Neurturin Research Articles

PD36-05 PIOGLITAZONE ENHANCES SURVIVAL AND REGENERATION OF PELVIC GANGLIA NEURONS AFTER CAVERNOSAL NERVE INJURY

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2015PD36-05 PIOGLITAZONE ENHANCES SURVIVAL AND REGENERATION OF PELVIC GANGLIA NEURONS AFTER CAVERNOSAL NERVE INJURY Eric Katz, Daniel Heidenberg, Nora Haney, Taylor Peak, George Lasker, Margaret Knoedler, Daniel Rittenberg, Bashir Rezk, Ahmed Moustafa, Zakaria Abd Elmageed, Faysal Yafi, Suresh Sikka, Asim Abdel Mageed, and Wayne Hellstrom Eric KatzEric Katz More articles by this author , Daniel HeidenbergDaniel Heidenberg More articles by this author , Nora HaneyNora Haney More articles by this author , Taylor PeakTaylor Peak More articles by this author , George LaskerGeorge Lasker More articles by this author , Margaret KnoedlerMargaret Knoedler More articles by this author , Daniel RittenbergDaniel Rittenberg More articles by this author , Bashir RezkBashir Rezk More articles by this author , Ahmed MoustafaAhmed Moustafa More articles by this author , Zakaria Abd ElmageedZakaria Abd Elmageed More articles by this author , Faysal YafiFaysal Yafi More articles by this author , Suresh SikkaSuresh Sikka More articles by this author , Asim Abdel MageedAsim Abdel Mageed More articles by this author , and Wayne HellstromWayne Hellstrom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2254AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Pioglitazone has neuroprotective effects in models of sciatic nerve ischemia, optic nerve crush injury, and traumatic brain injury. Our group previously demonstrated that it restores erectile function in a model of cavernosal nerve (CN) crush injury. In this study, we investigated the mechanism of this physiological recovery with a molecular analysis of the rat pelvic ganglia, thereby evaluating its utility for prevention of post-prostatectomy neurogenic erectile dysfunction (ED). METHODS Sprague-Dawley rats (n=32) aged 12 weeks were divided into four groups (n=8 each). Groups A, B, and C underwent bilateral CN crush injury. Group D underwent sham operations. Pre-operative injection of Fluorogold (FG) fluorescent tracer into the cavernosal tissue was performed. Group A received pre and post-surgical treatment with pioglitazone (6.5 mg/kg); Group B only received post-surgical treatment. Pre-surgical treatment of pioglitazone was four days and post-surgical treatment was two weeks following crush injury. Pelvic ganglia were resected from all rats after two weeks and processed for reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB) for the expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), beta-III tubulin, neurturin (NTN), GDNF family receptor alpha-2 (GFRa2), and FG. RESULTS Pre-treatment with pioglitazone (Group A) induced significantly higher expression of nNOS, NTN, GFRa2, and FG in the pelvic ganglia compared to that in Group C rats. Post-surgical treatment (Group B) induced statistically insignificant improvements in nNOS and FG compared to no treatment (Group C). The sham group (Group D) showed moderate expression of these markers, but not to the same level of rats in Group A. CONCLUSIONS Pre-treatment with pioglitazone has neuroprotective and neuroregenerative effects on the pelvic ganglia; this may signify a novel therapeutic avenue for preventing neurogenic ED after radical prostatectomy. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e766 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric Katz More articles by this author Daniel Heidenberg More articles by this author Nora Haney More articles by this author Taylor Peak More articles by this author George Lasker More articles by this author Margaret Knoedler More articles by this author Daniel Rittenberg More articles by this author Bashir Rezk More articles by this author Ahmed Moustafa More articles by this author Zakaria Abd Elmageed More articles by this author Faysal Yafi More articles by this author Suresh Sikka More articles by this author Asim Abdel Mageed More articles by this author Wayne Hellstrom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Neurturin influences inflammatory responses and airway remodeling in different mouse asthma models.

