Modulating Wnt signaling to improve cell replacement therapy for Parkinson＇s disease

Clinical trials have demonstrated the capacity for dopamine neurons, transplanted ectopicaUy into the striatum, to structurally inte- grate, restore dopamine transmission, and induce long-term functional benefits for Parkinson＇s disease （PD） patients. Despite this proof of principle, a number of limitations have hindered the development of cell replacement therapy over the past 20 years, particu- larly tissue availability, graft survival, and adequate reinnervation of the host brain. With a greater understanding of failure in prior clinical trials, increased knowledge of midbrain dopamine development （now including Wnts）, and the development of pluripotent stem cell technologies, we are better equipped than ever to re-address a number of these challenges. This review summarizes the trials, tribulations, and progress in cell replacement therapy for PD. We discuss the prospects of modulating canonical and non-canon- ical Wnt signalingto improve cell therapy based upon their roles in dopamine neural development and the adult brain. This will include the potential of Wnts to （i） expand fetaUy derived tissue in vitro and foUowing transplantation, （ii） promote the differentiation of pluripotent stem cells, （iii） increase graft integration and restoration of neural circuitry, and finally （iv） enhance graft survival.     

Targeting Wnt signaling at the neuroimmune interface for dopaminergic neuroprotectionJrepair in Parkinson＇s disease

During the past three decades, the Wingless-type MMTV integration site （Wnt） signaling cascade has emerged as an essential system regulating multiple processes in developing and adult brain. Accumulating evidence points to a dysregulation of Wnt signaling in major neurodegenerative pathologies including Parkinson＇s disease （PD）, a common neurodegenerative disorder characterized by the pro- gressive loss of midbrain dopaminergic （mDA） neurons and deregulated activation of astrocytes and microglia. This review highlights the emerging link between Wnt signaling and key inflammatory pathways during mDA neuron damage/repair in PD progression. In particular, we summarize recent evidence documenting that aging and neurotoxicant exposure strongly antagonize Wnt/β-catenin signaling in mDA neurons and subventricular zone （SVZ） neuroprogenitors via astrocyte-microglial interactions. Dysregulation of the crosstalk between Wnt/β-catenin signaling and anti-oxidant/anti-inflammatory pathways delineate novel mechanisms driving the decline of SVZ plasticity with age and the limited nigrostriatal dopaminergic self-repair in PD. These findings hold a promise in devetoping therapies that target Wnt/β-catenin signaling to enhance endogenous restoration and neuronal outcome in age-dependent diseases, such as PD.     

Mitochondrial dysfunction in Parkinson＇s disease： a possible target for neuroprotection

Mitochondria are dynamic organelles which are required for maintaining cellular homeostasis. Thus, it is not surprising that irregularities in mitochondrial function result in cellular damage and are linked with neurodegenerative diseases, such as Parkinson＇s disease. Evidence that mitochondrial dysfunction is key to the pathogenesis of Parkinson＇s disease is founded in studies in post-mortem tissue from patients with Parkinson＇s disease, and also from genetic studies stemming from patients with familial Parkinson＇s disease. Whether triggered by environmental or genetic factors, mitochondrial dysfunction occurs early in the pathogenic process, and is central to Parkinson＇s disease pathology. As such, targeting the mitochondria to slow or halt disease progression is an attractive strategy for disease-modifying agents in Parkinson＇s disease. Indeed, several therapies which target the mitochondria have been investigated as neuroproteetive treatments for Parkinson＇s disease. This review will discuss the evidence supporting mitochondrial dysfunction in Parkinson＇s disease pathology as well as treatment strategies that target the mitochondria.     

The lysosome and neurodegenerative diseases

It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer＇s disease, Parkinson＇s disease, Huntington＇s disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by iysosomal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.     

