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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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Loss of Ikbkap in RGCs caused slow, progressive RGC degeneration. A, Representative Brn3 (RGC marker) staining in the temporal and nasal retinas at 6 months. Images were taken at 1 mm from ONH. B, The number of Brn3+ RGCs in Ikbkap CKO (mutant) retinas was counted. Significant loss of Brn3+ cells was observed in temporal and superior retinas at 6 months, which progressively spread into entire retinas by 14 months. C, The numbers of Brn3+ RGCs were counted in each quadrant of 6- to 8-month-old Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas at 1 mm from the ONH. There was no significant decrease in the number of RGCs in Cre+;Ikbkapf/+ compared to Cre-;Ikbkapf/+retinas, demonstrating that Cre expression itself and/or loss of one Ikbkap allele did not cause RGC degeneration. S, superior; N, nasal; I, inferior; T, temporal. Error bars represent SEM (n = ≥4 per point for B; n = 3 for C). *p < 0.05 with t-test. Scale bars, 100 μm (A).
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Figure 5: Loss of Ikbkap in RGCs caused slow, progressive RGC degeneration. A, Representative Brn3 (RGC marker) staining in the temporal and nasal retinas at 6 months. Images were taken at 1 mm from ONH. B, The number of Brn3+ RGCs in Ikbkap CKO (mutant) retinas was counted. Significant loss of Brn3+ cells was observed in temporal and superior retinas at 6 months, which progressively spread into entire retinas by 14 months. C, The numbers of Brn3+ RGCs were counted in each quadrant of 6- to 8-month-old Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas at 1 mm from the ONH. There was no significant decrease in the number of RGCs in Cre+;Ikbkapf/+ compared to Cre-;Ikbkapf/+retinas, demonstrating that Cre expression itself and/or loss of one Ikbkap allele did not cause RGC degeneration. S, superior; N, nasal; I, inferior; T, temporal. Error bars represent SEM (n = ≥4 per point for B; n = 3 for C). *p < 0.05 with t-test. Scale bars, 100 μm (A).

Mentions: Because Tα1-Cre is expressed predominantly in RGCs in the retina (Fig. 2) and human FD patients show RGC defects (Mendoza-Santiesteban et al., 2012, 2014), we tracked spatial and temporal changes in RGCs in the Tα1-Cre Ikbkap CKO retinas. Retinas were harvested at 1, 3, 6, and 14 months of age, and the number of RGCs 1 mm from the ONH in temporal, nasal, superior, and inferior retinas were counted using the RGC nuclear marker Brn3 (Fig. 5). At 1 month, the mutant and control retinas contained similar numbers of Brn3+ RGCs in all quadrants, suggesting that RGC development was not affected by the absence of Ikbkap. In contrast, by 6 months, a significant loss of RGCs was observed in the mutant retinas. The reduction in RGCs was greatest in the temporal retina (∼50% reduction; Fig. 5A, B), which corresponds to the clinical observation of FD patients (Mendoza-Santiesteban et al., 2012, 2014). Superior and inferior mutant retinas had an approximately 30% reduction in RGCs compared with control retinas at 6 months. RGC degeneration continued with age, and by 14 months, panretinal loss of RGCs was observed, with the most dramatic loss of RGCs, greater than 60%, in the temporal retina. To rule out a possibility that Cre expression itself causes RGC degeneration, the number of Brn3+ RGCs in 6- to 8-month Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas was counted at 1 mm from the ONH (Fig. 5C). The results showed there was no loss of RGCs, indicating that neither Cre expression itself nor loss of one Ikbkap allele causes RGC degeneration. We conclude from these results that in the absence of Ikbkap, RGCs undergo a slow but progressive degeneration, with the same spatial pattern as observed in FD patients.


Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia
Loss of Ikbkap in RGCs caused slow, progressive RGC degeneration. A, Representative Brn3 (RGC marker) staining in the temporal and nasal retinas at 6 months. Images were taken at 1 mm from ONH. B, The number of Brn3+ RGCs in Ikbkap CKO (mutant) retinas was counted. Significant loss of Brn3+ cells was observed in temporal and superior retinas at 6 months, which progressively spread into entire retinas by 14 months. C, The numbers of Brn3+ RGCs were counted in each quadrant of 6- to 8-month-old Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas at 1 mm from the ONH. There was no significant decrease in the number of RGCs in Cre+;Ikbkapf/+ compared to Cre-;Ikbkapf/+retinas, demonstrating that Cre expression itself and/or loss of one Ikbkap allele did not cause RGC degeneration. S, superior; N, nasal; I, inferior; T, temporal. Error bars represent SEM (n = ≥4 per point for B; n = 3 for C). *p < 0.05 with t-test. Scale bars, 100 μm (A).
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Figure 5: Loss of Ikbkap in RGCs caused slow, progressive RGC degeneration. A, Representative Brn3 (RGC marker) staining in the temporal and nasal retinas at 6 months. Images were taken at 1 mm from ONH. B, The number of Brn3+ RGCs in Ikbkap CKO (mutant) retinas was counted. Significant loss of Brn3+ cells was observed in temporal and superior retinas at 6 months, which progressively spread into entire retinas by 14 months. C, The numbers of Brn3+ RGCs were counted in each quadrant of 6- to 8-month-old Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas at 1 mm from the ONH. There was no significant decrease in the number of RGCs in Cre+;Ikbkapf/+ compared to Cre-;Ikbkapf/+retinas, demonstrating that Cre expression itself and/or loss of one Ikbkap allele did not cause RGC degeneration. S, superior; N, nasal; I, inferior; T, temporal. Error bars represent SEM (n = ≥4 per point for B; n = 3 for C). *p < 0.05 with t-test. Scale bars, 100 μm (A).
Mentions: Because Tα1-Cre is expressed predominantly in RGCs in the retina (Fig. 2) and human FD patients show RGC defects (Mendoza-Santiesteban et al., 2012, 2014), we tracked spatial and temporal changes in RGCs in the Tα1-Cre Ikbkap CKO retinas. Retinas were harvested at 1, 3, 6, and 14 months of age, and the number of RGCs 1 mm from the ONH in temporal, nasal, superior, and inferior retinas were counted using the RGC nuclear marker Brn3 (Fig. 5). At 1 month, the mutant and control retinas contained similar numbers of Brn3+ RGCs in all quadrants, suggesting that RGC development was not affected by the absence of Ikbkap. In contrast, by 6 months, a significant loss of RGCs was observed in the mutant retinas. The reduction in RGCs was greatest in the temporal retina (∼50% reduction; Fig. 5A, B), which corresponds to the clinical observation of FD patients (Mendoza-Santiesteban et al., 2012, 2014). Superior and inferior mutant retinas had an approximately 30% reduction in RGCs compared with control retinas at 6 months. RGC degeneration continued with age, and by 14 months, panretinal loss of RGCs was observed, with the most dramatic loss of RGCs, greater than 60%, in the temporal retina. To rule out a possibility that Cre expression itself causes RGC degeneration, the number of Brn3+ RGCs in 6- to 8-month Cre-;Ikbkapf/+ and Cre+;Ikbkapf/+ retinas was counted at 1 mm from the ONH (Fig. 5C). The results showed there was no loss of RGCs, indicating that neither Cre expression itself nor loss of one Ikbkap allele causes RGC degeneration. We conclude from these results that in the absence of Ikbkap, RGCs undergo a slow but progressive degeneration, with the same spatial pattern as observed in FD patients.

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 (T&alpha;1-Cre). In the retina, T&alpha;1-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, M&uuml;ller 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