AANAT1 functions in astrocytes to regulate sleep homeostasis

Abstract

The monoamine catabolic enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by astrocytes and subsets of serotonergic, glutamatergic, GABAergic and cholinergic neurons in the adult brain of Drosophila.AANAT1 limits accumulation of serotonin and dopamine in the brain upon sleep deprivation.Loss of AANAT1 from astrocytes, but not from neurons, causes flies to increase their daytime rebound sleep in response to overnight sleep deprivation. 3 Summary Characteristic features of sleep are conserved among species [1], and from humans to insects sleep is influenced by neural circuits involving monoamines such as serotonin and dopamine [2]. Glial cells have been increasingly implicated in mechanisms of baseline and homeostatic sleep regulation in mammals and flies [3-11], but it remains unknown whether and how glia might influence monoaminergic control of sleep. Sleep is regulated by circadian rhythms and a homeostatic drive to compensate for prolonged wakefulness, andgrowing evidence suggests that neural mechanisms controlling homeostatic sleep can be discriminated from those controlling baseline sleep [12][13][14][15]. In Drosophila, mutants of arylalkylamine N-acetyltransferase 1 (AANAT1 lo ) have normal baseline amounts of sleep and motor activity, but increased rebound sleep following deprivation [16]. AANAT1 can acetylate and inactivate monoamines in vitro [17], but the role of AANAT1 in vivo remains poorly understood. We find AANAT1 to be expressed in astrocytes and subsets of neurons in the adult Drosophila brain, with levels in astrocytes declining markedly overnight. In sleep-deprived AANAT1 mutant flies, heightened rebound sleep is accompanied by increased serotonin and dopamine levels in the brain. In neurons, AANAT1 functions to limit the quantity and consolidation of nighttime sleep, but in astrocytes AANAT1 constrains the amount of rebound sleep that flies take in response to sleep deprivation.These findings distinguish sleep-control functions of AANAT1 in neurons and astrocytes, and identify a critical role for astrocytes in the regulation of monoamine bioavailability and calibration of the response to sleep need. 4 Results and DiscussionWe generated antiserum to AANAT1 (known previously as Dopamine acetyltransferase (Dat) and confirmed its specificity with immunohistochemistry (IHC) in the embryonic CNS. AANAT1 immunoreactivity was observed in the cytoplasm of many cells (Fig. 1A) but was absent in age-matched embryos that were homozygous for a deletion of the entire AANAT1 gene (Fig. 1B). In adult brains, AANAT1 was co-labeled with Bruchpilot (nc82, Fig. 1C), a presynaptic marker that labels neuropil regions. We found that AANAT1 was expressed in neurons (Elav + , Fig. 1D,D') and glia (Repo + , Fig.

speck, First Identified in Drosophila melanogaster in 1910, Is Encoded by the Arylalkalamine N-Acetyltransferase (AANAT1) Gene

“…In vertebrates, dopamine signaling is inactivated by a class of enzymes called monoamine oxidases (MAOs) that have, so far, not been identified in insects (reviewed in Yamamoto and Seto 2014). Localization of transcripts and protein has placed speck in astrocytes in adult Drosophila brains, where it could function to inactivate monoamine signaling in a similar mechanism to ebony (Croset et al 2018;Suh and Jackson 2007;Davla et al 2019). With the publication of the adult Drosophila brain "connectome" (Zheng et al 2018;Xu et al 2020), the identification of which synapses inactivate biogenic amines via AANAT1 acetylation may soon be deduced.…”

“…With the publication of the adult Drosophila brain "connectome" (Zheng et al 2018;Xu et al 2020), the identification of which synapses inactivate biogenic amines via AANAT1 acetylation may soon be deduced. Weak alleles of speck show a defect in sleep (Shaw et al 2000;Ganguly-Fitzgerald et al 2006;Davla et al 2019), as well as changes in monoamine levels (Davla et al 2019), but it is not known how its expression pattern relates to the emerging network of sleep in Drosophila (Bringmann 2018).…”

“…Neither speck mRNA or protein undergoes daily expression cycles in whole-head extracts (Brodbeck et al 1998); however, melatonin synthesis might only occur in a small subset of cells, and cycling might be undetectable in whole head extracts. Both speck mRNA and protein have been identified in a small subset of serotonergic neurons in adult brains (Croset et al 2018;Davla et al 2019), but it is not known if these neurons synthesize melatonin. Interestingly, melatonin synthesis is lost in mutants of the circadian rhythm gene period (Callebert et al 1991).…”

“…The Drosophila melanogaster AANAT1 enzyme is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily and transfers an acetyl group from acetyl-CoA to biogenic amines including dopamine, serotonin, and others via a sequential binding mechanism (Cheng et al 2012;Dempsey et al 2014a). The AANAT1 locus has two splice isoforms (RA and RB) and is expressed throughout development in a subset of gut, neuronal, and glial cells (Brodbeck et al 1998;Hintermann et al 1995;Hintermann et al 1996;Croset et al 2018;Davla et al 2019). A partial loss of function allele (AANAT1 lo ), which shows decreased levels of isoform RA, shows no pigmentation phenotype but has behavioral defects in sleep (Ganguly-Fitzgerald et al 2006;Shaw et al 2000;Davla et al 2019).…”

“…The AANAT1 locus has two splice isoforms (RA and RB) and is expressed throughout development in a subset of gut, neuronal, and glial cells (Brodbeck et al 1998;Hintermann et al 1995;Hintermann et al 1996;Croset et al 2018;Davla et al 2019). A partial loss of function allele (AANAT1 lo ), which shows decreased levels of isoform RA, shows no pigmentation phenotype but has behavioral defects in sleep (Ganguly-Fitzgerald et al 2006;Shaw et al 2000;Davla et al 2019). Strong loss of function phenotypes in Tribolium (flour beetle) (Noh et al 2016), Bombyx (silk moth) (Dai et al 2010;Zhan et al 2010), Zootermopsis (termite) (Masuoka and Maekawa 2016), and Oncopeltus (milkweed bug) (Liu et al 2016) show increased melanization, and AANAT1 has also been identified as the locus responsible for interspecies pigment differences in Drosophila (Ahmed-Braimah and Sweigart 2015).…”