A molecular biological study of Alzheimer's disease : Investigation of amyloid precursor protein, amyloid precursor-like protein 2 and presenilin-1
Author: Johnston, Janet A.
Location: Hörsalen plan 4, Novum, Huddinge sjukhus
The devastating symptoms of Alzheimer's disease result from a progressive regional neurodegenerative process, the cause of which is unknown. Current research suggests that several different mechanisms may be involved, perhaps acting at different points in a common pathological pathway. This thesis explores potential levels of involvement of three molecules likely to be important in this pathway, namely amyloid precursor protein (APP), amyloid precursor-like protein 2 (APLP2) and presenilin- 1 (PS- 1). A metabolite of APP, B-amyloid (AB), is found aggregated in abnormal extracellular amyloid plaques in Alzheimer's disease brains. APP missense mutations which can cause Alzheimer's disease have been identified. To investigate the prevalence of such mutations in Sweden, APP exons 16 and 17 were sequenced in 46 individuals affected by Alzheimer's disease (Paper I). The previously identified APP670/671 double mutation was the only abnormality present, demonstrating that these mutations are a not a major cause of Alzheimer's disease in Sweden. Although relatively rare, the APP mutations opened up an important new line of inquiry into the aetiology of Alzheimer's disease. In order to investigate the mechanism(s) of action of the APP670/671 mutation, we determined APP mRNA, protein and AB levels in fibroblast cell lines from 6 mutation-bearing and 6 control family members (Paper II). Mutation-bearing cells released approximately threefold more AB than controls, suggesting a mechanism by which this mutation could lead to the increased AB deposition characteristic of Alzheimer's disease. In order to investigate whether altered mRNA expression of APP or the homologous protein, APLP2, could be involved in the pathogenesis of Alzheimer's disease, quantitative solution hybridisation assays were developed for these mRNAs (Paper III). These assays were characterised and shown to quantify the target mRNAs reproducibly in tissue samples and cell culture systems, before they were used to determine APP and APLP2 mRNA levels in post mortem brains (mid-temporal and superior frontal cortices) from a series of 14 Apolipoprotein E (APOE) genotyped sporadic Alzheimer's disease and 14 control individuals (Paper IV). Inheritance of the APOE 4 allele increases susceptibility to Alzheimer's disease by an unknown mechanism and we wished to determine whether it affected APP or APLP2 mRNA levels. Total APP and APLP2 mRNA levels were reduced in mid-temporal cortex, while the proportion of APP splice forms containing a kunitz-type protease inhibitor insert increased significantly in both brain regions studied. APOE genotype had no effect on target mRNA levels. The accumulation of AB in sporadic Alzheimer's disease cannot therefore be attributed to over-expression of the precursor protein, although APP splicing may be altered in the disease. The PS-I gene, identified in 1995, also carries mutations which are pathogenic for Alzheimer's disease. A quantitative solution hybridisation assay was established and used to determine PS-I mRNA levels in the series of post mortem brains described above, to determine whether the expression of this gene was altered in sporadic Alzheimer's disease (Paper V). There were no significant differences between the levels of PS- I mRNA in the Alzheimer's disease and control groups. PS-I mRNA levels were around 10% that of APP and 30% that of APLP2 mRNA in both cortical regions studied. Significant positive correlations between the levels of PS- I mRNA, APP and APLP2 mRNAs were identified, perhaps indicating that the expression of these genes is co-regulated, or that they are expressed in similar neuronal populations. There is evidence that cerebral cortex affected by Alzheimer's disease is subjected to levels of stress sufficient to induce a heat shock response. Since APP expression is increased in response to heat shock, an altered APP stress response could predispose to AB accumulation. Several PS-I mutations increase AB42/43 production, and we investigated whether they also affected basal APP levels or response to stress by studying the APP heat shock response in Iymphoblastoid cell lines from 8 PS-I mutation-bearing and 9 control members of Alzheimer's disease families (Paper VI). APP mRNA levels increased reversibly following heat shock, followed by a similar increase in APP protein levels. No significant differences were observed between cell lines derived from mutation-bearers and controls, indicating that the five PS-I mutations studied did not affect the normal APP stress response in these cell lines. The studies presented in this thesis indicate that APP mutations causing Alzheimer's disease are rare and that their effects are exerted at the level of protein processing so as to increase the accumulation and deposition of AB. Accumulation of AB in sporadic Alzheimer's disease could not be attributed to over expression of APP or APLP2 mRNA, although APP RNA splicing was altered in affected post mortem brain. PS-I mRNA levels, which were unchanged in Alzheimer's disease post mortem brain, showed positive correlations with cortical APP and APLP2 mRNA levels. PS-I mutations which are pathogenic for Alzheimer's disease did not affect the normal APP stress response in Iymphoblastoid cell lines.
Issue date: 1997-05-02
Publication year: 1997
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