Daniela Sanges

Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors

Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum in a degenerating neuron.

Cross-talk between two apoptotic pathways activated by endoplasmic reticulum stress: differential contribution of caspase-12 and AIF

Co-activation and cross-talk of different apoptotic pathways have been described in several systems however, the differential contributions of the different executors have not been well characterized. Here we report the co-translocation to the nucleus of caspase-12 and AIF in response to two endoplasmic reticulum (ER) stresses: protein misfolding and disruption of calcium homeostasis. As seen by treatment with pan-caspase inhibitor and calpain inhibitors, apoptosis is not mediated by executor caspases but by calpains. By reduction of AIF or caspase-12 expression we unraveled that AIF primarily controls apoptosis caused by changes in calcium homeostasis while caspase-12 has a main role in programmed cell death induced by protein misfolding. Nevertheless, the two apoptotic factors appear to reinforce each other during the apoptotic process, confirming that while the first response primarily involves one organelle, mitochondria and ER can influence each other in the apoptotic event.

Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Despite the recent success of gene‐based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain‐of‐function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post‐transcriptional gene regulation (RNA silencing). Engineered zinc‐finger (ZF) protein‐based repression of transcription may represent a novel approach for treating gain‐of‐function mutations, although proof‐of‐concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild‐type hRHO allele in a mutational‐independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc‐finger‐based mutation‐independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain‐of‐function mutations.

Photoreceptor rescue and toxicity induced by different calpain inhibitors

J. Neurochem. (2010) 115, 930–940.

Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors

Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration.

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.