Derepression of a neuronal inhibitor due to miRNA dysregulation in a schizophrenia-related microdeletion.

22q11.2 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.

TIMELESS mutation alters phase responsiveness and causes advanced sleep phase.

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human TIMELESS (hTIM) gene that causes familial advanced sleep phase (FASP). Tim CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.

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.

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.

The BDNF Val66Met variant affects gene expression through miR-146b.

Variation in gene expression is an important mechanism underlying susceptibility to complex disease and traits. Single nucleotide polymorphisms (SNPs) account for a substantial portion of the total detected genetic variation in gene expression but how exactly variants acting in trans modulate gene expression and disease susceptibility remains largely unknown. The BDNF Val66Met SNP has been associated with a number of psychiatric disorders such as depression, anxiety disorders, schizophrenia and related traits. Using global microRNA expression profiling in hippocampus of humanized BDNF Val66Met knock-in mice we showed that this variant results in dysregulation of at least one microRNA, which in turn affects downstream target genes. Specifically, we show that reduced levels of miR-146b (mir146b), lead to increased Per1 and Npas4 mRNA levels and increased Irak1 protein levels in vitro and are associated with similar changes in the hippocampus of hBDNF(Met/Met) mice. Our findings highlight trans effects of common variants on microRNA-mediated gene expression as an integral part of the genetic architecture of complex disorders and traits.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex.

Individuals with 22q11.2 microdeletions have cognitive and behavioral impairments and the highest known genetic risk for developing schizophrenia. One gene disrupted by the 22q11.2 microdeletion is DGCR8, a component of the "microprocessor" complex that is essential for microRNA production, resulting in abnormal processing of specific brain miRNAs and working memory deficits. Here, we determine the effect of Dgcr8 deficiency on the structure and function of cortical circuits by assessing their laminar organization, as well as the neuronal morphology, and intrinsic and synaptic properties of layer 5 pyramidal neurons in the prefrontal cortex of Dgcr8(+/-) mutant mice. We found that heterozygous Dgcr8 mutant mice have slightly fewer cortical layer 2/4 neurons and that the basal dendrites of layer 5 pyramidal neurons have slightly smaller spines. In addition to the modest structural changes, field potential and whole-cell electrophysiological recordings performed in layer 5 of the prefrontal cortex revealed greater short-term synaptic depression during brief stimulation trains applied at 50 Hz to superficial cortical layers. This finding was accompanied by a decrease in the initial phase of synaptic potentiation. Our results identify altered short-term plasticity as a neural substrate underlying the cognitive dysfunction and the increased risk for schizophrenia associated with the 22q11.2 microdeletions.

MicroRNA dysregulation in neuropsychiatric disorders and cognitive dysfunction.

MicroRNAs (miRNA), a class of non-coding RNAs, are emerging as important modulators of neuronal development, structure and function. A connection has been established between abnormalities in miRNA expression and miRNA-mediated gene regulation and psychiatric and neurodevelopmental disorders as well as cognitive dysfunction. Establishment of this connection has been driven by progress in elucidating the genetic etiology of these phenotypes and has provided a context to interpret additional supporting evidence accumulating from parallel expression profiling studies in brains and peripheral blood of patients. Here we review relevant evidence that supports this connection and explore possible mechanisms that underlie the contribution of individual miRNAs and miRNA-related pathways to the pathogenesis and pathophysiology of these complex clinical phenotypes. The existing evidence provides useful hypotheses for further investigation as well as important clues for identifying novel therapeutic targets.

PRISM: A Progenitor-Restricted Intersectional Fate Mapping Approach Redefines Forebrain Lineages.

Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages-Shh and Cux2.

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO.

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.

Genetics of human sleep behavioral phenotypes.

Quality sleep is critical for daily functions of human beings and thus the timing and duration of sleep are tightly controlled. However, rare genetic variants affecting sleep regulatory mechanisms can result in sleep phenotypes of extremely deviated sleep/wake onset time or duration. Using genetic analyses in families with multiple members expressing particular sleep phenotypes, these sleep-associated genetic variants can be identified. Deciphering the nature of these genetic variants using animal models or biochemical methods helps further our understanding of sleep processes. In this chapter, we describe the methods for studying genetics of human sleep behavioral phenotypes.

The 22q11.2 microdeletion: fifteen years of insights into the genetic and neural complexity of psychiatric disorders.

Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25-30% and such deletions account for as many as 1-2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.

Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12.

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.