ROS produced by NOX2 control in vitro development of cerebellar granule neurons development

Abstract

Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H2O2) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development.

Keywords: NADPH-oxidases; axonal morphogenesis; cerebellar granule neurons; glutathione; neuronal development; reactive oxygen species.

Figure 1.

ROS are differentially produced during CGN development. (a) Representative micrographs of CGN grown in depolarizing conditions (K25) and nondepolarizing conditions (K5) from 1 to 5 DIV. Calcein-positive cells are marked in green, and propidium iodide-positive cells are marked in red (scale bar, 100 µm). (b) Cell viability is expressed as the percentage of calcein-positive cells from the total number of cells, which was estimated as the sum of calcein-positive cells plus propidium iodide-positive cells. #, ## are significantly different from K25 at 4 and 5 DIV (p < .01, p < .001, ANOVA, n = 6). Data are mean ± SEM. (c) Metabolic activity was determined by MTT transformation of CGN grown in K25 or K5 from 1 to 5 DIV. *is significantly different from K25 at 1 DIV (p < .001, ANOVA, n = 6). # is significantly different from K25 at 5 DIV (p < .001, ANOVA, n = 6). Data are mean ± SEM. (d) Phase contrast and fluorescence micrographs of CGN grown in K25 or K5 during 1 to 5 DIV (scale bar, 100 µm). (E) ROS levels of CGN grown in K5 or K25 from 1 to 5 DIV. ROS levels are expressed as mean values of the mean fluorescence intensity of ethidium cation, which is the product of the dihydroethidium oxidation. * is significantly different from K25 at 1 DIV (p < .05, ANOVA, n = 4). # is significantly different from K25 at 4 and 5 DIV (#p < .001, ANOVA, n = 4). Data are mean ± SEM. (f) ROS levels of CGN grown in K25 from 0 to 8 DIV. ROS levels produced from 1 to 8 DIV were higher compared to 0 DIV (p < .001, ANOVA, n = 3–9). ROS levels produced at 2 and 3 DIV were higher compared to 0, 1, 4–8 DIV (p < .001, ANOVA, n = 3–9). Data are normalized with respect to 2 DIV and are presented as mean ± SD. DIV = days in vitro; ROS = reactive oxygen species; CGN = cerebellar granule neurons; ANOVA = analysis of variance.

Figure 1.

ROS are differentially produced during CGN development. (a) Representative micrographs of CGN grown in depolarizing conditions (K25) and nondepolarizing conditions (K5) from 1 to 5 DIV. Calcein-positive cells are marked in green, and propidium iodide-positive cells are marked in red (scale bar, 100 µm). (b) Cell viability is expressed as the percentage of calcein-positive cells from the total number of cells, which was estimated as the sum of calcein-positive cells plus propidium iodide-positive cells. #, ## are significantly different from K25 at 4 and 5 DIV (p < .01, p < .001, ANOVA, n = 6). Data are mean ± SEM. (c) Metabolic activity was determined by MTT transformation of CGN grown in K25 or K5 from 1 to 5 DIV. *is significantly different from K25 at 1 DIV (p < .001, ANOVA, n = 6). # is significantly different from K25 at 5 DIV (p < .001, ANOVA, n = 6). Data are mean ± SEM. (d) Phase contrast and fluorescence micrographs of CGN grown in K25 or K5 during 1 to 5 DIV (scale bar, 100 µm). (E) ROS levels of CGN grown in K5 or K25 from 1 to 5 DIV. ROS levels are expressed as mean values of the mean fluorescence intensity of ethidium cation, which is the product of the dihydroethidium oxidation. * is significantly different from K25 at 1 DIV (p < .05, ANOVA, n = 4). # is significantly different from K25 at 4 and 5 DIV (#p < .001, ANOVA, n = 4). Data are mean ± SEM. (f) ROS levels of CGN grown in K25 from 0 to 8 DIV. ROS levels produced from 1 to 8 DIV were higher compared to 0 DIV (p < .001, ANOVA, n = 3–9). ROS levels produced at 2 and 3 DIV were higher compared to 0, 1, 4–8 DIV (p < .001, ANOVA, n = 3–9). Data are normalized with respect to 2 DIV and are presented as mean ± SD. DIV = days in vitro; ROS = reactive oxygen species; CGN = cerebellar granule neurons; ANOVA = analysis of variance.