LRRK2基因与帕金森病

帕金森病是一种复杂的多因素共同作用的神经变性性疾病,遗传因素和环境因素被认为是发病的重要原因。越来越多的研究发现遗传因素在其发病中起着重要作用,而最近LRRK2基因的发现更加确定了遗传因素在帕金森发病中不可忽视的作用。本文对LRRK2基因以及LRRK2基因在帕金森病中的研究做一综述。     

Wnt signaling in the nervous system and in Alzheimer＇s disease

Wnts comprise a large family of proteins that have shown to be part of a signaling cascade that regulates several aspects of develop- ment including organogenesis, mid brain development as welt as stem cell proliferation. Wnt signaling pathway plays different roles in the development of neuronal circuits and also in the adult brain, where it regulates synaptic transmission and plasticity. It has been also implicated in various diseases including cancer and neurodegenerative diseases, reflecting its relevance in fundamental biological pro- cesses. This review summarizes the progress about Wnts function in mature nervous system with a focus on Alzheimer＇s disease （AD）. We discuss the prospects of modulating canonical and non-canonical Wnt signaling as a strategy for neuroprotection. This will include the potential of Wnts to： （i） act as potent regulators of hippocampai synapses and impact in learning and memory; （ii） regulate adult neurogenesis; and finally （iii） control AD pathogenesis.     

蝎毒耐热蛋白对MPTP诱导的帕金森病小鼠脑内小胶质细胞的影响

目的：进一步探讨蝎毒耐热蛋白（SVHRP）改善MPTP（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP）小鼠伴有空间学习记忆障碍的机制。方法：给予C57BL／6小鼠颈部皮下注射MPTP（20mg／kg）,连续8d,同时设立SVHRP治疗纽,观察小胶质细胞免疫反应活性的改变。结果：与盐水对照组相比,MPTP小鼠脑区OX-42免疫反应阳性小胶质细胞免疫反应活性明显增强。模型给药组与模型组相比OX-42免疫反应阳性小胶质细胞免疫反应活性明显降低。结论：SVHRP可以抑制MPTP诱发的小鼠脑内小胶质细胞的激活以减轻脑内神经炎症。     

Dysfunction of Wnt signaling and synaptic ：lisassembly in neurodegenerative diseases

The molecular mechanisms that regulate synapse formation have been well documented. However, little is known about the factors that modulate synaptic stability. Synapse loss is an early and invariant feature of neurodegenerative diseases including Alzheimer＇s lAD） and Parkinson＇s disease. Notably, in AD the extent of synapse loss correlates with the severity of the disease. Hence, understanding the molecular mechanisms that underlie synaptic maintenance is crucial to reveal potential targets that will allow the development of ther- apies to protect synapses. Writs play a central role in the formation and function of neuronal circuits. Moreover, Wnt signaling compo- nents are expressed in the adult brain suggesting their role in synaptic maintenance in the adult. Indeed, blockade of Wnts with the Wnt antagonist Dickkopf-1 （Dkkl） causes synapse disassembly in mature hippocampal cells. Dkkl is elevated in brain biopsies from AD patients and animal models. Consistent with these findings, Amyloid-β （Aβ） oUgomers induce the rapid expression of Dkkl. Importantly, Dkkl neutralizing antibodies protect synapses against Aβ toxicity, indicating that Dkkl is required for Aβ-mediated synapse loss. In this review, we discuss the role of Wnt signaling in synapse maintenance in the adult brain, particularly in relation to synaptic loss in neurodegenerative diseases.     

突触囊泡循环障碍与帕金森病

帕金森病是人类第二大神经退行性疾病,虽然通过对帕金森病遗传家族的研究已发现不少与之相关的致病基因,然而其病因尚不清楚。在已发现的与帕金森病相关基因中,α-synuclein、LRRK2、Parkin、PINK1以及DJ-1也在调节神经元突触前囊泡的神经递质释放以及突触前囊泡循环过程中发挥重要作用。最近的一些研究表明突触前囊泡循环障碍在帕金森病发病过程中扮演重要角色。本文对上述基因在突触囊泡循环以及其突变导致帕金森病病程中的作用作一概述,并推测囊泡循环障碍在帕金森病发病过程中的作用机理,最后指出目前在该研究领域需要解决的一些问题。     

TRPC1 protects dopaminergic SH-SY5Y cells from MPP＋, salsolinol,and N-methyl-（R）-salsolinol-induced cytotoxicity

