Ruolo delle alterazioni mitocondriali nel processo di morte cellulare in patologie neurodegenerative

I processi di morte cellulare sono caratterizzati da profondi cambiamenti morfologico-funzionali delle diverse componenti cellulari. In queste condizioni i mitocondri vanno incontro ad una serie di evidenti alterazioni, tra le quali la perdita del potenziale di membrana mitocondriale (MMP) (Reed and Kroemer, 2000) può rappresentare sia un evento precoce dei processi di apoptosi sia un segnale associato all’autofagocitosi degli organelli stessi (Rodriguez-Enriquez et al., 2004). Nel nostro gruppo è stato osservato che cellule di neuroblastoma umano (TGA) iper-esprimenti la Ttransglutaminasi di tipo 2 (TG2), presentavano alterazioni sia nell’ultrastuttura sia nella fisiologia dei mitocondri (Piacentini et al., 2002), tra cui una iperpolarizzazione costitutiva della membrana interna. Benché l’espressione della TG2 venga indotta durante i processi di morte cellulare per apoptosi, il ruolo di questo enzima nella cascata di eventi che inducono la cellula a morire, non è stata ancora completamente chiarito. Lo scopo di parte di questa tesi di dottorato è stato quello di verificare, a livello molecolare, quali fossero le modificazioni TG2-dipendenti dei mitocondri e le eventuali interazioni con proteine implicate nella regolazione della via mitocondriale dell’apoptosi. L’analisi della sequenza primaria della TG2 ha portato all’identificazione di una regione con un’elevata omologia col dominio BH3 delle proteine della famiglia di Bcl-2, le quali controllano l’omeostasi mitocondriale e mediano il rilascio dei fattori pro- ed anti-apoptotici dai mitocondri. Il ruolo biologico di questo dominio della TG2 è stato studiato mediante l’utilizzo di peptidi in grado di penetrare la cellula e mimare l’azione del dominio stesso. Questa strategia ha rivelato come la TG2 sia in grado di interagire con Bax a livello mitocondriale e mediante questa interazione provocare la fuoriuscita del citocromo c dagli organelli. La prima parte dei risultati presentati in questa tesi, analizzano l’azione della TG2 e suggeriscono che essa possa essere un nuovo tipo di proteina “BH3-only” e le modalità della sua interazione e/o attivazione di Bax, la collocano esattamente tra le così dette “Bid-like proteins”. Queste proteine, tramite il loro dominio BH3, sono in grado di attivare in modo diretto Bax e sono potenzialmente in grado di regolare la funzionalità si altri membri della famiglia di Bcl-2. Contestualmente, il nostro gruppo si è occupato dello studio del possibile coinvolgimento della TG2 come modulatore della morte cellulare nella corea di Huntington (HD), una patologia caratterizzata da notevoli disfunzioni mitocondriali. Malgrado l’iper-espressione della TG2 induca alterazioni mitocondriali e stress ossidativo favorendo la morte cellulare (Piacentini et al., 2002), l’incrocio di topi transgenici (HD) R6/1 con i topi knock out per la TG2, evidenzia una riduzione della morte cellulare, un significativo miglioramento della motilità degli animali ed una prolungata sopravvivenza (Mastroberardino et al., 2002). Questo effetto è comunque indipendente dalla capacità dell’attività transamidasica della TG2 a formare le caratteristiche inclusioni intranucleari osservate nella patologia (Bailey and Johnson, 2005). Nella corea di Huntington, la degenerazione cellulare sembra dipendere dall’acquisizione di funzione tossica da parte dell’huntingtina mutata e la perdita di funzione protettiva da parte della proteina normale. Entrambi questi eventi portano a numerose alterazioni cellulari, che coinvolgono anche i mitocondri. Tali alterazioni possono indurre morte cellulare, che può manifestarsi sia come apoptosi sia come autofagia. La seconda parte di questa tesi riguarda lo studio dell’effetto delle alterazioni mitocondriali che si riscontrano in linfociti derivati da pazienti affetti da HD. I linfoblasti presentano alcune delle caratteristiche osservate nei neuroni HD, d'altra parte, non essendo interessati dal processo degenerativo, permettono sia un'analisi diretta degli effetti dell'htt mutata rispetto a quella wild-type sia l’effetto della mutazione in etero- ed omo-zigosi. Infatti, recenti evidenze, hanno evidenziato una più rapida progressione della malattia nei pazienti omozigoti (Squitieri et al., 2003) probabilmente imputabile alla presenza in doppia dose della proteina mutata ed alla totale mancanza del ruolo protettivo svolto dall'htt normale. Nella seconda