Amyloid precursor protein with the Alzheimer´s disease 670/671 mutation : Animal and cellular models

Sammanfattning: The amyloid precursor protein (APP) and it s derivatives play a key role in the pathogenesis of Alzheimer s disease (AD), which is characterized by the presence of multiple aggregates of an APP proteolytic product, Ab, and tau protein in the brains of affected patients. Clinically the disease is manifested by a progressive loss of memory and executive functions. Several mutations within APP or in proteins involved in APP processing cause an inherited form of AD. Our goal was to recapitulate the features of the disease in a rat model. We have established a transgenic rat expressing human APP with the so called Swedish mutation (APPswe). All human carriers of this mutation develop AD with an onset of clinical manifestation between the age of 44 and 61 years. The first paper of this thesis describes the generation of the Tg6590 APPswe transgenic rat. A cDNA construct carrying human APPswe and an ubiquitin promotor was injected into the pronucleus of rat oocytes. After confirming the expression of the human protein in the transgenic founder, the rat offspring were breed to homozygosity. The Tg6590 rat line shows mainly neuronal APPswe expression, with the highest levels found in the cortex, hippocampus and cerebellum. APPswe is processed in the rat brain, as it s secreted fragments can be found in the cerebrospinal fluid. Homozygous Tg6590 rats begin to show Ab accumulation, mainly in the cerebral blood vessels, starting from 15 months of age. At 11 months of age, the Ab peptide levels are elevated by 65% in the hippocampus and by 40% in the cortex of transgenic animals, as compared to control animals (paper II). In paper II the animals were characterized further by behavioral testing and magnetic resonance imaging (MRI) of the brain. At the age of 9 months, but possibly even earlier, the Tg6590 male rats show inferior spatial memory (assessed by Morris water maze) and altered spontaneous behavior (measured in open-field test), as compared to control animals. We have not detected any gross degeneration of the hippocampus or cortex of the 9 months old rats by MRI, but preliminary results suggest diminution of the cortex thickness in older animals. Since destabilization of calcium homeostasis has been implied as one of the proximal events leading to neuronal degeneration in AD, in the last two papers we focus on calcium signaling in primary hippocampal neurons derived from heterozygous Tg6590 rats. Cytosolic free calcium levels ([Ca2+]i) were imaged by confocal microscopy using the fluorescent dye fluo-3AM. In paper III we demonstrate that transgenic hippocampal cultures show increased frequency but unaltered amplitude of spontaneous [Ca2+]i oscillations as compared to wild-type neurons. The altered calcium signaling in transgenic neurons seems unlikely to be due to modulation of the N-methyl-D-aspartate or nicotinic neurotransmitter systems, nor to depend on secreted APP derivatives, suggesting that either the fulllength (non-processed) APP protein or intracellular APP derivatives are responsible for this effect. In paper IV we show, that transgenic neurons have increased basal [Ca2+]i and altered response to hyperosmotic stress, but no perturbations in endoplasmic reticulum calcium loading. Increased osmolarity can be encountered by neurons during diabetic hyperglycemia or after ischemic stroke, which in their turn are associated with an increased risk of developing AD in later life. We found that the altered response to hypersomotic stress could involve aberrant activation of L-type calcium channels, since transgenic neurons showed significantly greater sensitivity to the L-type calcium channel antagonist, nimodipine. In summary, we have demonstrated that APPswe induces complex alterations of calcium homeostasis in hippocampal neurons, which might at least partly be responsible for the memory deficits seen in the Tg6590 rats. We believe that the Tg6590 rat is a suitable model of early AD and should prove useful for testing new therapeutic strategies aimed at improving memory in AD patients.