Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila
Introduction
Alzheimer's disease (AD) is characterized by progressive neurodegeneration resulting in the loss of cognitive ability. The exact pathology of AD is not well understood and is widely debated. The amyloid cascade hypothesis suggests that abnormal production of neurotoxic Amyloid-beta (Aβ) in combination with tangles of phosphorylated Tau-microtubule associated protein lead to neuronal dysfunction, cell loss and thus cognitive decline (Hardy and Selkoe, 2002, Mandelkow and Mandelkow, 1998, Selkoe, 2000). The crucial enzyme in the production of Aβ is the rate-limiting Beta-site Amyloid Precursor Protein Cleaving Enzyme (BACE), which shows elevated expression in AD patients (Fukumoto et al., 2002, Vassar et al., 2009). Recently, knock-in of human BACE was shown to recapitulate many behavioral and physiological phenotypes of AD in a murine model (Plucinska et al., 2014). In addition to BACE, Amyloid Precursor Protein (APP) is cleaved by an α-secretase. Both cleavage pathways yield a secreted N-terminal fragment known as sAPP, sAPPβ for β-cleavage and sAPPα for α-cleavage. Subsequent ϒ-cleavage following β or α-cleavage results in either the Aβ or P3 fragment, respectively (De Strooper and Annaert, 2000, Selkoe, 2000, Turner et al., 2003). In addition to those two fragments, ϒ-cleavage also produces the APP intracellular domain (AICD), which has been shown to affect transcriptional regulation (Belyaev et al., 2010, Kimberly et al., 2001, Pardossi-Piquard and Checler, 2012, Turner et al., 2003). Both detrimental and positive effects of the AICD have been reported in cultured cells (Lu et al., 2000, Zhou et al., 2012); however, the physiological function of the AICD remains poorly understood.
One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps (Harper et al., 2005, Volicer et al., 2001, Wu and Swaab, 2007). Impaired rest-activity rhythms have also been reported in experimental AD model mice (Roh et al., 2012, Sterniczuk et al., 2010). Rest-activity cycles are generated by a molecular system known as the circadian clock. The circadian clock mechanism is based on negative feedback loops involving transcriptional activators and repressors, which are largely conserved from Drosophila to humans (Hardin and Panda, 2013). Circadian clocks generate daily rhythms in expression of many output genes leading to cellular, physiological, and behavioral rhythms. Loss of circadian rhythms is detrimental to health and adversely affects neuronal homeostasis in both murine (Hastings and Goedert, 2013, Kondratova and Kondratov, 2012, Reddy and O'Neill, 2010) and Drosophila models (Krishnan et al., 2012). Therefore, it is important to understand the connections between the circadian system and AD pathology.
The links between AD and circadian rhythms were inferred from transgenic model animals expressing pathogenic versions of human genes (Rezaval et al., 2008, Roh et al., 2012, Sterniczuk et al., 2010). Recent reports showed that pathogenic human amyloid peptides disrupt behavioral rest-activity rhythms in Drosophila but are not sufficient to disrupt molecular oscillations of PERIOD (PER) in central pacemaker neurons (Chen et al., 2014, Long et al., 2014). Here, we manipulated AD-related genes in the fruit fly, Drosophila melanogaster in order to understand endogenous pathways that may interfere with the circadian system. APP has a functional ortholog in Drosophila called Amyloid Precursor Protein-like (APPL) (Luo et al., 1990, Luo et al., 1992, Rosen et al., 1989). In addition, orthologs of BACE, the α-secretase, and the ϒ-secretase complexes have been identified in Drosophila. They are known as dBACE, Kuzbanian (KUZ) and Presenilin (PSN), respectively, and were shown to process APPL resulting in peptide fragments comparable to APP fragments (Bolkan et al., 2012, Carmine-Simmen et al., 2009, Greeve et al., 2004). This conservation made Drosophila a suitable model for our study. Because BACE is the rate-limiting enzyme in the production of Aβ, we first investigated if over-expression of dBACE is involved in the rest-activity disturbances characteristic of AD.
We found that increasing dBACE expression disrupted endogenous rest-activity rhythms. This effect was most severe in aged flies suggesting an age-dependent mechanism. Furthermore, dBACE expression resulted in the dampened oscillation of the core clock protein PER in central pacemaker neurons, which are master regulators of rest-activity rhythms. Surprisingly, over-expression of Kuzbanian (KUZ) also disrupted rest-activity rhythms suggesting a mechanism independent of endogenous Drosophila Aβ peptide (dAβ) production, which like its vertebrate ortholog has been shown to be neurotoxic (Carmine-Simmen et al., 2009). This suggested that rhythm deficits were due to a cleavage product of APPL conserved in both α and β cleavage pathways. Therefore, we expressed the APPL intracellular domain (dAICD) and this caused severe disruption of behavioral rest-activity rhythms. In contrast, expression of full-length APPL in central clock neurons protected against an age-dependent decline in rest-activity rhythms. Taken together, these findings suggest that APPL and specifically the dAICD can affect circadian behavior by interfering with molecular clock oscillations.
