Satinder S. Sarang

Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice

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.

Ascorbic Acid Enhances Differentiation of Embryonic Stem Cells Into Cardiac Myocytes

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 &agr;-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 &agr;-actinin. Furthermore, ascorbic acid induced the expression of cardiac genes, including GATA4, &agr;-MHC, and &bgr;-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 acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells.

Alzheimer’s disease drug discovery targeted to the APP mRNA 5′Untranslated region

We performed a screen for drugs that specifically interact with the 5′ untranslated region of the mRNA coding for the Alzheimer’s Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5′UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5′UTR-directed luciferase expression (>95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involed in lipid metabolism, and metal chelators. These APP 5′UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and Aβ peptide output for Alzheimer’s disease therapeutics.

Identification, Coassembly, and Activity of γ-Aminobutyric Acid Receptor Subunits in Renal Proximal Tubular Cells

Although the properties and functions of GABAA receptors in the mammalian central nervous system have been well studied, the presence and significance of GABAA receptors in non-neural tissues are less clear. The goal of this study was to examine the expression of GABAA receptor α1, α2, α4, α5, β1, γ1, γ2, and δ subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABAA receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the α1, α5, β1, γ1, and γ2 subunits and that they were similar to the neuronal subunits. Sequencing of the PCR products revealed that the rabbit kidney cortex contained the α1 and γ2 subunits and that they were similar to their neuronal counterparts. Immunoprecipitation and immunoblot studies using GABAA receptor subunit-specific antibodies and detergent-solubilized rat kidney cortex membranes identified a GABAA receptor complex containing α5, β1, and γ1. Isolated rat renal proximal tubular cells exhibited GABA-mediated, picrotoxin-sensitive 36Cl- uptake. These studies demonstrate the presence of numerous GABAA receptor subunits in the kidneys of two species, the assembly of the subunits into at least one novel receptor complex, and an active GABAA receptor in renal proximal tubular cells.