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. 2013 Jul 11;22(23):4698–4705. doi: 10.1093/hmg/ddt318

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

Yuji Okamoto 1, Davut Pehlivan 1, Wojciech Wiszniewski 1, Christine R Beck 1, G Jackson Snipes 2, James R Lupski 1,3,4,*, Mehrdad Khajavi 1,†
PMCID: PMC3820132  PMID: 23847051

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.

INTRODUCTION

There have been tremendous advances in our understanding the molecular genetic bases of inherited peripheral neuropathies (1). This has revolutionized diagnostics, enabling an accurate and secure molecular diagnosis, precise recurrent risk estimates, prenatal diagnosis and better management of patients (2). However, there has been a partial failure to translate these findings into molecular based therapeutics. Currently, there is no effective small molecule or drug therapy for Charcot-Marie-Tooth (CMT) disease (3,4); supportive treatment is limited to rehabilitative therapy and surgical correction of skeletal deformities and soft-tissue abnormalities (5). Limited studies exist for treatments targeting different mutational mechanisms (Table 1).

Table 1.

Ongoing CMT studies based on novel therapeutic approaches [adapted from Fledrich et al.(37)]

Human disease Mutation in patients Clinical trial (for human) Experimental therapy in vivo Experimental therapy in vitro Comment
CMT1A PMP22 duplication Ascorbic acid Ascorbic acid None of the clinical trials reported beneficial effects of ascorbic acid to patients with CMT1A.
P2X7 inhibition
CMT1A/1E PMP22 point mutation Neurotrophin-3 (for CMT1A) Neurotrophin-3 Slight improvement of sensory and reflex scores but not of motor function. Number of patients’ samples is small.
Curcumin Curcumin
Rapamycin
Geldanamycin
Caloric restriction
CMT1B MPZ point mutation CHOP ablation
Curcumin Curcumin
Phosphatidylcoline curcumin derivative
CMT2F/HMN2B HPSB1; HSP27 point mutation HDAC6 inhibition HDAC6 inhibition
CMTX Cx32/GJB1 point mutation RAG-1 ablation
MCP-1 ablation
CSF-1 ablation
DSS/CHN PMP22 duplication Ascorbic acid Ascorbic acid
Rapamycin
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Some myelin gene mutants (e.g. MPZ and PMP22) can cause severe disease (Dejerine-Sottas neuropathy, DSN; congenital hypomyelinating neuropathy, CHN) apparently by protein accumulation within the endoplasmic reticulum (ER), causing Schwann cell apoptosis, and subsequently peripheral neuropathy (6,7). Previous studies have shown that PMP22 is associated with ER chaperones during normal folding and such interactions are more prolonged with PMP22 mutants during quality control causing retention in the ER of Schwann cells (8–10). Accumulation of mutant PMP22 in the ER could be potentially accounted for by its interaction with calnexin (10) and perhaps triggering the unfolded protein response (UPR), resulting in Schwann cell death by apoptosis and subsequently, causing peripheral neuropathy (7,11). Previous studies in MpzS63del mice, a CMT1B mouse model, have shown that there is UPR activation in MPZS63del transgenic Schwann cells (12). Mutant MpzS63del is also retained in the ER, triggering the UPR as evidenced by significant changes in the UPR markers BiP [GRP78, a member of the heat-shock protein-70 (HSP70) family], CHOP and Downstream of CHOP genes (DOCs) (12). Although both MPZ and PMP22 are components of compact myelin and play major structural roles, MPZ accounts for more than 50% of the protein content of peripheral myelin (13); therefore, Schwann cells may be required to process higher levels of misfolded mutant MPZ compared with Tr-J mutant protein.

