Laboratory  of   signal  transduction

470 citations
690 citations
720 citations

Friedreich's Ataxia, a degenerative disease due to accelerated cell death of sensory neurons. We selected a human monogenic disease in which accelerated cell death in specific tissues has pathogenic relevance. Friedreich's Ataxia (FRDA) is caused by the defective expression of frataxin, a mitochondrial protein involved in iron homeostasis. We defined the precise size of mature frataxin in human cells, and suggested the presence of alternative processing pathways. We provided evidence that frataxin expression confers stress protection to a variety of cell types and, importantly, we demonstrated the existence of an extramitochondrial pool of mature frataxin that can replace the mitochondrial form in granting stress protection and promoting cell survival. Extramitocondrial frataxin interacts with and regulates the activity of cytosolic aconitase/IRP1, a major player in iron metabolism.

We unvealed that the Ubiquitin-Proteasome system controls frataxin stability and we provided evidence for the therapeutic potential of small molecule chemical compounds that prevent frataxin ubiquitination. A new series of such compounds (named Ubiquitin Competing Molecules) was developed that shows higher potency and specificity. More recently, we identified the E3 ligase that directly ubiquitinates the frataxin precursor, RNF126.

C.elegans as an animal model for FRDA and metabolic control of lifespan. We generated frataxin-deficient C.elegans, in order to investigate the metabolic consequences of frataxin deficiency at the organismal level. We found that mildly frataxin-deficient nematodes live 25% longer than normal. Interestingly, on the other hand, strong frataxin suppression results in shortening of life span, clearly suggesting a “threshold effect” already observed for other mitochondrial mutants. Mild frataxin suppression may therefore represent a useful model to mimick the early stages of FRDA, where a protective stress response is still at play. We observed that both lifespan-extending and lifespan-shortening responses, depending on the severity of frataxin suppression, are controlled by CEP1/p53, and that this is critically dependent on the strenght of the stress response induced by frataxin deficiency. Moreover, the extent of autophagy directly control lifespan extension in frataxin-deficient C.elegans.



Lipid and glycolipid mediators of programmed cell death. We have provided evidence suggesting that surface crosslinking of the receptor Fas generates ceramide accumulation through the activation of an acidic sphingomyelinase (ASM), a type-C phospholipase responsible for membrane sphingomyelin hydrolysis. Although we showed that multiple signaling pathways originate from Fas, ceramide produced by ASM appears relevant for the progression of the apoptotic signal, as cellular mutants defective in Fas "death domain" expression, fail to activate ASM and are resistant to Fas-induced death signals. We have investigated Fas-induced upstream activators of ASM, as well as downstream targets of ceramide. We identified GD3 ganglioside as a key mediator for the progression of ceramide and Fas-induced apoptotic signals in hematopoietic cells. By adoptive transfer of ASM into ASM-deficient cells we could show that ASM-deirived ceramide is required for GD3 accumulation and efficent apoptosis of lymphoid cells. GD3 can directly target mitochondria causing mitochondrial damage, in a bcl2-controlled manner. Importantly, sialic acid acetylation of GD3 completely suppresses the ability of GD3 to induce mitochondrial damage and apoptosis. This might represent an important mechanism for tumor cells to escape apoptosis.


