Enhanced Expression of Trib3 during the Development of Myelin Breakdown in dmy Myelin Mutant Rats

The demyelination (dmy) rat exhibits hind limb ataxia and severe myelin breakdown in the central nervous system. The causative gene of dmy rats is the MRS2 magnesium transporter gene. Tribbles homolog 3 (Trib3) is a pseudokinase molecule that modifies certain signal pathways, and its expression is increased in response to various stresses. Here we sought to clarify the mechanism of myelin breakdown by focusing Trib3, which is remarkably up-regulated in dmy rats. The expression of Trib3 mRNA was significantly increased at 4, 5, 6, 7 and 8 weeks of age in the dmy rats, prior to the prominent myelin breakdown between 7 and 10 weeks of age. The expression level of Trib3 was increased concurrently with the progression of the clinical and pathological conditions in the dmy rats. Double immunofluorescence demonstrated that TRIB3 was mainly expressed in neurons and oligodendrocytes and localized in the Golgi apparatus. Our findings indicate that Trib3 may be associated with the pathogenic mechanism of dmy rats.


Introduction
Myelin mutants are extremely useful as tools for clarifying the complex process of myelination, the maintenance of myelin, and myelin diseases. Many myelin mutants have been established, including the jimpy (jp) mouse [1] which has X-linked mutations affecting the proteolipid -protein gene; the myelin-deficient (md) rat [2]; the shiverer (shi) mouse [3]; the Long Evans Shaker (les) rat [4], which has a genetic mutation in myelin basic protein and the taiep rat [5], which has a microtubule abnormality.
The demyelination (dmy) rat is a unique spontaneous myelin mutation that exhibits severe myelin breakdown with a late onset of clinical signs [6]. The causative autosomal recessive mutation has been identified at the MRS2 magnesium transporter (Mrs2) gene, which encodes an essential component of the major Mg 2+ influx system in mitochondria; Mrs2 gene is expressed mainly in neurons [7]. The dmy rat is characterized by severe myelin destruction throughout the white matter of the central nervous system (CNS) during the late stage of myelination, and the distribution progresses rapidly [8]. Hypertrophic oligodendrocytes were frequently observed in the ventral funiculus of the spinal cord of dmy rats, and the cytoplasm was intensely stained with mitochondrial markers [9]. These findings indicate that the Mrs2 gene plays a prominent role in the development and maintenance of myelin. However, the detailed pathogenesis including the relationship between mitochondrial dysfunction and myelin disorder remains uncertain.
Tribbles homolog 3 (Trib3) has been reported as a pseudokinase with scaffold-like regulatory functions related to inflammation and certain signaling. Trib3 expression is increased in response to various stresses, such as nutritional deficiencies [10,11], endoplasmic reticulum stress [12][13][14][15], hypoxia [16] and oxidant stress [17]. Trib3 has also been reported to be an important regulatory protein involved in signal pathways; for example, the inhibition of mitosis [18], the inhibition of insulin signaling by binding directly to Akt [19], the modulation of mitogen-activated protein kinase activity [20] and more. However, there are few reports describing the function of Trib3 in the CNS [21,22]. To address the detailed pathomechanism of myelin destruction in dmy rats, we performed a microarray analysis using spinal cord samples and observed a remarkably up-regulated expression of Trib3 gene. The purpose of this study is to shed light on the mechanism of myelin destruction in the dmy rat by focusing on the expression of Trib3.