Neurotoxins and alterations in Ca2＋ homeostasis have been associated with Parkinson＇s disease （PD）, but the role of store-operated Ca2＋ entry channels is not well understood. Previous studies have shown the neurotoxicity of salsolinol and 1-methyl-4-phenylpyridinium ion on SH-SY5Y cells and cytoprotection induced by transient receptor potential protein 1 （TRPC1）. In the present study, N-methyl-（R）-salsolinol was tested for its cellular toxicity and effects on TRPC1 expression. MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-dipbenyl- tetrazolium bromide） assays, DAPI （4＇,6-diamidino-2-pheny- lindole）, fluorescein isothiocyanate-Annexin-V/propidium iodide, western blot analysis, and JC-1 labeling revealed that the three indicated drugs could induce caspase-dependent, mitochondrial-mediated apoptosis. Exposure of SH-SY5Y cells to the indicated drugs resulted in a significant decrease in thapsigargin-mediated Ca2＋ influx and TRPC1 expression. Immnnocytochemistry experiments revealed that neurotoxins treatment induced TRPC1 translocation to the cytoplasm. Taken together, our results indicate that treatment with neurotoxins may alter Ca2＋ homeostasis and induce mitochondrial-mediated caspase-dependent cytotoxicity, an important characteristic of PD.     

Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1

Several mutations have been found in the leucine-rich repeat kinase 2 gene (LRRK2), encoding the protein dardarin, which are associated with autosomal dominant Parkinson disease. We have previously shown that mutant LRRK2/dardarin is toxic to neurons and neuron-like cell lines in culture and that some mutations are also associated with an inclusion-body phenotype. There is a homologous kinase, LRRK1, which has a similar domain structure but is not known to carry mutations causing Parkinson disease. In the current study, we introduced mutations at equivalent residues in both LRRK2 and LRRK1 to determine their effects in cells. We show that mutations in dardarin are more prone to form inclusion bodies in transfected cells and are more toxic than equivalent mutations in LRRK1. This work suggests that dardarin/LRRK2 is inherently more damaging than LRRK1.     

G蛋白偶联受体激酶5在稳定表达α-Synuclein的SHSY5Y细胞中的基因表达调控功能

目的观测G蛋白偶联受体激酶5（G protein-coupled receptor kinase,GRK5）在稳定表达hα-synuclein（人突触核蛋白）的SHSY5Y细胞的胞核、胞浆中的表达情况并对其在帕金森病中的可能作用进行研究。方法应用Western blotting、组蛋白去乙酰化酶（histone deacetylase,HDAC）活性检测技术以及shRNA干扰技术等对稳定表达hα-synuclein的SHSY5Y细胞中GRK5的表达及其亚细胞分布、胞核内GRK5蛋白的功能进行研究。结果发现GRK5蛋白在过表达hα-synuclein的细胞核以及细胞浆内均表达增加,胞核中的GRK5蛋白通过影响组蛋白去乙酰化酶的活性对bcl-2基因的转录和表达进行调控。结论帕金森模型中GRK5通过对bcl-2基因的表达进行调控发挥作用。     

尼古丁在帕金森病中的神经保护作用

帕金森病(Parkinson’s disease,PD)是常见的中老年神经退行性疾病。研究表明,尼古丁具有抵抗黑质多巴胺神经元损伤的作用,其通过烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)途径与非受体途径抑制帕金森病的发生与发展。本文就尼古丁在帕金森病中的神经保护作用以及保护机制的相关研究进行综述。     

