Biology subjects

Rihel, J.

Publications and source records attributed to Rihel, J..

18 recordsLinked to original sources

bioRxiv2026

DPYD links glial lipids and myelination to sleep duration

The regulation of sleep is heritable and conserved across species1,2, but the governing genetic mechanisms remain poorly understood. Here, we investigate the role of DPYD--which encodes the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase--in sleep. Using a cross-species approach, we show that DPYD and its orthologs act specifically in glia to promote sleep through the regulation of brain lipids. In mouse and zebrafish, we find that DPYD orthologs are enriched in oligodendrocyte lineage cells, and their loss reduces myelination and sleep independent of DPYDs established role in uracil catabolism to {beta}-alanine. Genetic and pharmacological manipulations of myelin in zebrafish show that altering myelination is sufficient to perturb sleep duration. In Drosophila, which lack myelin, we find that the DPYD ortholog acts in ensheathing glia to promote sleep through formation of glial lipid droplets, a proposed evolutionary precursor to myelin. Furthermore, a human GWAS-identified sleep variant2 resides in a conserved cis-regulatory element that selectively contacts the promoter of DPYD within oligodendrocytes, connecting the human genetic association to oligodendrocyte-specific control of DPYD. Together, these results identify a deeply conserved role for glial lipids in sleep regulation and establish DPYD as a mechanistic link between myelin biology and sleep.

neuroscience↗

bioRxiv2026

A novel pipeline for the validation of manganese chelators for the treatment of manganese overload

Manganese neurotoxicity, arising from environmental overexposure or inherited transporter disorders due to pathogenic variants in SLC30A10 and SLC39A14, leads to manganism, a debilitating Parkinsonian movement disorder. Alhtough chelation therapy can partially reverse neuropathology, current clinical practice relies on intravenous CaNa2EDTA, which is burdensome and poorly suited for long-term use. Consequently, there remains a significant unmet need for more effective, orally bioavailable chelators. This study aimed to establish and validate a pipeline for identifying and assessing novel ligands that attenuate manganese neurotoxicity and support preclinical translational development. Based on the structural features of manganese-based MRI contrast agents, we selected two chelators, N-picolyl-N,N',N'-trans-1,2-cyclohexylenediaminetriacetic acid (H3PyC3A) and ethylenediaminetetraacetic acid-benzothiazole aniline (H4EDTA-BTA), and their methyl ester derivatives, Me3PyC3A and Me4EDTA-BTA. These were evaluated in vivo using zebrafish (slc39a14U801/U801) and mouse (Slc30a10KO/KO) models of manganese overload. H3PyC3A and Me3PyC3A demonstrated greater manganese-mobilizing efficacy than CaNa2EDTA, improving locomotor behavior in slc39a14U801/U801 zebrafish. In Slc30a10KO/KO mice, intravenous administration confirmed selective in vivo chelation of excess manganese over physiological concentrations of zinc and copper. Although oral bioavailability was low (<1%), long-term oral administration of H3PyC3A modestly reduced liver and brain Mn accumulation, suggesting an added benefit of oral administration via gastrointestinal chelation. This integrated in vitro to in vivo pipeline provides a robust and scaleable approach for the development of next-generation Mn chelators. Slc39a14U801 loss-of-function zebrafish enable high throughput identification of candidate compounds while Slc30a10KO/KO mice offer a clinically relevant disease model for pharmacokinetic profiling and proof-of-concept validation.

pharmacology and toxicology↗

bioRxiv2025

A meningeal circadian clock and brain state co-ordinately enhance macromolecular clearance during wake

The removal of brain waste is thought to be regulated by the circadian clock and sleep-wake cycle. A meningeal scavenger cell population, called mural Lymphatic Endothelial Cells (muLECs), uptake macromolecules from the cerebrospinal fluid (CSF) and are poised to clear soluble brain waste. Whether CSF clearance is regulated across the 24-hour day is unknown. Using a quantitative, in vivo assay in zebrafish, we show that the capacity of muLECs to remove macromolecules is highest during the waking day. The muLEC protein uptake is regulated by a cell-autonomous circadian oscillator that drives rhythmic protein levels of Mannose Receptor 1a (Mrc1a); in contrast, lipoprotein uptake increases as a function of time spent awake. Thus, circadian and brain state cues independently maximize muLECs function during wake. Consistently, muLECs are required for normal recovery after the heightened metabolic demand of a seizure, suggesting that muLEC clearance contributes to both normal and disrupted brain homeostasis. Impact StatementA meningeal scavenger cell population called Mural Lymphatic Endothelial Cells preferentially clear macromolecules from the CSF during the waking day, and this uptake is regulated independently by brain state and circadian cues in a substrate-specific manner.

