What are the physiological roles of connexin channels?
How are their mutations involved in neurodegeneration?

Connexins are the building blocks of intercellular communication in vertebrates. Hence, they determine tissue homeostasis and frequently underlie human pathogenesis. We are interested in understanding the molecular function of connexin channels in physiology and pathology, in particular associated with neurodegeneration. Our investigation is conceived to attain groundbreaking findings using an innovative approach which combines Physics, Biology and Medicine.

Background
Research
Group Members
Key Publications

The Bortolozzi lab is currently focusing on the study of connexin 32 (Cx32) protein, whose mutations cause the X-linked form of Charcot-Marie-Tooth peripheral neuropathy (CMTX1), a degenerative motor and sensory disorder for which there is no cure. Since the first mutations were reported in 1993, over 450 different mutations associated with CMTX1 have been identified in the GJB1 gene, which encodes Cx32. Despite the availability of numerous studies of normal and mutant Cx32 investigated under a variety of conditions, the molecular function of Cx32 channels in health and disease of the peripheral nervous remains unknown. Bortolozzi lab’s research points towards clarifying this function by (i) a combination of in silico, in vitro and in vivo techniques; (ii) cellular models based on human stem cells to mimic the peripheral nervous system as well as CMTX1 features; (iii) therapeutic strategies investigated at the single Cx32 channel level. We recently suggested a possible key role of Cx32 hemichannels in the molecular pathogenesis of CMTX1 and proposed a therapeutic strategy based on mimetic peptides (Carrer et al., Human Molecular Genetics, 2017).

What is the molecular function of Cx32 in the peripheral nervous system?
Cx32 is a 32 kDa protein of the connexin family that is abundantly found in liver, but it is also expressed in many other tissues, including the central and peripheral nervous systems. In the peripheral nervous system, Cx32 localizes only in myelinating Schwann cells, mainly to the paranodes, the periodic interruptions in the compact myelin called Schmidt–Lanterman incisures, and the two outer layers of myelin (Figure 1). Elucidation of the molecular function of Cx32 in myelinating Schwann cells is a requirement for understanding how different mutations lead to the sequence of events that end in demyelination and axonal loss in CMTX1 patients.


Figure.1 Diagram showing one Schwann cell and its myelin sheath unrolled from a peripheral axon (top) and a longitudinal section of the physiological Schwann cell rolled configuration (bottom), including Cx32 channel location (Adapted from [1]).

What biophysical properties of Cx32 channels are altered by CMTX1 mutations?
HeLa cells provide a useful in vitro system to perform biophysical experiments for the study of Cx32 mutations up to the single channel level. In our lab, we combine electrophysiology and optical fluorescence microscopy to analyze permeability properties of normal (wild-type, WT) and mutant Cx32 channels expressed in HeLa cells. The comparison between biophysical properties of WT and CMTX1 mutant channels facilitates the identification of specific molecular interactions that underlie permeability and gating dysfunctions and provides critical input for understanding the role of Cx32 in the peripheral nervous system.


Figure 2. Two adjacent HeLa cells (left), transfected with an expression vector carrying the coding region of Cx32, reconstructed in 3-D from a confocal microscope z-stack. Cx32 junctional channels aggregate in gap junction (GJ) plaques (red), which can be measured in size and number. Ionic currents (i, right panel) flowing through GJ plaques can be measured by the dual patch clamp by applying a voltage difference between the two adjacent cells. Scale bar 5 microns.

Is it possible to study CMTX1 in a human system?
We are developing an innovative in vitro model of motor neurons co-cultured with Schwann cells, both derived from human stem cells of healthy donors or CMTX1 patients. Skin biopsies are minimally invasive and can be used to isolate fibroblasts that can be reprogrammed to a pluripotent stem cell condition (iPSCs, Figure 3).


Figure 3. The human skin is an accessible source of fibroblasts which can be reprogrammed to a pluripotent state. Neurons and glial cells are obtained from these cell types by ad hoc differentiation strategies.

