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Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia

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ABSTRACT

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

No MeSH data available.


Endogenous Ikbkap expression in the retina. Representative LacZ staining on Ikbkap:β-gal retina or β-galactosidase IHC images are shown. A, At E15.5, Ikbkap expression was detected in developing RGCs in the GCL. N, nasal; T, temporal. B, P7 retina showed strong expression in the GCL. There was no regional bias or asymmetry in the expression pattern. C, At 1 month, RGCs, amacrine cells, subset of bipolar cells, and photoreceptors (IS and outer plexiform layer) expressed IKAP. D, At 1 month, antibody staining showed the same pattern of Ikbkap expression as LacZ staining. E, All RGCs (RBPMS+, green) expressed Ikbkap (red; bottom). F, Many amacrine cells (AP2α+) expressed Ikbkap (red; arrowheads). G, A subset of bipolar cells (Otx2+ in INL) expressed Ikbkap (red; arrows). H, Müller glial marker Sox2 (green) did not colocalize with β-gal (red; arrows), suggesting that they do not express Ikbkap. Some of the Sox2+ amacrine cells expressed Ikbkap (arrowheads). NBL, neuroblastic layer. Scale bars, 1 mm (A, top), 50 μm (A, bottom), 500 μm (B), 250 μm (C, top), 25 μm (C, bottom), and 50 μm (D–H).
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Figure 1: Endogenous Ikbkap expression in the retina. Representative LacZ staining on Ikbkap:β-gal retina or β-galactosidase IHC images are shown. A, At E15.5, Ikbkap expression was detected in developing RGCs in the GCL. N, nasal; T, temporal. B, P7 retina showed strong expression in the GCL. There was no regional bias or asymmetry in the expression pattern. C, At 1 month, RGCs, amacrine cells, subset of bipolar cells, and photoreceptors (IS and outer plexiform layer) expressed IKAP. D, At 1 month, antibody staining showed the same pattern of Ikbkap expression as LacZ staining. E, All RGCs (RBPMS+, green) expressed Ikbkap (red; bottom). F, Many amacrine cells (AP2α+) expressed Ikbkap (red; arrowheads). G, A subset of bipolar cells (Otx2+ in INL) expressed Ikbkap (red; arrows). H, Müller glial marker Sox2 (green) did not colocalize with β-gal (red; arrows), suggesting that they do not express Ikbkap. Some of the Sox2+ amacrine cells expressed Ikbkap (arrowheads). NBL, neuroblastic layer. Scale bars, 1 mm (A, top), 50 μm (A, bottom), 500 μm (B), 250 μm (C, top), 25 μm (C, bottom), and 50 μm (D–H).

Mentions: Toward this end, we first determined the expression pattern of Ikbkap in wild-type retinas at various developmental ages using LacZ reporter mice (Ikbkap:β-gal; George et al., 2013). Retinas at embryonic day 15 (E15; peak of RGC generation), postnatal day 7 (P7), P14 (completion of retinal development), 1 month, and 2 months (adult) were collected, and LacZ staining was performed (Fig. 1A–C). At E15, Ikbkap expression was detected in postmitotic neurons in the GCL but not in retinal progenitors (Fig. 1A). We did not observe any regional bias or asymmetry in the expression pattern at any developmental stage (Fig. 1A, B). At P14 and older, the mature expression pattern was established: LacZ staining was detected in RGCs, in a subset of cells in the inner nuclear layer (INL), photoreceptor inner segment (IS), and outer plexiform layer (Fig. 1C, 1 month shown). IHC analyses using anti–β-galactosidase antibody at 1 month showed the same pattern as the LacZ staining (Fig. 1D top) and revealed that all RGCs express Ikbkap, indicated by the colocalization of β-galactosidase and a pan-RGC marker, RBPMS (Rodriguez et al., 2014; Fig. 1E). In addition, Ikbkap expression was detected in many amacrine cells in both the INL and GCL (AP2α+ or Sox2+; Fig. 1F, H, arrowheads) as well as in a subset of bipolar cells (Otx2+ in INL; Fig. 1G, arrows). Interestingly, Sox2+ Müller glial nuclei did not colocalize with β-galactosidase (Fig. 1H, arrows), suggesting that Müller glia do not express Ikbkap in the adult retina.


Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia
Endogenous Ikbkap expression in the retina. Representative LacZ staining on Ikbkap:β-gal retina or β-galactosidase IHC images are shown. A, At E15.5, Ikbkap expression was detected in developing RGCs in the GCL. N, nasal; T, temporal. B, P7 retina showed strong expression in the GCL. There was no regional bias or asymmetry in the expression pattern. C, At 1 month, RGCs, amacrine cells, subset of bipolar cells, and photoreceptors (IS and outer plexiform layer) expressed IKAP. D, At 1 month, antibody staining showed the same pattern of Ikbkap expression as LacZ staining. E, All RGCs (RBPMS+, green) expressed Ikbkap (red; bottom). F, Many amacrine cells (AP2α+) expressed Ikbkap (red; arrowheads). G, A subset of bipolar cells (Otx2+ in INL) expressed Ikbkap (red; arrows). H, Müller glial marker Sox2 (green) did not colocalize with β-gal (red; arrows), suggesting that they do not express Ikbkap. Some of the Sox2+ amacrine cells expressed Ikbkap (arrowheads). NBL, neuroblastic layer. Scale bars, 1 mm (A, top), 50 μm (A, bottom), 500 μm (B), 250 μm (C, top), 25 μm (C, bottom), and 50 μm (D–H).
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Related In: Results  -  Collection

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Figure 1: Endogenous Ikbkap expression in the retina. Representative LacZ staining on Ikbkap:β-gal retina or β-galactosidase IHC images are shown. A, At E15.5, Ikbkap expression was detected in developing RGCs in the GCL. N, nasal; T, temporal. B, P7 retina showed strong expression in the GCL. There was no regional bias or asymmetry in the expression pattern. C, At 1 month, RGCs, amacrine cells, subset of bipolar cells, and photoreceptors (IS and outer plexiform layer) expressed IKAP. D, At 1 month, antibody staining showed the same pattern of Ikbkap expression as LacZ staining. E, All RGCs (RBPMS+, green) expressed Ikbkap (red; bottom). F, Many amacrine cells (AP2α+) expressed Ikbkap (red; arrowheads). G, A subset of bipolar cells (Otx2+ in INL) expressed Ikbkap (red; arrows). H, Müller glial marker Sox2 (green) did not colocalize with β-gal (red; arrows), suggesting that they do not express Ikbkap. Some of the Sox2+ amacrine cells expressed Ikbkap (arrowheads). NBL, neuroblastic layer. Scale bars, 1 mm (A, top), 50 μm (A, bottom), 500 μm (B), 250 μm (C, top), 25 μm (C, bottom), and 50 μm (D–H).
Mentions: Toward this end, we first determined the expression pattern of Ikbkap in wild-type retinas at various developmental ages using LacZ reporter mice (Ikbkap:β-gal; George et al., 2013). Retinas at embryonic day 15 (E15; peak of RGC generation), postnatal day 7 (P7), P14 (completion of retinal development), 1 month, and 2 months (adult) were collected, and LacZ staining was performed (Fig. 1A–C). At E15, Ikbkap expression was detected in postmitotic neurons in the GCL but not in retinal progenitors (Fig. 1A). We did not observe any regional bias or asymmetry in the expression pattern at any developmental stage (Fig. 1A, B). At P14 and older, the mature expression pattern was established: LacZ staining was detected in RGCs, in a subset of cells in the inner nuclear layer (INL), photoreceptor inner segment (IS), and outer plexiform layer (Fig. 1C, 1 month shown). IHC analyses using anti–β-galactosidase antibody at 1 month showed the same pattern as the LacZ staining (Fig. 1D top) and revealed that all RGCs express Ikbkap, indicated by the colocalization of β-galactosidase and a pan-RGC marker, RBPMS (Rodriguez et al., 2014; Fig. 1E). In addition, Ikbkap expression was detected in many amacrine cells in both the INL and GCL (AP2α+ or Sox2+; Fig. 1F, H, arrowheads) as well as in a subset of bipolar cells (Otx2+ in INL; Fig. 1G, arrows). Interestingly, Sox2+ Müller glial nuclei did not colocalize with β-galactosidase (Fig. 1H, arrows), suggesting that Müller glia do not express Ikbkap in the adult retina.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

No MeSH data available.