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Retinitis pigmentosa: a new form of cell death

What form of cell death do the photoreceptors of people suffering from retinitis pigmentosa undergo and how can their degeneration be prevented or delayed with pharmaceutical substances? Dr. Paquet-Durand from the Institute for Ophthalmic Research in Tübingen is working to resolve these questions.

Dr. Paquet-Durand, a biochemist at the Institute for Ophthalmic Research in Tübingen, is investigating the cell death mechanisms associated with the eye disease retinitis pigmentosa. © Paquet-Durand

One interesting discovery resulting from Dr. François Paquet-Durand’s research is that there are different forms of cell death. The biochemist has been investigating cell death mechanisms associated with photoreceptor neurodegeneration in retinitis pigmentosa for around four years now. Retinitis pigmentosa is a genetic eye condition that leads to blindness. The disorder is characterised by the progressive peripheral loss of photoreceptor cells (i.e. the rods concentrated at the outer edges of the retina which make night vision possible) that can lead to a loss of central vision. Although the cones that are responsible for colour vision do not usually have a genetic defect, they eventually also degenerate as the disease progresses. Retinitis pigmentosa symptoms progress from night blindness to the reduction of the peripheral visual field (tunnel vision) leading eventually to complete blindness. The development of effective medical treatments requires the investigation and clarification of the cell death mechanisms of the photoreceptor cells.

Neither apoptosis nor necrosis

Around 45 rod-related genes are known which, when mutated, can lead to retinitis pigmentosa. However, Paquet-Durand’s investigations found that most of the biochemical processes involved in photoreceptor degeneration are not associated with established cell death mechanisms such as apoptosis or necrosis. “We found that some processes are associated with apoptosis, i.e. programmed cell death, and some with necrosis. However, we also found processes that are not associated with either of these two forms of cell death.” Although there is little evidence that substantiates the presence of apoptotic mechanisms, the degeneration of the rods was initially thought to be an apoptotic process because some parts of the process were identical.

In the classical process of apoptosis, mitochondrial cytochrome C is released into the cytosol where it induces the formation of apoptosomes, protein complexes that boost the death of cells. Proteolytic enzymes, i.e. caspases, play a key role in apoptosis where they are overexpressed and activated. “We found no evidence that this is the case in photoreceptor cells. We did not find any cytochrome C in the cytosol nor did we find any up-regulated and activated caspases. In addition, apoptosis inhibitors had no effect. Although all this has been known for around 15 years, little attention has been given to these findings, explained the researcher.

“We want to understand the process of cell death in order to find new ways to prevent it.”

Overview of cell death mechanisms in photoreceptors (preliminary): an elevated cGMP level leads to an excessive influx of calcium (Ca2+) by way of cGMP-regulated ion channels as well as to the activation of the enzyme protein kinase G, which in turn induces further degenerative processes. The sum of these processes leads to the death of the photoreceptors. © Paquet-Durand
If the photoreceptors do not undergo apoptosis, what form of cell death do they undergo instead? It is known certain enzymes display a higher metabolic activity when the rods degenerate. For example, a higher enzymatic activity of proteolytic calpains that are associated with necrosis has been found. “Necrosis is often associated with traumata or intoxications that lead to acute, sometimes also chaotic processes that induce inflammatory processes. However, in this case, no inflammatory processes were found. There is no conclusive evidence that the death of the photoreceptors is a necrotic process,” said Paquet-Durand who hypothesizes that the degeneration of the photoreceptor cells is a programmed cell death mechanism, but one that is very different from classical apoptosis. The researchers from Tübingen focus predominantly on enzymes that have an effect on the histones, i.e. proteins that package the DNA in the cell nucleus. “We were able to show for the very first time that histone deacetylases and cGMP-dependent protein kinases are associated with the degeneration of the retinal photoreceptors,” said Paquet-Durand summarising the greatest milestone in his scientific career in Tübingen so far. Paquet-Durand investigates the enzymes on the expression level as well as on the metabolic level by comparing the enzymatic activity of individual photoreceptor cells in wild-type retinas and diseased retinas. Paquet-Durand believes that all proteins that are overexpressed during cell-death processes represent potential targets for medical therapies. “Our observations have shown that cGMP-dependent protein kinase G inhibitors have some protective effect. Inhibitors such as calpain blockers might under certain circumstances have a toxic effect. However, the most important questions relate to the connection between these processes as well as their temporal course and which of the processes are slow and which are relatively quick. This would provide us with information on different approaches and therapeutic time slots,” said Paquet-Durand explaining the ambitious programme of his group of researchers.

Goal: Developing cell degeneration markers

Retinal cross section. The cGMP (red) level increases when the rod photoreceptors die. © Paquet-Durand

Paquet-Durand mainly uses tissue slices and cultures of explanted retinas that can be kept in the laboratory for up to six weeks. “The advantage of this procedure is that we can monitor changes under controlled conditions,” said Paquet-Durand. Since at the moment there are hardly any markers for degenerating photoreceptor cells available, reliably identifying these cells is a huge challenge. Amongst other things, his team is looking for characteristic morphological changes such as signs of cell nucleus decondensation. Besides these investigations, the group of researchers is also aiming to develop markers to work on in the future, which would also benefit the search for effective drugs.

But Paquet-Durand does not want to raise unrealistic hopes, as he is aware that the processes that need to be targeted are highly complex. “Realistically, I believe that due to the genetic heterogeneity of retinitis pigmentosa, it will not be possible to fully cure the disease in the near future. I do not believe that it is possible to completely prevent the degeneration of the photoreceptors in the near future, but I do believe that we will be able to slow down the progression of the disease.”

A coherent global approach: medical therapy and retinal implants

Enabling medical treatment would be an important piece in the jigsaw puzzle in terms of the effort to save, or even restore, the vision of retinitis pigmentosa sufferers as much as possible. Such medical treatment would be used in combination with other therapeutic approaches. A completely different approach is the electronic retinal implant that was also developed at the Institute for Ophthalmic Research at the University of Tübingen by researchers led by Prof. Dr. Eberhart Zrenner. The implant has already been used to treat 18 blind people. The retinal implant replaces degenerated photoreceptors with technical light sensors. However, such an implant can only be used for the treatment of patients who are completely blind and, as such, cannot be treated with drugs. “Our approaches are very different, but could complement each other. I believe that in about ten to twenty years’ time we will be capable of delaying the degeneration of photoreceptors for a period of time that would allow us to further develop the retinal implant,” said Paquet-Durand.

Over the next few years, Paquet-Durand and his team will focus on increasing their knowledge of the specific cell death of photoreceptors and in particular investigating the role of the epigenetic process in the degeneration of photoreceptors. He also hopes to be able to find out why retinitis pigmentosa also leads to the degeneration of the cone photoreceptors, although the genetic mutations that have been discovered only affect the rods. The secondary degeneration of the cones is not yet understood in detail and further research is necessary to obtain further insights into the process. “One reason might be related to changes in oxygen supply. But we do not know for sure and want to focus on this in greater detail. In animal experiments, we have already found that cones with mutations undergo similar cell death processes to rods,” said Paquet-Durand.

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