Genetic and chemical disruption of amyloid precursor protein processing impairs zebrafish sleep maintenance.

Amyloid precursor protein (APP) is a brain-rich, single pass transmembrane protein that is proteolytically processed into multiple products, including amyloid-beta (Aß), a major driver of Alzheimer disease (AD). Although both overexpression of APP and exogenously delivered Aß lead to changes in sleep, whether APP processing plays an endogenous role in regulating sleep is unknown. Here, we demonstrate that APP processing into Aß40 and Aß42 is conserved in zebrafish and then describe sleep/wake phenotypes in loss-of-function appa and appb mutants. Larvae with mutations in appa had reduced waking activity, whereas larvae that lacked appb had shortened sleep bout durations at night. Treatment with the γ-secretase inhibitor DAPT also shortened night sleep bouts, whereas the BACE-1 inhibitor lanabecestat lengthened sleep bouts. Intraventricular injection of P3 also shortened night sleep bouts, suggesting that the proper balance of Appb proteolytic processing is required for normal sleep maintenance in zebrafish.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Sleep is bi-directionally modified by amyloid beta oligomers.

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 (Aß) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aß affects sleep are unknown. We demonstrate in zebrafish that Aß acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aß oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aß forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signaling cascade. Our data indicate that Aß can trigger a bi-directional sleep/wake switch. Alterations to the brain's Aß oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signaling events.

Wnt/ß-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro.

Here, we investigated the role of the Wnt/ß-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of ß-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a ß-catenin-mediated Wnt signaling activator (6-bromoindirubin-3'-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of ß-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that ß-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of ß-catenin and activation of its downstream genes.