Molecular Analysis of Homocysteic Acid-Induced Neuronal Stress

Abstract

Hyperhomocysteinemia is a risk factor for vascular and neuronal lesions often observed with concomitant high levels of homocysteic acid. In contrast to homocysteine, homocysteic acid induces calcium influx into neurons, with characteristics of an excitotoxic glutamatergic agonist at elevated concentrations. On the molecular level this is correlated to fast modifications of proteins (phosphorylation and proteolysis). Within the homocysteic acid induced molecular signature we focused in more detail on phosphorylation of two proteins implicated as risk factors in schizophrenia and neurodegeneration: Dihydropyrimidinase related protein and 14-3-3 protein isoforms. Among the identified proteins there are known chaperones and oxidative metabolism enzymes, but a few are new in context of neuronal stress: Lasp-1, a vitamin D associated factor and an expressed sequence with features of a Rho GDP dissociation inhibitor. Moreover, we detect a specific proteolytic processing of heat shock protein 70 and proteindisulfide isomerase, which is abolished by vitamins (folic acid, vitamin B12, and vitamin B6), which also decrease elevated intracellular calcium levels induced by homocysteic acid.

Investigating the neuroprotective mechanism of action of a CDK5 inhibitor by phosphoproteome analysis

“…An increase in tyrosine-phosphorylated protein spots following Indolinone A administration was also demonstrated using an anti-phosphotyrosine antibody. Additionally, phosphorylation of dihydropyrimidinase related proteins was confirmed in a parallel study by MS/MS analysis [Sommer et al, 2004]. It should be mentioned however, that other methods for phosphoproteome analysis have been developed, which may show higher sensitivity and accuracy [Mann et al, 2002].…”

“…Subsequent analysis was performed at ProteoSys AG (Mainz, Germany) as has been described in detail elsewhere [Sommer et al, 2004;Wang et al, 2004]. Phosphoproteomes were isolated by Fe-agarose columns equilibrated with binding buffer (50 mM bis-(hydroxyethyl)-piperazine-HCl, pH 3.4, 2% Triton X-100) [Muszynska et al, 1986].…”

Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model

“…These modifications can occur multiply, thus creating the often enormous complexity of protein biomarker signatures under a given physiological condition [1]. We have shown previously that neural derivatives of stem cells are an appropriate model to correlate the functional and molecular parameters [2,3], using Ca-imaging to quantify functional responses to excitotoxic concentrations of homocysteic acid and a subsequent differential Proteomics approach, to understand molecular details on the level of fast posttranslational modifications of proteins. Here we use a similar approach to investigate chemical ischemia.…”

“…Moreover, we correlate the molecular level to changes of intracellular calcium concentrations on the functional level, because many stress responses are mediated by calcium in connection with reversible phosphorylation [6][7][8][9]. Neural embryonic stem cells provide a suitable model, integrating differential molecular analysis and a functional paradigm for hypoxic stress [2,3]. Among the proteins identified, some are known to be responsive to oxidative stress, like isoforms of superoxide dismutase, chaperonin, heat shock protein 47 and acidic calcium-independent phospholipase A2, which thus are part of the immediate molecular response to chemically induced ischemia in cell culture [10,11].…”

Stammzellbasierte In-vitro-Modelle als Ersatz für Tiermodelle bei Toxizitäts- und Wirksamkeitsprüfungen

“…Abbildung 2 zeigt, dass aus mESC differenzierte Neurone (mESC-Neurone) auf die Stimulation mit einer niedrigen Glutamatkonzentration (10 μM) mit einem deutlichen Kalziumeinstrom reagieren, der -wie bereits früher gezeigt wurde [10,11] -im Wesentlichen von NMDA-Rezeptoren (N-Methyl-D-Aspartat-Rezeptoren) getragen wird. Dies zeigt die Vitalität der Zellen.…”