Here we highlighted a novel pathogenic trait of retinal degenerative diseases by analysing a series of knock-in mice with Sema4A
mutations, which are thought to contribute to human retinal degenerative disease11
. Among these mice, only Sema4AF350C/F350C
mice exhibited light-induced retinal degeneration that occurred immediately after birth, which is similar to that observed in Sema4A−/−
mice (). In addition, a protein structural modelling and mutational analysis of the Sema4A protein revealed that the side-chain volume of the 350th amino acid is critical for its proper conformation and function in the endosomal sorting of molecules indispensable for photoreceptor survival (). We further determined that Sema4A
has therapeutic effects in retinal degenerative diseases using virus-mediated gene therapy ().
Recently, we reported that Sema4A mediated the exosomal release of prosaposin and endosomal sorting of retinoid-binding proteins, including CRALBP, in RPE cells10
. The finding that Sema4A functions as an intracellular guide for specific molecules was highly significant because semaphorins and their receptor plexins were previously shown to function as extracellular guidance molecules3
. Indeed, our structural modelling of the Sema4A protein indicated that the plexin-binding site is distant from the 350th amino acid (), suggesting that this mutation does not block this ligand–receptor interaction. This notion is consistent with our previous finding that mice lacking Sema4A receptors exhibited no apparent retinal defects10
. However, as Plexin-D1-deficient mice die soon after birth21
, we cannot exclude a possibility that the defects are potentially mediated by interacting receptors. Further careful evaluation would be required to determine the pathogenesis.
In addition, BN-PAGE analysis showed that the F350C mutation causes intracellular aggregation of the Sema4A protein, probably within the endoplasmic reticulum, in RPE cells (). Thus, it appears that this structural defect prevents the protein from being properly transported to its cellular compartments. In this context, the present findings support the notion that Sema4A can function as an ‘intracellular navigator’ that releases molecules essential for photoreceptor survival. It is also noteworthy that the Sema4AF350C protein did not affect the expression of the Sema4AWT and Sema4AD345H proteins (), indicating that this mutated protein does not function in a dominant-negative manner.
mice did not show a disease phenotype in our study (). In our mouse Sema4A ectodomain model, D345 is located in an α-helix with its side chain well exposed to the solvent and R713 is located in the short cytoplasmic tail region, which is unlikely to form a structural domain. Therefore, both D345H and R713Q do not seem to cause major structural destabilization, which is further supported by the normal cell surface expression of these mutants (). However, we cannot completely exclude the possibility that these mutations contribute to the pathogenicity of human retinal degenerative diseases. There are several possibilities for the differences between our study using mutant mice and a previous human study11
. First, humans have a longer lifespan than mice, and thus F350C heterozygosity (with D345H) in humans may ultimately induce retinal degenerative disease owing to reduced expression of functional Sema4A proteins. Second, we should carefully evaluate the findings of the human study because this report only sequenced the Sema4A
gene but did not definitively exclude the possible involvement of other genetic factors. In addition, the human report did not include the phenotypes of F350C homozygotes. Third, slight amino-acid differences between human and mouse Sema4A might contribute to the fragility of the human Sema4A protein structure with a D345H mutation. It has been recently reported that transiently expressed human Sema4AD345H
mutant protein showed altered intracellular localization in human RPE cell lines22
. Additionally, as large-scale sequential analyses of patient DNA have not been performed except for Pakistani individuals11
, further studies will be required to determine whether patients in other racial groups possess the same mutation.
We presented evidence that virus-mediated Sema4A
gene transfer was successful in an animal model (). Thus, it is theoretically possible to treat this type of retinal degenerative disease with gene therapy, if performed immediately after birth. Recently, considerable progress has been made in the development of gene therapy for retinal degenerative diseases using recombinant adeno-associated virus or lentivirus-based vectors23
. Currently, RPE65
, which encodes the retinoid isomerase enzyme and causes Leber congenital amaurosis, is the first and only gene that has been successfully treated by gene transfer therapy in human eyes24
. As Sema4A
gene transfer displayed a strong curative effect that was comparable to that of RPE65
at least in the histology in animal models, it appears that Sema4A
might be a candidate therapy for retinal degenerative diseases. However, although ERG responses could be detected after gene transfer (), the levels of responses were relatively low compared to those in WT retinas (). Deeper investigations would be necessary to reveal the extent of the gene transfer efficacy. As retinal degenerative diseases are caused by many genetic changes, Sema4A
gene therapy might be limited to a subset of patients carrying Sema4A
genetic changes, such as the F350C mutation. However, considering the endosomal sorting function of Sema4A for various molecules that are indispensable for retinal homeostasis, it is possible that Sema4A
replacement gene therapy might be efficacious in a wider subset of patients with retinal degenerative diseases. Further studies are required to assess the potential of Sema4A
Collectively, we demonstrated that Sema4A is required for photoreceptor survival. We determined that a point mutation in the Sema4A
gene causes retinal degenerative disease, which was further supported by structural modelling analyses. The F350C mutation reduces the amino-acid side-chain volume and creates a significant vacant space in the protein interior, which is known to affect the overall stability of the protein19
and may lead to the precipitation of the protein into non-functional aggregates. Furthermore, photoreceptor degeneration could be rescued by Sema4A
gene supplementation in an animal model. Our findings provide a novel therapeutic target for retinal degenerative diseases.