parte di questa tesi sono state analizzate le diverse vie di morte cellulare e le alterazioni mitocondriali attraverso uno studio comparativo tra eterozigoti e omozigoti. In entrambi i casi si osservano evidenti e significative alterazioni morfologico-funzionali dei mitocondri che correlano con il genotipo delle linee cellulari.Apoptosis and autophagy are cell death pathways characterised by great modification of the cell structure, among which morphological and functional alterations of mitochondria. The loss of mitochondrial membrane potential (MMP) (Reed and Kroemer, 2000) could be considered both an early event towards apoptosis or a signal associated with autophagy of the organelles (Rodriguez-Enriquez et al., 2004). We previously observed that TG2 (Transglutaminase 2) over-expressing cells showed mitochondria that were greatly modified with respect to both their ultrastructure and physiology (Piacentini et al., 2002), with a constitutive hyperpolarization of the inner membrane. Even if TG2 expression is increased during apoptosis, the exact role of the enzyme in the cascade of events leading to cell death remain to be clarified. The first part of this thesis investigated, at a molecular level, the extension of TG2- mitochondrial modification as well as the possible interaction between TG2 and the proteins involved in the regulation of the mitochondrial pathway of apoptosis. The analysis of the TG2 primary sequence lead to the identification of a region with high homology with the BH3 domain of Bcl-2 family, to which belongs pro- and anti-apoptotic proteins. The biological role of the TG2 domain has been studied by means of cell-permeable peptides, mimicking the domain sequence. This kind of approach allow us to characterise the effect of TG2 at mitochondrial level, where it interacts with Bax and causes cytochrome c release. The obtained results suggest that TG2 might be a new kind of BH3-only protein and its modality of interaction and/or activation of Bax, place it in the so called “Bid-like” proteins. These proteins, by means of their BH3 domain, are direct activators of Bax and potentially able to regulate others Bcl-2 family members. At the same time our group had been studying the possible involvement of TG2 as a cell death modulator in Huntington’s disease (HD). In fact, it is known that over-expression of TG2 leads to cell death in human neuronal cells by inducing mitochondrial alterations and oxidative stress (Piacentini et al., 2002), but also by crossing HD R6/1 transgenic mice with TG2 knockout mice, a marked reduction in cell death is observed in R6/1, TG2 null when compared withR6/1 transgenic mice. The reduction in cell death is paralleled by a significant improvement in motor performances of R6/1, TG2 null versus R6/1 mice and extended survival (Mastroberardino et al., 2002). However this is independent from TG2 capability to make intranuclear inclusions by its cross-linking activity (Bailey and Johnson, 2005). The degenerative process of Huntington disease, relies both on the acquisition of toxic function by mutated huntingtin (htt) as well as on the loss of protection exerted by the wild type protein. Both these events lead to a lot of cellular alterations, which involve also mitochondria and they lead to cell death by apoptosis and/or autophagy. The second part of this thesis deal with the study of mitochondrial alterations observed in lymphocyte derived from HD patients. Lymphoblasts display some characteristics observed in HD neurons, otherwise, as they are not involved in neurodegenerative process, they allow a direct analysis of mutated htt effects in respect to the wild-type one as well as the effect of the mutation in hetero- and homo-zygosis. Recent evidences suggested that, the faster progression of the disease observed in homozygous patients, might be due to the presence in double dose of the mutated protein, coupled to the complete loss of the wild type one’s protective role (Squitieri et al., 2003). The second part of this thesis analyzes the different cell death pathways and the mitochondrial alterations in HD cell lines, through comparative study between heterozygous and homozygous patients. The results obtained show that in both cases we can observe plain and remarkable morfologial-functional alterations of mitochondria that correlates with the genotype of the patient

Sirtuins and redox signaling interplay in neurogenesis, neurodegenerative diseases, and neural cell reprogramming

