF-promoted growth, suggesting that neurite growth inhibition is mediated via HVEM. Blocking HVEM signaling by a function-blocking anti-HVEM antibody significantly enhanced neurite growth from nodose neurons grown both with and without BDNF. Likewise, neurons from LIGHT-deficient neonates exhibited significantly greater neurite growth than neurons from wild-type littermates in both the presence and absence of BDNF. LIGHT overexpression significantly inhibited NF-kappaB activity, while preventing LIGHT-induced NF-kappaB inhibition by overexpressing the p65 and p50 NF-kappaB subunits prevented LIGHT-mediated growth inhibition. Together, these findings show that LIGHT/HVEM signaling negatively regulates neurite growth from developing sensory neurons via NF-kappaB inhibition.

Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA.

Nuclear factor kappaB (NF-kappaB) signaling is known to promote neurite growth from developing sensory neurons and to enhance the size and complexity of pyramidal neuron dendritic arbors in the developing cerebral cortex. In marked contrast, here we show that NF-kappaB signaling can also exert a potent inhibitory influence on neurite growth in certain neurons, and can either promote or inhibit neurite growth in the same neurons depending on the mechanism of NF-kappaB activation. In neonatal superior cervical ganglion sympathetic neurons, enhancing NF-kappaB transcriptional activity by overexpressing either the p65 NF-kappaB subunit or the IkappaB kinase-beta (IKKbeta) subunit of the IkappaB kinase complex, or by tumor necrosis factor alpha (TNFalpha) treatment, strongly inhibits neurite growth. Paradoxically in neonatal nodose ganglion sensory neurons, enhancing NF-kappaB transcriptional activity by p65/p50 overexpression increases neurite growth, whereas enhancing NF-kappaB transcriptional activity by IKKbeta overexpression inhibits neurite growth. In addition to activating NF-kappaB, IKKbeta overexpression leads to phosphorylation of p65 on serine 536. Blockade of serine 536 phosphorylation by a S536A-p65 mutant protein prevents the growth-inhibitory effects of IKKbeta overexpression in both sensory and sympathetic neurons and the growth-inhibitory effects of TNFalpha on sympathetic neurons. Furthermore, expression of a p65 S536D phosphomimetic mutant inhibits neurite growth from sensory neurons. These results demonstrate that NF-kappaB can either stimulate or inhibit neurite growth in developing neurons depending on the phosphorylation status of p65.

Nuclear factor-kappaB activation via tyrosine phosphorylation of inhibitor kappaB-alpha is crucial for ciliary neurotrophic factor-promoted neurite growth from developing neurons.

The cytokine ciliary neurotrophic factor (CNTF) promotes the growth of neural processes from many kinds of neurons in the developing and regenerating adult nervous system, but the intracellular signaling mechanisms mediating this important function of CNTF are poorly understood. Here, we show that CNTF activates the nuclear factor-kappaB (NF-kappaB) transcriptional system in neonatal sensory neurons and that blocking NF-kappaB-dependent transcription inhibits CNTF-promoted neurite growth. Selectively blocking NF-kappaB activation by the noncanonical pathway that requires tyrosine phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha), but not by the canonical pathway that requires serine phosphorylation of IkappaB-alpha, also effectively inhibits CNTF-promoted neurite growth. CNTF treatment activates spleen tyrosine kinase (SYK) whose substrates include IkappaB-alpha. CNTF-induced SYK phosphorylation is rapidly followed by increased tyrosine phosphorylation of IkappaB-alpha, and blocking SYK activation or tyrosine phosphorylation of IkappaB-alpha prevents CNTF-induced NF-kappaB activation and CNTF-promoted neurite growth. These findings demonstrate that NF-kappaB signaling by an unusual activation mechanism is essential for the ability of CNTF to promote the growth of neural processes in the developing nervous system.

Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain.

Deficits of neurotrophic support caused by reduced levels of brain-derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntington's disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon-1 and full-length knock-in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down-regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/-) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.

