The complex lipid constituents of the eukaryotic plasma membrane are precisely controlled in a cell-type-specific manner, suggesting an important, but as yet, unknown cellular function. Neuronal membranes are enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and alterations in LC-PUFA metabolism cause debilitating neuronal pathologies. However, the physiological role of LC-PUFAs in neurons is unknown. We have characterized the neuronal phenotype of C. elegans mutants depleted of LC-PUFAs. The C. elegans genome encodes a single Δ6-desaturase gene (fat-3), an essential enzyme for LC-PUFA biosynthesis. Animals lacking fat-3 function do not synthesize LC-PUFAs and show movement and egg-laying abnormalities associated with neuronal impairment. Expression of functional fat-3 in neurons, or application of exogenous LC-PUFAs to adult animals rescues these defects. Pharmacological, ultrastructural and electrophysiological analyses demonstrate that fat-3 mutant animals are depleted of synaptic vesicles and release abnormally low levels of neurotransmitter at cholinergic and serotonergic neuromuscular junctions. These data indicate that LC-PUFAs are essential for efficient neurotransmission in C. elegans and may account for the clinical conditions associated with mis-regulation of LC-PUFAs in humans.
Mutations in the human presenilin genes cause the most frequent and aggressive forms of familial Alzheimer's disease (FAD). Here we show that in addition to its role in cell fate decisions in non-neuronal tissues, presenilin activity is required in terminally differentiated neurons in vivo. Mutations in the Caenorhabditis elegans presenilin genes sel-12 and hop-1 result in a defect in the temperature memory of the animals. This defect is caused by the loss of presenilin function in two cholinergic interneurons that display neurite morphology defects in presenilin mutants. The morphology and function of the affected neurons in sel-12 mutant animals can be restored by expressing sel-12 only in these cells. The wild-type human presenilin PS1, but not the FAD mutant PS1 A246E, can also rescue these morphological defects. As lin-12 mutant animals display similar morphological and functional defects to presenilin mutants, we suggest that presenilins mediate their activity in postmitotic neurons by facilitating Notch signalling. These data indicate cell-autonomous and evolutionarily conserved control of neural morphology and function by presenilins.
The gene for a novel extracellular metalioprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amnmo acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-arol region of the chromosome and was not required for growth or sporulation.The gram-positive, spore-forming bacterium Bacillus subtilis produces and secretes proteases, esterases, and other types of exoenzymes at the end of the exponential phase of growth (15). The principal extracellular proteolytic enzymes, alkaline (subtilisin) and neutral (metallo-) proteases, are encoded by the apr and npr genes, respectively (11,24,26,29). In addition, the genes for two minor extracellular proteases, epr (21) and bpr (encoding bacillopeptidase F; A. Sloma, G. A. Rufo, Jr., C. F. Rudolph, B. J. Sullivan, K. A. Theriault, and J. Pero, submitted for publication) have been identified. Strains bearing null mutations in these four protease genes and the major intracellular protease gene isp-i (12) still produce extracellular protease activity. The majority of the residual protease activity can be attributed to a novel cysteine-containing metalloprotease (Mpr) that has been purified to apparent homogeneity (17). Here we report the cloning of the gene encoding this cysteine-containing protease. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis strains are listed in Table 1. Plasmid pBs81/6 is a derivative of pBD64 (8). Plasmids pBR322 and pUC19 were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. The cat gene was obtained from plasmid pMI1101 (30), and the ble gene was obtained from pUB110 (20). B. subtilis strains were grown on tryptose blood agar base or minimal glucose medium and were made competent by the procedure of Anagnostopoulos and Spizizen (1). Selection for phleomycin resistance was carried out on tryptose blood agar base by the overlayer method after a 1.5-h delay at 37°C to allow for expression.The final concentration of phleomycin was 2 jig/ml. Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Birnboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. Amino acid sequence determination. The N-terminal amino acid sequence of purified Mpr was determined by automated sequential Edman degradation with subsequent identification and quantification of phenylthiohydantoin-labeled amino acids by reverse-phase high-performance liquid chromatography. Additional amino acid sequences were obtained from internal fragments of Mpr generated by trypsin digestion. The purified enzyme was incubated with trypsin, and t...
ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor-activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3-K/Akt) and the mitogen-activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor-mediated astrogliosis after traumatic injury and after microinfusion of ADP beta S (P2Y(1,12,13) receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADP beta S, enhanced P2Y(1) receptor-expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADP beta S-microinjection. Pretreatment with PPADS or wortmannin abolished the up-regulation of pAkt by injury alone or ADP beta S-treatment. The ADP beta S-enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time-dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y(1) receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3-K/Akt-pathway in processes of injury-induced astroglial proliferation and anti-apoptosis via activation of P2Y(1) receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases.
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