Towards new treatments for hereditary photoreceptor degeneration: Pharmacological and genetic approaches to restore Ca2+ homeostasis and Pde6a function

Abstract

Retinitis pigmentosa (RP) is a rare hereditary retinopathy that begins with the loss of peripheral vision and gradually progresses towards the centre causing blindness. The responsible factor behind this pattern is the primary degeneration of rod photoreceptors (rods) followed by the secondary degeneration of cone photoreceptors (cones). About 1 in 4,000 individuals are affected worldwide with no effective cure currently available. RP is genetically very heterogeneous with about 65 causative genes and numerous underlying mutations. In this study, two different RP causing mutations have been investigated to understand the cell death mechanisms and explore possible therapeutic interventions. This work focuses on mutations in phosphodiesterase-6 (PDE6) enzyme, which leads to the elevated accumulation of intracellular cGMP in photoreceptors and in-turn is responsible for high Ca2+ influx into the photoreceptors. One of the considered consequences of high Ca2+ is photoreceptor degeneration by the activation of Ca2+-dependent cysteine type proteases, calpain. Primarily two approaches were employed to investigate the consequences of the PDE6 mutations: (1) interfering with the downstream cascade following a Pde6b mutation. This was achieved by targeting Cyclic Nucleotide Gated Channels (CNGC) and the lowering of intracellular Ca2+ concentration and (2) by editing the mutant mRNA of Pde6a via site-directed RNA editing to restore functional PDE6A subunit of PDE6 enzyme. Earlier studies have highlighted that the high Ca2+ influx in RP is caused via CNGC, suggesting it as the target candidate for reducing the intracellular Ca2+ concentration. This study applied the possibility of reducing intracellular Ca2+ concentration by: (A) targeting CNGC using pharmacological inhibition (diltiazem enantiomers and cGMP analogues) and (B) designing Antisense Oligonucleotides (ASO) to down-regulate Cngb1. Diltiazem is a well known hypertension drug where L- and D-cis-diltiazem are known to target CNGC and Voltage Gated Calcium Channels (VGCC) respectively, the two important channels responsible for Ca2+ influx in the photoreceptors. To understand the downstream effects of Ca2+ channel inhibition, the activity of calpain and Poly(ADP-ribose) Polymerase (PARP) were investigated. Activated PARP uses Nicotinamide Adenine Dinucleotide (NAD+) and generates Poly (ADP-Ribose) (PAR) polymer as a response to DNA damage during cell death. Thus, PAR was also investigated with the treatment conditions. Further the blocking of VGCC with D-cis-diltiazem showed a lowering of the PARP activity in rd1 however, blocking CNGC with L-cis-diltiazem increased the PARP activity in wild-type (wt). These indicate that level of intracellular Ca2+ could modulate the PARP activity. Lowering of the Ca2+ influx via CNGC (L-cis-diltiazem) showed elevated calpain-2 in wt to the level of rd1 retina but did not change the calpain activity. The inhibition of photoreceptor Ca2+ influx via VGCC showed lowered calpain-2 in rd1, however showed no change in the calpain activity. Moreover, the photoreceptor degeneration got elevated on CNGC inhibition using L-cis-diltiazem and cGMP analogue (DF156). Thus, the CNGC blocking and inhibition of Ca2+ influx via CNGC did not prevent or slow the course of photoreceptor degeneration. Consequences of mutation in PDE6 can also be prevented by replacing it with a functional PDE6. The restoration of functional PDE6A in animal model Pde6a V685M was achieved with site-directed RNA editing on the mouse retinal explant cultures. The editing was achieved by using ASO that exploits naturally occurring enzymatic activity of Adenosine Deaminase that acts on RNA (ADAR) in the cell. It acts on the primary transcript (mRNA) to perform site-directed editing and is highly targeted and reversible approach with reduced off-target effects. Site-directed RNA editing in Pde6a V685M retinal explants showed expression of PDE6A in retinal histological sections, indicating a possible functional restoration of PDE6A. NAD+ consumption leads to energy depletion and Glucose transporters help in meeting the energy demand of photoreceptors. The localisation of Glucose Transporter 3 (GLUT 3), a neuronal glucose transporter, was investigated to indicate its possible role in the degeneration. GLUT 3 showed a continuous, but likely a structurally independent expression between OS and IS across the adult mouse retina. Their active role is unclear. Retinal degeneration resulting due to exogenous factors such as, malarial parasite (Plasmodium berghei) was investigated on the wt retina. Infiltration of the malarial parasites were found in the neuroretina by crossing the blood-retinal-barrier. Muller cells are likely to play an important role during parasitaemia indicated by their co-localisation with the parasites.

Therefore in the cell death pathway, PARP activity was found to be modulated by Ca2+ concentration and photoreceptor degeneration in rd1 does not appear to be directly influenced by the CNGC Ca2+ influx. Importantly, the pharmacological inhibition of CNGC was not protective for degenerating the rod photoreceptors, providing a new insight on rd1 cell death mechanisms.