Beta/A4-Amyloid increases nerve growth factor production in rat primary hippocampal astrocyte cultures.

Nerve growth factor (NGF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity of basal forebrain cholinergic neurons (BFCN). In processes of chronic degeneration of BFCN like in Alzheimer's disease (AD), characterized among others by amyloid containing plaques, NGF has been shown to improve cognitive decline and rescue BFCN but also to reduce survival of hippocampal neurons via p75 neurotrophin receptor (p75). Little is known about the mechanisms of NGF regulation in glial cells under pathological conditions in AD. This study investigates the influence of amyloid administration on the NGF protein secretion in rat primary hippocampal astrocytes. Astrocytes were stimulated with "aged" beta/A4-Amyloid (1-40), and NGF was measured in different fractions, such as supernatant, vesicles, and cytosol fraction. Treatment with amyloid at a final concentration of 10 microM for 72 h led to increased NGF protein levels up to 30-fold increase compared to unstimulated controls. This observation may be an endogenous neuroprotective mechanism possibly contributing to a delay of amyloid-dependent loss of cholinergic neurons or contribute to accelerated neuronal death by activation of p75 within Alzheimer pathology.

A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain.

DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.

Apolipoprotein E and beta A4-amyloid: signals and effects.

In humans, the apolipoprotein E gene (APOE) is polymorphic with the alleles APOE epsilon 2, 3 and 4 coding for apolipoproteins (Apo) E2, 3 and 4. Apart from age, the APOE epsilon 4 allele represents the most important risk factor in sporadic Alzheimer's disease (AD). Compared to APOE epsilon 3 homozygotes, the histopathological onset of tau pathology is found 1-2 decades earlier but progresses with the same speed. ApoE dose-dependently and specifically increases free intraneuronal calcium levels in the order ApoE4 > ApoE3 > ApoE2. This effect is amplified in the presence of beta A4-peptide. The ApoE effects on calcium are not affected by the blockade of action potentials with tetrodotoxin, or by inhibition of common ApoE binding sites. The calcium channel involved has been identified as a P/Q-type-like channel. Brain tissue ApoE levels differ with respect to APOE alleles and Braak-stage for Alzheimer-histopathology. The production of ApoE in astrocytes is controlled by several receptor/effector systems such as adrenoceptors and cAMP. In the presence of beta A4-peptide fragments, astrocytes stop their synthesis of ApoE resulting in a massive reduction in the bioavailability of ApoE. In the periphery, ApoE directs cholesterol transport and thereby influences its cellular concentrations. In neurons, changes in the concentration of cholesterol influence the phosphorylation status of the microtubule-associated protein tau at sites known to be altered in AD.

The effects of beta/A4-amyloid and its fragments on calcium homeostasis, glial fibrillary acidic protein and S100beta staining, morphology and survival of cultured hippocampal astrocytes.

Aggregated beta/A4-amyloid is known to increase intraneuronal calcium by various mechanisms and to lead eventually to the death of the cultured neuron. This study deals with the role of beta/A4-amyloid and several of its fragments in calcium homeostasis, glial fibrillary acid protein and S100beta staining, morphology and survival of cultured rat hippocampal astrocytes as determined by Fura imaging, indirect immunofluorescence and life/death assays. In contrast to cultured neurons, none of the 12 different beta/A4 fragments tested caused an increase in intra-astrocytic free calcium. However, among the compounds evaluated, the fragments 10-20mer, 25-35mer and the full-length peptides (1-40, 1-42 and 1-43mer), at 5 and 10 microM, decreased free intra-astrocytic calcium statistically significantly after the cells had been incubated for 48 and 72 h. This occurred both for astrocytes treated with vehicle alone or the reversed sequence of the 1-40mer, i.e. the 40-1mer. However, survival was not altered under the conditions examined, even when there was a change in free intracellular calcium. Concomitant with the decrease in intracellular free calcium, the shape of the astrocytes became more spider-like, normally an indication of activated astrocytes, and markedly more intense anti-S100beta and anti-glial fibrillary acidic protein staining was seen. The functional relevance of altered calcium homeostasis for apolipoprotein E secretion, potentially relevant for neuronal plasticity in general and in Alzheimer's disease, is discussed.

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures.

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease. We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis. We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.

Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum.

We describe two modifications of the popular reporter green fluorescent protein (GFP) which have short half-lives in our system, the cellular slime mould Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP was originally planned to be a substrate of the "N-end-rule" pathway, but deubiquitination does not seem to occur, and a degradation by the UFD (ubiquitin-fusion-degradation pathway seems more probable. The protein half-life is about 3-5 h. The second construct has an N-terminus derived from the L11 ribosomal protein; it is transported to the nucleus and broken down much more rapidly than the ubiquitin fusion (protein half-life about 30 min). We show examples of the use of these reporters in the study of gene expression in Dictyostelium.

Wild-type strains of Dictyostelium discoideum can be transformed using a novel selection cassette driven by the promoter of the ribosomal V18 gene.

Almost all methods for transformation of the social ameba Dictyostelium discoideum rely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10(-5) with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several beta-galactosidase reporter cassettes. Our vector can also be used in axenic transformations.