January 1, 2012 (Vol. 32, No. 1)
Vicki Glaser Writer GEN
A growing understanding of the role microRNAs (miRNAs) play in brain development and function and in synaptic plasticity, combined with the recognition that a single miRNA can regulate the expression of multiple genes across networks of biochemical pathways, is fueling interest in miRNAs as targets for intervening in psychiatric disorders.
At the recent Society for Neuroscience annual meeting in Washington, D.C., researchers presented studies evaluating the association between miRNA dysregulation, neurodevelopment, schizophrenia risk, and anxiety disorders.
Schizophrenia and bipolar disorder each affect about 1% of the population in the U.S., costing more than $100 billion/year and taking a high toll on families of affected individuals.
Although both disorders tend to have high heritability, they are not strictly genetic diseases and disease development may depend on a complex interplay between genes and environmental triggers. Further complicating the picture, both diseases involve dysregulation of multiple signaling pathways; additionally, different molecular aberrations can result in a similar phenotype.
The predominant mechanism by which a miRNA silences a target gene in mammals is through post-transcriptional regulation, preventing a strand of messenger RNA (mRNA) from being translated into a protein.
Claes Wahlestedt, M.D., Ph.D., professor of psychiatry and behavioral sciences, Miller School of Medicine, University of Miami, described miRNAs as “master regulators” due to the ability of a single miRNA to target hundreds of mRNAs and the emerging consensus that miRNAs regulate more than half of all protein-coding genes.
This regulatory layer “may account for some of the missing genetic/epigenetic variability in the etiology of psychiatric disease,” he said.
At present, the most significant known risk factors for schizophrenia are copy number variations (CNVs), with the 22q11 deletion being the most common. It is present in about 1 in 4,000 people and in 1% of people with schizophrenia.
Most cases of 22q11 deletion syndrome (DS) involve a de novo deletion, typically about 3 million base pairs in size, and are not inherited. The disorder has variable presentations and individuals carrying the deletion have about a 25% risk of developing schizophrenia.
Included in the 22q11 deletion is the DGCR8 gene, which has a key role in the biogenesis and maturation of miRNAs to their mature, shortened form. Linda Brzustowicz, M.D., professor, department of genetics, Rutgers University, introduced the concept of canalization, which refers to the robustness of a trait or phenotype and the ability of a phenotype to be expressed regardless of genotypic or environmental variability. She proposed that “miRNAs may be a mechanism of canalization.”
The fact that schizophrenia does not develop in about 75% of people with 22q11-DS may be attributable to the increased capacity of canalized traits to absorb mutational variance. Dr. Brzustowicz presented a model in which 22q11-DS results in DGCR8 haplotype insufficiency, which perturbs the miRNA regulatory system, allowing previously silenced regulatory mutations to alter gene expression. DGCR8 mutations result in a reduction in one or more subsets of miRNA, with the consequence of increased gene expression.
By determining which miRNAs are reduced in the brains of patients with 22q11-DS and applying the canalization hypothesis, it may be possible to predict which genes are likely to have increased expression and to have a role in the development of schizophrenia.
Maria Karayiorgou, M.D., professor of psychiatry at Columbia University, is also studying 22q11 mutations, and specifically the 22q11.2 microdeletion.
Dr. Karayiorgou uses a mouse model of 22q11 CNVs, characterized by deletions on mouse chromosome 16 that correlate to 22q11 CNVs in humans. She applies behavioral assays to study cognitive deficits and has reported deficits in spatial working memory tasks indicative of decreased working memory capacity. These behavioral deficits are associated with DGCR8 deficiency in DGCR8 knockout mice.
The 22q11.2 microdeletion does not appear to affect basal synaptic transmission in mice, but it may affect the activity of neuronal networks in the prefrontal cortex. The mutated mice exhibited reduced synaptic memory in this region of the brain. Dr. Karayiorgou concluded that miRNA dysregulation likely contributes to the cognitive impairment seen in the mouse model by altering short-term synaptic plasticity.
A presentation by Brooke Miller, Ph.D., research associate at Scripps Research Institute, demonstrated that “miR-132 dysregulation in schizophrenia has adult and neurodevelopmental implications.” She described the analysis of human brain tissue samples from 34 control subjects, 35 patients with schizophrenia, and 31 individuals with bipolar disease, evaluating each for 854 miRNAs using microarrays. Only 2 of the 854 miRNAs were differentially expressed in schizophrenia and 10 in bipolar disease; miR-132 overlapped both.
Dr. Miller gave several reasons why miR-132 is of particular interest: its transcription is directly regulated by the cAMP-response element binding protein; it regulates synaptic outgrowth; it potentiates N-methyl-d-aspartate (NMDA) receptor signaling; and its targets are significantly upregulated in tissue samples from individuals with schizophrenia.
