STAT3 integrates cytokine and neurotrophin signals to promote sympathetic axon regeneration.

The transcription factor STAT3 has been implicated in axon regeneration. Here we investigate a role for STAT3 in sympathetic nerve sprouting after myocardial infarction (MI) - a common injury in humans. We show that NGF stimulates serine phosphorylation (S727) of STAT3 in sympathetic neurons via ERK1/2, in contrast to cytokine phosphorylation of Y705. Maximal sympathetic axon regeneration in vitro requires phosphorylation of both S727 and Y705. Furthermore, cytokine signaling is necessary for NGF-induced sympathetic nerve sprouting in the heart after MI. Transfection studies in neurons lacking STAT3 suggest two independent pools of STAT3, phosphorylated on either S727 or Y705, that regulate sympathetic regeneration via both transcriptional and non-transcriptional means. Additional data identify STAT3-microtubule interactions that may complement the well-characterized role of STAT3 stimulating regeneration associated genes. These data show that STAT3 is critical for sympathetic axon regeneration in vitro and in vivo, and identify a novel non-transcriptional mode of action.

Reliability and Validity of Shoulder and Handgrip Strength Testing.

Purpose: This study aimed to estimate the intra- and inter-rater reliability of the JTECH computerized, wireless apparatus and its validity (compared to established devices) for measurements of maximal shoulder isometric strength and handgrip strength among healthy adults with no shoulder pathology. Methods: Twenty healthy young adults had shoulder strength tested with JTECH and Micro-FET2 hand-held dynamometers, and the handgrip strength was tested using JTECH and Jamar handgrip dynamometers. Assessments were performed by the same rater to determine intra-rater reliability and convergent validity, at least two days apart, while on a third visit, the other rater performed measures to determine inter-rater reliability. Results: The JTECH computerized, wireless devices demonstrated good to excellent intra-rater reliability (ICCs (2,1) = 0.78-0.97) and inter-rater reliability for strength measures (ICCs (2,1) = 0.76-0.95). The JTECH computerized device compared to the Micro-FET2 hand-held dynamometer showed substantial concurrent validity for shoulder flexion (R2 = 0.87), extension (R2 = 0.87), abduction (R2 = 0.88), and adduction (R2 = 0.85). Substantial concurrent validity was shown for the JTECH computerized device and Jamar handgrip dynamometers (R2 = 0.92). Conclusions: The JTECH computerized, wireless devices demonstrated high intra- and inter-rater reliability and substantial concurrent validity for shoulder isometric strength and handgrip strength in healthy adults.

Objectif : évaluer la fiabilité intraévaluateur et interévaluateur et la validité (par rapport à des appareils éprouvés) du dispositif informatisé sans fil JTECH pour mesurer la force isométrique de l'épaule et la force de préhension maximales chez des adultes en santé sans pathologie de l'épaule. Méthodologie : vingt jeunes adultes en bonne santé ont fait vérifier la force de leur épaule à l'aide des dynamomètres manuels JTECH et Micro-FET2, et leur force de préhension à l'aide des dynamomètres à poignée JTECH et Jamar. Le même évaluateur a procédé aux évaluations pour déterminer la fiabilité intraévaluateur et la validité convergente, à au moins deux jours d'intervalle, tandis qu'à un troisième rendez-vous, l'autre évaluateur a effectué des mesures pour établir la fiabilité interévaluateur. Résultats : les dispositifs informatisés sans fil JTECH possèdent une fiabilité intraévaluateur (coefficient de corrélation intraclasse, ou CCI [2,1] = 0,78 à 0,97) et une fiabilité interévaluateur pour mesurer la force (CCI [2,1] = 0,76 à 0,95) bonnes à excellentes. Par rapport au dynamomètre manuel Micro-FET2, le dispositif informatisé sans fil JTECH a démontré une validité convergente importante à l'égard de la flexion (R2 = 0,87), de l'extension (R2 = 0,87), de l'abduction (R2 = 0,88) et de l'adduction (R2 = 0,85) de l'épaule. Le dispositif informatisé JTECH et le dynamomètre manuel Jamar ont également démontré une validité convergente importante (R2 = 0,92). Conclusions : les dispositifs informatisés sans fil JTECH ont démontré une fiabilité intraévaluateur et interévaluateur élevée et une validité convergente importante pour la force isométrique de l'épaule et la force de préhension chez des adultes en santé.

