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Researchers Report Neublastin Virtually Restores Complete Long-Term Sensory Motor Fun

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  • Researchers Report Neublastin Virtually Restores Complete Long-Term Sensory Motor Fun

    Researchers Report Neublastin Virtually Restores Complete Long-Term Sensory Motor Function in Preclinical Studies

    Findings by Biogen Idec, University of Arizona and Tufts University Reported in Nature Neuroscience

    CAMBRIDGE, Mass.--(BUSINESS WIRE)--Biogen Idec (Nasdaq: BIIB), in collaboration with scientists at the University of Arizona and Tufts University reported in the April issue of the journal Nature Neuroscience that in preclinical studies, injections of the protein neublastin promoted the regeneration of damaged sensory nerve cells and produced virtually complete, long-term restoration of sensory and motor function. These studies suggest neublastin has potential for further development as a treatment for traumatic nerve injury.
    Neublastin, also known as artemin, belongs to a family of proteins, called glial-derived neurotrophic factors (GDNF), which promote nerve cell survival. The protein is unique because it acts selectively on sensory neurons. In previous preclinical studies, neublastin reversed a number of features of chronic pain associated with peripheral nerve injury.
    Specifically in the studies, six neublastin injections were administered over 11 days following injury to the dorsal root, a bundle of peripheral nerve fibers adjacent to the spinal cord that transmit sensory information to the central nervous system. The injections promoted nerve growth into the spinal cord and restored the ability to respond normally to a variety of sensory stimuli and perform complex motor activities such as grasping an object on contact. The functional recovery occurred even after a two-day delay in administering neublastin and lasted for more than six months.
    “Sensory nerves entering the spinal cord have minimal capacity to regenerate and severe injury often results in permanent

    http://www.businesswire.com/portal/s...86&newsLang=en
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  • #2
    great news max.....a cure is all we need.

    Comment


    • #3
      Key word "peripheral nerve injury" we all have Central nerve injury damage, big difference.
      "Life is about how you
      respond to not only the
      challenges you're dealt but
      the challenges you seek...If
      you have no goals, no
      mountains to climb, your
      soul dies".~Liz Fordred

      Comment


      • #4
        Originally posted by Curt Leatherbee
        Key word "peripheral nerve injury" we all have Central nerve injury damage, big difference.
        ouch curt ...you are right.sci is dffr.

        Comment


        • #5
          Originally posted by Curt Leatherbee
          Key word "peripheral nerve injury" we all have Central nerve injury damage, big difference.
          Yeah. I'm not sure what to make of this.

          I thought the peripheral nervous system was already capable of regeneration.

          Hopefully Wise will chime in.

          Comment


          • #6
            Biogen Idec - Neublastin

            CAMBRIDGE, Mass.--(BUSINESS WIRE)--Biogen Idec (Nasdaq: BIIB), in collaboration with scientists at the University of Arizona and Tufts University reported in the April issue of the journal Nature Neuroscience that in preclinical studies, injections of the protein neublastin promoted the regeneration of damaged sensory nerve cells and produced virtually complete, long-term restoration of sensory and motor function. These studies suggest neublastin has potential for further development as a treatment for traumatic nerve injury.

            Neublastin, also known as artemin, belongs to a family of proteins, called glial-derived neurotrophic factors (GDNF), which promote nerve cell survival. The protein is unique because it acts selectively on sensory neurons. In previous preclinical studies, neublastin reversed a number of features of chronic pain associated with peripheral nerve injury.

            Specifically in the studies, six neublastin injections were administered over 11 days following injury to the dorsal root, a bundle of peripheral nerve fibers adjacent to the spinal cord that transmit sensory information to the central nervous system. The injections promoted nerve growth into the spinal cord and restored the ability to respond normally to a variety of sensory stimuli and perform complex motor activities such as grasping an object on contact. The functional recovery occurred even after a two-day delay in administering neublastin and lasted for more than six months.

            http://www.businesswire.com/portal/s...86&newsLang=en

            Comment


            • #7
              Dont get to excited everyone, dont mean to be the bearer of bad news but these are peripheral nerves they are talking about not central nerves like the ones SCI people injure, although some SCI's do involve peripheral nerve damage in addition to the central nerve damage.
              "Life is about how you
              respond to not only the
              challenges you're dealt but
              the challenges you seek...If
              you have no goals, no
              mountains to climb, your
              soul dies".~Liz Fordred

              Comment


              • #8
                Roger,
                Thanks for the post!

                Christopher

                Comment


                • #9
                  Researchers Report Neublastin Virtually Restores Complete Long Term Sensory Motor Fun

                  Researchers Report Neublastin Virtually Restores Complete Long Term Sensory Motor Function In Preclinical Studies


                  Biogen Idec (Nasdaq: BIIB), in collaboration with scientists at the University of Arizona and Tufts University reported in the April issue of the journal Nature Neuroscience that in preclinical studies, injections of the protein neublastin promoted the regeneration of damaged sensory nerve cells and produced virtually complete, long-term restoration of sensory and motor function. These studies suggest neublastin has potential for further development as a treatment for traumatic nerve injury.

                  Neublastin, also known as artemin, belongs to a family of proteins, called glial-derived neurotrophic factors (GDNF), which promote nerve cell survival. The protein is unique because it acts selectively on sensory neurons. In previous preclinical studies, neublastin reversed a number of features of chronic pain associated with peripheral nerve injury.

                  Specifically in the studies, six neublastin injections were administered over 11 days following injury to the dorsal root, a bundle of peripheral nerve fibers adjacent to the spinal cord that transmit sensory information to the central nervous system. The injections promoted nerve growth into the spinal cord and restored the ability to respond normally to a variety of sensory stimuli and perform complex motor activities such as grasping an object on contact. The functional recovery occurred even after a two-day delay in administering neublastin and lasted for more than six months.

                  more....

                  http://www.medicalnewstoday.com/articles/102947.php

                  Comment


                  • #10
                    Im not an expert and would like to hear Dr Young's take on it, but conus and cauda equina injuries could be helped by this type of treatment, if the nerve roots have been injured. Am I correct?

                    Comment


                    • #11
                      Originally posted by Curt Leatherbee
                      Dont get to excited everyone, dont mean to be the bearer of bad news but these are peripheral nerves they are talking about not central nerves like the ones SCI people injure, although some SCI's do involve peripheral nerve damage in addition to the central nerve damage.
                      Does anybody honestly get excited?

                      f
                      ight

                      Comment


                      • #12
                        Originally posted by Curt
                        Dont get to excited everyone, dont mean to be the bearer of bad news but these are peripheral nerves they are talking about not central nerves like the ones SCI people injure, although some SCI's do involve peripheral nerve damage in addition to the central nerve damage.
                        Curt's right on the money. Neublastin cannot overome an inhibitory lesion.

