CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Second messenger homeostasis in subcellular compartments

Methodological aspects

One of the major topics of the lab over the last years has been the development and use of organelle targeted GFP-based fluorescent indicators for monitoring second messenger levels in living cells. In the last years, novel Ca2+ indicators for peroxisomes, Golgi apparatus, and mitochondria have been generated and characterized. These novel tools have allowed for the first time to address the study of Ca2+ homeostasis in subcellular compartments for which until now only indirect information was available. In addition to Ca2+, much of the interest of the lab is concentrated on cAMP probes and novel tools, again targeted to subcellular regions (plasma membrane, mitochondria, nucleus), have been generated or are under development. These methodologies are instrumental for addressing with novel approaches the role of second messenger heterogeneity in physiological and pathological conditions. The final goal of this part of the research is that of using the already available probes (and the new ones that are being developed) not only in cell lines or primary cell cultures, but also in more intact tissue preparations (acute slices) and in vivo. To this end, various approaches are being pursued, from generation of transgenic animals expressing the probes (in collaboration with the group of Prof. P. Bonaldo), to the development and utilization of viral vectors, to in vivo electroporation.

New insights into Alzheimer's Disease

 

Evidence has accumulated showing that Familial Alzheimer's Disease (FAD) is linked to an imbalance of cellular Ca2+ homeostasis. It has been suggested that presenilins (PSs), the catalytic core of γ-secretase, the enzyme responsible of β-amyloid (Aβ production, play a key role in the control of Ca2+ leak out of the endoplasmic reticulum (ER). We are studying the mechanisms through which PSs and their FAD mutants affect not only store (ER and Golgi) but also mitochondria Ca2+ handling in model cells ranging from fibroblasts of FAD patients to neurons from transgenic AD mouse models based on mutated PS2 or PS1. In particular, we are testing the hypothesis, presented by our group, that FAD-PS2 mutations decrease, instead of increasing, the store Ca2+ content, partially contrasting the damaging effect of an increased Aβ production, caused by mutated PSs. Instrumental to this issue are two main strategies: a) The understanding of cytosolic and organelle Ca2+ dynamics by means of classical Ca2+ probes and recombinant, organelle-targeted aequorins, in model cells carrying mutated PSs. b) Developing and applying new FRET-based Ca2+ probes (cameleons) to monitor Ca2+ concentration at the level of single cell microdomains, such as mitochondrial matrix, outer mitochondrial membrane, trans-Golgi apparatus and peroxisomes.

Cardiac cell physiopathology

The group has a long standing interest in the study of cardiac cell physiopathology. In collaboration with Dr. M. Zaccolo (now at the University of Glasgow) we have focused our attention on the role of cAMP microdomains in the control of cardiac cell excitability and in the process of cardiac hypertrophy in vitro. More recently, we have focused our attention on the role of cAMP in the control of mitochondrial function in cardiomyocytes. To this end, we are using some of the novel Ca2+ and cAMP probes mentioned above. The goal is to move from in vitro models (cardiomyocytes from newborn animal or isolated cardiomyocytes) to the intact tissue.

Physiopathology of second messengers in the olfactory system

 

In collaboration with Dr. C. Lodovichi, we have focused our attention on the role of second messengers in the formation of the sensory map in the olfactory bulb. We have recently addressed the problem of the distribution and functional characterization of the mechanisms controlling second messenger regulation (cAMP, cGMP and Ca2+) in olfactory sensory neurons, with special attention to their production in the growth cone-terminal synapse. Primary neuronal cultures, as well as semintact preparations (hemiheads), are being used for this studies. The final goal is that of understanding how second messenger gradients within these neurons can influence axonal convergence in the olfactory bulb and control the specificity of olfactory circuit formation.

Publications

  • Brunello L, Zampese E, Florean C, Pozzan T, Pizzo P, Fasolato C (2009) Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake. J. Cell. Mol. Med. 13:3358-69.
  • Maritan M, Monaco G, Zamparo I, Zaccolo M, Pozzan T, Lodovichi C (2009) Odorant receptors at the growth cone are coupled to localized cAMP and Ca2+ increases. Proc. Natl. Acad. Sci. U.S.A. 106:3537-42.
  • Drago I, Giacomello M, Pizzo P, Pozzan T (2008) Calcium dynamics in the peroxisomal lumen of living cells. J. Biol. Chem. 283:14384-90.
  • Colella M, Grisan F, Robert V, Turner JD, Thomas AP, Pozzan T (2008) Ca2+ oscillation frequency decoding in cardiac cell hypertrophy: role of calcineurin/NFAT as Ca2+ signal integrators. Proc. Natl. Acad. Sci. U.S.A. 105:2859-64.
  • Pizzo P, Pozzan T (2007) Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics. Trends Cell Biol. 17:511-7.

Grants

FIRB

Fondazione Cassa di Risparmio di Padova e Rovigo

PRIN

Progetto Strategico dell'Università degli Studi di Padova

 

PI photo

Tullio Pozzan

Contact information

email  E-mail

email  +39 049 827 6070

Participating staff

Cristina Fasolato

Marco Mongillo

Paola Pizzo