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

Immunohistochemistry for Golgi 58K.

<p>The immunohistochemistry for Golgi 58K in the lumbar spinal cords from the <i>dmy</i> rats at 4, 6, 7 and 8 weeks of age. The immunoreactivity for the Golgi apparatus is elevated in the ventral funiculus of the <i>dmy</i> rats at 6–8 weeks of age. Bars: 20 μm.</p

Real-time PCR for <i>Trib3</i> (VF, <i>mv</i> and <i>dmy</i> rats).

<p>Relative expression of <i>Trib3</i> in the white matter (A) and the gray matter (B) of the cervical spinal cords from the <i>dmy</i>, <i>mv</i> and VF rats by the real-time PCR. The data are presented as the mean ratio against expression in the control rats. Although expression levels of <i>Trib3</i> mRNA is significantly increased in the <i>dmy</i> rats, no significant differences are seen in the VF and <i>mv</i> rats. *, <i>p</i> <0.05, **, <i>p</i> <0.01, Tukey-Kramer test (<i>n</i> = 3 in VF and <i>dmy</i> rats. <i>n</i> = 2 in <i>mv</i> rats.).</p

Immunohistochemistry for TRIB3 in the <i>dmy</i> rat.

<p>The immunohistochemistry for TRIB3 in the lumbar spinal cords from <i>dmy</i> rats at 7-week-old. Cytoplasm of neurons and glial cells are stained with TRIB3 in the gray (A) and white matter (B) of the <i>dmy</i> rats. Bars: 20 μm.</p

Real-time PCR for <i>Trib3</i>.

<p>Expression of <i>Trib3</i> gene in the ventral funiculus in the <i>dmy</i> rats (A), gray matter (B) and dorsal funiculus (C) of the cervical spinal cords from the control (+/+ and <i>dmy</i>/+) and the <i>dmy</i> (<i>dmy</i>/<i>dmy</i>) rats. The data are presented as the mean ratio of target to reference gene. β-actin is used as an internal control. In the <i>dmy</i> rats, expression levels of <i>Trib3</i> mRNA is significantly increased in all areas at 4, 5, 6, 7 and 8 weeks of age. *, <i>p</i> <0.05, **, <i>p</i> <0.01, Tukey-Kramer test (<i>n</i> = 3 in each group).</p