Cytokines inhibit norepinephrine transporter expression by decreasing Hand2.

Functional noradrenergic transmission requires the coordinate expression of enzymes involved in norepinephrine (NE) synthesis, as well as the norepinephrine transporter (NET) which removes NE from the synapse. Inflammatory cytokines acting through gp130 can suppress the noradrenergic phenotype in sympathetic neurons. This occurs in a subset of sympathetic neurons during development and also occurs in adult neurons after injury. For example, cytokines suppress noradrenergic function in sympathetic neurons after axotomy and during heart failure. The molecular basis for suppression of noradrenergic genes is not well understood, but previous studies implicated a reduction of Phox2a in cytokine suppression of dopamine beta hydroxylase. We used sympathetic neurons and neuroblastoma cells to investigate the role of Phox2a in cytokine suppression of NET transcription. Chromatin immunoprecipitation experiments revealed that Phox2a did not bind the NET promoter, and overexpression of Phox2a did not prevent cytokine suppression of NET transcription. Hand2 and Gata3 are transcription factors that induce noradrenergic genes during development and are present in mature sympathetic neurons. Both Hand2 and Gata3 were decreased by cytokines in sympathetic neurons and neuroblastoma cells. Overexpression of either Hand2 or Gata3 was sufficient to rescue NET transcription following suppression by cytokines. We examined expression of these genes following axotomy to determine if their expression was altered following nerve injury. NET and Hand2 mRNAs decreased significantly in sympathetic neurons 48 h after axotomy, but Gata3 mRNA was unchanged. These data suggest that cytokines can inhibit NET expression through downregulation of Hand2 or Gata3 in cultured sympathetic neurons, but axotomy in adult animals selectively suppresses Hand2 expression.

ERK5 induces ankrd1 for catecholamine biosynthesis and homeostasis in adrenal medullary cells.

Extracellular signal-regulated kinases (ERKs) play important roles in proliferation, differentiation and gene expression. In our previous study, we demonstrated that both ERK5 and ERK1/2 were responsible for neurite outgrowth and tyrosine hydroxylase (TH) expression in rat pheochromocytoma cells (PC12) (J Biol Chem 284, 23,564-23,573, 2009). However, the functional differences between ERK5 and ERK1/2 signaling in neural differentiation remain unclear. In the present study, we show that ERK5, but not ERK1/2 regulates TH levels in rat sympathetic neurons. Furthermore, microarray analysis performed in PC12 cells using ERK5 and ERK1/2-specific inhibitors, identified ankyrin repeat domain 1 (ankrd1) as an ERK5-dependent and ERK1/2-independent gene. Here, we report a novel role of the ERK5/ankrd1 signaling in regulating TH levels and catecholamine biosynthesis. Ankrd1 mRNA was induced by nerve growth factor in time- and concentration-dependent manners. TH levels were reduced by ankrd1 knockdown with no changes in the mRNA levels, suggesting that ankrd1 was involved in stabilization of TH protein. Interestingly, ubiquitination of TH was enhanced and catecholamine biosynthesis was reduced by ankrd1 knockdown. Finally, we examined the relationship of ERK5 to TH levels in human adrenal pheochromocytomas. Whereas TH levels were correlated with ERK5 levels in normal adrenal medullas, ERK5 was down-regulated and TH was up-regulated in pheochromocytomas, indicating that TH levels are regulated by alternative mechanisms in tumors. Taken together, ERK5 signaling is required for catecholamine biosynthesis during neural differentiation, in part to induce ankrd1, and to maintain appropriate TH levels. This pathway is disrupted in pathological conditions.

HIV type 1-specific IgE in serum of long-term surviving children inhibits HIV type 1 production in vitro.

