CFTR-deficient pigs display peripheral nervous system defects at birth

Abstract

Peripheral nervous system abnormalities, including neuropathy, have been reported in people with cystic fibrosis. These abnormalities have largely been attributed to secondary manifestations of the disease. We tested the hypothesis that disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) gene directly influences nervous system function by studying newborn CFTR(-/-) pigs. We discovered CFTR expression and activity in Schwann cells, and loss of CFTR caused ultrastructural myelin sheath abnormalities similar to those in known neuropathies. Consistent with neuropathic changes, we found increased transcripts for myelin protein zero, a gene that, when mutated, can cause axonal and/or demyelinating neuropathy. In addition, axon density was reduced and conduction velocities of the trigeminal and sciatic nerves were decreased. Moreover, in vivo auditory brainstem evoked potentials revealed delayed conduction of the vestibulocochlear nerve. Our data suggest that loss of CFTR directly alters Schwann cell function and that some nervous system defects in people with cystic fibrosis are likely primary.

Fig. 1.

CFTR is expressed in trigeminal nerve Schwann cells. Data are confocal microscopic images of trigeminal nerve immunostained for CFTR (green) and marker indicated above middle panels (red). Nuclei were stained with DAPI (blue). (A) Transverse cross-section with axons stained with β-tubulin III antibody. (B) CFTR^−/− trigeminal nerves immunostained for CFTR and β-tubulin III. Other controls in CFTR^−/− nerves are in Fig. S2. (C) Sagittal cross-section immunostained with fluoromyelin. (D) Section stained with S100 antibody, a marker of Schwann cells. (E) Section stained with p75 antibody, also a marker of Schwann cells. (Scale bar, 20 μm.)

Fig. 2.

CFTR is functionally active in Schwann cells. (A) Primary cultures of porcine Schwann cells were used 4 wk after seeding when they had developed the specific bipolar morphology and a phase-bright cell body under differential interference contrast microscopy. Schwann cells were positive for the phenotypic markers S100 and p75. (B) Whole-cell current recorded in the presence of PKA and ATP in the pipette solution and 1 min after adding 100 μM of CFTR inhibitor GlyH-101 to the bath solution. (Left) Example of currents from one cell; Inset shows voltage-pulse protocol. (Upper Right) Example of current-voltage relationship. (Lower Right) Data from five CFTR^+/+ Schwann cells and seven CFTR^−/− Schwann cells. *P = 0.003 (Mann–Whitney rank sum test).

Fig. 3.

Loss of CFTR alters myelin sheath structure. Images are transmission electron photomicrographs of myelin sheath in trigeminal nerve of CFTR^+/+ (A), CFTR^+/− (B), and CFTR^−/− (C and D) pigs. Arrows in B and C point to the ringed appearance of the myelin sheath. Star in D indicates myelin infolding. Myelin infoldings (E) were observed in a greater percentage of CF axons. n = 385 axons total from four CFTR^+/+ pigs; n = 404 axons total from four CFTR^+/− pigs; and n = 602 axons total from four CFTR^−/− pigs. (Scale bars, 1.5 μm.)

Fig. 4.

Myelin gene transcripts are altered in trigeminal nerve of newborn CFTR^−/− pigs. Data are qRT-PCR for transcripts of MPZ (A), MBP (B), CNX32 (C), and ANX2 (D). For CFTR^+/+, n = 5 animals; CFTR^+/−, n = 4 animals; CFTR^−/−, n = 6 animals. Data are expressed relative to average CFTR^+/+ levels. *P < 0.05 compared with CFTR^+/+.

Fig. 5.

Lack of CFTR decreases axon density and increases axon diameter in the trigeminal nerve. Data are from five CFTR^+/+ newborn pigs (7,210 total myelinated axons), seven CFTR^+/− newborn pigs (8,229 total myelinated axons), and six CFTR^−/− newborn pigs (6,127 total myelinated axons). In A–D, each point represents data from an individual animal (800–1,500 axons per animal). *P < 0.05 compared with CFTR^+/+. (A) Axon density. (B) Trigeminal nerve circumference. (C) Inner diameter or axonal diameter. (D) Outer diameter (the axon plus the myelin sheath). (E) Distribution of outer diameters as a percentage of total axons. (F) Curve fitting of the outer diameter of two main populations of myelinated axons. Dashed lines are the total populations, and the two curves are Gaussian peaks (labeled peak 1 and peak 2). Vertical blue lines highlight peaks in CFTR^+/+ newborn pigs. (G) Peaks from Gaussian curve fitting.

Fig. 6.

Conduction velocity is reduced in the trigeminal, sciatic, and vestibulocochlear nerves of newborn CFTR^−/− pigs. Trigeminal nerve showed reduced conduction velocity (A) whereas the sciatic nerve (B) showed a strong trend for reduced conduction velocity (P = 0.084). The compound action potential amplitude and the area under the curve are shown in Table S1. (C–E) Latencies of wave I and wave II and the wave I–wave II interval of in vivo auditory brainstem responses. Latency of other waves and other intervals did not differ between CFTR^+/+ and CFTR^−/− pigs (Table S2). *P < 0.05 compared with CFTR^+/+. Each point represents the data collected from an individual animal.