Bruce S. Kristal

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.

Metabolomics: A Global Biochemical Approach to Drug Response and Disease

Metabolomics is the study of metabolism at the global level. This rapidly developing new discipline has important potential implications for pharmacologic science. The concept that metabolic state is representative of the overall physiologic status of the organism lies at the heart of metabolomics. Metabolomic studies capture global biochemical events by assaying thousands of small molecules in cells, tissues, organs, or biological fluids-followed by the application of informatic techniques to define metabolomic signatures. Metabolomic studies can lead to enhanced understanding of disease mechanisms and to new diagnostic markers as well as enhanced understanding of mechanisms for drug or xenobiotic effect and increased ability to predict individual variation in drug response phenotypes (pharmacometabolomics). This review outlines the conceptual basis for metabolomics as well as analytical and informatic techniques used to study the metabolome and to define metabolomic signatures. It also highlights potential metabolomic applications to pharmacology and clinical pharmacology.

Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease

Minocycline is broadly protective in neurologic disease models featuring cell death and is being evaluated in clinical trials. We previously demonstrated that minocycline-mediated protection against caspase-dependent cell death related to its ability to prevent mitochondrial cytochrome c release. These results do not explain whether or how minocycline protects against caspase-independent cell death. Furthermore, there is no information on whether Smac/Diablo or apoptosis-inducing factor might play a role in chronic neurodegeneration. In a striatal cell model of Huntingtons disease and in R6/2 mice, we demonstrate the association of cell death/disease progression with the recruitment of mitochondrial caspase-independent (apoptosis-inducing factor) and caspase-dependent (Smac/Diablo and cytochrome c) triggers. We show that minocycline is a drug that directly inhibits both caspase-independent and -dependent mitochondrial cell death pathways. Furthermore, this report demonstrates recruitment of Smac/Diablo and apoptosis-inducing factor in chronic neurodegeneration. Our results further delineate the mechanism by which minocycline mediates its remarkably broad neuroprotective effects.

Mass-spectrometry-based metabolomics: limitations and recommendations for future progress with particular focus on nutrition research

Mass spectrometry (MS) techniques, because of their sensitivity and selectivity, have become methods of choice to characterize the human metabolome and MS-based metabolomics is increasingly used to characterize the complex metabolic effects of nutrients or foods. However progress is still hampered by many unsolved problems and most notably the lack of well established and standardized methods or procedures, and the difficulties still met in the identification of the metabolites influenced by a given nutritional intervention. The purpose of this paper is to review the main obstacles limiting progress and to make recommendations to overcome them. Propositions are made to improve the mode of collection and preparation of biological samples, the coverage and quality of mass spectrometry analyses, the extraction and exploitation of the raw data, the identification of the metabolites and the biological interpretation of the results.

Proposed minimum reporting standards for data analysis in metabolomics

The goal of this group is to define the reporting requirements associated with the statistical analysis (including univariate, multivariate, informatics, machine learning etc.) of metabolite data with respect to other measured/collected experimental data (often called meta-data). These definitions will embrace as many aspects of a complete metabolomics study as possible at this time. In chronological order this will include: Experimental Design, both in terms of sample collection/matching, and data acquisition scheduling of samples through whichever spectroscopic technology used; Deconvolution (if required); Pre-processing, for example, data cleaning, outlier detection, row/column scaling, or other transformations; Definition and parameterization of subsequent visualizations and Statistical/Machine learning Methods applied to the dataset; If required, a clear definition of the Model Validation Scheme used (including how data are split into training/validation/test sets); Formal indication on whether the data analysis has been Independently Tested (either by experimental reproduction, or blind hold out test set). Finally, data interpretation and the visual representations and hypotheses obtained from the data analyses.

The metabolomics standards initiative (MSI)

In 2005, the Metabolomics Standards Initiative has been formed. An outline and general introduction is provided to inform about the history, structure, working plan and intentions of this initiative. Comments on any of the suggested minimal reporting standards are welcome to be sent to the open email list Msi-workgroups-feedback@lists.sourceforge.net

Mitochondrial Permeability Transition in the Central Nervous System: Induction by Calcium Cycling‐Dependent and ‐Independent Pathways

Abstract: Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg2+ and reduced by cyclosporin A, dithiothreitol, and N‐ethylmaleimide. Ruthenium red prevented calcium cycling‐induced, but only attenuated phosphate‐induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4‐bromo‐A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC‐1, from rod‐like to rounded, swollen structures. Similar changes were observed in digitonin‐permeabilized astrocytes exposed to phosphate. The incidence of calcium‐induced changes in astrocyte mitochondria was prevented by Mg2+ and pretreatment with dithiothreitol and N‐ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na+/Ca2+ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin‐treated, but not ionophore‐treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling‐dependent and ‐independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.

The Metabolomics Standards Initiative

In 2005, the Metabolomics Standards Initiative has been formed. An outline and general introduction is provided to inform about the history, structure, working plan and intentions of this initiative. Comments on any of the suggested minimal reporting standards are welcome to be sent to the open email list Msi-workgroups-feedback@lists.sourceforge.net

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two‐dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor‐intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simulataneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high‐throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimers disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.

Chemotherapy for the Brain: The Antitumor Antibiotic Mithramycin Prolongs Survival in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.