                        Comment


                        • #13
                          Originally posted by agnes
                          Im not an expert and would like to hear Dr Young's take on it, but conus and cauda equina injuries could be helped by this type of treatment, if the nerve roots have been injured. Am I correct?
                          Well I agree it looks like Neuroblastin is the perfect target for cauda equina injuries.. I'm optimistic because it looks like for chronicals and I understand that sensory nerves roots are sprouting back from the PNS to the CNS:
                          The injections promoted nerve growth into the spinal cord and restored the ability to respond normally to a variety of sensory stimuli and perform complex motor activities such as grasping an object on contact.
                          Besides, the last words are strange; "lasted for more than six months". Does that mean that sensory recoveries are going away after 6 months?.. What do you think?.. :-)

                          Comment


                          • #14
                            Basically, as I understand it, neublastin is a member of the GDNF family of growth factors that have already by reported by a number of different groups to be useful for regenerating the spinal cord. There is not much published under the name neublastin (ref 1-3). But there is much more published under an older name Artemin. The specific study referred to in this thread is in reference 4, I believe.

                            Wise.
                            1. Bonde C, Kristensen BW, Blaabjerg M, Johansen TE, Zimmer J and Meyer M (2000). GDNF and neublastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures. Neuroreport. 11: 4069-73. Anatomy and Neurobiology, SDU-Odense University, Ballerup, Denmark. The potential neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) and neublastin (NBN) against NMDA-induced excitotoxicity were examined in hippocampal brain slice cultures. Recombinant human GDNF (25-100 ng/ ml) or NBN, in medium conditioned by growth of transfected, NBN-producing HiB5 cells, were added to slice cultures I h before exposure to 10 microM NMDA for 48h. Neuronal cell death was monitored, before and during the NMDA exposure, by densitometric measurements of propidium iodide (PI) uptake and loss of Nissl staining. Both the addition of rhGDNF and NBN-containing medium significantly reduced the NMDA-induced PI uptake in the CA1 (p < 0.01), suggesting neuroprotective effects of these factors, beyond their well-known trophic effects on dopaminergic neurons.
                            2. Baudet C, Mikaels A, Westphal H, Johansen J, Johansen TE and Ernfors P (2000). Positive and negative interactions of GDNF, NTN and ART in developing sensory neuron subpopulations, and their collaboration with neurotrophins. Development. 127: 4335-44. Laboratory of Molecular Neurobiology, Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor beta family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that coexpress GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.
                            3. Rosenblad C, Gronborg M, Hansen C, Blom N, Meyer M, Johansen J, Dago L, Kirik D, Patel UA, Lundberg C, Trono D, Bjorklund A and Johansen TE (2000). In vivo protection of nigral dopamine neurons by lentiviral gene transfer of the novel GDNF-family member neublastin/artemin. Mol Cell Neurosci. 15: 199-214. Wallenberg Neuroscience Center, Lund University, Solvegatan 17, Lund, S-223 62, Sweden. carl.rosenblad@mphy.lu.se. The glial cell line-derived neurotrophic factor (GDNF)-family of neurotrophic factors consisted until recently of three members, GDNF, neurturin, and persephin. We describe here the cloning of a new GDNF-family member, neublastin (NBN), identical to artemin (ART), recently published (Baloh et al., 1998). Addition of NBN/ART to cultures of fetal mesencephalic dopamine (DA) neurons increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons by approximately 70%, similar to the maximal effect obtained with GDNF. To investigate the neuroprotective effects in vivo, lentiviral vectors carrying the cDNA for NBN/ART or GDNF were injected into the striatum and ventral midbrain. Three weeks after an intrastriatal 6-hydroxydopamine lesion only about 20% of the nigral DA neurons were left in the control group, while 80-90% of the DA neurons remained in the NBN/ART and GDNF treatment groups, and the striatal TH-immunoreactive innervation was partly spared. We conclude that NBN/ART, similarly to GDNF, is a potent neuroprotective factor for the nigrostriatal DA neurons in vivo.
                            4. Wang R, King T, Ossipov MH, Rossomando AJ, Vanderah TW, Harvey P, Cariani P, Frank E, Sah DW and Porreca F (2008). Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury. Nat Neurosci. 11: 488-96. Department of Pharmacology, University of Arizona, College of Medicine, 1501 N Campbell Avenue, Tucson, Arizona 85724-5050, USA. Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.
                            5. Otsuki K, Uchida S, Watanuki T, Wakabayashi Y, Fujimoto M, Matsubara T, Funato H and Watanabe Y (2008). Altered expression of neurotrophic factors in patients with major depression. J Psychiatr Res. Division of Neuropsychiatry, Department of Neuroscience, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan. There is an abundance of evidence suggesting the involvement of altered levels of expression of neurotrophic factors in the pathophysiology of neuropsychiatric disorders. Although postmortem brain studies have indicated the alterations in the expression levels of neurotrophic factors in mood disorder patients, it is unclear whether these changes are state- or trait-dependent. In this study, we examined the expression levels of the members of the glial cell line-derived neurotrophic factor (GDNF) family (GDNF, artemin (ARTN), neurturin, and persephin), brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3), and neurotrophin-4 mRNAs by using quantitative real-time PCR method in peripheral blood cells of patients with major depressive and bipolar disorders in both a current depressive and a remissive states. Reduced expression levels of GDNF, ARTN, and NT-3 mRNAs were found in patients with major depressive disorder in a current depressive state, but not in a remissive state. Altered expressions of these mRNAs were not found in patients with bipolar disorder. Our results suggest that the changes in the expression levels of GDNF, ARTN, and NT-3 mRNAs might be state-dependent and associated with the pathophysiology of major depression.
                            6. Murata T, Tsuboi M, Koide N, Hikita K, Kohno S and Kaneda N (2008). Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen. J Neurosci Res. Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya, Japan. To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation. (c) 2008 Wiley-Liss, Inc.
