Journal: Cell Transplantation

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells

doi: 10.1177/0963689718774030

Figure Lengend Snippet: Correlation of decline in DG NSPC regeneration to Nrf2 expression. Immunohistochemical analysis by age group (N, YA, A, MA and O) illustrating MCM2 staining (for proliferation) in A–E and its quantification is in F ( p < 0.01, N versus YA and p < 0.05, A versus MA; one-way ANOVA with Tukey’s post-hoc test). Qualitative assessment of hippocampal Sox2 + NSPCs and their expression of Nrf2 across the five age-groups is in G–Z, with quantification in UU ( p < 0.01, N versus YA and p < 0.01, A versus MA; one-way ANOVA with Tukey’s post-hoc test), and VV ( p < 0.001, N versus YA and p < 0.05, A versus MA; one-way ANOVA with Tukey’s post-hoc test) are shown. Similarly, qualitative and quantitative analysis of Dcx + cells is in AA–TT, WW ( p < 0.0001, N versus YA and p < 0.001, A versus MA; one-way ANOVA with Tukey’s post-hoc test) and XX ( p < 0.0001, N versus YA and p < 0.01, A versus MA; one-way ANOVA with Tukey’s post-hoc test). Expression of Nrf2 and GCLM in cultured hippocampal NSPCs across the five age-groups is shown in a–e and g–k, with quantification in f ( p < 0.001, N versus YA and p < 0.001, A versus MA; one-way ANOVA with Tukey’s post-hoc test) and l ( p < 0.01, N versus YA and p < 0.001, A versus MA; one-way ANOVA with Tukey’s post-hoc test). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: A–E; G–Z, AA–TT: 25 µm, a–e: 15 µm. A: adult; ANOVA: analysis of variance; DG: dentate gyrus; N: newborn; GCLM: glutamate–cysteine ligase modifier subunit; MA: middle-aged; NSPC: neural stem progenitor cell; O: old; YA: young adult.

Article Snippet: For the overexpression studies, a rat Nrf2 expression plasmid (CMV promoter, Creative Biogene Technology, Shirley, NY, USA) was transfected into NSPCs using Lipofectamine ® LTX Reagent (Life Technologies).

Techniques: Expressing, Immunohistochemical staining, Staining, Cell Culture

Journal: Cell Transplantation

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells

doi: 10.1177/0963689718774030

Figure Lengend Snippet: Effects of altered Nrf2 expression on DG NSPC regeneration in vitro. Graphs A and B show results from live-dead (viability) and BrdU (proliferation) assays performed on untreated, control siRNA, and Nrf2 siRNA-treated (siNrf2) newborn rat hippocampal NSPCs ( p < 0.05, p < 0.001, U/siC versus siNrf2, unpaired t tests). Panels C and D show the viability and proliferation of untreated middle-aged cells compared with those transfected with Nrf2 ( p < 0.001, U versus Nrf2). The in vitro survival and proliferative function of DG NSPCs isolated from Nrf2-/- mice compared with WT mice is depicted in E and F ( p < 0.01, unpaired t tests). The capacity of newborn Nrf2 WT and Nrf2-/- NSPCs to differentiate into Tuj1 + neurons ( p < 0.05, unpaired t test), GFAP + astrocytes ( p < 0.05, unpaired t test), and RIP + oligodendrocytes is in (G). * p < 0.05, ** p < 0.01, *** p < 0.001. BrdU: bromodeoxyuridine; DG: dentate gyrus; GFAP: glial fibrillary acidic protein; NSPC: neural stem progenitor cell.

Article Snippet: For the overexpression studies, a rat Nrf2 expression plasmid (CMV promoter, Creative Biogene Technology, Shirley, NY, USA) was transfected into NSPCs using Lipofectamine ® LTX Reagent (Life Technologies).

