Effect of treatment on serum glial cell line-derived neurotrophic factor in depressed patients

Post-mortem studies have demonstrated a decreased number of glia, reduced glial density, and a decreased glia/neuron ratio in different brain areas of patients diagnosed with a major depressive disorder (MDD). Researchers have therefore suggested that neurotrophic growth factor systems might be involved in the aetiology of MDD. This study aimed to test whether glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor beta family, in serum was associated with MDD. Serum concentrations were measured in MDD patients before treatment (n=76), after 8 weeks of antidepressant treatment (n=39), and in control subjects (n=50) using a sandwich ELISA method. Serum GDNF was significantly lower in MDD patients before treatment than in control subjects (P<0.001). From baseline to remission after 8 weeks of treatment, the increase in serum GDNF was statistically significant (P<0.001). The present study suggests that lower serum GDNF might be involved in the pathophysiology of MDD and antidepressant treatment increases the GDNF in MDD.     

Ciliary neurotrophic factor and interleukin-6 differentially activate microglia

Studies have shown that cytokines released following CNS injury can affect the supportive or cytotoxic functions of microglia. Interleukin-6 (IL-6)-family cytokines are among the injury factors released. To understand how microglia respond to IL-6 family cytokines, we examined the effects of ciliary neurotrophic factor (CNTF) and IL-6 on primary cultures of rat microglia. To assess the functional state of the cells, we assayed the expression of tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and cyclooxygenase 2 (COX-2) following stimulation. We show that CNTF reduces COX-2 levels, whereas IL-6 increases the expression of IL-1beta, TNFalpha, and Cox-2. We also examined trophic factor expression and found that CNTF enhances glial cell-line derived neurotrophic factor (GDNF) mRNA and protein secretion, whereas IL-6 has no effect. Correspondingly, conditioned media from CNTF-stimulated microglia promote motor neuron survival threefold beyond controls, whereas IL-6-stimulated microglia decrease neuronal survival twofold. To understand better the signaling mechanisms responsible for the opposite responses of these IL-6-family cytokines, we examined STAT-3 and ERK phosphorylation in CNTF- and IL-6-stimulated microglia. IL-6 markedly increases STAT-3 and ERK phosphorylation after 20 min of treatment, whereas these signal transducers are weakly stimulated by CNTF across a range of doses. We conclude that CNTF modifies microglial activation to support neuronal survival and that IL-6 enhances their capacity to do harm, as a result of different modes of intracellular signaling.     

Neurturin regulates postnatal differentiation of parasympathetic pelvic ganglion neurons, initial axonal projections, and maintenance of terminal fields in male urogenital organs

We have investigated the development of autonomic nerves in the urogenital tract of male mice and the effect of neurturin gene deletion on this process. At birth, autonomic innervation of the reproductive organs was sparse, but urinary bladder smooth muscle was well innervated. Further innervation of reproductive tissues occurred until P21, but noradrenergic axons established their complete terminal field later than nitrergic cholinergic axons: in adults the former are more prevalent, yet this became apparent only at P7 (vas deferens, seminal vesicles), P14 (prostate) or after P14 (penis). Neurturin was essential for initial projection of axons (mucosa of vas deferens), maintenance of terminal fields (prostate and seminal vesicles), or both functions (cavernosum of penis). In contrast, some targets (e.g., bladder muscle and suburothelium, vas deferens smooth muscle) were unaffected by neurturin gene deletion. Pelvic ganglion neurons more than doubled between birth and adulthood, probably as aresult of continued maturation of p75-positive undifferentiated neuronal precursors rather than cell division. The adult number of neurons was achieved by P7 (sympathetic) or P21 (parasympathetic). In adult neurturin knockout mice, there were approximately 25% fewer parasympathetic neurons compared with wild types, because of failure of differentiation after P14. This study revealed the complexity of postnatal maturation of urogenital innervation, with each organ showing a distinct chronology of innervation and different requirement for neurturin. Our results also indicate that in adults there will be distinct differences in neurturin dependence between organs, such that proregenerative therapies may have to be tailored specifically for the nerve pathway of interest.     

GFRα-1 is expressed in parvalbumin GABAergic neurons in the hippocampus

Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to GDNF-family receptor α-1 (GFRα-1). This receptor is expressed in different areas of the brain, including the hippocampus and dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase (GAD) expressing neurons co-expressed GFRα-1 in the hippocampus. GFRα-1/GAD co-expression was found mainly in the stratum (s) pyramidale (29–37%) and s. oriens (20–25%). Further characterization of GFRα-1 expressing interneurons, based on their calcium-binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFRα-1 in the s. pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFRα-1/PV double labeled neurons were also detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFRα-1 in principal neurons and in a specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner, functions of the entire adult hippocampus.     

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.     

