SVZ neurosphere formation
Neural progenitor cells give rise to clonal aggregates of undifferentiated neural precursors, called neurospheres. These cells also reaggregate from dispersed cells in suspension culture and regenerate into histotypical three-dimensional spheres. SVZ cells are self-renewing and muti-potential progenitor cells (Reynolds and Weiss, 1996
). In order to determine whether multi-potentiality and self-renewal of SVZ cells are altered with age, floating SVZ neurosphere formation was measured. shows that SVZ cells from both young and retired breeder rats cultured in the growth medium (containing heparin, bFGF and EGF) form floating multicellular spheres (A and D). A significant decrease in neurosphere size and number was observed in SVZ neurosphere cultures from 14 month old retired breeder rats (D, G and H) compared with culture from 3 month young adult rats (A, G and H). DETA-NONO-ate treatment of neurospheres derived from both young (both doses, i.e. 0.1 μ
M and 0.4 μ
M) and retired breeder (0.1 and 0.4 μ
M) rats significantly decreases floating neurosphere formation number and size, and induces neurosphere attachment to the flask. Both young (B and C) and retired breeder (E and F) rat SVZ neurospheres subsequently disperse and grow into sheets of largely flattened cells when cultured in growth medium.
Fig. 1 SVZ neurosphere formation. Panels A–C show young rat SVZ neurosphere formation in non-treated control (A), treated with 0.1 μM DETA-NONOate (B) and treated with 0.4 μM DETA-NONOate (C). Panels D–F show retired breeder rat (more ...)
Neurosphere cell viability
Decreased floating neurosphere formation number may be caused by an increase of cell adhesion, decrease of cell survival and cell proliferation (Chen et al., 2005
). To measure whether DETA-NONOate treatment affects cell viability, total live and dead cells were measured. Neurosphere cells were mechanically dissociated to single-cells and were plated onto laminin-coated well. Neurosphere cells attached and dispersed. Cell viability was measured. shows no significant difference between neurosphere cell viability in the DETA-NONOate-treated groups compared with the control group in both young adult (A–C) and retired breeder neurospheres (E–G). shows the quantitative data of cell viability in young (D) and retired breeder (H) neurospheres. shows that total live cell numbers were significantly increased in young adult neurosphere cells compared with retired breeder neurosphere cells (I, P
Fig. 2 SVZ neurosphere cell viability, proliferation (BrdU ELISA) and telomerase activity measurements. Panels A–D show young adult SVZ neurosphere cell live–dead viability measurement (A for young adult neurosphere control; B for young neurosphere (more ...)
SVZ neurosphere cell proliferation
To test SVZ neurosphere cell proliferation, BrdU ELISA assays were performed. There are no significant treatment group by age interactions (P=0.414) or treatment group main effects (P=0.132) observed in BrdU incorporation. shows that retired breeder SVZ neurosphere cell proliferation significantly decreased compared with young adult rat SVZ neurospheres (J, P<0.05). DETA-NONOate added to young adult and retired breeder rat SVZ neurosphere culture did not alter cell proliferation compared with control non-treated SVZ neurospheres. This is consistent with BrdU expression measured by cell flow cytometry (data not shown).
SVZ neurosphere cell telomerase activity
Telomerase activity increases with cell proliferation, and rapidly decreases as cells differentiate (Cherif et al., 2003
). To test whether the DETA-NONOate-induced decreases of young and aged SVZ neurosphere formation are related to the regulation of telomerase activity, telomerase activity was measured in both young adult and retired breeder rat SVZ neurospheres with or without DETA-NONOate. There is a significant age by treatment interaction (P
<0.05) observed in the telomerase activity assay data. shows that telomerase activity was significantly decreased in retired breeder neurospheres compared with young adult neurospheres (P
<0.05). In addition, DETA-NONO-ate treatment significantly decreases telomerase activity in both young and retired breeder neurospheres compared with non-treated neurospheres ().