Glial cell line-derived neurotrophic factor promotes the arborization of cultured striatal neurons through the p42/p44 mitogen-activated protein kinase pathway.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival or differentiation of several types of neurons. This study examines GDNF-induced signal transduction and biological effects in cultured striatal neurons. Results show that GDNF addition to striatal cultures transiently increased the protein levels of phosphorylated p42/p44, but did not change the levels of phosphorylated Akt. GDNF effects on phosphorylated p42/p44 levels were blocked by the mitogen-activated protein kinase (MAPK) pathway specific inhibitors (PD98059 and U0126). Activation of the p42/p44 MAPK pathway by GDNF led to an increase in the degree of dendritic arborization and axon length of both GABA- and calbindin-positive neurons but had no effect on their survival and maturation. These GDNF-mediated effects were suppressed in the presence of the inhibitor of the MAPK pathway (PD98059). Furthermore, the addition of the phosphatidylinositol 3-kinase pathway specific inhibitor (LY294002) blocked GDNF-mediated striatal cell differentiation suggesting that the basal activity of this pathway is needed for the effects of GDNF. Our results indicate that treatment of cultured striatal cells with GDNF specifically activates the p42/p44 MAPK pathway, leading to an increase in the arborization of GABA- and calbindin-positive neurons.

Brain-derived neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum.

Brain-derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl-2 family members. We first analyzed the involvement of the phosphatidylinositol 3-kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p-Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non-NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl-2, Bcl-x(L) and Bax protein levels and heterodimerization are selectively regulated by NMDA and non-NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl-2 protein levels, leading to reduced levels of Bax:Bcl-2 heterodimers. In contrast, changes in Bax protein levels are not required for KA-induced apoptotic cell death, but decreased levels of both Bax:Bcl-2 and Bax:Bcl-x(L) heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase-3. Intrastriatal grafting of a BDNF-secreting cell line counter-regulated p-AKT, Bcl-2, Bcl-x(L) and Bax protein levels, prevented changes in the heterodimerization between Bax and pro-survival proteins, and blocked caspase-3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti-apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl-2 family members, which is probably mediated by Akt activation.

Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons.

Brain-derived neurotrophic factor (BDNF) is a potent trophic factor for striatal cells that promotes survival and/or differentiation of GABAergic neurons in vitro. In the present study, we show that the stimulation of cultured striatal cells with BDNF increased the phosphorylation of Akt and p42/p44. This effect was specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) pathways (LY294002 and wortmannin) or p42/p44 mitogen-activated protein (MAP) kinase (PD98059 and U0126). BDNF treatment induced an increase in the number of calbindin-positive neurons but not in the number of GABAergic or total cells. Furthermore, BDNF increased the degree of dendritic arborization, soma area and axon length of striatal neurons. However, PD98059 was more effective blocking BDNF effects on calbindin- than on GABA-positive neurons, whereas LY294002 inhibited morphological differentiation in both neuronal populations. Moreover, BDNF induced neuronal survival only through the activation of the PI3-K pathway.

Excitatory amino acids differentially regulate the expression of GDNF, neurturin, and their receptors in the adult rat striatum.

Glial cell line-derived neurotrophic factor (GDNF) family ligands are important regulators of neuronal development and maintenance of the connectivity in the basal ganglia and show neuroprotective activities in several paradigms of brain injury. The mRNAs of two members of this family, GDNF and neurturin, and also their receptors have been detected in the basal ganglia. In the present work, we analyzed the time course changes in the expression of these neurotrophic factors and receptors in the adult rat striatum, induced by quinolinate or kainate excitotoxicity. Our results show that stimulation of NMDA or non-NMDA receptors induced different effects on the mRNA levels analyzed. Expression of GDNF and its preferred receptor, GDNF family receptor-alpha1 (GFRalpha1), was transiently up-regulated by quinolinate and kainate, but with differing intensity and temporal pattern. Immunohistochemical analysis showed that, although GDNF and GFRalpha1 were initially localized in neurons, excitotoxicity induced the expression of these proteins in astrocyte-like cells. Neurturin mRNA levels were only up-regulated after quinolinate injection, whereas quinolinate or kainate injection did not modify GFRalpha2 mRNA. The mRNA for the common receptor, c-Ret, was up-regulated by both agonists with similar temporal pattern but with differing intensity. Immunohistochemical analysis showed that c-Ret protein was located on neurons. These changes in mRNA levels and protein localization of GDNF family components could reflect an endogenous trophic response of striatal cells to different excitotoxic insults.