Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

Therapeutic potential of glial cell line-derived neurotrophic factor and cell reprogramming for hippocampal-related neurological disorders

Hippocampus serves as a pivotal role in cognitive and emotional processes,as well as in the regulation of the hypothalamus-pituitary axis.It is known to undergo mild neurodegenerative changes during normal aging and severe atrophy in Alzheimer's disease.Furthermore,dysregulation in the hippocampal function leads to epilepsy and mood disorders.In the first section,we summarized the most salient knowledge on the role of glial cell-line-derived neurotrophic factor and its receptors focused on aging,cognition and neurodegenerative and hippocampal-related neurological diseases mentioned above.In the second section,we reviewed the therapeutic approaches,particularly gene therapy,using glial cell-line-derived neurotrophic factor or its gene,as a key molecule in the development of neurological disorders.In the third section,we pointed at the potential of regenerative medicine,as an emerging and less explored strategy for the treatment of hippocampal disorders.We briefly reviewed the use of partial reprogramming to restore brain functions,non-neuronal cell reprogramming to generate neural stem cells,and neural progenitor cells as source-specific neuronal types to be implanted in animal models of specific neurodegenerative disorders.

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND： Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor （GDNF） injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE： To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease （PD）, and analyze the action pathway. DESIGN： A controlled observation. SETTING： Neurobiological Laboratory of Xuzhou Medical College. MATERIALS： Twenty-four male Kunming mice of 7 - 8 weeks old were used. GDNF, 1-methy1-4-pheny1-1,2,3,6-tetrahydropyridine （MPTP） were purchased from Sigma Company （USA）; LEICAQWin image processing and analytical system. METHODS： The experiments were carded out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group： GDNF 4-day group, phosphate buffer solution （PSB） 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μ L GDNF solution （20 μ g/L, dispensed with 0.01 mol/L PBS） injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12^th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase （TH） and calcium binding protein （CB） with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES： Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS： All the 24 mice were involved in the analysis of results. The numbers of TH^＋ and CB^＋ neurons in the GDNF 4-day group （54.33±6.92, 46.33±5.54） were obviously more than those in the PBS 4-day group （27.67±5.01, 21.50±5.96, P 〈 0.01）. The numbers of TH^＋ and CB^＋ neurons in the GDNF 6-day group （75.67±5.39, 69.67±8.69） were obviously more than those in the PBS 6-day group （27.17±4.50, 21.33 ±5.72, P 〈 0.01） and those in the GDNF 4-day group （P 〈 0.01 ）. CONCLUSION： Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.

Secretion of nerve growth factor,brain-derived neurotrophic factor,and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

The present study co-cultured human embryonic olfactory ensheathJng cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7-10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid （10%, 20% and 30%） in the growth medium caused a decrease of neurotrophic factor secretion Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Advances in treatment of neurodegenerative diseases: Perspectives for combination of stem cells with neurotrophic factors

Neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease,Huntington’s disease and amyotrophic lateral sclerosis,are a group of incurable neurological disorders,characterized by the chronic progressive loss of different neuronal subtypes.However,despite its increasing prevalence among the everincreasing aging population,little progress has been made in the coincident immense efforts towards development of therapeutic agents.Research interest has recently turned towards stem cells including stem cells-derived exosomes,neurotrophic factors,and their combination as potential therapeutic agents in neurodegenerative diseases.In this review,we summarize the progress in therapeutic strategies based on stem cells combined with neurotrophic factors and mesenchymal stem cells-derived exosomes for neurodegenerative diseases,with an emphasis on the combination therapy.

Establishment and expression of recombinant human glial cell linederived neurotrophic factor and TNF α receptor in human neural stem cells

Objective:To investigate the interference and expression of human glial cell line-derived neurotrophic factor(hCDNF) and soluble TNF alpha(sTMFRⅠ) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.Methods:Full-length of GDNF cDNA(538 bp) and sTMFRⅠcDNA(504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus(gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem ceils.The infection and expression of gene were tested under immunofluorescence.ELISA and Westem-blot after 48 hours.Results:Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP and KFP were observed in neural stem cells,which indicated the expression of GDNF and sTMFRⅠ.After transfection of GDNF and sTMFRⅠgenes,many neural stem cells show GFAP and tubulin immunofluorescence positive staining,which meant that most neural stem cells differentiated into neuron at that condition.Conclusions:The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF and sTMFRⅠgenes expressing in neural stem cells,which is useful for differentiation of neural stem cell.

