Rapid Transgene Expression in Multiple Precursor Cell Types of Adult Rat Subventricular Zone Mediated by Adeno-Associated Type 1 Vectors

The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1+ precursors (30% of the GFP+ cells), Dlx2+ neuronal progenitors (55%), Olig2+ oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx+) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP+) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP+ precursors were unaltered. Indeed, the proportion of Dcx+ cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP+ cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy

Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

Abstract The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e. adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease

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

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

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

<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