Neurturin (NTN) was previously described for its neuronal activities, but recently, we have shown that this factor is also involved in asthma physiopathology. However, the underlying mechanisms of NTN are unclear. The aim of this study was to investigate NTN involvement in acute bronchial Th2 responses, to analyze its interaction with airway structural cells, and to study its implication in remodeling during acute and chronic bronchial inflammation in C57BL/6 mice. We analyzed the features of allergic airway inflammation in wild-type and NTN(-/-) mice after sensitization with two different allergens, OVA and house dust mite. We showed that NTN(-/-) dendritic cells and T cells had a stronger tendency to activate the Th2 pathway in vitro than similar wild-type cells. Furthermore, NTN(-/-) mice had significantly increased markers of airway remodeling like collagen deposition. NTN(-/-) lung tissues showed higher levels of neutrophils, cytokine-induced neutrophil chemoattractant, matrix metalloproteinase 9, TNF-α, and IL-6. Finally, NTN had the capacity to decrease IL-6 and TNF-α production by immune and epithelial cells, showing a direct anti-inflammatory activity on these cells. Our findings support the hypothesis that NTN could modulate the allergic inflammation in different mouse asthma models.

Enhanced delivery and bioactivity of the neurturin neurotrophic factor through focused ultrasound-mediated blood--brain barrier opening in vivo.

The blood-brain barrier (BBB) constitutes a major obstacle in brain drug delivery. Focused ultrasound (FUS) in conjunction with microbubbles has been shown to open the BBB noninvasively, locally, and transiently to allow large molecules diffusion. Neurturin (NTN), a member of the glial-derived neurotrophic factor (GDNF) family, has been demonstrated to have neuroprotective and regenerative effects on dopaminergic neurons in vivo using invasive drug delivery methods. The brain's ascending nigrostriatal pathway is severely damaged in Parkinson's disease (PD), and therefore the substantia nigra (SN) and striatal caudoputamen (CP) were selected as the target areas. The objective of the study was to investigate whether safe and efficient NTN delivery can be achieved through FUS-induced BBB opening via intravenous administration, and thus trigger the neuroregeneration cascade in the nigrostriatal pathway. After the optimization of FUS parameters and target locations in the murine brain, NTN bioavailability and downstream signaling were detected and characterized through immunostaining. FUS significantly enhanced the delivery of NTN compared with the direct injection technique, whereas triggering of the signaling cascade was detected downstream to the neuronal nuclei. These findings thus indicate the potential of the FUS method to mediate transport of proteins through the blood-brain barrier in a PD animal model.

Glial Cell Line-Derived Neurotrophic Factor Family Members Reduce Microglial Activation via Inhibiting p38MAPKs-Mediated Inflammatory Responses.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. However, little is known about direct influences of the GFL on microglia function, which are known to express part of the GDNF receptor system. Using RT-PCR and immunohistochemistrym we investigated the expression of the GDNF family receptor alpha 1 (GFR alpha) and the coreceptor transmembrane receptor tyrosine kinase (RET) in rat microglia in vitro as well as the effect of GFL on the expression of proinflammatory molecules in LPS activated microglia. We could show that GFL are able to regulate microglia functions and suggest that part of the well known neuroprotective action may be related to the suppression of microglial activation. We further elucidated the functional significance and pathophysiological implications of these findings and demonstrate that microglia are target cells of members of the GFL (GDNF and the structurally related neurotrophic factors neurturin (NRTN), artemin (ARTN), and persephin (PSPN)).

Effect of neurturin deficiency on cholinergic and catecholaminergic innervation of the murine eye

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123–4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4–6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 μm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

The neurotrophic factor neurturin contributes toward an aggressive cancer cell phenotype, neuropathic pain and neuronal plasticity in pancreatic cancer

