Sodium-dependent vitamin C transporter 2 deficiency causes hypomyelination and extracellular matrix defects in the peripheral nervous system

Abstract

Ascorbic acid (vitamin C) is necessary for myelination of Schwann cell/neuron cocultures and has shown beneficial effects in the treatment of a Charcot-Marie-Tooth neuropathy 1A (CMT1A) mouse model. Although clinical studies revealed that ascorbic acid treatment had no impact on CMT1A, it is assumed to have an important function in peripheral nerve myelination and possibly in remyelination. However, the transport pathway of ascorbic acid into peripheral nerves and the mechanism of ascorbic acid function in peripheral nerves in vivo remained unclear. In this study, we used sodium-dependent vitamin C transporter 2-heterozygous (SVCT2(+/-)) mice to elucidate the functions of SVCT2 and ascorbic acid in the murine peripheral nervous system. SVCT2 and ascorbic acid levels were reduced in SVCT2(+/-) peripheral nerves. Morphometry of sciatic nerve fibers revealed a decrease in myelin thickness and an increase in G-ratios in SVCT2(+/-) mice. Nerve conduction velocities and sensorimotor performance in functional tests were reduced in SVCT2(+/-) mice. To investigate the mechanism of ascorbic acid function, we studied the expression of collagens in the extracellular matrix of peripheral nerves. Here, we show that expression of various collagen types was reduced in sciatic nerves of SVCT2(+/-) mice. We found that collagen gene transcription was reduced in SVCT2(+/-) mice but hydroxyproline levels were not, indicating that collagen formation was regulated on the transcriptional and not the posttranslational level. These results help to clarify the transport pathway and mechanism of action of ascorbic acid in the peripheral nervous system and may lead to novel therapeutic approaches to peripheral neuropathies by manipulation of SVCT2 function.

Figure 1.

SVCT2 expression and vitamin C concentrations are reduced in SVCT2^+/− sciatic nerves. A, Real-time reverse transcription PCR of mRNA extracts of sciatic nerves from SVCT2^+/− mice and wild-type controls at ages P60 and P300 was performed for SVCT2 and GAPDH for normalization. Results are shown as relative changes (rel.) of GAPDH-normalized SVCT2 expression ratios in SVCT2^+/− mice compared (comp.) to wild-type mice. qRT-PCR showed significant downregulation of SVCT2 at both P60 and P300 (P60 and P300: **p < 0.01, n = 3). B, Western blots of sciatic nerve lysates of SVCT2^+/− and wild-type control mice at ages P60 and P300 was performed and membranes were incubated with SVCT1 and SVCT2 antibodies. Actin was used as a marker of protein loading. Western blots showed reduced SVCT2 protein expression in SVCT2^+/− compared to wild-type sciatic nerves. SVCT1 was not changed, showing that SVCT1 is not compensatorily upregulated. C, Cross-sections of sciatic nerves of wild-type (left) and SVCT2^+/− (right) mice were stained with SVCT2 (green) and neurofilament (SMI32, red) antibodies. Immunohistochemistry showed reduced expression of SVCT2 in SVCT2^+/− sciatic nerves (scale bar, 5 μm). D, Ascorbic acid concentrations (asc. acid conc.) in peripheral nerve lysates from SVCT2^+/− and wild-type mice at ages P20, 60, and 300 were measured by high-pressure liquid chromatography. Ascorbic acid levels were markedly reduced in SVCT2^+/− compared to wild type at all time points (P20: ***p < 0.001, n = 3; P60: **p < 0.01, n = 3; P300: *p < 0.05, n = 3).

Figure 2.

