Mario Bortolozzi

How are genetic and environmental factors involved in neurodegeneration?

We are interested in understanding the molecular mechanisms underlying pathology of the central and peripheral nervous systems. In particular, we focused on the study of Charcot-Marie-Tooth and Parkinson’s diseases using an innovative combination of Physics, Bioinformatics and Biology.
Background

Research

Group Members

Key Publications

The Bortolozzi lab is currently working on three main projects: (i) the study of connexin 32 (Cx32), whose mutations cause the X-linked form of Charcot-Marie-Tooth peripheral neuropathy (CMT1X), a degenerative motor and sensory disorder; (ii) the study of pathological variants of the parkin protein which cause the autosomal recessive juvenile parkinsonism (ARJP); (iii) the study of the effects of perfluoroalkyl substances (PFAS) in the physiology and pathology of brain neurons. Both CMT1X and ARJP diseases are associated to dysfunctional ion channels and lack an effective treatment. Bortolozzi lab’s research points towards clarifying the molecular mechanisms underlying pathology by a combination of biophysics and cellular models, including the most recent technology derived from induced pluripotent stem cells, such as human brain organoids. Therapeutic strategies are investigated up to the single channel level. We recently suggested a possible key role of Cx32 hemichannels in the molecular pathogenesis of CMT1X and proposed a therapeutic strategy based on mimetic peptides (Patent nr. 102017000084299 deposited on 24^th July 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 CMT1X patients.

Figure.1 Basic structure of peripheral nerves. 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.

What biophysical properties of Cx32 channels are altered by CMT1X mutations?

Recent work, carried out also by our group, suggests a new paradigm for the molecular pathogenesis of CMT1X as being no longer linked to Cx32 gap junctions (GJs) but rather to Cx32 hemichannels, which could regulate the ATP-mediated paracrine signaling that is critical for the myelination process. Demonstrating this hypothesis would strongly focus the therapeutic strategy on a class of chemical and biological agents (peptides and antibodies) which can selectively bind mutant Cx32 hemichannels modulating their gain/loss of function. The known R220X mutation of Cx32 was selected as an animal model benchmark to investigate the pathogenesis of CMT1X and to develop a therapeutic strategy based on a mimetic peptide (GAP24) that we found to be able to restore the inhibited function of R220X hemichannels expressed in vitro.

Is it possible to recapitulate Parkinson’s disease features in a dish?

In vitro approaches to model human brain development and disease are a promising area of research. In the context of Parkinson's disease, human midbrain organoids (hMOs) have been used to recapitulate pathological aspects of the disease such as α-synuclein aggregation and mitochondrial impairment. The hMOs investigated in our project in collaboration with Dr. Alessio Di Fonzo (Policlinico of Milan) are a new model obtained from patients carrying different mutations of the PARK2 gene which encodes the parkin protein. PARK2 mutations are known to cause autosomal recessive juvenile parkinsonism (ARJP), but the molecular mechanisms underlying the neuronal degeneration in the substantia nigra are still unknown.

Figure.2 Generating hMOs from patients with PARK2 gene mutations. (Left) Numerous substrates for parkin have been identified, indicating that it is a multifunctional protein involved in many intracellular processes, including the ubitiquination of the glutamate kainate receptor (KAR). The hyperactivation of this ion channel by mutant parkin may contribute to the death of nigral dopaminergic neurons and the pathogenesis of ARJP. (Right) A 154 days old hMO studied by two-photon microscopy in our lab and expressing a complex glial architecture positive to the GFAP marker (green, blue are nuclei).

Are parkin mutations altering the kainate receptor function causing ARJP?

A combination of 2-photon Ca²⁺ imaging, patch-clamp and multielectrode arrays (MEAs) permitted us to measure a highly coordinated neuronal activity in hMOs which supported the notion that dopaminergic neurons can act as pacemakers in the substantia nigra. According to previous observations that parkin protein regulates the expression of KARs and may be involved in ARJP pathogenesis, we were able to modulate the spontaneous Ca²⁺ activity in both healthy and diseased hMOs by pharmacological intervention with agonists and antagonists of KARs. Our results suggest that hMOs can be a useful tool to model the mechanisms leading to nigrostriatal degeneration and for personalized medicine.

