Involvement of glutaredoxin-1 and thioredoxin-1 in beta-amyloid toxicity and Alzheimer's disease.

Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.

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 Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's 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 Alzheimer's disease cases. In contrast, familial Alzheimer's 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.

Amyloid precursor protein heat shock response in lymphoblastoid cell lines bearing presenilin-1 mutations.

The amyloid precursor protein (APP) gene promoter contains a heat shock element. An abnormal APP heat shock response could increase accumulation of A beta, the APP metabolite found in Alzheimer's disease amyloid plaques. Since A beta production is affected by presenilin-1 (PS-1) mutations, we investigated whether basal APP levels or response to heat shock were altered in lymphoblastoid cell lines from 8 PS-1 mutation-bearers and 9 control members of Alzheimer's disease families. Lymphoblastoid cell lines were incubated at 42 degrees C for 35 min and allowed to recover at 37 degrees C for 1, 3, 8, 24 and 48 h. APP mRNA levels, quantified using RNA-RNA solution hybridisation, increased significantly at 1 and 3 h post-heat shock to between 123% and 163% of pre-heat shock (0 h) levels and returned to normal by 8 h. Semi-quantitative Western immunoblotting of cell lysates using the 22C11 antibody detected two major bands, migrating at approximately 145 and approximately 120 kDa. Band optical densities increased significantly at 3 h to approximately 155% of 0 h levels, following the increase in APP mRNA levels and showing a similar reversibility. APP mRNA and protein responses were comparable in the PS-1 mutation-bearing and control cell lines. This study shows that both APP mRNA and protein are induced in lymphoblastoid cell lines following heat shock and that this response is not affected by PS-1 mutations which are pathogenic for Alzheimer's disease.

Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes.

Accumulation of paired helical filaments (PHFs) in neurofibrillary tangles, neuropil threads, and dystrophic neurites is one of the major neuropathological hallmarks of Alzheimer disease (AD). The principal protein subunit of PHFs is the abnormally hyperphosphorylated tau. Glycogen synthase kinase 3beta (GSK-3beta) is one of the candidate kinases involved in PHF-tau formation. To play a role in PHF-tau formation, it would be expected that GSK-3beta is active in tangle bearing neurons. In the present study, we investigated the regional and intracellular distributions of active and inactive forms of GSK-3beta in brains staged for neurofibrillary changes. We found that neurons with tangle-like inclusions positive for active, but not inactive, GSK-3beta appear initially in the Pre-alpha layer of the entorhinal cortex and extend to other brain regions, coincident with the sequence of the development of neurofibrillary changes. Active, but not inactive, GSK-3beta was found to initially accumulate in the cytoplasm of pretangle neurons. These data provide direct in situ evidence that is consistent with the involvement of GSK-3beta in PHF-tau formation.

The neurochemistry of Alzheimer's disease.

The last 15-20 years have seen a wealth of studies to characterize the neurochemical abnormalities of Alzheimer's disease, in particular those involving the beta-amyloid and tau proteins, as well as more recently, apolipoprotein E4. This article provides a summary of the evidence for the involvement of these proteins in Alzheimer's disease pathogenesis based on postmortem brain and CSF studies.

Neurotransmitters, signal transduction and second-messengers in Alzheimer's disease.

It has long been assumed that widespread changes in postsynaptic neurotransmitter receptor function are not a feature of the disrupted neurotransmission seen in the brains with Alzheimer's disease (AD). However, recent evidence from postmortem brain and fibroblast studies suggests that both the neurotransmitter receptor/G-protein-modulated adenylyl cyclase and the phosphatidylinositol hydrolysis signal transduction cascades are disrupted in AD. Such disruptions may severely limit the functional integrity of key receptor types and undermine pharmacological attempts to ameliorate disease symptomatology through neurotransmitter replacement strategies. The involvement of some signalling mechanisms in the regulation of beta-amyloid precursor protein metabolism suggests also that disrupted signal transduction may exacerbate AD pathology.