含CpG基元核酸疫苗免疫慢性MPTP帕金森病小鼠的治疗作用

目的探讨含有CpG基元的α-突触核蛋白（α-synuclein,α-syn）核酸疫苗免疫慢性帕金森病小鼠的治疗效果。方法将本实验室成功构建的含有CpG基元的α-syn核酸疫苗-pVAX1-hα-Syn140用Qiagen试剂盒大量制备α-syn质粒;24只慢性MPTP帕金森病小鼠随机分为3组：实验组、空质粒对照组和PBS对照组,各组小鼠分别肌注pVAX1-hα-Syn140重组质粒、pVAX1空质粒和PBS各100μL,共免疫3次,每次间隔3周,末次免疫后2周,观察小鼠行为学变化及中脑黑质α-syn表达和多巴胺能神经元数目变化。结果pVAX1-hα-Syn140核酸疫苗免疫组小鼠的类帕金森病样症状与pVAX1空质粒和PBS对照组相比减轻,有显著差异（P〈0．01）;小鼠中脑黑质α-syn表达较对照组减少约39％（P〈0．01）,且多巴胺能神经元数目较空质粒和PBS对照组增多了46％～55％（P〈0．01）。结论pVAX1-hα-Syn140核酸疫苗具有较强的免疫原性,能对帕金森病小鼠产生较好的免疫治疗作用。     

The Role of LRRK2 in Intracellular Organelle Dynamics

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene hyperactivate LRRK2 kinase activity and lead to the development of Parkinson’s disease (PD). Membrane recruitment of LRRK2 and the identification of RAB GTPases as bona fide LRRK2 substrates strongly indicate that LRRK2 regulates intracellular trafficking. This review highlights the current literature on the role of LRRK2 in intracellular organelle dynamics. With a focus on the effects of LRRK2 on microtubule function, mitochondrial dynamics, the autophagy-lysosomal pathway, and synaptic vesicle trafficking, it summarizes our current understanding of how intracellular dynamics are altered upon pathogenic LRRK2 hyperactivation.     

帕金森病α-Synuclein转基因小鼠模型中G蛋白偶联受体激酶5的表达

目的观测G蛋白偶联受体激酶5（G protein-coupled receptor kinase,GRK5）在帕金森病α-synuclein转基因小鼠模型中的表达变化情况,了解GRK5在帕金森病中的可能作用,为发现帕金森病发病机制和探索更好的治疗方法提供新的方向。方法采用Western blotting和实时荧光定量PCR技术对具有不同的人alpha synuclein（hα--syn）表达水平的帕金森病α-synuclein转基因模型小鼠以及3月龄,6月龄以及9月龄A53T突变型帕金森病α-synuclein转基因模型小鼠脑组织进行GRK5的RNA和蛋白水平检测,与同窝阴性对照小鼠进行比较。结果各组帕金森病α-synuclein转基因小鼠与阴性对照小鼠相比,GRK5蛋白表达水平均有不同程度的增加,并且随着转入的hα--syn蛋白表达水平的高低而有所变化。3月龄和6月龄帕金森病转基因模型小鼠与同月龄阴性对照组小鼠相比,GRK5的mRNA和蛋白水平没有变化;而9月龄帕金森病转基因模型小鼠与同月龄阴性对照组小鼠相比,GRK5的mRNA和蛋白水平都有所增加。结论帕金森病α-synuclein转基因模型小鼠具有更高表达水平的GRK5。     

帕金森病的研究进展

帕金森病是一种中老年人常见的运动障碍性疾病,年龄是最主要的危险因素,已经显示出遗传倾向性的一些易感基因包括α-突触核蛋白、LRRK-2、GBA等也是重要的发病因素。多巴胺的替代治疗是帕金森病最重要的治疗办法,可以显著减轻病人的运动障碍,胚胎干细胞移植及基因治疗都是在探索中及具广阔前景的治疗办法。     

TRPC1和Ca2＋在帕金森病内质网应激中的作用

帕金森病是一种常见的神经系统退行性疾病,内质网应激在帕金森病发病过程中具有重要的作用。瞬时受体电位阳离子通道1（transient receptor potential channel 1,TRPCI）是非选择性的阳离子通道,主要位于细胞膜上,叮以调节Ca2＋的内流和抑制内质网应激所致的神经元凋亡。Ca2＋是细胞内重要的第二信使,其浓度的稳定对维持细胞的功能起着重要作用。内质网是细胞内Ca2＋储存的重要细胞器。因此,TRPCI和Ca2＋在帕金森病中起着重要作用。综述了TRPCI和Ca2＋与帕金森病内质网应激的相关进展。     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.