                            7. Forrest SL and Keast JR (2008). Expression of receptors for glial cell line-derived neurotrophic factor family ligands in sacral spinal cord reveals separate targets of pelvic afferent fibers. J Comp Neurol. 506: 989-1002. Pain Management Research Institute, Kolling Institute, University of Sydney at Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. Nerve growth factor has been proposed to mediate many structural and chemical changes in bladder sensory neurons after injury or inflammation. We have examined the expression of receptors for the glial cell line-derived neurotrophic factor (GDNF) family within sensory terminals located in the sacral spinal cord and in bladder-projecting sacral dorsal root ganglion neurons of adult female Sprague-Dawley rats. Nerve fibers immunolabelled for GFRalpha1 (GDNF receptor), GFRalpha2 (neurturin receptor), or GFRalpha3 (artemin receptor) showed distinct distribution patterns in the spinal cord, suggesting separate populations of sensory fibers with different functions: GFRalpha1-labeled fibers were in outer lamina II and the lateral-collateral pathway and associated with autonomic interneurons and preganglionic neurons; GFRalpha2-labeled fibers were only in inner lamina II; GFRalpha3-labeled fibers were in lamina I, the lateral-collateral pathway, and areas surrounding dorsal groups of preganglionic neurons and associated interneurons. Immunofluorescence studies of retrogradely labelled bladder-projecting neurons in sacral dorsal root ganglia showed that approximately 25% expressed GFRalpha1 or GFRalpha3 immunoreactivity, the preferred receptors for GDNF and artemin, respectively. After cyclophosphamide-induced bladder inflammation, fluorescence intensity of GFRalpha1-positive fibers increased within the dorsal horn, but there was no change in the GFRalpha2- or GFRalpha3-positive fibers. These studies have shown that GDNF and artemin may target bladder sensory neurons and potentially mediate plasticity of sacral visceral afferent neurons following inflammation. Our results have also revealed three distinct subpopulations of sensory fibers within the sacral spinal cord, which have not been identified previously using other markers.
                            8. Peterziel H, Paech T, Strelau J, Unsicker K and Krieglstein K (2007). Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors. J Neurochem. 103: 2491-504. Department of Neuroanatomy, IZN, University of Heidelberg, Heidelberg, Germany. heike.peterziel@urz.uni-heidelberg.de. Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor beta (TGFbeta). We have provided evidence that TGFbeta regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRalpha1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFbeta, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFbeta. Additional treatment with TGFbeta does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRalpha2 and GFRalpha1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRalpha1, TGFbeta is not required for recruitment of GFRalpha2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFbeta. Disruption of TGFbeta signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRalpha1 crystallization and ontogenetic development of the CG that may explain the differences in TGFbeta-dependence of GDNF and NRTN.
                            9. Jeong DG, Park WK and Park S (2008). Artemin activates axonal growth via SFK and ERK-dependent signalling pathways in mature dorsal root ganglia neurons. Cell Biochem Funct. 26: 210-20. Department of Anesthesiology and Pain Medicine, Anesthesia and Pain Research Institute, BK21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea. 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.
                            10. Damon DH, Teriele JA and Marko SB (2007). Vascular-derived artemin: a determinant of vascular sympathetic innervation? Am J Physiol Heart Circ Physiol. 293: H266-73. Department of Pharmacology, University of Vermont, 89 Beaumont Avenue, Burlington, VT 05405, USA. Deborah.Damon@uvm.edu. Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor alpha3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 +/- 11.9 mm, and that in the absence of FA was 26.3 +/- 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.
                            11. Quartu M, Serra MP, Boi M, Sestu N, Lai ML and Del Fiacco M (2007). Tissue distribution of neurturin, persephin and artemin in the human brainstem at fetal, neonatal and adult age. Brain Res. 1143: 102-15. Department of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy. The occurrence of the glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) was examined by immunohistochemistry in the normal human brainstem at pre-, perinatal and adult age. Immunolabelled neurons were unevenly distributed and each trophin had a consistent distribution pattern. As a rule, the NTN antiserum produced the most abundant and diffuse tissue labelling, whereas the lowest density of positive elements was observed after ART immunostaining. Labelling for NTN, PSP, and ART occurred at all examined ages. For each trophin, neuronal perikarya were observed within sensory and motor nuclei of cranial nerves, dorsal column nuclei, olivary nuclear complex, reticular formation, pontine nuclei, locus caeruleus, raphe nuclei, substantia nigra, and quadrigeminal plate. Nerve fibers occurred within gracile and cuneate fasciculi, trigeminal spinal tract and nucleus, oculomotor and facial nerves, solitary tract, vestibular nerve, medial longitudinal fasciculus, medial and lateral lemnisci, and inferior and superior cerebellar peduncles. Age changes were detected in the distribution pattern for each trophin. On the whole, in the grey matter, labelled perikarya were more frequently observed in pre- and perinatal than in adult specimens; on the other hand, in discrete regions, nerve fibers and terminals were abundant and showed a definite arrangement only in adult tissue; finally, distinct fiber systems in the white matter were immunolabelled only at pre- and perinatal ages. The results support the concept of a trophic involvement of NTN, PSP, and ART in the development, functional activity and maintenance of a variety of human brainstem neuronal systems.
                            12. Alfano I, Vora P, Mummery RS, Mulloy B and Rider CC (2007). The major determinant of the heparin binding of glial cell-line-derived neurotrophic factor is near the N-terminus and is dispensable for receptor binding. Biochem J. 404: 131-40. School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 OEX, UK. GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor beta domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRalpha1 (GDNF family receptor alpha1), and heparin-bound GDNF is able to bind GFRalpha1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRalpha1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF-GFRalpha1 interaction, and the extracellular domain of GFRalpha1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF-GFRalpha1 engagement.
                            13. Bespalov MM and Saarma M (2007). GDNF family receptor complexes are emerging drug targets. Trends Pharmacol Sci. 28: 68-74. Institute of Biotechnology, University of Helsinki, Viikinkaari 9, PO Box 56, FIN-00014 Helsinki, Finland. Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), which consist of GDNF, neurturin, artemin and persephin, regulate the development and maintenance of the nervous system. GDNF protects and repairs dopamine-containing neurons, which degenerate in Parkinson's disease, and motoneurons, which die in amyotrophic lateral sclerosis. GDNF and neurturin have shown promise in clinical trials of Parkinson's disease, and artemin is currently undergoing clinical trials for chronic pain treatment. However, the delivery of GFLs into the brain through invasive approaches such as neurosurgery, viral vectors or by the use of encapsulated cells is associated with multiple obstacles. The development of small molecules that specifically activate GFL receptors and that can be applied systemically would overcome most of these problems. The unique nature of the GFL receptors, recent progress in elucidation of the 3D structures of GFLs and GFL-receptor complexes and the use of high-throughput screening have resulted in the development of the first small molecules that mimic the effects of the different GFLs.
