Disrupted sleep is a major feature of Alzheimer’s disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (Aβ) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aβ affects sleep are unknown. We demonstrate in zebrafish that Aβ acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aβ oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aβ forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signaling cascade. Our data indicate that Aβ can trigger a bi-directional sleep/wake switch. Alterations to the brain’s Aβ oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signaling events.
Accumulation of amyloid-beta (Aβ) in plaques, along with tau tangles, is one of the two pathological hallmarks of Alzheimer’s disease (AD). Change in Aβ levels in the brain is one of the earliest known pathological events in AD and is detectable years before the development of Aβ plaques and decades before the clinical onset of AD (Bateman et al., 2007; Jack et al., 2013). Because of its importance in AD progression, Aβ has been mostly characterized as a functionless, pathological, intrinsically neurotoxic peptide (Moir and Tanzi, 2019). However, Aβ is an ancient neuropeptide conserved across vertebrates through at least 400 million years of evolution (Moir and Tanzi, 2019). Aβ’s cleavage from amyloid precursor protein (APP) is tightly regulated by multiple enzymatic reactions (O’Brien and Wong, 2011), and its release from neurons is carefully controlled (Kamenetz et al., 2003). Aβ interacts with numerous surface receptors and can activate intrinsic cellular signalling cascades to alter neuronal and synaptic function (Jarosz-Griffiths et al., 2016). More recently, Aβ has been suggested to act as an antimicrobial peptide (Soscia et al., 2010), and the deposition of Aβ may be induced as an innate immune defence mechanism against microbial pathogens (Kumar et al., 2016). However, the various biological effects of Aβ in health or disease remain obscure.
One of the earliest symptoms of AD is the disruption of sleep, and AD patients have sleep-wake abnormalities, including insomnia at night and increased napping during the day (Allen et al., 1987; Loewenstein et al., 1982; Moran et al., 2005; Prinz et al., 1982). Multiple transgenic AD mouse models that overproduce Aβ also show disrupted sleep phenotypes (Roh et al., 2012; Sterniczuk et al., 2010; Wang et al., 2002), often in the absence of neuronal loss and preceding impairments of learning and memory (Irizarry et al., 1997). In non-pathological conditions, Aβ levels in the cerebrospinal fluid (CSF) are modulated by the sleep-wake cycle (Kang et al., 2009; Xie et al., 2013). Aβ generation and release are controlled by electrical and synaptic activity (Cirrito et al., 2005; Kamenetz et al., 2003), leading to increased extracellular Aβ levels during wakefulness and decreased levels during sleep (Kang et al., 2009; Xie et al., 2013). These observations have led to the proposal that sleep and Aβ dynamics create a vicious feed-forward cycle, wherein increases in wakefulness result in increased extracellular Aβ and aggregation, which then dysregulates sleep, further exacerbating pathogenic Aβ production (Roh et al., 2012). How increased Aβ burden leads to disruptions in sleep remains unknown, although AD-related cell death of critical sleep/wake regulatory neurons has been suggested as a possible mechanism (Fronczek et al., 2012; Lim et al., 2014; Manaye et al., 2013).
Given the relationship between Aβ and sleep, we hypothesized that Aβ may directly modulate sleep-regulatory pathways independently of neuronal cell death. To test this, we took advantage of the ability to directly deliver small molecules and Aβ peptides to the brain of larval zebrafish, which have conserved APP processing machinery and Aβ peptides (Newman et al., 2014) and share genetic, pharmacological, and neuronal sleep-regulatory mechanisms with mammals (Barlow and Rihel, 2017). We found that Aβ size-dependently and reversibly modulates behavior through two distinct genetic, pharmacologically tractable pathways that regulate sleep in opposing directions.
Aβ dose-dependently modifies zebrafish sleep and wake behavior
Isolating the specific biological effects of Aβ has been experimentally difficult. One challenge is that Aβ is processed from a series of complex cleavage steps of a longer transmembrane protein, APP, which also produces other protein products with a variety of functions (O’Brien and Wong, 2011). This restricts the utility of genetic manipulations to tease out Aβ-specific roles from the other APP components. Another challenge is that Aβ forms, in vitro and in vivo, a variety of oligomeric species (e.g. dimers, longer oligomers, or large fibrils) with diverse structures, binding affinities, and signalling properties (Benilova et al., 2012; Jarosz-Griffiths et al., 2016). Teasing out the biological signalling capabilities of these diverse oligomeric species requires selective manipulation of Aβ oligomeric states, which is difficult in vitro and is currently nearly impossible endogenously in vivo.
To overcome some of these barriers, we developed an injection assay in which the amount and type of the Aβ oligomers can be controlled and then tested the acute signaling effects of Aβ on sleep and wake behavior. Our minimally invasive intra-cardiac injection assay in 5 days post fertilization (5 dpf) larval zebrafish avoids direct damage to brain tissue (Figure 1A and B). This technique rapidly (