We previously demonstrated that curcumin, a small molecule compound derived from the curry spice tumeric, ameliorates the severity of the Pmp22 Tr-J neuropathy; an effect that correlates with decreased Schwann cell apoptosis (7). Curcumin has been reported to have anti-inflammatory and antitumor properties. Experiments in diverse disease animal models suggest that curcumin enables misfolded proteins to traverse from the ER to the plasma membrane concurrently reducing the cytotoxicity of the mutant protein (14–16). Although the exact mechanism by which curcumin corrects the processing of misfolded or aggregated mutant proteins in cells remains obscure, it appears that it modulates a number of cellular messenger/signaling pathways including NF-kB and intracellular calcium, interfering with the function of the ER calcium-dependent chaperones (15,17).

To elucidate the pathophysiology of the peripheral neuropathy disorder caused by Tr-J mutation, we investigated signal-transduction and stress pathways using focused gene expression arrays and compared gene expressions in sciatic nerves of 2-week-old pups and 4-month-old adult Tr-J with the same age groups of wild-type mice. Our data suggest that hypoxia induced, oxidative/metabolic stress, inflammatory response, heat-shock proteins, specifically the Hsp70 family, and branches of UPRs are significantly increased in Tr-J mice. We then detected a decrease in the expression level of UPR components in curcumin-treated mice compared with untreated Tr-J mice suggesting that curcumin facilitates a transitory cellular ER stress response in Tr-J sciatic nerves. We also tested whether Hsp70 levels could influence the severity of the Tr-J neuropathy. Remarkably, we found that Tr-J neuropathy was exacerbated by heterozygous or homozygous absence of Hsp70, whereas the absence of Hsp70i has no identifiable effect in wild- type mice.

RESULTS

Increase in heat-shock stress, apoptosis and inflammatory response signaling pathway genes in Tr-J sciatic nerves

From both human and mouse studies, it is clear that mutations affecting the PMP22 gene cause demyelinating neuropathies; however, the normal biological functions of PMP22 and how mutants lead to disease remain poorly understood. Severely affected CMT1 patients with identical PMP22 mutations and nerve biopsies show alterations analogous to those detected in heterozygous Tr-J mice (18). Thus, a better understanding of the pathophysiology of peripheral neuropathies caused by Tr-J could provide the basis for broadly applicable therapies. Here, we compared the patterns of gene expression in Tr-J and wild-type sciatic nerves of both 2-week-old pups and 4-month-old adult mice using stress and toxicity-specific pathway arrays. We identified 29 genes that are either up-regulated or down-regulated by Tr-J in pups and 28 genes in adult mice compared with wild-type mice (Table 2). The expressions of three groups of genes were significantly different: (i) apoptosis signaling genes such as Fas, Casp1; (ii) heat-shock proteins such as Hspa4 (Hsp70) and (iii) inflammatory response genes such as Cd40Ig, Ifng, Il1a, Il1b and Tnf. These changes are in agreement with earlier findings of PMP22 mutant aggregation-induced apoptosis (7,11).

Table 2.

Significant increases in hypoxia, heat shock, apoptosis and inflammatory response signaling genes in Tr-J sciatic nerves from both pups and adult mice.

Pathways Tr-J pup Tr-J adult
Apoptosis signaling Fas, Casp1 Fas, Tnfrsf1a
Cell-cycle check point/Arrest Hus1 Chek1, Chek2, Hus1, Rad51, Nbn
Heat-shock proteins/UPR Hspa41, Atf4, Ddit3, Dnajc3 Hspa41, Atf6b, Bid, Dnajc3
Hypoxia Car9, Epo, Serpine, Slc2a1, Vegfa Car9, Epo, Mmp9, Serpine, Vegfa
Inflammatory response Cd40Ig, Crp, Ifng, Il1a, Il1b, Il6, Tnf Cd40Ig, Crp, Ifng, Il1a, Il1b, Il6,
Oxidative/Osmotic stress Fth1, Akr1b3, Aqp2, Aqp4, Cftr, Edn1, Nfat5 Txn1, Aqp2, Aqp4, Cftr, Edn1
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Pmp22 mutation causes ER stress in sciatic nerves

