Survival Of Sympathetic Neurons Research Articles

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Oxygen deprived (The zebrafish embryo as a dynamic model of anoxia tolerance by Bryce A. Mendelsohn, Bethany L. Kassebaum, and Jonathan D. Gitlin, Dev Dyn 237:1780–1788; and Coordination of development and metabolism in the pre-midblastula transition zebrafish embryo by Bryce A. Mendelsohn and Jonathan D. Gitlin, Dev Dyn 237:1789–1798) It is hard to grow when you cannot breath—consequently, zebrafish embryos postpone development in an anoxic environment. Depending on the embryonic stage and the metabolic state, development proceeds when oxygen becomes available. The authors characterized this intriguing tolerance phenomenon in back-to-back studies. One question addressed is, how might anoxia trigger arrest? Mendelsohn, Kassebaum, and Gitilin tested if energy homeostasis was involved. The 24-hour-old embryos were incubated in normal oxygen with chemicals such as cyanide that block oxidative phosphorylation—causing a shift from aerobic to anaerobic energy production. This shift, leads to developmental arrest similar to that seen under anoxic conditions. What mechanism is involved in sensing the change in energy metabolism? One contender is the energy sensing AMP activated protein kinase (AMPK) pathway. Consistent with this supposition, the authors show, using phosphorylation assays, that AMPK and downstream factors are activated in embryos exposed to anoxia or inhibitors of oxidative phosphorylation. In their second study, Mendelsohn and Gitilin show that younger embryos also stall development upon chemical induced anoxia. Developmental arrest was initiated before the mid blastula transition, when maternal components rule, revealing that in this case zygotic transcription is not required. Maternal AMPK is activated by anoxia, suggesting a conservation of response between young and old embryos. The AMPK pathway regulates essential processes including translation, cell cycle control, and glycolysis that are required for development to proceed making the pathway a good candidate for the link from energy sensing to anoxia tolerance. Sympathetic development (Loss of the Prader-Willi Syndrome protein necdin causes defective migration, axonal outgrowth, and survival of embryonic sympathetic neurons by Alysa A. Tennese, Christopher B. Gee, and Rachel Wevrick, Dev Dyn 237:1935–1943) Prader-Willi syndrome is a neurodevelopmental disorder with multiple symptoms including low muscle tone, learning disability, excessive hunger leading to obesity, and difficulty swallowing. The disease is caused by deletion of several imprinted genes on chromosome 15. One, Ndn, encodes necdin—a protein previously shown in mice to be required for normal development of sensory and central neurons. Tennese, Gee, and Wevrick now show that Ndn knock out mice also have developmental defects in a subset of sympathetic neurons, primarily in the superior cervical ganglia (SCG), which innervate salivary, parotid, submandibular glands, and nasal mucosa. Phox2B and tyrosine hydroxylase mark sympathetic neurons, and were used to follow SCG during proliferation and migration. They show that a subset of SCG neurons failed to migrate to their final rostral position between vertebral levels C1 and C4. Moreover, SCG neurons were reduced in size and number, which corresponds to an observed increase in cell death. Finally, defects in SCG axon outgrowth and neuronal morphology likely contribute to an observed reduction in innervation of target tissues. Some Prader-Willi patients have difficulty swallowing due to decreased saliva production, suggesting an analogous disruption could be the cause. Exit the pool (Effect of canonical Wnt inhibition in the neurogenic cortex, hippocampus, and premigratory dentate gyrus progenitor pool by Nina Solberg, Ondrej Machon, and Stefan Krauss, Dev Dyn 237:1799–1811) Canonical Wnt signaling, through β-catenin, is required for hippocampal and dentate gyrus (DG) development. But its precise role in the development of these tissues is not clear. Just before formation of DG progenitor cells, Solberg, Machon, and Krauss, decreased Wnt signaling in mice by driving expression of the inhibitor, Dickkopf1 (Dkk1) in the developing cortex and hippocampus. They found, similar to conditional mutants in Wnt3A and β-catenin, that there was a diminution of the hippocampus and a severe reduction in granule cells of the DG. Furthermore, they determined that reduced cell number in the DG was caused by an increase in cell cycle length and a decrease in proliferation of the progenitor pool. Importantly, adults lack two DG structures, the internal blade and part of the external blade. This finding shows that there was no compensation as development proceeded, suggesting the progenitor pool was depleted during embryogenesis.