DETA-NONOate increases SVZ cell neuronal differentiation, but decreases glial differentiation
To test whether DETA-NONOate regulates SVZ neurosphere cell differentiation, RT-PCR was used to measure TUJ1 and GFAP mRNA expression in young adult and retired breeder rat SVZ neurospheres treated with or without DETA-NONOate (0.1 μM and 0.4 μM). A significant treatment by age interaction is found in the analysis of GFAP gene expression (P=0.096), but not in TUJ1 gene expression (P=0.813). shows that there are no significant differences in TUJ1 mRNA expression in retired breeder neurospheres compared with young adult neurospheres. DETA-NONOate treatment significantly increases TUJ1 (A) mRNA expression in both young adult and retired breeder rats SVZ neurospheres. However, a significant increase of GFAP mRNA expression is found in retired breeder neurospheres compared with young adult neurospheres (B). DETA-NONOate significantly decreases GFAP mRNA expression in retired breeder neurospheres, but does not alter GFAP mRNA in young adult neurospheres compared with respective non-treatment group (B). These data suggest that DETA-NONOate promotes neuronal differentiation in both young and retired breeder neurospheres. However, DETA-NONOate selectively decreases the glial differentiation in retired breeder neurospheres.
Fig. 3 SVZ neurosphere cell differentiation and neurite outgrowth. Panels A and B show data from young and retired breeder neurospheres treated with or without DETA-NONOate regulate TUJ1 (A), and GFAP (B) mRNA expression. Panels C–H show TUJ1 (TUJ1 positive (more ...)
To confirm that DETA-NONOate promotes neuronal differentiation in young and retired breeder rat SVZ neurospheres, neurospheres were passaged by mechanical dissociation and reseeded as single cells at a density of 2.0×104 cells/ml in laminin-coated glass coverslips and treated with or without DETA-NONOate (0, 0.1 and 0.4 μM) for 7 days in differentiation medium. TUJ1 and GFAP protein immunostaining were performed. shows that both doses of DETA-NONOate treatment (0.1 μM and 0.4 μM) significantly increase TUJ1 positive cell numbers compared with the respective non-treatment control group in both young adult and retired breeder neurospheres. In addition, GFAP expression significantly increases in retired breeder control neurospheres compared with young adult control neurospheres (J). shows that DETA-NONOate decreases GFAP positive cells number in retired breeder neurospheres (K–M), but not in young adult neurospheres compared with respective non-treated controls (J). These data confirm that DETA-NONOate promotes neuronal differentiation in both young and retired breeder neurospheres, as well as decreases glial differentiation in retired breeder neurospheres.
DETA-NONOate promotes neurite outgrowth
To test whether DETA-NONOate regulates neurite outgrowth, total length of neurite outgrowth was measured from TUJ1 positive cells. shows that neurite outgrowth significantly decreases in retired breeder control neurospheres compared with young adult control neurospheres. DETA-NONOate-treated groups show significantly increased neurite outgrowth compared with non-treatment controls in both young adult and retired breeder rat neurospheres, respectively.
N-cadherin mediates DETA-NONOate-induced cell adhesion, neuronal differentiation and neurite outgrowth
The above data show that DETA-NONOate decreases floating neurosphere formation and induces neurosphere adhesion to the flask and promotes neuronal differentiation. N-cadherin regulates cell adhesion and modulates the Wnt-induced nuclear activity of β
-catenin (Modarresi et al., 2005
), and β
-catenin is required for neuronal differentiation (Otero et al., 2004
). Therefore, we first tested whether DETA-NONOate regulates N-cadherin mRNA expression. shows that there are no significant differences in N-cadherin mRNA expression in retired breeder neurospheres compared with young adult neurospheres. However, DETA-NONOate treatment in both young adult and retired breeder rat neurospheres significantly increased N-cadherin mRNA expression (A) compared with the respective non-treatment controls.
Fig. 4 N-cadherin regulates neurosphere formation and cell adhesion. Panel A shows quantitative data of N-cadherin mRNA expression in young and retired breeder rat SVZ neurospheres treated with or without DETA-NONOate for 7 days. Panels B–F show young (more ...)
To further investigate whether N-cadherin participates in DETA-NONOate-induced neuronal differentiation, N-cadherin (5 μg/cm2) (recombinant human N-cadherin/Fc chimera, R&D System) and neutralized anti-N-cadherin antibody (monoclonal anti-N-cadherin/A-CAM; Sigma, St. Louis, MO, USA) treatment was employed. The young adult and retired breeder SVZ neurospheres were treated with or without DETA-NONOate, recombinant human N-cadherin/Fc chimera-coated (5 μg/cm2) or an anti-N-cadherin antibody (80 ng/ml) in growth medium for 7 days. Neurosphere formation and cell differentiation were measured. shows that N-cadherin significantly inhibits floating neurosphere formation (D), and significantly promotes neurosphere cell adhesion to the flask, dispersion and growth into sheets of largely flattened cells (D) compared with control young adult neurospheres (B). Anti-N-cadherin (F) reverses DETA-NONOate-induced neurosphere adhesion, and promotes neurosphere formation compared with DETA-NONOate (0.4 μM NONOate) treatment alone (C). shows the quantitative data of neurosphere formation from the different treatment groups. The retired breeder neurospheres have the same pattern of change as young adult neurospheres when treated with or without N-cadherin, or anti-N-cadherin (data not shown).
