Processing and metabolism of beta-amyloid precursor protein (APP) and generation of a variety of beta-amyloid (Abeta) peptides in the human brain is essentially associated with pathophysiology of Alzheimer's disease (AD). APP degradation activity of the 68 kDa serine protease, which was originally prepared from familial AD lymphoblastoid cells and harbors beta-secretase-like activity, was analyzed by Western blot using anti Abeta 1/40 antibody and anti APP cytoplasmic domain (CT) antibody. Native lymphocyte APP (LAPP) prepared from normal or AD-derived lymphoblastoid cells was degraded by the protease, generating a 16 kDa Abeta-bearing C-terminal fragment of APP. N-terminal amino acid sequencing of the fragment indicated that the protease cleaves LAPP at the Abeta-N-terminus. When the LAPP was treated with chondroitinase ABC prior to proteolysis, the activity to generate the fragment was inhibited, but pretreatment with heparitinase resulted in no effect. Native hippocampal APP prepared from normal brain, however, did not generate the 16 kDa peptide by the protease treatment. These results suggest that the process of APP degradation and Abeta-peptides generation, including beta-secretase activity, is associated with tissue specificity of both APP substrate and proteases. They also indicate that sulfated glycoconjugates attached to a portion of APP isoforms may play a role as a molecular determinant in the proteolysis.
Archival cerebral tissue from infants whose deaths were attributed to sudden infant death syndrome (SIDS) from South Australia and Western Denmark were stained for ß-amyloid precursor protein (ß-APP) and graded according to a simple scoring chart. The resulting APP scores were correlated with sleeping situation (shared vs. alone) showing a significantly higher amount of ß-APP staining in the non-bed-sharing, than in the bed-sharing infants (Mann-Whitney, Australia: p = 0.0128, Denmark: p = 0.0014, Combined: p = 0.0031). There was also a marked but non-significant difference in sex distribution between bed-sharers and non-bed-sharers with a male to female ratio of 1:1 in the first group and 2:1 in the latter. Of 48 Australian and 76 Danish SIDS infants, ß-APP staining was present in 116 (94%) cases. The eight negative cases were all from the Danish cohort. This study has shown that the amount of ß-APP staining was significantly higher in infants who were sleeping alone compared to those who were bed-sharing with one or more adults, in both an Australian and Danish cohort of SIDS infants. Whether this results from differences in the speed with which these infants die, differences in lethal mechanisms involving possible accidental asphyxiation in shared sleepers, or differences in the number of previous hypoxic-ischemic events, remains to be clarified.
Abeta peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Abeta42 specifically in adult neurons, to avoid developmental effects. Abeta42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Abeta42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Abeta42 toxicity. Abeta42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Abeta42. The GSK-3-mediated effects on Abeta42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Abeta42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Abeta42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Abeta42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.
Disruption of the circadian rhythms is a frequent preclinical and clinical manifestation of Alzheimer's disease. Furthermore, it has been suggested that shift work is a risk factor for Alzheimer's disease. Previously, we have reported association of intolerance to shift work (job-related exhaustion in shift workers) with a variant rs12506228A, which is situated close to melatonin receptor type 1A gene (MTNR1A) and linked to MTNR1A brain expression levels. Here, we studied association of that variant with clinical and neuropathological Alzheimer's disease in a Finnish whole-population cohort Vantaa 85+ (n = 512, participants over 85 years) and two follow-up cohorts. Rs12506228A was associated with clinical Alzheimer's disease (p = 0.000073). Analysis of post-mortem brain tissues showed association with higher amount of neurofibrillary tangles (p = 0.0039) and amyloid beta plaques (p = 0.0041). We then followed up the associations in two independent replication samples. Replication for the association with clinical Alzheimer's disease was detected in Kuopio 75+ (p = 0.012, n = 574), but not in the younger case-control sample (n = 651 + 669). While melatonin has been established in regulation of circadian rhythms, an independent role has been also shown for neuroprotection and specifically for anti-amyloidogenic effects. Indeed, in vitro, RNAi mediated silencing of MTNR1A increased the amyloidogenic processing of amyloid precursor protein (APP) in neurons, whereas overexpression decreased it. Our findings suggest variation close to MTNR1A as a shared genetic risk factor for intolerance to shift work and Alzheimer's disease in old age. The genetic associations are likely to be mediated by differences in MTNR1A expression, which, in turn, modulate APP metabolism.