Dopamine D1and D2Receptor-Mediated Acute and Long-Lasting Behavioral Effects of Glial Cell Line-Derived Neurotrophic Factor Administered into the Striatum

To determine the differences in behavioral effects between intrastriatal and intracerebroventricular glial cell-derived neurotrophic factor (GDNF) administration, spontaneous locomotor activity was measured after intrastriatal or intracerebroventricular injection of GDNF (10 μg) in normal adult rats with implanted guide cannulae. In addition, the distribution of GDNF after intracerebral injection was studied immunohistochemically. Intrastriatal administration of GDNF significantly increased rearing behavior 3–4 h after injection. Increases in all three aspects of locomotor activity (motility, locomotion, and rearing) were most pronounced 3 days after intrastriatal injection, and they lasted for several days. This hyperactivity was blocked by the selective dopamine D₁receptor antagonist SCH22390 and by the selective D₂receptor antagonist raclopride at doses of the dopamine receptor antagonists, which by themselves did not affect spontaneous locomotor activity. These results suggest that GDNF has both acute and long-lasting pharmacological effects on dopamine neurons in adult animals and stimulates locomotor activity by activating both dopamine D₁and D₂receptors. On the other hand, intracerebroventricular administration of the same dose of GDNF failed to increase locomotor activity at any time during the test period (12 days). The immunohistochemical study demonstrated widespread distribution of GDNF in the entire body of the striatum within 24 h after intrastriatal injection. It also revealed deep penetration of GDNF from the ventricular space into the brain parenchyma after intracerebroventricular injection. GDNF-immunoreactive neuronal cell bodies were seen in the ipsilateral substantia nigra pars compacta most frequently 6 h after intrastriatal injection. The number of such cell bodies after intracerebroventricular administration, on the other hand, was much lower than that seen after intrastriatal administration. Taken together, these data suggest that intrastriatal administration of GDNF is an effective approach for affecting DA transmission. Long-lasting behavior effects are mediated via dopamine D1 and D2 receptors. Higher doses of GDNF would probably be needed using the intracerebroventricular route as compared to intraparenchymal delivery to exert effects on the nigrostriatal system in Parkinson's disease patients.     

COMMENTS

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

GDNF基因修饰的骨髓基质干细胞分泌物对多巴胺能神经元的营养作用

目的:克隆GDNF基因并修饰骨髓基质干细胞,观察该工程细胞分泌物对多巴胺能神经元的营养作用。方法:应用逆转录聚合酶链反应(RT-PCR)方法从新生小鼠大脑皮层细胞克隆出GDNFcDNA片断,以pECPP-Cl为载体导入骨髓基质干细胞,制备稳定表达GDNF基因的MSCs工程细胞,收集并浓缩MSCs和GDNF’基因修饰的MSCs工程细胞的条件培养液,通过MTT、倒置显微镜和免疫组织化学的方法观察MSCs和GDNF基因修饰的MSCs工程细胞分泌物对多巴胺能神经元的营养作用。结果:MSCs和GDNF基因修饰的MSC工程细胞分泌物均能促进多巴胺能神经元的存活和生长,MSCs工程细胞作用更强。结论:成功构建了GDNF基因修饰的MSCs工程细胞,该细胞对多巴胺能神经元有明显营养保护作用,在帕金森病治疗中可能有重要价值。     

TH和GDNF双转基因工程细胞对多巴胺能神经元的保护作用

目的将人的外源基因TH和GDNF共同转染SH-SY5Y细胞,建立一种可同时高效稳定表达TH和GDNF的工程细胞,探讨其在帕金森病(PD)基因治疗中的作用。方法将人GDNF和TH的cDNA构建于表达载体PcDNA3.0和PcDNA3.1,形成重组真核表达载体PcDNA3.1/hGDNF、和PcDNA3.0/hTH,同时转染至SH-SY5Y细胞系,利用RT-PCR鉴定筛选出高效稳定表达阳性克隆,将此工程细胞与大鼠原代多巴胺(DA)能神经元共培养,免疫细胞化学检测观察DA能神经元的数目和生长状态。结果成功建立了可同时高效稳定表达人TH和GDNF双基因的工程细胞,并且该细胞可防止DA能神经元退变死亡,有助于DA能神经元抵抗MPP+的毒性损伤。结论人TH和GDNF双转基因工程细胞对DA能神经原有防治兼顾的保护作用。、     

稳定表达GDNF基因的MN9D细胞可保护多巴胺能神经元

目的：建立一种可同时分泌多巴胺（DA）和GDNF的工程细胞，并研究其在帕金森病（PD）基因治疗中的可能作用。方法：克隆携带Kozak序列的GDNFcDNA，转染至可分泌DA的MN9D细胞系，将此工程细胞和大鼠原代多巴胶能神经元共培养，免疫组化检测DA能神经元。结果：发现该工程细胞可防止DA能神经元退变死亡，并有助于DA能神经元抵抗MPP+的毒性损伤。结论：本文首次将PD防治兼顾策略结合起来构建MN9D工程细胞，结果表明其在PD的基因治疗中可能具有重要的应用价值。