Since the discovery of Neural Stem Cells (NSCs) there are still mechanism to be clarified, such as the role of mitochondrial metabolism in the regulation of endogenous adult neurogenesis and its implication in neurodegeneration. Although stem cells require glycolysis to maintain their stemness, they can perform oxidative phosphorylation and it is becoming more and more evident that mitochondria are central players, not only for ATP production but also for neuronal differentiation's steps regulation, through their ability to handle cellular redox state, intracellular signaling, epigenetic state of the cell, as well as the gut microbiota-brain axis, upon dietary influences. In this scenario, the 8-oxoguanine DNA glycosylase (OGG1) repair system would link mitochondrial DNA integrity to the modulation of neural differentiation. On the other side, there is an increasing interest in NSCs generation, from induced pluripotent stem cells, as a clinical model for neurodegenerative diseases (NDs), although this methodology still presents several drawbacks, mainly related to the reprogramming process. Indeed, high levels of reactive oxygen species (ROS), associated with telomere shortening, genomic instability, and defective mitochondrial dynamics, lead to pluripotency limitation and reprogramming efficiency's reduction. Moreover, while a physiological or moderate ROS increase serves as a signaling mechanism, to activate differentiation and suppress self-renewal, excessive oxidative stress is a common feature of NDs and aging. This ROS-dependent regulatory effect might be modulated by newly identified ROS suppressors, including the NAD(+)-dependent deacetylase enzymes family called Sirtuins (SIRTs). Recently, the importance of subcellular localization of NAD synthesis has been coupled to different roles for NAD in chromatin stability, DNA repair, circadian rhythms, and longevity. SIRTs have been described as involved in the control of both telomere's chromatin state and expression of nuclear gene involved in the regulation of mitochondrial gene expression, as well as in several NDs and aging. SIRTs are ubiquitously expressed in the mammalian brain, where they play important roles. In this review we summarize the current knowledge on how SIRTs-dependent modulation of mitochondrial metabolism could impact on neurogenesis and neurodegeneration, focusing mainly on ROS function and their role in SIRTs-mediated cell reprogramming and telomere protection

Galectin-1 sensitizes resting human T lymphocytes to Fas (CD95)-mediated cell death via mitochondrial hyperpolarization, budding, and fission.

Galectins have emerged as a novel family of immunoregulatory proteins implicated in T cell homeostasis. Recent studies showed that galectin-1 (Gal-1) plays a key role in tumor-immune escape by killing antitumor effector T cells. Here we found that Gal-1 sensitizes human resting T cells to Fas (CD95)/caspase-8-mediated cell death. Furthermore, this protein triggers an apoptotic program involving an increase of mitochondrial membrane potential and participation of the ceramide pathway. In addition, Gal-1 induces mitochondrial coalescence, budding, and fission accompanied by an increase and/or redistribution of fission-associated molecules h-Fis and DRP-1. Importantly, these changes are detected in both resting and activated human T cells, suggesting that Gal-1-induced cell death might become an excellent model to analyze the morphogenetic changes of mitochondria during the execution of cell death. This is the first association among Gal-1, Fas/Fas ligand-mediated cell death, and the mitochondrial pathway, providing a rational basis for the immunoregulatory properties of Gal-1 in experimental models of chronic inflammation and cancer.Fil: Matarrese, Paola. Istituto Superiore di Sanità; ItaliaFil: Tinari, Antonella. Istituto Superiore di Sanità; ItaliaFil: Mormone, Elisabetta. Istituto Superiore di Sanità; ItaliaFil: Bianco, German Ariel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Toscano, Marta Alicia. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ascione, Barbara. Istituto Superiore di Sanità; ItaliaFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Malorni, Walter. Istituto Superiore di Sanità; Itali

The Model of Interstitial Cystitis for Evaluating New Molecular Strategies of Interstitial Regeneration in Humans

