Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy

Abstract

Mutations in myelin genes cause inherited peripheral neuropathies that range in severity from adult-onset Charcot-Marie-Tooth disease type 1 to childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Many myelin gene mutants that cause severe disease, such as those in the myelin protein zero gene (MPZ) and the peripheral myelin protein 22 gene (PMP22), appear to make aberrant proteins that accumulate primarily within the endoplasmic reticulum (ER), resulting in Schwann cell death by apoptosis and, subsequently, peripheral neuropathy. We previously showed that curcumin supplementation could abrogate ER retention and aggregation-induced apoptosis associated with neuropathy-causing MPZ mutants. We now show reduced apoptosis after curcumin treatment of cells in tissue culture that express PMP22 mutants. Furthermore, we demonstrate that oral administration of curcumin partially mitigates the severe neuropathy phenotype of the Trembler-J mouse model in a dose-dependent manner. Administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and results in increased number and size of myelinated axons in sciatic nerves, leading to improved motor performance. Our findings indicate that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and improves the neuropathologic phenotype in an animal model of human neuropathy, suggesting a potential therapeutic role in selected forms of inherited peripheral neuropathies.

Figure 1.

Release of ER-retained Tr-J and Tr mutants with curcumin treatment. A, Subcellular localization of wild-type (Wt) PMP22, Tr-J, and Tr in transiently transfected HeLa cells. Tr-J and Tr are extensively retained in the ER, as evidenced by colocalization with PDI (arrows in f and i). B, Curcumin treatment of HeLa cells rescues Tr-J and Tr mutants (green) retained in the ER (PDI markers [red]), as revealed by increased cytoplasmic staining. Cells treated with 40 μM curcumin showed reduced ER retention (arrows in f and i). Scale bars = 25 μm.

Figure 2.

Increased cell death in HeLa cells after transfection with Tr-J and Tr mutations. A, Apoptosis induced in HeLa cell lines after transient transfection (scale bar = 25 μm). Cell lines transfected with wild-type (Wt) PMP22 (b) show a significantly lower number of positive cells. Data from TUNEL assays revealed the presence of more TUNEL-positive cells only after transfection with Tr-J (c) and Tr (d) mutations. Negative control cells (a) transfected with empty plasmid are also shown. B, Western-blot analysis showing similar PMP22 protein levels (arrow) for both wild-type and mutants in transiently transfected HeLa cell lines. Quantitative RT-PCR experiments revealed equal amounts of mRNA in the experimental and control samples, which is consistent with the western blots (not shown). C, Representative study of the flow-cytometric analysis of apoptosis after transfection of cells with wild-type PMP22, Tr-J, and Tr. Significant differences were observed in the percentage of cells undergoing apoptosis when transfected with Tr-J and Tr, showing a higher toxic effect of those mutations on cells compared with that of wild-type PMP22 (representative data are from one of four independent experiments with comparable results). FS = flow-sorted cells; PI = propidium iodide.

Figure 3.

Curcumin's reversal of induction of apoptosis. Shown are results of apoptosis analysis of cells transiently transfected with wild-type (Wt) PMP22, Tr-J, and Tr mutations after curcumin treatment. Note that the percentage of apoptotic cells after curcumin treatment is significantly different from that observed for no treatment (fig. 2) (representative data are from one of four independent experiments with comparable results). FS = flow-sorted cells; PI = propidium iodide.

Figure 4.

Improved neuromotor behavior in treated Tr-J mice. A, Rotarod test performed with 3-mo-old wild-type (Wt), Tr-J, curcumin-treated Tr-J, and placebo-treated mice. In three series and 10 trials, the time that animals remained on a rod was measured and plotted. The rotation speed was increased every minute, from 16 to 36 rpm, in steps of 4 rpm. The mean holding time of curcumin-treated Tr-J mice (n=20) was significantly higher than that of placebo and Tr-J mice (n=10). All animals were allowed to stay on the rod for a maximum of 270 s. B, Curcumin treatment was discontinued for a group of Tr-J mice (n=5) after 3 mo of treatment. The rotarod test was performed similar to as described above, after curcumin treatment followed by discontinuation of treatment (marked on the X-axis with an asterisk [*]). We did not observe significant difference in the motor performance of Tr-J mice at 1 wk (trials 12–14), 2 wk (trials 15–18), and 1 mo (trials 19–22) after withdrawal of curcumin. Tr-J mice started to weaken significantly at 2 mo after treatment was ceased (trials 23–26) (P<.001). A double asterisk (**) denotes the group of mice (n=5) that were originally treated with curcumin for 3 mo and then had the treatment discontinued.

