The Role of Connexin32 in the Pathogensis of CMTX
Connexin32在CMTX发病机制中的作用
基本信息
- 批准号:8732705
- 负责人:
- 金额:$ 34.65万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2007
- 资助国家:美国
- 起止时间:2007-02-01 至 2015-08-31
- 项目状态:已结题
- 来源:
- 关键词:AcuteAffectAnimal ModelAreaAstrocytesAutopsyAxonBindingBiologyBiopsyBrainBuffersCell membraneCell modelCentral Nervous System DiseasesCervical spinal cord structureClinicalCo-ImmunoprecipitationsCommunicationConnexin 43ConnexinsConnexonCorpus CallosumCouplingDataDefectDemyelinating DiseasesDemyelinationsDiffusionDiseaseDominant-Negative MutationElectrophysiology (science)Fluorescence Recovery After PhotobleachingFrameshift MutationGap JunctionsGene FamilyGene MutationGenesGlucoseGoalsGrantHereditary Spastic ParaparesisHumanIntegral Membrane ProteinInvestigationIonsKnockout MiceLeadLymphedemaMagnetic Resonance ImagingMeasuresMetabolicMissense MutationMolecularMultiple SclerosisMusMutationMyelinNeurogliaNeuronsNeuropilOligodendrogliaOptic NervePaintPatientsPhenotypeProteinsProteolipidsRoleSliceStaining methodStainsTransgenic MiceVertebratesdysmyelinationexperimental analysisgap junction channelglucose analogin vivomolecular massmutantnovelnovel therapeutic interventionsmall moleculetraffickingwhite matter
项目摘要
DESCRIPTION (provided by applicant): Recessive mutations in GJC2, the human gene that encodes Cx47, cause Pelizaeus-Merzbacher-like disease (PMLD) and hereditary spastic paraparesis (HSP), presumably related their lack of Cx47 function in oligodendrocytes. How the loss of Cx47 function results in the clinical picture of PMLD is unknown. MRI paints the picture of profound dysmyelination, but this has yet to be confirmed in an autopsy or biopsy. In cellular and animal models, the mutants associated with PMLD impair GJ communication, but we do not yet know why GJ communication is essential for the proper functioning of oligodendrocytes. In this competing renewal, we will determine whether novel Cx47 mutants affect GJ coupling, explore whether O: O coupling a general feature of oligodendrocytes, and the functional significance of O: O coupling. 1. Investigate the molecular defects of hCx47 mutants causing PMLD or familial lymphedema. In the last grant cycle of this grant, we showed that 3 recessive Cx47 mutants associated with PMLD have defective trafficking and do not form functional channels with either Cx47 or Cx43. We will build on this observation by investigating 2 additional missense mutations, 2 frameshift mutations that affect the C-terminus of Cx47, deletion of the PDZ-binding domain (del433-437; a relevant, but not a naturally-occurring mutation), and 6 dominant mutations that cause a completely different phenotype, Familial Lymphedema. The ability of each mutant to form functional GJ plaques will be investigated - by immunostaining, scrape loading, fluorescence recovery after photobleaching (FRAP), and electrophysiology. Whether the dominant mutants have dominant-negative effects on WT Cx47 will also be investigated (by co-staining, co-immunoprecipitation, and electrophysiology). We will also generate lines of transgenic mice that express the Cx47del433-437 or R257C, one of the dominant mutations causing Familial Lymphedema. We will assess the ability of these mutants to "rescue" the phenotype of Gjc2-null (Gjc2-/-) mice, and of the R257C mutant to worsen the phenotype of Gjc2-heterozygous (Gjc2+/-) mice. 2. Is O: O coupling a general feature of oligodendrocytes? In the last grant cycle of this grant, we showed that O: A coupling in lost in mice lacking both Cx32 and Cx47. We also showed, expectedly, O: O coupling was prominent in the corpus callosum, that O: O coupling was also lost in mice lacking both Cx32 and Cx47, that the GJs directly join intrafascicular oligodendrocytes. The goal of this aim is to determine whether O: O coupling is found in other white matter tracts, and hence is a general feature of intrafascicular oligodendrocytes. To that end, we will measure the diffusion of sulforhodamine-B (SR- B), examine the ultrastructure of O: O junctions, and examine the expression of glial connexins (Cx30, Cx32, Cx43, Cx47) in two tracts - the optic nerve and the ventral funiculus of the cervical spinal cord. 3. The functional significance of O: O coupling. The function of O: A and O: O coupling is uncertain. The experimental results to date support two, non-mutually exclusive, functions - spatial buffering of K+ and metabolic cooperation. K+ clearance is modestly decreased in acute brain slices from mice in which astrocytes lack both Cx43 and Cx30. These mice also provide the best evidence for metabolic coupling, demonstrating that Cx30 and Cx43 are required for the intracellular diffusion of a fluorescent glucose analogue (2-NDBG) to areas of neuropil with increased neuronal activity. We reasoned that O: O coupling might serve a similar purpose for myelinated axons, and propose an experimental analysis of this possibility in this Aim. We will determine whether (a) activity increases the extent of 2-NDBG diffusion, and (b) whether glucose infused into a single oligodendrocyte can "rescue" axonal conduction in glucose-deprived conditions.
