Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina

Abstract

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants

“…In any case animals were treated according to European Union standards and were sacrificed according to the current legislation (CE 1099/2009). Cultures of neuroretina explants were carried out as previously described [38]. Explants were cultured at 37°C under normoxia (21% of oxygen) or mild hypoxia conditions (5% of oxygen) for 24h.…”

“…Explants were cultured at 37°C under normoxia (21% of oxygen) or mild hypoxia conditions (5% of oxygen) for 24h. To evaluate the possible protective effect of cGMP accumulation, Zaprinast was used at 100 nmol/L as cGMP-specific phosphodiesterase inhibitor of PDE6 [38, 39]. Zaprinast can inhibit both PDE6 (IC50 = 150 nmol/L) and PDE5 (IC50 = 500–700 nmol/L), however, the inhibitory potency of Zaprinast for rod PDE6 and cone PDE6 (Ki = 30 nM and Ki = 32 nM, respectively) is four times higher than for PDE5 (Ki = 130 nM).…”

“…To evaluate cell death, the terminal deoxynucleotidyl transferase dUTP nick and labelling (TUNEL) assay was used as previously described [38]. TUNEL-, PAR- and caspase -3 positive cells per visual field were counted in at least three visual fields per each retinal explant using software ImageJ software.…”

Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

“…Interestingly, in long term organotypic retinal explant cultures, at concentrations of up to 200 M, Zaprinast induces selective photoreceptor death without affecting other retinal neurons (37). Why does Zaprinast not cause generalized cell death and instead appears to affect only PDE6 under culture conditions (37)(38)(39)(40)? Tissue culture medium, such as R16 (41), used for organotypic retinal culture contains both fatty acids and glutamine.…”

Calpain: a molecule to induce AIF-mediated necroptosis in RGC-5 following elevated hydrostatic pressure

“…Recent studies show that calpains, caspase-3, caspase-8 and caspase-9 are all up-regulated in experimental retinal detachment, which suggests calpains are involved in caspase-dependent photoreceptor death [7]. Pharmacological inhibition of phosphodiesterase 6 (PDE6) induces retinal degeneration in rod and cone-enriched retinal explants with activation of caspase-3, calpain and poly (ADP-ribose) accumulation, which suggests a potential connection between calpain activation and apoptosis [8]. However, besides its role in apoptosis, a new feature of calpains has been found recently.…”