ABSTRACT
The function of sleep remains one of biology's biggest mysteries. A solution to this problem is likely to come from a better understanding of sleep homeostasis, and in particular of the cellular and molecular processes that sense sleep need and settle sleep debt. Here, we highlight recent work in the fruit fly showing that changes in the mitochondrial redox state of sleep-promoting neurons lie at the heart of a homeostatic sleep-regulatory mechanism. Since the function of homeostatically controlled behaviours is often linked to the regulated variable itself, these findings corroborate with the hypothesis that sleep serves a metabolic function.
Subject(s)
Mitochondria , Sleep , Animals , Sleep/physiology , Mitochondria/metabolism , Drosophila/physiology , Sleep Deprivation , Homeostasis/physiologyABSTRACT
The essential but enigmatic functions of sleep1,2 must be reflected in molecular changes sensed by the brain's sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need4, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep5. Here we show that oxidative byproducts of mitochondrial electron transport6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain8,9 of Shaker's KVß subunit, Hyperkinetic10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP+-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep-three processes implicated independently in lifespan, ageing, and degenerative disease6,12-14-are thus mechanistically connected. KVß substrates8,15,16 or inhibitors that alter the ratio of bound NADPH to NADP+ (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs.
Subject(s)
Drosophila melanogaster/physiology , Mitochondria/metabolism , Potassium Channels/chemistry , Potassium Channels/metabolism , Protein Subunits/metabolism , Sleep/physiology , Action Potentials , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Electron Transport , Energy Metabolism , Female , Luminescent Proteins/metabolism , NADP/metabolism , Neurons/metabolism , Optogenetics , Oxidation-Reduction , Oxidative Stress , Oxidoreductases/metabolism , Protein Subunits/chemistry , Reactive Oxygen Species , Recombinant Fusion Proteins/metabolism , Shaker Superfamily of Potassium Channels/metabolism , Sleep Aids, Pharmaceutical , Time FactorsABSTRACT
Sleep-promoting neurons in the dorsal fan-shaped body (dFB) of Drosophila are integral to sleep homeostasis, but how these cells impose sleep on the organism is unknown. We report that dFB neurons communicate via inhibitory transmitters, including allatostatin-A (AstA), with interneurons connecting the superior arch with the ellipsoid body of the central complex. These "helicon cells" express the galanin receptor homolog AstA-R1, respond to visual input, gate locomotion, and are inhibited by AstA, suggesting that dFB neurons promote rest by suppressing visually guided movement. Sleep changes caused by enhanced or diminished allatostatinergic transmission from dFB neurons and by inhibition or optogenetic stimulation of helicon cells support this notion. Helicon cells provide excitation to R2 neurons of the ellipsoid body, whose activity-dependent plasticity signals rising sleep pressure to the dFB. By virtue of this autoregulatory loop, dFB-mediated inhibition interrupts processes that incur a sleep debt, allowing restorative sleep to rebalance the books. VIDEO ABSTRACT.
Subject(s)
Drosophila melanogaster/physiology , Interneurons/physiology , Sleep/physiology , Animals , Brain/physiology , Circadian Rhythm , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Excitatory Postsynaptic Potentials/physiology , Female , Homeostasis , Insect Hormones/physiology , Light , Locomotion/radiation effects , Male , Membrane Potentials , Nerve Tissue Proteins/physiology , Neurons/physiology , Optogenetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/physiology , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/physiology , Recombinant Fusion Proteins/metabolism , Vision, OcularABSTRACT
Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.
Subject(s)
Cell Movement/physiology , Cell Shape/physiology , Heparan Sulfate Proteoglycans/metabolism , Neurites/metabolism , Neuronal Outgrowth/physiology , Nogo Proteins/metabolism , Animals , Carrier Proteins/metabolism , Cell Line , Cells, Cultured , Heparitin Sulfate/metabolism , Mice , Protein Binding , Proteoglycans/metabolism , Receptors, Lysosphingolipid/metabolismABSTRACT
Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A(-/-) mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity.
Subject(s)
Hippocampus/metabolism , Motor Cortex/metabolism , Myelin Proteins/genetics , Neuronal Plasticity/genetics , Receptors, Lysosphingolipid/genetics , Animals , Cell Proliferation , GTP-Binding Protein alpha Subunits, G12-G13/genetics , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , Gene Expression Regulation , Hippocampus/cytology , Long-Term Potentiation , Lysophospholipids/metabolism , Mice , Mice, Knockout , Motor Cortex/cytology , Myelin Proteins/deficiency , Myelin Sheath/genetics , Myelin Sheath/metabolism , Neurites/metabolism , Nogo Proteins , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine-1-Phosphate Receptors , Synapses/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding ProteinABSTRACT
Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.
Subject(s)
Axons/physiology , Cerebral Cortex/metabolism , Ganglia, Spinal/metabolism , Myelin Proteins/metabolism , Spinal Cord Regeneration , Up-Regulation , Animals , Axons/metabolism , Cells, Cultured , Cerebral Cortex/pathology , Cerebral Cortex/physiology , Ephrin-A3/genetics , Ephrin-A3/metabolism , Ephrin-A4/genetics , Ephrin-A4/metabolism , Ganglia, Spinal/pathology , Ganglia, Spinal/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Proteins/genetics , Myelin Sheath/genetics , Myelin Sheath/metabolism , Nogo Proteins , Pyramidal Tracts/metabolism , Pyramidal Tracts/pathology , Pyramidal Tracts/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spinal Cord Injuries/metabolismABSTRACT
Nogo-A was initially discovered as a myelin-associated growth inhibitory protein limiting axonal regeneration after central nervous system (CNS) injury. This review summarizes current knowledge on how myelin and neuronal Nogo-A and its receptors exert physiological functions ranging from the regulation of growth suppression to synaptic plasticity in the developing and adult intact CNS.
Subject(s)
Central Nervous System/metabolism , Myelin Proteins/metabolism , Neuronal Plasticity , Neurons/metabolism , Synaptic Transmission , Animals , Central Nervous System/growth & development , Central Nervous System/injuries , Central Nervous System/pathology , Humans , Myelin Sheath/metabolism , Nerve Regeneration , Nogo Proteins , Receptors, Cell Surface/metabolism , Trauma, Nervous System/metabolism , Trauma, Nervous System/pathologyABSTRACT
Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.
