Dynamic and Assembly Characteristics of Deep-Cavity Basket Acting as a Host for Inclusion Complexation of Mitoxantrone in Biotic and Abiotic Systems.

We describe the preparation, dynamic, assembly characteristics of vase-shaped basket 13- along with its ability to form an inclusion complex with anticancer drug mitoxantrone in abiotic and biotic systems. This novel cavitand has a deep nonpolar pocket consisting of three naphthalimide sides fused to a bicyclic platform at the bottom while carrying polar glycines at the top. The results of 1 H Nuclear Magnetic Resonance (NMR), 1 H NMR Chemical Exchange Saturation Transfer (CEST), Calorimetry, Hybrid Replica Exchange Molecular Dynamics (REMD), and Microcrystal Electron Diffraction (MicroED) measurements are in line with 1 forming dimer [12 ]6- , to be in equilibrium with monomers 1(R) 3- (relaxed) and 1(S) 3- (squeezed). Through simultaneous line-shape analysis of 1 H NMR data, kinetic and thermodynamic parameters characterizing these equilibria were quantified. Basket 1(R) 3- includes anticancer drug mitoxantrone (MTO2+ ) in its pocket to give stable binary complex [MTO⊂1]- (Kd =2.1 µM) that can be precipitated in vitro with UV light or pH as stimuli. Both in vitro and in vivo studies showed that the basket is nontoxic, while at a higher proportion with respect to MTO it reduced its cytotoxicity in vitro. With well-characterized internal dynamics and dimerization, the ability to include mitoxantrone, and biocompatibility, the stage is set to develop sequestering agents from deep-cavity baskets.

Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

miRNA-132 orchestrates chromatin remodeling and translational control of the circadian clock.

Mammalian circadian rhythms are synchronized to the external time by daily resetting of the suprachiasmatic nucleus (SCN) in response to light. As the master circadian pacemaker, the SCN coordinates the timing of diverse cellular oscillators in multiple tissues. Aberrant regulation of clock timing is linked to numerous human conditions, including cancer, cardiovascular disease, obesity, various neurological disorders and the hereditary disorder familial advanced sleep phase syndrome. Additionally, mechanisms that underlie clock resetting factor into the sleep and physiological disturbances experienced by night-shift workers and travelers with jet lag. The Ca(2+)/cAMP response element-binding protein-regulated microRNA, miR-132, is induced by light within the SCN and attenuates its capacity to reset, or entrain, the clock. However, the specific targets that are regulated by miR-132 and underlie its effects on clock entrainment remained elusive until now. Here, we show that genes involved in chromatin remodeling (Mecp2, Ep300, Jarid1a) and translational control (Btg2, Paip2a) are direct targets of miR-132 in the mouse SCN. Coordinated regulation of these targets underlies miR-132-dependent modulation of Period gene expression and clock entrainment: the mPer1 and mPer2 promoters are bound to and transcriptionally activated by MeCP2, whereas PAIP2A and BTG2 suppress the translation of the PERIOD proteins by enhancing mRNA decay. We propose that miR-132 is selectively enriched for chromatin- and translation-associated target genes and is an orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment. These findings will further our understanding of mechanisms governing clock entrainment and its involvement in human diseases.

Uncovering the proteome response of the master circadian clock to light using an AutoProteome system.

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 µg and 200 µg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.

Elevated expression of MeCP2 in cardiac and skeletal tissues is detrimental for normal development.

MeCP2 plays a critical role in interpreting epigenetic signatures that command chromatin conformation and regulation of gene transcription. In spite of MeCP2's ubiquitous expression, its functions have always been considered in the context of brain physiology. In this study, we demonstrate that alterations of the normal pattern of expression of MeCP2 in cardiac and skeletal tissues are detrimental for normal development. Overexpression of MeCP2 in the mouse heart leads to embryonic lethality with cardiac septum hypertrophy and dysregulated expression of MeCP2 in skeletal tissue produces severe malformations. We further show that MeCP2's expression in the heart is developmentally regulated; further suggesting that it plays a key role in regulating transcriptional programs in non-neural tissues.

Segregation of expression of mPeriod gene homologs in neurons and glia: possible divergent roles of mPeriod1 and mPeriod2 in the brain.

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus function as the master circadian clock, coordinating the timing of diverse cell populations and organ systems. Dysregulation of clock timing is linked to a broad range of human conditions, including obesity, cardiovascular disease and a wide spectrum of neurological disorders. Aberrant regulation of expression of the PERIOD genes has been associated with improper cell division and human cancers, while the autosomal dominant disorder familial advanced sleep phase syndrome has been mapped to a single missense mutation within the critical clock gene hPERIOD2. An essential tool to begin to dissect the inherent molecular timing process is the clock gene reporter. Here, we functionally characterize two new mouse transgenic clock reporters, mPeriod1-Venus and mPeriod2-DsRED. Venus and DsRED are fluorescent proteins that can be used to monitor transcription in individual cells in real-time. Imaging of the SCN revealed oscillations, as well as light inducibility, in Venus and DsRED expression. Rhythmic Venus and DsRED expression was observed in distinct SCN cell populations, suggesting the existence of discrete cellular SCN clocks. Outside of the SCN, mPeriod1-Venus expression was broadly expressed in neuronal and non-neuronal populations. Conversely, mPeriod2-DsRED was expressed in glial populations and progenitor cells of the dentate gyrus; limited expression was detected in neurons. This distinct expression pattern of the two reporters reveals that the central nervous system possesses mechanistically distinct subpopulations of neuronal and non-neuronal cellular clocks. These novel mouse models will facilitate our understanding of clock timing and its role in human diseases.

