Elsevier

Neurobiology of Aging

Volume 27, Issue 1, January 2006, Pages 67-77
Neurobiology of Aging

The Arctic Alzheimer mutation facilitates early intraneuronal Aβ aggregation and senile plaque formation in transgenic mice

https://doi.org/10.1016/j.neurobiolaging.2004.12.007Get rights and content

Abstract

The Arctic mutation (APP E693G) is unique, since it is located within the amyloid-β (Aβ) sequence and leads to Alzheimer's disease (AD). Arctic Aβ peptides more easily form Aβ protofibrils in vitro, but little is known about the pathogenic mechanism of the Arctic mutation in vivo. Here, we analyzed APP transgenic mice with both the Swedish and Arctic mutations (tg-APPArcSwe) and transgenic mice with the Swedish mutation alone (tg-APPSwe). Intense intraneuronal Aβ-immunoreactive staining was present in young tg-APPArcSwe mice, but not in tg-APPSwe mice. Intracellular Aβ aggregates in tg-APPArcSwe were strongly stained by antibodies recognizing the N-terminus of Aβ, while those recognizing the C-terminus of Aβ stained weakly. The Aβ aggregates inside neurons increased with age and predated extracellular Aβ deposition in both tg-APPArcSwe and tg-APPSwe mice. Senile plaque deposition was markedly accelerated in tg-APPArcSwe mice, as compared to tg-APPSwe mice. We conclude that the Arctic mutation causes AD by facilitating amyloidosis through early accumulation of intracellular Aβ aggregates in association with a rapid onset of senile plaque deposition.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, abundant senile plaque and neurofibrillary tangle formation in the brain. Aβ peptides aggregate into crossed β-sheet fibrils which accumulate in the extracellular space as highly protease resistant material with distinct tinctorial properties. The “amyloid cascade hypothesis”, which states that Aβ is central to the disease, has been criticized for its inability to explain how Aβ confer neurodegeneration and dementia. Cognitive decline correlates better with synapse loss or tau pathology than with Aβ plaque burden [2], [39]. A possible explanation could be that soluble Aβ, rather than insoluble Aβ, confer neurotoxicity [24]. Indeed, certain species of soluble Aβ, e.g. Aβ oligomers, Aβ diffusible ligands (ADDLs), Aβ protofibrils and amylospheroids are neurotoxic in vitro or disrupt long-term potentiation (LTP) [13], [19], [40], [41]. The accumulation of Aβ oligomers has recently been reported to occur inside neurons in association with morphological abnormalities [37]. The Arctic mutation, which leads to AD, could be an excellent tool to better understand Aβ oligomerization, since Arctic Aβ peptides more easily form soluble Aβ protofibrils in vitro [30]. The Arctic mutation is located within the Aβ domain, in contrast to other APP mutations linked to AD which frame the Aβ sequence. We sought to use the Arctic mutation in transgenic mice to study its pathogenic mechanism and the role of soluble Aβ aggregates in vivo. Our idea was that the Arctic and Swedish mutations, when used together, would give rise to high levels of Arctic Aβ peptides resulting in an abundance of Aβ protofibrils. Human APP containing the Swedish (KM670/671NL) and Arctic (E693G) mutations was inserted into a Thy-1 expression vector [26], [30], [36]. Here, we show that young tg-APPArcSwe mice develop strong intraneuronal Aβ aggregation prior to extracellular Aβ deposition and that tg-APPArcSwe show much earlier onset of senile plaque formation than tg-APPSwe. The intraneuronal Aβ aggregates in APPArcSwe mice did not stain with thioflavine S and Aβ antibodies reacted differently with intraneuronal Aβ than with fibrillar Aβ in extracellular senile plaques. This suggests that intraneuronal Aβ aggregates predate Aβ fibrils and that the structure of these species differ from those of fibrillar Aβ. Further investigation of intraneuronal Aβ could improve our understanding of early stage AD and the mechanistic links between intraneuronal Aβ and tau pathology, neurodegeneration and dementia.

Section snippets

Expression vectors, transgenic mice and tissue preparation

Human APP cDNA clones (3′-UTR extended to SmaI at +3100) with the Swedish mutation (KM670/671NL) and with or without the Arctic mutation (E693G) were generated, attached to a modified Kozak sequence, inserted into the murine Thy-1323-cassette and sequenced [22]. The constructs were linearized with NotI and purified with β-agarase, microinjected (2 μg/ml) into pronuclear oocytes of C57BL/6-CBA-F1 mice and implanted into pseudopregnant foster mothers at the two-cell stage. Founders were screened

Strong intraneuronal Aβ immunostaining in young tg-APPArcSwe mice

Founder mice were initially screened with PCR using two sets of primer pairs that framed the Thy-1 basal promoter region and the APP coding region. We identified one founder line with both Swedish and Arctic mutations (called tg-APPArcSwe mice) and two founder lines with Swedish mutation alone (called tg-APPSwe mice). Brain tissue of 1-month-old mice was examined for APP protein expression. Human APP expression was measured with the 6E10 antibody (epitope 1–16 in Aβ) and total (human + murine)

Discussion

The most prominent phenotype in tg-APPArcSwe mice, as compared to tg-APPSwe mice, is strong intraneuronal Aβ aggregation which is associated with an early onset of parenchymal amyloid deposition. The Arctic mutation does not appear to alter Aβ levels when measured with ELISA in young mice. It should be noted that our Aβ analysis was limited to ELISA measurements of 2-month-old mice with C-terminal directed Aβ antibodies. Thus, C-terminal truncated/modified Aβ peptides will not be detected by

Acknowledgements

We thank Dr. Herman van der Putten at Novartis for providing the murine Thy-1 expression cassette. Aβ40- and Aβ42-specific antibodies were generously provided by Dr. Jan Näslund (Karolinska Institutet) and the 3D6 antibody by Dr. Dale Schenk (Elan Pharmaceuticals). Support by Uppsala University Transgenic Facility for this project is greatly acknowledged. The research was funded by grants from Uppsala University, Landstinget i Uppsala län, Alzheimerfonden, Hjärnfonden, Bertil Hållstens

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