Previous studies have shown that both over-expression of wild-type Pmp22 and mutant Tr-J protein form a complex with calnexin, a Ca2+-binding chaperone, that contributes to ER retention (10). Accumulation of Pmp22 mutant Tr-J can also potentially trigger ER stress, resulting in Schwann cell death by apoptosis, and subsequently causing peripheral neuropathy (7,11). The mechanism by which the processing and function of misfolded or aggregated Pmp22, Tr-J mutant proteins in cells can be associated with apoptosis has not been determined. Nevertheless, it is hypothesized that it may include interference with the function of the ER chaperones. To determine the role of UPR in Tr-J mice, we analyzed different UPR markers including Bip, Chop, Atf3 and the oxidoreductase Ero1-lβ in sciatic nerves of both wild-type and Tr-J mice in two different age groups (2-week-old pups and 4-month-old adult mice, n = 20 in each group). We detected an increase of 1.34-, 1.54- and 1.29-fold of BiP, Atf3 and Ero1-lβ mRNA levels in Tr-J pups and similarly 2.12-, 2.06-, 2.1- and 1.5-fold of BiP, Atf3, Ero1-lβ and Chop expression levels in Tr-J adults compared with wild-type pups and adults, respectively (Fig. 1A–D). We, however, did not detect significant increase in Chop mRNA expression levels in Tr-J pups (Fig. 1D, P = 0.2). We further verified and independently confirmed our findings by western analysis (Fig. 1E) in each group. In addition, we evaluated the possibility of Tr-J mutation inducing X-box binding protein (XBP-1) splicing in sciatic nerves. IRE-1, a serine/threonine protein kinase that possesses endonuclease activity, senses the accumulation of unfolded proteins in the lumen of the ER that leads to enzyme auto-activation (19). The active IRE-1 splices XBP-1 mRNA and converts it into a potent UPR transcriptional activator (19). We observed a high ratio of spliced XBP-1 in adult Tr-J mice (Fig. 1F) indicating a canonical UPR. Induction of multiple markers associated with activation of UPR leads to the up-regulation of certain ER chaperones and interference with folding of newly synthesized proteins suggesting a persistent ER stress that could be detrimental to cell growth and survival (20,21).

Figure 1.

Figure 1.

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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.

Curcumin relieves ER stress in sciatic nerves of the Tr-J mouse

We previously showed that curcumin relieves the toxicity and/or cell stress associated with the Tr-J mutation and potentially enabling a more efficient protein-trafficking process in the ER that can result in increased axonal size and myelination that is sufficient to improve the clinical phenotype of Tr-J mice (7). Here, we analyzed the overall effect of curcumin on UPR components in sciatic nerves of curcumin-treated Tr-J mice. As shown in Figure 2, even though Bip and Chop mRNA levels were not reduced to wild-type level, the expression of UPR markers such as Atf3 and Ero1-lβ was decreased significantly to levels comparable with wild-type mice. We also measured the expression level of Pmp22 in both curcumin-treated wild-type and Tr-J mice and found that Pmp22 expression levels increased 1.4- and 1.5-fold in curcumin-treated wild-type and Tr-J mice, respectively (unpublished data). These findings support the contention that curcumin protects cells from ER stress-induced apoptosis by facilitating the cellular stress response yielding an overall reduction of stress, as evidenced by mitigation of marker gene expression, in Tr-J sciatic nerves.

Figure 2.

Figure 2.

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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.

Lack of Hsp70 expression strongly potentiates the severity of the Tr-J neuropathy