Techniques: Expressing, In Vitro, Control, Transfection, Isolation

Journal: Cell Transplantation

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells

doi: 10.1177/0963689718774030

Figure Lengend Snippet: In vivo assessment of DG NSPCs from Nrf2 knockout mice. In vivo immunohistochemical analysis of the DG NSPCs in newborn Nrf2 WT and Nrf2-/- mice using antibodies targeting MCM2 (proliferation; A–B), Sox2 (proliferating neural progenitors; C–D), GFAP (astrocytes; E–F) and Dcx (neuroblasts; G–H) was performed. NSPCs from adult Nrf2 WT and knockout animals were also assessed: MCM2 (I–K), Sox2 (L–N), GFAP/nestin (O–Q) and Dcx (R–T). Behavioral analysis of young adult Nrf2 WT and knockout mice through the Morris water maze task is shown in U, and the number of platform entries in the probe trial is in V ( p < 0.05, unpaired t tests). Behavioral results from the pattern separation task is in W ( p < 0.05, unpaired t tests). * p < 0.05, ** p < 0.01. Scale bars: A–H: 60 µm, I–S: 30 µm. DG: dentate gyrus; GFAP: glial fibrillary acidic protein; NSPC: neural stem progenitor cell.

Article Snippet: For the overexpression studies, a rat Nrf2 expression plasmid (CMV promoter, Creative Biogene Technology, Shirley, NY, USA) was transfected into NSPCs using Lipofectamine ® LTX Reagent (Life Technologies).

Techniques: In Vivo, Knock-Out, Immunohistochemical staining

Journal: Cell Transplantation

Article Title: A Role for Nrf2 Expression in Defining the Aging of Hippocampal Neural Stem Cells

doi: 10.1177/0963689718774030

Figure Lengend Snippet: Characterization of NSPC transplants overexpressing Nrf2 and their behavioral effects across the critical period. (A) Schematic of the experimental design illustrating that newborn and middle-aged NSPCs were transfected with an eGFP tagged AAV2/1 virus with or without Nrf2. These cells were transplanted into the DG of 11-month-old rats and the animals aged through the CP of NSPC decline. Behavioral and histological analysis was performed at age 15 months of age. Stereotaxic transplantation sites are noted in B with corresponding fluorescence confirmation of GFP + graft locations. Nrf2 expression in newborn (N) and middle-aged (MA) NPSCs with or without viral Nrf2 transduction is in A–D. Representation of AAV2/1 transduced NSPC cultures, as single-cell and neurospheres, before grafting is in E and F. In vivo Nrf2 expression of GFP + transplants are in G–J (newborn grafts (G–H) and middle-aged grafts (I–J)). Quantification of grafted cells in the different experimental groups is in K ( p < 0.01 N-eGFP versus N-Nrf2-eGFP; p < 0.01, N-eGFP versus MA-eGFP; p < 0.001, N-Nrf2-eGFP versus MA-Nrf2-eGFP; one-way ANOVA with post-hoc Tukey’s test). Results from the pattern separation task, conducted on naïve 11-month-old animals before transplantation (baseline) are in L, N, and after the CP at 15 mo are in M, O (* p < 0.05, novel versus familiar in animals implanted with newborn grafts overexpressing Nrf2, # p < 0.05 compared with control). * p < 0.05, ** p < 0.01, # p < 0.05. Scale bars: B: 200 µm, A, C–D: 20 µm, E: 25 µm, F: 100 µm, G–J: 50 µm. ANOVA: analysis of variance; CP: critical period; DAPI: 4’,6’-diamidino-2-phenylindole, dihydrochloride; DG: dentate gyrus; GFP: green fluorescent protein; NSPC: neural stem progenitor cell; eGFP: enhanced green fluorescent protein.

Article Snippet: For the overexpression studies, a rat Nrf2 expression plasmid (CMV promoter, Creative Biogene Technology, Shirley, NY, USA) was transfected into NSPCs using Lipofectamine ® LTX Reagent (Life Technologies).

Techniques: Transfection, Virus, Transplantation Assay, Fluorescence, Expressing, Transduction, In Vivo, Control