Expression and significance of GDNF gene in recovery of spermatogenesis in mice

Objectives: To analyze the expression and significance of glial cell derived neurotrophic factor (GDNF) in the recovery of spermato genesis in mice. Methods: Adult Kunming mice were injected in traperitoneally with 2 doses of busulfan (10 mg/kg) 24 days apart so as to establish the spermatogenesis recovery modei. Testes were harvested at weeks l, 2, 3,4, 6, 8 and 10 after the second injection. Eight normai mice served as the controls. Recovery of spermatoge nesis was observed by light and electron microscopy and the GDNF mRNA measured by semi-quantitative RT-PCR and in situ hybridization. Results: After the second injection the expression of GDNF mRNA was increased significantly at week l and reached its peak at week 2. It was then decreased significantly at week 3 and reached its valley at week 4. After that it was increased gradu ally and recovered at week 10. GDNF mRNA was mainly ex pressed by the Sertoli celis. Conclusion: In the course of recovery of spermatogenesis, a high Ievel of GDNF expression plays a key role in the promotion of self-renewal and maintenance of the num ber of spermatogonial stem celis.

Antenatal taurine reduces cerebral cell apoptosis in fetal rats with intrauterine growth restriction

From pregnancy to parturition, Sprague-Dawley rats were daily administered a low protein diet to establish a model of intrauterine growth restriction. From the 12th day of pregnancy, 300 mg/kg taurine was daily added to food until spontaneous delivery occurred. Brain tissues from normal neonatal rats at 6 hours after delivery, neonatal rats with intrauterine growth restriction, and neo- natal rats with intrauterine growth restriction undergoing taurine supplement were obtained for fur- ther experiments. The terminal deoxyribonucleotidyl transferase （TdT）-mediated biotin-16-dUTP nick-end labeling assay revealed that the number of apoptotic cells in the brain tissue of neonatal rats with intrauterine growth restriction significantly increased. Taurine supplement in pregnant rats reduced cell apoptosis in brain tissue from neonatal rats with intrauterine growth restriction. Immu- nohistochemical staining revealed that taurine supplement increased glial cell line-derived neuro- trophic factor expression and decreased caspase-3 expression in the cerebral cortex of intrauterine growth-restricted fetal rats. These results indicate that taurine supplement reduces cell apoptosis through the glial cell line-derived neurotrophic factor-caspase-3 signaling pathway, resulting in a protective effect on the intrauterine growth-restricted fetal rat brain.

GDNF基因逆转录病毒表达载体的构建及转染神经干细胞的研究

应用基因重组技术将大鼠GDNF cDNA克隆到逆转录病毒载体pLXSN中,经酶切及PCR对重组质粒进行鉴定。将重组体包装到PA317细胞中,并感染增殖旺盛的大鼠神经干细胞,通过免疫细胞化学、RT-PCR和western-blot等方法鉴定转染效率。结果显示GDNF cDNA正确地克隆到逆转录病毒载体pLXSN中,该真核细胞表达载体成功转染增殖旺盛的神经干细胞并有效表达。因此本研究成功构建了GDNF真核细胞表达载体,并成功转染到神经干细胞中。为移植替代和基因治疗神经系统疾病提供了实验基础。

GDNF转染雪旺氏细胞构建神经导管复合体

目的利用GDNF(胶质神经源性神经营养因子)基因转染的雪旺氏细胞和PLGA(聚乳酸/聚乙醇酸共聚物)管共同培养构建神经导管复合体。方法利用乳鼠臂丛神经和坐骨神经培养雪旺氏细胞,再把pcDNA3.1(+)/GDNF质粒通过脂质体转入细胞内。利用PCR检测转染雪旺氏细胞的GDNF表达。将转染雪旺氏细胞扩增达到一定数量后,用微注射和共培养法,与PLGA管构建神经导管。通过光镜和扫描电镜检测雪旺氏细胞的生长情况。结果成功培养出雪旺氏细胞,并转入GDNF基因。转染的雪旺氏细胞可表达GDNF,PCR显示转染的雪旺氏细胞GDNF表达较正常细胞明显提高。转染的雪旺氏细胞可在PLGA管表面贴壁、生长。结论GDNF基因转染的雪旺氏细胞可与PLGA管共同构建神经导管复合体。