Proteomic assessment of sympathetic ganglia from adult mice that possess null mutations of ExonIII or ExonIV in the p75 neurotrophin receptor gene

Neurotrophins, such as nerve growth factor (NGF), are capable of binding to the transmembrane p75 neurotrophin receptor (p75NTR), which regulates a variety of cellular responses including apoptosis and axonal elongation. While the development of mutant mouse strains that lack functional p75NTR expression has provided further insight into the importance of this neurotrophin receptor, there remains a paucity of information concerning how the loss of p75NTR expression may alter neural phenotypes. To address this issue, we assessed the proteome of the cervical sympathetic ganglia from two mutant lines of mice, which were compared to the ganglionic proteome of age-matched wild type mice. The ganglionic proteome of mice possessing two mutant alleles of either exonIII or exonIV for the p75NTR gene displayed detectable alterations in levels of Lamin A, tyrosine hydroxylase, and Annexin V, as compared to ganglionic proteome of wild type mice. Decreased expression of the basic isoform of tyrosine hydroxylase may be linked to perturbed NGF signaling in the absence of p75NTR in mutant mice. Stereological measurement showed significant increases in the number of sympathetic neurons in both lines of p75NTR-deficient mice, relative to wild type mice. This enhanced survival of sympathetic neurons coincides with shifts toward the more basic isoforms of Annexin V in mutant mice. This study, in addition to providing the first comparative proteomic assessment of sympathetic ganglia, sheds new light onto the phenotypic changes that occur as a consequence of a loss of p75NTR expression in adult mice.

HAND transcription factors are required for neonatal sympathetic neuron survival

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Abnormal sympathetic nervous system development and physiological dysautonomia in Egr3-deficient mice

Sympathetic nervous system development depends upon many factors that mediate neuron migration, differentiation and survival. Target tissue-derived nerve growth factor (NGF) signaling-induced gene expression is required for survival, differentiation and target tissue innervation of post-migratory sympathetic neurons. However, the transcriptional regulatory mechanisms mediated by NGF signaling are very poorly defined. Here, we identify Egr3, a member of the early growth response (Egr) family of transcriptional regulators, as having an important role in sympathetic nervous system development. Egr3 is regulated by NGF signaling and it is expressed in sympathetic neurons during development when they depend upon NGF for survival and target tissue innervation. Egr3-deficient mice have severe sympathetic target tissue innervation abnormalities and profound physiological dysautonomia. Unlike NGF, which is essential for sympathetic neuron survival and for axon branching within target tissues, Egr3 is required for normal terminal axon extension and branching, but not for neuron survival. The results indicate that Egr3 is a novel NGF signaling effector that regulates sympathetic neuron gene expression required for normal target tissue innervation and function. Egr3-deficient mice have a phenotype that is remarkably similar to humans with sympathetic nervous system disease, raising the possibility that it may have a role in some forms of human dysautonomia, most of which have no known cause.

Loss of the Prader‐Willi syndrome protein necdin causes defective migration, axonal outgrowth, and survival of embryonic sympathetic neurons

Prader-Willi syndrome is a neurodevelopmental disorder marked by abnormalities in feeding, drinking, thermoregulation, intestinal motility, and reproduction, suggesting disruption of the autonomic nervous system. Necdin, one of several proteins genetically inactivated in individuals with Prader-Willi syndrome, is important for the differentiation of central and sensory neurons. We now show that formation, migration, and survival of sympathetic superior cervical ganglion neurons are impaired in Ndn-null embryos. We observed reduced innervation of superior cervical ganglion target organs, including the submandibular gland, parotid gland, and nasal mucosa. While the formation of other sympathetic chain ganglia is unaffected, axonal extension is impaired throughout the sympathetic nervous system. These results demonstrate a novel role for necdin in cellular migration, in addition to its roles in survival and axon outgrowth. Furthermore, reduced sympathetic function provides a plausible explanation for deficiencies of salivary gland function in individuals with congenital necdin deficiency consequent to Prader-Willi syndrome.

Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.