Section snippets
Fly stocks
D. melanogaster were reared on diet containing 1% agar, 6.25% cornmeal, 6.25% molasses, and 3.5% Red Star yeast at 25 °C. Flies were entrained to 12-hour light:dark (LD, 12:12) cycles (with an average light intensity of ~ 1500 lx). All experiments were performed on mated male flies of different ages, as specified in the results. We used the binary UAS–GAL4 system to express specific genes using transgenic flies carrying the following constructs: UAS–dBACE, UAS–APPL, UAS–dAICD (Carmine-Simmen et
Over-expression of dBACE in clock cells accelerates aging phenotypes and disrupts rest-activity rhythms
To examine effects of elevated dBACE in flies, we expressed dBACE in all clock cells using the timeless (tim-GAL4) driver. We first tested whether this affected lifespan, and found a significant reduction (p < 0.0001) in tim > dBACE flies with a median lifespan of 61d compared to 82d for tim > GFP controls (Fig. 1A). Aging also was accelerated in tim > dBACE flies as they had significantly reduced climbing ability (p < 0.0001) at 35d and 50d compared to age-matched controls (Fig. 1B). We next
Discussion
Loss of rest-activity rhythms is a well-established early symptom of AD in humans. Because disruption of circadian rhythms is detrimental to neuronal homeostasis (Kondratova and Kondratov, 2012, Krishnan et al., 2012), it is important to understand relationships between AD and circadian rhythms at the cellular and molecular levels. To address this question, we examined how manipulations of the fly ortholog of APP and its cleaving enzymes affect endogenous rest-activity rhythms and clock
Acknowledgments
We thank Dr. Taishi Yoshii for advice on immunocytochemistry, Dr. R Stanewsky for anti-PER antibody, Dr. Philip Copenhaver for anti-AICD antibody, and Dani Long for reading the manuscript. Research reported in this publication was supported by the National Institute of Aging of the National Institutes of Health under award number R01 AG045830 to JMG and by a pilot project grant from the Oregon Institute of Occupational Health Sciences to DK.
References (53)
The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a {beta}-secretase-dependent pathway
J. Biol. Chem.
(2010)Neurotoxic effects induced by the Drosophila amyloid-beta peptide suggest a conserved toxic function
Neurobiol. Dis.
(2009)Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila
Exp. Gerontol.
(2005)Systems approaches to biological rhythms in Drosophila
Methods Enzymol.
(2005)- et al.
Circadian timekeeping and output mechanisms in animals
Curr. Opin. Neurobiol.
(2013) Disturbance of endogenous circadian rhythm in aging and Alzheimer disease
Am. J. Geriatr. Psychiatr.
(2005)- et al.
Circadian clocks and neurodegenerative diseases: time to aggregate?
Curr. Opin. Neurobiol
(2013) The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner
J. Biol. Chem.
(2001)Loss of circadian clock accelerates aging in neurodegeneration-prone mutants
Neurobiol. Dis.
(2012)Human amyloid precursor protein ameliorates behavioral deficit of flies deleted for APPL gene
Neuron
(1992)
Tau in Alzheimer's disease
Trends Cell Biol.
A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock
Cell
Healthy clocks, healthy body, healthy mind
Trends Cell Biol.
A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila [published erratum appears in Cell 2000 Mar 31;101(1): following 113]
Cell
Characterization of the 3xTg-AD mouse model of Alzheimer's disease: Part 1. Circadian changes
Brain Res.
Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory
Prog. Neurobiol.
Amyloid precursor proteins are protective in Drosophila models of progressive neurodegeneration
Neurobiol. Dis.
Disturbance and strategies for reactivation of the circadian rhythm system in aging and Alzheimer's disease
Sleep Med.
The APP intracellular domain (AICD) inhibits Wnt signalling and promotes neurite outgrowth
Biochim. Biophys. Acta
Beta-secretase cleavage of the fly amyloid precursor protein is required for glial survival
J. Neurosci.
The central molecular clock is robust in the face of behavioural arrhythmia in a Drosophila model of Alzheimer's disease
Dis. Model. Mech.
Proteolytic processing and cell biological functions of the amyloid precursor protein
J. Cell Sci.
Alpha, beta- and gamma-secretases in Alzheimer's disease
Front. Biosci. (Schol. Ed.)
Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease
Arch. Neurol.
APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation
PLoS One
Drosophila amyloid precursor protein-like is required for long-term memory
J. Neurosci.
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Present address: Baxter Lab for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
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Present address: Department of Animal Physiology, Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland.