Neurology Clinic E
Generating cellular and animal models of inherited neuropathies: We have generated and published the first transgenic mice in Cyprus serving as models of inherited neuropathy and encephalopathy, and have been able to clarify the cellular mechanisms of the disease.
• Studying mechanisms of axonal degeneration in disease models of neuropathy.
• Generating disease models of multiple sclerosis: We have generated for the first time in Cyprus Experimental Autoimmune encephalomyelitis mouse models of Multiple Sclerosis, in order to study cellular and molecular mechanisms of the disease. We also use post‐mortem human brain tissue to confirm these findings.
• Developing new therapeutic approaches with gene replacement for inherited neuropathies and leukodystrophies: we have just started a gene therapy project using lentiviral vectors to deliver genes to peripheral nerves and the central nervous system.
• Studying disorders of nerve and brain excitability: we have made significant contributions in the last 5 years in the clarification of antigenic targets that cause autoimmune limbic encephalitis, neuromyotonia and Morvan's syndrome.
• Investigating molecular mechanisms of neuronal dysfunction caused by ion channel and cell adhesion molecule defects.
• Clinical and genetic investigation of families with inherited neuropathies and other neurogenetic disorders.
• Clinical and epidemiological study of myasthenia in Cyprus and investigation of new treatments.
Most of these research projects are based on on‐going collaborations with leading scientists at academic institutions in Europe and the USA, including the University of Pennsylvania, USA, Imperial College London and University of Oxford, UK, the University of Crete, Greece, and San Rafaelle Scientific Institute, Milan, Italy.In the past several years in research funded by the National Multiple Sclerosis Society of the USA, the Cyprus Research Promotion foundation, and the Cyprus Telethon, in collaboration with scientists at the University of Pennsylvania and Harvard Medical School, as well as other institutions, we have focused on the clinical aspects as well as cellular and molecular mechanisms of inherited peripheral neuropathies (Kleopa and Scherer, 2002), especially the X-linked form (X-linked Charcot-Marie-Tooth Disease, CMT1X), which is caused by mutations in the gap junction protein conexin32 (Yum et al., 2002; Kleopa et al., 2006b).A question that has driven the creation of cellular and animal models of the disease in our lab was the mechanisms leading to the CNS phenotype associated with several of the connexin32 mutations (Kleopa et al., 2002). Additional gap junction proteins in oligodendroglia were characterized and possible interactions of mutant connexin32 with these gap junction proteins in the CNS are under investigation (Altevogt et al., 2002; Kleopa et al., 2004). We recently published our work on the expression of Cx31.3 in human oligodendrocytes and showed that Cx32 mutants with CNS manifestations did not affect the function of this gap junction in vitro (Sargiannidou et al., 2008).
To investigate further the molecular basis of the phenotypes caused by connexin32 mutations we have generated for the first time transgenic mice expressing representative CMT1X mutations in both central and peripheral myelinating cells, and have created mutant lines both on wild type as well as on knockout background. We have shown that the transgene is expressed in oligodendrocytes throughout the CNS and in Schwann cells. The connexin32 mutants (T55I and R75W) are localized in the perinuclear cytoplasm, do not form GJ plaques, and do not alter the expression and localization of two other oligodendrocytic GJ proteins, Cx47 and Cx29, or the expression of Cx29 in Schwann cells. On wild type background, the expression of endogenous mCx32 is unaffected by the T55I mutant, but is partly impaired by R75W. Transgenic mice with the R75W mutation and all mutant animals with Gjb1-null background develop a progressive demyelinating peripheral neuropathy along with CNS myelination defects. These findings suggest that Cx32 mutations result in loss of function in myelinated cells without trans-dominant negative effects on other GJ proteins (Sargiannidou et al., 2009).
Semithin sections of femoral motor nerves from transgenic mice expressing connexin32 mutations on wild type (B-C) and on knockout (E-F) background compared to wild type (WT) and knockout (KO) mice at 8 months of age. There is significant de- and remyealination in all KO lines, and to a milder degree also in the R75W mutant, due to dominant effect of the R75W on endogenous connexin32 (Sargiannidou et al, 2009).
Expression of connexin32 mutations that cause CMT1X in Schwann cells. Both the T55I and R75W mutants are abnormally localized in the perinuclear cytoplasm. While the R75W mutant impairs the expression of the endogenous Cx32, neither T55I nor R75W affect the expression of another gap junction protein, Cx29 (Sargiannidou et al., 2009).
Expression of Cx32 mutations in oligodendrocytes shows that they are abnormally localized in the perinuclear cytoplasm and do not affect the expression of Cx47, the other major gap junction protein in these cells. The R75W mutant impairs the expression of endogenous Cx32 as it does in the peripheral nerves (Sargiannidou et al., 2009).
The molecular architecture as well as genetic and aquired-autoimmune disorders of neuronal voltage gated ion channels has also been a main focus of his research. Especially the expression and characterization of novel potassium channels in the central and peripheral nervous system (Devaux et al., 2004), and the study of the molecular mechanisms in patients with acquired hyperexcitability of the peripheral and/or central nervous system, in the forms of neuromyotonia and limbic encephalitis (Kleopa et al., 2006a; Vincent et al., 2006; Bataller et al., 2007) has been pursuit in collaboration with scientists form the University of Oxford and University of Pennsylvania.
We have also studied the expression of ion channels in transgenic mice that lack the cell adhesion molecule TAG-1, in collaboration with Scientists at the University of Crete and the University of Athens. This work has shown that deficiency of TAG-1 results in altered expression of voltage gated potassium channels, leading to hyperexcitability and deficits in memory and learning (Savvaki et al., 2008). We currently analyze the alterations of sodium channels in the same model.
Disrupted clustering of Kv1.1 and Kv1.2 Shaker-type potassium channels in juxtaparanodes of the corpus callosum in TAG-1 deficient mice, and increased density of nodes of Ranvier (Savvaki et al., 2008).
Ongoing and future research projects focus on the further study of molecular mechanisms in animal models of CMTX with the aim to develop possibilities for future therapies; on the role of gap junctions in central myelin in health and disease, especially multiple sclerosis, in collaboration with Imperial College of Medicine; and on the study of autoimmune mechanisms involving ion channels throughout the nervous system in a variety of clinical conditions including chronic pain.