(A) L1-Fc was bound to Oli- cells in the presence of control or F3 siRNA and detected with an anti–human Fc Cy2 antibody (left)

F3 knockdown was analyzed by Western blotting of the cells used for L1-Fc binding. Mouse brain lysate was used as control and GAPDH served as a loading control (right). Bars, 10 μm. (B) Differentiated Oli- cells were incubated with 75 nM of control Fc (human IgG, C-Fc) or L1-Fc and 75 nM L1-Fc in the presence of monoclonal F3 or O4 (control) antibodies. Binding was quantified by cell ELISA (see Materials and methods; = 6). (C) Oli- cells were treated with 0 or 25 nM L1-Fc after treatment with control or F3 siRNA. Binding was quantified by cell ELISA and the ratio of 25 nM Li-Fc–treated cells/0 nM L1-Fc–treated cells was plotted to express the relative amount of bound L1-Fc in control and F3 siRNA-treated cells. Note that because of a different experimental setup, the reduction of F3 protein levels was not as efficient as in the experiment shown in A ( = 9). Error bars indicate SEM. Significance was assessed by tests: *, P < 0.02; **, P < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Differentiated Oli- cells or primary oligodendrocytes were treated with the indicated concentrations of L1-Fc

Equal amounts of cell lysates were analyzed by Western blotting using the indicated antibodies. Numbers on top indicate L1-Fc concentration in nM. (B, left) Oli- cells were incubated with control Fc (C-Fc, human IgG) or L1-Fc in the presence of control or Fyn siRNA. Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) and analyzed by Western blotting for hnRNP A2 (P-Tyr A2). Levels of tyrosine-phosphorylated A2 are strongly increased in L1-Fc–treated cells compared with control Fc–treated cells, and this effect is reduced in cells treated with Fyn siRNA. Total lysates (before IP) were analyzed by Western blotting with hnRNP A2, Fyn, and GAPDH antibodies and demonstrated unchanged levels of total hnRNP A2 and a reduction of Fyn protein by Fyn siRNA. GAPDH served as a loading control. (B, right) The diagram represents the data from three such experiments. Protein bands of tyrosine-phosphorylated hnRNP A2 were densitometrically quantified and the values of control Fc–treated cells were set to 1. The relative increase of tyrosine-phosphorylated hnRNP A2 in L1-Fc–treated cells in the presence of control and Fyn siRNA was plotted. Error bars indicate SEM; = 3.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) An A2RE-containing region of the 3′ UTR of MBP14 mRNA was cloned downstream of the luciferase coding sequence (CDS) and used as a translational reporter in the DualGlo luciferase assay

(B) Oli- cells were nucleofected with an A2RE containing luciferase reporter, a luciferase control, and either wild-type Fyn (Fyn WT), constitutively active Fyn (Fyn), or EGFP. was normalized to luciferase activity for every measurement in all experiments ( = 3). (C) Total RNA was isolated from the cells used in the luciferase assay shown in B. qRT-PCR was performed to compare A2RE luciferase mRNA from Fyn (WT and Fyn)-transfected cells with EGFP-transfected cells ( was used for normalization). Error bars indicate SEM. Significance was assessed with tests: *, P < 0.05; **, P < 0.01; ns, not significant.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Oli- cells and primary oligodendrocytes were transfected with full-length hnRNP A2 and immunostained for hnRNP A2 or MBP

Images were acquired by confocal microscopy, and either a single slice (Oli-) or the complete stack (primary oligodendrocytes) is depicted. HnRNP A2–containing granules are present in the processes of Oli- cells as well as primary oligodendrocytes. Insets show an enlarged view of the boxed sections. Bars, 10 μm. (B) A granule-free supernatant was analyzed by Western blotting for hnRNP E1 and A2 proteins after RNase A treatment or L1-Fc binding. Western blot bands were analyzed densitometrically from 8 and 15 experiments for RNase treatment and L1-Fc binding, respectively. The control values (RNase A and C-Fc) were set to 1 and the mean relative increase of hnRNP E1 and A2 in the granule-free fraction was plotted in response to RNase A treatment or L1-Fc binding. Error bars indicate SEM; significance was tested with tests: *, P ≤ 0.05; **, P ≤ 0.01. = 8 (RNase A) and = 15 (L1-Fc). (C) The model illustrates the proposed events: During initial axon–glial contacts, neuronal L1 binds glial F3 (1), leading to an activation of Fyn (2), which phosphorylates hnRNP A2 (3). This leads to a release of hnRNP A2 and E1 from the granule and liberation of MBP mRNA (4) at the axon–glial contact site, allowing localized synthesis of the MBP protein (5) required for generation of the myelin sheath. The dotted lines illustrate potential alternative activation pathways of Fyn kinase mediated by L1 binding.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Oli- cells were transfected with control EGFP or a constitutively active Fyn (Fyn) construct

Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) from cell lysates (lysate) and analyzed for hnRNP A2 and hnRNP E1/E2. HnRNP A2 immunoprecipitates from cells transfected with active Fyn, whereas hnRNP E1/E2 is absent. Mouse brain lysate and antibody beads alone served as blotting controls. Note that the hnRNP A2 antibody also recognizes the splice variants hnRNP B1 and B0a. HC and LC, heavy and light chain of mouse anti-phosphotyrosine antibody used in the immunopreciptiation. Black lines indicate that intervening lines have been spliced out. (B) Endogenous hnRNP A2 was immunoprecipitated from Oli- cells and subjected to an in vitro kinase assay using purified recombinant Fyn kinase. The top shows a phosphotyrosine blot and a band at 36 kD only in the presence of recombinant Fyn. This was identified as hnRNP A2 by reblotting with an hnRNP A2 antibody (bottom). Numbers to the right of the gel blots indicate molecular mass in kD. (C) HnRNP A2 coimmunoprecipitates with Fyn from Oli- cells transfected with wild-type Fyn, whereas a control protein (α-tubulin) does not.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

Glial Promoter Selectivity following AAV-Delivery to the Immature Brain

<div><p>Recombinant adeno-associated virus (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP) expression driven by the myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA)-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.</p></div

Promoter selectivity targets transgene expression to specific neural cell types <i>in vitro</i> and in the adult brain.

<p>AAV vectors (1×10⁹ vg) were used to express GFP driven by the indicated promoters in enriched primary oligodendrocyte cultures (A–C). For in vivo studies vectors (2×10⁹ vg) were injected in the striatum of adult mice (D–F). Representative images of results by double-immunocytochemistry for GFP (green) and cell-type specific markers (red) illustrate promoter selectivity. A, In primary cultures AAV-CBA-GFP expressed in all NeuN⁺ neurons. In addition, NeuN-negative astrocytes (top in picture) showed GFP immunoreactivity. B, AAV-MBP-GFP-mediated GFP-expression was restricted to ASPA⁺ oligodendrocytes in vitro. C, AAV-GFAP-GFP transduction resulted in GFP immunoreactivity limited to cultured GFAP⁺ astrocytes. D, CBA promoter-controlled GFP expression was highly specific to neurons in vivo. E, The MBP promoter was selective for forebrain oligodendrocytes. F, The GFAP promoter drove GFP specifically in astrocytes. Representative results from three independent experiments are shown. Bars: A–C, 50 µm; D–F, 100 µm.</p

Quantification of glial promoter selectivity and vector spread after AAV delivery to adult mice.

<p>Three weeks prior to analysis 2×10⁹ vg AAV-MBP-GFP (A–E) or AAV-GFAP-GFP (F–J) were injected into the dorsal striatum of adult mice (n = 3). Higher magnification views of the striatum following detection of GFP with either ASPA (A,F), NeuN (B,G) or ALDH1L1 (C,H) was performed to identify the GFP⁺ cell-types. Quantification of results after AAV-MBP-GFP injection showed the vast majority of transgene-expressing cells were ASPA⁺ oligodendrocytes, followed by a smaller fraction representing NeuN⁺ neurons (D). In contrast, AAV-GFAP-GFP-mediated transgene expression was strictly astrocytic (I). The vector spread, determined by monitoring transgene expression in the rostro-caudal extension, was comparable for both vectors (E,J). Arrows in A,B,H indicate co-labelling. Bars: 50 µm.</p

Promoter specificity and volume of GFP-expression after AAV injection at different postnatal stages.

<p>A, For each promoter and time point used, the proportion of neurons, oligodendrocytes, and astrocytes was calculated as a percentage of the total number of cells expressing GFP (n = 3). B, Summary of the percentage of GFP-expressing cells relative to the three different neural populations in the target area. Contrary to neonatal AAV-delivery, the MBP promoter robustly targeted the oligodendrocyte population following injection at P10 and P90 (MBP-P0: 3.0±0.2%, MBP-P10: 68.3±9.2%, MBP-P90: 53.3±12.5%). The GFAP promoter resulted in robust transgene expression in the astrocyte population regardless of the time point of AAV-injection (GFAP-P0: 52.0±5.6%, GFAP-P90: 65.4±3.1%). C, Volume showing GFP expression after AAV-MBP-GFP or AAV-GFAP-GFP delivery. Vector delivery at early time points result in higher efficacy compared to the adult stage (MBP-P0: 23.5±1.7 mm³, MBP-P10: 23.1±1.8 mm³, MBP-P90: 8.3±1.2 mm³, GFAP-P0: 26.7±5.8 mm³, GFAP-P90: 5.8±1.2 mm³). D, Vector spread relative to the whole brain volume (MBP-P0: 5.9±0.4%, MBP-P10: 4.6±0.4%, MBP-P90: 1.7±0.2%, GFAP-P0: 6.7±50.7%, GFAP-P90: 1.2±0.2%). p<0.001, 2-way ANOVA and Bonferroni post-test.</p

Spread of GFP expression after AAV-MBP-GFP delivery to the neonatal or P10 striatum.

<p>Vectors (2×10⁹ vg) were delivered unilaterally to the striatum (n = 3) at P0 (left) or P10 (right). Three weeks later brain sections were immunostained for GFP and every 8^th section was used to determine the area showing GFP immunoreactivity. The graph shows quantitative results after plotting the area covered by GFP immunoreactivity as a function of the distance from the injection site. An arrowhead labels the approximate injection site. The spread of transgene expression is comparable after neonatal or P10 delivery. Bars: 1 mm.</p