New in vitro cellular model for molecular studies of retinitis pigmentosa

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening

Activation of Bax in Three Models of Retinitis Pigmentosa

PURPOSE:The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas. METHODS:Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin. RESULTS:We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina. CONCLUSIONS:Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration

Mutations in splicing factor PRPF3, causing retinal degeneration, form detrimental aggregates in photoreceptor cells.

PRPF3 is an element of the splicing machinery ubiquitously expressed, yet mutations in this gene are associated with a tissue-specific phenotype: autosomal dominant retinitis pigmentosa (RP). Here, we studied the subcellular localization of endogenous- and mutant-transfected PRPF3. We found that (i) subcellular distribution of the endogenous wild-type protein co-localizes with small nuclear ribonucleoproteins, partially with a nucleolar marker and accumulates in speckles labeled by SC35; (ii) in human retinas, PRPF3 does not show a distinctive abundance in photoreceptors, the cells affected in RP and (iii) the RP causing mutant PRPF3, differently from the wild-type protein, forms abnormally big aggregates in transfected photoreceptor cells. Aggregation of T494M mutant PRPF3 inside the nucleus triggers apoptosis only in photoreceptor cells. On the basis of the observation that mutant PRPF3 accumulates in the nucleolus and that transcriptional, translational and proteasome inhibition can induce this phenomenon in non-photoreceptor cells, we hypothesize that mutation affects splicing factor recycling. Noteworthy, accumulation of the mutant protein in big aggregates also affects distribution of some other splicing factors. Our data suggest that the mutant protein has a cell-specific dominant effect in rod photoreceptors while appears not to be harmful to epithelial and fibroblast cells

Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis

Systemic Complement Activation in Age-Related Macular Degeneration

Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes

Donne e teatro

Il volume "Donne e teatro" riporta atti, documenti ed immagini relativi al Convegno che si è svolto a Venezia, il 6 ottobre 2003 in Auditorium Santa Margherita. Il tema di questo incontro è stato principalmente il teatro e l’arte, temi al centro dell’attenzione dell’Università e della città di Venezia. Durante l'incontro sono state analizzate figure come quelle delle attrici italiane Virginia Ramponi, Adelaide Ristori e Marta Abba. I Relatori del Convegno sono Daria Perocco, Piermario Vescovo, Carmelo Alberti, Teresa Viziano, Maria Ida Biggi, Paola Daniela Giovanelli

Biografie difficili

Il volume "Biografie difficili" raccoglie documenti, ricerche ed immagini relativi al Convegno svolto a Venezia, in Auditorium Santa Margherita, il 30 novembre 2001. Il Convegno vede la presenza del Rettore Maurizio Rispoli i Relatori Anna Laura Bellina, Gino Benzoni, Paola Bruna, Daniela M. Ciani Forza, Alberta Fabris Grube, Augusto Gentili, Rosella Mamoli Zorzi, Berndt Ostendorf e Franco Rella. Il Convegno nasce come prosecuzione della giornata di studio "Voci dal Silenzio", organizzata nel 1999, e vede la partecipazione del Soroptimist Club di Venezia, il quale ha accolto la proposta di collaborazione del CPO. Il Soroptimist Club ha appoggiato l’iniziativa secondo le proprie finalità di valorizzare la donna nei campi professionali e sociali e di ribadire i suoi diritti/doveri di collaborare al progresso della società e al riconoscimento obiettivo del suo impegno. Il volume riporta il saluto del Rettore Maurizio Rispoli, l'intervento della Coordinatrice Area Educazione - Cultura Soroptimist Marina Magrini e, a seguire, gli interventi dei vari Relatori

Mobbing . Molestie morali nel luogo di lavoro

Il volume "Mobbing - Molestie morali nel luogo di lavoro" raccoglie documenti, ricerche ed interventi relativi al Convegno svolto a Venezia, in Auditorium Santa Margherita a Venezia, il l'8 marzo 2002. Il Convegno nasce come prosecuzione delle giornate di studio "Voci dal Silenzio" e "Biografie difficili", organizzate rispettivamente nel 1999 e nel 2001. Il CPO ha organizzato tale Convegno al fine di interrogare alcuni esperti per chiarire e approfondire il fenomeno del mobbing e di ottenere informazioni sugli strumenti atti a rimuoverlo e prevenirlo. Il volume riporta la presentazione di Romana Frattini, gli interventi di Renato Gilioli, Annalisa Lantermo, Michele Cangiani e Martina Pampaloni