                            14. Bennett DL, Boucher TJ, Michael GJ, Popat RJ, Malcangio M, Averill SA, Poulsen KT, Priestley JV, Shelton DL and McMahon SB (2006). Artemin has potent neurotrophic actions on injured C-fibres. J Peripher Nerv Syst. 11: 330-45. Wolfson Centre for Age Related Disease, King's College London, Guy's Campus, London, UK. david.bennett@kcl.ac.uk. In this study, we have investigated the effects of artemin (ARTN), one of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors, on C-fibres following nerve injury in the adult rat. GDNF family receptor alpha (GFRalpha) 3, the ligand binding domain of the ARTN receptor, is expressed in 34% of dorsal root ganglion (DRG) cells, predominantly in the peptidergic population of C-fibres and in a proportion of the isolectin B4 (IB4)-binding population. Interestingly, only 30% of GFRalpha3-expressing DRG cells co-expressed RET (the signal transducing domain). In agreement with previous studies, treatment with ARTN prevented many of the nerve injury-induced changes in the histochemistry of both the peptidergic and the IB4-binding populations of small, but not large, diameter DRG cells. In addition, ARTN treatment maintained C-fibre conduction velocity, and C-fibre evoked substance P release within the dorsal horn following nerve injury. ARTN was also protective following capsaicin treatment, which produces selective C-fibre injury. Given the potent neurotrophic actions of ARTN on C-fibres, it may therefore provide potential for the treatment of nerve injury, particularly in the maintenance of small fibre function.
                            15. Cass WA, Peters LE, Harned ME and Seroogy KB (2006). Protection by GDNF and other trophic factors against the dopamine-depleting effects of neurotoxic doses of methamphetamine. Ann N Y Acad Sci. 1074: 272-81. Department of Anatomy and Neurobiology, MN-225 Chandler Medical Center, University of Kentucky, Lexington, KY 40536-0298, USA. wacass1@uky.edu. Repeated methamphetamine (METH) administration to animals can result in long-lasting decreases in striatal dopamine (DA) content. It has previously been shown that glial cell line-derived neurotrophic factor (GDNF) can reduce the DA-depleting effects of neurotoxic doses of METH. However, there are several other trophic factors that are protective against dopaminergic toxins. Thus, the present experiments further investigated the protective effect of GDNF as well as the protective effects of several other trophic factors. Male Fischer-344 rats were given an intracerebral injection of trophic factor (2-10 microg) 1 day before METH (5 mg/kg, s.c., 4 injections at 2-h intervals). Seven days later DA levels in the striatum were measured using high-performance liquid chromatography (HPLC). Initial experiments indicated that only intrastriatal GDNF, and not intranigral GDNF, was protective. Thereafter, all other trophic factors were administered into the striatum. Members of the GDNF family (GDNF, neurturin, and artemin) all provided significant protection against the DA-depleting effects of METH, with GDNF providing the greatest protection. Brain-derived neurotrophic factor, neurotrophin-3, acidic fibroblast growth factor, basic fibroblast growth factor, ciliary neurotrophic factor, transforming growth factor-alpha (TGF-alpha), heregulin beta1 (HRG-beta1), and amphiregulin (AR) provided no significant protection at the doses examined. These results suggest that the GDNF family of trophic factors can provide significant protection against the DA-depleting effects of neurotoxic doses of METH.
                            16. Hamra FK, Chapman KM, Nguyen D and Garbers DL (2007). Identification of neuregulin as a factor required for formation of aligned spermatogonia. J Biol Chem. 282: 721-30. Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. kent.hamra@utsouthwestern.edu. In the absence of somatic cells, medium conditioned by the SNL fibroblast line (SNL-CM) is able to stimulate primary cultures of rat type-A single spermatogonia to develop into chains of aligned spermatogonia at the 8-, 16-, and 32-cell stages. By comparison, medium conditioned by an MSC-1 Sertoli cell line is ineffective. Glial cell line-derived neurotrophic factor (GDNF)-like molecules were identified in SNL-CM and recombinant forms of GDNF, neurturin, and artemin were shown to stimulate formation of aligned spermatogonia, but principally to only the 4- and 8-cell stages. Because SNL-CM and GDNF-like molecules stimulated the formation of spermatogonial chain length differently, we purified components of SNL-CM to identify the additional contributing factor(s). A fraction was isolated that was dependent on GDNF, but required for effective formation of 16- and 32-cell chain lengths. Sequence analysis identified the factor as mouse neuregulin-1. At picomolar concentrations, recombinant neuregulin-1 in combination with GDNF effectively stimulated formation of aligned spermatogonia up to the 32-cell stage. Neuregulin in the absence of GDNF was relatively ineffective. Soluble receptors for neuregulins blocked the effects of GDNF and SNL-CM, suggesting that both neuregulin and GDNF are required for effective formation of long spermatogonial chains. Addition of neuregulin-1 to cultures on MSC-1 feeder layers resulted in spermatogonial behavior similar to that seen on feeder layers of SNL fibroblasts. In fact, SNL cells were found to express 100-fold higher levels of neuregulin-1 transcripts than MSC-1 cells. Thus, we identify neuregulin as a factor required for spermatogonial amplification and differentiation in culture.
                            17. Ceyhan GO, Bergmann F, Kadihasanoglu M, Erkan M, Park W, Hinz U, Giese T, Muller MW, Buchler MW, Giese NA and Friess H (2007). The neurotrophic factor artemin influences the extent of neural damage and growth in chronic pancreatitis. Gut. 56: 534-44. Department of General Surgery, University of Heidelberg, Heidelberg, Germany. BACKGROUND AND AIMS: Chronic pancreatitis is characterised by severe abdominal neuropathic pain, perineural inflammatory cell infiltrations and intrapancreatic neural growth. Artemin was recently shown to eliminate neuropathic pain and reverse neurochemical damage after nerve injury. The role of artemin and its receptor GFRalpha3 was investigated in patients with chronic pancreatitis. METHODS: Expression of artemin and its receptor GFRalpha3 was studied in chronic pancreatitis (n = 66) and normal (n = 22) pancreatic tissues by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and western blot analysis. Artemin expression was correlated with pain and pathomorphological changes (inflammation, perineural inflammatory cell infiltration, neural alterations and fibrosis). Immunohistochemistry was used to localise artemin and GFRalpha3 in the tissues. To detect sources of artemin, primary human pancreatic stellate cells (hPSCs) were isolated and analysed by QRT-PCR and immunocytology analysis. RESULTS: In chronic pancreatitis, artemin and GFRalpha3 were significantly overexpressed and located in smooth muscle cells of arteries, Schwann cells and neural ganglia. Increased levels of artemin mRNA correlated with pain severity, inflammation, perineural inflammatory cell infiltration, neural density and hypertrophy. Furthermore, the severity of fibrosis was positively related with artemin expression and neural alterations. Activated hPSCs expressed low basal levels of artemin mRNA which were upregulated by exposure to transforming growth factor (TGF)beta1. CONCLUSIONS: Overexpression of artemin in chronic pancreatitis might function as a compensatory upregulation in order to repair neural damage incurred by ongoing pancreatic inflammation. Upregulation of TGFbeta1 seems not only to increase pancreatic fibrosis but also to contribute to neural alteration by stimulating artemin expression in hPSCs. However, overexpression of endogenous artemin does not seem to be sufficient to prevent pain in chronic pancreatitis.