Figure 2.

Glutathione is differentially produced and is necessary for CGN survival. (a, b) Reduced glutathione (GSH) and oxidized glutathione (GSSG) content were determined in CGN grown in K25 and K5 from 0 to 8 DIV by a modification of the Tietze recycling assay as detailed in Methods. (a) * is significantly different from K25 at 0 DIV (p < .001, ANOVA, n = 4). # is significantly different from K25 at 5 DIV (p < .001, ANOVA, n = 4). Data are mean ± SEM. (b) * is significantly different from K25 at 0 DIV (p < .001, ANOVA, n = 4). Data are mean ± SEM. (c) GSH and GSSG were determined in CGN grown in K25 at 2 DIV and treated with BSO (100 µM) for 48 hr and Euk-134 (10 µM) for 24 hr. BSO treatments reduced the levels of GSH and GSSG (p < .05, ANOVA nonparametric test, n = 5). Data are mean ± SEM. (d to f) Cell viability was determined by calcein and propidium iodide. Data are expressed as the percentage of calcein-positive cells from the total number of cells, which was estimated as the sum of calcein-positive cells plus propidium iodide-positive cells. (d) Cell viability was determined in CGN grown in K25 and treated with BSO (100 µM) for 24 hr at 1, 2, 3, 4, 5, and 8 DIV (no statistical differences were found, ANOVA, n = 4). (e) Cell viability was determined in CGN grown in K25 and treated with BSO (100 µM) for 48 hr at 2, 3, 4, 5, and 8 DIV. * is significantly different from Control at 2 DIV (*p < .001, ANOVA, n = 4). (f) Cell viability was determined in CGN grown in K25 at 2 DIV and treated with BSO (100 µM) for 48 hr and Euk-134 (10 µM) for 24 hr. * is significantly different from BSO at 2 DIV (p < .001, ANOVA, n = 4). Data are mean ± SEM. (g) Representative micrographs of CGN grown in K25 and treated with BSO (100 µM) for 48 hr and Euk-134 (10 µM) for 24 hr. Calcein-positive cells are marked in green and propidium iodide-positive cells are marked in red (scale bar, 100 µm). ANOVA = analysis of variance; BSO = buthionine sulphoximine; CGN = cerebellar granule neurons; DIV = days in vitro.

Figure 3.

NOX-produced ROS promote CGN maturation. The levels of Tau and MAP2 were determined by Western blot in homogenates of CGN grown in K25 from 0 to 3 DIV and cultured cerebellar astrocytes. (a) Representative blots of Tau (∼70 kDa) and MAP2 (∼280 kDa) with their respective densitometric analysis of Tau (*p < .001, ANOVA, n = 3) and MAP2 (**p < .005, *p < .001, ANOVA, n = 4). Data were normalized with respect to 3 DIV and are mean ± SEM. (b to d) Representative blots of Tau and MAP2 with their respective densitometric analysis of CGN treated with the antioxidants Ebselen (10 µM) or Euk-134 (20 µM) or the NOX inhibitors AEBSF (50 µM) or Apocynin (400 µM) for 24 hr. (b) CGN at 1 DIV, Tau (*p < .001, ANOVA, n = 5), MAP2 (*p < .01, ANOVA, n = 4). (c) CGN at 2 DIV, Tau (*p < .05, ANOVA, n = 4), MAP2 (*p < .01, ** p < .001, ANOVA, n = 4). (d) CGN at 3 DIV, Tau, and MAP2, no statistical differences were found (ANOVA, n = 5). Densitometric values are the ratio of Tau/GADPH or MAP2/GAPDH and are normalized with respect to control. Data are mean ± SEM. NOX = NADPH-oxidase; ROS = reactive oxygen species; CGN = cerebellar granule neurons; ANOVA = analysis of variance; DIV = days in vitro.

Figure 4.

