Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant alpha-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

<div><p>Glial fibrillary acidic protein (GFAP) is the main astrocytic intermediate filament (IF). GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ), whereas GFAP⁺¹ is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD) mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-β, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS), neurogenic astrocytes of the subgranular zone (SGZ), and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP⁺¹ staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain.</p> </div

Immunocytochemical stainings for GFAP in cortex of 3xTgAD mice.

<p>(<b>A</b>) Intraneuronal APP/Aβ staining in neocortical neurons in layer 4/5 of 3xTgAD at 18 months. GFAPpan immunostaining does not show any reactive astrocytes. (<b>B</b>) Diffuse plaques (arrows) in the cortex of an 18 month old 3xTgAD female mouse are not surrounded by GFAPpan-positive reactive astrocytes. (<b>C</b>) Higher magnification of a cortical plaque (arrow) illustrating the absence of reactive gliosis. (<b>D</b>) Double staining for Aβ and microglia (Iba1) demonstrates the absence of microgliosis around a diffuse plaque (arrow). Arrowheads indicate the positions of individual Iba1-positive microglia without aggregation around plaques. (<b>E</b>) Some plaques in the cortex have a more compact amyloid structure (arrowhead) than the more diffuse plaques (arrows) and these deposits are surrounded by GFAPpan-positive reactive astrocytes. (<b>F</b>) Higher magnification of gliosis, demonstrated by GFAPc-term immunostaining around a compact plaque. (<b>G,G’</b>) Triple staining for Aβ and GFAPc-term and GFAPδ. Reactive astrocytes contacting plaques in 3xTgAD cortex are immunopositive for GFAPc-term and GFAPδ. (<b>H</b>) Hippocampal neurons with accumulated tau-protein (HT7 antibody) in 3xTgAD are not associated with reactive astrocytes.</p

Table <b>3.</b> Mouse GFAP-isoform specific antibodies and other antibodies used.

*<p>TGl  =  thyroglobulin; note the erroneous insertion of <i>E</i> when designing the epitope sequence.</p>**<p>Keyhole limpet hemocyanin.</p>***<p>Tetanus Toxoid.</p><p>ip, immunopurified.</p

Quantification of fold change in hippocampal and cortical transcript levels in 3xTgAD mice compared age-matched WT.

*<p><i>P</i><0.05;</p>**<p><i>P</i><0.01;</p>***<p><i>P</i><0.001;</p><p>Pooled data from 9–18 month old mice: n = 25 to AD; n = 38 to WT; n.d., not detectable. HPC, hippocampus; CX, cortex.</p>1<p>Gfap-Δ135 and Gfap-dEx6 assays lack sensitivity to detect levels lower than 0.003% of Gfap-α.</p>2<p>Gfap-ΔEx7 was not detectable in all of the samples; indicated number of mice (TG+WT) with detectable signal over all mice studied. No change in AD vs WT was detected.</p><p>Data are presented as fold change of <b>3xTgAD</b> mice compared to expression levels found in WT mice of the same age.</p

Co-expression of GFAPα and GFAPδ at different ratios results in different IF network morphologies.

<p>Co-transfection of SW13/cl.2 cells with different ratios of GFAPα and GFAPδ encoding vectors. Transfected cultures fixed 24 h after transfection and stained with GFAPpan to study the morphology of the resulting IF networks. Transfection of GFAPα without GFAPδ yielded complex networks composed of long filaments present throughout the cell (<b>A</b>), whereas 75% GFAPα/25% GFAPδ results in condensed networks (<b>B</b>). A 50% GFAPα/50% GFAPδ ratio yields small networks or just isolated short filaments (<b>C</b>). At 25% GFAPα/75% GFAPδ and 100% GFAPδ only short “squiggles” were observed (<b>D–F</b>).</p

Schematic representation of the different mouse GFAP isoforms studied.

<p>The scheme illustrates the differential splicing routes resulting in 10 different Gfap transcript isoforms. The 9 exons containing the canonical Gfap-α isoform is shown on top. Size of the depicted exons is to scale except for exon 1 and 9, indicated by breaks. The target position of primers used for qPCR assays are indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823.s002" target="_blank">Table S1</a> for their sequences). The position of the epitope for the isoform-specific antibodies generated by us is indicated. Note that only the full-length sequences of mouse Gfap-α, Gfap-δ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823-Roelofs1" target="_blank">[25]</a>, and Gfap-κ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823-Blechingberg1" target="_blank">[26]</a> were identified by us. Transcripts encoding for GfapΔ135, and the GFAP+1 variants (GfapΔ164 and GfapΔEx6), as found in human brain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823-Hol1" target="_blank">[24]</a>, were not detected by qPCR. We found evidence for the existence of GfapΔEx7, a potential GFAP+1 variant, but no effort was made to clone the full-length sequence. Gfap-δ and Gfap-κ each encode for a unique C-terminal amino acid sequence of 41 aa and 46 aa, respectively, different from the Gfap-α encoded C-terminus. Gfap-β was decribed for rat brain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823-Feinstein1" target="_blank">[21]</a>, Gfap-γ and Gfap-ζ were isolated from mouse brain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042823#pone.0042823-Zelenika1" target="_blank">[23]</a>.</p

Specificity of antibodies directed against mouse GFAP isoforms tested on SW13/cl.2 cells transfected with mouse full length GFAP isoform cDNAs.

*<p>Transfection with human GFAPδ showed panGFAP positive cells that were negative for msGFAPδ.</p>**<p>GFAPd135 is expected to have a conventional GFAPα C-terminus and the observed absence of staining.</p