MicroRNA Dysregulation in Neuropsychiatric Disorders and Cognitive Dysfunction

MicroRNAs (miRNAs) are evolutionarily-conserved small non-coding RNAs that are important posttranscriptional regulators of gene expression. Genetic Variants may cause microRNA dysregulation and the concomitant aberrant target expression. The dysregulation of one or a few targets may in turn lead to functional consequences ranging from phenotypic variations to disease conditions. In this thesis, I present our studies of mouse models of two human genetic variants - a rare copy number variant (CNV), 22q11.2 microdeletions, and a common single nucleotide polymorphism (SNP), BDNF Val66Met. 22q11.2 microdeletions result in specific cognitive deficits and high risk to develop schizophrenia. Analysis of Df(16)A+/- mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines as well as microRNA dysregulation in brain. We show a drastic reduction of miR- 185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion and demonstrate that this reduction impairs dendritic and spine development. miR-185 targets and represses, through an evolutionary conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A+/- mice and could affect the formation and maintenance of neural circuits. Reduction of miR-185 also has milder effects on the expression of a group of Golgi-related genes. One the other hand, BNDF Val66Met results in impaired activity-dependent secretion of BDNF from neuronal terminals and affects episodic memory and affective behaviors. We found a modest reduction of miR-146b which causes derepression of mRNA and/or protein levels of a few targets. Our findings add to the growing evidence of the pivotal involvement of miRNAs in the development of neuropsychiatric disorders and cognitive dysfunction. In addition, the identification of key players in miRNA dysregulation has implications for both basic and translational research in psychiatric disorders and cognitive dysfunction

DEC2 modulates orexin expression and regulates sleep.

Adequate sleep is essential for physical and mental health. We previously identified a missense mutation in the human DEC2 gene (BHLHE41) leading to the familial natural short sleep behavioral trait. DEC2 is a transcription factor regulating the circadian clock in mammals, although its role in sleep regulation has been unclear. Here we report that prepro-orexin, also known as hypocretin (Hcrt), gene expression is increased in the mouse model expressing the mutant hDEC2 transgene (hDEC2-P384R). Prepro-orexin encodes a precursor protein of a neuropeptide producing orexin A and B (hcrt1 and hcrt2), which is enriched in the hypothalamus and regulates maintenance of arousal. In cell culture, DEC2 suppressed prepro-orexin promoter-luc (ore-luc) expression through cis-acting E-box elements. The mutant DEC2 has less repressor activity than WT-DEC2, resulting in increased orexin expression. DEC2-binding affinity for the prepro-orexin gene promoter is decreased by the P384R mutation, likely due to weakened interaction with other transcription factors. In vivo, the decreased immobility time of the mutant transgenic mice is attenuated by an orexin receptor antagonist. Our results suggested that DEC2 regulates sleep/wake duration, at least in part, by modulating the neuropeptide hormone orexin

Derepression of a Neuronal Inhibitor due to miRNA Dysregulation in a Schizophrenia-Related Microdeletion

Summary22q11.2 microdeletions result in specific cognitive deficits and schizophrenia. Analysis of Df(16)A+/− mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines, as well as altered brain microRNAs. Here, we show a drastic reduction of miR-185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion, and we demonstrate that this reduction alters dendritic and spine development. miR-185 represses, through an evolutionarily conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus and shows higher prenatal brain expression. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A+/− mice and results in structural alterations in the hippocampus. Reduction of miR-185 also has milder age- and region-specific effects on the expression of some Golgi-related genes. Our findings illuminate the contribution of microRNAs in psychiatric disorders and cognitive dysfunction

A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait.

In humans, the connection between sleep and mood has long been recognized, although direct molecular evidence is lacking. We identified two rare variants in the circadian clock gene PERIOD3 (PER3-P415A/H417R) in humans with familial advanced sleep phase accompanied by higher Beck Depression Inventory and seasonality scores. hPER3-P415A/H417R transgenic mice showed an altered circadian period under constant light and exhibited phase shifts of the sleep-wake cycle in a short light period (photoperiod) paradigm. Molecular characterization revealed that the rare variants destabilized PER3 and failed to stabilize PERIOD1/2 proteins, which play critical roles in circadian timing. Although hPER3-P415A/H417R-Tg mice showed a mild depression-like phenotype, Per3 knockout mice demonstrated consistent depression-like behavior, particularly when studied under a short photoperiod, supporting a possible role for PER3 in mood regulation. These findings suggest that PER3 may be a nexus for sleep and mood regulation while fine-tuning these processes to adapt to seasonal changes

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The forward physics facility at the high-luminosity LHC