Abnormalities in Alzheimer's disease fibroblasts bearing the APP670/671 mutation.

Abnormalities in cultured fibroblasts from familial Alzheimer's 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.

Apolipoprotein E isoform-specific disruption of phosphoinositide hydrolysis: protection by estrogen and glutathione.

The mechanism(s) by which the E4 isoform of apolipoprotein E (apoE4) influences Alzheimer's disease (AD) are not fully known. We report that apoE4, but not apoE3, disrupts carbachol-stimulated phosphoinositide (PI) hydrolysis in SH-SY5Y neuroblastoma cells. Carbachol responses were also disrupted by beta-amyloid (Abeta) (1-42) and apoE4/Abeta(1-42) complexes, but not by apoE3/Abeta(1-42). Glutathione and estrogen protected against apoE4 and Abeta(1-42) effects, as well as those of H(2)O(2). Estrogen protection was partially blocked by wortmannin, suggesting the involvement of phosphatidylinositol 3-kinase. An apoE4-induced disruption of acetylcholine muscarinic receptor-mediated signalling may explain the lower effectiveness of cholinergic replacement treatments in apoE4 AD patients. Also, the beneficial effect of estrogen in AD may be partially due to its ability to protect against apoE4- and Abeta(1-42)-mediated disruption of PI hydrolysis.

Quantification of presenilin-1 mRNA in Alzheimer's disease brains.

The presenilin-1 (PS-1) gene on chromosome 14 carries mutations which cosegregate with early-onset familial Alzheimer's disease. We quantified PS-1 mRNA in post-mortem mid-temporal and superior frontal cortices from 14 Alzheimer's disease subjects, 9 non-demented controls and 5 subjects with other neurological diseases using solution hybridisation-RNase protection assay. APP and APLP2 mRNAs had previously been quantified in these samples (Johnston et al. (1996) Mol. Brain Res., in press) and subjects were apolipoprotein E (APOE) genotyped. There were no significant differences between PS-1 mRNA levels per pg total RNA in mid-temporal or superior frontal cortices of the Alzheimer's disease subjects, compared to controls. PS-1 mRNA levels corresponded to 10% of total APP and 30% of APLP2 mRNA levels, and were not significantly affected by age, post-mortem delay, tissue pH, or APOE genotype. PS-1 mRNA showed significant positive correlations with APP and APLP2 mRNA levels in mid-temporal cortex and with APP mRNA in superior frontal cortex. This may reflect a co-regulation of the expression of these genes, or the fact that they are expressed in similar neuronal populations.

Increased beta-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation.

Cell lines transfected with the Swedish Alzheimer's disease amyloid precursor protein APP670/671 mutation release significantly more beta-amyloid than wild-type cells. Citron et al. [Proc. Natl. Acad. Sci. USA (1994) in press] have recently shown that fibroblasts carrying the APP670/671 mutation also release more beta-amyloid than control cells [1]. The present study confirms a ca. threefold increase in beta-amyloid release from mutation-bearing fibroblasts. APP mRNA levels did not differ between mutation-bearing and control cells, although mutation-bearing fibroblasts contained significantly more APP751/770 than controls. Mild stress decreased beta-amyloid secretion and increased APP751/770 levels in all cell lines. In conclusion, the proportion of APP committed to amyloidogenic processing is increased in fibroblasts from family members with the APP670/671 mutation, and this mutation may also compromise the APP stress response.

Receptor-G-protein signalling in Alzheimer's disease.

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

Dysfunctional intracellular calcium homoeostasis: a central cause of neurodegeneration in Alzheimer's disease.

The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of beta-amyloid (A beta) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive A beta to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.

Expression of presenilin-1 and Notch-1 receptor in human embryonic CNS.

