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      Smoothened Agonist Reduces Human Immunodeficiency Virus Type-1-Induced Blood-Brain Barrier Breakdown in Humanized Mice

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          Abstract

          Human Immunodeficiency Virus type-1 (HIV)-associated neurocognitive disorder is characterized by recruitment of activated/infected leukocytes into the CNS via disrupted Blood Brain Barrier (BBB) that contributes to persistent neuro-inflammation. In this report, humanized NOD/scid-IL2Rγ c null mice were used to establish that impaired Sonic hedgehog (Shh) signaling is associated with loss of BBB function and neurological damage, and that modulating Shh signaling can rescue these detrimental effects. Plasma viral load, p24 levels and CD4 + T cells were measured as markers of productive HIV infection. These mice also showed impaired exclusion of Evans blue dye from the brain, increased plasma levels of S100B, an astrocytic protein, and down-regulation of tight junction proteins Occludin and Claudin5, collectively indicating BBB dysfunction. Further, brain tissue from HIV + mice indicated reduced synaptic density, neuronal atrophy, microglial activation, and astrocytosis. Importantly, reduced expression of Shh and Gli1 was also observed in these mice, demonstrating diminished Shh signaling. Administration of Shh mimetic, smoothened agonist (SAG) restored BBB integrity and also abated the neuropathology in infected mice. Together, our results suggest a neuroprotective role for Shh signaling in the context of HIV infection, underscoring the therapeutic potential of SAG in controlling HAND pathogenesis.

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          Most cited references48

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          Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.

          Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.
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            Molecular profile of reactive astrocytes—Implications for their role in neurologic disease

            The central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. While this phenomenon is found in many different species, its function is obscure. Understanding the molecular profile characteristic of reactive astrocytes should help define their function. The purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. In vivo, this increase in glial fibrillary acidic protein-positive cells reflects predominantly phenotypic changes of resident astroglia rather than migration or proliferation of such cells. Upon activation, astrocytes upmodulate the expression of a large number of molecules. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. For example, upregulation of proteases and protease inhibitors could help remodel the extracellular matrix, regulate the concentration of different proteins in the neuropil and clear up debris from degenerating cells. Cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood-central nervous system interface. Neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostasis within the central nervous system. Therefore, an impairment of astroglial performance has the potential to exacerbate neuronal dysfunction. Based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. Among them are the relative contribution of microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive astrocytes with techniques that can resolve fast physiologic processes. Differences between reactive astrocytes in vivo and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism.
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              ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression.

              Extracellular ATP released from axons is known to assist activity-dependent signaling between neurons and Schwann cells in the peripheral nervous system. Here we report that ATP released from astrocytes as a result of neuronal activity can also modulate central synaptic transmission. In cultures of hippocampal neurons, endogenously released ATP tonically suppresses glutamatergic synapses via presynaptic P2Y receptors, an effect that depends on the presence of cocultured astrocytes. Glutamate release accompanying neuronal activity also activates non-NMDA receptors of nearby astrocytes and triggers ATP release from these cells, which in turn causes homo- and heterosynaptic suppression. In CA1 pyramidal neurons of hippocampal slices, a similar synaptic suppression was also produced by adenosine, an immediate degradation product of ATP released by glial cells. Thus, neuron-glia crosstalk may participate in activity-dependent synaptic modulation.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                31 May 2016
                2016
                : 6
                : 26876
                Affiliations
                [1 ]Department of Microbiology and Immunology, University of Rochester Medical Center , 601 Elmwood Avenue, Box 672, Rochester, NY 14642, USA
                [2 ]Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center , Omaha, Nebraska 68198, USA.
                Author notes
                Article
                srep26876
                10.1038/srep26876
                4886511
                27241024
                1b6e39f6-bfa5-477a-a5ff-2df509deb46b
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 23 February 2016
                : 10 May 2016
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