Monika Vestling

Abnormalities in Alzheimer's Disease Fibroblasts Bearing the APP670/671 Mutation

Abnormalities in cultured fibroblasts from familial Alzheimers Disease (FAD) cases uniquely enable the determination of how gene defects alter cell biology in living tissue from affected individuals. The current study focused on measures of calcium regulation and oxidative metabolism in fibroblast lines from controls and FAD individuals with the Swedish APP670/671 mutation. Bombesin-induced elevations in calcium in APP670/671 mutation-bearing lines were reduced by 40% (p < 0.05), a striking contrast to the 100% increase seen in sporadic AD and presenilin-1 (PS1) mutation-bearing cells in previously published studies. The APP670/671 mutation-bearing lines did not exhibit the exaggerated 4-bromo-A23187 releasable pool of calcium following 10 nM bradykinin, the enhanced sensitivity of calcium stores to low concentrations of bradykinin, nor the reduced activity of alpha-ketoglutarate dehydrogenase previously reported in cells from sporadic AD and mutant PS1 FAD. Thus, an altered regulation of internal calcium stores is common to all AD lines, but the calcium pool affected and the polarity of the alteration varies, apparently in association with particular gene mutations. Comparison of signal transduction in cell lines from multiple, genetically characterized AD families will allow testing of the hypothesis that these various pathogenic FAD abnormalities that lead to AD converge at the level of abnormal signal transduction.

Disrupted β1-adrenoceptor—G protein coupling in the temporal cortex of patients with Alzheimer's disease

Abstract The efficacy of β 1 -adrenoceptor—G protein coupling was studied in postmortem temporal cortex synaptic membranes from a series of control and Alzheimers disease subjects. For the control cases, the non-hydrolysable GTP analogue 5′-guanylylimidodiphosphate (Gpp[NH]p) gave a significant reduction in the affinity of the agonist isoprenaline to displace binding of the radiolabelled antagonist (±)-4-(3- t -butylamino-2-hydroxypropoxy)-[5,7- 3 H]benzimidazol-2-one ([ 3 H]CGP-12177). This effect was attributed to the conversion of high agonist-affinity sites to a lower-affinity state and was not found for the Alzheimers disease cases. These data indicate that a disruption of β 1 -adrenoceptor—G protein coupling occurs in the temporal cortex of Alzheimers disease patients.

A novel pathogenic mutation (Leu262Phe) found in the presenilin 1 gene in early-onset Alzheimer's disease

Several mutations causing early-onset familial Alzheimers disease (AD) have been detected in the presenilin 1 (PS-1) gene. Pathogenic mutations have also been described in an homologous gene, presenilin 2 (PS-2). In order to screen for mutations in these genes, cDNA samples from early-onset AD cases were analysed, using single strand conformation polymorphism (SSCP) and direct cDNA sequencing. Two missense mutations in the PS-1 gene were detected, a previously unidentified amino acid substitution Leu262Phe and an earlier reported amino acid substitution Glu318Gly. No disease-related mutations were found in the PS-2 gene.

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases.

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimers disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimers disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimers disease cases. In contrast, familial Alzheimers disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.

Differential regulation of adenylyl cyclase in fibroblasts from sporadic and familial Alzheimer's disease cases with PS1 and APP mutations.

β-ADRENOCEPTOR- and forskolin-45 cyclase activities were determined in primary skin fibroblasts established from patients with sporadic Alzheimers disease (AD) and from individuals with familial APP KM670/671NL, PS1 M146V and PS1 H163Y mutations. Our data showed a significantly decreased β-adrenoceptor-stimulated adenylyl cyclase activity in fibroblasts from sporadic AD compared with age-matched controls (p < 0.001, Students unpaired t-test). In contrast, both β-adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were significantly increased in fibroblasts bearing PS1 M146V and PS1 H163Y mutations compared with controls (p < 0.01 and p < 0.05, respectively). No differences were seen between cell lines with and without the Swedish APP KM670/671NL double mutation. We suggest that various gene mutations associated with AD have different consequences for the regulation of adenylyl cyclase signal transduction in this disorder.

Akt activity in presenilin 1 wild-type and mutation transfected human SH-SY5Y neuroblastoma cells after serum deprivation and high glucose stress.

The majority of early‐onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 ΔE9) and L250S mutations predispose SH‐SY5Y neuroblastoma cells to high glucose stress‐induced apoptosis and that the anti‐apoptotic effect of insulin‐like growth factor I (IGF‐I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH‐SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)‐kinase arm of the IGF‐I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro‐apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF‐I. We also looked at the phosphorylatiom state of GSK‐3β at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF‐I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6‐ or 24‐hr post‐treatment. Immunoblotting for Phospho‐Akt as a ratio of total Akt, as well as for GSK‐3β phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro‐apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF‐I. It is suggested that another component of IGF‐I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis. J. Neurosci. Res. 66:448–456, 2001.

Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: Effects of the swedish Alzheimer's disease APP 670/671 mutation on binding levels

SummaryWe have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimers disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [3H] quinuclidinyl benzilate ([3H]QNB) and the M2/M4 selective antagonist [3H](±)-5,11-dihydro-11-{[(2-[(di-propylamino)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one ([3H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [3H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine>himbacine>methoctramine>(±)-p-fluoro-hexahydro-sila-difenidol hydrochloride>pirenzepine>muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25–35% lower levels of muscarinic receptors labelled with [3H]QNB, [3H]N-methyl scopolamine and [3H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.

Alzheimerʼs disease: mutations, apoptosis and cellular signalling

Purpose of review Mutations in the amyloid precursor protein, presenilin 1 and presenilin 2 genes are believed to cause early onset familial Alzheimers disease by increasing the production of more amyloidogenic forms of the β-amyloid peptide (so called Aβ42). However, the phenotypes of familial Alzheimers disease mutations are more complex than can be explained only by effects on amyloid precursor protein processing and β-amyloid production. This review summarises alternative ideas suggesting that familial Alzheimers disease mutations cause disease by altering cellular signalling systems to increase the vulnerability of neurons to die by apoptosis. Recent findings The themes covered by this article include the consequences of altered amyloid precursor protein and presenilin function for apoptotic signalling, as well as the neurotoxic mechanism of action of the β-amyloid peptide. We also review the recent findings implicating altered insulin/insulin growth factor I signal transduction as a recurrent feature in many of the processes considered to result in apoptotic cell death in Alzheimers disease. Summary Insulin growth factor I has previously received much attention as a potential therapeutic agent for Alzheimers disease due to its multiple neuroprotective and neurotrophic effects. New findings indicating that altered insulin growth factor I signalling may be involved in the mechanism of action of β-amyloid and famililal Alzheimers disease-causing gene mutations have reawakened interest in this molecule and its signalling pathways. The identification of insulin/insulin growth factor I signalling alterations in sporadic and familial Alzheimers disease may provide novel targets for therapies designed to slow or prevent the neurodegeneration associated with this devastating disorder.