Our gene expression analysis and previous immunocytochemical studies (22) suggest that heat-shock proteins, specifically Hsp70 and Hsp90, might be involved in the processing of impaired intracellular trafficking of mutant Pmp22. To test this directly, we crossed Hsp70i knockout mice (Hsp70.1/3−/−) with Tr-J mice and evaluated the effects of reduced Hsp70i levels on the severity of the Tr-J neuropathy in adult animals using the rotarod test. As predicted, we found that the neuropathy in the Tr-J mice was severely aggravated by heterozygous or homozygous absence of Hsp70i, though the absence of Hsp70i had little effect on the rotarod performance of wild-type mice. In fact, double heterozygous mice (Tr-J+/−/Hsp70.1/3+/−) and Hsp70i null Tr-J (Tr-J+/−/Hsp70.1/3−/−) mice were unable to stay on the rotating rod, even at low angular velocity and after many trials (Fig. 3). This finding strongly suggests that reduced or absent Hsp70i may potentiate Tr-J mutation effects and that Hsp70 may directly be involved in intracellular processing of mutant Tr-J. We then sought to evaluate the effect of oral curcumin supplementation in mice with reduced Hsp70 gene dosage. Unlike Tr-J mice, curcumin treatment did not improve the neuropathy of either Tr-J+/−/Hsp70.1/3+/− or Tr-J+/−/Hsp70.1/3−/−. In fact, curcumin treatment was discontinued after 1 week because it had an apparent lethal side effect in this group of mice perhaps by potentiating the neuropathy. These findings suggest that curcumin may facilitate the stress response through potentiating the levels or function of HSP70.

Figure 3.

Figure 3.

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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).

UPR in low dosage of Hsp70.1/3

We analyzed different branches of UPR pathways in two different groups of mice: the first group was Tr-J+/−/Hsp70.1/3+/− and Tr-J+/−/Hsp70.1/3−/− and the second group included Pmp22+/+/Hsp70.1/3+/− and Pmp22+/+/Hsp70.1/3−/−. We compared both groups with wild-type mice. As shown in Figure 4, BiP and Chop were increased 7.5- and 4.3-fold, respectively, in double heterozygous mice while an increase of 8.3- and 6.6-fold was observed in Tr-J+/−/Hsp70.1/3−/− mice suggesting that UPR is activated as a protein quality control in this group. We also observed an increase of 5.3- and 2.4-fold in the expression of Atf3 and Ero1-lβ, respectively, in double heterozygous mice while an increase of 6.4 and 2.6 of Atf3 and Ero1-lβ, respectively, in Hsp70i null Tr-J mice (Fig. 4). Our data suggest an activation of UPR pathways in both double heterozygous and Hsp70 null Tr-J mice as evidenced by significant increase in the expression of UPR markers; comparison between the groups suggest an Hsp70 gene dosage-dependent effect. Notably, we did not observe any significant changes of UPR markers in Pmp22+/+/Hsp70+/− and Pmp22+/+/Hsp70−/− when compared with the wild-type.

Figure 4.

Figure 4.

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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.

DISCUSSION

We investigated specific molecular and cellular pathways through which curcumin may function to ameliorate the neuropathy in Tr-J mice and evaluated the effect of curcumin supplementation on the expression of genes associated with UPR pathways in sciatic nerves of Tr-J mice. We initially found that accumulation of Tr-J in the Schwann cell ER triggers UPR that leads to up-regulation of heat-shock proteins in Tr-J mice. The persistent ER stress activates apoptosis signaling genes such as Caspase1 and Fas (TNF receptor superfamily member 6) consequently resulting in Schwann cell death by apoptosis. Recent studies have shown that ablation of Chop decreases demyelination in ER-stress-mediated neuropathy (12). We did not detect a significant increase (P = 0.23) in Chop levels between pups Tr-J and wild-type mice; however, adult Tr-J showed a significant increase in Chop compared with wild-type suggesting a potential time-dependent accumulation of stress. Similar to our results, Sancho et al. (11) showed that Schwann cell apoptosis occurs naturally in developing nerves of wild-type mice during the first postnatal 10 days and did not show significant differences between wild-type and Tr mice. However, at the age of 10 weeks, Tr mice showed a significant increase in Schwann cell density/proliferation and apoptosis at the same time.