Materials and Methods Animals
The dmy rats were supplied by the National BioResource Project-Rat, Kyoto University (Kyoto, Japan). The rats were maintained at the Education and Research Center for Experimental Animal Science at Osaka Prefecture University. We mated heterozygous (dmy/+) females with heterozygous (dmy/+) males and obtained the affected (dmy/dmy) and control (+/+ or dmy/+) rats. We examined these rats at 4, 5, 6, 7 and 8 weeks of age.
For the comparison of Trib3 expression in myelin lesions, we also examined two other myelin mutant rats, myelin vacuolation (mv) and vacuole formation (VF) rats. The mv rat is a spontaneous tremor mutant with a null mutation in the attractin gene; these rats show hypomyelination and vacuole formation in the myelin throughout the CNS from the early stage of myelination [23]. The VF rat shows tremor behavior (especially in the caudal body) from the age of approx. 10 days. The peak of tremor is observed around 4 weeks of age, and then, tremor behavior gradually improves [24]. Hypomyelination and abnormal vacuoles around the axons in VF rats were observed mainly in the white matter of the spinal cord, and the vacuoles were gradually reduced [24]. The VF rat has a mutation in Dopey1 gene, which is likely to be involved in the traffic of myelin proteins [25]. We mated heterozygous (mv/+, vf/+) females with heterozygous (mv/+) or homozygous (vf/vf) males and obtained the affected (mv/mv, vf/vf) and control (+/+ or mv/+, vf/+) rats. As soon as the rats were weaned, we carried out genotyping by using the Amp-FTA method as described [26]. In the present study, we examined the affected homozygous and wild type control rats of mv and VF mutants at 4 and 6 weeks of age and at 4, 10, 20 weeks of age, respectively.
All of the rats were maintained in a room with controlled temperature and a 12-hr lightdark cycle. Food and water were provided ad libitum. This study was carried out in strict accordance with the recommendations in the Guidelines for Animal Experimentation of Osaka Prefecture University. The protocol was approved by the Animal Experimentation Committee of Osaka Prefecture University (Permit nos. 25-68 and 27-54). All rats used in this study were deeply anesthetized with isoflurane and exsanguinated from abdominal aorta, and all efforts were made to minimize suffering.

Microarray analysis
For the microarray analysis, 6-week-old control and homozygous rats of each mutant were deeply anesthetized and exsanguinated from the abdominal aorta. Total RNA was isolated from the cervical spinal cord with the SV Total RNA isolation system (Promega, Madison, WI, USA). The quality of the RNA was checked with Lab Chips (Agilent technologies, Santa Clara, CA) on an Agilent 2100 Bioanalyzer (Agilent Technologies) and NanoDrop spectrophotometer ND-1000 (nanoDrop Technologies, Wilmington, DE). The RNA was then prepared and hybridized to the rat Genome 230 2.0 Array (Affymetrix, Santa Clara, CA) by Bio Matrix Research, Inc. (Nagareyama, Japan.), following the protocols provided by Affymetrix. The data analysis was done using Gene-Spring GX software [26].

Real-time RT-PCR
Cervical spinal cords from dmy homozygous and control rats at 4, 5, 6, 7 and 8 weeks of age were dissected into the ventral funiculus, dorsal funiculus and gray matter. The mv and control rats at 4 and 6 weeks old and VF and control rats at 4, 10 and 20 weeks old were also necropsied, and the cervical spinal cords were dissected into the white and gray matter. Total RNA was isolated with the SV Total RNA isolation system (Promega) according to the manufacturer's instructions. A real-time PCR was performed with SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) with the following setting: 40 cycles of amplification, 95˚C for 5 s, 60˚C for 30 s. The data were normalized to β-actin mRNA using the following primer pairs: Trib3 forward 5'-TCATCTTGCGCGACCTCA-3' and reverse 5'-GTCCAGTCATCACACAG GCATC-3'; β-actin forward 5'-TAAAGACCTCTATGCCAACAC-3' and reverse 5'-CTCC TGCTTGCTGATCCACAT-3'.The relative expression levels were calculated using the comparative Ct (threshold cycle) method.