GDNF基因多态性与精神分裂症临床特征的相关性

目的探讨GDNF基因多态性与精神分裂症临床特征的相关性。方法对符合纳入标准的精神分裂症病例组及健康对照组进行临床资料收集及血样的采集,采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法检测GDNF基因多态性。选取GDNF基因2个SNP位点:rs2973050,rs2910702。所有数据应用SSPS13.0软件包处理。结果①哈迪温伯格平衡(Hardy-Weinberg s equilibrium)结果显示,GDNF基因rs2910702在病例组中偏离哈迪温伯格平衡(χ2=24.983,P=0.000);②GDNF等位基因频率在病例组与对照组中的分布无统计学差异(P>0.05),但基因型频率分布有统计学差异(P<0.05);③GDNF各基因型与精神分裂症的临床分型、PANSS量表各因子分无明显相关性。结论 rs2973050基因型C/C、rs2910702基因型G/G可能与精神分裂症的发生有关,为精神分裂症的危险基因型。

阳离子脂质体介导GDNF体内转基因对脊髓损伤后运动神经元的保护作用

目的 :观察阳离子脂质体介导的胶质细胞源性神经营养因子 (GDNF)体内转基因对大鼠脊髓损伤 (SCI)后伤区脊髓前角运动神经元的影响。方法 :采用改良Nystr m法制备大鼠胸段脊髓后路压迫损伤模型 ,将阳离子脂质体DC Chol和重组质粒 pEGFP GDNFcDNA混合后直接注入大鼠损伤脊髓。利用RT PCR技术和荧光显微镜检测GDNF体内转基因的表达。应用尼氏染色、酶组织化学染色方法观察前角运动神经元死亡的数目和胆碱酯酶(CHE)及酸性磷酸酶 (ACP)的变化。结果 :1周后在注射局部GDNFmRNA和GDNF有较高表达。GDNF体内转基因早期 (1～ 4周 )SCI区前角运动神经元死亡的数目减少 ,CHE和ACP变化的幅度降低。结论 :GDNF体内转基因能减少脊髓不完全性损伤后引起的神经元坏死 ,阳离子脂质体介导GDNF体内转基因治疗创伤性SCI的方法是可行的。

Type-B monoamine oxidase inhibitors in neurological diseases:clinical applications based on preclinical findings

Type-B monoamine oxidase inhibitors,encompassing selegiline,rasagiline,and safinamide,are available to treat Parkinson's disease.These drugs ameliorate motor symptoms and improve motor fluctuation in the advanced stages of the disease.There is also evidence suppo rting the benefit of type-B monoamine oxidase inhibitors on non-motor symptoms of Parkinson's disease,such as mood deflection,cognitive impairment,sleep disturbances,and fatigue.Preclinical studies indicate that type-B monoamine oxidase inhibitors hold a strong neuroprotective potential in Parkinson's disease and other neurodegenerative diseases for reducing oxidative stress and stimulating the production and release of neurotrophic factors,particularly glial cell line-derived neurotrophic factor,which suppo rt dopaminergic neurons.Besides,safinamide may interfere with neurodegenerative mechanisms,countera cting excessive glutamate overdrive in basal ganglia motor circuit and reducing death from excitotoxicity.Due to the dual mechanism of action,the new generation of type-B monoamine oxidase inhibitors,including safinamide,is gaining interest in other neurological pathologies,and many supporting preclinical studies are now available.The potential fields of application concern epilepsy,Duchenne muscular dystrophy,multiple scle rosis,and above all,ischemic brain injury.The purpose of this review is to investigate the preclinical and clinical pharmacology of selegiline,rasagiline,and safinamide in Parkinson's disease and beyond,focusing on possible future therapeutic applications.