Figure 5.

In vivo TUNEL staining of longitudinal sections of Tr-J and treated Tr-J sciatic nerves. A, Longitudinal sections of sciated nerve of wild-type (a), Tr-J (b), curcumin-treated Tr-J (c), and placebo-treated Tr-J (d) mice. The scale bar is 40 μm for the inset and 100 μm for the lower-power resolution. Arrows indicate TUNEL-positive cells. B, Western-blot analysis showing similar PMP22 protein levels for both wild-type (Wt) and Tr-J mice before and after curcumin treatment. C, Quantification of apoptosis in the sciatic nerves of wild-type, curcumin-treated, and placebo-treated Tr-J mutants. D, Quantitative analysis of cell density per mm² in wild-type control, Tr-J, placebo-treated Tr-J, and curcumin-treated Tr-J sciatic nerves. An asterisk (*) and a dagger (†) denote statistically significant differences from the wild-type and curcumin-treated Tr-J mice, respectively. Note that there is no statistically significant difference between the Tr-J mice and the Tr-J mice treated with placebo.

Figure 6.

HPLC and mass-spectrometry analyses of curcumin. A, Curcumin detected with UV/VIS (190–800 nm) or fluorescence (excitation at 430 nm; emission at 540 nm) detectors. B, Verification of the peak corresponding to curcumin by mass spectrometry (MS) analysis. C, HPLC analysis of sciatic nerves after curcumin treatment. Because of the low levels of curcumin in sciatic nerves, curcumin was not detectable by UV/VIS; instead, fluorescence detectors were used. The curcumin peak (arrow) in the sciatic nerves had the same retention time as in the curcumin standard. No peak was observed at this position in samples from mice that did not receive curcumin treatment.

Figure 7.

Curcumin bioavailability assayed by HPLC analysis in multiple tissues after curcumin treatment. Curcumin has a retention time of 20 min in the standard (A). Curcumin was detected in liver (B), blood (C), and brain (D) of 2-wk-old pups after curcumin treatment. We also detected curcumin in treated adult mice in a similar pattern.

Figure 8.

Histopathology and quantitative analyses of normal, Tr-J, and treated Tr-J sciatic nerves. A, Note the difference in morphology and mean axonal diameter of myelinated fibers in wild-type normal (a), Tr-J (b), curcumin-treated Tr-J (c), and placebo-treated Tr-J (d) sciatic nerves from 3-mo-old mice. Scale bars = 10 μm. B, Distribution of fiber size (based on diameter) in Tr-J (red), curcumin-treated Tr-J (white), and wild-type control (blue) mice. C, Distribution of axonal size in Tr-J (red), curcumin-treated Tr-J (white), and wild-type control (blue) mice. D, Normalized plot of g ratios within each group, to account for the trends (nonlinear) between axon size and the ratio observed in the wild-type mice.

Figure 9.

A, Electron micrographs of normal, Tr-J, and treated Tr-J sciatic nerves. In wild-type control mice (a), NFs are relatively sparse (arrows), whereas, in Tr-J mice (b), the NFs are increased in number. In curcumin-treated Tr-J mice (c), NFs (arrows) become less compact compared with those in untreated Tr-J mice (b). Scale bars = 0.2 μm. B, Calculation of the average number of NF per axonal diameter in wild-type control, Tr-J, and curcumin-treated Tr-J mice. NFs are increased significantly (P<.001) in number in Tr-J mice compared with in curcumin-treated Tr-J mice and wild-type mice.