NOX1 and NOX2 are differentially expressed during CGN development. (a) NOX activity was determined in CGN from 1 to 5 DIV by cytochrome c reduction as detailed in Methods. * is significantly different from 1 DIV (p < .01, ANOVA, n = 5). Data were calculated as nmol min⁻¹ per mg protein and were normalized with respect to 3 DIV and are presented as mean ± SEM. (b to e) Relative mRNA levels of NOX1 and NOX2 were determined in CGN from 0 to 5 DIV by the 2^−ΔΔCt method of relative quantification as detailed in Methods. Data were normalized with respect to the time in which the expression reaches its maximum level. (b) * is significantly different from 1 DIV (p < .01, ANOVA, n = 3). Data are mean ± SEM. (c) NOX2 mRNA levels at 0, 1, 2, 4, and 5 DIV were significantly different with respect to 3 DIV (*p < .05, ** p < .001, ANOVA, n = 3). Data are mean ± SEM. (d) NOX2 mRNA levels at 1 DIV are significantly different from NOX1 mRNA levels at 1 DIV (p < .05, Mann–Whitney U Test, n = 4). Data are mean ± SEM. (e) NOX2 mRNA levels at 3 DIV are significantly different from NOX1 mRNA levels at 3 DIV (p < .05, Mann–Whitney U Test, n = 4). Data are mean ± SEM. NOX = NADPH-oxidase; CGN = cerebellar granule neurons; DIV = days in vitro; ANOVA = analysis of variance.

Figure 5.

NOX2 is expressed in filopodia and axonal growth cones in developing CGN. Representative confocal micrographs of NOX2 (green) and Tau (red) distribution and phase contrast (PC) micrographs at 0 and 3 DIV. (a) Two representative images of CGN at 0 DIV. White arrows indicate small protrusions and white arrowheads indicate growth cones. (b) A representative micrograph of CGN at 3 DIV. White squares (1 to 3) are shown below as magnified images. CGN were seeded at low density. White arrowheads indicate growth cones, black arrowheads indicate filopodia, and black arrows indicate varicosities (White scale bar, 20 µm; black scale bars, 5 µm). NOX = NADPH-oxidase; CGN = cerebellar granule neurons; DIV = days in vitro.

Figure 6.

H₂O₂ is produced in specific regions of developing CGN. (a to f) Representative micrographs of CGN of 2 DIV transfected with the plasmid HyPer and H₂O₂ levels were detected as detailed in Methods. The emission fluorescence was recorded from the excitation wavelengths 480 nm and 395 nm in time-lapse imaging (Supplementary videos). Color scale bars represent the ratio between the excitation wavelengths 480 nm and 395 nm, which represents the regions in the cell where H₂O₂ is being produced. Arrowheads indicate: soma (S), axon (A), axonal growth cone (AGC), dendritic growth cone (DGC), dynamic zone (DZ), and varicosities (V) (scale bar, 50 µm). (g) Magnified time-lapse images of the dynamic zone marked in (f). (a) Cell soma of (b). (e) Cell soma of (f). (f) Was captured previously (e) to allow H₂O₂ (500 µM) perfusion shown in (f). White arrowheads indicate a region of the axonal shaft previous or posterior to the filopodium formation. Red arrowheads indicate a region of the axonal shaft where a filopodium is present (ASF) and also corresponds to a relative high H₂O₂ production area. Arrows indicate filopodia (F´) with relative high H₂O₂ production (scale bar, 5 µm). (h) Quantification of H₂O₂ levels normalized with respect to the soma (*p < .05, ANOVA nonparametric test, n = 42 (A), n = 52 (AGC), n = 12 (DGC), n = 14 (DZ), n = 106 (ASF), n = 26 (F), n = 93 (V)). Data are mean ± SEM of 42 neurons registered in time-lapse imaging. (i) Quantification of the fluorescence recorded during filopodia formation normalized with respect to the soma. The fluorescence was measured in ASF during the time before filopodium formation, during the time the filopodium is present and after filopodia retraction. The mean fluorescence in ASF during filopodium formation is significantly different from the mean fluorescence recorded in ASF after and before filopodium formation (*p < .001, Paired t test, n = 21). H₂O₂ = hydrogen peroxide; CGN = cerebellar granule neurons; DIV = days in vitro; ANOVA = analysis of variance.

Figure 6.