In fact, more than 25 (13%) of putative miR-132 targets are significantly upregulated in schizophrenia. miR-132 also has a role in regulating gene expression in mice during postnatal weeks 2 to 4, which corresponds to adolescence in mice, a crucial period of neurodevelopment.
“Of 201 miR-132 targets, 57 overlap with gene-expression changes in that developmental period,” she said.
Stephen Magill, an M.D., Ph.D. student at Oregon Health and Science University, provided evidence to support the hypothesis that “miR-132 regulates dendritic growth and arborization of adult newborn hippocampal neurons.” He studied the role of miR-132 at the cellular level.
In mouse studies, using sensors of miRNA activity that have single-cell resolution, Magill and colleagues showed that ablation of miR-212/132 resulted in significantly decreased dendrite outgrowth in vivo, including reductions in both the length and branching of dendrites. miR-132 is the predominant functional product of miR-132/212.
Ilris Hovatta, Ph.D., University of Helsinki, looked at differences between inbred mouse strains as a means of studying mRNA and miRNA networks and defining mechanisms that regulate genes associated with anxiety disorders. Dr. Hovatta presented data on miRNA expression in various brain regions, including the prefrontal cortex, hippocampus, and hypothalamus, and identified anticorrelations between miRNA and mRNA pathways. As expression of a miRNA increases, expression of its target mRNA decreases.
An unbiased genome-wide screen led to the identification of 69 miRNAs with expression levels that correlated with anxiety-like behavior. The researchers used pathway analysis to predict their target mRNAs and the gene-regulatory networks that control anxiety in mice.
Schahram Akbarian, M.D., Ph.D., associate professor of psychiatry, University of Massachusetts Medical School, described “Neural epigenomes in developing and diseased prefrontal cortex.”
Epigenomic mapping studies, with a focus on hydroxymethylation of cytosine, have identified more than 100 site-specific covalent modifications affecting nucleosome core histone proteins that are important for normal brain development. As part of the Brain Epigenome Project at UMass Medical School, researchers have studied more than 80 billion base pairs of DNA from nucleosomes derived from neurons in the prefrontal cortex. A comparison of 15 epigenomes identified differences between neuronal and non-neuronal epigenomic signatures.
Zooming In on the Brain for a Greater Understanding of Neurology
- Nikon launched the Eclipse Ni-E motorized microscope system with CFI Plan Apochromat Lambda objectives and Nano Crystal Coat technology at the recent “Neuroscience” meeting. The Eclipse Ni-E features automatic adjustment with objective changeover.Also new from Nikon is the Ni-E fixed-stage multiphoton confocal system, capable of whole-animal and deep-tissue imaging. The Ni-U upright microscopy has fly-eye optics built into the transmitted-light illumination system, noise terminator technology to eliminate stray light, and an image capture button.
- Olympus presented its new DP73, 17.28 megapixel cooled digital color camera, capable of capturing 15 frames/section in brightfield and widefield fluorescence imaging. Also new is the BX63 automated upright microscope, which features a fixed-stage design and focusing nosepiece to ensure a stable specimen surface. The company’s Andor™ Revolution® XD Spinning Disk Confocal microscope, designed for live-cell imaging, integrates Andor custom coupling optics to optimize optical alignment and minimize chromatic aberration, allows for the use of up to six lasers, and is compatible with 30x and 60x silicone immersion objectives.To enhance 3-D imaging within tissues and organs, Olympus offers the Scaleview 25x objective for use with the Scaleview-A2 clearing reagent, enabling microscope imaging to a depth of 4 mm.
- Carl Zeiss has integrated its Microimaging and Nano Technology Systems business units into a unified division called Carl Zeiss Microscopy. At “Neuroscience”, Zeiss showcased its expanded ZEN software, including a free trial of the entry-level version ZEN lite 2011.The company’s Shuttle & Find interface for performing correlative microscopy combines the resolution of electron microscopy with the labeling capabilities of fluorescence laser-scanning confocal microscopy.Zeiss’ Axio Zoom.V16 microscope system offers a 16x zoom range and 2.5 higher resolution and 10x brighter fluorescence than a conventional stereomicroscope, according to the company.Also newly introduced was a prototype of Zeiss’ redesigned plan-apochromatic 20x/1.0 VIS-IR objective lens for use with the Scale clearing reagent to enable 3-D imaging at depths down to 5.6 mm in intact brain tissue using a confocal laser scanning or multiphoton microscope.
- The New B-Scope in vivo imaging system from Thorlabs allows users to perform multiphoton microscopy on live animals. By eliminating the need for a traditional microscope, the B-Scope can provide 10 inches of course travel in the z-axis and can operate in an upright or tilted position, enabling rotation of the imaging plane and image capture from above or from the side of an animal.