                            18. Malin SA, Molliver DC, Koerber HR, Cornuet P, Frye R, Albers KM and Davis BM (2006). Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo. J Neurosci. 26: 8588-99. Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10-100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund's adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRalpha3 (for GDNF family receptor alpha) was seen in DRG, and GFRalpha3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.
                            19. Elitt CM, McIlwrath SL, Lawson JJ, Malin SA, Molliver DC, Cornuet PK, Koerber HR, Davis BM and Albers KM (2006). Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold. J Neurosci. 26: 8578-87. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.
                            20. Airaksinen MS, Holm L and Hatinen T (2006). Evolution of the GDNF family ligands and receptors. Brain Behav Evol. 68: 181-90. Neuroscience Center, University of Helsinki, Helsinki, Finland. mairaksi@operoni.helsinki.fi. Four different ligand-receptor binding pairs of the GDNF (glial cell line-derived neurotrophic factor) family exist in mammals, and they all signal via the transmembrane RET receptor tyrosine kinase. In addition, GRAL (GDNF Receptor Alpha-Like) protein of unknown function and Gas1 (growth arrest specific 1) have GDNF family receptor (GFR)-like domains. Orthologs of the four GFRalpha receptors, GRAL and Gas1 are present in all vertebrate classes. In contrast, although bony fishes have orthologs of all four GDNF family ligands (GFLs), one of the ligands, neurturin, is absent in clawed frog and another, persephin, is absent in the chicken genome. Frog GFRalpha2 has selectively evolved possibly to accommodate GDNF as a ligand. The key role of GDNF and its receptor GFRalpha1 in enteric nervous system development is conserved from zebrafish to humans. The role of neurturin, signaling via GFRalpha2, for parasympathetic neuron development is conserved between chicken and mice. The role of artemin and persephin that signal via GFRalpha3 and GFRalpha4, respectively, is unknown in non-mammals. The presence of RET- and GFR-like genes in insects suggests that a ProtoGFR and a ProtoRET arose early in the evolution of bilaterian animals, but when the ProtoGFL diverged from existing transforming growth factor (TGFbeta)-like proteins remains unclear. The four GFLs and GFRalphas were presumably generated by genome duplications at the origin of vertebrates. Loss of neurturin in frog and persephin in chicken suggests functional redundancy in early tetrapods. Functions of non-mammalian GFLs and prechordate RET and GFR-like proteins remain to be explored.
                            21. Ceyhan GO, Giese NA, Erkan M, Kerscher AG, Wente MN, Giese T, Buchler MW and Friess H (2006). The neurotrophic factor artemin promotes pancreatic cancer invasion. Ann Surg. 244: 274-81. Department of General Surgery, University of Heidelberg, Heidelberg, Germany. OBJECTIVE: To analyze the role of Artemin in pancreatic ductal adenocarcinoma (PDAC) in terms of expression, influence on cancer cell behavior, and pain correlation. SUMMARY BACKGROUND DATA: PDAC is characterized by prominent local nerve alterations and perineural invasion, which frequently affects the extrapancreatic nerve plexus, causing severe pain and precluding curative resection. Artemin, a neurotrophic protein controlling growth, regeneration, and survival of neurons was analyzed to highlight the neuro-cancer interactions in PDAC. METHODS: Artemin and its receptors (GFRalpha3/RET) were studied in PDAC tissues and normal pancreas by Western blot analysis and immunohistochemistry. RNA expression was analyzed in pancreatic tissues (normal, cancer) and pancreatic cancer cell lines by QRT-PCR. To evaluate whether Artemin influences cancer cell proliferation and invasion, MTT-growth and Matrigel-invasion assays were used. In addition, the tissue expression of Artemin was correlated with pain in PDAC. RESULTS: Artemin and GFRalpha3/RET were both detected at enhanced levels in PDAC compared with normal pancreas, localizing predominantly in hypertrophic nerves and arterial walls, as well as in cancer cells of primary and metastatic lesions. The levels of Artemin and GFRalpha3 did not correlate with pain in PDAC patients. However, Artemin promoted pancreatic cancer cell invasion up to 5-fold, without affecting cancer cell proliferation. CONCLUSION: Artemin expression was not associated with pain in PDAC. However by increasing cancer cell invasion, Artemin seems to influence neural invasion and thereby contribute to cancer cell spreading along pancreatic nerves.
                            22. Maroldt H, Kaplinovsky T and Cunningham AM (2005). Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system. J Neurocytol. 34: 241-55. Developmental Neurosciences Program, School of Women's and Children's Health, Faculty of Medicine, Sydney Children's Hospital, University of New South Wales, High St, Randwick, NSW, 2031, Australia. The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON).In this immunohistochemical study, we localized GDNF, neurturin, GFRalpha1, GFRalpha2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRalpha1, whereas progenitors and immature neurons more avidly expressed GFRalpha2.In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRalpha2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.
                            23. Park S and Hong YW (2006). Transcriptional regulation of artemin is related to neurite outgrowth and actin polymerization in mature DRG neurons. Neurosci Lett. 404: 61-6. Department of Applied Chemistry, Dongduk Women's University, Sungbuk-ku, Seoul, Korea. sypark21@dongduk.ac.kr. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands that helps to ensure the survival of sensory neurons. We used an in vitro isolated dorsal root ganglia model to study the effects of artemin on the adult rat neuronal system and investigate differentially regulated genes. We found that 285 genes were differentially transcribed by artemin after 3 h of treatment, including genes related to cell adhesion and actin polymerization. A series of genes involved in the regulation of actin dynamics, including coronin, Myr 5, Wiskott-Aldrich syndrome protein interacting protein, cofilin, drebrin and dynamin were down-regulated by artemin, suggesting that it plays a previously undefined role in the regulation of actin polymerization and synaptic vesicle movement. Artemin also down-regulated the expression of genes related to cell adhesion and matrix assembly, including biglycan, plectin, nestin, neuronatin and the neuron-glia-CAM-related cell adhesion molecule, which is functionally relevant to neurite elongation in DRG neurons. Artemin resulted in increases in total neurite length and branching of the DRG neurons. Also artemin caused an increase of synaptic vesicle clustering. Our results showed that the inhibition of DNA methylation suppressed the artemin-dependent neurite growth, suggesting that the genetic regulation could be relevant to neurite elongation in mature DRG.
