Retinitis pigmentosa: New treatment – Restoring the molecular zipper prevent cell death


Retinitis pigmentosa, a degenerative genetic disease of the eye, is characterized by progressive vision loss, usually leading to blindness. In some patients, structural defects in the photoreceptor cells have been observed, but the molecular mechanisms involved are not understood.

A team from the University of Geneva (UNIGE), in collaboration with the University of Lausanne (UNIL), has identified the essential role played by a molecular zipper formed by four proteins. The absence of this zipper leads to cell death in retinal cells. This discovery could lead to the development of therapeutic approaches for retinitis pigmentosa. This work can be read in the journal PLOS Biology.

Retinitis pigmentosa is the most common hereditary retinal disease in humans, with a prevalence of one in every 4,000 people worldwide. The first symptoms usually appear between the ages of 10 and 20 with a loss of night vision.

Thereafter, the visual field narrows into a “tunnel vision” to finally lead to blindness around the age of 40. This disease is characterized by a degeneration of the light sensitive cells, the photoreceptors.

These specialized neuronal cells of the retina are responsible for the conversion of light into a nerve signal. The outer segment of the cell is made up of stacks of disks on which the light-sensitive pigments are located. The inner segment contains all the metabolic machinery essential to the functioning of the cell and is linked to the outer segment by the connecting cilium.

A molecular zipper

Mutations in the genes of four proteins located in this connecting cilium are all associated with retinal pathologies presenting degeneration of photoreceptors. These four proteins had been identified by the laboratory of Paul Guichard and Virginie Hamel of the Department of Molecular and Cellular Biology of the Faculty of Science. They are located in centrioles, cylindrical structures made of microtubules and present in all animal cells.

“In the centriole, these proteins ensure the cohesion of the different microtubules by acting like a zipper. We wondered if they did not play the same role in the tubular structures of the connecting cilium,” explains Virginie Hamel, last author of the study.

Observations with unprecedented precision

Thanks to an expansion microscopy technique optimized by the group of Virginie Hamel and Paul Guichard, which allow cells to be inflated without deforming them, the scientists were able to observe retinal tissue with a resolution never achieved. The biologists focused on the structure of connecting cilia from mice that had – or did not have – a mutation in the gene for one of the four mentioned proteins. These observations were conducted at different life stages.

“In the absence of the mutation, we found that these proteins ensure, just as we had previously seen in centrioles, the cohesion between microtubules by forming a zipper that closes as development proceeds,” explains Olivier Mercey, researcher in the Department of Molecular and Cellular Biology and first author of the study.

On the other hand, when the gene for this protein is mutated, although the structure of the microtubules appears normal in the first days, the microtubules gradually become less and less attached to each other. In adulthood, the affected mice have microtubules that are no longer “zipped” together at all and eventually collapse, leading to cell death of the photoreceptors.

Restoring the ‘molecular zipper’ to prevent cell death

This work has led to a better understanding at the molecular and structural level of retinitis pigmentosa, which allows to consider therapeutic treatments that act upstream of cell degeneration.

“By injecting the protein into patients suffering from certain types of retinitis pigmentosa, we can imagine that the molecular zipper could be restored to ensure the structural integrity of the microtubules of the connecting cilia, thus preventing the death of photoreceptor cells. We are evaluating this approach in collaboration with our colleagues from UNIL and the Jules-Gonin Ophthalmic Hospital, Yvan Arsenijevic and Corinne Kostic,” says Paul Guichard, coauthor of the study.

The retina is a thin tissue lining the back of the eyeball that contains photoreceptor cells, which convert light inputs into electrical signals, a process crucial for the detection of visual stimuli. These highly specialized ciliated cells are partitioned into 2 main regions, a photosensitive outer segment (OS) and a photoreceptor inner segment, which are connected via a thin bridging structure known as the connecting cilium (CC) with its underlying ciliary basal body.

This structural linker, emanating from a mature centriole, is made of 9 microtubule doublets (MTDs) that extend distally to form the axonemal stalk, the basis of the OS onto which hundreds of stacked membrane discs that contain phototransduction proteins are positioned [1–3]. Mutations in the gene that encodes the microtubule-binding protein FAM161A, which localizes at the CC, have been associated with the human pathology retinitis pigmentosa 28 (RP28), a subtype of retinitis pigmentosa, the most prevalent human inherited retinal disease with an incidence of 1/4,000 worldwide [4–11].

Mouse models of FAM161A-associated RP28 have revealed structural defects in the CC, with spread MTDs and disturbed membrane disc organization that underlie photoreceptor loss [9,12]. Similarly, mutations in the genes for the CC-localized proteins POC5, CENTRIN, and POC1B have all been associated with retinal pathologies displaying photoreceptor degeneration [13–15].

Recently, we localized these 4 proteins at the level of centrioles and composing the so-called inner scaffold, a structure connecting neighboring microtubule blades [16]. Depletion of inner scaffold components leads to centriole architectural defects, hinting at a role for this structure in the structural cohesion of the entire organelle [16,17]. These 4 inner scaffold proteins also being present in the CC, we hypothesized that a similar inner scaffold structure might exist at the level of the CC to provide the structural cohesion of the MTDs, thus ensuring OS integrity

Fig 1. Molecular mapping of the mammalian photoreceptor connecting cilium.
(a) Low magnification U-ExM image of a P14 mouse retina highlighting the preservation of the different retina layers. Note that the RPE layer is removed with the dissection. Scale bar: 10 μm. (b) Expanded P14 photoreceptor layer (equivalent region to the white dashed line depicted in (a)). Inset shows details of 1 photoreceptor cell. Arrowheads indicate centrioles. Arrow depicts the gap between centriole and CC POC5 signals. Scale bar: 5 μm (inset: 500 nm). (c–g) Confocal U-ExM images of adult photoreceptors stained for tubulin (magenta) and POC5 (c: green) or CENTRIN (d: yellow), FAM161A (e: gray), CEP290 (f: cyan), and LCA5 (g: orange). Lower panels show transversal views of the CC for each staining. Arrowheads indicate centrioles. Scale bars: side view = 500 nm; transversal view = 200 nm. (h) Distances between the maximum intensity of POC5 (green), CENTRIN (dark yellow), and CEP290 (cyan) compared to tubulin (magenta) calculated from transversal view images. Gray bars indicate the values obtained from the simulation in S4 Fig. n ≥ 2 animals per staining. See S1 Table. (i) Transversal view (left) and polar transform (right) of GT335 (magenta) and CEP290 (cyan) signals revealing overlapping 9-fold symmetry. Scale bar: 200 nm. (j) Plot profiles of CEP290 (cyan) and GT335 (magenta) polar transform of (i). (k) Symmetrized EM image of a P14 CC transversal section revealing an inner ring decorating MTDs (green arrowhead) and Y-links bridging MTDs to the membrane (blue arrowhead). Scale bar: 50 nm. (l) Model representing relative positions calculated in (h) and (j) of POC5 (green line), CENTRIN (dark yellow line), CEP290 (cyan dot and line) to tubulin (magenta) on a contrasted symmetrized EM picture of a CC. Light color lines represent the SD for each protein. Scale bar: 50 nm. The data underlying all the graphs shown in the figure are included in the S1 Data file. CC, connecting cilium; DC, daughter centriole; GCL, ganglion cells layer; IPL, inner nuclear layer; MC, mother centriole; MTD, microtubule doublet; ONL, outer nuclear layer; POS, photoreceptor outer segment.

More information: Olivier Mercey et al, The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration, PLOS Biology (2022). DOI: 10.1371/journal.pbio.3001649


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