There were a number of other markers such as Bip, Atf3 and Erol-1β that were significantly increased in sciatic nerves of both pup and adult Tr-J mice indicating the partial activation of UPR pathways. In fact, previous studies (12,23) have reported activation of UPR as the pathogenic mechanism for demyelination by misfolded Mpz mutants in different CMT1B mouse models. In a recent study of a more severe form of early onset CMT, a misfolded R98C Mpz mutant causes the activation of a ‘canonical’ UPR response (23). Interestingly, regular curcumin treatment showed a minor effect in improving neuropathy symptoms in the R98C CMT1B mouse model, whereas potentially more potent curcumin derivatives, dissolved in sesame oil or phophatidylcholine, rescue the motor deficit and ameliorated the neuropathy phenotype evidently by reducing the UPR activation (24). Mpz, however, is highly expressed in peripheral myelin and processing higher levels of misfolded mutant Mpz compared with Tr-J mutant proteins may potentially require a more potent treatment. We also observed a decrease in the expression level of UPR components in regular curcumin-treated mice compared with untreated Tr-J mice and did not detect any difference in the treatment outcome using curcumin dissolved in sesame oil in Tr-J mice (unpublished data).

There are a number of cellular mechanisms by which curcumin could act on Tr-J nerves; first, it is possible that curcumin might modulate signal-transduction pathways that regulate myelin gene expression. Alternatively, curcumin may act directly on neurons by acting on signal-transduction pathways, affecting the growth of myelinating Schwann cells potentially causing it to stimulate maturing axons to increase their caliber. Although this hypothesis may serve to explain the clinical effects of curcumin on the Tr-J neuropathy, it does not explain the reduced rate of apoptosis observed in curcumin-treated Tr-J sciatic nerves. Administering curcumin apparently results in the reduction of ER stress sensors in sciatic nerves of treated Tr-J mice. We have previously shown that curcumin treatment partially mitigates the clinical and neuropathological phenotype of Tr-J mice by relieving the toxic effect of the mutant PMP22, thereby inhibiting Schwann cell apoptosis and increasing axonal caliber and myelin thickness (7). The positive clinical response to curcumin occurs in a dose-dependent manner and is reversed after withdrawal of treatment. The ability of curcumin to increase axonal caliber and myelin thickness (7), without tomacula formation, is consistent with the hypothesis that curcumin relieves ER-associated degradation (ERAD) stress caused by abnormal processing of Pmp22 and/or other Schwann cell proteins that are required for myelination. We now show that this rescue is not due to reduced expression of the Tr-J, but due to reduced activation of the UPR. While the level of heat-shock proteins in curcumin-treated Tr-J stays comparable to untreated Tr-J mice, this could be expected in order to stabilize the re-translocated mutant Pmp22 protein to the cytoplasm and potentially facilitate the Tr-J protein for subsequent degradation. Additionally, Patzko et al. (24) recently showed that curcumin decreases promyelinating transcription factors including c-Jun and SCIP (Oct6, Pou3f1) beside the UPR markers in heterozygous and homozygous R98C CMT1B mice. The heat-shock protein genes, specifically the Hsp70 family, showed the largest differences in our gene expression analyses of Tr-J nerves. The over-expression of heat-shock protein (Hsp70) has been shown to have a cytoprotective role against ischemia and other misfolding protein disorders such as spinal cerebellar ataxia 1 (25,26).