Peripheral nerves of SVCT2^+/− mice show thinner myelin and increased G-ratios but unaffected axon diameters. Sciatic nerves of SVCT2^+/− mice and wild-type controls were processed for histology by semi-thin toluidine blue-stained sections, ultrathin electron microscopy sections, and teased fiber analysis. A–D, SVCT2^+/− nerves showed thinner myelin sheaths (B, D) compared to wild type nerves (A, C) in semi-thin toluidine-blue stained sections (A, B) and electron micrographs (C, D). E–G, Semi-thin toluidine blue-stained sections at P60 and P300 were analyzed by morphometry. E, Morphometry demonstrated significantly reduced myelin thickness in SVCT2^+/− compared to wild-type mice at both time points (P60: *p < 0.05, n = 3; P300: **p < 0.01, n = 3). F, Axon diameters were unchanged in SVCT2^+/− nerves (P60: nonsignificant, n = 3; P300: nonsignificant, n = 3). G, Corresponding to reduced myelin thickness, G-ratios (axon diameter/fiber diameter) were increased in SVCT2^+/− compared to wild-type mice (P60: *p < 0.05, n = 3; P300: **p < 0.01, n = 3). H–J, Distributions of myelin thickness, axon diameters, and G-ratios of representative nerves from P300 mice are shown. H, The distribution of myelin thickness was skewed to the left in SVCT2^+/− nerves (*p < 0.001, n = 3). I, The distribution of axon diameters was not affected in SVCT2^+/− compared to controls. J, The scatter plot of G-ratios over axon diameters showed a shift toward higher G-ratios in SVCT2^+/− nerves while the slope of the linear regression was unchanged. K, L, Individual myelin wraps within myelin sheaths could be observed and counted on high-magnification electron micrographs (36,000×) from wild-type (K) and SVCT2^+/− (L) sciatic nerves. M, Quantification of myelin wraps demonstrated significant reductions in SVCT2^+/− mice compared to wild-type mice at both P60 and P300 (P60: *p < 0.05, n = 3; P300: **p < 0.01, n = 3). N, The number of axons per mm² was not significantly different between SVCT2^+/− and wild-type nerves (P300: p = nonsignificant, n = 3). O, The percentage of myelinated fibers was reduced in SVCT2^+/− nerves (P300: *p < 0.05, n = 3). P, The number of Remak bundles per visual field was not significantly different between SVCT2^+/− and wild-type nerves (P300: p = nonsignificant, n = 3). Q, The number of axons per Remak bundle was increased in SVCT2^+/− nerves (P300: *p < 0.05, n = 3). R–Y, Single fiber analysis of teased fibers. Merged images of phase contrast and fluorescence-labeled markers (green) are shown in R–W; merged images of myelin basic protein (green) and neurofilament (SMI32, red) are shown in X and Y. Immunoreactivities to sodium channels (Na-Ch.) (R, S), Kv1.2-channels (T, U), and Caspr (V, W) in nodes of Ranvier, paranodes, and juxtaparanodes, respectively, were unchanged between SVCT2^+/− and wild-types. MBP stainings of teased fibers confirmed the observation of thinner myelin sheaths (X, Y). On MBP stainings or phase contrast images of teased fibers, no focal demyelinations or tomaculae could be observed. For morphometry, an average of 300 fibers per section were counted on three sections per nerve. For myelin wraps, an average of 100 fibers per nerve were analyzed. Three animals were analyzed per genotype and age group. Scale bars: A–D, R–Y, 10 μm; L, M: 1 μm.

Figure 3.

