Striatal delivery of CERE-120, an AAV2 vector encoding human neurturin, enhances activity of the dopaminergic nigrostriatal system in aged monkeys.

Neurturin (NTN) is a potent survival factor for midbrain dopaminergic neurons. CERE-120, an adeno-associated virus type 2 (AAV2) vector encoding human NTN (AAV2-NTN), is currently being developed as a potential therapy for Parkinson's disease. This study examined the bioactivity and safety/tolerability of AAV2-NTN in the aged monkey model of nigrostriatal dopamine insufficiency. Aged rhesus monkeys received unilateral injections of AAV2-NTN into the caudate and putamen, with each animal therefore serving as its own control. Robust expression of NTN within the nigrostriatal system was observed 8 months postadministration. (18)F-fluorodopa imaging using positron emission tomography revealed statistically significant increases in (18)F-fluorodopa uptake in the injected striatum compared with the uninjected side at 4 and 8 months. In addition, at 8 months postadministration, a significant enhancement in tyrosine hydroxylase immunoreactive fibers and an increase in the number of tyrosine hydroxylase immunoreactive cells was observed in the AAV2-NTN injected striatum compared with the uninjected side. Robust activation of phosphorylated extracellular signal-regulated kinase immunoreactivity in the substantia nigra was also observed. Histopathological analyses revealed no adverse effects of AAV2-NTN in the brain. Collectively, these results are consistent with the neurotrophic effects of NTN on the dopaminergic nigrostriatal system and extend the growing body of evidence supporting the concept that AAV2-NTN may have therapeutic benefit for Parkinson's disease.     

Distinct roles for GFRalpha1 and GFRalpha2 signalling in different cranial parasympathetic ganglia in vivo

Neurturin (NRTN), signalling via the GDNF family receptor alpha2 (GFRalpha2) and Ret tyrosine kinase, has recently been identified as an essential target-derived factor for many parasympathetic neurons. NRTN is expressed in salivary and lacrimal glands, while GFRalpha2 and Ret are expressed in the corresponding submandibular, otic and sphenopalatine ganglia. Here, we have characterized in more detail the role of GDNF and NRTN signalling in the development of cranial parasympathetic neurons and their target innervation. Gfra1 mRNA was expressed at E12 but not in newborn cranial parasympathetic ganglia, while Gfra2 mRNA and protein were strongly expressed in newborn and adult cranial parasympathetic neurons and their projections, respectively. In newborn GFRalpha1- or Ret-deficient mice, where many submandibular ganglion neurons were still present, the otic and sphenopalatine ganglia were completely missing. In contrast, in newborn GFRalpha2-deficient mice, most neurons in all these ganglia were present. In these mice, the loss and atrophy of the submandibular and otic neurons were amplified postnatally, accompanied by complete loss of innervation in some target regions and preservation in others. Surprisingly, GFRalpha2-deficient sphenopalatine neurons, whose targets were completely uninnervated, were not reduced in number and only slightly atrophied. Thus, GDNF signalling via GFRalpha1/Ret is essential in the early gangliogenesis of some, but not all, cranial parasympathetic neurons, whereas NRTN signalling through GFRalpha2/Ret is essential for the development and maintenance of parasympathetic target innervation. These results indicate that GDNF and NRTN have distinct functions in developing parasympathetic neurons, and suggest heterogeneity among and within different parasympathetic ganglia.     

NGF but not GDNF or neurturin enhance acetylcholine tissue levels in striatal organotypic brain slices

Trophic factors play important roles in survival and nerve fiber growth of cholinergic interneurons in the striatum in vivo and in vitro. In this study an organotypic slice model was used to investigate the effects of nerve growth factor and the novel factors glial cell line-derived neurotrophic factor and neurturin as well as other trophic factors on the striatal acetylcholine tissue levels. During culturing over 2 weeks acetylcholine tissue levels markedly decreased, representing degeneration of cholinergic neurons. When striatal slices were cultured for 2 weeks in the presence of 100 ng\ml nerve growth factor tissue levels of acetylcholine and the expression of choline acetyltransferase-like immunoreactivity and mRNA, as well as the muscarinic M2 autoreceptor mRNA were markedly enhanced compared to slices cultured without or with 10 ng\ml nerve growth factor. A single administration of nerve growth factor had no effect on acetylcholine tissue levels suggesting that nerve growth factor does not directly increase acetylcholine synthesis. All other trophic factors (glial cell line-derived neurotrophic factor, neurturin, brain-derived neurotrophic factor, neurotrophin-3 and -4\5, fibroblast growth factor-2, insulin like growth factor-I) had no effects on acetylcholine tissue levels. Thus, the organotypic slice model is a suitable system to study the effects of trophic factors and it is concluded that nerve growth factor selectively enhances acetylcholine tissue levels, indicating protection of cholinergic interneurons in the dorsal striatum.     

