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

BNP signaling is crucial for embryonic stem cell proliferation.

Embryonic stem (ES) cells have unlimited proliferation potential, and can differentiate into several cell types, which represent ideal sources for cell-based therapy. This high-level proliferative ability is attributed to an unusual type of cell cycle. The Signaling pathways that regulate the proliferation of ES cells are of great interest.In this study, we show that murine ES cells specifically express brain natriuretic peptide (BNP), and its signaling is essential for ES cell proliferation. We found that BNP and its receptor (NPR-A, natriuretic peptide receptor-A) were highly expressed in self-renewing murine ES cells, whereas the levels were markedly reduced after ES cell differentiation by the withdrawal of LIF. Targeting of BNP with short interfering RNA (siRNA) resulted in the inhibition of ES cell proliferation, as indicated by a marked reduction in the cell number and colony size, a significant reduction in DNA synthesis, and decreased numbers of cells in S phase. BNP knockdown in ES cells led to the up-regulation of gamma-aminobutyric acid receptor A (GABA(A)R) genes, and activation of phosphorylated histone (gamma-H2AX), which negatively affects ES cell proliferation. In addition, knockdown of BNP increased the rate of apoptosis and reduced the expression of the transcription factor Ets-1.Appropriate BNP expression is essential for the maintenance of ES cell propagation. These findings establish BNP as a novel endogenous regulator of ES cell proliferation

Fourier-Transform Infrared Imaging Spectroscopy and Laser Ablation -ICPMS New Vistas for Biochemical Analyses of Ischemic Stroke in Rat Brain

Objective: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Understanding the post-stroke biochemical and molecular changes are critical for patient survival and stroke management. The aim of this work was to investigate the photo-thrombotic ischemic stroke in male rats with particular focus on biochemical and elemental changes in the primary stroke lesion in the somatosensory cortex and surrounding areas, including the corpus callosum.Materials and Methods: FT-IR imaging spectroscopy and LA-ICPMS techniques examined stroke brain samples, which were compared with standard immunohistochemistry studies.Results: The FTIR results revealed that in the lesioned gray matter the relative distribution of lipid, lipid acyl and protein contents decreased significantly. Also at this locus, there was a significant increase in aggregated protein as detected by high-levels Aβ1-42. Areas close to the stroke focus experienced decrease in the lipid and lipid acyl contents associated with an increase in lipid ester, olefin, and methyl bio-contents with a novel finding of Aβ1-42 in the PL-GM and L-WM. Elemental analyses realized major changes in the different brain structures that may underscore functionality.Conclusion: In conclusion, FTIR bio-spectroscopy is a non-destructive, rapid, and a refined technique to characterize oxidative stress markers associated with lipid degradation and protein denaturation not characterized by routine approaches. This technique may expedite research into stroke and offer new approaches for neurodegenerative disorders. The results suggest that a good therapeutic strategy should include a mechanism that provides protective effect from brain swelling (edema) and neurotoxicity by scavenging the lipid peroxidation end products

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

<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

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