In vitro studies have shown that the Alzheimer's disease-related presenilin-1 protein can mediate Notch-1 receptor cleavage during signalling. In the present study, we compared the distribution of presenilin-1 and Notch-1 receptor immunoreactivities in human embryonic CNS tissue during the first trimester of development. Our aim was to gain insight into whether these proteins are likely to interact functionally during human fetal brain development. CNS material was obtained from routine abortions, cryosectioned and studied by means of immunohistochemistry with antibodies to presenilin-1 and Notch-1. At very early stages of embryonic development (four to five gestational weeks) intensive presenilin-1 immunoreactivity could be seen predominantly in neurites in the ventral horn of the spinal cord, where it overlapped with 200-kDa neurofilament immunoreactivity. Presenilin-1 immunoreactivity was also seen in neuroblasts of the ventricular zone of the tel- and mesencephalon, as well as of the brainstem. Notch-1 receptor appeared in neuronal and ependymal cells throughout the CNS. Seven- to eight-week CNS tissue showed similar patterns of presenilin-1 and Notch-1 receptor expression in the spinal cord and cerebral cortex as was seen at five weeks. Both proteins were localised in the neuroepithelial cell layer lining the ventricles, as well as in the cortical plate layer, where immunoreactivity was seen in the cell bodies. In addition, presenilin-1 immunoreactivity was seen in thin neurites in the subplate of the developing cortex. At 10 weeks, presenilin-1 immunoreactivity was reduced in the spinal cord. These results show that, although presenilin-1 and Notch-1 receptor are localised to the same differentiating cell populations in the human cerebral cortex, making a direct interaction possible, these proteins are otherwise confined to different neurons or neuronal compartments, suggesting a role for presenilin-1 during early CNS differentiation that does not involve Notch-1 receptor processing. Double staining for presenilin-1 in the endoplasmic reticulum and presenilin-1 in the Golgi showed overlap to some extent in investigated CNS regions, but not in neurites. This suggests that presenilin-1 function during neurogenesis is not exclusively correlated to protein processing within the endoplasmic reticulum and Golgi, but that presenilin-1 may also be involved in other processes, such as axonal and dendritic outgrowth or synaptic formation. In summary, our findings provide supportive evidence that the presenilin-1 protein is involved in the development and maturation of the human fetal CNS. The presence of presenilin-1 immunoreactivity in both the cell bodies and neurites of developing neurons strongly suggests divergent mechanisms of function for presenilin-1 during human brain development. These may include interactions with any of the Notch receptor proteins, as well as Notch-independent mechanisms.

Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis.