Neurotrophic factors possess an emerging role in the pathophysiology of several gastrointestinal disorders, regulating innervation, pain sensation and disease-associated neuroplasticity. Here, we aimed at characterizing the role of the neurotrophic factor neurturin (NRTN) and its receptor glial-cell-line-derived neurotrophic factor receptor alpha-2 (GFRα-2) in pancreatic cancer (PCa) and pancreatic neuropathy. For this purpose, NRTN and GFRα-2 were studied in normal human pancreas and PCa tissues via immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, immunoblotting and correlated to abdominal pain. The impact of NRTN/GFRα-2 on PCa cell (PCC) biology was investigated via exposure to hypoxia, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide viability and matrigel invasion assays in native and specific small interfering RNA-silenced PCCs. To assess the influence of NRTN on pancreatic neuroplasticity and neural invasion (NI), its impact was explored via an in vitro 'neuroplasticity assay' and a 3D neural migration assay. NRTN and GFRα-2 demonstrated a site-specific upregulation in PCa, predominantly in nerves, PCCs and extracellular matrix. Patients with severe pain demonstrated higher intraneural GFRα-2 immunoreactivity than patients with no pain. PCa tissue and PCCs contained increased amounts of NRTN, which was suppressed under hypoxia. NRTN promoted PCC invasiveness, and silencing of NRTN limited both PCC proliferation and invasion. Depletion of NRTN from PCa tissue extracts and PCC supernatants decreased axonal sprouting in neuronal cultures but did not influence glial density. Silencing of NRTN in PCCs boosted NI. We conclude that increased NRTN/GFRα-2 in PCa seems to promote an aggressive PCC phenotype and neuroplasticity in PCa. Accelerated NI following NRTN suppression constitutes a novel explanation for the attraction of PCC to nerves in the hypoxic PCa tumor microenvironment. PCa is characterized by intrapancreatic neuroplasticity and NI. Here, we show that PCC produce the neurotrophic factor NRTN, which reinforces their biological properties, triggers neuroplastic alterations, NI and influences pain sensation via the GFRα-2 receptor.

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.

Pharmacotherapy of Parkinson’s disease: Progress or regress?

Parkinson's disease (PD) is a chronic, progressive disease of the central nervous system (CNS), characterized by a slow loss of dopaminergic neurons in the substantia nigra, leading to significant decrease in dopamine (DA) levels in the striatum. Currently used drugs, such as levodopa (L-DOPA), amantadine, dopamine agonists (D) or anticholinergic drugs, are not effective enough, and do not eliminate the causes of disease. Many research centers are conducting research on new forms of currently used drugs (e.g. Duodopa, XP21279, IPX066), new drugs of already known groups (e.g. safinamide), medicines that suppress side effects of L-DOPA (e.g. AFQ056, fipamezole), and, finally, compounds with a novel mechanism of action (e.g. PMY50028, A2A receptor antagonists). A lot of scientific reports indicate an important role of A2A receptors in the regulation of the central movement system, so a new group of compounds - selective antagonists of A2A receptors (e.g. istradefylline, preladenant, SYN115) - has been developed and their potential use in PD has been examined. Clinical studies of A2A receptor antagonists have shown that this group of compounds can shorten off periods and at the same time they do not worsen dyskinesias in patients with PD. Moreover, there is ongoing research on new forms of treatment, such as gene therapy. Attempts to apply the viral vector AAV-2, which will be able to infect neurons with a variety of genes, including the gene of glutamate decarboxylase (GAD), neurturin (NTN), or aromatic L-amino acid decarboxylase, are currently being carried out. The results of phase I and II clinical studies showed some efficacy of this form of treatment, but the method requires further studies. An analysis of potential future therapies of Parkinson's disease suggests that some progress in this field has been made.

GDNF family ligand dependent STAT3 activation is mediated by specific alternatively spliced isoforms of GFRα2 and RET

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinson's disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine727 but not tyrosine705 phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

Evidence for increased tissue androgen sensitivity in neurturin knockout mice

Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family and signals through GDNF family receptor alpha 2 (GFRα2). We hypothesised that epithelial atrophy reported in the reproductive organs of Ntn (Nrtn)- and Gfrα2 (Gfra2)-deficient mice could be due to NTN affecting the hormonal environment. To investigate this, we compared the reproductive organs of Ntn- and Gfrα2-deficient male mice in parallel with an analysis of their circulating reproductive hormone levels. There were no significant structural changes within the organs of the knockout mice; however, serum and intratesticular testosterone and serum LH levels were very low. To reconcile these observations, we tested androgen sensitivity by creating a dihydrotestosterone (DHT) clamp (castration plus DHT implant) to create fixed circulating levels of androgens, allowing the evaluation of androgen-sensitive endpoints. At the same serum DHT levels, serum LH levels were lower and prostate and seminal vesicle weights were higher in the Ntn knockout (NTNKO) mice than in the wild-type mice, suggesting an increased response to androgens in the accessory glands and hypothalamus and pituitary of the NTNKO mice. Testicular and pituitary responsiveness was unaffected in the NTNKO males, as determined by the response to the human chorionic gonadotrophin or GNRH analogue, leuprolide, respectively. In conclusion, our results suggest that NTN inactivation enhances androgen sensitivity in reproductive and neuroendocrine tissues, revealing a novel mechanism to influence reproductive function and the activity of other androgen-dependent tissues.