描述(由申请人提供):编码Cx47的人类基因GJC2的隐性突变可导致pelizaeus - merzbacer样疾病(PMLD)和遗传性痉挛性麻痹(HSP),可能与它们在少突胶质细胞中缺乏Cx47功能有关。Cx47功能的丧失如何导致PMLD的临床表现尚不清楚。核磁共振成像描绘了严重的髓鞘异常,但这还有待于尸检或活组织检查的证实。在细胞和动物模型中,与PMLD相关的突变体损害GJ通讯,但我们还不知道为什么GJ通讯对少突胶质细胞的正常功能至关重要。在这个竞争性更新中,我们将确定新的Cx47突变体是否影响GJ偶联,探索O: O偶联是否是少突胶质细胞的一般特征,以及O: O偶联的功能意义。1. 研究导致PMLD或家族性淋巴水肿的hCx47突变体的分子缺陷。在本研究的最后一个资助周期中,我们发现与PMLD相关的3个隐性Cx47突变体具有缺陷的转运,并且不能与Cx47或Cx43形成功能通道。在此基础上,我们将研究另外2个错义突变,2个影响Cx47 c端的移码突变,pdz结合域的缺失(del433-437,一种相关的,但不是自然发生的突变),以及6个导致完全不同表型的显性突变,家族性淋巴水肿。每个突变体形成功能性GJ斑块的能力将通过免疫染色、刮片加载、光漂白后荧光恢复(FRAP)和电生理学来研究。我们还将研究显性突变体是否对WT Cx47具有显性-负性影响(通过共染色、共免疫沉淀和电生理)。我们还将产生表达Cx47del433-437或R257C的转基因小鼠,这是导致家族性淋巴水肿的主要突变之一。我们将评估这些突变体“拯救”gj2 -null (Gjc2-/-)小鼠表型的能力,以及R257C突变体恶化gj2 -杂合(Gjc2+/-)小鼠表型的能力。2. O: O偶联是少突胶质细胞的普遍特征吗?在本基金的最后一个资助周期中,我们发现O: A偶联在缺乏Cx32和Cx47的小鼠中丢失。我们还发现,意料之中的是,O: O偶联在胼胝体中很突出,在缺乏Cx32和Cx47的小鼠中O: O偶联也缺失,GJs直接连接束内少突胶质细胞。本目的目的是确定O: O偶联是否存在于其他白质束中,因此是束内少突胶质细胞的普遍特征。为此,我们将测量硫代丹-B (SR- B)的扩散,检查O: O连接的超微结构,并检查神经胶质连接蛋白(Cx30, Cx32, Cx43, Cx47)在视神经和颈脊髓腹侧索两个束中的表达。3. O: O耦合的功能意义。O: A和O: O耦合的函数是不确定的。迄今为止的实验结果支持两个不相互排斥的功能- K+的空间缓冲和代谢合作。在星形胶质细胞缺乏Cx43和Cx30的小鼠急性脑切片中,K+清除率适度降低。这些小鼠也提供了代谢偶联的最佳证据,表明Cx30和Cx43是荧光葡萄糖类似物(2-NDBG)细胞内扩散到神经元活性增加的神经细胞区域所必需的。我们推断O: O耦合可能在髓鞘轴突中起到类似的作用,并在本研究中提出了这种可能性的实验分析。我们将确定(a)活性是否增加2-NDBG扩散的程度,以及(b)在葡萄糖缺乏的情况下,注入单个少突胶质细胞的葡萄糖是否可以“拯救”轴突传导。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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STEVEN Simon Scherer其他文献
STEVEN Simon Scherer的其他文献
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{{ truncateString('STEVEN Simon Scherer', 18)}}的其他基金
Autoimmune Mechanisms in Peripheral Neuropathy
周围神经病变的自身免疫机制
- 批准号:
10239173 - 财政年份:2018
- 资助金额:
$ 34.65万 - 项目类别:
How do dominant PMP2 mutations cause demyelinating neuropathy?
PMP2 显性突变如何导致脱髓鞘性神经病?
- 批准号:
9437210 - 财政年份:2017
- 资助金额:
$ 34.65万 - 项目类别:
How do dominant PMP2 mutations cause demyelinating neuropathy?
PMP2 显性突变如何导致脱髓鞘性神经病?
- 批准号:
9572452 - 财政年份:2017
- 资助金额:
$ 34.65万 - 项目类别:
The Role of Connexin32 in the Pathogensis of CMTX
Connexin32在CMTX发病机制中的作用
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8337714 - 财政年份:2007
- 资助金额:
$ 34.65万 - 项目类别:
The Role of Connexin32 in the Pathogensis of CMTX
Connexin32在CMTX发病机制中的作用
- 批准号:
8186867 - 财政年份:2007
- 资助金额:
$ 34.65万 - 项目类别:
The role of connexin32 in the pathogenesis of CMTX
Connexin32在CMTX发病机制中的作用
- 批准号:
7213822 - 财政年份:2007
- 资助金额:
$ 34.65万 - 项目类别:
The role of connexin32 in the pathogenesis of CMTX
Connexin32在CMTX发病机制中的作用
- 批准号:
7342822 - 财政年份:2007
- 资助金额:
$ 34.65万 - 项目类别:
The role of connexin32 in the pathogenesis of CMTX
Connexin32在CMTX发病机制中的作用
- 批准号:
7730833 - 财政年份:2007
- 资助金额:
$ 34.65万 - 项目类别:
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