Müller glia factors induce survival and neuritogenesis of peripheral and central neurons

We have examined the trophic effects of conditioned media obtained from purified murine Müller glia cells on chick purified sympathetic or dorsal root ganglia (DRG) neurons and on Retinal Ganglion Cells (RGC) from postnatal mice. Purified murine Müller glia cultures stained positively for vimentin, GFAP or S-100, but were negative for neuronal markers. Murine Müller glial conditioned medium (MMG) was concentrated and at 1:1 dilution supported 100% survival of chick or rat sympathetic neurons after 48 h compared to < 5% in controls. Partial purification of the MMG using centriprep concentrators showed that trophic activity is from molecules above 10 kDa. MMG stimulated AKT, ERK and pStat3 in sympathetic neurons. Sympathetic or DRG neuronal survival induced by MMG was blocked by anti-human NGF, but not by anti-human CNTF (sympathetic) or by anti-BDNF (DRGs) neutralizing antibodies. MMG also induced neurite outgrowth in P4 mice retinal explants and on isolated RGC. RGCs plated on top of Müller glia cells had a much better survival rate (> 80%, 96 h) compared to laminin + poly- l-lysine substrates. In conclusion, we show that purified mice Müller glia cultures secrete NGF that support peripheral neuronal survival and other unidentified trophic molecules that induce RGC survival and neuritogenesis.

Sympathetic dysautonomia despite the absence of sympathetic neuron loss in Egr3‐deficient mice

The sympathetic nervous system (SNS) is essential for tissue and organ homeostasis. Despite the fact that it is the target of a wide variety of congenital and degenerative diseases, the mechanisms involved in sympathetic neuron differentiation and target tissue innervation are still very poorly characterized. Sympathetic neurons depend upon target tissue‐derived trophic factors such as nerve growth factor (NGF) for survival and differentiation, but the downstream signaling molecules that regulate new gene expression have not been fully defined. We recently identified the NGF‐regulated transcription factor Egr3 as an essential mediator of normal SNS development. Egr3‐deficient mice can survive to adulthood but they have profound physiologic dysautonomia reminiscent of human disease. The dysautonomia is associated with sympathetic neuron loss and abnormal target tissue innervation. Thus, it has not been clear whether Egr3 is primarily involved in gene regulation related to sympathetic neuron survival, differentiation or both. To examine whether Egr3 has a direct role in sympathetic neuron survival, Egr3 knockout mice were mated to mice lacking the pro‐apoptotic molecule Bax. We found that both physiologic and Egr3‐dependent sympathetic neuron death was completely inhibited in Egr3−/−:Bax−/− double knockout mice, yet the mice still had dysautonomia and target tissue innervation defects. These results demonstrate that Egr3 has an essential role in sympathetic axon outgrowth and target tissue innervation that is independent of any role it may have in sympathetic neuron survival. Moreover, the results reaffirm the concept that human dysautonomias, which are virtually always associated with neuron loss, are not necessarily caused by primary defects in neuron survival. Support: NIH and HHMI.

NGF-promoted axon growth and target innervation requires GITRL-GITR signaling

Nerve growth factor (NGF) has an important role in regulating sympathetic neuron survival and target field innervation during development. Here we show that glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), a member of the TNF superfamily, and its ligand (GITRL) are co-expressed in mouse sympathetic neurons when their axons are innervating their targets under the influence of target-derived NGF. In culture, GITRL enhanced NGF-promoted neurite growth from neonatal sympathetic neurons, and preventing GITR-GITRL interaction in these neurons or knocking down GITR inhibited NGF-promoted neurite growth without affecting neuronal survival. Tnfrsf18(-/-) (Gitr) neonates have reduced sympathetic innervation density in vivo compared with Gitr(+/+) littermates. GITR activation is required for the phosphorylation of extracellular signal-regulated kinases 1 and 2 by NGF that is necessary for neurite growth. Our results reveal a previously unknown signaling loop in developing sympathetic neurons that is crucial for NGF-dependent axon growth and target innervation.

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3

Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF−/−, NT3−/− and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine β hydroxylase (DβH) mRNA levels were normal in NGF−/− embryos, but significantly reduced in NT3−/− embryos. In contrast, the β2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3−/− embryos, but significantly reduced in NGF−/− embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75NTR. These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors.

