Expression and Localization of Mitochondrial Ferritin mRNA in Alzheimer's Disease Cerebral Cortex

Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and β-amyloid peptide (Aβ) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with Aβ, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of Aβ and H2O2, but not with Aβ alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress

Immunohistochemical Localization of an Isoform of TRK-Fused Gene-Like Protein in the Rat Retina

The TRK-fused gene (TFG) was originally identified in chromosome translocation events, creating a pair of oncogenes in some cancers, and was recently demonstrated as the causal gene of hereditary motor and sensory neuropathy with proximal dominant involvement. Recently, we cloned an alternative splicing variant of Tfg from a cDNA library of the rat retina, tentatively naming it retinal Tfg (rTfg). Although the common form of Tfg is ubiquitously expressed in most rat tissues, rTfg expression is localized to the central nervous system. In this study, we produced an antibody against an rTFG-specific amino acid sequence and used it to examine the localization of rTFG-like protein in the rat retina by immunohistochemistry and Western blots. Western blot analysis showed that the antibody detected a single band of 24 kDa in the rat retina. When we examined rTFG recombinant protein, the antibody detected two bands of about 42 kDa and 24 kDa. The results suggest that the 24 kDa rTFG-like protein is a fragment of rTFG. In our immunohistochemical studies of the rat retina, rTFG-like immunoreactivity was observed in all calbindin D-28K-positive horizontal cells and in some syntaxin 1-positive amacrine cells (ACs). In addition, the rTFG-like immunopositive ACs were actually glycine transporter 1-positive glycinergic or glutamate decarboxylase-positive GABAergic ACs. Our findings indicate that this novel 24 kDa rTFG-like protein may play a specific role in retinal inhibitory interneurons

Effect of MtF expression on cell viability after treatment with H₂O₂.

<p>An MtF protein band exists with an apparent molecular mass of 22 kDa on SDS-PAGE; weak bands were detected in IMR-32 and vector-IMR-32 cells (B). Wild-type IMR-32 cells, empty vector transfectants (Vector-IMR-32), and MtF transfectants (MtF-IMR-32) were treated with 300 µM H₂O₂ for 30 min (A). Cell viability was measured by MTT assay. A remarkable decrease in the viability of IMR-32 and Vector- IMR-32 cells (about 50%; p<0.01, compared with control groups) was observed after treatment with 300 µM H2O₂ for 30 min (A). The viability of MtF- IMR-32 cells under treatment decreased about 30%, however, the cell viability was much higher than in the control group (p<0.05). <i>** p<0.01</i> vs. non-treated cells; <i>* p<0.05</i> vs. the H₂O₂-treated control cells.</p

Western-blot analysis of MtF expression in human brain.

<p>(A) Three AD cases (lanes marked A) and three control cases (lanes marked C) were probed with anti β-actin antibody and anti MtF antibody. (B) When the expression level of MtF is normalized to β-actin, the expression level of MtF in the temporal cortex of AD patients is higher than in control cases (**<i>p<0.01</i>).</p

Mitochondrial ferritin mRNA expression levels in cultured cells.

<p>(A) Compared to the control, the MtF mRNA expression level is significantly high after H2O2 treatment but not after treatment with Aβ or DMSO. The group treated with both of H2O2 and Aβ displays the highest level of MtF mRNA expression among all groups. Asterisks and double asterisks indicate <i>p</i><0.05 and <i>p</i><0.01, respectively. (B) Results of real-time PCR analysis of MtF mRNA using total RNA from cultured cells in the peroxynitrite treatment group and the control groups. The MtF mRNA expression level was not changed significantly by peroxynitrite treatment.</p

Western-blot analysis of MtF expression in each <i>in vitro</i> experiment group.

<p>(A) An immunoreactive band of approximately 22 kD probed with an anti-MtF antibody is present in all lanes. (B) When the expression level of MtF is normalized to β-actin, MtF expression after treatment with H₂O₂ or with a combination of Aβ and H₂O₂ was significantly greater than that in control cases (**<i>p<0.01</i>. <i>*** p<0.001</i>). However, treatment with Aβ or DMSO alone shows no significant effect on MtF expression.</p

Formation of oligomeric Aβ1–42 assemblies.

<p>(A) Profiles of Aβ1–42 oligomers before (0 h) and after 24-h incubation (24 h) on SDS-PAGE followed by silver staining. M: Molecular weight markers. 0 h: Aβ1–42 preparation 0 h at 4°C. 24 h: Initial ADDL preparation 24 h later at 4°C. (B) Electrophoretic pattern of Aβ1–42 oligomeric preparations separated with centrifugal filters before electrophoresis. Representative photographs showing Aβ1–42 preparations before (0 h) and after incubation (24 h). Arrowheads in B indicate Aβ1–42 oligomers. Scale bar: 50 nm.</p

<i>In situ</i> hybridization histochemistry of the cerebral cortex of control (A–C) and Alzheimer's disease (AD) cases (D–F) using antisense (A, B, D, E) and sense (C, F) probes.

<p>Mitochondrial ferritin mRNA localizes mainly in neurons. Both the number and intensity of positive neurons increase in AD cases (D) compared to controls (A). Using sense probes (C and F), no signals are detected in the cortex. Bars  = 200 µm in A, C, D, F, and 50 µm in B, E.</p