We previously identified a group of long-term pediatric survivors who had acquired HIV-1 through maternal transmission; had not received antiretroviral therapy; are now >8 years old, in good health, and with no opportunistic infections; and have not failed to thrive, although they have greatly decreased numbers of blood CD4+ T cells (<500/mm(3)). All the children have elevated total serum IgE levels (210-2475 IU/ml) and make anti-HIV-1 IgE or IgE directed against non-HIV-1 specificities (radioimmunoassay, Western blot assay); they have no detectable antigenemia. We have now studied the ability of anti-HIV-1 IgE in serum obtained from these children to regulate (1) production of HIV-1 by interleukin 2/phytohemagglutinin (IL-2/PHA)-stimulated peripheral blood mononuclear cells (PBMCs) taken from HIV-1-seronegative donors and infected with a T cell-tropic clone of HIV-1, and (2) transmission of a primary HIV-1 strain from adult AIDS patients to uninfected IL-2/PHA-stimulated PBMCs (p24 core antigen production). High levels of HIV-1 production were observed when PBMCs were cultured for 5 days in the presence of HIV-1-seronegative donor serum that was either IgE positive or IgE negative (IgE, >100 or <100 IU/ml, respectively). HIV-1 production also was observed when PBMCs were cultured with HIV-1-infected donor serum that either contained IgE directed against non-HIV-1 specificities or was IgE negative; these levels were 40% less than those seen with sera from the HIV-1-seronegative donors. Far greater inhibition of virus production was observed if the serum in culture contained anti-HIV-1 IgE (>95%). Virus neutralization did not appear to account for the inhibition obtained with anti-HIV-1 IgE-containing serum because virus production was not suppressed in cultures to which serum was added immediately preinfection (<10%), but was strongly suppressed when serum was added 1.5 hr postinfection (>95%). The inhibition of virus production obtained with serum containing anti-HIV-1 IgE was reversed when (1) serum was depleted of IgE (immunoaffinity), but not when it was depleted of IgG (protein G-Sepharose) before inclusion in culture postinfection, (2) anti-IgE, but not anti-IgG, was included in culture, or (3) serum was heat treated before culture. The results indicate that serum from certain HIV-1-infected pediatric long-term survivors contains agents that inhibit HIV-1 production in vitro, and that these agents include anti-HIV-1 IgE. They suggest that a cytotoxic event, rather than virus neutralization, plays an important role in anti-HIV-1 IgE-mediated inhibition of virus production.

Leptin stimulates sympathetic axon outgrowth.

The neurohormone leptin regulates energy homeostasis. Circulating levels of leptin secreted by adipose tissue act on hypothalamic neurons in the brain leading to decreased appetite and increased energy expenditure. Although leptin signaling in the central nervous system (CNS) is fundamental to its ability to regulate the body's metabolic balance, leptin also has a variety of effects in many peripheral tissues including the heart, the liver, and the sympathetic nervous system. Leptin stimulation of the hypothalamus can stimulate glucose uptake via the sympathetic nervous system in heart, muscle, and brown adipose tissue. Leptin receptors (Ob-Rb) are also expressed by peripheral sympathetic neurons, but their functional role is not clear. In this study, we found that leptin stimulates axonal growth of both adult and neonatal sympathetic neurons in vitro. Leptin stimulates acute activation of the transcription factor STAT3 via phosphorylation of tyrosine 705. STAT3 phosphorylation is required for leptin-stimulated sympathetic axon outgrowth. Thus, circulating levels of leptin may enhance sympathetic nerve innervation of peripheral tissues.

Unusual Stüve-Wiedemann syndrome with complete maternal chromosome 5 isodisomy.

A woman was isozygous for a novel mutation in the leukemia inhibitory factor receptor gene (LIFR) (c.2170C>G; p.Pro724Ala) which disrupts LIFR downstream signaling and results in Stüve-Wiedemann syndrome (STWS). She inherited two identical chromosomes 5 from her mother, heterozygous for the LIFR mutation. The presentation was typical for STWS, except there was no long bone dysplasia. Prominent cold-induced sweating and heat intolerance lead to an initial diagnosis of cold-induced sweating syndrome, excluded by exome sequencing. Skin biopsies provide the first human evidence of failed postnatal cholinergic differentiation of sympathetic neurons innervating sweat glands in cold-induced sweating, and of a neuropathy.

Sympathetic denervation of peri-infarct myocardium requires the p75 neurotrophin receptor.