We further tested if Hsp70 levels could influence the severity of the Tr-J neuropathy. Remarkably, we found that Tr-J neuropathy was severely affected by heterozygous or homozygous absence of Hsp70, even though the absence of Hsp70 has no detectable effect in wild-type mice. Although morphological analysis could potentially provide additional insight into the worsening effect from lack of Hsp70 in sciatic nerves, our data provides evidence that this effect may be due to a general protective function of Hsp70, such as the one found in cancer cells fighting against cytotoxicity induced by anticancer agents that trigger the caspase-independent lysosomal cell-death pathway (27). Alternatively, Hsp70 may function as a necessary chaperone for Pmp22 prior to its degradation. In fact, in our curcumin treatment of Tr-J+/−/Hsp70.1/3+/− mice, we found that the level of Hsp70 is critical after treatment with curcumin to process re-translocated Tr-J mutant in cytoplasm. In the absence of Hsp70, curcumin treatment had an apparent lethal side effect in Tr-J+/−/Hsp70.1/3+/− mice presumably by potentiating the neuropathy. In this case, Hsp70 may help stabilize aggregation-prone regions of Pmp22, as it traverses the cytoplasm to the proteasome. It has been shown that Hsp70 chaperones function as facilitators for degradation either by preventing aggregation and/or actively transferring them to the proteolytic system (28). Other heat-shock proteins have been shown to play a role in different types of CMT disease such as mutations in the 27 kDa small heat-shock protein gene (HSPB1, also called HSP27) causing axonal CMT type 2F and distal hereditary motor neuropathy (distal HMN) (29). HSPB1 acts as a chaperone by binding misfolded or denatured proteins, and prevents them from forming toxic aggregates (30,31). Mutant HSPB1 decreases acetylated α-tubulin levels and decreases axonal transport. In addition, another study has shown that heat-shock protein 90 (HSP90) inhibitors enhance cytosolic chaperones, including HSP27 and HSP70, and improve myelination in neuropathic mouse cell lines (32).

Studies that increase expression of Hsp70 by using transgenic mouse models could potentially identify novel factors involved in glycoprotein ERAD of transmembrane folding defects. It is critical to understand the differences in how various myelin gene mutant proteins interact with the protein quality control machinery, because this could provide the basis for determining which chemical chaperone therapeutic strategies might be useful for specific protein-folding disorders. Rational drug design could be based on different principles, such as interfering with chaperone activity and allowing misfolded proteins to make their way out of the ER, or involving upregulation at the transcriptional level and modulation of protein-folding steps (33). Interestingly, one of the reported effects of curcumin is to induce the expression of Hsp70 (28,34). This may partially explain the mechanism by which curcumin acts on Schwann cell apoptosis and ameliorates the Tr-J neuropathy (7). It also provides further evidence for the importance of ERAD pathways in the pathogenesis of CMT.

Together, these data provide insights into the pathological disease mechanisms caused by myelin gene mutations and identify new candidate genes that are likely to play important roles in disease process and may provide potential targets for therapeutic approaches to ER-retained mutants.

MATERIALS AND METHODS

Sample collection, RNA extraction and western blot analysis

Matings between Tr-J heterozygous females and males in the C57BL/6 genetic background were implemented to generate Tr-J heterozygous and wild-type controls. Tail DNAs were used for genotyping. We also crossed Hsp70i knockout mice (Hsp70.1/3−/−) (35) with heterozygous Tr-J mice. These mice were obtained from a breeding colony of Tr-J and Hsp70.1/3−/− mice that we established in collaboration with G. Jackson Snipes, MD, PhD in our mouse facility. The sciatic nerves were dissected and frozen immediately in liquid nitrogen, and stored in –80°C. For RNA extraction, the sciatic nerves were homogenized in Trizol and RNA extracted according to the manufacturer's instruction (Invitrogen, Carlsbad, CA, USA) followed by purification on columns from RNeasy mini kit (Qiagen Sciences, Germantown, MD, USA). RNA quality was assessed using Agilent Bioanalyzer 2100 and the NanoDrop ND-1000 spectrophotometer. For western blot analysis, frozen sciatic nerves were pulverized, dissolved in lysis buffer containing 5 mm Tris–HCl (pH 7.5), 1.5 mm KCl, 2.5 mm MgCl2, 1% deoxycholic acid, 1% Triton X-100, supplement with protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The protein content of supernatant was determined using a BSA standard curve (BCA Protein Assay Kit, Thermo, Rockford, IL, USA). Equal amounts of the protein were electrophoresed on 12% SDS–polyacrylamide gels, and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked for overnight at 4°C with 5% blotting-grade blocker (Bip-rad, Hercules, CA, USA) in TBS and then incubated with specific primary antibody, Gapdh (1:2000, MILLPORE, Temecula, CA, USA), Atf-3 (1:1000, Sigma, St Louis, MO, USA), Bip (1:2000, Sigma), Chop (1:2000 Thermo, Rockford, IL,USA) and Ero1-LB (1:200 Proteintech, Chicago, IL, USA) in PBST with 5% blotting-grade blocker. Following three washes with PBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000–1:10000 dilution; Bio-rad). After three washes with PBST, the transferred proteins were detected using chemiluminescent (ECL) substrate for HRP (Supersignal West Pico Chemiluminescent Substrate, Thermo) with X-ray film.

