OBJECTIVE:The circadian clock anticipates daily repetitive events to adapt physiological processes. In mammals, the circadian system consists of a master clock in the suprachiasmatic nucleus (SCN), which synchronizes subordinate tissue clocks, including extra-SCN central nervous system (CNS) clocks involved in functions such as sleep and appetite regulation. Appetite is controlled by both homeostatic and non-homeostatic (hedonic) circuits. Homeostatic appetite addresses energy needs, while hedonic feeding targets cravings for palatable, calorie-dense foods. The adipokine leptin is a major appetite regulator, interacting with the circadian clock. Although leptin's role in satiation through its action in the mediobasal hypothalamus (MBH) is well established, its involvement in the circadian regulation of feeding remains poorly understood. We hypothesized that circadian gating of leptin signaling in the CNS controls homeostatic and hedonic appetite across the day. METHODS:We analyzed food intake rhythms in mice with a loss of leptin (ob/ob mice) or clock function (Per1/2 or Bmal1 KO) and in mice with specific disruption of leptin circadian gating in the CNS (ObRb.Bmal1). RESULTS:We found that in leptin-deficient mice hedonic appetite increases specifically in the early rest phase. In contrast, clock-deficient Per1/2 mutant mice exhibit blunted rhythms in both hedonic and homeostatic appetite control. Finally, when clock function is disrupted in leptin-sensitive neurons only, mice display a lower sensitivity to palatable food, along with reduced initial weight gain and adipose hypertrophy under obesogenic diet conditions. CONCLUSIONS:Our data describe a local clock-controlled central leptin gating mechanism that modulates hedonic food intake rhythms and impacts metabolic homeostasis.

Zooplankton are important eukaryotic constituents of marine ecosystems characterized by limited motility in the water. These metazoans predominantly occupy intermediate trophic levels and energetically link primary producers to higher trophic levels. Through processes including diel vertical migration (DVM) and production of sinking pellets they also contribute to the biological carbon pump which regulates atmospheric CO2 levels. Despite their prominent role in marine ecosystems, and perhaps, because of their staggering diversity, much remains to be discovered about zooplankton biology. In particular, the circadian clock, which is known to affect important processes such as DVM has been characterized only in a handful of zooplankton species. We present annotated de novo assembled transcriptomes from a diverse, representative cohort of 17 marine zooplankton representing six phyla and eight classes. These transcriptomes represent the first sequencing data for a number of these species. Subsequently, using translated proteomes derived from this data, we demonstrate in silico the presence of orthologs to most core circadian clock proteins from model metazoans in all sequenced species. Our findings, bolstered by sequence searches against publicly available data, indicate that the molecular machinery underpinning endogenous circadian clocks is widespread and potentially well conserved across marine zooplankton taxa.

The choroid plexus secrets cerebrospinal fluid (CSF) composed of electrolytes, cytokines, growth factors, metabolites and extracellular vesicles (EVs) that flow through the interconnected brain ventricles. On their course, CSF components can act as signals that affect, for example, neural stem cells (NSCs) residing in niches of the ventricular wall. We studied EV-born CSF signals in an in vitro culture system. We purified EVs from the secretome of a choroid plexus cell line (Z310 cells), and from primary choroid plexus cultures and co-cultured those EVs with NSCs isolated from the niche of the lateral and the third ventricle. EVs(Z310) and EVs(CHP) were purified by differential centrifugation. This yielded fractions of EVs of 50-150-nm diameter that induced a complex multicellular network formation and NSC differentiation. Both types of EV converted the round NSCs to cells that extended long processes that contacted nearby, alike-shaped cells. Mass spectrometry showed that the differentiation-inducing EVZ310 were enriched for membrane and membrane-associated proteins involved in cell differentiation, membrane trafficking, and membrane organization. We hypothesize that this type of EV (Z310) cargo causes changes of stem cell morphology that leads to multicellular networks in the niches. This cell-shape transition may represent an initial step in NSC differentiation.

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron-specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked h αSyn TP or h αSyn Δ119 (h stands for “human”) into the murine αSyn locus. hαSyn TP is a structure-based mutant with t riple alanine to p roline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster . hαSyn Δ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of h αSyn TP or h αSyn Δ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that h αSyn TP and h αSyn Δ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSyn TP and hαSyn Δ119 mice, despite the lower levels of hαSyn TP and hαSyn Δ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSyn Δ119 mice, while a compulsive behavior was exclusively detected in hαSyn TP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.