Mutations in the presenilin-1 (PS1) and presenilin-2 (PS2) genes account for the majority of early-onset familial Alzheimer's disease cases. Recent studies suggest that presenilin gene mutations predispose cells to apoptosis by mechanisms involving altered calcium homeostasis and oxidative damage. In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed to an overnight (17 h) serum deprivation, followed by a 30 min treatment with either 20 mM glucose, 10 nM insulin-like growth factor-1 or 20 mM glucose + 10 nM insulin-like growth factor-1. Cells were then cultured for a further 3, 6 or 24 h and stained for apoptotic condensed nuclei using propidium iodide. Confirmation that cells were undergoing an active apoptotic process was achieved by labelling of DNA strand breaks using the terminal dUTP nick end labelling (TUNEL) technique. We also determined cell viability using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Propidium iodide staining revealed that all cell lines and controls showed an increased number of apoptotic cells appearing with condensed nuclei at 24 h compared with 6 h and 3 h. High glucose-induced hyperosmotic stress resulted in significantly more apoptotic cells in the PS1 deltaE9 and PS1 L250S mutation cell lines at 24 h, compared with the wild-type PS1 lines (P < 0.001, ANOVA for both comparisons). Mean values (+/-S.D.) for the percentage number of apoptotic cells at 24 h following high glucose treatment were 16.1 +/- 3.5%, 26.7 +/- 5.5% and 31.0 +/- 5.7% for the wild-type PS1, PS1 deltaE9 and PS1 L250S lines, respectively. The pro-apoptotic effects of high glucose treatment were reversed by 10 nM insulin-like growth factor-1, although to a lesser extent in the mutation cell lines (5.8 +/- 2.4%, 15.2 +/- 7.3% and 13.2 +/- 2.0% for the wild-type PS1, PS1 deltaE9 (P < 0.01 for comparison with wild-type PS1) and PS1 L250S (P < 0.01 for comparison with wild-type PS1) transfected lines, respectively. TUNEL labelling of cells at 24 h following treatment gave essentially the same results pattern as obtained using propidium iodide. The percentage number of apoptotic cells with DNA strand breaks (means +/- S.D.) following high glucose treatment was 15.4 +/- 2.6% for the wild-type PS1, 26.8 +/- 3.2% for the PS1 deltaE9 (P < 0.001 for comparison with wild-type PS1) and 29.7 +/- 6.1% for the PS1 L250S transfected lines (P < 0.001 for comparison with wild-type PS1). The PS1 deltaE9 and PS1 L250S transfected lines also showed a higher number of apoptotic cells with DNA strand breaks at 24 h following high glucose plus insulin-like growth factor-1 treatment (11.4 +/- 2.0% and 14.3 +/- 2.8%, respectively), compared with values for the wild-type PS1 lines (8.5 +/- 2.4%). These differences were significant (P < 0.01) for the comparison of wild-type PS1 and PS1 L250S, but not PS1 deltaE9 lines. The mutation-related increases in number of apoptotic cells at 24 h following high glucose treatment were not accompanied by significant differences in cell viability at this time-point. Our results indicate that PS1 mutations predispose to hyperosmotic stress-induced apoptosis and that the anti-apoptotic effects of insulin-like growth factor-1 are compromised by these mutations. Perturbations of insulin-like growth factor-1 signalling may be involved in PS1 mutation-related apoptotic neuronal cell death in Alzheimer's disease.

Alterations in the ryanodine receptor calcium release channel correlate with Alzheimer's disease neurofibrillary and beta-amyloid pathologies.

Investigation of the integrity of the ryanodine receptor in Alzheimer's disease is important because it plays a critical role in the regulation of calcium release from the endoplasmic reticulum in brain, impairment of which is believed to contribute to the pathogenesis of Alzheimer's disease. The present study compared ryanodine receptor levels and their functional modulation in particulate fractions from control and Alzheimer's disease temporal cortex, occipital cortex and putamen. Relationships between ryanodine receptor changes and the progression of Alzheimer's disease pathology were determined by examining autoradiographic [3H]ryanodine binding in entorhinal cortex/anterior hippocampus sections from 22 cases that had been staged for neurofibrillary changes and beta-amyloid deposition. A significant (P < 0.02) 40% decrease in the Bmax for [3H]ryanodine binding and significantly higher IC50 values for both magnesium and Ruthenium Red inhibition of [3H]ryanodine binding were detected in Alzheimer's disease temporal cortex particulate fractions compared to controls. Immunoblot analyses showed Type 2 ryanodine receptor holoprotein levels to be decreased by 20% (P < 0.05) in these Alzheimer's disease cases compared to controls. No significant differences were detected in [3H]ryanodine binding comparing control and Alzheimer's disease occipital cortex or putamen samples. The autoradiography study detected increased [3H]ryanodine binding in the subiculum, CA2 and CA1 regions in cases with early (stage I-II) neurofibrillary pathology when compared to Stage 0 cases. Analysis of variance of data with respect to the different stages of neurofibrillary pathology revealed significant stage-related declines of [3H]ryanodine binding in the subiculum (P < 0.02) with trends towards significant decreases in CA1, CA2 and CA4. Post-hoc testing with Fisher's PLSD showed significant reductions (74-94%) of [3H]ryanodine binding in the subiculum, and CA1-CA4 regions of the late isocortical stage (V-VI) cases compared to the early entorhinal stage I-II cases. [3H]Ryanodine binding also showed significant declines with staging for beta-amyloid deposition in the entorhinal cortex (P < 0.01) and CA4 (P < 0.05) with trends towards a significant decrease in the dentate gyrus. We conclude that alterations in ryanodine receptor binding and function are very early events in the pathogenesis of Alzheimer's disease, and may be fundamental to the progression of both neurofibrillary and beta-amyloid pathologies.