The processing of the Amyloid Precursor Protein (APP) is a critical event in the formation of amyloid plaques which are composed of the 4kDa amyloid beta-peptide (Abeta). Processing of APP occurs through a non-amyloidogenic pathway, mediated by initial alpha-secretase cleavage or through an amyloidogenic pathway via sequential cleavage by beta- and gamma-secretase enzymes, which produces Abeta peptides. Currently, the diagnosis of probable or possible Alzheimer's disease (AD) is primarily based on neuropsychological and neuroradiological assessment. Recent reports indicate that platelet beta-secretase activity is moderately increased in patients with AD and mild cognitive impairment (MCI). To our knowledge platelet alpha-secretase activity has not yet been explored in this context and estimation of the ratio of the activities of alpha- and beta-secretase in platelets may represent a useful surrogate marker of the balance between the two pathways of APP metabolism and be of importance for the diagnosis of AD. We therefore considered it of interest to develop assays of platelet alpha- and beta-secretase activities suitable for such clinical investigations. Application of these assays to a Swedish population failed to uncover an effect of AD or MCI on individual platelet secretase activities or the secretase ratio. However, we did observe an inverse correlation between plasma triacylglycerol levels and the secretase ratio. The results are discussed in the context of the clinical usefulness of the secretase ratio as a biochemical adjunct to the diagnosis of AD.
In this study, we screened for the amyloid beta precursor protein (APP) 665 (glutamic acid to aspartic acid), 670/671 (lysine to asparagine and methionine to leucine) and 717 (valine to isoleucine) mutations in 34 persons affected with familial Alzheimer's disease (AD) and 139 with sporadic AD, originating from eastern Finland, using polymerase chain reaction amplification and restriction enzyme digestion. We did not find any of these mutations in the APP gene in our study. We conclude that these mutations in the APP gene may be a very rare cause of AD in Eastern Finland and thus most Finnish familial AD will likely be due to other gene defects.
Cerebrovascular lesions related to congophilic amyloid angiopathy (CAA) often accompany deposition of ?-amyloid (A?) in Alzheimer's disease (AD), leading to disturbed cerebral blood flow and cognitive dysfunction, posing the question how cerebrovascular pathology contributes to the pathology of AD. To address this question, we characterised the morphology, biochemistry and functionality of brain blood vessels in transgenic arctic ?-amyloid (arcA?) mice expressing human amyloid precursor protein (APP) with both the familial AD-causing Swedish and Arctic mutations; these mice are characterised by strong CAA pathology. Mice were analysed at early, mid and late-stage pathology. Expression of the glucose transporter GLUT1 at the blood-brain barrier (BBB) was significantly decreased and paralleled by impaired in vivo blood-to-brain glucose transport and reduced cerebral lactate release during neuronal activation from mid-stage pathology onwards. Reductions in astrocytic GLUT1 and lactate transporters, as well as retraction of astrocyte endfeet and swelling consistent with neurovascular uncoupling, preceded wide-spread ?-amyloid plaque pathology. We show that CAA at later disease stages is accompanied by severe morphological alterations of brain blood vessels including stenoses, BBB leakages and the loss of vascular smooth muscle cells (SMCs). Together, our data establish that cerebrovascular and astrocytic pathology are paralleled by impaired cerebral metabolism in arcA? mice, and that astrocyte alterations occur already at premature stages of pathology, suggesting that astrocyte dysfunction can contribute to early behavioural and cognitive impairments seen in these mice.