IKAP expression levels modulate disease severity in a mouse model of familial dysautonomia

Abstract

Hereditary sensory and autonomic neuropathies (HSANs) encompass a group of genetically inherited disorders characterized by sensory and autonomic dysfunctions. Familial dysautonomia (FD), also known as HSAN type III, is an autosomal recessive disorder that affects 1/3600 live births in the Ashkenazi Jewish population. The disease is caused by abnormal development and progressive degeneration of the sensory and autonomic nervous systems and is inevitably fatal, with only 50% of patients reaching the age of 40. FD is caused by a mutation in intron 20 of the Ikbkap gene that results in severe reduction in the expression of its encoded protein, inhibitor of kappaB kinase complex-associated protein (IKAP). Although the mutation that causes FD was identified in 2001, so far there is no appropriate animal model that recapitulates the disorder. Here, we report the generation and characterization of the first mouse models for FD that recapitulate the molecular and pathological features of the disease. Important for therapeutic interventions is also our finding that a slight increase in IKAP levels is enough to ameliorate the phenotype and increase the life span. Understanding the mechanisms underlying FD will provide insights for potential new therapeutic interventions not only for FD, but also for other peripheral neuropathies.

Figure 1.

Generation and molecular characterization of FD mouse models. (A) Schematic representation of the wild-type allele (WT), Ikbkapflox allele (flox) and Ikbkap allele lacking exon 20 (Δ20). Exons are represented by black rectangles, and the loxP sites by black ovals. Restriction sites shown on the schematic are: Bam HI (B), HindIII (H), BstXI (BX) and BstEII (BE). (B) Western analyses of total protein lysates from the forebrain of 11-month-old WT, Ikbkap^Δ20/+, Ikbkap^flox/+ and Ikbkap^flox/flox mice. The upper panel shows the detection of IKAP with the polyclonal anti-IKAP antibody (AnaSpec), and the lower panel shows anti-β-tubulin staining for loading control. Note that the IKAP protein level is severely reduced in  Ikbkap^flox/flox brain. (C) Western analyses of total protein lysates from the whole brain of E16.5 Ikbkap^flox/flox and Ikbkap^Δ20/flox embryos. The upper panel shows the detection of IKAP with the polyclonal anti-IKAP antibody (AnaSpec), and the lower panel shows anti-β-tubulin staining for loading control. Note that in the Ikbkap^Δ20/flox brain IKAP protein expression is reduced compared with the Ikbkap^flox/flox brain. (D and E) Quantitative analyses of IKAP expression levels in mouse brains of different genotypes. (D) IKAP expression levels were normalized over tubulin levels and are expressed as percentage of WT. (E) IKAP expression levels were normalized over tubulin levels and are expressed as percentages of Ikbkap^flox/flox. Data are represented as mean ± SD; n = 3 experiments. *P < 0.05, ***P < 0.001.

Figure 2.

Postnatal characteristics of Ikbkap^Δ20/flox and Ikbkap^flox/flox mice. (A) Appearance of Ikbkap^Δ20/flox mice at P18. WT (top) and Ikbkap^Δ20/flox (bottom) littermates were photographed side by side. Note that the mutant Ikbkap^Δ20/flox mouse is significantly smaller than its WT littermate and exhibits abnormal posture and puffy feet. (B and C) Representative histological examinations of tongue fungiform papillae. Tongues of P18 WT (B) and Ikbkap^Δ20/flox (C) littermates were processed for paraffin embedding, and coronal sections of the anterior part of the tongue were stained with H&E. Note that the three fungiform papillae of the WT littermates appear normal (B), whereas in the mutant the fungiform papilla shown is degenerating (C). (D) Postnatal growth curves of control (blue, n = 37) and Ikbkap^flox/flox (red, n = 14) male mice. Similar results were found for female mice. Data are represented as mean ± SEM. (E and F) MicroCT scans of 11-month-old WT (E) and Ikbkap^Δ20/flox (F) littermate male mice. Note that the mutant is significantly smaller than the control and displays a severe curvature of the spine (kyphosis). In this scan, a significant enlargement of the bladder is also observed in the mutant (arrowhead). (G–I) 16-month-old WT (G) and Ikbkap^flox/flox (H and I) female littermates. Note the spinal curvature of the mutant (H) and the sitting-up posture (I) that it assumes frequently.

Figure 3.

Sensory ganglia deficits in FD mouse models. (A–C) Representative H&E-stained cross sections through thoracic DRGs of 18-month-old WT (A), Ikbkap^flox/flox (B) and Ikbkap^Δ20/flox (C) mice. Note that the DRGs of WT and Ikbkap^flox/flox mice contain numerous small dark neurons (arrowheads), which are virtually absent in Ikbkap^Δ20/flox DRGs. (D) Quantification of small dark (type B) and large light (type A) neuronal profiles in 18-month-old WT, Ikbkap^flox/flox and Ikbkap^Δ20/flox DRGs (n = 3). Percentages of type A and type B neuronal cells over the total number of cells are displayed (n = 3). (E and F) Transverse paraffin sections of E18.5 WT (E) and Ikbkap^Δ20/flox (F) embryos were immunostained with anti-CGRP antibody. Note the large number of CGRP-positive neuronal cells in WT DRG in contrast to their near absence in Ikbkap^Δ20/flox mutant DRG. (G and H) H&E-stained transverse sections of WT (G) and Ikbkap^Δ20/flox (H) E18.5 embryos show that the DRG is significantly reduced in the size in the mutants already at this stage. (I) Volumes of lumbar L1 DRG (L1) and thoracic T2 (T2) of E18.5 WT (n = 4), Ikbkap^flox/flox (n = 3) and Ikbkap^Δ20/flox embryos (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Autonomic deficits in FD mouse models. (A–C) Representative coronal H&E-stained sections of SCGs of E18.5 WT (A), Ikbkap^flox/flox (B) and Ikbkap^Δ20/flox (C) at their largest dimensions. (D) Volumes of E18.5 stellate and superior cervical sympathetic ganglia of WT, Ikbkap^flox/flox and Ikbkap^Δ20/flox embryos, displayed as the percentage of controls (n = 3–5). (E) Neuronal counts of E18.5 SCGs of WT, Ikbkap^flox/flox and Ikbkap^Δ20/flox embryos are displayed as percentages of controls (n = 3). (F and G) H&E-stained representative cross sections through SCGs of 10-month-old WT (F) and Ikbkap^flox/flox (G) mice. Note the smaller size of the mutant SCG as well as the sparse distribution of neurons. (H) Neuronal counts of SCGs from P21 and 10-month-old WT and Ikbkap^flox/flox mice are displayed as percentages of controls. Note that there is a significant decline in neuronal numbers from P21 to 10 months of age in Ikbkap^flox/flox SCGs relative to controls (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.