Development of cardiac sympathetic heterogeneity after myocardial infarction contributes to ventricular arrhythmias and sudden cardiac death. Regions of sympathetic hyperinnervation and denervation appear in the viable myocardium beyond the infarcted area. While elevated nerve growth factor (NGF) is implicated in sympathetic hyperinnervation, the mechanisms underlying denervation are unknown. Recent studies show that selective activation of the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons causes axon degeneration. We used mice that lack p75(NTR) to test the hypothesis that activation of p75(NTR) causes peri-infarct sympathetic denervation after cardiac ischemia-reperfusion. Wild type hearts exhibited sympathetic denervation adjacent to the infarct 24h and 3 days after ischemia-reperfusion, but no peri-infarct sympathetic denervation occurred in p75(NTR)-/- mice. Sympathetic hyperinnervation was found in the distal peri-infarct myocardium in both genotypes 3 days after MI, and hyperinnervation was increased in the p75(NTR)-/- mice. By 7 days after ischemia-reperfusion, cardiac sympathetic innervation density returned back to sham-operated levels in both genotypes, indicating that axonal pruning did not require p75(NTR). Prior studies revealed that proNGF is elevated in the damaged left ventricle after ischemia-reperfusion, as is mRNA encoding brain-derived neurotrophic factor (BDNF). ProNGF and BDNF preferentially bind p75(NTR) rather than TrkA on sympathetic neurons. Immunohistochemistry using Bdnf-HA mice confirmed the presence of BDNF or proBDNF in the infarct after ischemia-reperfusion. Thus, at least two p75(NTR) ligands are elevated in the left ventricle after ischemia-reperfusion where they may stimulate p75(NTR)-dependent denervation of peri-infarct myocardium. In contrast, NGF-induced sympathetic hyperinnervation in the distal peri-infarct ventricle is attenuated by p75(NTR).

Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells.

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.

Chronic morphine treatment alters endogenous opioid control of hippocampal mossy fiber synaptic transmission.

Synaptic adaptations are thought to be an important component of the consequences of drug abuse. One such adaptation is an up-regulation of adenylyl cyclase that has been shown to increase transmitter release at several inhibitory synapses. In this study the effects of chronic morphine treatment were studied on mossy fiber synapses in the guinea pig hippocampus using extracellular field potential recordings. This opioid-sensitive synapse was chosen because of the known role of the adenylyl cyclase cascade in the regulation of glutamate release. Long-term potentiation (LTP) at the mossy fiber synapse was enhanced after chronic morphine treatment. In control animals, opioid antagonists increased LTP but had no effect in morphine-treated guinea pigs. In contrast, the long-lasting depression of transmission induced by a mGluR agonist and CA1 LTP were not altered. Chronic morphine treatment neither caused tolerance to mu- and kappa-receptor-mediated inhibition at the mossy fiber synapse nor modified total hippocampal dynorphin levels. The results suggest that the phasic inhibition of glutamate transmission mediated by endogenous opioids is reduced after chronic exposure to morphine.

Characterization of rat prepro-orphanin FQ/nociceptin((154-181)): nociceptive processing in supraspinal sites.

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand for the orphan receptor-like/kappa(3)-like opioid receptor clone, produces a variety of behavioral responses, including those associated with pronociception and antinociception. The OFQ/N precursor rattus-proOFQ (rppOFQ/N) contains several paired basic amino acids, which raises the possibility that post-translational processing can be responsible for the production of a number of additional biologically active peptide fragments. One of these putative peptides, rppOFQ/N (rppOFQ/N(154-181)), was examined for antinociceptive and pronociceptive processes in four brain sites involved in pain inhibition: the ventrolateral periaqueductal gray (vlPAG), the amygdala, the locus coeruleus (LC), and the rostroventromedial medulla (RVM). Endogenous rppOFQ/N(154-181) was identified in each region. rppOFQ/N(154-181) produced a dose-dependent antinociception in all four sites using the tailflick assay. Injections into misplaced cannula sites failed to exert effects. Antinociception in the four sites differed in their response to the opioid antagonist naloxone. Naloxone pretreatment completely blocked rppOFQ/N(154-181)-induced antinociception in the vlPAG and the amygdala, but not in the LC or RVM. In contrast rppOFQ/N(154-181) was hyperalgesic in the LC and RVM, but not in the vlPAG or amygdala. rppOFQ/N(154-181) also was compared with either its N-terminal 17-amino acid peptide (rppOFQ/N(154-170), also known as OFQ2) or its 8-amino acid C-terminal fragment (rppOFQ/N(174-181)). Although both rppOFQ/N(154-181) and rppOFQ/N(154-170) produced antinociception, the latter was less effective because the C-terminal fragment was inactive. Thus, rppOFQ/N(154-181) has complex antinociceptive and pronociceptive actions within the brain, and the pharmacological specificity of its actions differs among supraspinal sites.