Enhanced neurotrophic distribution, cell signaling and neuroprotection following substantia nigral versus striatal delivery of AAV2-NRTN (CERE-120)

This paper reassesses the currently accepted viewpoint that targeting the terminal fields (i.e. striatum) of degenerating nigrostriatal dopamine neurons with neurotrophic factors in Parkinson's disease (PD) is sufficient for achieving an optimal neurotrophic response. Recent insight indicating that PD is an axonopathy characterized by axonal transport deficits prompted this effort. We tested whether a significantly greater neurotrophic response might be induced in SN neurons when the neurotrophic factor neurturin (NRTN) is also targeted to the substantia nigra (SN), compared to the more conventional, striatum-only target. While recognizing the importance of maintaining the integrity of nigrostriatal fibers and terminals (especially for achieving optimal function), we refocused attention to the fate of SN neurons. Under conditions of axonal degeneration and neuronal transport deficits, this component of the nigrostriatal system is most vulnerable to the lack of neurotrophic exposure following striatal-only delivery. Given the location of repair genes induced by neurotrophic factors, achieving adequate neurotrophic exposure to the SN neurons is essential for an optimal neurotrophic response, while the survival of these neurons is essential to the very survival of the fibers. Two separate studies were performed using the 6-OHDA model of nigrostriatal degeneration, in conjunction with delivery of the viral vector AAV2-NRTN (CERE-120) to continuously express NRTN to either striatum or nigra alone or combined striatal/nigral exposure, including conditions of ongoing axonopathy. These studies provide additional insight for reinterpreting past animal neurotrophic/6-OHDA studies conducted under conditions where axon transport deficiencies were generally not accounted for, which suggested that targeting the striatum was both necessary and sufficient. The current data demonstrate that delivering NRTN directly to the SN produces 1) expanded NRTN distribution within the terminal field and cell bodies of targeted nigrostriatal neurons, 2) enhanced intracellular neurotrophic factor signaling in the nigrostriatal neurons, and 3) produced greater numbers of surviving dopamine neurons against 6-OHDA-induced toxicity, particularly under the conditions of active axonopathy. Thus, these data provide empirical support that targeting the SN with neurotrophic factors (in addition to striatum) may help enhance the neurotrophic response in midbrain neurons, particularly under conditions of active neurodegeneration which occurs in PD patients.

Recovery of behavioral symptoms in hemi-parkinsonian rhesus monkeys through combined gene and stem cell therapy

The use of adipose mesenchymal stromal cells (ASCs) in cellular and genic therapy has attracted considerable attention as a possible treatment for neurodegenerative disorders, including Parkinson disease. However, the effects of gene therapy combined with intracerebral cell transplantation have not been well defined. Recent studies have demonstrated the respective roles of LIM homeobox transcription factor 1, alpha (LMX1A) and Neurturin (NTN) in the commitment of embryonic stem cells (ESCs) to a midbrain dopaminergic neuronal fate and the commitment of mesenchymal stromal cells to cells supporting the nutrition and protection of neurons. We investigated a novel in vitro neuronal differentiation strategy with the use of LMX1A and Neurturin. We were able to elicit a neural phenotype regarding cell morphology, specific gene/protein expression and physiological function. Neuronal-primed ASCs derived from rhesus monkey (rASCs) combined with adenovirus containing NTN and tyrosine hydroxylase (TH) (Ad-NTN-TH) were implanted into the striatum and substantia nigra of methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored with the use of behavioral tests and health measures until the fourth month after implantation. The differentiated cells transcribed and expressed a variety of dopaminergic neuron-specific genes involved in the SHH/LMX1A pathway. Single-photon emission computed tomography analysis and postmortem analysis revealed that the grafting of rASCs combined with Ad-NTN-TH had neuroprotective effects compared with Ad-NTN-TH or rASCs alone. Behavioral measures demonstrated autograft survival and symptom amelioration. These findings may lead to cellular sources for autologous transplantation of Parkinson disease. Combined transplantation of Ad-NTN-TH and induced rASCs expressing LMX1A and NTN may be a better therapy candidate for the treatment of Parkinson disease.