neuroscience↗

bioRxiv2025

Neural microexons modulate arousal states via cAMP signalling in zebrafish

Arousal states, often dysregulated in neurodevelopmental disorders, shape how organisms perceive and respond to their environment. Here, we show that srrm3, a master regulator of neural microexons, is essential for normal arousal in zebrafish larvae. srrm3 mutants exhibit persistent hyperarousal, including sleep loss, sensory hypersensitivity, anxiety-like behaviour, and heightened neural and behavioural activity. Elevated cAMP signalling likely drives this hyperarousal, as pharmacologically reducing cAMP rescues mutant behaviour, while increasing cAMP in wild-type larvae phenocopies the mutant hyperaroused state. Pharmacological cAMP modulation also mimics and reverses srrm3-dependent transcriptional changes. These include immediate early gene downregulation, which, together with altered activity-dependent transcription factor motif occupancy, suggest adaptation to sustained neuronal hyperactivity. Additionally, srrm3 mutants show upregulation of microexon- containing genes, likely compensating for microexon loss. Together, these findings reveal a role for neural microexons in shaping arousal via cAMP signalling, providing insight into how splicing defects may underlie sensory and sleep disturbances.

neuroscience↗

bioRxiv2025

JACUZI-SD: An automated, high-throughput, minimally stressful approach to sleep depriving larval zebrafish.

While sleep deprivation broadly disrupts health and well-being, the neural and molecular mechanisms that signal increased sleep pressure remain poorly understood. A key obstacle to progress is the fact that traditional methods for inducing sleep deprivation (SD) in animal models often introduce confounding stress or disrupt circadian rhythms. Here, we present JACUZI-SD (Jetting Automated Currents Under Zebrafish to Induce Sleep Deprivation), a fully automated, high-throughput system designed to induce sleep deprivation in larval zebrafish with minimal stress. By delivering randomized water pulses via a custom milli-fluidic device integrated with a 96-well plate and controlled by an Arduino, JACUZI-SD promotes wakefulness during the natural dark cycle without the stress associated with existing SD methods. Our results demonstrate that JACUZI-SD reduces total sleep time by 41-64% and elicits a robust rebound sleep characterized by increased sleep bout length following deprivation. Importantly, this method avoids activating the hypothalamic-pituitary-interrenal (HPI) stress axis, as evidenced by reduced stress marker expression compared to other deprivation methods. Additionally, the system reliably activates established sleep pressure pathways, including the upregulation of galanin in the neurosecretory preoptic area, while also revealing biologically relevant inter-individual variability in homeostatic rebound responses. JACUZI-SD provides a powerful, minimally invasive platform for dissecting the neural and molecular underpinnings of sleep homeostasis in vertebrates.

neuroscience↗

bioRxiv2024

Sleep Disruption Improves Performance in Simple Olfactory and Visual Decision-Making Tasks

Sleep disruption drastically impacts cognitive functions including decision-making and attention across many different species. In this study, we leveraged the small size and conserved vertebrate brain structure of larval zebrafish to investigate how sleep disruption modulates visual-and olfactory-decision-making. Strikingly, sleep disruption improved performance in both paradigms. Specifically, sleep disruption lengthens reaction times and increases correct decisions in a visual motion discrimination task, an effect that we attribute to longer integration periods in disrupted animals. Using a drift diffusion model, we predict specific circuit changes underlying these effects. Additionally, we demonstrate that sleep disruption heightens odor sensitivity in an olfactory decision-making task, likely mediated by cortisol. Our findings lay essential groundwork for investigating the brain circuit changes that arise from sleep disruption across species.

neuroscience↗

bioRxiv2024

Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

neuroscience↗

bioRxiv2023

Behavioural pharmacology predicts disrupted signalling pathways and candidate therapeutics from zebrafish mutants of Alzheimer's disease risk genes