                            24. Yang C, Hutto D and Sah DW (2006). Distribution of GDNF family receptor alpha3 and RET in rat and human non-neural tissues. J Mol Histol. 37: 69-77. BiogenIdec, Inc., 14 Cambridge Center, Cambridge, MA, 02142, USA, Chunhua.Yang@biogenidec.com. The neurotrophic growth factor artemin binds selectively to GDNF family receptor alpha3 (GFRalpha3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date. To study the distribution of GFRalpha3 and RET in adult rat and human non-neural tissues, we carried out a comprehensive immunohistochemical study. We stained major organs from the digestive, urinary, reproductive, immune, respiratory and endocrine systems, and from other systems (cardiovascular, skeletal muscle), as well as regions of the nervous system for comparison. In both rat and human, the majority of non-neural cells did not exhibit detectable GFRalpha3-like immunoreactivity. In the rat, GFRalpha3- and RET-like staining were found in the same non-neural cell type only in kidney. In the human digestive and reproductive systems, a subset of epithelial cells exhibited GFRalpha3- and RET-like staining, suggesting co-localization. In other tissues, sub-populations of cells expressed either GFRalpha3- or RET-like immunoreactivity. The functional consequences of GFRalpha3 expression in non-neural cells remain to be determined.
                            25. Wang X, Baloh RH, Milbrandt J and Garcia KC (2006). Structure of artemin complexed with its receptor GFRalpha3: convergent recognition of glial cell line-derived neurotrophic factors. Structure. 14: 1083-92. Howard Hughes Medical Institute, Stanford University School of Medicine, Department of Microbiology and Immunology, Stanford, California 94305-5124, USA. Artemin (ARTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) which regulate the development and maintenance of many neuronal populations in the mammalian nervous system. Here we report the 1.92 A crystal structure of the complex formed between ARTN and its receptor GFRalpha3, which is the initiating step in the formation of a ternary signaling complex containing the shared RET receptor. It represents a new receptor-ligand interaction mode for the TGF-beta superfamily that reveals both conserved and specificity-determining anchor points for all GFL-GFRalpha pairs. In tandem with the complex structure, cellular studies using receptor chimeras implicate dyad-symmetric composite interfaces for recruitment and dimerization of RET, leading to intracellular signaling. These studies should facilitate the functional dissection of the specific versus pleiotropic roles of this system in neurobiology, as well as its exploitation for therapeutic applications.
                            26. Wissel K, Wefstaedt P, Rieger H, Miller JM, Lenarz T and Stover T (2006). Upregulation of glial cell line-derived neurotrophic factor and artemin mRNA in the auditory nerve of deafened rats. Neuroreport. 17: 875-8. Department of Otolaryngology, Medical University of Hannover, Hannover, Germany. Nerve growth factors play key roles in spiral ganglion cells survival and excitability. Our aim was to determine gene expression patterns of glial cell line-derived neurotrophic factor family (GDNF) members and their receptors in the auditory nerve and inferior colliculus of deafened rats. The gene expression of GDNF, persephin, artemin and neurturin, and their receptors GFRalpha1, GFRalpha2, GFRalpha3 and Ret, was determined by semiquantitative reverse transcriptase-polymerase chain reaction using GAPDH expression as an internal standard. Following deafness, no significant changes in expression of GDNF family genes were found in inferior colliculus. In contrast, artemin, GDNF, GFRalpha1-3 and Ret RNA expression were strongly upregulated in the auditory nerve following deafness, indicating their importance in protecting the auditory nerve against cell damage.
                            27. Silvian L, Jin P, Carmillo P, Boriack-Sjodin PA, Pelletier C, Rushe M, Gong B, Sah D, Pepinsky B and Rossomando A (2006). Artemin crystal structure reveals insights into heparan sulfate binding. Biochemistry. 45: 6801-12. Department of Drug Discovery, Biogen Idec, Inc., 12 Cambridge Center, Cambridge, Massachusetts 02142, USA. laura.silvian@biogenidec.com. Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFRalpha3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.
                            28. Wefstaedt P, Scheper V, Rieger H, Lenarz T and Stover T (2006). [Neurotrophic factors of the GDNF family and their receptors are detectable in spiral ganglion cells of normal hearing as well as of deafened rats]. Laryngorhinootologie. 85: 802-8. Hals-Nasen-Ohrenklinik der Medizinischen Hochschule Hannover. BACKGROUND: Recent studies have shown that neurotrophic factors like BDNF, NT-3 and GDNF induce protective effects on spiral ganglion cells after noise- or drug-induced hearing loss. According to these studies it is suggested that deafness leads to a lack of neurotrophic factor or relating receptor expression in spiral ganglion cells, that has to be compensated by local cochlear application of these factors. METHODS: In the present study we examined the expression pattern of members of the GDNF family (GDNF, Neurturin, Artemin, Persephin) and their relating receptors (Ret, GFRalpha1 - 3) as well as BDNF and trkB on spiral ganglion cells of normal hearing and experimentally deafened rats (10 % neomycine). Indirect immunofluorescence was carried out to determine protein expression of these factors and their receptors 26 days following deafening. RESULTS: Our results demonstrate neurotrophic factor and receptor expression on spiral ganglion cells of normal hearing as well as experimentally deafened animals. CONCLUSIONS: Our data indicate that within a period of 26 days after deafening no detectable reduction of the GDNF-family member expression and their receptors was ascertainable on spiral ganglion cells by immunohistochemistry. Thus, a lack of neurotrophic factor expression is unlikely to be the only cause of spiral ganglion cell loss following deafening.
