Enhanced neuronal survival in senescent cultures of cortical neurons mediated by rAAV2/1-NF8d1-driven GDNF transgene expression.

<p>(<b>A</b>) Embryonic day 16 cortical neurons were maintained in 96 wells plates for 60 days. Cultures were fixed and stained with NeuN (a neuronal specific nuclear protein) or PSD95 (a neuronal postsynaptic density protein) a class of anchoring proteins which serve to localize various neuronal ion channels to post-synaptic densities antibodies. The number of NeuN-positive or PSD95-positive neurons is significantly higher in cultures treated with rAAV2/1-NF8d1-GDNF as compared to rAAV2/1-NF8d1-EGFP (CtrL) (p<0.05; student test, n = 10). Bars represent means ± SEM. (<b>B</b>) Similar cultures seeded on coated glass coverslips were fixed at day 10, day 21 and day 47 and stained with antibodies directed against activated NFkB followed by a secondary antibody coupled with cyanine 2. Shown are confocal microscopy photographs. Bars represent 50 µM. The mean optical density of the nuclei was measured using the Image J software. Differences between day 10 (n = 39) and day 21 (n = 59) or day 47 (n = 57) were respectively highly significant (**; p<0.001) and very highly significant (###; p<0.0001). The experiment was repeated twice with similar results. (<b>C</b>) Culture media from rAAV1-NF8-d1-GDNF- and rAAV1-NF8-d1-EGFP-infected cultures were harvested from day 5 to day 42 post-infection and GDNF concentrations (pg/ml) were measured using ELISA assay. For rAAV1-NF8-d1-GDNF-infected cultures, all values, except at day 5, were significantly higher (***; p<0.0001; n = 10 for rAAV1-NF8-d1-hGDNF and n = 8 for rAAV1-NF8-d1-EGFP) than the basal level (medium from rAAV1-NF8-d1-EGFP-infected cultures). The experiment was repeated twice with similar results.</p

Hippocampal sclerosis and upregulation of inflammatory markers in the hippocampus of kainic acid-treated rats.

<p><b>A.</b> Haematoxylin-eosin staining showing typical hippocampal sclerosis in kainic acid-treated rats (KA) (left) as compared to controls (right). Bar represents 200 µm. <b>B.</b> Comparison of the intensity of the GFAP astrocytic marker staining in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using mouse anti-GFAP followed by a secondary antibody coupled to Cy3 fluorophore. The intensity of the staining of three random areas in the hipocampal layers on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 8 for each group of animals). The difference between kainic acid and saline-treated groups was highly significant (student test, P = 0,00018). Bar represents 500 µm. <b>C.</b> Comparison of the intensity of the CD11b staining of activated microglia in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using mouse anti-CD11b followed by streptavidin-biotin-peroxidase staining. The intensity of the staining of three random areas within the hippocampus on every section was quantified using the Image J program. Arrow shows localized staining at the level of the needle tract. Data are expressed as the mean optical density ± SEM (n = 4 for each group of animals). The difference between kainic acid and saline-treated groups was significant (student test, P = 0,0068). Bar represents 200 µm. <b>D.</b> Comparison of the intensity of the IbaI microglial staining in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using goat anti-IbaI followed by anti-goat antibody coupled with cyanine 3. The intensity of the staining of three random areas within the hippocampus on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 6 for each group of animals). The difference between kainic acid and saline-treated groups was significant (student test, p = 0,00121). Bar represents 500 µm.</p

Absence of transgene upregulation in rAAV2/1-NF8d1-EGFP-mediated gene transfer in the cerebellum of kainic acid-treated rats.

<p>Kainic acid (5 mg/kg) was injected intraperitoneally (ip) one month after stereotaxic injection of rAAV2/1-NF8d1-EGFP into the cerebellum. Fifty µm coronal sections were labeled with GFP or GFAP antibodies. Confocal pictures showing GFP (A) or GFAP (B). Bars represent 50 µm. GFAP staining was quantified by measuring the optical density of three area in all sections stained per animal (6 rats for each condition). The difference between KA- and saline-treated rats is not significant (student test, p = 0,8612) (B).</p

Disease-inducible rAAV1-NF-mediated transgene expression in the hippocampus of kainic acid-treated rats.

