INTRODUCTION:

Eye is the window to the soul. It is one of the God’s amazing creations that helps one to see and experience his other creations. The complexity of the physiologic processes taking place in the human eye is just unimaginable. Even an absence of a single entity can lead to its pathologic condition. The major process of perception of an image takes place in the retina. Retina is a light sensitive layer present at the back of the eye.

PHYSIOLOGY OF PHOTOTRANSDUCTION:

The process of phototransduction occurs in the photoreceptor layer in the retina which involves changes in potential of a photoreceptor cell on exposure to a photon. In the dark there is a high concentration of cGMP in the photoreceptor cells. This cGMP causes the ion channels to open which in turn causes the entry of positive ions like sodium and calcium resulting in depolarisation. Hence these cells release glutamate in the dark. By these processes the photoreceptor cells remain depolarised in the dark. On exposure to light, the retinal is converted from its cis form into its trans form. This structural change of retinal causes the production of a protein called transducin, which has the capability to increase the level of phosphodiesterase.

The main function of this phosphodiesterase is to convert cGMP into 5GMP which is the inactive form of cGMP. Thus this decrease in level of cGMP causes the closure of the ion channels and preventing the entry of positive ions causing hyperpolarisation. These processes cause the hyperpolarisation of the photoreceptor cells on exposure to light. These changes in potential of a photoreceptor cell causes the transmission of electric signals from the retina to the processing centre in the occipital lobe of cerebral cortex through the optic nerve. Any changes in this normal physiology of phototransduction inhibits the normal perception of images. This disruption in the normal physiology of phototransduction may be due to a wide range of causes including mutations in a particular gene that may lead to production of misfolded proteins or may inhibit the production of a particular protein involved in the pathway of phototransduction. One such disease that arises due to multiple genes that get mutated causing the production of many defective proteins is retinitis pigmentosa (RP).

RETINITIS PIGMENTOSA- GENETIC BACKGROUND:

Retinitis pigmentosa is basically an inherited disease leading to degeneration of the photoreceptor cells disrupting the normal physiology of phototransduction. This may be autosomal dominant, autosomal recessive, X-linked or maternally acquired. Mutations in pre-mRNA splicing causes autosomal dominant retinitis pigmentosa. Autosomal recessive RP is caused when two unaffected individuals who are carriers of the same RP inducing gene in diallelic form can produce offspring with RP. X-linked RP is identified with mutations of 6 genes most commonly occurring at a specific loci in the RPGR and RP2 genes. These multiple mutations are produced causing the degeneration of photoreceptor cells. About 150 mutations has been identified till date. The mutations of various genes disrupts the normal pathway in various methods depending upon the misfolded proteins that are produced..

The mutation may also be caused in the rhodopsin gene which helps in the production and biochemical pathways involving rhodopsin, a pigment helping for vision in the dark. Rhodopsin is basically made up of opsin and retinine. This mutation in the opsin gene is found in all the three domains of the protein: intradiscal, transmembrane and cytoplasmic. This mutation in the rhodopsin gene disrupts the normal rod-opsin pathways which helps in the conversion of light into electric signals. Class I mutant protein’s activity is due to specific point mutations in protein coding amino acid sequence that affect the pigment protein’s transportation to theouter segment of the eye where the phototransduction occurs. Misfolding of class II rhodopsin gene mutations disrupts the protein’s conjunction with 11cis-retinal to induce normal chromophore formation [2].

Fig (1)

Some mutations can also decrease protein stability and the activation rates of opsin and transducin. Some models suggest that the retinal pigment epithelium fails to phagocytosethe aged rod cells. This inturn causes the accumulation of rod debris. A defect in phosphodiesterase has also been noted. This defect may lead to toxic levels of cGMP as it cannot be converted into 5GMP due to the defective production of phosphodiesterase. Therefore there is continuous entry of positive ions like calcium leading to apoptosis [3].

Fig (2).