What are the atomic alterations of Cx32 channels carrying CMTX1 mutations?
Our experimental studies are complemented by the development and study of in silico models of Cx32 WT channel and its CMTX1 mutants, based on Homology Modelling and Molecular Dynamics techniques. Our objective is to clarify, at the molecular level, the relationship between Cx32 channel structure and permeability/gating properties observed experimentally in GJ channels and hemichannels.


Figure 4. Side and front view of a WT connexon formed by six Cx32 WT connexins, depicted with different colours. Permeation of a single Lucifer yellow molecule is also shown.

References
1. Richard H. Quarles, W.B.M., Pierre Morell, Myelin Formation, Structure and Biochemistry, in Chapter 4 of Basic Neurochemistry: Molecular, Cellular and Medical Aspects2006, Elsevier. p. 51-71.

Saima Imran

Postdoc

Giulia Crispino

Postdoc

Angela Pellizzon

Master degree student

  1. A Carrer, A Leparulo, G Crispino, CD Ciubotaru, O Marin, F Zonta and M Bortolozzi. Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X mutation that causes Charcot-Marie-Tooth disease. Human Molecular Genetics; 27:80–94 (2017).
  2. Monterisi S, Lobo MJ, Livie C, Castle JC, Weinberger M, Baillie GS, Surdo N, Musheshe N, Stangherlin A, Gottlieb E, Maizels R J, Bortolozzi M, Micaroni M and Zaccolo M. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling. eLife, 6, e21374 (2017).
  3. Ford KL, Moorhouse EL, Bortolozzi M, Richards M, Swietach P and Vaughan-Jones RD. Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes. Cardiovascular Research; 113: 984–995 (2017).
  4. B Cali, S Ceolin, F Ceriani, M Bortolozzi, A Agnellini, V Zorzi, A Predonzani, V Bronte, F Mammano. Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury. Oncotarget; 6: 10161-10174 (2015).
  5. AKC Wong, P Capitanio, V Lissandron, M Bortolozzi, T Pozzan, P Pizzo. Heterogeneity of Ca2+ handling among and within Golgi compartments. Journal of Molecular Cell Biology; 5:266-76 (2013).
  6. Rodriguez L, Simeonato E, Scimemi P, Anselmi F, Calì B, Crispino G, Ciubotaru CD, Bortolozzi M, Ramirez FG, Majumder P, Arslan E, De Camilli P, Pozzan T, Mammano F. Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition. Proc Natl Acad Sci U S A; 109:14013-8 (2012).
  7. Zampese E, Fasolato C, Kipanyula M, Bortolozzi M, Pozzan T and Pizzo P. Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca2+ cross-talk. Proc Natl Acad Sci U S A; 108: 2777-2782 (2011).
  8. M Bortolozzi, M Brini, N Parkinson, G Crispino, P Scimemi, RD De Siati, F Di Leva, A Parker, S Ortolano, E Arslan, SD Brown, E Carafoli and F Mammano. The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice. The Journal of Biological Chemistry; 285: 37693-37703 (2010).
  9. M Giacomello, I Drago, M Bortolozzi, M Scorzeto, A Gianelle, P Pizzo and T Pozzan. Ca2+ hot spots on the mitochondrial surface are generated by Ca2+ mobilization from stores, but not by activation of store operated Ca2+ channels. Molecular Cell; 38:1097-2765 (2010).
  10. M Bortolozzi, A Lelli and F Mammano. Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution. Cell Calcium; 44: 158-168 (2008)

MARIO BORTOLOZZI

  • Master Degree: Physics, University of Padova, Italy (2004).
  • PhD: Neurobiology, Biosciences School, University of Padova, Italy (2008).
  • Postdoc: Venetian Institute of Molecular Medicine (VIMM), Padova, Italy (2008-2010).
  • Assistant Professor: Dept. Physics and Astronomy, University of Padova, Italy (2010-2017).
  • Visiting Scientist: Dept. of Physiology, Anatomy and Genetics, University of Oxford, UK (2012-2013).
  • Group leader: Venetian Institute of Molecular Medicine (VIMM), Padova, Italy (since 2013).
  • Associate professor: Dept. Physics and Astronomy, University of Padova, Italy (since 2017).