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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.

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Six-month Ikbkap CKO retinas were grossly normal except for the reduction in RGCs. A, H&E staining showing control and mutant (CKO) eyes. The mutant eyes at 6 months appear normal. B, Retinal cross sections at central (>0.25 mm from ONH), middle (1 mm from ONH), and peripheral (1.75 mm from ONH) retinas in the temporal hemisphere. Retinal structure of the mutant was grossly normal, except for the reduction in cell number in the GCL that is apparent toward the peripheral retinas. C, Reduction in cell number was observed in the mutant GCL at 1 mm from the ONH (middle). Images of the temporal retina are shown. D, Mutant retinas showed reduced numbers of RGC marker, Brn3 (red). Islet1 (green) IHC showed normal cholinergic amacrine and optic nerve bipolar cells. E, Choline acetyltransferase (green) IHC showed normal number and structure of cholinergic amacrine cells. PKD2L-1 (red) IHC indicated normal photoreceptor cilia structure. F, Müller glial marker Sox9 (green), Sox2 (red), and GFAP (blue) showed normal, nonactivated Müller glia in the mutant retinas. G, Because of variability in the phenotype, degenerating mutant retinas were occasionally observed at 6 months. In these retinas, Müller glia were activated (GFAP upregulation), and photoreceptor rosettes were seen. D–G, Images represent 1 mm from the ONH at the temporal retina. Scale bars, 250 μm (A) and 50 μm (B–G).
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Figure 4: Six-month Ikbkap CKO retinas were grossly normal except for the reduction in RGCs. A, H&E staining showing control and mutant (CKO) eyes. The mutant eyes at 6 months appear normal. B, Retinal cross sections at central (>0.25 mm from ONH), middle (1 mm from ONH), and peripheral (1.75 mm from ONH) retinas in the temporal hemisphere. Retinal structure of the mutant was grossly normal, except for the reduction in cell number in the GCL that is apparent toward the peripheral retinas. C, Reduction in cell number was observed in the mutant GCL at 1 mm from the ONH (middle). Images of the temporal retina are shown. D, Mutant retinas showed reduced numbers of RGC marker, Brn3 (red). Islet1 (green) IHC showed normal cholinergic amacrine and optic nerve bipolar cells. E, Choline acetyltransferase (green) IHC showed normal number and structure of cholinergic amacrine cells. PKD2L-1 (red) IHC indicated normal photoreceptor cilia structure. F, Müller glial marker Sox9 (green), Sox2 (red), and GFAP (blue) showed normal, nonactivated Müller glia in the mutant retinas. G, Because of variability in the phenotype, degenerating mutant retinas were occasionally observed at 6 months. In these retinas, Müller glia were activated (GFAP upregulation), and photoreceptor rosettes were seen. D–G, Images represent 1 mm from the ONH at the temporal retina. Scale bars, 250 μm (A) and 50 μm (B–G).

Mentions: We analyzed 6-month-old mutant retinas for any sign of retinal degeneration. Although mutant eyes were occasionally smaller compared with their littermate controls, overall morphology of the eye appeared grossly normal (Fig. 4A). Analysis of 6-month retinal cross sections by H&E staining showed no abnormal lamination or thickness of retinal layers in the mutants (Fig. 4B). However, loss of cells in the middle (1 mm from the ONH) to peripheral retinas was observed in the RGC layer of the mutant retina (Fig. 4B, C). IHC analyses revealed reduction in Brn3+ RGCs in the mutants (Fig. 4D, red; see below for quantitation), whereas staining for Islet1 (a marker for cholinergic amacrine, optic nerve bipolar, and RGCs; Fig. 4D, green) and choline acetyltransferase (a marker for cholinergic amacrine cells; Fig. 4E, green) showed comparable cell numbers, morphology, and location between the mutant and littermate controls. Sox9+/Sox2+ Müller glial nuclei were localized in a single layer in the INL, and the activation marker GFAP was not observed in either mutant or control retinas (Fig. 4F). The number of photoreceptor nuclei appeared normal (Fig. 4B), and the photoreceptor cilia marker PKD2L-1 showed a normal pattern in the mutant (Fig. 4F). Because of the high variability in mutant phenotypes, we occasionally observed severe retinal degeneration as early as 6 months (Fig. 4G), and those retinas had photoreceptor rosette formation, Müller glial activation (GFAP upregulation), and loss of RGCs, as seen in older mutant retinas (Fig. 3).


Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia
Six-month Ikbkap CKO retinas were grossly normal except for the reduction in RGCs. A, H&E staining showing control and mutant (CKO) eyes. The mutant eyes at 6 months appear normal. B, Retinal cross sections at central (>0.25 mm from ONH), middle (1 mm from ONH), and peripheral (1.75 mm from ONH) retinas in the temporal hemisphere. Retinal structure of the mutant was grossly normal, except for the reduction in cell number in the GCL that is apparent toward the peripheral retinas. C, Reduction in cell number was observed in the mutant GCL at 1 mm from the ONH (middle). Images of the temporal retina are shown. D, Mutant retinas showed reduced numbers of RGC marker, Brn3 (red). Islet1 (green) IHC showed normal cholinergic amacrine and optic nerve bipolar cells. E, Choline acetyltransferase (green) IHC showed normal number and structure of cholinergic amacrine cells. PKD2L-1 (red) IHC indicated normal photoreceptor cilia structure. F, Müller glial marker Sox9 (green), Sox2 (red), and GFAP (blue) showed normal, nonactivated Müller glia in the mutant retinas. G, Because of variability in the phenotype, degenerating mutant retinas were occasionally observed at 6 months. In these retinas, Müller glia were activated (GFAP upregulation), and photoreceptor rosettes were seen. D–G, Images represent 1 mm from the ONH at the temporal retina. Scale bars, 250 μm (A) and 50 μm (B–G).
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Figure 4: Six-month Ikbkap CKO retinas were grossly normal except for the reduction in RGCs. A, H&E staining showing control and mutant (CKO) eyes. The mutant eyes at 6 months appear normal. B, Retinal cross sections at central (>0.25 mm from ONH), middle (1 mm from ONH), and peripheral (1.75 mm from ONH) retinas in the temporal hemisphere. Retinal structure of the mutant was grossly normal, except for the reduction in cell number in the GCL that is apparent toward the peripheral retinas. C, Reduction in cell number was observed in the mutant GCL at 1 mm from the ONH (middle). Images of the temporal retina are shown. D, Mutant retinas showed reduced numbers of RGC marker, Brn3 (red). Islet1 (green) IHC showed normal cholinergic amacrine and optic nerve bipolar cells. E, Choline acetyltransferase (green) IHC showed normal number and structure of cholinergic amacrine cells. PKD2L-1 (red) IHC indicated normal photoreceptor cilia structure. F, Müller glial marker Sox9 (green), Sox2 (red), and GFAP (blue) showed normal, nonactivated Müller glia in the mutant retinas. G, Because of variability in the phenotype, degenerating mutant retinas were occasionally observed at 6 months. In these retinas, Müller glia were activated (GFAP upregulation), and photoreceptor rosettes were seen. D–G, Images represent 1 mm from the ONH at the temporal retina. Scale bars, 250 μm (A) and 50 μm (B–G).
Mentions: We analyzed 6-month-old mutant retinas for any sign of retinal degeneration. Although mutant eyes were occasionally smaller compared with their littermate controls, overall morphology of the eye appeared grossly normal (Fig. 4A). Analysis of 6-month retinal cross sections by H&E staining showed no abnormal lamination or thickness of retinal layers in the mutants (Fig. 4B). However, loss of cells in the middle (1 mm from the ONH) to peripheral retinas was observed in the RGC layer of the mutant retina (Fig. 4B, C). IHC analyses revealed reduction in Brn3+ RGCs in the mutants (Fig. 4D, red; see below for quantitation), whereas staining for Islet1 (a marker for cholinergic amacrine, optic nerve bipolar, and RGCs; Fig. 4D, green) and choline acetyltransferase (a marker for cholinergic amacrine cells; Fig. 4E, green) showed comparable cell numbers, morphology, and location between the mutant and littermate controls. Sox9+/Sox2+ Müller glial nuclei were localized in a single layer in the INL, and the activation marker GFAP was not observed in either mutant or control retinas (Fig. 4F). The number of photoreceptor nuclei appeared normal (Fig. 4B), and the photoreceptor cilia marker PKD2L-1 showed a normal pattern in the mutant (Fig. 4F). Because of the high variability in mutant phenotypes, we occasionally observed severe retinal degeneration as early as 6 months (Fig. 4G), and those retinas had photoreceptor rosette formation, Müller glial activation (GFAP upregulation), and loss of RGCs, as seen in older mutant retinas (Fig. 3).

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.


Related in: MedlinePlus