Neurturin Overexpression in Skin Enhances Expression of TRPM8 in Cutaneous Sensory Neurons and Leads to Behavioral Sensitivity to Cool and Menthol

Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret-GFRα2 coreceptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B(4) (IB(4)). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide-positive, free nerve endings in footpad epidermis. GFRα2-immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X(3) (found in IB(4)-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hindpaw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical, and mechanical stimuli.

Bone marrow-derived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson's disease

Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson's disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.

Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRα2 axis

The glial cell line-derived neurotrophic factor (GDNF) family member neurturin (NRTN) and its receptor GFRα2 play a deciding role in the normal development of pancreatic parasympathetic innervation. In this study, we aimed at investigating the role of NRTN/GFRα2 axis in pancreatic neuropathy in human chronic pancreatitis (CP). Expression of NRTN/GFRα2 was compared between normal human pancreas (NP) and CP tissues via immunohistochemistry, immunoblotting, and quantitative RT-PCR and correlated to abdominal pain sensation. To elucidate the impact of NRTN in pancreatic neuroplasticity, neuronal phenotype and glial density were quantified via an in vitro neuroplasticity assay in dissociated newborn rat dorsal root ganglia (DRG) cultured 1) in CP tissue extracts depleted from NRTN, 2) in NP, 3) in untreated CP tissue extracts, and 4) CP extracts in which nerve growth factor, glial cell derived-neurotrophic factor, or TGF-β(1) was depleted. NRTN and GFRα2 were highly upregulated in CP, especially in intrapancreatic nerves and the extracellular matrix. CP tissue demonstrated increased amounts of mature multimeric NRTN and elevated levels of GFRα2. The noticeable neurotrophic effect of CP tissue extracts on DRG neurons was diminished upon blockade of NRTN from these extracts. However, blockade of NRTN from CP extracts did not influence the density of DRG glia cells. In conclusion, the NRTN/GFRα2 axis is activated during the course of CP and represents a major key player in the reactive neural alterations in CP. This is the first study to provide functional evidence for the contribution of neurotrophic factors to neuroplasticity in CP.

The delivery of tyrosine hydroxylase accelerates the neurorestoration of Macaca Rhesus model of Parkinson's disease provided by Neurturin

Neurturin (NTN) is a desired candidate therapeutic gene for PD treatment, however, only neuroprotective effect may not work for PD clinical remission due to nearly 50% of the dopaminergic neurons have died way when symptoms appear. In this study, we constructed a bicistronic adenovirus expressing both Neurturin and tyrosine hydroxylase (TH). We hypothesized that the expression of NTN could provide neuroprotection to dopaminergic neurons and stop progressive neurodegeneration, while TH enhanced the synthesis of dopamine and accelerated the recovery of Parkinsonism. The chimeric adenovirus have been prepared and assayed in vitro and delivered into the target nucleuses of PD Macaca Rhesus models by MRI based stereotaxic injection. The observation assessments and physiological results indicated that compared to the group treated with NTN only, instant behavior recovery was seen after bicistronic adenovirus infusion. Although both groups displayed neuroprotection of dopaminergic neurons finally, the addition of TH genuinely accelerated animal behavior recovery, which showed great potential for clinical application.

Human-protein-derived peptides for intracellular delivery of biomolecules

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.

Phenotypic Switching of Nonpeptidergic Cutaneous Sensory Neurons following Peripheral Nerve Injury

In adult mammals, the phenotype of half of all pain-sensing (nociceptive) sensory neurons is tonically modulated by growth factors in the glial cell line-derived neurotrophic factor (GDNF) family that includes GDNF, artemin (ARTN) and neurturin (NRTN). Each family member binds a distinct GFRα family co-receptor, such that GDNF, NRTN and ARTN bind GFRα1, -α2, and -α3, respectively. Previous studies revealed transcriptional regulation of all three receptors in following axotomy, possibly in response to changes in growth factor availability. Here, we examined changes in the expression of GFRα1-3 in response to injury in vivo and in vitro. We found that after dissociation of adult sensory ganglia, up to 27% of neurons die within 4 days (d) in culture and this can be prevented by nerve growth factor (NGF), GDNF and ARTN, but not NRTN. Moreover, up-regulation of ATF3 (a marker of neuronal injury) in vitro could be prevented by NGF and ARTN, but not by GDNF or NRTN. The lack of NRTN efficacy was correlated with rapid and near-complete loss of GFRα2 immunoreactivity. By retrogradely-labeling cutaneous afferents in vivo prior to nerve cut, we demonstrated that GFRα2-positive neurons switch phenotype following injury and begin to express GFRα3 as well as the capsaicin receptor, transient receptor potential vanilloid 1(TRPV1), an important transducer of noxious stimuli. This switch was correlated with down-regulation of Runt-related transcription factor 1 (Runx1), a transcription factor that controls expression of GFRα2 and TRPV1 during development. These studies show that NRTN-responsive neurons are unique with respect to their plasticity and response to injury, and suggest that Runx1 plays an ongoing modulatory role in the adult.