Immunohistochemistry
Lumbar spinal cords from 7-week-old dmy and control rats were removed and frozen at −80˚C. Ten-μm frozen sections were cut using a cryostat. After drying, the sections were fixed in diethyl ether at 4˚C for 10 min. The tissues were then rinsed in phosphate-buffered saline (PBS) and treated with 10% normal goat serum in PBS for 30 min. The sections were then incubated with mouse anti-TRIB3 antibody (clone 1H2) (1:100; Sigma-Aldrich, St. Louis, MO) at 4˚C overnight. For the detection of Golgi apparatus, dmy and control rats were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH 7.4). Tissue samples from lumbar spinal cords were routinely processed and embedded in paraffin. Four-μm sections were dewaxed, pretreated in a microwave with citrate buffer (pH 6.0, 10 mM) for 20 min, and incubated in 5% skim milk for 30 min. The sections were then incubated overnight at 4˚C with anti-Golgi 58K mouse monoclonal antibody (1:1000; Sigma-Aldrich). After incubation with the primary antibody, the sections were treated with 0.3% hydrogen peroxide/0.065% sodium azide in PBS to quench endogenous peroxidase activity. The sections were then treated with a peroxidase-conjugated anti-mouse IgG antibody (Histofine Simplestain MAX PO; Nichirei, Tokyo) for 60 min. Signals were visualized with a 3, 3'-diaminobenzidine (DAB) substrate kit (Nichirei).
For the immunofluorescence analysis, sections were rinsed in PBS containing 0.3% Triton X-100 (Sigma-Aldrich), and then treated with 10% normal goat serum in PBS for 30 min. The sections were then incubated with mouse anti-TRIB3 antibody at 4˚C overnight and treated with the Alexa Fluor 488-labeled secondary antibody against mouse IgG (1:1000; Invitrogen, Carlsbad, CA) for 45 min and coverslipped by mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Signals were detected with a confocal imaging system C1Si (Nikon, Tokyo).

Statistical analysis
The data are presented as means ± standard deviation. The statistical analysis was performed using Tukey-Kramer tests. A p-value <0.05 was considered significant.

Results
Trib3 mRNA expression was significantly increased in the dmy rats but not in the other myelin mutant rats The microarray analysis revealed a 6.2-fold higher expression of Trib3 in the dmy rats compared to the control rats (Gene Expression Omnibus, accession no. GSE88855). We next investigated the Trib3 mRNA expression in more detail by the real-time PCR analysis. The expression of Trib3 mRNA in the dmy rat was significantly increased in all areas of the spinal cord (i.e. the ventral funiculus, dorsal funiculus and gray matter) at all time points examined (4, 5, 6, 7 and 8 weeks); the expression in the ventral funiculus was the most prominent (Fig 1). We also observed that the increased expression became conspicuous with age in the ventral funiculus and gray matter. For the comparison of Trib3 expression in the myelin lesions of the other myelin mutants, we also conducted a real-time PCR analysis in the mv and VF rats using samples from the white and gray matter of the spinal cord. The expression level of Trib3 of the dmy rats was approx. 12-hold higher in the white matter compared to that in the controls at 4 and 6 weeks of age, and approx. 8-fold higher in the gray matter at 6 weeks of age (Fig 2). In the white matter of the VF rats at 4 weeks of age, a significant increase in the expression level of Trib3 was seen; however, it was not as remarkable as that of the dmy rats (Fig 2A). No significant differences in the relative expression levels of Trib3 were detected in the gray matter between the mv and VF rats (Fig 2B).