SVCT2^+/− mice show deficits in sensorimotor function and motor nerve conduction velocity. SVCT2^+/− and wild-type control mice were tested by Rotarod, gait analysis, hot plate test, and nerve conduction studies for functional assessment. A, Rotarod testing showed significant reduction of performance in SVCT2^+/− animals at P60 (P60: *p < 0.05, n = 8). At the age of P300 both wild-type and SVCT2^+/− mice performed worse than at P60. There was no significant difference between the two genotypes at the age of P300 (P300: p = nonsignificant, n = 8). B, Gait analysis revealed significant reductions in stride length in SVCT2^+/− compared to wild-type mice at both P60 and P300 (P60: **p < 0.01, n = 8; P300: ***p < 0.001, n = 8). C, Gait analysis further showed a significant increase in the stride variability at both time points measured as difference between longest and shortest stride in a run (P60: *p < 0.05, n = 8; P300: ***p < 0.01, n = 8). D, E, Representative walking tracks of a wild-type mouse (D) and an SVCT2^+/− mouse (E) at the age of P300 are shown with lines marking the foot-to-walk axis. F, Measurements of foot-to-walk axis in P300 mice showed significantly wider stance in SVCT2^+/− mice compared to controls (*p < 0.05, n = 8). G, In the hot plate test, SVCT2^+/− mice responded to the heat stimulus later than controls (**p < 0.01, n = 6), indicating a defect in nociception. H–J, Nerve conduction studies were conducted to assess neurophysiological changes in SVCT2^+/− and wild-type peripheral nerves. H, I, Representative traces of sciatic nerve compound motor action potentials, CMAP, recorded from plantar foot muscles after proximal and distal stimulation (upward arrowheads) are shown. The latency until start of the CMAP is indicated by downward arrowheads. Compared to wild-type mice at P100 (H), age-matched SVCT2^+/− mice (I) showed temporal dispersion of CMAP amplitudes and a decrease in motor nerve conduction velocity (mNCV) (**p < 0.01, n = 6), whereas there was no change in CMAP amplitude size (p = nonsignificant, n = 6). Partial conduction block (p. cond. block) defined as ≥50% reduction of the proximal compared to the distal CMAP amplitude was found in two of six SVCT2^+/− and none of six wild-type mice (p = nonsignificant). Results of nerve conduction studies are summarized in table J.

Figure 4.

Formation of collagen (Coll)-containing and laminin (Lam)-containing extracellular matrix is reduced in SVCT2^+/− peripheral nerves. To investigate the extracellular matrix of SVCT2^+/− peripheral nerve tissue, we studied sciatic nerves by light microscopy, immunofluorescence microscopy, and Western blots. A, Light microscopic imaging following Gomori's staining showed only mild changes in SVCT2^+/− nerves (bottom) compared to wild-type nerves (top). SVCT2^+/− nerves showed slightly disorganized collagen fibrils (green), particularly in the epineurium (arrowheads), which appeared partially disrupted (bottom image, asterisk). B, Western blots of sciatic nerve lysates showed reduced band intensities for collagen IV, V, and XXVIII and for laminin-2. Actin was used as a marker for protein loading. C, Densitometric analysis of Western blot bands confirmed significant downregulation of collagens IV, V, and XXVII, as well as laminin-2 (***p < 0.001, **p < 0.01, *p < 0.05, n = 3). D–U′, Immunohistochemical analysis of collagen types XXVIII, V, and laminin-2 (green) and merged images with counterstaining using neurofilament antibodies (SMI32, red), fibronectin (Fib) antibodies (red), and E-cadherin (E-Cad) antibodies (red), as well as DAPI (blue) on cryosections of sciatic nerves from SVCT2^+/− and wild-type mice, are shown. D–I′, Immunostainings of the peripheral nerve-specific collagen type XXVIII showed reduced expression in SVCT2^+/− nerves (D–I) compared to wild-type nerves (D′–I′). In the wild-type nerves, collagen XXVIII was localized in the extracellular matrix as shown by double-labeling with fibronectin (F, G, insets) and in the paranodal region of nodes of Ranvier, as shown by double-labeling with E-cadherin (H, I, insets). In both locations, collagen XXVIII was reduced in SVCT2^+/− mice (F′, G′, D′, I′, insets). J–O′, Immunohistochemistry of collagen type V showed strong expression in wild-type nerves (J–O), which was markedly reduced in SVCT2^+/− animals (J′–O′). Collagen V in the extracellular matrix was strongly reduced (compare M to M′, insets). Accumulation of collagen V at nodes of Ranvier was also reduced in SVCT2^+/− nerves (compare O to O′, insets). P–U′, Stainings with antibodies to laminin-2 showed a relative reduction in intensity in SVCT2^+/− nerves (P′–U′) compared to wild-types (P–U). Scale bars: 20 μm.

Figure 5.

