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:

1. 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.
2. 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 Prof. M. Zaccolo (now at the University of Glasgow, UK) 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

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.

Other projects

In addition to the above projects, the lab is involved in several collaborative projects within the Department, the CNR Neuroscience Institute, and with other groups in Italy and abroad. Of particular relevance are the projects in collaboration with Dr. G. Carmignoto on neuron-astrocyte crosstalk, with Prof. R. Rizzuto on the role of mitochondria in apoptosis and in the identification of the molecular components of the mitochondrial Ca²⁺ handling machinery, and with Prof. C.B. Wollheim (University of Geneva, Switzerland) in studies concerning the role of mitochondria in the control of insulin secretion in pancreatic β cells.