Distribution and subcellular localization of TRIB3 in the dmy rat
To identify the localization of TRIB3 in the spinal cord of the dmy rats, we conducted immunohistochemistry for TRIB3 in 7-week-old rats. The cytoplasm of the neurons and glial cells in the white matter were positively stained for TRIB3 in a dot-like pattern (Fig 3). To identify the TRIB3-positive cells in the dmy rats, we performed double immunofluorescence using specific markers for neurons, oligodendrocytes, microglial cells and astrocytes. TRIB3 was coexpressed with neurons and some of the oligodendrocytes, but not with microglial cells or astrocytes (Fig  4). We investigated the subcellular localization of TRIB3 by double immunofluorescence. It revealed the co-localization of TRIB3 and Golgi apparatus in neurons and oligodendrocytes (Fig 5). The immunohistochemistry for the Golgi apparatus marker revealed that immunoreactivity was elevated in the ventral funiculus of the dmy rats at 6-8 weeks of age (Fig 6).  7-10 weeks of age [8]. In agreement with a previous study's finding that myelin lesions in dmy rats deteriorate with advancing age [8], we observed in the present study that the expression level of Trib3 gradually increased from 4 to 8 weeks of age. Although no remarkable histopathological myelin-related lesions were found in the dmy rats up to 6 weeks old [8], in the present study we observed an increased level of Trib3 expression in the 4-5 week-old rats, suggesting that Trib3 may play some roles in the mechanism of myelin destruction in dmy rats from an early period. The double immunofluorescence demonstrated that TRIB3 was expressed mainly in neurons and oligodendrocytes in the dmy rats. An up-regulated expression of TRIB3 may be related to a potential degeneration of neurons and/or oligodendrocytes in these rats. Mrs2, a causative gene for dmy rats encodes a component of the Mg 2+ influx system in mitochondria, and in our analysis this gene was expressed mainly in neurons. No apparent alteration was found in neurons of the dmy rats, and the detailed mechanism underlying how dysfunction in mitochondria leads to the destruction of myelin remains unclear. Trib3 is induced in multiple cellular models of Parkinson's disease (PD) and the dopaminergic neurons of human PD patients, and it is thus considered a potential mediator of neuronal death and degeneration in PD [27]. Oligodendrocytes are known to be a target of the autoimmune attack in demyelinating diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis, an animal model of MS [28]. Moreover, oligodendrocytes in culture are more sensitive to oxidative stress exerted by H 2 O 2 compared to astrocytes and microglial cells [29]. In the present study's microarray analysis, the expression levels of hemeoxygenase-1 (HO-1) and NAD(P)H dehydrogenase, antioxidant enzymes, were increased in the dmy rats. This is consistent with previous studies' findings that HO-1 was predominantly induced in oligodendrocytes during the early stage of demyelination in the dmy rat but not in the mv rat, and it suggests that oxidative stress is likely involved in the pathogenesis of demyelination in the dmy rat [30].
Our immunohistochemistry for Golgi 58K has demonstrated that immunoreactivity for the Golgi apparatus was elevated in the ventral funiculus in the dmy rats between 6 and 8 weeks of age. Our double immunofluorescence also demonstrated that TRIB3 is localized in the Golgi  apparatus. Oxidative stress enhances C/EBP homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, and increases the binding of CHOP to the Trib3 promoter in murine diabetic kidneys [31]. The overexpression of Trib3 is thus likely to be promoted by oxidative stress, and ER stress may be a possible reason for the myelin destruction in the dmy rat.
Several studies have shown that oxidative stress and ER stress induce the apoptosis of oligodendrocytes and suppress remyelination in demyelinating lesions in MS [29,[32][33][34]. Trib3 is considered to be a target of CHOP/ATF4 and works as a sensor for ER stress-induced apoptosis [14]. If the ER stress is transient and mild, the induced Trib3 blocks the CHOP and ATF4 function via a negative feedback mechanism and represses apoptosis. However, when the ER stress is potent and prolonged, excess Trib3 will be produced and lead to apoptosis by dephosphorylating Akt and inducing the translocation of Bax to mitochondria, which accelerates programmed cell death. Trib3 is also involved in apoptosis via Caspase 3 (CASP3) [35]. Caspases play a central role in the execution-phase of apoptosis, and Trib3 prevents the activation of CASP3 by the nuclear translocation of proCASP3. However, in conditions of prolonged stress, Trib3 is cleaved by CASP3 and could be a trigger for the further activation of caspases and induce apoptosis. This indicates that Trib3 functions as a molecular switch between the cell survival and apoptotic pathways under stressful conditions. Aime et al. considered that an elevated expression of Trib3 may enhance the proapoptotic activities and be related to the promotion of neuronal death in PD and its models [27]. Using a TUNEL assay and electron microscopy, we did not observe any apparent increased apoptosis in the spinal cord of the dmy rats at 7 weeks of age. However, dmy rats exhibit severe myelin destruction throughout the  Enhanced Trib3 Expression in Myelin Mutant dmy Rats white matter of the CNS during the late stage of myelination, and it might thus be difficult to detect apoptotic cells. Further research is needed to understand the precise role of increased Trib3 expression in the dmy rat.