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      Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

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          Abstract

          Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.

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          Most cited references 30

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          Oligodendroglia metabolically support axons and contribute to neurodegeneration

          Summary Oligodendroglia support axon survival and function through mechanisms independent of myelination and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been hypothesized. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and utilization. We show the most abundant lactate transporter in the CNS, monocarboxylate transporter 1 (MCT1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and mouse models of, amyotrophic lateral sclerosis (ALS), suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.
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            Cre-lox-regulated conditional RNA interference from transgenes.

            We have generated two lentiviral vectors for conditional, Cre-lox-regulated, RNA interference. One vector allows for conditional activation, whereas the other permits conditional inactivation of short hairpin RNA (shRNA) expression. The former is based on a strategy in which the mouse U6 promoter has been modified by including a hybrid between a LoxP site and a TATA box. The ability to efficiently control shRNA expression by using these vectors was shown in cell-based experiments by knocking down p53, nucleophosmin and DNA methyltransferase 1. We also demonstrate the usefulness of this approach to achieve conditional, tissue-specific RNA interference in Cre-expressing transgenic mice. Combined with the growing array of Cre expression strategies, these vectors allow spatial and temporal control of shRNA expression in vivo and should facilitate functional genetic analysis in mammals.
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              GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease.

              In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.
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                Author and article information

                Journal
                Exp Neurobiol
                Exp Neurobiol
                EN
                Experimental Neurobiology
                The Korean Society for Brain and Neural Science
                1226-2560
                2093-8144
                June 2016
                22 June 2016
                : 25
                : 3
                : 120-129
                Affiliations
                [1 ]Center for Neuroscience and Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
                [2 ]Research Animal Resource Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
                [3 ]Neuroscience Program, University of Science and Technology (UST), Daejeon 34113, Korea.
                [4 ]KU-KIST School of Converging Science and Technology, Korea University, Seoul 02841, Korea.
                Author notes
                To whom correspondence should be addressed. TEL: 82-2-958-6940, FAX: 82-2-958-6919, cjl@ 123456kist.re.kr
                Article
                10.5607/en.2016.25.3.120
                4923356
                27358580
                Copyright © Experimental Neurobiology 2016.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Funding
                Funded by: National Research Foundation of Korea, CrossRef http://dx.doi.org/10.13039/501100003725;
                Award ID: 2015R1A3A2066619
                Funded by: KIST, CrossRef http://dx.doi.org/10.13039/501100003693;
                Award ID: 2E26662
                Funded by: KU-KIST Graduate School of Science and Technology;
                Award ID: R1435281
                Categories
                Original Article

                Neurosciences

                parvalbumin, dlx-cre, virus, psico, thalamic reticular nucleus, bestrophin-1

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