Artemin activates axonal growth via SFK and ERK‐dependent signalling pathways in mature dorsal root ganglia neurons

Artemin, one of the glial cell line-derived neurotrophic factor (GDNF) family, enhances the generation and survival of early sympathetic neurons and superior cervical ganglion (SCG) neurons. Src-family kinases (SFK) are involved in the growth and differentiation of cells, which are composed of unique Src homology 2 (SH2), Src homology 3 (SH3) and kinase domains. Various extra-cellular molecules containing growth factors and G-protein coupled receptors stimulate SFK. In this report, artemin is shown to have a significant effect on the neurite growth of dorsal root ganglia (DRG) neurons. Also, artemin triggers Src-family kinase activation and the phosphorylation of extra-cellular signal-regulated kinases (ERK) mitogen-activated protein kinase (MAPK). Artemin also regulated actin polymerization. There are several indications that another SH3-containing protein, Hck, and an SH3-containing adaptor protein, Nck1, play an important role in the organization of the actin cytoskeleton by cellular signalling. These findings suggest that the exploration of binding partners for the SH3 domain could provide an insight into regulation between the microtubule and actin networks. The binding partners for the SH3 domains of Nck, Src and Hck that we identified were Smc chromosome segregation ATPases, FOG Zn-finger protein and the FYVE zinc-binding domain, respectively.

NT-3 Expression in Spared DRG and the Associated Spinal Laminae as well as Its Anterograde Transport in Sensory Neurons Following Removal of Adjacent DRG in Cats

Neurotrophin-3 plays an important role in survival and differentiation of sensory and sympathetic neurons, sprouting of neurites, synaptic reorganization, and axonal growth. The present study evaluated changes in expression of NT-3 in the spinal cord and L6 dorsal root ganglion (DRG), after ganglionectomy of adjacent dorsal roots in cats. NT-3 immunoreactivity increased at 3 days post-operation (dpo), but decreased at 10 dpo in spinal lamina II after ganglionectomy of L1-L5 and L7-S2 (leaving L6 DRG intact). Conversely, NT-3 immunoreactivity decreased on 3 dpo, but increased on 10 dpo in the nucleus dorsalis. Very little NT-3 mRNA signal was detected in the spinal cord, despite the changes in NT-3 expression. The above changes may be related to changes in NT-3 expression in the DRG. Ganglionectomy of L1-L5 and L7-S2 resulted in increase in NT-3 immunoreactivity and mRNA in small and medium-sized neurons, but decreased expression in large neurons of L6 DRG at 3 dpo. It is possible that increased NT-3 in spinal lamina II is derived from anterograde transport from small- and medium-sized neurons of L6 DRG, whereas decreased NT-3 immunoreactivity in the nucleus dorsalis is due to decreased transport of NT-3 from large neurons in the DRG at this time. This notion is supported by observations on NT-3 distribution in the dorsal root of L6 after ligation of the nerve root. The above results indicate that DRG may be a source of neurotrophic factors such as NT-3 to the spinal cord, and may contribute to plasticity in the spinal cord after injury.

One at a time, live tracking of NGF axonal transport using quantum dots

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50-150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body.

Estrogen regulation of neurotrophin expression in sympathetic neurons and vascular targets

We hypothesize that estrogen exerts a modulatory effect on sympathetic neurons to reduce neural cardiovascular tone and that these effects are modulated by nerve growth factor (NGF), a neurotrophin that regulates sympathetic neuron survival and maintenance. We examined the effects of estrogen on NGF and tyrosine hydroxylase (TH) protein content in specific vascular targets. Ovariectomized, adult Sprague–Dawley rats were implanted with placebo or 17β-estradiol (release rate, 0.05 mg/day). Fourteen days later, NGF levels in the superior cervical ganglia (SCG) and its targets, the heart, external carotid artery, and the extracerebral blood vessels, as well as estrogen receptor α (ERα) content levels in the heart, were determined using semi-quantitative Western blot analysis. TH levels in the SCG and extracerebral blood vessels were determined by Western blotting and immunocytochemistry, respectively. Circulating levels of 17β-estradiol and prolactin (PRL) were quantified by RIA. Estrogen replacement significantly decreased NGF protein in the SCG and its targets, the external carotid artery, heart and extracerebral blood vessels. TH protein associated with the extracerebral blood vessels was also significantly decreased, but ERα levels were significantly increased in the heart following estrogen replacement. These results indicate that estrogen reduces NGF protein content in sympathetic vascular targets, which may lead to decreased sympathetic innervations to these targets, and therefore reduced sympathetic regulation. In addition, the estrogen-induced increase in ERα levels in the heart, a target tissue of the SCG, suggests that estrogen may sensitize the heart to further estrogen modulation, and possibly increase vasodilation of the coronary vasculature.

