Exploring the physiopathological mechanisms of connexinopathies

General objective: We focus our research into elucidate the role of cell communication by connexins in the physiology of different tissues upon normal and pathological conditions. Physiopathological mechanisms of several human diseases associated with dysfunction in connexins are largely unknown. Currently our activity is covering several lines of research:

1. Role of Connexin-36 in Epilepsy and genesis of Brain Rhythms upon Physiological and Pathological conditions. We study the contribution of electrical synapses mediated by Connexin-36 to the generation and maintenance of electrical activity in the brain upon normal and pathologic conditions such as the epilepsy.

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Electroencephalography (EEG) recordings in mice. (A) Picture showing a mouse with electrodes stereotaxically implanted in brain cortex and hippocampus. (B) Examples of EEG recordings in mice. Note in middle and bottom traces an example of seizure activity induced by pentylenetetrazole (arrows).

2. Therapeutic approaches for stroke in preclinical models. Connexin deficiency is associated with better outcome after stroke in animal models. We are testing Connexin inhibitors for stroke treatment in preclinical phase. In parallel we are using Bone Marrow (BM) Mesenchymal Stem Cells encapsulated in silk fibroins to promote functional recovery in a model of focal ischemia.

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Stem Cell Therapy for Stroke. (A) Longitudinal femoral section, a tissue that is preferentially used as donor for BM cells. (B) BM stroma expanded ex_vivo is also used as source for transplantation in mice. (C) Focal ischemia is induced in mice by middle cerebral artery occlusion (dMCAO). (D) Focal ischemia by dMCAO causes brain damage mainly at the somatosensoral cortex. (E) Electroencephalography recordings in mice are used as a method to evaluate functional recovery after stem cell transplantation in mice. Current focus is on the use of bone marrow (BM) mesenchymal stem cells in preclinical models of focal brain ischemia (longitudinal femoral BM section and EGFP mesenchymal cells on left side). Functional recovery is evaluated by different neuro-physiological tests including spontaneous or evoked potentials recordings in mice (right side).

3. Connexins in the Bone Marrow Hematopoietic Niche. Our aim is to study whether Connexin signaling is essential in the interaction between hematopoietic stem cells and the stroma microenvironment, and analyze the relevance of such interaction in the context of hematopoietic stem expansion and traffic.

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Cell signaling through Connexin channels in the Bone Marrow. We are using different cell, molecular and animal biology techniques to study the role of Connexins in the BM microenvironment. (A) Example of a confocal microscope image showing stromal cells cultures transduced with a vector containing the Connexin-43 cDNA linked with the EGFP sequence as reporter gene. (B) Cx43 Gap Junctions channels between apposed stromal cells can be detected by inmunofluorescence. (C) y (D) Flow cytometry techniques are commonly used in our lab to inmunophenotipically identify the different BM cell populations. (D) Deficiency of Connexin-43 causes bone marrow failure during serial transplantation in mice, emphasizing the importance of these proteins in the hematopoietic function.

4. Pathogenic Mechanisms in Myelin Disorders caused by Mutations in Connexin-46.6 and Connexin-43. We are investigated the role of connexins in the pan-glial syncitium and their function on myelin regulation and maintenance in peripheral and central nervous system.

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Electrophysiological recordings in Xenopus Oocytes. We use electrophysiological techniques to detect intercellular and transmembrane currents associated with different Connexins expressed in glial cells. We evaluate functional alterations caused by Connexin-46.6 and Connexin-43 mutations identified in patients with myelin disorders. (A) Picture showing an single oocyte injected with the RNA encoding Connexin-46.6. (B) A second stage of the experimental procedure; Oocytes are putting together to favor the formation of intercellular channels between them.  (C) Example of electrophysiological recordings in the voltage-clamp configuration to detect and quantify the magnitude of intercellular current flowing between both oocytes in response to intercellular voltage pulses.

Laboratories:

  • Experimental and Computational Neurology
  • Biomaterial and Tissue Engineering
  • Molecular Biology and Biochemistry: Biofunctionalization

Productivity

Contact: Daniel Gonzalez Nieto

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