The GDNF/Neurturin-Ret multicomponent receptor system

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related neurotrophic factors that play crucial roles in the survival of peripheral sensory, sympathetic and dopaminergic neurons as well as motoneurons. Glial cell line-derived neurotrophic factordeficient mice showed lack of enteric neurons and renal agenesis or dysgenesis. Surprisingly, this phenotype was strikingly similar to that of ret proto-oncogene-deficient mice, suggesting that Ret tyrosine kinase might be a functional receptor for GDNF. Recent studies demonstrated that both GDNF and NTN were able to induce Ret tyrosine phosphorylation although they did not bind to Ret with high affinity, but novel glycosyl-phosphatidylinositol (GPI)-linked cell-surface proteins as ligand-binding components were required for Ret activation. Since GDNF and NTN are expected as therapeutic agents in neurodegenerative disorders such as Parkinson's disease and amyotrophic lateral sclerosis, the studies on the mechanisms of their biological actions through Ret would contribute to the development of new therapeutic strategies for these diseases.     

Expression of Glial Cell Line-Derived Neurotrophic Factor and Neurturin in Mature Kidney,Nephrogenic Rests,and Nephroblastoma: Possible Role as Differentiating Factors

Kidney development involves a series of complex interactions between the ureteric bud and undifferentiated mesenchyme, resulting in the production of the nephron unit. Among locally derived soluble factors, a particular relevance has been recognized to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) for the mesenchyme-to-epithelial conversion of a metanephron. Nephroblastoma is a developmental tumor of the kidney deriving from metanephric blastema that mimics renal development and may offer an adequate model of human nephrogenesis. We investigated the immunohistochemical expression of GDNF, NTN, and their receptors (GFR1, 2, and 3, and Ret) in normal human kidney and in 42 nephroblastomas, 20 of which were associated with nephrogenic rests (group A) and 22 were not (group B). We compared the immunostaining pattern in group A vs. group B and correlated clinical course with stage, grade, presence of nephrogenic rests, and immunohistochemical findings. GDNF, NTN, and their receptors were expressed in mature kidney and in 67% (GDNF) and 33% (NTN) of tumors, particularly in the epithelial component; precursor lesions were negative. No significant differences of expression were observed between groups A and B tumors. Low stage (P = 0.012), absence of nephrogenic rests (P = 0.016), intense expression of GDNF (P = 0.034), and NTN (P = 0.05) were associated with a more favorable outcome. Besides inductive activity in nephrogenesis, GDNF and NTN may play a role in maintaining differentiation and survival functions in mature kidney and may contribute to induce differentiation of nephroblastoma cells toward the less aggressive epithelial component. The pathway of activation seems to follow an autocrine/paracrine mechanism, as neurotrophic factors, GFR1-2-3 receptors and Ret are coexpressed.     

Neurturin和NGF联合应用对AD模型鼠基底前脑NGFR阳性神经元的保护作用

本研究目的是探讨neurturin和神经生长因子(NGF)联合应用对穹窿-海马伞(FF)切断后基底前脑胆碱能神经元的保护作用。将这两种因子联合注射到FF切断的大鼠侧脑室,注射一个月后,取脑进行免疫组化结合图像分析方法对内侧隔核(MS)和斜角带核垂直支(VDB)的神经生长因子受体(NGFR)阳性神经元存活情况进行比较分析。结果表明:FF切断一个月后,损伤组损伤侧MS和VDB的NGFR阳性神经元大量减少(分别减少64.8％和51.4％),MS和VDB的NGFR阳性细胞的面积和周长显著降低(P<0.01),OD值显著增高(P<0.01);NGF组损伤侧NGFR阳性神经元受到保护,NGFR阳性经元数显著高于损伤组损伤侧(P<0.01);联合组损伤侧NGFR阳性神经元也受到保护(MS和VDB细胞数分别只下降7.3％和15.4％),与损伤组损伤侧比较NGFR阳性细胞数增加了57.4％和36.0％(P<0.01),也明显高于NGF组(P<0.05);细胞形态学参数明显改善(P<0.05),细胞膜受体含量显著提高(P<0.05)。结果提示,neurturin和NGF联合应用和NGF单独应用均能不同程度地保护基底前脑胆碱能神经元,联合应用效果更佳。     

Neurturin和NGF对老年性痴呆模型鼠海马突触素表达的影响

为了探讨联合应用 Neurturin和神经生长因子对老年性痴呆模型鼠海马突触素的影响 ,本研究采用切断成年 SD大鼠左侧穹窿海马伞 ,建立隔 -海马胆碱能系统损害的痴呆模型 ,损伤 4周后 ,用免疫组化和图象分析技术等方法对大鼠海马突触素进行定量分析。结果显示 ,损伤对照组损伤侧海马 CA1 区多形层、辐射层、腔隙分子层和齿状回分子层突触素含量分别减少了42 .60 %、46.0 4%、5 7.3 6%和 5 5 .2 2 % ,损伤对照组与正常对照组之间有高度显著性差异 ( P<0 .0 1) ;NGF治疗组损伤侧海马CA1 区多形层、辐射层、腔隙分子层和齿状回分子层突触素含量分别只减少了 3 3 .64 %、3 8.2 5 %、3 8.42 %和 2 7.64 % ,NGF治疗组与损伤对照组之间有显著性差异 ( P<0 .0 5 ) ;联合治疗组损伤侧海马 CA1 区多形层、辐射层、腔隙分子层和齿状回分子层突触素含量分别只减少了 9.97%、8.0 5 %、14 .70 %和 4.2 8% ,联合治疗组与损伤对照组之间有高度显著性差异 ( P<0 .0 1)。结论 :联合应用 neurturin和 NGF促使老年性痴呆模型鼠海马突触素含量增多的效果较单用 NGF治疗好     

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.