Expression of Myelin Protein Zero and collagen IV, V, and XXVIII mRNA is reduced in SVCT2^+/− sciatic nerves. To study the effect of ascorbic acid reduction on myelin and collagen gene expression, we analyzed RNA extracts of sciatic nerves by quantitative RT-PCR. Relative changes in SVCT2^+/− compared to wild-type nerves are shown for each gene. A, qRT-PCR of the main myelin genes MPZ and PMP22 showed significant reductions of MPZ mRNA in SVCT2^+/− nerves compared to wild-type nerves at both P60 and P300 (relative change: −4.7-fold and −3.1-fold, respectively). PMP22 expression showed an insignificant trend toward an upregulation at P60 (2.8-fold) and essentially no change at P300. B, qRT-PCR of collagen types IV, V, XV, and XXVIII (α1 chains) showed downregulation of types IV, V, and XXVIII in SVCT2^+/− nerves compared to wild-types. Collagen XV mRNA concentration was not significantly changed between the two genotypes. Significance is given in the graphs; n = 3 for each genotype and age.

Figure 6.

Malondialdehyde and hydroxyproline levels are not significantly changed in SVCT2^+/− compared to wild-type peripheral nerves. To assess the level of proline hydroxylation and lipid peroxidation, hydroxyproline and malondialdehyde assays were performed. A, Hydroxyproline assays, performed by spectrophotometry after reaction with chloramine T and Ehrlich's reagent, showed no significant differences between SVCT2^+/− and wild-type sciatic nerves (P60: p = nonsignificant, n = 3; P300: p = nonsignificant, n = 3). B, Malondialdehyde concentrations, as a measure for lipid peroxidation, were not significantly different between SVCT2^+/− and wild-type controls (P60: p = nonsignificant, n = 3; P300: p = nonsignificant, n = 3).

Figure 7.

Extracellular collagen and myelinated segments are reduced in cell culture under SVCT2-inhibiting conditions. To test the effect of SVCT2 inhibition on collagen formation and myelination in vitro, we used primary Schwann cell cultures and Schwann cell/DRG cocultures. A, Western blots of supernatants from Schwann cells treated with serum-free media (ctr, control), ascorbic acid (AA; 50 μg/ml), and ascorbic acid plus phloretin (AA + Phil;100 μm) showed increased collagen bands after ascorbic acid treatment that were reduced by addition of phloretin. Densitometry confirmed the increase in collagen bands by ascorbic acid treatment (**p < 0.01, n = 3), which was reduced to control levels by phloretin (***p < 0.001, n = 3). B, Western blots of Schwann cell cultures treated with siRNA against SVCT2 and scrambled control siRNA showed reduced collagen bands in SVCT2-siRNA treated cultures but not in scrambled control cultures. SVCT2 bands confirmed knockdown of SVCT2 by siRNA. Densitometry confirmed these observations (collagen bands: *p < 0.05; SVCT2 bands: ***p < 0.001; n = 3). C, Real-time PCR also confirmed the knockdown of SVCT2 on the mRNA level (*p < 0.05, n = 5) D–I, Cocultures of Schwann cells and DRG neurons treated with ascorbic acid (Asc. acid; 50 μg/ml, D–F) and ascorbic acid plus phloretin (Asc. acid + phl.; 100 μm, G–I) were fixed and stained with MBP antibodies (D, G, red) and neurofilament antibodies (E, H; green). Merged images with DAPI are shown on the right (F, I). J, Myelinated segments as assessed by MBP staining were longer in ascorbic acid treated than ascorbic acid plus phloretin-treated cultures (***p < 0.001, n = 5). K, The number of myelinated segments was not reduced by phloretin (p = nonsignificant, n = 5). L, The number of axon break-off points, as an indicator for damage to the neuritic network, was not different between ascorbic acid or ascorbic acid plus phloretin (p = nonsignificant, n = 5). M, The viability of both Schwann cells (SC) and DRG neurons, as assessed by Trypan blue exclusion, was not affected by phloretin (p = nonsignificant, n = 6). Scale bar: 20 μm.