To test whether N-cadherin regulates β-catenin, and whether the N-cadherin/β-catenin pathway plays a role in DETA-NONOate-induced neuronal differentiation, β-catenin and TUJ1 mRNA expression was measured using RT-PCR. shows that N-cadherin promotes TUJ1 (A: retired breeder neurosphere C: young neurosphere) mRNA expression as well as upregulates β-catenin (B: retired breeder neurospheres; D: young neurospheres) mRNA expression in both young adult and retired breeder neurospheres. Anti-N-cadherin attenuates DETA-NONOate-induced TUJ1 (A and C) and β-catenin (B and D) mRNA expression. However, GFAP mRNA expression was not significantly different in N-cadherin- and anti-N-cadherin-treated groups (E).
Fig. 5 N-cadherin regulates SVZ neurosphere β-catenin mRNA expression, cell differentiation and neurite outgrowth. Panels A–D show quantitative data of TUJ1 (A for retired breeder neurosphere; C for young adult neurosphere), β-catenin (more ...)
DETA-NONOate has similar effects on regulating neuronal differentiation, and neurite outgrowth in both young and retired breeder neurospheres. Therefore, to test whether N-cadherin may also play a role in DETA-NONOate-induced neurite outgrowth, only young neurospheres were employed. Young adult neurospheres passaged by mechanical dissociation were reseeded as single cells at a density of 2.0×104 cells/ml in laminin-coated glass cover-slips and treated with or without N-cadherin (5 μg/cm2) and neutralizing anti-N-cadherin antibody for 7 days in differentiation medium. TUJ1 protein immunostaining was performed. shows that N-cadherin treatment significantly increases TUJ1 positive cell neurite outgrowth compared with the control group. Anti-N-cadherin significantly decreases DETA-NONOate-induced neurite outgrowth (F) compared with DETA-NONOate treatment alone.
DETA-NONOate regulates Ngn1 gene expression ()
bHLH transcription factors such as Ngn1 play important roles in the regulation of neuronal differentiation and the inhibition glial differentiation (Sauvageot and Stiles, 2002
). To evaluate the possible signal transduction pathways that may be involved in DETA-NONOate-induced neuronal differentiation and decreased glial differentiation, Ngn1 gene expression was measured in both young and retired breeder rat SVZ neurospheres. A significant two-way interaction between treatment groups (P
<0.05) was found. shows that Ngn1 mRNA expression is significantly decreased in retired breeder neurospheres compared with young adult rat neurospheres. The decreased Ngn1 expression parallels the increase of GFAP mRNA expression in retired breeder neurospheres (). DETA-NONO-ate treatment significantly increases Ngn1 gene expression in both young adult (0.4 μ
M NONOate treatment) and retired breeder rat SVZ neurospheres (0.1 μ
M and 0.4 μ
M NONOate treatment) compared with non-treated controls.
To test whether N-cadherin also regulates Ngn1 expression, SVZ neurospheres derived from both young adult and retired breeder rats were cultured in growth medium for 7 days and were treated with or without DETA-NONOate (0.4 μM), N-cadherin (5 μg/cm2) and anti-N-cadherin antibody (80 ng/ml) with DETA-NONOate (0.4 μM). Ngn1 mRNA expression was measured. shows that N-cadherin and DETA-NONOate significantly increase Ngn1 mRNA expression compared with control non-treatment neurospheres in both young adult and retired breeder rats. Anti-N-cadherin significantly inhibits DETA-NONOate-induced Ngn1 mRNA expression in both young adult and retired breeder neurospheres compared with DETA-NONOate (0.4 μM) treatment alone.
Fig. 6 Ngn1 mRNA expression decreases with age; N-cadherin regulates Ngn1 mRNA expression. Panel A shows Ngn1 mRNA expression measured by RT-PCR in young adult and retired breeder neurospheres treated with or without DETA-NONOate for 7 days. Panels B and C show (more ...)