APOPTOSIS AND PHOTORECEPTOR DEGENERATION:

Apoptosis is the common final pathway in all kinds of RP. The process of apoptosis can occur either by mitochondrial pathway or cell death receptor pathway, that is, either by caspase dependant or caspase independent pathway. There is presence of pro or anti-apoptotic bcl2 family of proteins. These proteins play a major role in the pathogenesis of apoptosis in retinal disorders. This kind of apoptosis can be treated by delivering anti-apoptotic molecules targeting the bcl2 pathway through the use of cell membrane permeable transport peptides. Calpins also play a major role in this process of apoptosis causing degeneration of photoreceptors.

Fig (3).

Apoptosis is usually associated with increased intracellular calcium ion levels. This elevated calcium ion levels is caused by transporters and channels present in the cell membrane, mitochondria and the endoplasmic reticulum. Cyclic Nucleotide Gated ion Channels (CNGC’s) is present on the outer segment and Voltage Gated Calcium Channels (VGCC’s) are located in the cell body and synaptic terminal and they play an important role in increasing intracellular calcium levels. These channels are closed by hyperpolarisation of the cell. Steele et al suggested that the average level of intracellular calcium varies from approximately 350nM in hyperpolarised light adapted cells to more than 39µM in depolarised cells of salamander rods and cones. But in case of RP there is degeneration of photoreceptor cells by apoptosis causing an increase in calcium levels. This elevated calcium level is provided by plasma membrane calcium ATPase, store operated calcium entry and the calcium stores present within mitochondria and endoplasmic reticulum. One of the multiple mutations is the mutation in β-subunit of cGMP phosphodieserase causing rd1. This mutation causes the defective production of phosphodiesterase, responsible for the closure of ion channels by converting cGMP into 5GMP when the photoreceptors are exposed to light. Because of this, the ion channels are not closed even on light exposure. This in turn causes the continuous inflow of calcium ions. Hence this toxic levels of calcium ions in the photoreceptors can be detected even before the detection of apoptosis. Now this increased calcium ion levels trigger a protein called calpins. From then, apoptosis can be caused by either of the two pathways, that is caspase dependant or caspase independent pathways. Caspase dependant apoptosis takes place when these calpins activates caspases 3, 4, 7, 9 which causes further downstream signalling of proteins resulting in caspase dependant apoptosis. These calpins induces proteins like cathepsins and mitochondrial calpins activatesAIF, which is translocated from mitochondria to the nucleus resulting in caspase independent apoptosis and finally causing the degeneration of photoreceptors causing RP[5].

The basic idea to prevent this form of apoptosis occurring due to elevated calcium levels is by the action of calcium channel antagonists like D-cis-diltiazem, a benzothiazepin which can block the CNGC’s and VGCC’s through which the calcium ions enter. D-cis-diltiazem reduced intracellular calcium levels, down regulating calpins and further processes in apoptosis in rd1 mice. It showed inhibitory effects on CNGC’s and L-type VGCC’s. L-cis isomer of diltiazem inhibits L-type VGCC’s. The difference in action between D-cis form and L-cis form of diltiazem shows that CNGC is also important for photoreceptor neuroprotection [4].

However it is important to note that this process of mutations occurs mainly in the rod cells due to rod-specific mutations. There is no clear explanation for the death of the cone cells. But, however, in case of RP there is degeneration of both rods and cones. Recent studies show that the apoptosis of cone cells occurs due to cell starvation and changes in the insulin/ MTOR pathway. In a recent research conducted in four mouse models, having mutations in rod-specific genes, the non-autonomous cone death occurs due to changes in the insulin/mammalian target of rapamycin pathway which coincides with the autophagy during the period of cone death. Basically this cell starvation of cones leads to autophagy. Studies has been done in this case by increasing or decreasing the insulin levels and measured the survival of cones in one of the models. Mice that were treated systemically had prolonged cone survival because of that fact that insulin prevents cell starvation. The depletion of endogenous insulin level had the opposite effect. Hence, this non-autonomous cone death in RP, atleast in part, occurs as a result of starvation of cones.

Fig (4).