By exposing genes associated with disease, genomic studies provide hundreds of starting points that should lead to druggable processes. However, our ability to systematically translate these genomic findings into biological pathways remains limited. Here, we combine rapid loss-of-function mutagenesis of Alzheimers risk genes and behavioural pharmacology in zebrafish to predict disrupted processes and candidate therapeutics. FramebyFrame, our expanded package for the analysis of larval behaviours, revealed that decreased night-time sleep was common to F0 knockouts of all four late-onset Alzheimers risk genes tested. We developed an online tool, ZOLTAR, which compares any behavioural fingerprint to a library of fingerprints from larvae treated with 3,677 compounds. ZOLTAR successfully predicted that sorl1 mutants have disrupted serotonin signalling and identified betamethasone as a drug which normalises the excessive day-time sleep of presenilin-2 knockout larvae with minimal side effects. Predictive behavioural pharmacology offers a general framework to rapidly link disease-associated genes to druggable pathways.

neuroscience↗

bioRxiv2023

Sleep pressure modulates single-neuron synapse dynamics in zebrafish

Sleep is a nearly universal behaviour with unclear functions1. The Synaptic Homeostasis Hypothesis (SHY) proposes that sleep is required to renormalize the increases in synaptic number and strength that occur during wakefulness2. Some studies examining either large neuronal populations3 or small patches of dendrites4 have found evidence consistent with SHY, but whether sleep merely serves as a permissive state or actively promotes synaptic downregulation at the scale of whole neurons is unknown. Here, by repeatedly imaging all excitatory synapses on single neurons across sleep/wake states of zebrafish larvae, we show that synapses are gained during periods of wake (either spontaneous or forced) and lost during sleep in a neuron-subtype dependent manner. However, synapse loss is greatest during sleep associated with high sleep pressure following prolonged wakefulness and low in the latter half of the night. Conversely, sleep induced pharmacologically during periods of low sleep pressure is insufficient to trigger synapse loss unless adenosine levels are boosted while noradrenergic tone is inhibited. We conclude that sleep-dependent synapse loss is regulated by sleep pressure at the level of the single neuron and that not all sleep periods are equally capable of fulfilling the functions of synaptic homeostasis.

neuroscience↗

bioRxiv2023

Noradrenergic Tone is Not Required for Neuronal Activity-Induced Rebound Sleep in Zebrafish

Sleep pressure builds during wakefulness, but the mechanisms underlying this homeostatic process are poorly understood. One zebrafish model suggests that sleep pressure increases as a function of global neuronal activity, such as during sleep deprivation or acute exposure to drugs that induce widespread brain activation. Given that the arousal-promoting noradrenergic system is important for maintaining heightened neuronal activity during wakefulness, we hypothesised that genetic and pharmacological reduction of noradrenergic tone during drug-induced neuronal activation would dampen subsequent rebound sleep in zebrafish larvae. Unexpectedly, dampening noradrenergic tone with the 2-adrenoceptor agonist clonidine during acute caffeine or pentylenetetrazol treatment enhanced subsequent rebound sleep, while stimulating noradrenergic signalling during caffeine exposure with a cocktail of 1- and {beta}-adrenoceptor agonists did not enhance sleep. Similarly, CRISPR/Cas9-mediated elimination of the dopamine {beta}-hydroxylase (dbh) gene, which encodes an enzyme required for noradrenalin synthesis, enhanced baseline sleep in larvae but did not prevent additional rebound sleep following acute induction of neuronal activity. Across all drug conditions, c-fos expression immediately after drug exposure varied inversely with noradrenergic tone and correlated strongly with the amount of induced rebound sleep. These results are consistent with a model in which increases in neuronal activity, as reflected by brain-wide levels of c-fos induction, drive a sleep pressure signal that promotes rebound sleep independently of noradrenergic tone.

