Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood-brain barrier, this drug may be a novel therapy for ALS.

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5-untranslated region (5-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (؉51 to ؉94 from the 5-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (؉101 to ؉146). APP 5-UTR conferred translation was selectively downregulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (⌬4 bases:⌬83AGAG86). Iron regulation of APP mRNA through the APP 5-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce A␤ peptide burden during Alzheimer's disease. The amyloid precursor protein (APP)1 is cleaved into the 40 -42-amino acid A␤ peptides that constitute the main component of the neurotoxic amyloid plaques formed during the progression of Alzheimer's disease (AD) and Down's syndrome (1, 2). In healthy individuals, APP holoprotein is expressed ubiquitously as a protein resembling a type I transmembrane receptor and metal-binding protein (3-6). Secreted APP (APP(s)) is neurotrophic (7).There are now several reports supporting an important role for translational regulatory mechanisms to control APP synthesis and probably A␤ peptide secretion in biologically relevant circumstances (8). First, interleukin-1 (IL-1), the first cytokine released during the acute phase response, significantly increases APP protein synthesis in astrocytes without altering APP mRNA levels (9). IL-1 acts by regulating APP and ferritin genes at the level of message translation (9). Second, reversible ischemic assault significantly increases APP levels without any alteration in the steady-state levels of APP mRNA in rabbit spinal cord neurons (10). Third, APP mRNA 3Ј-UTR sequences located between alternative poly(A) selection sites maintain efficient translation of microinjected APP in Xenopus oocytes and in Chinese hamster ovary transfectants (11).Iron-responsive elements (IREs) are RNA stem loops...

Background-Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions, ES cells can differentiate into specialized cells, including cardiac myocytes, but the efficiency is typically low and the process is incompletely understood. Methods and Results-We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific ␣-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP), 880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid, also known as vitamin C, markedly increased the number of EGFP-positive cells, which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and ␣-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, ␣-MHC, and ␤-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine, Tiron, or vitamin E. Conclusions-Ascorbic