The latency-associated transcript of herpes simplex virus type 1 promotes survival and stimulates axonal regeneration in sympathetic and trigeminal neurons

Herpes simplex virus type 1 (HSV-1) primarily infects mucoepithelial tissues of the eye, the orofacial region, and to a lesser extent the genitalia. The virus is retrogradely transported through the axons of sensory and sympathetic neurons to their cell bodies to establishe a life-long latent infection. Throughout this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). The function of LAT is still largely unknown. To understand how HSV-1 latency might affect neurons, the authors transfected primary cultures of sympathetic neurons and trigeminal sensory neurons obtained from rat embryos with LAT-expressing plasmids. LAT increased the survival of both sympathetic and trigeminal neurons after induction of cell death by nerve growth factor (NGF) deprivation. Because HSV-1 is transported through axons both after initial infection and during reactivation, the authors considered the possibility that LAT may affect axonal growth. They found that LAT expression increased axonal regeneration by twofold in both types of neurons. Inhibition of the mitogen-activated protein kinase (MAPK) pathway reverses stimulation of both neuronal survival and axonal regeneration, which indicates that these effects are mediated through the MAPK pathway. These data provide evidence that HSV-1 LAT promotes survival of sympathetic as well as trigeminal neurons. The authors show for the first time that LAT stimulates axonal regeneration in both sympathetic and trigeminal neurons.

Genetic Evidence for Adenylyl Cyclase 1 as a Target for Preventing Neuronal Excitotoxicity Mediated by N-Methyl-D-aspartate Receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.

BDNF: A missing link between sympathetic dysfunction and inflammatory disease?

Nerve growth factor (NGF) plays a role in sympathetic neuron integrity and survival. Brain-derived neurotrophic factor (BDNF) also has trophic effects on sympathetic neurons. We report here the serendipitous finding that co-treatment of hippocampus with BDNF and the NGF antagonist TrkA-Fc leads to perivascular inflammation and marked vasoconstriction. This effect is not observed with either reagent alone or in combination with other control proteins. Because NGF supports sympathetic neuron health, we tested the hypothesis that BDNF combined with sympathetic compromise caused this effect. Superior cervical ganglia were removed bilaterally with concurrent BDNF infusion into hippocampus. Perivascular inflammation was observed at 3 days, but not 12 days post treatment, when sympathetic terminals had receded, suggesting that the presence of these terminals was necessary for inflammation. Since sympathetic dysfunction may lead to compensatory overactivity of norepinephrine (NE) signaling, we co-infused BDNF with NE in the hippocampus and observed perivascular inflammation. In humans, sympathetic overactivity has been reported in a variety of vascular diseases. Some of these diseases, e.g. primary Raynaud's, are not accompanied by serious inflammatory disease whereas others, such as scleroderma and systemic lupus, are. We speculate that BDNF may contribute to the transformation of sympathetic dysfunction to inflammatory disease.

Glial Cell Line-Derived Neurotrophic Factor-Dependent Recruitment of Ret into Lipid Rafts Enhances Signaling by Partitioning Ret from Proteasome-Dependent Degradation

The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFRalphas. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

Increased NGF proforms in aged sympathetic neurons and their targets

Target-derived neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3) regulate sympathetic neuron survival. Here, NGF and NT-3 protein and transcript were examined in sympathetic neurons and targets in order to determine their role in age-related neuronal atrophy. One obvious alteration was a dramatic increase (up to 50-fold) in NGF protein forms, corresponding to proNGF-B, in the superior cervical ganglion (SCG) and targets where sympathetic innervation shows atrophy. In the iris, where sympathetic innervation is protected into old age, proNGF-B was decreased. Alterations in NGF transcript paralleled changes in NGF protein, albeit to a lesser degree. Though significantly increased in aged SCG, NT-3 protein, found primarily as the ‘mature’ form, showed only minor changes in most tissues, though NT-3 mRNA generally was decreased. In contrast, both NT-3 transcript and NT-3 precursors were increased in iris. The dramatic increases in proNGF, together with minimal changes in NT-3, suggest that alterations in NGF regulation may contribute to the loss of sympathetic innervation observed in many aged peripheral targets.