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.

A new quantitative solution hybridisation-RNase protection assay for APP and APLP2 mRNA.

Amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) are members of a multigene family of proteins implicated in the pathogenesis of Alzheimer's disease. We describe the development of an RNA-RNA solution hybridisation-RNase protection assay to quantify APP mRNA. APP mRNA splice forms containing the Kunitz-type protease inhibitor (KPI) insert, and APLP2 mRNA in total nucleic acid extracts from a range of tissue types. Solution hybridisation-RNase protection assay enables absolute quantification of target mRNA, by conversion of the hybridisation signal to pg mRNA using a standard curve. The assay is sensitive, capable of detecting 1 pg target mRNA, and reproducible, with an inter-assay variability of less than 10% and an intra-assay variability of 3-4%. We quantified APP and APLP2 mRNA in cell lines and post-mortem human brain tissue samples. To test whether we could detect physiological differences in APP mRNA levels, a fibroblast cell line with a paternal chromosome 21 deletion of the region including the APP gene was analysed and found to express half as much APP mRNA as control fibroblasts. In addition, a reversible, approx. 30% increase in APP mRNA levels was detected in human lymphoblastoid cell lines following heat shock, a physical stimulus previously shown to increase APP expression. Regional differences in the expression of APP and APLP2 were seen in human post-mortem cerebral cortex and cerebellum. Levels of APP and APLP2 mRNA were highest in the temporal cortex, slightly lower in frontal and occipital cortices, and lowest in the cerebellum. The highest proportion of KPI-containing APP was seen in the frontal and temporal cortices. The ratio of APP:APLP2 mRNA was 1:0.3 in the cortical tissue and 1:0.8 in the cerebellum. In conclusion, quantitative solution hybridisation-RNase protection assay of total APP. APP KPI and APLP2 mRNA provides a new tool to improve the resolution of studies of potentially subtle alterations in the expression of these genes in both cell culture model systems and Alzheimer's disease post-mortem human brain tissue.

Quantification of APP and APLP2 mRNA in APOE genotyped Alzheimer's disease brains.

Amyloid precursor protein (APP) is metabolised to produce A beta, a peptide found aggregated in Alzheimer's disease neuritic plaques. APP is a member of a multigene protein family which includes amyloid precursor-like protein 2 (APLP2). Since A beta accumulation can be triggered by factors acting up- or downstream of APP processing, we investigated whether APP mRNA expression was altered in Alzheimer's disease post-mortem cerebral cortex. In addition, we characterised cortical APLP2 mRNA levels. Quantitative RNA-RNA solution hybridisation-RNase protection was used to assay total APP. APP containing the Kunitz-type protease inhibitor (KPI) insert and APLP2 mRNA in mid-temporal and superior frontal cortices from apolipoprotein E-genotyped subjects with Alzheimer's disease, other neurological diseases and non-demented controls. Approximately 3 times more APP than APLP2 mRNA was detected and about 70% of total APP mRNA contained the KPI insert in the control subjects. Total APP and APLP2 mRNA levels were significantly reduced in Alzheimer's disease mid-temporal, but not superior frontal cortex, suggesting that regional reductions in these mRNA correlate with severity of disease pathology. A small significant increase in the proportion of APP KPI mRNA was seen in both cortical regions in Alzheimer's disease. Apolipoprotein E genotype did not influence cortical levels of total APP, APP KPI or APLP2 mRNA. Alzheimer's disease-related increases in tissue DNA content were seen in both regions studied, while tissue RNA levels were reduced in the positive disease controls. In summary, these results indicate that Alzheimer's disease is not associated with over-expression of either APP or APLP2 mRNA. Our findings reveal a disease-associated increase in the proportion of APP KPI-containing isoforms, and further investigation should clarify whether this predisposes affected individuals to A beta production and aggregation, or reflects later events such as gliosis and neuronal cell death.

