A translational continuum of model systems for evaluating treatment strategies in Alzheimer's disease: isradipine as a candidate drug.

A growing body of evidence supports the 'calcium hypothesis' of Alzheimer's disease (AD), which postulates that a variety of insults might disrupt the homeostatic regulation of neuronal calcium (Ca(2+)) in the brain, resulting in the progressive symptoms that typify the disease. However, despite ongoing efforts to develop new methods for testing therapeutic compounds that might be beneficial in AD, no single bioassay permits both rapid screening and in vivo validation of candidate drugs that target specific components of the Ca(2+) regulatory machinery. To address this issue, we have integrated four distinct model systems that provide complementary information about a trial compound: the human neuroblastoma MC65 line, which provides an in vitro model of amyloid toxicity; a transgenic Drosophila model, which develops age-dependent pathologies associated with AD; the 3×TgAD transgenic mouse, which recapitulates many of the neuropathological features that typify AD; and the embryonic nervous system of Manduca, which provides a novel in vivo assay for the acute effects of amyloid peptides on neuronal motility.

MR imaging of inflammation during myelin-specific T cell-mediated autoimmune attack in the EAE mouse spinal cord.

Purpose: The purpose of this study is to detect myelin-specific T cells, key pathological mediators in early multiple sclerosis, and the corresponding animal model, experimental autoimmune encephalomyelitis (EAE), in the mouse spinal cord.

Procedures: T cells were labeled with the iron-based, magnetic resonance (MR) contrast reagent, Feridex, and the transfection reagent, protamine sulfate, resulting in approximately 100% iron-labeling efficiency. Feridex-labeling did not alter the induction of EAE by T cells, and recipients were imaged by a 12-T MR instrument.

Bone marrow lacking integrin expression facilitates an enhanced susceptibility to EAE in the xenogeneic bone marrow chimeras.

Xenotransplantation of rat bone marrow cells (BMC) into immunodeficient (SCID) mice generates chimeric mice susceptible to paralytic autoimmune CNS inflammation. Herein, we identified a disease relevant subset of transplantable BMC lacking expression of CD11b/c and CD49d. Moreover, disease susceptibility was enhanced in the presence of non-myelin specific T-cells.

The selective detection of mitochondrial superoxide by live cell imaging.

A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine (HE) or its mitochondrially targeted derivative Mito-HE (MitoSOX Red). Two different excitation wavelengths are used to distinguish the superoxide-dependent hydroxylation of Mito-HE (385-405 nm) from the nonspecific formation of ethidium (480-520 nm). Furthermore, the dual wavelength imaging in live cells can be combined with immunocolocalization, which allows superoxide formation to be compared simultaneously in cocultures of two types of genetically manipulated cells in the same microscopic field.

Mitochondrial dysfunction in SOD1G93A-bearing astrocytes promotes motor neuron degeneration: prevention by mitochondrial-targeted antioxidants.

Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons.

Mitochondrial superoxide production and nuclear factor erythroid 2-related factor 2 activation in p75 neurotrophin receptor-induced motor neuron apoptosis.

Nerve growth factor (NGF) can induce apoptosis by signaling through the p75 neurotrophin receptor (p75(NTR)) in several nerve cell populations. Cultured embryonic motor neurons expressing p75(NTR) are not vulnerable to NGF unless they are exposed to an exogenous flux of nitric oxide (*NO). In the present study, we show that p75(NTR)-mediated apoptosis in motor neurons involved neutral sphingomyelinase activation, increased mitochondrial superoxide production, and cytochrome c release to the cytosol.

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures.

Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons.

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations.

Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular processes that can oxidize HE probes to ethidium (Etd).

Synthesis of peroxynitrite from nitrite and hydrogen peroxide.

We report a simple method for the synthesis of peroxynitrite from nitrite and hydrogen peroxide that can generate hundreds of milliliters of 180 mM peroxynitrite within 1 h from start to finish. It can be scaled down to make small quantities of isotope-labeled peroxynitrite. The method requires only a syringe pump and tubing connectors and is feasible for any biochemical laboratory.

Triuret: a novel product of peroxynitrite-mediated oxidation of urate.

Urate is an efficient antioxidant and has recently emerged as a competitive inhibitor of tyrosine nitration by peroxynitrite. In vivo and in vitro studies demonstrate the large extent to which urate prevents nitration and establish the biological importance of the reaction between urate and peroxynitrite. The existing lack of characterization of this reaction has led us to focus our studies upon the mechanism of urate oxidation and the products formed.