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.

Public Health Relevance

Mutations in GJB1 (encodes Cx32) and GJC2 (encodes Cx47) cause demyelinating diseases in humans. To devise rational treatments for these diseases, it is imperative to determine how and why these mutations cause demyelination. The investigations proposed in this grant will examine how recessive and dominant GJC2 mutations affect the ability of the mutant proteins to form functional gap junctions, including their effects on the ability of gap junction coupling between glial cells in vivo. These data will enhance our understanding of the biology and pathobiology of oligodendrocytes, and may elucidate important aspects of other demyelinating diseases such as multiple sclerosis, and may lead to novel therapeutic approaches.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Cellular and Molecular Biology of Glia Study Section (CMBG)
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Morris, Jill A
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University of Pennsylvania
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Wasseff, Sameh K; Scherer, Steven S (2015) Activated immune response in an inherited leukodystrophy disease caused by the loss of oligodendrocyte gap junctions. Neurobiol Dis 82:86-98
Abrams, Charles K; Scherer, Steven S; Flores-Obando, Rafael et al. (2014) A new mutation in GJC2 associated with subclinical leukodystrophy. J Neurol 261:1929-38
Wasseff, Sameh K; Scherer, Steven S (2014) Activated microglia do not form functional gap junctions in vivo. J Neuroimmunol 269:90-3
Abrams, Charles K; Scherer, Steven S (2012) Gap junctions in inherited human disorders of the central nervous system. Biochim Biophys Acta 1818:2030-47
Ferguson, Toby A; Scherer, Steven S (2012) Neuronal cadherin (NCAD) increases sensory neurite formation and outgrowth on astrocytes. Neurosci Lett 522:108-12
Kleopa, Kleopas A; Abrams, Charles K; Scherer, Steven S (2012) How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease? Brain Res 1487:198-205
Scherer, Steven S; Kleopa, Kleopas A (2012) X-linked Charcot-Marie-Tooth disease. J Peripher Nerv Syst 17 Suppl 3:9-13
Zhang, Junxian; Scherer, Steven S; Yum, Sabrina W (2011) Dominant Cx26 mutants associated with hearing loss have dominant-negative effects on wild type Cx26. Mol Cell Neurosci 47:71-8
Holzbaur, Erika L F; Scherer, Steven S (2011) Microtubules, axonal transport, and neuropathy. N Engl J Med 365:2330-2
Wasseff, Sameh K; Scherer, Steven S (2011) Cx32 and Cx47 mediate oligodendrocyte:astrocyte and oligodendrocyte:oligodendrocyte gap junction coupling. Neurobiol Dis 42:506-13

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