                            29. Hauck SM, Kinkl N, Deeg CA, Swiatek-de Lange M, Schoffmann S and Ueffing M (2006). GDNF family ligands trigger indirect neuroprotective signaling in retinal glial cells. Mol Cell Biol. 26: 2746-57. GSF-National Research Center for Environment and Health, Institute of Human Genetics, Ingolstaedter Landstrasse 1, 85764 Munich-Neuherberg, Germany. Apoptotic cell death of photoreceptors is the final event leading to blindness in the heterogeneous group of inherited retinal degenerations. GDNF (glial cell-line-derived neurotrophic factor) was found to rescue photoreceptor function and survival very effectively in an animal model of retinal degeneration (M. Frasson, S. Picaud, T. Leveillard, M. Simonutti, S. Mohand-Said, H. Dreyfus, D. Hicks, and J. Sahel, Investig. Ophthalmol. Vis. Sci. 40:2724-2734, 1999). However, the cellular mechanism of GDNF action remained unresolved. We show here that in porcine retina, GDNF receptors GFRalpha-1 and RET are expressed on retinal Mueller glial cells (RMG) but not on photoreceptors. Additionally, RMG express the receptors for the GDNF family members artemin and neurturin (GFRalpha-2 and GFRalpha-3). We further investigated GDNF-, artemin-, and neurturin-induced signaling in isolated primary RMG and demonstrate three intracellular cascades, which are activated in vitro: MEK/ERK, stress-activated protein kinase (SAPK), and PKB/AKT pathways with different kinetics in dependence on stimulating GFL. We correlate the findings to intact porcine retina, where GDNF induces phosphorylation of ERK in the perinuclear region of RMG located in the inner nuclear layer. GDNF signaling resulted in transcriptional upregulation of FGF-2, which in turn was found to support photoreceptor survival in an in vitro assay. We provide here a detailed model of GDNF-induced signaling in mammalian retina and propose that the GDNF-induced rescue effect on mutated photoreceptors is an indirect effect mediated by retinal Mueller glial cells.
                            30. Schueler-Furman O, Glick E, Segovia J and Linial M (2006). Is GAS1 a co-receptor for the GDNF family of ligands? Trends Pharmacol Sci. 27: 72-7. Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Glial-cell-line-derived neurotrophic factor (GDNF) is a survival and maintenance factor for dopamine-containing neurons and motoneurons. GDNF belongs to a family of structurally related factors that includes neurturin (NRTN), artemin (ARTN) and persephin (PSPN). An initial step in the activation of signaling via the GDNF family of ligands (GFLs) is their binding to their cognate co-receptor GFR alpha. GAS1, an apparently unrelated protein, exhibits homology to GFR alpha and thus we hypothesize that GAS1 can serve as an alternative receptor for GFLs. The functional similarity between GFR alpha and GAS1 extends to their role in embryogenesis, differentiation and glia maintenance, and is substantiated by overlap in their expression profile, subcellular localization and structural details. We propose that the relative expression and localization of the two remote receptors, GFR alpha and GAS1, on the membranes of neuronal and glial cells determines whether these cells survive or undergo apoptotic death.
                            31. Ducray A, Krebs SH, Schaller B, Seiler RW, Meyer M and Widmer HR (2006). GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon. Brain Res. 1069: 104-12. Department of Neurosurgery, University of Bern, CH-3010 Bern, Switzerland. Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none of the GFLs affected soma size of GABA-ir neurons. In contrast, only NRTN treatment significantly increased 5-HT-ir cells densities at 10 ng/ml (1.3-fold), while an augmentation was seen for GDNF and PSPN at 100 ng/ml (2.4-fold and 1.7-fold, respectively). ARTN had no effect on 5-HT-ir cell densities. Morphological analysis of 5-HT-ir neurons revealed a significant increase of soma size, number of primary neurites/neuron and neurite length/neuron after GDNF exposure, while PSPN only affected soma size, and NRTN and ARTN failed to exert any effect. In conclusion, we identified GFLs as effective neurotrophic factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development.
                            32. Zihlmann KB, Ducray AD, Schaller B, Huber AW, Krebs SH, Andres RH, Seiler RW, Meyer M and Widmer HR (2005). The GDNF family members neurturin, artemin and persephin promote the morphological differentiation of cultured ventral mesencephalic dopaminergic neurons. Brain Res Bull. 68: 42-53. Department of Neurosurgery, University of Bern, CH-3010 Bern, Switzerland. Neurturin (NRTN), artemin (ARTN), persephin (PSPN) and glial cell line-derived neurotrophic factor (GDNF) form a group of neurotrophic factors, also known as the GDNF family ligands (GFLs). They signal through a receptor complex composed of a high-affinity ligand binding subunit, postulated ligand specific, and a common membrane-bound tyrosine kinase RET. Recently, also NCAM has been identified as an alternative signaling receptor. GFLs have been reported to promote survival of cultured dopaminergic neurons. In addition, GDNF treatments have been shown to increase morphological differentiation of tyrosine hydroxylase immunoreactive (TH-ir) neurons. The present comparative study investigated the dose-dependent effects of GFLs on survival and morphological differentiation of TH-ir neurons in primary cultures of E14 rat ventral mesencephalon. Both NRTN and ARTN chronically administered for 5 days significantly increased survival and morphological differentiation of TH-ir cells at all doses investigated [0.1-100 ng/ml], whereas PSPN was found to be slightly less potent with effects on TH-ir cell numbers and morphology at 1.6-100 ng/ml and 6.3-100 ng/ml, respectively. In conclusion, our findings identify NRTN, ARTN and PSPN as potent neurotrophic factors that may play an important role in the structural development and plasticity of ventral mesencephalic dopaminergic neurons.
                            33. Lee RH, Wong WL, Chan CH and Chan SY (2006). Differential effects of glial cell line-derived neurotrophic factor and neurturin in RET/GFRalpha1-expressing cells. J Neurosci Res. 83: 80-90. Department of Paediatrics and Adolescent Medicine, the University of Hong Kong, Pokfulam, Hong Kong SAR, People's Republic of China. The c-ret protooncogene, RET, encodes a receptor tyrosine kinase. RET is activated by members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands, which include GDNF, neurturin, artemin, and persephin. The ligands bind RET through GDNF family receptor alpha, termed GFRalpha1-4. Despite the importance of RET signaling in the development of the enteric nervous system and the kidney, the differential signaling mechanisms between RET ligands are poorly established. It has been suggested that signal specificity is achieved through binding of the ligand to its preferred GFRalpha. To compare the signaling profiles of GDNF and neurturin, we have identified a cell line, NG108-15, which endogenously expresses RET and GFRalpha1 but not GFRalpha2-4. Immunoblot data showed that GDNF caused a transient activation, whereas neurturin caused a sustained activation, of both p44/p42 MAP kinases and PLCgamma. Under serum starvation, NG108-15 cells differentiate and form neurites. Neurturin but not GDNF stimulated neurite outgrowth, which could be blocked by the selective PLC inhibitor U73122. On the other hand, GDNF but not neurturin promoted cell survival, and this could be blocked by the p44/p42 MAP kinase inhibitor PD98059. Our findings not only show the differential signaling of GDNF and neurturin but also suggest that this can be achieved through binding to the same GFRalpha subtype, leading to distinct biological responses.