H₂O₂ is produced in specific regions of developing CGN. (a to f) Representative micrographs of CGN of 2 DIV transfected with the plasmid HyPer and H₂O₂ levels were detected as detailed in Methods. The emission fluorescence was recorded from the excitation wavelengths 480 nm and 395 nm in time-lapse imaging (Supplementary videos). Color scale bars represent the ratio between the excitation wavelengths 480 nm and 395 nm, which represents the regions in the cell where H₂O₂ is being produced. Arrowheads indicate: soma (S), axon (A), axonal growth cone (AGC), dendritic growth cone (DGC), dynamic zone (DZ), and varicosities (V) (scale bar, 50 µm). (g) Magnified time-lapse images of the dynamic zone marked in (f). (a) Cell soma of (b). (e) Cell soma of (f). (f) Was captured previously (e) to allow H₂O₂ (500 µM) perfusion shown in (f). White arrowheads indicate a region of the axonal shaft previous or posterior to the filopodium formation. Red arrowheads indicate a region of the axonal shaft where a filopodium is present (ASF) and also corresponds to a relative high H₂O₂ production area. Arrows indicate filopodia (F´) with relative high H₂O₂ production (scale bar, 5 µm). (h) Quantification of H₂O₂ levels normalized with respect to the soma (*p < .05, ANOVA nonparametric test, n = 42 (A), n = 52 (AGC), n = 12 (DGC), n = 14 (DZ), n = 106 (ASF), n = 26 (F), n = 93 (V)). Data are mean ± SEM of 42 neurons registered in time-lapse imaging. (i) Quantification of the fluorescence recorded during filopodia formation normalized with respect to the soma. The fluorescence was measured in ASF during the time before filopodium formation, during the time the filopodium is present and after filopodia retraction. The mean fluorescence in ASF during filopodium formation is significantly different from the mean fluorescence recorded in ASF after and before filopodium formation (*p < .001, Paired t test, n = 21). H₂O₂ = hydrogen peroxide; CGN = cerebellar granule neurons; DIV = days in vitro; ANOVA = analysis of variance.

Figure 7.

Axonal morphology is altered by glutathione depletion. (a) Representative micrographs of CGN at 2 DIV treated with BSO (100 µM) for 42 hr or with Euk-134 (10 µM) for 18 hr. Cells were labeled with PKH67 (3 µM) before plating and neurites were visualized as detailed in Methods. Two different axonal morphologies were found in CGN treated with BSO, axons containing multiple spheroids, and collapsed axons (scale bar, 100 µm). (b) Magnified images of the indicated areas by white squares in (a) (scale bar, 20 µm). (c) Quantification of axons with morphology altered by BSO treatments. CGN treated with BSO showed a higher percentage of axons with alterations as compared to Control and BSO + Euk-134 (p < .05, ANOVA nonparametric test, n = 4). (d) Representative micrographs of CGN transfected with the plasmid HyPer as detailed in methods. CGN were treated with BSO (100 µM) for 42 hr and then cells were recorded in time-lapse imaging (scale bar, 50 µm). CGN = cerebellar granule neurons; DIV = days in vitro; BSO = buthionine sulphoximine; ANOVA = analysis of variance.

Figure 7.

Axonal morphology is altered by glutathione depletion. (a) Representative micrographs of CGN at 2 DIV treated with BSO (100 µM) for 42 hr or with Euk-134 (10 µM) for 18 hr. Cells were labeled with PKH67 (3 µM) before plating and neurites were visualized as detailed in Methods. Two different axonal morphologies were found in CGN treated with BSO, axons containing multiple spheroids, and collapsed axons (scale bar, 100 µm). (b) Magnified images of the indicated areas by white squares in (a) (scale bar, 20 µm). (c) Quantification of axons with morphology altered by BSO treatments. CGN treated with BSO showed a higher percentage of axons with alterations as compared to Control and BSO + Euk-134 (p < .05, ANOVA nonparametric test, n = 4). (d) Representative micrographs of CGN transfected with the plasmid HyPer as detailed in methods. CGN were treated with BSO (100 µM) for 42 hr and then cells were recorded in time-lapse imaging (scale bar, 50 µm). CGN = cerebellar granule neurons; DIV = days in vitro; BSO = buthionine sulphoximine; ANOVA = analysis of variance.

Figure 8.

