An integrated high-performance liquid chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases.

Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 microL crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.

Chronic social stress induces peripheral and central immune activation, blunted mesolimbic dopamine function, and reduced reward-directed behaviour in mice.

Psychosocial stress is a major risk factor for depression, stress leads to peripheral and central immune activation, immune activation is associated with blunted dopamine (DA) neural function, DA function underlies reward interest, and reduced reward interest is a core symptom of depression. These states might be inter-independent in a complex causal pathway. Whilst animal-model evidence exists for some specific steps in the pathway, there is currently no animal model in which it has been demonstrated that social stress leads to each of these immune, neural and behavioural states. Such a model would provide important existential evidence for the complex pathway and would enable the study of causality and mediating mechanisms at specific steps in the pathway. Therefore, in the present mouse study we investigated for effects of 15-day resident-intruder chronic social stress (CSS) on each of these states. Relative to controls, CSS mice exhibited higher spleen levels of granulocytes, inflammatory monocytes and T helper 17 cells; plasma levels of inducible nitric oxide synthase; and liver expression of genes encoding kynurenine pathway enzymes. CSS led in the ventral tegmental area to higher levels of kynurenine and the microglia markers Iba1 and Cd11b and higher binding activity of DA D1 receptor; and in the nucleus accumbens (NAcc) to higher kynurenine, lower DA turnover and lower c-fos expression. Pharmacological challenge with DA reuptake inhibitor identified attenuation of DA stimulatory effects on locomotor activity and NAcc c-fos expression in CSS mice. In behavioural tests of operant responding for sucrose reward validated as sensitive assays for NAcc DA function, CSS mice exhibited less reward-directed behaviour. Therefore, this mouse study demonstrates that a chronic social stressor leads to changes in each of the immune, neural and behavioural states proposed to mediate between stress and disruption of DA-dependent reward processing. The model can now be applied to investigate causality and, if demonstrated, underlying mechanisms in specific steps of this immune-neural-behavioural pathway, and thereby to identify potential therapeutic targets.

Depletion of high-abundance proteins from serum by immunoaffinity chromatography: a MALDI-FT-MS study.

Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease.

Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.