High energy collisions at the High-Luminosity Large Hadron Collider (LHC) produce a large number of particles along the beam collision axis, outside of the acceptance of existing LHC experiments. The proposed Forward Physics Facility (FPF), to be located several hundred meters from the ATLAS interaction point and shielded by concrete and rock, will host a suite of experiments to probe standard model (SM) processes and search for physics beyond the standard model (BSM). In this report, we review the status of the civil engineering plans and the experiments to explore the diverse physics signals that can be uniquely probed in the forward region. FPF experiments will be sensitive to a broad range of BSM physics through searches for new particle scattering or decay signatures and deviations from SM expectations in high statistics analyses with TeV neutrinos in this low-background environment. High statistics neutrino detection will also provide valuable data for fundamental topics in perturbative and non-perturbative QCD and in weak interactions. Experiments at the FPF will enable synergies between forward particle production at the LHC and astroparticle physics to be exploited. We report here on these physics topics, on infrastructure, detector, and simulation studies, and on future directions to realize the FPF's physics potential

Molecular and Genetic Analysis of C. elegans psr-1 in the Cell-Corpse Engulfment Process

在多細胞生物中，正確的清除細胞自伐所造成的死亡細胞對於個體的生存十分重要。在線蟲死亡細胞的清除過程中，PSR-1被認為是作用在CED-2、CED-5、CED-10及CED-12上游的受器，調控這個過程。我們證實PSR-1會直接與CED-5及CED-12有直接的交互作用(Wang et al., 2003)，並且找出PSR-1中參與作用的區域。PSR-1中另外可能有2個NLS，所以我們產生α-GST-PSR-1-IN的多株抗體，希望能分析PSR-1在細胞內的分佈。但是沒有抗體可以辨識線蟲lysate中自源性或是過量表現的PSR-1蛋白質。我們同時也在psr-1突變種的線蟲中表現不同片段的PSR-1蛋白質(dNLS1, dNLS-2, dNLS1&2, dTM)，發現只有PSR-1-dTM不能復原psr-1突變所造成的細胞死亡清除的缺失。因此PSR-1是作用在細胞膜上來促進死亡細胞的吞噬，並且可能是扮演受器的角色。 為了了解psr-1和其他參與吞噬作用的基因之間的關係，我們觀察雙重或三重突變種中的細胞屍體數目。我們也發現了psr-1及mer-1可能共同作用在吞噬的路徑並且兩者間無直接的交互作用。另外，我們發現unc-73及mig-2可能共同作用在一個psr-1沒有參與的吞噬路徑。根據我們的實驗，我們推測PSR-1是可能扮演吞噬受器的角色來調控細胞屍體的吞噬，並且可能經由與MER-1共同作用來達成。In multicellular organisms, the proper removal of apoptotic cell corpses prevents the possible dispersal of harmful cellular contents from dying cells. In C. elegans, PSR-1 is proposed to function as a receptor and act upstream of the pathway mediated by CED-2, CED-5, CED-10, and CED-12, possibly through physical interaction with CED-5 and CED-12 to control the engulfment process (Wang et al., 2003). We further mapped the binding region in PSR-1 for CED-5 and CED-12 interaction. To examine the expression pattern of PSR-1, we generated mouse and rabbit polyclonal antibodies against GST-PSR-1-IN fusion protein in order to test the subcellular localization of PSR-1. However, these antibodies and α-human PSR antibodies fail to recognize either bacterially-expressed GST-PSR-1-IN or endogenous or overexpressed PSR-1 in worm lysates. In addition to a transmembrane domain (TM), PSR-1 is predicted to possess two nuclear localization signals (NLSs). To test if any of these motifs is important for PSR-1 function, we performed deletion assays. We found that TM but not NLS is important for the engulfment function of PSR-1. These results support the previous hypothesis that PSR-1 functions on the cell membrane and possibly as a receptor to promote cell corpses uptake. In order to investigate the relationship between psr-1 and other possible engulfment genes, mer-1, unc-73, and mig-2, we analyzed the numbers of corpse in double and triple mutants. We showed that psr-1 and another engulfment receptor, mer-1, act in the same genetic pathway, and that there is no direct interaction between PSR-1 and MER-1 intracellular or extracellular domains. Besides, unc-73 and mig-2 may play minor roles and act in the same genetic pathway which does not involve psr-1 to control cell-corpse engulfment. Together our experiments suggested that PSR-1 may act as a membrane receptor to regulate cell-corpse engulfment and that it may function with MER-1 to achieve this process.Table of Contents Table of Contents………………………………………………………………….1 Abstract…………………………………………………………………………….4 中文摘要...........................................................................................................6 Introduction……………………………………………………………………..