Two main lines of research are pursued in our laboratory that can be ascribed to the broad fields of neuronal development and axoglial interactions during myelination. We use a combination of genetic, molecular, cellular and functional approaches: transgenic animal models, ex-vivo transplantation systems, proteomics, RNA microarray analysis, real time-PCR, imaging, cell and tissue culture, protein biochemistry, electrophysiology and behaviour.
Our main interest lies in the field of axon guidance and neuronal migration during development. It is well established that both of these events are crucial to the patterning of specific neural connections that are set-up during development. In particular we are working with signals that are mediating contact-dependent guidance and migration. We have been focusing on adhesion/recognition molecules of the Immunoglobulin superfamily (IgSF) and especially TAG-1/contactin-2 and its ligands. TAG-1 (transient axonal glycoprotein-1) is expressed transiently on the surface of several neuronal populations. TAG-1 promotes adhesion and neurite outgrowth in vitro and, in vivo, it serves as a contact-dependent growth cue. The protein displays both homophilic and heterophilic interactions with other members of the IgSF.
1. We have established a role for TAG-1 in the tangential migration of neurons of the caudal medulla and of cortical interneurons. In both systems blocking TAG-1 function results in altered and reduced migration in vitro. In vivo analysis of homozygous mutant mice for Tag-1 (Tag-1 -/- ) showed abnormalities in precerebellar nuclei that result form cell death. Ongoing analysis involves the role of TAG-1+ cells in tangential migrations in the cortex.
|Corticofugal axon tract (L1:red, TAG-1:green)||Dil tracing of the corticofugal axons in Tag-1 -/- and WT mice||Dil tracing of the corticofugal axons in Tag-1 -/- and WT mice|
|TAG-1 expression in developing telencephalon||Cortical interneurons growing on CHO-TAG-1 expressing cells as a substrate|
|GFP-expressing cortical interneurons||GFP-expressing proliferating cortical interneurons stained for GFP (green), Lhx6(red) and DAPI(blue)|
2. We have also established a role of small GTPases Rac1 and Rac3 in cortical interneuron development. We have identified Rac1 as an important intracellular mediator of interneuron development during the mitotic phase and also during the tangential migration to the cortex (Rac1 and 3). Although a multitude of extracellular cues are implicated in these processes, none of the intracellular components has been elucidated. We are currently engaged in understanding the downstream molecular pathways responsible for the morphological phenotypes of the single and double mutants; in addition, we are studying the functional development of cortical circuits in particular in the prefrontal cortex during postnatal development and in the two mutants, that is, in conditions of decreased inhibition. Interneurons have been implicated in many neurodevelopmental pathologies (epilepsy, autism, schizophrenia, bipolar disorder), thus their study, especially in the prefrontal cortex, is expected to provide important and timely information on the balance of excitation and inhibition.
|GFP- positive interneurons expressed in prefrontal cortex and barrel cortex of WT and Rac1/Rac3 KO animals|
|Representative voltage traces from spontaneous activity recordings from heterozygous (top) and Rac1 cKO (bottom) brain slices in control aCSF (Kalemaki et al., 2018)|
We are interested in the molecular organization and functional architecture of myelinated fibers. Myelinated fibers are organized into distinct functional domains (the node of Ranvier, the paranode/juxtaparanode, and the internode) characterized by molecular interactions between the axon and the overlying glial cell. Axons and myelinating glial cells reciprocally influence each other's development and trophism. The proper organization of myelinated fibers is crucial for the rapid propagation of action potentials along the axon. In pathological conditions such as multiple sclerosis or a number of neuropathies, the architecture of myelinated fibers is disrupted.
We have shown that TAG-1 is expressed by myelinating glia as well as axons in both the central and the peripheral nervous system. We have established that TAG-1 can directly interact with the axonal juxtaparanodal proteins Caspr2 and potassium channels. The formation of this tripartite protein complex is essential for the molecular organization of the juxtaparanodes, whereas it is disrupted in Tag-1 -/- mice. Glial TAG-1 is indispensable in juxtaparanodal complex formation and maintenance. We generated transgenic mice that exclusively express TAG-1 in glial cells and lack neuronal expression. Molecular, behavioral and ultrastructural analysis of those mice revealed the pivotal role of glial protein in juxtaparanodal protein organization, in myelination and axonal distribution in the optic nerve as well as oligodendrocyte maturation. Current projects involve:
1. The identification of new interactors or downstream effectors of glial TAG-1
2. Microneurotrophins in de/remyelination assays in vivo and in vitro
3. The role of autophagy in CNS myelin
|Teased sciatic nerve fibers||Paranodes (red) and juxtaparanodes in optic nerve|
|Staining of the corpus callosum with PLP (green), GFAP (red) and topro (blue) showing demyelination and accumulation of astrocytes in the lesion area|
|On the left oligodendrocyte precursor cell culture stained with PDGFRa (green) and P62 (red), while on the right, mature oligodendrocyte culture stained with MBP (green) and p62 (red)|
We participate in the following graduate programs:
- the interdepartmental graduate programs “Molecular Biology-Biomedicine” organized by the Department of Biology, the School of Medicine and the IMBB.
- the graduate program in Neuroscience organized by the School of Medicine of the University of Crete.
- the graduate program on "Cellular and Genetic Etiology, Diagnosis and Therapeutics of Human Disease ” organized by the School of Medicine of the University of Crete.