Figure.3 Optical microscopy combined with MEAs technology to study human brain organoids. (Left) A 32-electrode tip is inserted within a single hMO fixed at the bottom of special chamber superfused with culture solution at 37 °C. (Right) Local fileld potentials and single neuron action potentials can be recorded for several hours.

Is PFAS bioaccumulation negatively interfering with neurogenesis and/or Parkinson’s disease pathogenesis?

Perfluoroalkyl substances (PFAS) are a class of synthetic chemicals colloquially known as “forever chemicals” due to their long-term stability imparted by high-energy fluorine-carbon bonds. PFAS were introduced in the industry to make surfaces repellent to water and lipids. Because of their chemical properties, PFAS tend to bioaccumulation in the blood, liver, kidney, heart, muscle and brain of various species. Recently (Di Nisio et al. 2022), we evaluated for the first time the accumulation of PFOA (a group of PFAS) in subjects resident in an area recognized as hotspot of environmental pollution by PFAS in the Veneto region of Italy. The high content of PFOA accumulated in dopaminergic neurons suggested a possible link between Parkinson’s Disease and PFAS exposure. In the same study, we performed experiments in a 2-dimensional human model of dopaminergic neurons differentiated from induced pluripotent stem cells observing a decrease in the expression of dopaminergic markers in the cells exposed to PFOA. The next step will be extending the study to a 3-dimensional model, like our human brain organoids.

Figure 4. PFAS concentrations in the blood of residents from the most contaminated area of Veneto (Red Area). Data are taken from the health surveillance plan on population exposed to PFAS published in 2019.

Imran SJ, Vagaska B, Kriska J, Anderova M, Bortolozzi M, Gerosa G, Ferretti P, Vrzal R.Aryl Hydrocarbon receptor (AhR)-mediated signaling in iPSC-derived human motor neurons. Pharmaceuticals 2022; 15(7), 828;

Di Nisio A, Pannella M, Vogiatzis S, Sut S, Dall'Acqua S, Santa Rocca M, Antonini A, Porzionato A, De Caro R, Bortolozzi M, De Toni L, Foresta C. Impairment of human dopaminergic neurons at different developmental stages by perfluoro-octanoic acid (PFOA) and differential human brain areas accumulation of perfluoroalkyl chemicals. Environment International 2022; 106982(158).

Donati V, Peres C, Nardin C, Scavizzi F, Raspa M, Ciubotaru CD, Bortolozzi M*, Pedersen MG, Mammano F. Calcium Signaling in the Photodamaged Skin: In Vivo Experiments and Mathematical Modeling. Function 2021; 3(1): zqab064.

*Co-corresponding author.

Tedesco S, Scattolini V, Albiero M, Bortolozzi M, Avogaro A, Cignarella A, Fadini GP. Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity. Int J Mol Sci. 2019; 20(19)

Bortolozzi M.* What's the Function of Connexin 32 in the Peripheral Nervous System? Front Mol Neurosci. 2018; 11:227

*Corresponding author.

Burdyga A, Surdo NC, Monterisi S, Di Benedetto G, Grisan F, Penna E, Pellegrini L, Zaccolo M, Bortolozzi M, Swietach P, Pozzan T, Lefkimmiatis K. Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane. Proc Natl Acad Sci U S A. 2018;115(28): E6497-E6506.

Bortolozzi M*, Mammano F. PMCA2 pump mutations and hereditary deafness. Neurosci Lett. 2018; 663:18-24.

*Corresponding author.

Carrer A, Leparulo A, Crispino G, Ciubotaru CD, Marin O, Zonta F and Bortolozzi M*. 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).

*Corresponding author.

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).

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).

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).

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).

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).

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).

Victor HH^#, Bortolozzi M^#, Pertegato V, Beltramello M, Giarin M, Zaccolo M, Pantano S and Mammano F. Unitary permeability of gap junction channels to second messengers measured by FRET microscopy and dual whole-cell current recordings. Nature Methods; 2007 Apr 4(4): 353-358.

^# Equally contributed.

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).

Selected Awards

Current funding

PRIN

AFM Telethon

TIN Foundation