Given the recent evidence in the clinical application of regenerative medicine, mostly on integumentary systems, we focused our interests on recent bladder regeneration approaches based on mesenchymal stem cells (MSCs), platelet-rich plasma (PRP), and hyaluronic acid (HA) in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS) in humans. IC/BPS is a heterogeneous chronic disease with not-well-understood etiology, characterized by suprapubic pain related to bladder filling and urothelium dysfunction, in which the impairment of immunological processes seems to play an important role. The histopathological features of IC include ulceration of the mucosa, edema, denuded urothelium, and increased detection of mast cells and other inflammatory cells. A deeper understanding of the molecular mechanism underlying this disease is essential for the selection of the right therapeutic approach. In fact, although various therapeutic strategies exist, no efficient therapy for IC/BPS has been discovered yet. This review gives an overview of the clinical and pathological features of IC/BPS, with a particular focus on the molecular pathways involved and a special interest in the ongoing few investigational therapies in IC/BPS, which use new regenerative medicine approaches, and their synergetic combination. Good knowledge of the molecular aspects related to stem cell-, PRP-, and biomaterial-based treatments, as well as the understanding of the molecular mechanism of this pathology, will allow for the selection of the right and best use of regenerative approaches of structures involving connective tissue and epithelia, as well as in other diseases

Fibromodulin, an Oxidative Stress-Sensitive Proteoglycan, Regulates the Fibrogenic Response to Liver Injury in Mice

Background & Aims: Collagen I deposition contributes to liver fibrosis, yet little is known about other factors that mediate this process. Fibromodulin is a liver proteoglycan that regulates extracellular matrix organization and is induced by fibrogenic stimuli. We propose that fibromodulin contributes to the pathogenesis of fibrosis by regulating the fibrogenic phenotype of hepatic stellate cells (HSCs). Methods: We analyzed liver samples from patients with hepatitis C–associated cirrhosis and healthy individuals (controls). We used a coculture model to study interactions among rat HSCs, hepatocytes, and sinusoidal endothelial cells. We induced fibrosis in livers of wild-type and Fmod−/− mice by bile duct ligation, injection of CCl4, or administration of thioacetamide. Results: Liver samples from patients with cirrhosis had higher levels of fibromodulin messenger RNA and protein than controls. Bile duct ligation, CCl4, and thioacetamide each increased levels of fibromodulin protein in wild-type mice. HSCs, hepatocytes, and sinusoidal endothelial cells produced and secreted fibromodulin. Infection of HSCs with an adenovirus that expressed fibromodulin increased expression of collagen I and α–smooth muscle actin, indicating increased activation of HSCs and fibrogenic potential. Recombinant fibromodulin promoted proliferation, migration, and invasion of HSCs, contributing to their fibrogenic activity. Fibromodulin was sensitive to reactive oxygen species. HepG2 cells that express cytochrome P450 2E1 produced fibromodulin, and HSCs increased fibromodulin production in response to pro-oxidants. In mice, administration of an antioxidant prevented the increase in fibromodulin in response to CCl4. Coculture of hepatocytes or sinusoidal endothelial cells with HSCs increased the levels of reactive oxygen species in the culture medium, along with collagen I and fibromodulin proteins; this increase was prevented by catalase. Fibromodulin bound to collagen I, but the binding did not prevent collagen I degradation by matrix metalloproteinase 13. Bile duct ligation caused liver fibrosis in wild-type but not Fmod−/− mice. Conclusions: Fibromodulin levels are increased in livers of patients with cirrhosis. Hepatic fibromodulin activates HSCs and promotes collagen I deposition, which leads to liver fibrosis in mice

Nuova tecnica di imaging 3D CARTOSOUND applicata all'estrazione transvenosa di elettrocateteri

