Seifollah Azadi

Calpain is activated in degenerating photoreceptors in the rd1 mouse

The retinal degeneration (rd)1 mouse displays an inherited retinal degeneration and therefore allows studies of the molecular mechanisms behind the blinding disease retinitis pigmentosa. Activation of the calcium‐dependent protease calpain has been suggested to play an important role in cell death in various tissues, but little is known about the expression and activity of calpain during inherited retinal degeneration. Using microarray techniques, transcript levels of cyclic AMP response element‐binding protein (CREB)‐1, calpastatin and of various calpain genes were analysed in the rd1 mouse compared with its wild‐type control. Expression of distinct calpain isoforms and calpastatin was investigated using immunofluorescence and immunoblotting. Gene transcription and protein expression levels were compared with calpain activity using an enzymatic assay that allowed monitoring of calpain activity at the cellular level. We found that CREB‐1 and calpastatin expression was reduced in rd1 retinas, whereas calpain activity was substantially increased in rd1 photoreceptors. Calpain activity peaked at postnatal day 13, together with rd1 photoreceptor cell death. Calpain‐specific inhibitors decreased calpain activity in situ. These results indicate that activation of calpains correlates with rd1 photoreceptor cell death, which raises the possibility of using calpain inhibitors to prevent or delay photoreceptor degeneration.

Excessive Activation of Poly(ADP-Ribose) Polymerase Contributes to Inherited Photoreceptor Degeneration in the Retinal Degeneration 1 Mouse

Retinitis pigmentosa (RP) is an inherited blinding disease for which there is no treatment available. It is characterized by a progressive and neurodegenerative loss of photoreceptors but the underlying mechanisms are poorly understood. Excessive activation of the enzyme poly(ADP-ribose) polymerase (PARP) has recently been shown to be involved in several neuropathologies. To investigate the possible role of PARP in retinal photoreceptor degeneration, we used the retinal degeneration 1 (rd1) mouse RP model to study PARP expression, PARP activity, and to test the effects of PARP inhibition on photoreceptor viability. PARP expression was found to be equal between rd1 and wild-type counterpart retinas. In contrast to this, a dramatic increase in both PARP activity per se and PARP product formation was detected by in situ assays in rd1 photoreceptors actively undergoing cell death. Furthermore, PARP activity colabeled with oxidatively damaged DNA and nuclear translocation of AIF (apoptosis-inducing factor), suggesting activation of PARP as a bridge between these events in the degenerating photoreceptors. The PARP-specific inhibitor PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide·HCl[ reduced the number of cells exhibiting death markers in a short-term retinal culture paradigm, a protective effect that was translated into an increased number of surviving photoreceptors when the inhibitor was used in a long-term culture setting. Our results thus demonstrate an involvement of PARP activity in rd1 photoreceptor cell death, which could have a bearing on the understanding of neurodegenerations as such. The findings also suggest that the therapeutical possibilities of PARP inhibition should include retinal diseases like RP.

Mouse retina explants after long-term culture in serum free medium

The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

CNTF + BDNF treatment and neuroprotective pathways in the rd1 mouse retina

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

Excessive HDAC activation is critical for neurodegeneration in the rd1 mouse

Inherited retinal degenerations, collectively termed retinitis pigmentosa (RP), constitute one of the leading causes of blindness in the developed world. RP is at present untreatable and the underlying neurodegenerative mechanisms are unknown, even though the genetic causes are often established. Acetylation and deacetylation of histones, carried out by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, affects cellular division, differentiation, death and survival. We found acetylation of histones and probably other proteins to be dramatically reduced in degenerating photoreceptors in the rd1 human homologous mouse model for RP. Using a custom developed in situ HDAC activity assay, we show that overactivation of HDAC classes I/II temporally precedes photoreceptor degeneration. Moreover, pharmacological inhibition of HDACs I/II activity in rd1 organotypic retinal explants decreased activity of poly-ADP-ribose-polymerase and strongly reduced photoreceptor cell death. These findings highlight the importance of protein acetylation for photoreceptor cell death and survival and propose certain HDAC classes as novel targets for the pharmacological intervention in RP.

Up-regulation and increased phosphorylation of protein kinase C (PKC) delta, mu and theta in the degenerating rd1 mouse retina.

The rd1 mouse serves as a model for inherited photoreceptor degeneration: retinitis pigmentosa. Microarray techniques were employed to compare the transcriptomes of rd1 and congenic wild-type retinas at postnatal day 11, when degenerative processes have started but most photoreceptors are still present. Of the several genes that were differentially expressed, focus was put on those associated with the protein kinase C (PKC) signaling pathway, in particular PKCδ, μ and θ. Microarray identified these as being up-regulated in the rd1 retina, which was confirmed by QRT-PCR. Western blotting and immunostaining, using antibodies against either total or phosphorylated variants of the PKC isoforms, revealed increased expression and phosphorylation of PKCδ, μ and θ in the rd1 retina at the protein level as well. Our results suggest that these PKC isoforms are involved in rd1 degeneration.

Thyroid-beta2 and the retinoid RAR-alpha, RXR-gamma and ROR-beta2 receptor mRNAs; expression profiles in mouse retina, retinal explants and neocortex.

In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the context of human ageing disorders.