neuroscience↗

bioRxiv2022

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains has recently been found to contain cryptic open reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here we show that two putative lincRNAs (linc-mipep and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings highlight the power of screening for unexplored micropeptide functions by revealing a key missing link in the evolution of vertebrate brain cell development and illustrating a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

developmental biology↗

bioRxiv2022

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 (A{beta}), a major driver of Alzheimers disease (AD). Although both over-expression of APP, as in mouse models used to study AD, and exogenously delivered A{beta} lead to changes in sleep behaviors, whether APP processing plays an endogenous role in regulating sleep is unknown. Here, we demonstrate that APP processing into A{beta}40 and A{beta}42 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 but normal sleep patterns, while larvae that lacked appb had shortened sleep bout durations at night. Treatment with the {gamma}-secretase inhibitor DAPT also shortened sleep bouts at night, while the BACE-1 inhibitor lanabecestat lengthened sleep bouts. Since both drugs fail to further alter sleep in appb mutants, and intraventricular injection of the App cleavage product P3 also shortens night-time sleep bouts, we conclude that the proper balance of Appb proteolytic processing is required for normal sleep maintenance in zebrafish.

neuroscience↗

bioRxiv2020

A genetic screen identifies dreammist as a regulator of sleep

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. Significance statementSleep is an essential behavioral state, but the genes that regulate sleep and wake states are still being uncovered. A viral insertion screen in zebrafish identified a novel sleep mutant called dreammist, in which a small, highly-conserved transmembrane protein is disrupted. The discovery of dreammist highlights the importance of a class of small transmembrane-protein modulators of the sodium pump in setting appropriate sleep duration.

neuroscience↗

bioRxiv2020

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.

genetics↗

bioRxiv2019

Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

developmental biology↗

bioRxiv2019

Hierarchical Compression Reveals Sub-Second to Day-Long Structure in Larval Zebrafish Behaviour

Animal behaviour is dynamic, evolving over multiple timescales from milliseconds to days and even across a lifetime. To understand the mechanisms governing these dynamics, it is necessary to capture multi-timescale structure from behavioural data. Here, we develop computational tools and study the behaviour of hundreds of larval zebrafish tracked continuously across multiple 24-hour day/night cycles. We extracted millions of movements and pauses, termed bouts, and used unsupervised learning to reduce each larvas behaviour to an alternating sequence of active and inactive bout types, termed modules. Through hierarchical compression, we identified recurrent behavioural patterns, termed motifs. Module and motif usage varied across the day/night cycle, revealing structure at sub-second to day-long timescales. We further demonstrate that module and motif analysis can uncover novel pharmacological and genetic mutant phenotypes. Overall, our work reveals the organisation of larval zebrafish behaviour at multiple timescales and provides tools to identify structure from large-scale behavioural datasets.

neuroscience↗

bioRxiv2019

Bi-directional modification of sleep and wake by amyloid beta oligomers

Disrupted sleep is a major feature of Alzheimers Disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (A{beta}) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which A{beta} affects sleep are unknown. We demonstrate in zebrafish that A{beta} acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short A{beta} oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer A{beta} forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signalling cascade. Our data indicate that A{beta} can trigger a bi-directional sleep/wake switch. Alterations to the brains A{beta} oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signalling events. HIGHLIGHTSO_LIAmyloid beta oligomers can drive either sleep or wakefulness, depending on their size C_LIO_LIWakefulness driven by short amyloid beta oligomers requires binding partners Adrenergic Beta Receptor 2 and Pgrmc1 C_LIO_LILong amyloid beta oligomers drive sleep through interaction with Prion Protein C_LIO_LIThe in vivo sleep effects of amyloid beta can be pharmacologically blocked by targeting several steps of the Amyloid beta-Prion Protein signalling cascade. C_LI

neuroscience↗

bioRxiv2018

Neuronal activity drives homeostatic sleep through engagement of the hypothalamic neuropeptide galanin

Sleep pressure homeostatically increases during wake and dissipates during sleep, but the molecular signals and neuronal substrates that measure homeostatic sleep pressure remain poorly understood. A pharmacological assay that generates acute, short-term increases in wakefulness in larval zebrafish, revealed that subsequent rebound sleep is dependent on the intensity of global neuronal activity. Whole brain activity mapping identified preoptic Galanin expressing neurons as selectively active during rebound sleep, and the induction of galanin transcripts was predictive of total rebound sleep time. Galanin is required for sleep homeostasis, as galanin mutants almost completely lack rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galanin expressing neurons play a key role in integrating sleep pressure signals derived from global neuronal activity and act as an output arm for the vertebrate sleep homeostat. (word count: 129).

neuroscience↗