PICK1 expression in reactive astrocytes within the spinal cord of amyotrophic lateral sclerosis (ALS) rats.

AIMS: The protein interacting with C kinase 1 (PICK1), a PDZ domain-containing protein mainly expressed in the central nervous system, interacts with the glutamate receptor subunit GluR2, with the glutamate transporter GLT-1b and with the enzyme serine racemase. These three proteins appear as key actors in the glutamate-mediated excitotoxicity associated with amyotrophic lateral sclerosis (ALS), in both patients and animal models of the disease. In this study, we examined the expression of PICK1 in the spinal cord of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A) ) during the progression of the disease. METHODS: Expression of PICK1 was examined by real-time qPCR at presymptomatic and symptomatic stages as well as at end-stage. The expression of PICK1 in the different cell types of the spinal cord was examined by immunohistochemistry. RESULTS: The overall expression of PICK1 is not modified in cervical and lumbar spinal cord of transgenic (hSOD1(G93A) ) rats during the progression of the disease. Nonetheless, immunohistochemical studies of lumbar ventral horns revealed a shift of PICK1 expression from motor neurones in healthy rats to activated astrocytes in end-stage hSOD1(G93A) animals. CONCLUSIONS: Considering the documented influence of PICK1 expression on d-serine release and glutamate transport in astrocytes, these findings point to a potential implication of PICK1 in the progression of ALS.

Opposite regulation of metabotropic glutamate receptor 3 and metabotropic glutamate receptor 5 by inflammatory stimuli in cultured microglia and astrocytes.

Metabotropic glutamate receptors (mGluRs) were previously shown to modulate several essential functions in glial cells, including cell proliferation, glutamate uptake, neurotrophic support, and inflammatory responses. As these receptors are regularly proposed as promising targets for the treatment of a wide range of neurological disorders, we herein examined the reciprocal modulation of glial mGluRs by inflammation. Such regulation of mGluRs was also studied in cultures from an experimental model of amyotrophic lateral sclerosis (ALS). Indeed, ALS is characterized by increased neuroinflammation, and glial cell cultures derived from the animal model (rat expressing hSOD1(G93A)) show enhanced glial reactivity. Within 72 h, the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1ß (IL-1ß) induced an increase in mGluR3 and a decrease in mGluR5 gene expression. A similar regulation of these receptors was observed in microglia 48 h after an initial 4-h exposure to lipopolysaccharide. In hSOD1(G93A)-derived glial cultures, the gene up-regulation of mGluR3 (but not the gene down-regulation of mGluR5) was found to be enhanced in both astrocytes and microglia. Together, these results indicate that an inflammatory environment triggers an opposite regulation in the gene expression of the two predominant mGluR subtypes found in glial cells, and that these regulations were particularly robust in hSOD1(G93A) glial cultures. As neuroinflammation commonly occurs in several nervous diseases, its influence on mGluR expression should be taken into account when considering these receptors as future drug targets.

The polypeptide PHI discriminates a GTP-insensitive form of VIP receptor in liver membranes.

In early reports on 125I-VIP binding experiments in liver membranes, it has been proposed that, the VIP binding sites were partially sensitive to GTP. Here we confirm that the VIP binding sites of chicken liver membranes consisted mainly in bivalent VIP/PACAP receptors and that about 50% of the 125I-VIP binding capacity was not affected by the GTP analogue GppNHp. Part of these bivalent receptors also appeared to represent PHI binding sites. In GppNHp-treated membranes, the GTP-insensitive VIP binding sites displayed a 17-fold higher relative affinity than in control membranes for the VIP analogue PHI. Such data suggested that GTP-insensitive VIP receptors may correspond to a subclass of high-affinity PHI receptors. Cross-linking of 125 I-VIP or 125 I-PHI to their receptors, revealed 2 components of 48 and 60 kDa. The radiolabelling of the 60 kDa component was strongly affected by increasing concentrations of the GTP analogue but was modestly abolished by an excess of PHI. Conversely, the radiolabelling of the 48 kDa molecular form was not affected by the GTP analogue but was efficiently abolished by increasing concentrations of PHI. Taken together, the data suggest that the 48 kDa component expressed in chicken liver membranes display the properties of a GTP-insensitive VIP/PHI receptor that can be pharmacologically discriminated from the GTP-sensitive 60 kDa form, through its much higher affinity for PHI.

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry.

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.

Identification of phenolic acids and inositols in balms and tissues from an Egyptian mummy.

A number of samples taken from an Egyptian mummy (ca. 100 B.C.) from the Guimet Museum in Lyon have been analyzed by GC-MS. Derivatives of aromatic acids (hydroxyhydrocinnamic, vanillic, protocatechuic and gallic acids) and inositols (non-methylated and mono-O-methyl) have been found among the constituents of extracts prepared by methanolysis and trimethylsilylation. From the reported electron impact mass spectra, ion sets where proposed for a sensitive and selective profiling of these selected compounds by mass fragmentometry. The source of gallic acid and inositol was found to be a vegetable tannin, an ingredient which was not previously known to be used for mummification in ancient Egypt. The nature and abundance ratios of the detected inositols also appeared to be a promising criterion to further investigate the botanical source of the tannin employed.