Date Posted: 16 April 2010
Research conducted at Harvard Medical School has provided a fresh perspective on why healthy cells die in patients with retinitis pigmentosa (RP). This long-standing puzzle may now be resolved through an improved understanding of how cones receive nutrition within the retina. A comprehensive series of experiments have shown that disturbance of an insulin-associated pathway in cone photoreceptor cells can lead to their un-timely death. The finding is of major significance due to the potential to target this pathway and thereby devise a treatment strategy that could benefit an estimated 1.5 million people who lose their sight annually from RP.
While RP is a disorder arising mainly from genetic mutations within rod photoreceptors it has been widely observed that rod cell death is invariably followed by cone cell death. For decades however, researchers have wondered why? In nearly all cases the cones are completely healthy and it is their loss, rather than the loss of rods, that causes most of the devastation in patients with RP. As cones are responsible for our fine and colour vision their degeneration inevitably leads to a significant alteration in the quality of life. Life without cones means the simple pleasures of reading, watching a movie or having the independence to drive from A to B may no longer be available.
Further compounding the mystery of healthy cone cell death has been the equally puzzling observation that the reverse situation does not apply the same rule. In other words when one looks at disorders arising primarily from mutations in cone specific genes, cone cell death does not appear to be followed by wide spread rod cell death. Intrigued by these observations Drs. Claudio Punzo, Karl Kornacker and Constance Cepko of the Harvard Medical School in Massachusetts, set about on a series of experiments to explore how this apparent riddle might be resolved. Their findings are published in a recent edition of Nature Neuroscience (Vol. 11, pp 44-52).
Retinitis pigmentosa is a devastating retinal degenerative disease afflicting approximately 1 in 3,500 of the general population. The disorder is both genetically and clinically heterogeneous. Up to 40 genes have been found to be associated with RP to date and onset can occur in infancy, teens and adulthood with the rate of photoreceptor cell loss varying sometimes between individuals with the same genetic lesion from the same family. Such heterogeneity has severely complicated efforts to devise a therapeutic approach.
On a clinical level a significant proportion of sufferers present with symptoms in their late teens to early adult years. Initial symptoms include loss of peripheral vision and loss of night vision. This phase is followed by continual loss of rod photoreceptors and diminishing low light visual ability. Eventually, a similar demise of the cone cells is observed and in many cases complete or near-complete blindness is the outcome.
While modern life and technology provides the capacity to avoid dim lighting conditions the loss of rod photoreceptor cells may not appear as a major devastation. However, the inevitable loss of the cone cells following rod cell death is where the true impact of the disease is realised. The research team at Harvard led by Dr. Cepko wanted to understand what was causing this cone cell death and, by finding the cause, potentially explore if any therapeutic intervention might be possible to extend the life span of cone photoreceptor cells.
The researchers examined four mouse models of RP used in several research labs around the world. In each instance, as had been previously reported, a mutation in a rod specific gene leads to the death of rod photoreceptors. In each of the models cone cell death always started at the end of the rod cell death phase and additionally appeared to spread from the central retina to the periphery. Although the timing of the phases was different in the different models there were sufficient common features to suggest that an under-lying common mechanism might explain the kinetics of cell death.
To see if such a common mechanism could be found the research team decided to analyze global gene expression in the rod and cone photoreceptors. Using sophisticated gene chip technologies researchers could take a snapshot of both rod and cone cell populations at various time points in the demise of the retina and get a ringside view of the activity of nearly 200 genes during various stages of photoreceptor cell death. When the data was crunched almost 35% of the "hits" showed activity in genes involved with cellular metabolism and one in particular, the insulin/mTOR signalling pathway, clearly stood out.
The insulin/ mTOR signalling pathway is known to be a critical pathway in regulating a number of aspects of cellular metabolism and its identification in a global gene expression assay of dying rod and cone photoreceptors suggested that there may be a link between the pathway and cell death. Under normal conditions the mTOR protein interacts with several cell proteins to facilitate high-energy processes such as protein translation. However, under conditions of stress, such as nutrient deprivation, mTOR has the opposite effect. Dr. Cepko and colleagues observed that the active mTOR was progressively reduced in the retinas of the four RP animal models and that its depletion coincided with cone cell death. This certainly appeared to be a smoking gun but a clearer understanding of the mechanism would be required before all the dots could be joined together.
The observations around mTOR activity suggested that a nutritional imbalance, possibly caused by reduced glucose levels, was occurring in cones during degeneration. In support of this model additional assays showed the transcription factor HIF-1 alpha/beta (hypoxia inducible factor 1) and its target, GLUT-1 (glucose transporter 1) were up-regulated in the cone photoreceptors of all models, which is what one would expect in cells trying to overcome nutrient deprivation. A further consequence of such nutrient deprivation would be the activation of "autophagy" in which cells re-absorb proteins and organelles in an effort to retrieve cellular nutrients. One form of such autophagy, "chaperone-mediated autophagy", or CMA, can be detected by the expression of CMA related genes in dying cone cells and this is exactly what was found in each of the RP animal models. Now several lines of evidence were pointing to the idea that cone cells in the degenerating RP retina may be dying from starvation brought about through compromised glucose uptake and low mTOR activity.
The next obvious step, once it was found something was missing, was to re-introduce the putative missing part and see what happens. When one of the animal models of RP was administered with insulin over a 4-week period cone cell survival improved. It appeared that facilitating glucose uptake ameliorated cone cell death. Taken together these observations provide an entirely new mechanism for explaining cone cell death in retinitis pigmentosa, not to mention the identification of a potential pathway to target for the development of new therapeutics.
The model for resolving the mystery of healthy cone cell death may also prove to be remarkably elegant in that it simultaneously accounts for why cone led pathologies do not lead to rod photoreceptor cell death. Given the role of the retinal pigment epithelium (RPE) in shuttling nutrients and oxygen from the choroid to the photoreceptors the Harvard model of cone cell demise via starvation is entirely reasonable when one considers the number of rods to cones in the human (and mouse) retina. As approximately 95% of human photoreceptors are rods and approximately 20-30 outer segments of photoreceptors connect in with one RPE cell, a simple calculation shows that possibly one or two of the RPE/outer segment contacts are with cone cell outer segments. As the retina degenerates the outer nuclear layer (ONL) breaks down and consequently the number of RPE/cone connections becomes less. Dr. Cepko and her team suggest that as the number of RPE/cone connections falls below a certain threshold required for proper flow of nutrients the reduced supply of nutrients to the cones leads to cell starvation. In other words cell density may represent a critical threshold and it may be no coincidence that in all four models of RP, cone cell death occurred when there was a single layer of rods remaining in the ONL.
The mechanism of cone cell death proposed by the Harvard researchers also neatly explains the "reverse case", i.e., why the loss of cones in a cone-led degeneration does not lead to rod photoreceptor cell death. Again, a simple look at the numbers show that cones account for less than 5% of human photoreceptors and, even when the majority of cones are lost, significant cell density remains from the rod cell population. In essence, the "critical threshold" is never reached in cone-led degenerations and so rods never experience a comparable starvation phase.
While the simple administration of insulin in the RP animal models lead to cone cell survival it is unlikely that such a strategy could be advised as a human therapeutic approach. However, if the mechanism proposed by Dr. Cepko and colleagues for cone cell death in RP is validated, then there may be an abundance of therapeutic targets and opportunities available for medical intervention. Given the clinical and genetic heterogeneity of RP, the commonality of its final phase may provide an attractive opportunity to treat a very large market and extend the viability for fine and colour vision even as the rods are lost.
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