Characterization of [3H]inositol 1,4,5-trisphosphate binding sites in human temporal cortical and cerebellar membranes.

The characteristics of [3H]Ins(1,4,5)P3 binding to human temporal cortical and cerebellar membranes have been determined and compared with the binding to calf cerebellar membranes. Association and dissociation of ligand was very rapid, k1 and k-1 values of the order of 7 x 10(7) M-1 min-1 and 0.2 min-1, respectively. KD values were 2.7 and 3.5 nM for temporal cortex and cerebellum, respectively. The corresponding Bmax values were 165 and 482 fmol/mg protein. Binding was influenced in a biphasic manner by calcium. The temporal cortical binding was inhibited by Ins(1,4,5)P3 and analogues with the following IC50 values (nM): Ins(1,4,5)P3 9.5 and 6.2 (two different salts from different sources), Ins(2,4,5)P3 42, Ins(1,3,4,5)P4 670, Ins(1,2,5,6)P4 2620, Ins(3,4,5,6)P4 4300, Ins(1,3,4,5,6)P5 5490, InsP(6)5280, Ins(4,5)P2 2600, Ins(1)P 3300, with the IC50 values for Ins(1,5,6)P3, Ins(1,4)P2 and Ins(4)P being > 25 microM. The IC50 value for heparin was 2.1 micrograms/ml. A similar pattern was seen in the cerebellum. In both tissues, the Hill slopes were near unity for all compounds except Ins(3,4,5,6)P4, where the slope was 0.4. The calf cerebellum had a similar ligand specificity (although the potency was generally lower) when values were expressed relative to that of Ins(1,4,5)P3, with the possible exception of Ins(1,3,4,5)P4, which had a greater relative potency. These data would suggest that in the human temporal cortex and cerebellum, [3H]Ins(1,4,5)P3 binding sites are expressed in different densities, but have similar properties. There may, however, be species differences in the [3H]Ins(1,4,5)P3 recognition site.

Characterization and regional distribution of adenylyl cyclase activity from human brain.

The characteristics of the human brain adenylyl cyclase complex were studied in membranes from post-mortem frontal cortex. Basal, guanine nucleotide, sodium fluoride and forskolin stimulated activities were highly magnesium-dependent. Sodium fluoride and guanine nucleotides gave bi-phasic responses, with both stimulation and inhibition of enzyme activity, the latter being more pronounced at lower magnesium concentrations. Enzyme activity was stimulated to a similar extent by GTP and its non-hydrolysable analogue Gpp(NH)p, suggesting a low GTPase activity in human post-mortem brain preparations. Guanine nucleotide stimulated enzyme activity was potently antagonized by guanosine 5?-O-(thiodiphosphate). Sodium fluoride stimulated activity was enhanced by aluminium chloride. In contrast to the effects seen with guanine nucleotides, the inhibition of sodium fluoride/aluminium chloride stimulated activity by guanosine 5?-O-(thiodiphosphate) was dependent upon pre-incubation of membranes with a neurotransmitter agonist. Basal, guanine nucleotide and sodium fluoride/aluminium chloride stimulated activities showed a marked regional distribution. Stimulated activities were highest in frontal and parietal cortex, intermediate in the nucleus caudatus and cerebellar cortex and lowest in occipital cortex, putamen, globus pallidus and ventral hippocampus. It is concluded that the regulation of human post-mortem brain adenylyl cyclase by guanine nucleotides is similar to that reported for studies on experimental animals.