Curcumin facilitates a transitory cellular stress response in Trembler-J mice

Abstract

We have previously shown that oral administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and partially mitigates the severe neuropathy phenotype of the Trembler-J (Tr-J) mouse model in a dose-dependent manner. Here we compared the gene expression in sciatic nerves of 2-week-old pups and adult Tr-J with the same age groups of wild-type mice and found a significant increase in gene expression for hypoxia, inflammatory response and heat-shock proteins, the latter specifically the Hsp70 family, in Tr-J mice. We also detected an activation of different branches of unfolded protein responses (UPRs) in Tr-J mice. Administering curcumin results in lower expression of UPR markers suggesting it relieves endoplasmic reticulum (ER) cell stress sensors in sciatic nerves of Tr-J mice while the level of heat-shock proteins stays comparable to untreated Tr-J mice. We further tested if Hsp70 levels could influence the severity of the Tr-J neuropathy. Notably, reduced dosage of the Hsp70 strongly potentiates the severity of the Tr-J neuropathy, though the absence of Hsp70 had little effect in wild-type mice. In aggregate, these data provide further insights into the pathological disease mechanisms caused by myelin gene mutations and further support the exploration of curcumin as a therapeutic approach for selected forms of inherited neuropathy and potentially for other genetic diseases due to ER-retained mutants.

Figure 1.

Activation of BiP (A), Atf3 (B), Ero1-lβ (C), Chop (D) in Tr-J. Expression levels in sciatic nerves were measured by quantitative RT–PCR using SYBR® Green in triplicates and normalized to β-Actin. All UPR markers except Chop in pups were significantly increased in 2-week-old pups and 4-month-old adult Tr-J compared with WT (n = 20 in Tr-J and WT). Error bars are standard error of the mean (SEM). (E) Western blot analysis of sciatic nerves was performed three times in two different sample groups. Our data shows an increase of UPR markers in both pups and adult Tr-J compared with WT, without CHOP expression levels in pups. (F) RT–PCR for XBP-1 unspliced (uXBP-1), spliced (sXBP-1) and Gapdh shows a high ratio of sXBP-1 in sciatic nerves of adult Tr-J mouse. *P < 0.05 and **P < 0.005.

Figure 2.

Curcumin reverse activation of the UPR markers. Levels of mRNA were measured by quantitative RT–PCR and normalized to 18S rRNA. BiP is still induced in curcumin-treated mice, whereas Chop, Atf3 and Ero1-lβ returned towards normal level after curcumin treatment. The age of the animals is 3-month-old adults.

Figure 3.

Neuromotor behavior in double heterozygous Hsp70/Tr-J and Hsp70^−/−/Tr-J mice. Rotarod test performed with 3-month-old wild-type (Wt), Tr-J, Hsp70^+/−/Pmp22^+/+(Hsp70^+/−) Hsp70^−/−/Pmp22^+/+ (Hsp70^−/−), Hsp70^+/−/Tr-J and Hsp70^−/−/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. All animals were allowed to stay on the rod for a maximum of 270 s. The mean holding time of double heterozygous mice (Tr-J^+/−/Hsp70.1/3^+/−) and Hsp70i null Tr-J (Tr-J^+/−/Hsp70.1/3^−/−) mice was significantly lower than that of Wt and Tr-J (P < 0.0001 and P < 0.01).

Figure 4.

Activation of BiP and ER stress sensors-Chop, Atf3 and Ero1-lβin Hsp70^+/−/Tr-J and Hsp70^−/−/Tr-J. Expression levels in sciatic nerves were measured by quantitative RT–PCR and normalized to 18S rRNA. All UPR markers were significantly increased in Hsp70^+/−/Tr-J and Hsp70^−/−/Tr-J compared with wild-type (WT). There was no statistical difference in the expression levels of UPR between male and female mice.