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G. Inana, C. Murat, M. J. McLaren, Y. Ishiba; Tubulin Defect in Knock-out Model of HRG4(UNC119). Invest. Ophthalmol. Vis. Sci. 2008;49(13):1281.
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HRG4(UNC119) is a photoreceptor synaptic and inner segment (IS) protein, a truncation mutation in which causes synaptic degeneration and late-onset retinal degeneration (RD) in human and a mouse transgenic (TG) model. A knock-out (KO) model of HRG4 demonstrated a more severe RD and a functional defect involving the inner and outer segments (OS) without evidence of early synaptic degeneration. To understand the mechanism of the RD in the KO, the status of HRG4, its interactor ARL2, and its downstream proteins was investigated.
The retinas of HRG4 TG and KO mice of various ages (6, 12, 15, 22, 28 months) were subjected to immunofluorescent analysis for HRG4, ARL2, GFAP, and alpha and beta tubulin. The tubulins were examined because of the important role ARL2 plays in tubulin heterodimerization and the demonstrated interaction between HRG4 and ARL2.
Dramatic gliosis as reflected by an increase in GFAP staining of Muller cells was demonstrated in the KO starting at 6 months, but only a modest increase in GFAP staining was present in the TG even at 22 months. Colocalization of HRG4 and ARL2 was demonstrated in the outer plexiform layer and the IS, consistent with their interaction. Specific localization of ARL2 in the connecting cilium (CC) between the IS and OS was demonstrated. The levels of alpha and beta tubulin staining in the CC were approximately equal in the normal and KO retinas at 6 months, began to show a decrease in the KO at 12 months, and were essentially absent in the KO at 15 months, despite the presence of IS/OS and 6-7 rows of photoreceptor nuclei in the outer nuclear layer. In contrast, the CC in 28 month old TG with approximately the same degree of RD (6-7 rows of photoreceptor nuclei left) showed strong staining for both alpha and beta tubulin.
There is a defect in tubulins that make up the microtubular structure, CC, in the KO, and this defect is not present in the TG. The tubulin defect in the KO is most likely caused by the absence of HRG4 via its interactor, ARL2. The results confirmed the difference in the mechanism of RD in the HRG4 KO and TG models.
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