Relevance of the Fas system in tissue homeostasis and autoimmune diseases. We have investigated the role of the Fas system in the lymphoid tissue associated with the human intestinal mucosa, a major peripheral lymphoid compartment heavily and persistently challenged by antigen. Lamina propria T lymphocytes (T-LPL), unlike peripheral blood T lymphocytes (T-PBL), are extremely sensitive to Fas-mediated signals, express detectable amounts of Fas Ligand (FasL) in vivo and display significant "spontaneous apoptosis" in vitro. Exploiting the "natural" sensitivity of freshly isolated T-LPL to Fas-generated death signaling, we investigated soluble or cellular factors affecting the susceptibility to Fas-induced apoptosis in primary lymphoid cells. We also recently studied the expression and function of Fas and FasL in the thyroids from patients with Hashimoto's thyroiditis. The constitutive expression of FasL and the inducible expression of Fas by IL-1, revealed a novel apoptogenic mechanism based on omotypic cellular interactions leading to tissue destruction and desease. On the other hand, a minor role seems to be played by thyroid infiltrating cytotoxic T lymphocytes. A different mechanism is likely to be at work in autoimmune diabetes, where IL-1-induced upregulation of Fas via nitric oxide primes beta cells for destruction by FasL+ infiltrating T lymphocytes. A critical role for islet alpha cells in preventing the T cell infiltration is suggested by data gathered in the NOD mouse model. Fas/FasL interactions therefore contribute significantly to the pathogenesis of organ-specific autoimmunity.


Ceramide-mediated pathways and cell differentiation. We investigated the relevance of ceramides as intracellular messengers in a variety of cellular adaptive responses. We have suggested that different cytokines can modulate dendritic cells differentiation and function through a common ceramide-mediated pathway. Ceramide catabolism also controls survival of dendritic cells. In Salmonella-infected macrophages, an ASM-dependent ceramide-mediated pathway is responsible for SEK1 activation. When triggered by LPS, this pathway is regulated by PKCzeta. We also found that the accumulation of ceramide contributes to the senescence of cultured fibroblasts.


Caspases in cell differentation and death. We found a number of potential cellular substrates for caspases by "in silico" screening of public protein databases. One of them is the PML/RARalpha fusion protein responsible for the arrest of differentiation in acute promyeloblastic leukemia. Another substrate we identified is the proto-oncogene Abl. Abl cleavage promotes the re-localization of a fragment into the nucleus that cooperates to apoptosis. The role of other potential substrates, and of natural inhibitors of caspases, in the differentiation and death of selected cell types, is currently being investigated. In dendritic cells, caspases largely mediate UVB-induced apoptosis, although mitochondrial changes proceed in a caspase-independent manner. Interestingly, we found that LPS triggers the activation of a caspase-like protease that promotes survival of human dendritic cells. We also studied regulatory mechanisms of caspases. We found that the tyrosine kinase src phosphorylates caspase 8 thus preventing its activation and the progression of the apoptotic cascade.

340 citations
310 citations




Frataxin in cancer. Frataxin partecipates to the hypoxic stress response in tumors. In fact, we reported that frataxin accumulates in human tumors in vivo, and that it is upregulated in cancer cells under hypoxic conditions.

This project is being continued by Dr. Natascia Ventura at the University of Dusseldorf

Drug repositioning for Friedreich ataxia. In search for already approved drugs that could be repositioned as therapeutics for FRDA, we found that interferon gamma upregulates frataxin in multiple cell types, including FRDA cells, and that interferon gamma prevents DRG neuronal degeneration and impairment of sensorimotor performances in FRDA mice. A pilot Phase IIa clinical trial to test the safety of interferon gamma in FRDA patients, provided encouraging results. A second Phase II clinical trial clearly showed that interferon gamma is well tolerated by FRDA patients and that it can significantly prevent disease progression.

We also reported that frataxin is phosphorylated by the tyrosine kinase src and that fraaxin levels ca be increased in patient cells by src inhibitors.

More recently, the functional screening of a library of >800 FDA-approved drugs, revealed that the anti-viral etravirine upregulates frataxin, by enhancing frataxin mRNA translation. A pilot Phase II clinical trial, aimed at assessing the safety and efficacy of etravirine in FRDA patients has been launched.

Frataxin and cadiac disease. Hyprtrophic cardiomyopathy is often associated with Friedreich ataxia. We recently observed that levels of frataxin correlate with levels of HAX1, a key regulator of cardiomyocyte deadth and survival. HAX1 could represent a useful biomarker for cardiac degeneration in FRDA