Artificial lighting, day-length changes, shift work, and transmeridian travel all lead to sleep-wake disturbances. The nychthemeral sleep-wake cycle (SWc) is known to be controlled by output from the central circadian clock in the suprachiasmatic nuclei (SCN), which is entrained to the light-dark cycle. Additionally, via intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (Opn4), short-term light-dark alternations exert direct and acute influences on sleep and waking. However, the extent to which longer exposures typically experienced across the 24-h day exert such an effect has never been clarified or quantified, as disentangling sustained direct light effects (SDLE) from circadian effects is difficult. Recording sleep in mice lacking a circadian pacemaker, either through transgenesis (Syt10(cre/cre)Bmal1(fl/-)) or SCN lesioning and/or melanopsin-based phototransduction (Opn4(-/-)), we uncovered, contrary to prevailing assumptions, that the contribution of SDLE is as important as circadian-driven input in determining SWc amplitude. Specifically, SDLE were primarily mediated (>80%) through melanopsin, of which half were then relayed through the SCN, revealing an ancillary purpose for this structure, independent of its clock function in organizing SWc. Based on these findings, we designed a model to estimate the effect of atypical light-dark cycles on SWc. This model predicted SWc amplitude in mice exposed to simulated transequatorial or transmeridian paradigms. Taken together, we demonstrate this SDLE is a crucial mechanism influencing behavior on par with the circadian system. In a broader context, these findings mandate considering SDLE, in addition to circadian drive, for coping with health consequences of atypical light exposure in our society.

ABSTRACTHistone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Amongst these enzymes SET domain containing 1b (SETD1B) has been linked to syndromic intellectual disability but its role in the postnatal brain has not been studied yet. Here we employ mice that lackSetd1bfrom excitatory neurons of the postnatal forebrain and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that SETD1B controls the expression of genes with a broad H3K4me3 peak at their promoters that represent neuronal enriched genes linked to learning and memory function. Comparative analysis to corresponding data from conditionalKmt2aandKmt2bknockout mice suggests that this function is specific to SETD1B. Moreover, postnatal loss ofSetd1bleads to severe learning impairment, suggesting that SETD1B-mediated regulation of H3K4me levels in postnatal neurons is critical for cognitive function.

Cohesin is a protein complex whose core subunits, Smc1, Smc3, Scc1, and SA1/SA2 form a ring-like structure encircling the DNA. Cohesins play a key role in the expression, repair, and segregation of eukaryotic genomes. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate the cohesin subunit Smc3, thereby inducing stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute cohesin as tri- or tetrameric assemblies and use those as physiologically-relevant substrates for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the murine Esco2 active site cleft, are critical for catalysis. Our experiments support a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, also involving two nearby serine residues - S566 and S527- that possess a proton relay function.

In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditional Esco2 knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.

The brain ventricles are interconnected, elaborate cavities that traverse the brain. They are filled with cerebrospinal fluid (CSF) that is, to a large part, produced by the choroid plexus, a secretory epithelium that reaches into the ventricles. CSF is rich in cytokines, growth factors and extracellular vesicles that glide along the walls of ventricles, powered by bundles of motile cilia that coat the ventricular wall. We review the cellular and biochemical properties of the ventral part of the third ventricle that is surrounded by the hypothalamus. In particular, we consider the recently discovered intricate network of cilia-driven flows that characterize this ventricle and discuss the potential physiological significance of this flow for the directional transport of CSF signals to cellular targets located either within the third ventricle or in the adjacent hypothalamic brain parenchyma. Cilia-driven streams of signalling molecules offer an exciting perspective on how fluid-borne signals are dynamically transmitted in the brain. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

The main stem cell niche for neurogenesis in the adult mammalian brain is the subventricular zone (SVZ) that extends along the cerebral lateral ventricles. We aimed at characterizing the initial molecular responses of the macaque monkey SVZ to transient, global cerebral ischemia. We microdissected tissue lining the anterior horn of the lateral ventricle (SVZa) from 7 day post-ischemic and sham-operated monkeys. Transcriptomics shows that in ischemic SVZa, 541 genes were upregulated and 488 genes were down-regulated. The transcription data encompassing the upregulated genes revealed a profile typical for quiescent stem cells and astrocytes. In the primate brain the SVZ is morphologically subdivided in distinct and separate ependymal and subependymal regions. The subependymal contains predominantly neural stem cells (NSC) and differentiated progenitors. To determine in which SVZa region ischemia had evoked transcriptional upregulation, sections through control and ischemic SVZa were analyzed by high-throughputin situhybridization for a total of 150 upregulated genes shown in thewww.monkey-niche.orgimage database. The majority of the differentially expressed genes mapped to the subependymal layers on the striatal or callosal aspect of the SVZa. Moreover, a substantial number of upregulated genes was expressed in the ependymal layer, implicating a contribution of the ependyma to stem cell biology. The transcriptome analysis yielded several novel gene markers for primate SVZa including the apelin receptor that is strongly expressed in the primate SVZa niche upon ischemic insult.