<p>Recombinant AAV2/1-NF8-d1-EGFP (8×10⁶ vg) was stereotaxically injected in the right hippocampus of male Wistar rats. Animals were kept in two groups: one group was intraperitoneally-injected with kainic acid (n = 8) one month post stereotaxy and the other group received only saline (n = 8). The animals were sacrificed one week after injection. Vibratome brain sections (50 µm) were immunolabeled using an anti-GFP antibody, biotin-streptavidin amplification and Cy2 fluorophore. <b>A.</b> Representative examples of GFP-labeling of the CA hippocampal layers dentate gyrus post-kainic acid injection. Note the massive presence of GFP-positive cells in the CA1 and CA3 layers. Stereological counts of GFP-positive cell numbers per animal demonstrates a highly significant induction of the AAV-NF vector by kainic acid (P = 0,000875; student test; n = 8 for the saline group; n = 6 for the kainic acid group). KA, kainic acid; saline, 0.9% NaCl. The experiment was repeated 3 times with similar results. Bars represent 500 µm and 100µm in lower and higher (1,2,3 subpannels) magnification pictures, respectively. <b>B</b>. Comparison of the staining intensity for activated NFκB in kainic acid- and saline-treated rats. Vibratome brain sections (50 µm) were immunolabeled using anti-activated NFκB antibody followed by streptavidin-biotin-peroxidase staining. Note, in particular, the localized increased staining in the CA hippocampal layers. The intensity of the staining of three random areas in the hipocampal layers on every section was quantified using the Image J program. Data are expressed as the mean optical density ± SEM (n = 8 for each group of animals). The difference between KA and saline-treated groups was significant (student test, P = 0.0010). Bars represent 500 µm. <b>C.</b> Correlation between NFκB activation and GFP expression mediated by rAAV2/1-NF8-d1-EGFP in hippocampal layers. A group of 16 rats was injected with a rAAV1/2-NF8-d1-EGFP in the hippocampus. 8 rats were injected with KA and 8 other animals were injected with the saline solution. Two KA-treated rats were removed from the analysis since they either contained no GFP-positive cells (presumably due to a failure to inject the virus) or had a totally different profile of GFP-expression (no GFP-positive cells in the hippocampal layers, presumably due to wrong stereotaxic coordinates). The total number of GFP-immunoreactive cells per animal (as evaluated by stereology was correlated with the mean optical density of the NFκB stainings in the region containing the transduced cells. There was a significant correlation between the two parameters with a coefficient of 0.7001 (p = 0.0002; n = 6 for KA-treated rats and n = 8 for saline-treated rats). The experiment was repeated a second time (n = 8 for KA-treated rats and n = 7 for saline-treated rats) with similar results.</p

Cellular specificity of rAAV2/1-NF8d1-EGFP-mediated gene transfer in the hippocampus of kainic acid-treated rats.

<p>Kainic acid (5 mg/kg) was injected intraperitoneally (ip) one month after stereotaxic injection of rAAV2/1-NF8d1-EGFP into the hippocampus. Fifty µm coronal sections were co-labeled with GFP (green fluorescence) and NeuN, GFAP IbaI or Olig2. Confocal pictures showing GFP and cell-specific markers co-labeled cells (yellow). White arrows indicate co-labeled cells. Bars represent 50 µm. The mean percentage of GFP-positive cells co-labeling with the GFAP, NeuN and Olig2 markers are shown. No GFP-positive cell stained positive for IbaI. Analysis was performed using confocal microscopy and counting the number co-labeled cells on 5 sections per animal (n = 5 rats for GFP/NeuN and GFP/GFAP, n = 6 for GFP/IbaI and n = 7 for GFP/Olig2 co-labeling).</p

Description of the NF-κB-responsive AAV vectors.

<p>A. Schematic diagram of NF-κB-responsive AAV vectors used in this study. ITR: AAV Inverted Terminal Repeat; TB: Transcriptional Blocker; NFκBRE (4X): four copies of the JC virus NFκB consensus sequence; mTK: herpes simplex virus minimal thymidine kinase promoter; SD/SA: Splice Donor/Splice Acceptor sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053156#pone.0053156-Zolotukhin2" target="_blank">[72]</a>; mCMVΔ2: improved minimal CMV promoter; d1, d3: distance separating the last NFκB consensus sequence and the minimal promoter, respectively, 31 and 71 bp. B. Sequence analysis of the transcription factor binding sites within the minimal CMV and the minimal CMV Δ2 promoters. The mCMV promoter originates from position −52 to position +77 in the wild-type promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053156#pone.0053156-Boshart1" target="_blank">[45]</a>. Analysis of the mCMV sequences highlighted the presence of several transcription factor binding sites : AP2, LFA-1, CRE-1, IFNγ, XRE-1 and GCF. The mCMVΔ2 was obtained by deleting the sequence upstream of the TATA box (−52 to −32) and the sequence downstream of the IFNγ site (+17 to +77) resulting in the removal of AP2, XRE-1 and GCF sites.</p

<i>In vitro</i> TNFα-mediated induction of NF-κB-responsive AAV vectors.

<p>HEK-293T cells were transfected with AAV-NF-EGFP (<b>A</b>) or with AAV-NF-hGDNF (<b>B</b>) vectors using the calcium phosphate coprecipitation method. 5×10⁵ cells in 6-wells were transfected with 250 ng DNA. Forty-eight hrs later, transfected cells were treated or not with TNFα (100 ng/ml) for 5 hrs. HEK-293T transfected with AAV-NF-EGFP were analyzed for GFP expression on a FACStar analyser/sorter (Becton Dickinson) (<b>A</b>). To analyse the inducibility of AAV-NF-hGDNF vectors in HEK-293T, the supernatant were harvested 4 h after changing the medium to measure secreted GDNF concentrations by a commercial ELISA assay (Human GDNF CytoSets, catalog #CHC2423, BioSource, Nivelles,Belgium) (<b>B</b>). For both AAV-NF8-d1-hGDNF and AAV-NF12-d1-hGDNF vectors, the differences between the TNFα-treated and untreated cultures were highly significant. The differences between the basal levels and the induced levels for AAV-NF8-d1-hGDNF versus AAV-NF12-d1-hGDNF are not significant (<b>B</b>). (***, p<0.001; ns, p>0,05, one-way ANOVA Newman-Keuls Multiple Comparison Test). Data are from one representative out of three experiments (3 separate transfections). Bars represent means ± standard deviations (SD) (<b>A</b>) or means ± standard error of the means (SEM) (<b>B</b>) from triplicate wells. A.U., arbitrary units.</p

An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer. © 2013 Chtarto et al.SCOPUS: ar.jinfo:eu-repo/semantics/publishe