- A fully automated Total Internal Reflection Fluorescence Microscopy (TIRFM) system from Leica, the AM TIRF MC, allows for single-molecule detection and kinetic studies, as well as visualization and analysis of events such as vesicle transport and molecular signaling. It includes four lasers with wavelengths of 405 nm, 488 nm, 561 nm, and 635 nm, and provides a z-resolution of 100–300 nm.The company’s Stimulated Emission Depletion Microscopy super-resolution confocal/multiphoton system uses two synchronized, pulsed laser beams to illuminate the sample—a 635 nm wavelength laser to excite the fluorochromes followed by a Ti:Sapphire infrared laser pulse, which provides ring-shaped illumination to inhibit fluorescence along the outer edges of the illuminated spot.
- QImaging’s Rolera™ Bolt Scientific CMOS camera incorporates Pixel-Freeze Technology™ that minimizes dark current. The Rolera Bolt is capable of image capture at 60 frames per second and streaming at full resolution at 30 fps. It can perform imaging and quantitation at low light levels and track high-speed dynamic events.
- Lumenera introduced two new products at the meeting, including the Infinity HD 2 megapixel microscopy color camera that features a CMOS sensor, 1080p60 streaming video with color output, automatic intensity compensation, and a USB 2.0 port.The company also launched its redesigned Infinity2-IR camera, with 14-bit data output, reduced operating temperature, and a dark current rating of <1 e-/s.
- In the first quarter of 2012, Sigma-Aldrich will add an autism knockout rat model to its SAGE™ Labs line. Sigma also added a Panorama® Antibody Array for neurobiology, which includes 224 antibodies spotted in 32 subarrays, each containing duplicate antibody spots, duplicate positive control spots, and several negative controls.
- New from Carestream Molecular Imaging is the In-Vivo Xtreme optical and x-ray small-animal imaging system, capable of luminescence, fluorescence, radioisotopic, and radiographic imaging. It combines an f/1.1 lens and CCD sensor, and a back illumated 4 MP camera (with optional front illuminated 16 MP camera). Carestream also recently introduced its Gel Logic 6000 PRO in vitro imaging system for analyzing blots and gels.
- For cell-based imaging, the ImageXpress Micro Widefield High Content Screening System from Molecular Devices can locate and identify subcellular components and follow them over multiday time-lapse experiments. The system has a 3-log dynamic range and enables screening of >10 million cells/hour.
- EMD Millipore designed the Direct Detect™ infrared (IR)-based quantitation system to differentiate and quantify the protein, lipid, carbohydrate, and nucleic acid components of a 2 µL biological sample, in place of a Bradford assay.
- Eppendorf featured the BioPhotometer® plus, with nine wavelengths, preprogrammed methods, and the ability to determine enzyme kinetics, fluorescent dye incorporation rates, and direct absorbance measurements of single wavelengths.
- New from DAGE-MTI is the RealView™ HD-210D high-definition color microscope camera, which enables real-time imaging at 60 frames per second. The company added a new autocontrast feature to its IR1000 infrared camera that is available as a retrofit.
- ProteinSimple’s Simon™ gel-free Western blot system is fully automated, from sample loading to data analysis. It can analyze 12 samples per run, with a run time of 3–5 hours and sample volumes of about 3 µg.
- ProSieve™ EX buffers from Lonza are suitable for use for protein separation and transfer on polyacrylamide gels in place of tris-glycine, with shorter run times and 10-minute transfers, according to the firm.
- The Integra Biosciences ViaFlo96 is an automated 96-well pipettor with disposable tips. The company’s Voyager multichannel pipettor automatically adjusts the distance between tips to facilitate 96- to 384-well plate-to-plate transfer. With multistep pipetting capability, the Voyager allows users to pick up, for example, reagent, then air (creating an air pocket in the tip), and then sample, dispensing all of the contents at once and mixing them in a single well.
- Bio-Rad showcased the Droplet Digital™ PCR technology, recently acquired from QuantaLife, the Protean® i12™ IEF system for 2-D electrophoresis, and the ChemiDoc™ MP imaging system for Western blot quantification.
- The IPA® software application from Ingenuity Systems analyzes experimental data in the context of known biology. The company’s iReport™ series of interactive reports for analyzing raw gene-expression data is in early-access release and is available free to the first 5,000 researchers.
- The DetectX® Protein Kinase A activity assay from Arbor Assays quantifies PKA alpha, beta, and gamma activity within three hours. The kit includes a recombinant PKA standard.
- Enzo Life Sciences launched its PEGylated Protein ELISA kits for quantifying NBR1 and p62 autophagy biomarkers.
- The BD Falcon™ Cell Culture Multi-Flask from BD Biosciences comes in three- or five-layer versions with 525 cm2 or 875 cm2 surface area, respectively, for space-saving scalability of cell cultures.
- RapidDirect™ Western blot kits from Solulink include a labeled primary antibody-HRP/antigen complex that is immobilized by α-species IgG attached to NanoLink™ magnetic beads.