Microarray processing and quantitative RT–PCR

Five to 10 µg of total RNA from each individual sciatic nerves of 4 Tr-J at 2 weeks and 4 months of age and four wild-type controls were used to produce cDNA target for microarray. The target was generated using a reverse transcription reaction to produce cDNA (Supercript Choice System, Invitrogen, Carlsbad, CA, USA). For PCR array data (Qiagen Sciences, Germantown, MD, USA) analysis, the ΔΔCt method was used and each gene fold-change was calculated as difference in gene expression between Tr-J and wild-type samples. Data were normalized against two housekeeping genes that were included in the PCR array by subtracting the average Ct value for two housekeeping genes from the average Ct value of gene of interest. ΔΔCt were compared across groups using t-tests. The P-value is calculated based on whether the test sample score is from the extreme case of scores from control treatment samples with compound not causing the toxic effect under evaluation (Qiagen Sciences, Germantown, MD, USA). The P-value is considered significant when it is <0.05. To verify changes in the expression level of specific genes identified on the microarray, additional real-time RT–PCR analyses was performed. Total RNA of 2 µg for each sample was reverse transcribed with the Superscript First-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) using a random primer. The cDNA was then PCR-amplified in triplicates and confirmed in two independent experiments by SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) on different animal groups to avoid biases. The primers were designed by using Universal Library Assay Design Center of Roche Applied Science web site (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000) (Roche Diagnostics Corporation Roche Applied Science, Indianapolis, USA) and RT Primer database (http://medgen.ugent.be/rtprimerdb/). For XBP-1, RT–PCR was performed as described previously (36). The relative level of each RNA sample is calculated using the ΔΔCt method normalized either to the corresponding Gapdh or β-Actin levels.

Curcumin treatment and specific motor response tests (motor activity on a rotating rod)

Curcumin treatment was performed as described previously (7). The animals' locomotor performances were tested using a rotarod test, a motor co-ordination and balance control task. Briefly, in every test, the exams were masked to the specific genotype. Each mouse underwent the same 5-day procedure. The first 2 days were used to train the animals [five sessions of 2 min walking were run at a very low speed, i.e. 5 rotations per minute (rpm)] until a steady baseline level was reached in their performances. The mice were then subjected to a higher rotation speed to familiarize them with the forthcoming experimental conditions. The last 3 days were used to run the test sessions. Here the mice had to perform two series of five trials with a 1 h rest period between the two series. In each trial, the mouse was placed on the rod and the rotation speed was increased every minute from 16 to 36 rpm in steps of 4 rpm. The scores obtained in this test were measured based on the time spent on the rod without falling minus the time during which the mouse is rotating with the rod without falling (7). The mean walking time spent on the rod was then calculated for each genotype at each rotation speed. As this task is very demanding for the mice, we will make sure the results are not affected by fatigue. Each individual's performances were compared between the first and last trials at the maximum speed (paired t-test).

Conflict of Interest statement. J.R.L. is a paid consultant for Athena Diagnostics, has stock ownership in 23andMe and Ion Torrent Systems and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis offered in the Medical Genetics Laboratory.

FUNDING

This work was supported in part by US National Institute of Neurological Disorders and Stroke,National Institutes of Health(NINDS,NIH) grant (R01NS058529). W.W. is supported by a (K23NS078056) grant from the NINDS,NIH.

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