NOX2 regulates axon formation. (a, b) Wild-type (W) and NOX2 KO CGN were labeled with PKH67 (3 µM) before plating and neurites were visualized at 1 and 2 DIV as detailed in Methods. (a) Representative micrographs of CGN labeled with PKH67 at 1 and 2 DIV (scale bar, 100 µm). (b) Quantification of axonal growth of W and NOX2 KO CGN was performed as detailed in Methods. * is significantly different from W at 1 and 2 DIV (p < .001, Mann–Whitney U Test, n = 2067 and n = 1151, respectively). Data are mean ± SEM. (c) Representative micrographs of W and NOX2 KO CGN cultured for 1 and 2 DIV and incubated with dihydroethidium as detailed in Methods (scale bar, 100 µm). (d, e) Quantification of ROS levels in W and NOX2 KO CGN cultured from 1 and 2 DIV. * is significantly different from 1 DIV (p < .05, Mann-Whitney U Test, n = 4). Data were normalized with respect to 1 DIV and are mean ± SEM. (f) ROS levels of NOX2 KO CGN at 1 and 2 DIV were compared with respect to W. (No statistical differences were found, Mann–Whitney U Test, n = 5). Data were normalized with respect to W and are presented as mean ± SEM. NOX = NADPH-oxidase; CGN = cerebellar granule neurons; DIV = days in vitro; ROS = reactive oxygen species.

Figure 8.

NOX2 regulates axon formation. (a, b) Wild-type (W) and NOX2 KO CGN were labeled with PKH67 (3 µM) before plating and neurites were visualized at 1 and 2 DIV as detailed in Methods. (a) Representative micrographs of CGN labeled with PKH67 at 1 and 2 DIV (scale bar, 100 µm). (b) Quantification of axonal growth of W and NOX2 KO CGN was performed as detailed in Methods. * is significantly different from W at 1 and 2 DIV (p < .001, Mann–Whitney U Test, n = 2067 and n = 1151, respectively). Data are mean ± SEM. (c) Representative micrographs of W and NOX2 KO CGN cultured for 1 and 2 DIV and incubated with dihydroethidium as detailed in Methods (scale bar, 100 µm). (d, e) Quantification of ROS levels in W and NOX2 KO CGN cultured from 1 and 2 DIV. * is significantly different from 1 DIV (p < .05, Mann-Whitney U Test, n = 4). Data were normalized with respect to 1 DIV and are mean ± SEM. (f) ROS levels of NOX2 KO CGN at 1 and 2 DIV were compared with respect to W. (No statistical differences were found, Mann–Whitney U Test, n = 5). Data were normalized with respect to W and are presented as mean ± SEM. NOX = NADPH-oxidase; CGN = cerebellar granule neurons; DIV = days in vitro; ROS = reactive oxygen species.

Figure 9.

Summary of the principal findings. (a, b) Relationship between the levels of ROS, reduced glutathione and cell survival in CGN under depolarizing and nondepolarizing conditions. During the first 3 DIV, ROS and reduced glutathione increase, reaching the highest levels around 2 DIV. By the third DIV, CGN maintained under depolarizing conditions (a) show a significant decrease in the ROS levels, as well as a moderate reduction of glutathione levels. Cell viability remains unaltered. In CGN cultured in nondepolarizing conditions (b), the levels of ROS remain increasing, while the levels of reduced glutathione decrease and the cell survival is compromised. (c to e) Effect of ROS in the development of CGN. (c) During normal development, the levels of ROS are regulated by reduced glutathione. Also, (d) when ROS production is decreased by NOX2 inhibition, the development of CGN is altered, as indicated by a low expression of the neuronal markers, Tau and MAP2, as well as a reduced axonal growth. (e) In contrast, the reduction of the levels of glutathione leads to an alteration in the axonal development and cell death. (f) The H₂O₂ produced in CGN is mainly localized in the axons; this H₂O₂ is associated with the formation of filopodia and with axonal growth cone dynamics. (g) Glutathione depletion leads to the formation of multiple spheroid-like structures in the axon that are rich in H₂O₂. ROS = reactive oxygen species; CGN = cerebellar granule neurons; DIV = days in vitro; NOX = NADPH-oxidase; H₂O₂ = hydrogen peroxide.