INTRODUZIONE. La rimozione di elettrocateteri cardiaci viene eseguita in un numero sempre maggiore di ospedali con incidenza di complicanze significativa,soprattutto nei centri a basso volume. Le attuali tecniche estrattive sono limitate dall’impossibilità di visualizzare direttamente le struttura anatomiche,provocando ansia persino negli operatori più esperti. Inoltre,nessuna tecnica di estrazione transvenosa può essere considerata standard e applicabile in tutti i pazienti. Da questo ne deriva la necessità di dover indirizzare le varie tecniche estrattive con rischio/beneficio ottimizzato al singolo paziente. METODI. Registro prospettico monocentrico in cui tutti i pazienti sottoposti ad estrazione di elettrocateteri sono stati consecutivamente arruolati. Sono stati creati e confrontati due gruppi in base all’utilizzo di tecnologia CartoSoundTM (gruppo CARTOSOUND) o sola fluoroscopia convenzionale (gruppo CONTROLLO). Una mappa 3D CartoSound delle strutture venose,delle camere cardiache destre,del tessuto fibroso e degli elettrocateteri è stata creata prima e durante la procedura. Sono state utilizzate diverse tecniche di estrazione,in linea con un approccio principalmente step-by-step nel gruppo CONTROLLO e basato sulla valutazione individuale del rischio/beneficio delle tecniche estrattive,legato prevalentemente all’entità ed alla localizzazione di aderenze,nel gruppo CARTOSOUND. Gli end-point dello studio includono complicanze periprocedurali e tempi procedurali e di esposizione a raggi X. RISULTATI. Da Novembre 2009 a Luglio 2011 sono stati arruolati 53 pazienti:36 (67.9%) nel gruppo CONTROLLO e 17 (32.1%) nel gruppo CARTOSOUND. Le complicanze procedurali sono state maggiori nel gruppo CONTROLLO: 27.8% vs 5.9% per complicanze minori e 8.3% vs 0% per complicanze maggiori. I tempi procedurali e di esposizione a raggi X non differivano significativamente tra i due gruppi: 79,5 ± 67,5 vs 73,6 ± 47,7 min (P=0.88) e 16 ± 20 vs 24,7 ± 36,8 min (P=0.28). CONCLUSIONI. In questo registro prospettico di pazienti sottoposti ad estrazione di elettrocateteri,l’utilizzo di approccio ecografico intracardiaco in tempo reale mediante modulo 3D CartoSoundTM ha mostrato un potenziale vantaggio in termini di sicurezza senza determinare allungamento dei tempi procedurali e di esposizione a raggi X. L’estrazione di elettrocateteri assistita da CartoSound trae beneficio dalla valutazione 3D delle aderenze fibrose e dall’identificazione precoce delle possibili complicanze

Different methods of bone marrow harvesting influence cell characteristics and purity, affecting clinical outcomes

Background: Bone marrow (BM)-derived stem cells were implanted to induce angiogenesis in patients with no-option critical limb-threatening ischemia. Considering the potential for this therapy, conflicting results related to BM harvesting methods have been reported that could affect stem cell concentrations and quality. Methods: A total of 75 patients with no-option critical limb-threatening ischemia were treated with BM implantation. For 58 patients, BM was harvested using a BM aspirate concentrate system (Harvest Technologies; group HT) with a standard aspiration needle, followed by an automated centrifugation process, to produce BM aspirate concentrate. For 17 patients, BM was harvested using the Marrow Cellution system (Aspire Medical Innovation; group MC). CD34+ cells/mL, CD117+ cells/mL, CD133+ cells/mL, CD309+ cells/mL, hematocrit, and BM purity were compared between the two BM preparations. Results: The retrospective analysis of a subset group after adjustment for age shows that the quality of BM obtained using the Marrow Cellution system is better, in terms of purity, than the classic harvesting method before centrifugation. Harvested BM before centrifugation is characterized by a higher percentage of CD133+ cells compared with BM after centrifugation. In contrast, the MC aspirate had a larger amount of very small embryonic-like cells, as indicated by the higher percentage of CD133+, CD34+, and CD45− cells. These differences translated into an increased occurrence of leg amputations in group HT than in group MC and an increase in transcutaneous oxygen pressure in patients treated with BM aspirated using MC. Conclusions: BM manipulation, such as centrifugation, affects the quality and number of stem cells, with detrimental consequences on clinical outcomes, as reflected by the different amputation rates between the two groups. : Clinical Relevance: Critical limb-threatening ischemia is the most advanced form of peripheral arterial disease with major economic and social effects due to the high amputation rate and mortality. The problem is even greater for diabetic patients, for whom the expected incidence of amputation is ∼40% to 50%. Thus, the need for new therapeutic options is urgent. The present report highlights the striking effects of angiogenic therapy by bone marrow-derived stem cells obtained using a novel technology. We found that the choice of bone marrow harvesting method does influence the clinical outcome; however, further studies are needed. The present study presents a meaningful background for future development