Aβ dimers induce behavioral and neurochemical deficits of relevance to early Alzheimer's disease

Abstract

We examined behaviors and neurotransmitter levels in the tgDimer mouse, a model for early Alzheimer's disease, that expresses exclusively soluble amyloid beta (Aβ) dimers and is devoid of Aβ plaques, astrogliosis, and neuroinflammation. Seven-month-old mice were subjected to tests of motor activity, attention, anxiety, habituation learning, working memory, and depression-related behaviors. They were impaired in nonselective attention and motor learning and showed anxiety- and despair-related behaviors. In 7- and 12-month-old mice, levels of acetylcholine, dopamine, and serotonin were measured in neostriatum, ventral striatum, prefrontal cortex, hippocampus, amygdala, and entorhinal cortex by high-performance liquid chromatography. The tgDimer mice had lower serotonin turnover rates in hippocampus, ventral striatum, and amygdala relative to wild type controls. The aged tgDimer mice had less hippocampal acetylcholine than adult tgDimers. Stress-test results, based on corticosterone levels, indicated an intact hypothalamus-pituitary-adrenal axis in 12-month-old mice. Since neither Aβ plaques nor astrogliosis or neuroinflammation was responsible for these phenotypes, we conclude that Aβ dimers contribute to neurotransmitter dysfunction and behavioral impairments, characteristic for the early stages of Alzheimer's disease.

Introduction

An early diagnosis of Alzheimer's disease (AD) is pivotal because it is believed that pharmacotherapies are more efficient when initiated before the massive structural damage due to amyloid-β (Aβ) plaque formation. The early stages of AD are not well explored. It is known, however, that about 30% of individuals with mild cognitive impairment convert to early stages of AD (Langa and Levine, 2014). Cognitive deficiency correlates higher with synaptic density than with neuronal loss. This is supported by the findings that Aβ promotes axonal pruning (Roselli et al., 2005, Shankar et al., 2007) and disrupts cognitive functions in the absence of neuronal loss (Cleary et al., 2005, Maurice et al., 1996, McDonald et al., 1994, Stéphan et al., 2001). As a consequence, soluble Aβ oligomers rather than Aβ plaques are increasingly believed to be responsible for the progressive neuronal degeneration in AD (Cleary et al., 2005, Dickson et al., 1995, Haass and Selkoe, 2007, Krafft and Klein, 2010, Terry et al., 1991). Especially Aβ dimers, a form of single Aβ oligomers, have been shown to be synaptotoxic and prevalent in the brain tissue of AD patients (Klyubin et al., 2008, Mc Donald et al., 2010, Shankar et al., 2008). Aβ deposition has been linked to the massive loss of cholinergic neuron in the basal forebrain and other brain areas (Blusztajn and Berse, 2000). Accordingly, cholinergic deficits are considered to be the major pathological characteristic of AD and also represent the main target for current available pharmacological treatment (Geula et al., 2008, Schliebs and Arendt, 2006).

Soluble Aβ oligomers may be responsible for both early cognitive (mild cognitive impairment) and affective symptoms (depression, anxiety) (Hefti et al., 2013). Until recently, it has not been possible to investigate in vivo the roles of the Aβ dimer through the life span of the host, due to the existing kinetic equilibrium of different multimers and the simultaneous occurrence of different forms of amyloid deposits. Intraventricular (i.c.v.) infusion of Aβ molecules in rodents was shown to decrease DA (Wang et al., 2007), 5-HT (Colaianna et al., 2010), and nerve growth factors including nerve growth factor and brain-derived neurotrophic factor (Colaianna et al., 2010). There is also evidence of behavioral action after i.c.v. infusion of Aβ oligomers, such as disruption of sleep pattern (Kincheski et al., 2017), impairment of long-term object recognition (Balducci et al., 2010), forced swimming (Ledo et al., 2013, Ledo et al., 2016), and sucrose preference (Ledo et al., 2016). However, there is no study on transgenic animal models, which has been successful in specifically assessing the impact of the Aβ dimer on neurochemical, cognitive, and emotional behavioral parameters.

The development of the transgenic Aβ-S8C mouse (tgDimer mouse) enabled us to investigate the role of biological effects of single Aβ oligomers in early AD (Müller-Schiffmann et al., 2016). To stabilize Aβ dimers, 2 Aβ monomers were linked by a disulfide bridge that was incorporated into the amyloid precursor protein (APP) by the introduction of a cysteine amino acid at position 8 of the Aβ domain (corresponding to APP751-S679C). This led to the secretion of a neurotoxic Aβ dimer, which did not influence the processing of the APP by cellular secretases (Müller-Schiffmann et al., 2011). The Aβ-S8C mutation in the tgDimer mouse resulted in a high concentration of Aβ dimers but not of monomers. Moreover, no insoluble amyloid species or plaques were generated over its life span. This allowed to distinguish between Aβ-specific effects and other neuropathological alterations such as amyloid plaques, cerebral amyloid angiopathy, and neurofibrillary tangles, as associated with other transgenic models. So far, the tgDimer mouse has been found to exhibit deficits in Morris water maze (MWM) learning and memory as well as in hippocampal long-term potentiation, comparable to findings in classical AD mice (Müller-Schiffmann et al., 2016). These results suggest that soluble Aβ dimers, per se, can induce neurotoxicity and aberrant synaptic signaling and impair cognitive functions in the absence of plaque pathology or elicited effects, such as neuroinflammation and astrogliosis.

The rationale of the present study was the further behavioral and neurochemical characterization of the Aβ-S8C mutation in adult and aged (7 and 12 months old) tgDimer mice in comparison to age-matched wild type (WT) C57BL/6N controls. The 7-month-old mice underwent tests of motor activity, attention, emotionality, and habituation learning in the open field and radial arm maze (RAM), anxiety- and fear-related behaviors in the elevated plus-maze (EPM), depression-related behaviors in the forced swimming test (FST) and motor coordination and learning on the rotarod. Furthermore, in 12-month-old animals, blood corticosterone levels were determined after subjection to restraint stress to assess hypothalamus-pituitary-adrenal (HPA) axis functionality. In further batches of adult and aged tgDimer and WT mice, levels of acetylcholine (ACh), dopamine (DA), and serotonin (5-HT) were measured in neostriatum, ventral striatum, prefrontal cortex, hippocampus, amygdala, and entorhinal cortex via high-performance liquid chromatography with electrochemical detection.