大鼠脊髓损伤后坐骨神经GDNFmRNA表达变化及意义

目的 :探讨大鼠脊髓损伤后GDNFmRNA在坐骨神经的表达变化及其意义。方法 :Allen’s方法致大鼠T13段脊髓不完全损伤后 ,以 β Actin为内参照物 ,应用半定量RT PCR方法 ,观察大鼠坐骨神经在脊髓损伤前、后不同时间GDNFmRNA表达变化。结果 :脊髓损伤前GDNFmRNA在大鼠坐骨神经仅有微量表达 ,脊髓损伤后 2 4h表达增加 4倍 ,72h增加 15倍 ,7d增加 3倍 ,10d仍高于正常水平。结论 :脊髓损伤后坐骨神经高表达GDNFmRNA ,是神经元通过“胞体—轴突—靶器官”途径自我保护的一种反应形式 。

应用bcl-2基因及GDNF治疗大鼠脑缺血的研究

目的观察胶质细胞源性神经营养因子(GDNF)与bcl-2基因对大鼠局灶性脑缺血再灌注损伤(CIRI)的影响。方法取健康Wistar大鼠40只,采用Longa改良栓线法制成大脑中动脉闭塞(MCAO)模型,缺血2h后再灌注,随机分4组:对照组、bcl-2组、GDNF组、bcl-2+GDNF组,于再灌注3h后经侧脑室注射GDNF10μl、经颈动脉注射pLXSN-bcl-2质粒10μg,MCAO后3d(n=5),14d(n=5),通过免疫组化染色检测bcl-2蛋白和GDNF的表达情况,TUNEL法检测细胞凋亡情况。结果bcl-2+GDNF组bcl-2蛋白表达与GDNF表达水平均较其他组表达明显增加(P<0.05),bcl-2组、GDNF组较对照组明显增加(P<0.05);脑内细胞凋亡GDNF+bcl-2组较其他组明显减少(P<0.05),bcl-2组、GDNF组较对照组明显减少(P<0.05)。结论bcl-2基因和GDNF可能通过减少神经细胞凋亡,促进bcl-2与GDNF蛋白表达,增强对局灶性缺血脑组织的保护能力,加速中枢神经损伤的修复;bcl-2基因与GDNF协同作用,促使神经细胞在脑内的凋亡进一步减少,从而避免脑缺血再灌注后脑细胞进一步损伤。

Acupuncture promotes functional recovery after cerebral hemorrhage by upregulating neurotrophic factor expression

Acupuncture is widely used in the treatment of cerebral hemorrhage,and it improves outcomes in experimental animal models and patients.However,the mechanisms underlying the effectiveness of acupuncture treatment for cerebral hemorrhage are still unclear.In this study,a model of intracerebral hemorrhage was produced by injecting 50μL autologous blood into the caudate nucleus in Wistar rats.Acupuncture at Baihui(DU20)and Qubin(GB7)acupoints was performed at a depth of 1.0 inch,12 hours after blood injection,once every 24 hours.The needle was rotated at 200 r/min for 5 minutes,For each 30-minute session,needling at 200 r/min was performed for three sessions,each lasting 5 minutes.For the positive control group,at 6 hours,and 1,2,3 and 7 days after induction of hemorrhage,the rats were intraperitoneally injected with 1 mL aniracetam(0.75 mg/mL),three times a day.The Bederson behavioral test was used to assess palsy in the contralateral limbs.Western blot assay was used to examine the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia.Immunohistochemistry was performed to count the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Acupuncture effectively reduced hemorrhage and brain edema,elevated the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia,and increased the number of Nestin-and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia.Together,these findings suggest that acupuncture promotes functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors.The study was approved by the Committee for Experimental Animals of Heilongjiang Medical Laboratory Animal Center(approval No.2017061001)on June 10,2017.

Neurotrophic factors: from neurodevelopmental regulators to novel therapies for Parkinson's disease

Neuroprotection and neuroregeneration are two of the most promising disease-modifying ther- apies for the incurable and widespread Parkinson＇s disease. In Parkinson＇s disease, progressive degeneration of nigrostriatal dopaminergic neurons causes debilitating motor symptoms. Neurotrophic factors play important regulatory roles in the development, survival and maintenance of specific neuronal populations. These factors have the potential to slow down, halt or reverse the loss of nigrostriatal dopaminergic neurons in Parkinsoffs disease. Several neurotrophic fac- tors have been investigated in this regard. This review article discusses the neurodevelopmental roles and therapeutic potential of three dopaminergic neurotrophic factors： glial cell line-derived neurotrophic factor, neurturin and growth/differentiation factor 5.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.