…7 Results……………………………………………………………………………21 PSR-1 Is a Highly Conserved Novel Protein………………………………..21 PSR-1 Interacts with CED-5 and CED-12 via Its Intracellular C-terminal Regions………………………………………………………………………….22 PSR-1 Likely Function on Cell Membrane to Promote Cell-Corpse Engulfment……………………………………………………………………...24 PSR-1-GFP and GFP-PSR-1 Localization in Cell Nuclei and Deletion of the 2nd NLS Compromised the Nuclear Localization……................................26 psr-1 and mer-1 Possibly Act in the Same Genetic Pathway to Control Cell-Corpse Engulfment……………………………………………………….27 PSR-1 and MER-1 do not Interact through Their Intracellular Domains or Extracellular Domains………………………………………………………….28 unc-73 and mig-2 Possibly Act in the Same Genetic Pathway to Regulate Cell-Corpse Clearance………………………………………………………...29 psr-1 Likely Functions in Parallel with unc-73 and mig-2 to Control Cell-Corpse Engulfment……………………………………………………….30 Polyclonal α-PSR-1-IN or α-Human PSR Antibodies Failed to Recognize either Bacterial Expressed PSR-1-IN or Endogenous PSR-1 in Worm Lysates…………………………………………………………………………..31 2 Discussion………………………………………………………………………..32 PSR Promotes the Clearance of Apoptotic Cell Corpses………………….32 PSR-PS Ligation may be a Simple Tethering Mechanism…………………33 PSR Has Vital Functions in Fundamental Developmental Processes……34 The Subcellular Localization of PSR…………………………………………35 Cell-Corpse Engulfment Signaling Involving PSR-1, MER-1, UNC-73, and MIG-2……………………………………………………………………………37 Materials and Methods………………………………………………………….39 Strains and Genetics…………………………………………………………..39 Yeast-Two-Hybrid Assay………………………………………………………39 GST Pull-Down Assay (in vitro Binding Assay)……………………………...40 Antibodies and Western Blotting……………………………………………...41 References……………………………………….………………………………..42 Figures……………………………………………………………………………52 Figure 1 Molecules and Signaling Pathways Involved in Cell Corpse Engulfment in C. elegans and Their Mammalian Homologues……………52 Figure 2 Structural Features of PSR-1 and homologues in other species…………………………………………………………………………..53 Figure 3 The Intracellular Domain of PSR-1 Interacts with CED-5 and CED-12 in Yeast Two-Hybrid Assay………………………………………….54 Figure 4 Interaction of Various PSR-1 Intracellular Fragments with CED-5A …………………………………………………………………………55 Figure 5 Interaction of Various PSR-1 Intracellular Fragments with CED-5B………………………………………………………………………….56 Figure 6 Interaction of Various PSR-1 Intracellular Fragments with CED-12………………………………………………………………………….57 Figure 7 Relative Binding Abilities of PSR-1 Intracellular Fragments and human PSR to CED-5A, CED-5B and CED-12……………………………..58 Figure 8 Rescue of Cell-Corpse Engulfment Defect of the psr-1 Mutant by 3 Expressing PSR-1, PSR-1-dNLS1, PSR-1-dNLS2, and PSR-1- dNLS1&2, but not PSR-1-dTM Protein …………………………………………………..59 Figure 9 PSR-1-GFP Expression Patterns in 1.5-fold Embryos…………61 Figure 10 PSR-1-GFP Expression Patterns in 2-fold Embryos………….62 Figure 11 PSR-1-GFP Expression Patterns in Larva……………………..63 Figure 12 GFP-PSR-1 Expression Patterns in 1.5-fold Embryos……….64 Figure 13 GFP-PSR-1 Expression Patterns in 2-fold Embryos………….65 Figure 14 GFP-PSR-1 Expression Patterns in Larva…………………….66 Figure 15 psr-1 and mer-1 Possibly Act in the Same Genetic Pathway to Control Cell-Corpse Engulfment……………………………………………...67 Figure 16 The Intracellular domain of PSR-1 and MER-1 Fail to Self-Interact or Interact with Each Other in Yeast Two-Hybrid Assay……..68 Figure 17 The Extracellular domain of MER-1 Self-Interacts but Fails to Interact with the Extracellular domain of PSR-1 in Yeast Two-Hybrid Assay……………………………………………………………………………69 Figure 18 unc-73 and mig-2 may Play Minor Roles in Cell-Corpse Engulfment and Act in the Same Genetic Pathway…………………………70 Figure 19 unc-73 Enhances Cell Corpses in psr-1 Mutant Embryos at the Comma Stage…………………………………………………………………..71 Figure 20 mig-2 does not Enhance Cell Corpses in psr-1 Mutant………72 Figure 21 unc-73; mig-2 Enhances Cell Corpses in psr-1 Mutant Embryos at the Comma Stage…………………………………………………………...73 Figure 22 Western Blotting of GST-fused PSR-1 Proteins by Polyclonal Mouse and Rabbit α-PSR-1 Antibodies……………………………………...74 Figure 23 Western Blotting of GST-fused PSR-1 Proteins by α-Human PSR Antibodies…………………………………………………………………75 Figure 24 Polyclonal Mouse α-PSR-1 Antibodies Fail to Recognize Endogenous or Overexpress PSR-1 Protein in Worm Lysates……………76 Figure 25 PSR-1 and MER-1, Two Possible Cell-Corpse Receptors, may Cooperate to Promote the Clearance of Apoptotic Cell Corpse in Worm...7