TREATMENT FOR RETINITIS PIGMENTOSA:

ARGUS RETINAL PROSTHESIS –“BIONIC EYE”:

It is one of the recent research going on for the treatment of RP. It is basically a retinal implant which consists of two parts - a retinal implant and an external system. This external system is made up of an eye-glass mounted camera in combination with a small processor. This camera captures the real-time images which are processed by the processor and the processed signals are sent wirelessly to the retinal implant by a built-in video processor. Thus the image is transferred to the retinal implant. In the implant there are about 60 electrodes which makes use of the remaining healthy photoreceptor cells and transmits the signals to the brain via the optic nerve [6].

Human trials: The latest 3 year clinical trials have proven the long-term efficacy safety and reliability of the device. Studies show that 80 percent of the trial patients received benefit from this method. However, in this 3 year clinical trials, 11 patients have experienced severe adverse events which were then successfully treated. One of these treatments for an adverse event had to be done by removing the implant due to recurrent erosion. Adverse events maybe lower Intraocular Pressure (IOP) than normal, erosion of the conjunctiva, reopening of the surgical wound, inflammation of the eye and even lead to detachment of the retina [7].

Fig (5).

GENETIC THERAPY:

It is done by detaching the retina and injecting a virus particle in place of the retina. This virus has the capability to produce normal proteins that has to be produced by the normal photoreceptor cells. This type of gene therapy has been done in case of mutated RPGR gene in dogs. Studies show that when a normal RPGR gene is inoculated into the Adeno-associated virus particle (AAV) and is injected in-between the photoreceptor cell layer and the pigment epithelium layer of the retina, this normal human RPGR gene inoculated into the AAV produced normal RPGR protein in dogs preventing the degeneration of photoreceptor cells. Human trials: Doctors used a fine needle to inject the virus underneath the retina. Jonathan Wyatt, 65, had his weaker left eye treated as a part of the trial and was excited to notice his vision improving in a few days. He was amazed to see the green pitch much greener and the nearby objects much clearer [8].

Fig (6).

GENE EDITING TECHNIQUES – CRISPR:

CRISPR/ Cas 9 (Clustered Regulatory Interspaced Short Palindromis Repeat) has shown the ability to treat RP in test mice. It works by altering the genetic mutations that cause illness. The researchers used this CRISPR method to remove the mutated genes causing RP in mice. Then, a technique called optomotor reflex which involves head turns in response to moving stripes in varying brightness levels. This technique was used to measure the effect of CRISPR system single shot in the test mice. A single shot improved the vision of the mice when compared to the mice in the control group. This cas9 is based on a system used by a bacteria to terminate invasive viruses. Initially the bacteria copies a part of the virus’s genetic code. The code is copied into a special structure RNA that transmits the code’s instructions. When this invasive virus comes back, RNA attaches itself to cas9 protein. This RNA guides the cas9 protein to the similar gene in the invasive virus so that the protein can deactivate the matching gene present in the invasive virus. However research is still going on regarding the safety and reliability of this method to be used for human trials[8].

Fig (7).

ELECTRONIC CHIP – A RETINAL IMPLANT:

This project is funded by The National Institute for Health Research (NIHR) in association with Retinal Implant AG and the NIHR Oxford Biomedical Research Centre. It consists of a wafer-thin retinal implant chip measuring 3x3 mm²which is inserted into the back of the eye to replace the damaged photoreceptor cells. This process is done in a delicate 6-8 hour operation. This chip senses the light entering the eye and captures this light which in turn stimulates the nerve cells in the back of the retina thus transmitting the light to the brain through the optic nerve. This retinal implant chip is connected to a tiny computer places underneath the skin behind the ear, this is powered by a magnetic coil which is also present on the skin. This device is switched on once the healing occurs after surgery. It looks like a typical hearing aid. It will take some time for the users to interpret the signal provided by the implant as the part of the brain responsible for interpreting the visual signals would have remained dormant for a long time in the trial patients[8].

Fig (8).