Gene transfer provides a practical means for safe, long-term, targeted delivery of biologically active neurotrophic factor proteins for neurodegenerative diseases.

Efforts to develop neurotrophic factors to restore function and protect dying neurons in chronic neurodegenerative diseases like Alzheimer's (AD) and Parkinson's (PD) have been attempted for decades. Despite abundant data establishing nonclinical proof-of-concept, significant delivery issues have precluded the successful translation of this concept to the clinic. The development of AAV2 viral vectors to deliver therapeutic genes has emerged as a safe and effective means to achieve sustained, long-term, targeted, bioactive protein expression. Thus, it potentially offers a practical means to solve those long-standing delivery/translational issues associated with neurotrophic factors. Data are presented for two AAV2 viral vector constructs expressing one of two different neurotrophic factors: nerve growth factor (NGF) and neurturin (NRTN). One (AAV2-NGF; aka CERE-110) is being developed as a treatment to improve the function and delay further degeneration of cholinergic neurons in the nucleus basalis of Meynert, the degeneration of which has been linked to cognitive deficits in AD. The other (AAV2-NRTN; aka CERE-120) is similarly being developed to treat the degenerating nigrostriatal dopamine neurons and major motor deficits in PD. The data presented here demonstrate: (1) 2-year, targeted, bioactive-protein in monkeys, (2) persistent, bioactive-protein throughout the life-span of the rat, and (3) accurately targeted bioactive-protein in aged rats, with (4) no safety issues or antibodies to the protein detected. They also provide empirical guidance to establish parameters for human dosing and collectively support the idea that gene transfer may overcome key delivery obstacles that have precluded successful translation of neurotrophic factors to the clinic. More specifically, they also enabled the AAV-NGF and AAV-NRTN programs to advance into ongoing multi-center, double-blind clinical trials in AD and PD patients.

Cyclic AMP signalling through PKA but not Epac is essential for neurturin-induced biphasic ERK1/2 activation and neurite outgrowths through GFRα2 isoforms

Cyclic AMP (cAMP) and neurotrophic factors are known to interact closely to promote neurite outgrowth and neuronal regeneration. Glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NTN) transduce signal through a multi-component receptor complex consisting of GDNF family receptor alpha 2 (GFRα2) and Ret receptor tyrosine kinase. Neurons from GFRα2-deficient mice do not promote axonal initiation when stimulated by NTN, consistent with the role of GFRα2 in neuronal outgrowth. Multiple alternatively spliced isoforms of GFRα2 are known to be expressed in the nervous system. GFRα2a and GFRα2c but not GFRα2b promoted neurite outgrowth. It is currently unknown if cAMP signalling is differentially regulated by these isoforms. In this study, NTN activation of GFRα2a and GFRα2c but not GFRα2b induced biphasic ERK1/2 activation and phosphorylation of the major cAMP target CREB. Interestingly, inhibition of cAMP signalling significantly impaired GFRα2a and GFRα2c-mediated neurite outgrowth while cAMP agonists cooperated with GFRα2b to induce neurite outgrowth. Importantly, the specific cAMP effector PKA but not Epac was essential for NTN-induced neurite outgrowth, through transcription and translation-dependent activation of late phase ERK1/2. Taken together, these results not only demonstrated the essential role of cAMP-PKA signalling in NTN-induced biphasic ERK1/2 activation and neurite outgrowth, but also suggested cAMP-PKA signalling as a hitherto unrecognized underlying mechanism contributing to the differential neuritogenic activities of GFRα2 isoforms.