                            34. Ito Y, Okada Y, Sato M, Sawai H, Funahashi H, Murase T, Hayakawa T and Manabe T (2005). Expression of glial cell line-derived neurotrophic factor family members and their receptors in pancreatic cancers. Surgery. 138: 788-94. Department of Gastroenterological Surgery, Nagoya City University, Graduate School of Medical Sciences, Kawasumi, Japan. BACKGROUND: The glial cell line-derived neurotrophic factor (GDNF) is a member of neurotrophic polypeptide family, which promotes survival and rescue of various neural cells in the central and peripheral nerve systems. We previously reported that GDNF promotes tumor cell invasion in pancreatic cancer cell lines. The purpose of this study was to investigate GDNF family expression and the status of related receptors in actual cancer tissues, and assess correlations with clinicopathologic behavior. METHODS: Immunohistochemical assessment of GDNF, neurturin, persephin, artemin, GDNF family receptor alpha-1 and alpha-2, and RET was performed for 51 cases of surgically resected pancreatic cancer. RESULTS: In all intrapancreatic nerves, GDNF and artermin were expressed strongly. In pancreatic cancer tissues. The expression of RET was stronger than that seen in normal ductal cells and was significantly related to the survival rate after resection (P = .026) and lymphatic invasion (P = .014). Intrapancreatic neural invasion was significantly related to the expression of GDNF (P = .047). CONCLUSIONS: We conclude that the expression of RET in pancreatic cancer tissues may be a useful prognostic marker and GDNF may play an important role in neural invasion.
                            35. Lucini C, Maruccio L, Tafuri S, Bevaqua M, Staiano N and Castaldo L (2005). GDNF family ligand immunoreactivity in the gut of teleostean fish. Anat Embryol (Berl). 210: 265-74. Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Via Veterinaria 1, 80137 Napoli, Italy. lucini@unina.it. Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRalpha, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.
                            36. Sah DW, Ossipov MH, Rossomando A, Silvian L and Porreca F (2005). New approaches for the treatment of pain: the GDNF family of neurotrophic growth factors. Curr Top Med Chem. 5: 577-83. Alnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, MA 02142, USA. This article focuses on the GDNF family of neurotrophic factors as a potential new class of therapeutics for neuropathic pain, with a particular emphasis on the ligands, artemin and GDNF. In vivo activity of the ligands, expression of ligands and receptors after peripheral nerve injury, and modulation of nerve injury-induced changes by the ligands are reviewed in detail. Structural considerations, particularly with regard to implications for binding interactions and biological activity are discussed.
                            37. Wang S, Davis BM, Zwick M, Waxman SG and Albers KM (2006). Reduced thermal sensitivity and Nav1.8 and TRPV1 channel expression in sensory neurons of aged mice. Neurobiol Aging. 27: 895-903. Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Sensory neurons in aging mammals undergo changes in anatomy, physiology and gene expression that correlate with reduced sensory perception. In this study we compared young and aged mice to identify proteins that might contribute to this loss of sensation. We first show using behavioral testing that thermal sensitivity in aged male and female mice is reduced. Expression of sodium channel (Nav1.8 and Nav1.9) and transient receptor potential vanilloid (TRPV) channels in DRG and peripheral nerves of young and old male mice was then examined. Immunoblotting and RT-PCR assays showed reduced Nav1.8 levels in aged mice. No change was measured in TRPV1 mRNA levels in DRG though TRPV1 protein appeared reduced in the DRG and peripheral nerves. The GFRalpha3 receptor, which binds the growth factor artemin and is expressed by TRPV1-positive neurons, was also decreased in the DRG of aged animals. These findings indicate that loss of thermal sensitivity in aging animals may result from a decreased level of TRPV1 and Nav1.8 and decreased trophic support that inhibits efficient transport of channel proteins to peripheral afferents.
                            38. Quartu M, Serra MP, Manca A, Mascia F, Follesa P and Del Fiacco M (2005). Neurturin, persephin, and artemin in the human pre- and full-term newborn and adult hippocampus and fascia dentata. Brain Res. 1041: 157-66. Department of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Italy. The immunochemical occurrence and localization of the Glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) is described in the human postmortem hippocampus and fascia dentata from subjects aged 21 weeks of gestation to 88 years. The detectability of NTN, PSP, and ART is shown in the rat by Western blot and immunohistochemistry up to 70 h postmortem. In the human tissue, labeled neuronal perikarya were detectable for each trophin at all examined ages, with prevalent localization in the pyramidal layer of the Ammon's horn and hilus and granular layer of the fascia dentata. In the adult subjects, punctate elements were also present. Comparison of the pattern of immunoreactive structures among young and adult subjects suggests that intracellular distribution and/or trafficking of the GDNF family ligands may undergo age-related changes. Labeled glial elements were also identifiable. Western blot analysis indicates that the availability of the dimeric and monomeric forms of the trophins may vary with age and postmortem delay. The results obtained suggest the involvement of NTN, PSP, and ART in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature human hippocampal neurons.
                            39. Carmillo P, Dago L, Day ES, Worley DS, Rossomando A, Walus L, Orozco O, Buckley C, Miller S, Tse A, Cate RL, Rosenblad C, Sah DW, Gronborg M and Whitty A (2005). Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin. Biochemistry. 44: 2545-54. Biogen Idec, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, USA. To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.
                            40. Leitner ML, Wang LH, Osborne PA, Golden JP, Milbrandt J and Johnson EM, Jr. (2005). Expression and function of GDNF family ligands and receptors in the carotid body. Exp Neurol. 191 Suppl 1: S68-79. Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, MO 63110-1031, USA. The carotid body is a neural crest-derived neuroendocrine organ that detects the oxygen level in blood and regulates ventilation. Unlike many other neural crest derivatives, the trophic factors mediating survival and differentiation of neuroendocrine cells of the carotid body are unknown. Given that many neural crest derivatives rely on the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) for survival and function, we undertook an analysis of the carotid body as a potential site of GFL action. RET and GDNF family receptor alphas (GFRalpha) 1-3 are expressed in the developing carotid body as detected by RT-PCR and immunocytochemistry. mRNA for GDNF, and artemin (ARTN) were also present. In vitro, treatment with GDNF, neurturin (NRTN), or ARTN, individually or in combination, produced an increase in the number and length of processes of the Type-I glomus cells of the carotid body [embryonic day-17 (E17) rats]. However, GFLs alone or in combination had no effect on glomus cell survival in either postnatal day-1 (P1) or E17 carotid body cultures. These results suggest that one or more GFLs may have a role in carotid body function. In addition, the results of this study suggest that endogenous or exogenous GFLs may enhance target innervation by carotid body transplants.

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                            • #15

                              ... many thanks for clarifying the situation Dr. Young..
                              May be a bit hard for me to eat that but it looks like nothing is talking about human trials..
                              So let's keep in touch with that!..

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