Defective body-weight regulation, motor control and abnormal social interactions in Mecp2 hypomorphic mice.

MeCP2 is an abundant protein that binds to methylated cytosine residues in DNA and regulates transcription. Mutations in MECP2 cause Rett syndrome, a severe neurological disorder that affects approximately 1:10 000 females. Mice lacking MeCP2 have been generated and constitute important models of Rett syndrome. However, it is yet unclear whether certain physiological events are sensitive to a decrease, rather than a complete lack of MeCP2. Here we report that a Mecp2 floxed allele (Mecp2(lox)) that was generated to allow conditional mutagenesis behaves as a hypomorph and the corresponding mutant mice exhibit phenotypical alterations including body weight gain, motor abnormalities and altered social behavior. Our data reinforce the view that the central nervous system is extremely sensitive to MeCP2 expression levels and suggest that the 3'-UTR of Mecp2 might contain important elements that contribute to the regulation of its stability or processing.

Snf2h Drives Chromatin Remodeling to Prime Upper Layer Cortical Neuron Development.

Alterations in the homeostasis of either cortical progenitor pool, namely the apically located radial glial (RG) cells or the basal intermediate progenitors (IPCs) can severely impair cortical neuron production. Such changes are reflected by microcephaly and are often associated with cognitive defects. Genes encoding epigenetic regulators are a frequent cause of intellectual disability and many have been shown to regulate progenitor cell growth, including our inactivation of the Smarca1 gene encoding Snf2l, which is one of two ISWI mammalian orthologs. Loss of the Snf2l protein resulted in dysregulation of Foxg1 and IPC proliferation leading to macrocephaly. Here we show that inactivation of the closely related Smarca5 gene encoding the Snf2h chromatin remodeler is necessary for embryonic IPC expansion and subsequent specification of callosal projection neurons. Telencephalon-specific Smarca5 cKO embryos have impaired cell cycle kinetics and increased cell death, resulting in fewer Tbr2+ and FoxG1+ IPCs by mid-neurogenesis. These deficits give rise to adult mice with a dramatic reduction in Satb2+ upper layer neurons, and partial agenesis of the corpus callosum. Mice survive into adulthood but molecularly display reduced expression of the clustered protocadherin genes that may further contribute to altered dendritic arborization and a hyperactive behavioral phenotype. Our studies provide novel insight into the developmental function of Snf2h-dependent chromatin remodeling processes during brain development.

Voluntary Running Triggers VGF-Mediated Oligodendrogenesis to Prolong the Lifespan of Snf2h-Null Ataxic Mice.

Exercise has been argued to enhance cognitive function and slow progressive neurodegenerative disease. Although exercise promotes neurogenesis, oligodendrogenesis and adaptive myelination are also significant contributors to brain repair and brain health. Nonetheless, the molecular details underlying these effects remain poorly understood. Conditional ablation of the Snf2h gene impairs cerebellar development producing mice with poor motor function, progressive ataxia, and death between postnatal days 25-45. Here, we show that voluntary running induced an endogenous brain repair mechanism that resulted in a striking increase in hindbrain myelination and the long-term survival of Snf2h cKO mice. Further experiments identified the VGF growth factor as a major driver underlying this effect. VGF neuropeptides promote oligodendrogenesis in vitro, whereas Snf2h cKO mice treated with full-length VGF-encoding adenoviruses removed the requirement of exercise for survival. Together, these results suggest that VGF delivery could represent a therapeutic strategy for cerebellar ataxia and other pathologies of the CNS.

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation.

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.

Snf2l regulates Foxg1-dependent progenitor cell expansion in the developing brain.

Balancing progenitor cell self-renewal and differentiation is essential for brain development and is regulated by the activity of chromatin remodeling complexes. Nevertheless, linking chromatin changes to specific pathways that control cortical histogenesis remains a challenge. Here we identify a genetic interaction between the chromatin remodeler Snf2l and Foxg1, a key regulator of neurogenesis. Snf2l mutant mice exhibit forebrain hypercellularity arising from increased Foxg1 expression, increased progenitor cell expansion, and delayed differentiation. We demonstrate that Snf2l binds to the Foxg1 locus at midneurogenesis and that the phenotype is rescued by reducing Foxg1 dosage, thus revealing that Snf2l and Foxg1 function antagonistically to regulate brain size.

Cell-specific expression of wild-type MeCP2 in mouse models of Rett syndrome yields insight about pathogenesis.

Rett syndrome (RTT), a leading cause of mental retardation with autistic features in females, is caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). RTT is characterized by a diverse set of neurological features that includes cognitive, motor, behavioral and autonomic disturbances. The diverse features suggest that specific neurons contribute to particular phenotypes and raise the question whether restoring MeCP2 function in a cell-specific manner will rescue some of the phenotypes seen in RTT. To address this, we generated transgenic mice expressing inducible MeCP2 under the control of the brain-specific promoters calcium/calmodulin-dependent protein kinase II (CamKII) or neuron-specific enolase (Eno2) and bred them onto mouse models lacking functional MeCP2. Expression of normal MeCP2 in either CamKII or Eno2 distribution was unable to prevent the appearance of most of the phenotypes of the RTT mouse models. These results suggest that most RTT phenotypes are caused either by disruption of complex neural networks involving neurons throughout the brain or by disruption of the function of specific neurons outside of the broad CamKII or Eno2 distribution.