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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.


Tα1-Cre mice were crossed to mTmG Cre reporter, and GFP expression was analyzed at E17.5, P10, and 1 and 8 months. A and B, At E17.5, Cre expression (GFP+) was detected in the GCL without regional bias. GFP colocalized with pan-RGC marker RBPMS (white). C, GFP was detected in the optic nerve at E17.5, indicating Cre expression in RGCs. D, Very few cells other than RGCs expressed Cre (GFP+) at P10. E, Most RGCs (RBPMS+) expressed Cre (GFP+) at P10. Representative images at the temporal retina are shown. F, Approximately 90% of RGCs expressed Cre by P10, and there was no regional bias. Error bars represent SEM (n = 4). Central, >0.25 mm from ONH; temporal and nasal, 1 mm from ONH. G–K, Cell type–specific markers were used to analyze Cre expression in the retina at 1 month. Very few bipolar cells (BP; Otx2+ in INL; G, J), amacrine cells (AC; G), photoreceptors (PR; G, K), or Müller glia (MG; Sox9+; H, J) expressed Cre, whereas Cre expression was detected in ∼90% of RGCs (RBPMS+; I, J). Cre-expressing photoreceptor numbers did not increase in older retinas (8 months) compared with 1 month (K). Error bars in J and K represent SEM (n = 3). NBL, neuroblastic layer. Scale bars, 250 μm (A–C) and 50 μm (G–I).
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Figure 2: Tα1-Cre mice were crossed to mTmG Cre reporter, and GFP expression was analyzed at E17.5, P10, and 1 and 8 months. A and B, At E17.5, Cre expression (GFP+) was detected in the GCL without regional bias. GFP colocalized with pan-RGC marker RBPMS (white). C, GFP was detected in the optic nerve at E17.5, indicating Cre expression in RGCs. D, Very few cells other than RGCs expressed Cre (GFP+) at P10. E, Most RGCs (RBPMS+) expressed Cre (GFP+) at P10. Representative images at the temporal retina are shown. F, Approximately 90% of RGCs expressed Cre by P10, and there was no regional bias. Error bars represent SEM (n = 4). Central, >0.25 mm from ONH; temporal and nasal, 1 mm from ONH. G–K, Cell type–specific markers were used to analyze Cre expression in the retina at 1 month. Very few bipolar cells (BP; Otx2+ in INL; G, J), amacrine cells (AC; G), photoreceptors (PR; G, K), or Müller glia (MG; Sox9+; H, J) expressed Cre, whereas Cre expression was detected in ∼90% of RGCs (RBPMS+; I, J). Cre-expressing photoreceptor numbers did not increase in older retinas (8 months) compared with 1 month (K). Error bars in J and K represent SEM (n = 3). NBL, neuroblastic layer. Scale bars, 250 μm (A–C) and 50 μm (G–I).

Mentions: We generated Ikbkap CKO mice using Tα1-Cre, which targets postmitotic neurons (Chaverra et al., unpublished observations; Gloster et al., 1994; Coppola et al., 2004; Coksaygan et al., 2006). To measure Cre expression pattern in the retina, we crossed Tα1-Cre with mTmG Cre reporter mice (Muzumdar et al., 2007). At E17.5, Cre expression was detected in postmitotic cells in the GCL and optic nerve, but not in retinal progenitors (Fig. 2A–C). To our surprise, at P10, Cre expression was fairly limited to RGCs, with only a few other retinal neuron types and Müller glia being Cre+ (Fig. 2D). In the central (>0.25 mm from the ONH), temporal, and nasal (1 mm from the ONH) retina, ∼90% of RGCs (RBPMS+) expressed Cre by P10 without regional bias (Fig. 2E, F). The Cre reporter expression pattern did not change in 1- and 8-month retinas, and there was no increase in Cre+ cells compared to P10 retinas (Fig. 2G–K; 1 month images shown). Very few bipolar cells (1.6% Otx2+ cells in INL; Fig. 2G, J), photoreceptors (Fig. 2G, K), Müller glia (3.0% Sox9+ cells; Fig. 2H, J), or amacrine cells (Fig. 2G) expressed the GFP Cre reporter, whereas 89.2% of RGCs (Fig. 2I, J) expressed Cre at 1 month. At 8 months, the number of GFP+ photoreceptors did not differ significantly compared to younger (1 month) retinas (Fig. 2K).


Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia
Tα1-Cre mice were crossed to mTmG Cre reporter, and GFP expression was analyzed at E17.5, P10, and 1 and 8 months. A and B, At E17.5, Cre expression (GFP+) was detected in the GCL without regional bias. GFP colocalized with pan-RGC marker RBPMS (white). C, GFP was detected in the optic nerve at E17.5, indicating Cre expression in RGCs. D, Very few cells other than RGCs expressed Cre (GFP+) at P10. E, Most RGCs (RBPMS+) expressed Cre (GFP+) at P10. Representative images at the temporal retina are shown. F, Approximately 90% of RGCs expressed Cre by P10, and there was no regional bias. Error bars represent SEM (n = 4). Central, >0.25 mm from ONH; temporal and nasal, 1 mm from ONH. G–K, Cell type–specific markers were used to analyze Cre expression in the retina at 1 month. Very few bipolar cells (BP; Otx2+ in INL; G, J), amacrine cells (AC; G), photoreceptors (PR; G, K), or Müller glia (MG; Sox9+; H, J) expressed Cre, whereas Cre expression was detected in ∼90% of RGCs (RBPMS+; I, J). Cre-expressing photoreceptor numbers did not increase in older retinas (8 months) compared with 1 month (K). Error bars in J and K represent SEM (n = 3). NBL, neuroblastic layer. Scale bars, 250 μm (A–C) and 50 μm (G–I).
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Figure 2: Tα1-Cre mice were crossed to mTmG Cre reporter, and GFP expression was analyzed at E17.5, P10, and 1 and 8 months. A and B, At E17.5, Cre expression (GFP+) was detected in the GCL without regional bias. GFP colocalized with pan-RGC marker RBPMS (white). C, GFP was detected in the optic nerve at E17.5, indicating Cre expression in RGCs. D, Very few cells other than RGCs expressed Cre (GFP+) at P10. E, Most RGCs (RBPMS+) expressed Cre (GFP+) at P10. Representative images at the temporal retina are shown. F, Approximately 90% of RGCs expressed Cre by P10, and there was no regional bias. Error bars represent SEM (n = 4). Central, >0.25 mm from ONH; temporal and nasal, 1 mm from ONH. G–K, Cell type–specific markers were used to analyze Cre expression in the retina at 1 month. Very few bipolar cells (BP; Otx2+ in INL; G, J), amacrine cells (AC; G), photoreceptors (PR; G, K), or Müller glia (MG; Sox9+; H, J) expressed Cre, whereas Cre expression was detected in ∼90% of RGCs (RBPMS+; I, J). Cre-expressing photoreceptor numbers did not increase in older retinas (8 months) compared with 1 month (K). Error bars in J and K represent SEM (n = 3). NBL, neuroblastic layer. Scale bars, 250 μm (A–C) and 50 μm (G–I).
Mentions: We generated Ikbkap CKO mice using Tα1-Cre, which targets postmitotic neurons (Chaverra et al., unpublished observations; Gloster et al., 1994; Coppola et al., 2004; Coksaygan et al., 2006). To measure Cre expression pattern in the retina, we crossed Tα1-Cre with mTmG Cre reporter mice (Muzumdar et al., 2007). At E17.5, Cre expression was detected in postmitotic cells in the GCL and optic nerve, but not in retinal progenitors (Fig. 2A–C). To our surprise, at P10, Cre expression was fairly limited to RGCs, with only a few other retinal neuron types and Müller glia being Cre+ (Fig. 2D). In the central (>0.25 mm from the ONH), temporal, and nasal (1 mm from the ONH) retina, ∼90% of RGCs (RBPMS+) expressed Cre by P10 without regional bias (Fig. 2E, F). The Cre reporter expression pattern did not change in 1- and 8-month retinas, and there was no increase in Cre+ cells compared to P10 retinas (Fig. 2G–K; 1 month images shown). Very few bipolar cells (1.6% Otx2+ cells in INL; Fig. 2G, J), photoreceptors (Fig. 2G, K), Müller glia (3.0% Sox9+ cells; Fig. 2H, J), or amacrine cells (Fig. 2G) expressed the GFP Cre reporter, whereas 89.2% of RGCs (Fig. 2I, J) expressed Cre at 1 month. At 8 months, the number of GFP+ photoreceptors did not differ significantly compared to younger (1 month) retinas (Fig. 2K).

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.