SMART GLASSES:

This method was developed by doctor Stephen Hicks, A scientist at Oxford University in collaboration with the John Radcliffe Hospital, Oxford. It consist of a video camera mounted on the frame of the glasses, a computer processing unit that is small enough to be pocketed and a software that provides images of objects close by to the see through displays present in the eyepieces of the glasses. The transparent electronic displays in the glass lenses gives a simple image of people and nearby obstacles using the software enabling people to see nearby objects distinctly. The advantage is that these glasses are designed to work well in dim light helping people to cope up with night blindness. However the testing of this method is still going on[8].

Fig (9).

RECOMMENDATIONS:

The above mentioned methods help to improve the vision of an RP patient to a great extent but however complete cure has not been discovered yet. The treatment involving electronic implants provide a distinct image of the nearby objects but the processing time and heat generated may prove harmful to the eye. The process of injecting a virus particle into the retina and other genetic alterations must be done very carefully as retina is a very delicate layer. So it is better to treat RP by natural methods involving stem cells.

FUTURE:

The Embryonic Stem Cells (ESC’s) have the capability to differentiate into almost any cell type in our body when right signals are given at the right time. These cells can be grown in the lab and can be moulded into becoming retinal cells. This has been done by the scientists at RIKEN Centre for Developmental Biology, Kobe, Japan. This team grew the ESC’s in vitro and coaxed them into becoming retinal cells. When this new retinal tissue was transplanted into the monkeys with RP, they were able to regain their sight. The main drawbacks of this method are, The ESC’s need to differentiate into two types of photo receptor cells – rods and cones. They also need to wire themselves to the already existing network of supporting cells in the retina, but these ESC’s when differentiated into photoreceptor cells were capable of forming new connections within the already existing supporting tissue. Also these stem cells can be obtained from various sources provided they have the capacity to differentiate into retinal cells. The research team of ReNeuron has developed stem cells called CTX cells which release large quantities of exosomes. These exosomes are microscopic vesicles present in body fluids which contains proteins, lipids and RNA. The main activities of these exosomes include promoting the activation of regenerative program in diseased or injured cells of the body. Still Phase I clinical trials are going on to test the regenerative power of these exosomes. These cells can also be used for regeneration of damaged photoreceptor cells in the retina.

CONCLUSION:

These treatments are far safe and reliable than electronic implants and chips. But for now these electronic implants helps the RP patients to live their once not possible dreams by improving their vision to a significant extent. But a complete cure for RP can be provided by the stem cells which has the potential to completely replace the degenerated photoreceptors in the retina. This exciting research is still going on. Once declared safe it will be a new day in the life of RP patients and a great medical breakthrough.

REFERENCES:

1. G.Q. Chang, Y. Hao and F. Wang, “Apoptosis: Final common pathway of photoreceptor death in rd, rds and rhodopsin mutant mice”, Neuron, Vol.11, no.4,pp- 595-605, 1993

2. T.N. Dryja, T.L. McGee and T.L. McGee,” A point mutation of the rhodopsin gene in one form of retinitis pigmentosa”, Nature, Vol.343, no.6256, pp. 364-366, 1990

3. M.E. McLaughlin, M.A. Sanderberg, E.L. Berson and T.P. Dryja, “Recessive mutations in the gene encoding the β-subunit of rod phosphodiesterase in patients with RP”, Nature Genetics, Vol.4, no.2, pp. 130-134, 1993

4. Mitsuru Nakazawa, “Effects of calcium ion, calpins and calcium ion channel blockers on retinitis pigmentosa”, Journal of ophthalmology, Vol 2011, Article ID- 2920401, 7 pages.

5. Sandra Cottet and Daniel. F. Schorderet, “Mechanisms of apoptosis in Retinitis Pigmentosa”, Current Molecular Medicine 2009, 9, 375-383375

6. “A Bionic Eye comes to the market”, MIT Technology Review, 7 March 2011, Retrieved 17 Feb 2013.

7. “History”, Second Sight, Retrieved 17 Feb 2013.

8. “RP Fighting Blindness” [Internet] [Place: unknown], Updated 22 Jan 2016, Available from www.rpfightingblindness.org.uk