Mitochondrial DNA damage level determines neural stem cell differentiation fate

Abstract

The mitochondrial DNA (mtDNA) of neural stem cells (NSCs) is vulnerable to oxidation damage. Subtle manipulations of the cellular redox state affect mtDNA integrity in addition to regulating the NSC differentiation lineage, suggesting a molecular link between mtDNA integrity and regulation of differentiation. Here we show that 8-oxoguanine DNA glycosylase (OGG1) is essential for repair of mtDNA damage and NSC viability during mitochondrial oxidative stress. Differentiating neural cells from ogg1(-/-) knock-out mice spontaneously accumulate mtDNA damage and concomitantly shift their differentiation direction toward an astrocytic lineage, similar to wt NSCs subjected to mtDNA damaging insults. Antioxidant treatments reversed mtDNA damage accumulation and separately increased neurogenesis in ogg1(-/-) cells. NSCs from a transgenic ogg1(-/-) mouse expressing mitochondrially targeted human OGG1 were protected from mtDNA damage during differentiation, and displayed elevated neurogenesis. The underlying mechanisms for this shift in differentiation direction involve the astrogenesis promoting Sirt1 via an increased NAD/NADH ratio in ogg1(-/-) cells. Redox manipulations to alter mtDNA damage level correspondingly activated Sirt1 in both cell types. Our results demonstrate for the first time the interdependence between mtDNA integrity and NSC differentiation fate, suggesting that mtDNA damage is the primary signal for the elevated astrogliosis and lack of neurogenesis seen during repair of neuronal injury.

Figure 1.

DNA glycosylase OGG1 is essential for protection against mtDNA damage in NSCs. A, Sensitivity of wt and ogg1^−/− NSCs to pro-oxidants. Single NSCs were plated in differentiation medium for 4 h before exposure. B, mtDNA damage detection by the relative quantitative amplification of a small (117 bp) and large (10 kb) of mtDNA. Top, A representative result. Bottom, The mean with SD from more than three independent experiments. C, mtDNA damage detection by the ability to inhibit restriction cleavage, quantified by real-time PCR. Data are mean with SD from more than three independent experiments. *p < 0.05; **p < 0.01.

Figure 2.

Differentiation of wt and ogg1^−/− NSCs. A, Growth factor removal-induced differentiation of NSCs is manifested as reduced levels of nestin and corresponding elevations in the neuronal Tuj-1 and astrocytic GFAP markers. B, Progressive reduction of neurogenesis and increased astrogenesis from ogg1^−/− NSCs. Left, Differentiating cells from wt and ogg1^−/− NSCs are morphological similar (B-W, black and white phase-contrast images). Neurons and astrocytes at differentiation day 1 and 5 were identified by immunocytochemistry using antibodies against Tuj-1 (middle) and GFAP (right). C, Quantitative analysis of neurons and astrocytes at day 1 and day 5 in differentiation. Gene expression analyses, presented relative to wt at day 1. Data are mean with SD from more than three independent experiments. *p < 0.05; **p < 0.01.

Figure 3.

Altered redox levels and Sirt1 activation in ogg1^−/− cells. A, Cellular redox levels were analyzed as NAD/NADH ratio in differentiation cells, as described in Materials and Methods. B, Sirt1 expression in cell cultures from wt and ogg1^−/− NSC during differentiation. C, Redox manipulations correspondingly involve Sirt1. Sirt1 expression levels after treatment with oxidants [BSO and menadione (MEN)] and antioxidants (NAC and LA) were assessed by RT-PCR. All data are mean with SD from more than three independent experiments *p < 0.05.

Figure 4.

NSC mtDNA integrity determines differentiation capacity. A, Pro-oxidants shift differentiation toward astrogenesis in wt cells, but suppress total differentiation capacity in ogg1^−/− NSCs. Cells were treated with BSO or menadione and astrocytes/neurons determined by immunocytochemistry at day 5. B, Antioxidants suppress generation of astrocytes in ogg1^−/− NSCs with corresponding increase in neurogenesis. Cell were treated with NAC, LA and astrocytes/neurons identified by immunocytochemistry at day 5. C, Strong 8-oxoguanine DNA glycosylase activity in brain mitochondria from transgenic ogg1^−/− mice overexpressing mitochondrial hOGG1 (mtOGG1). Brain mitochondrial extracts (6 μg) from six different P5 littermates of mtOGG1 (1–6), adult wt (wt), and adult ogg1^−/− (ogg1^−/−) mice were assayed for OGG1 DNA glycosylase activity as described. Recombinant human OGG1 (25 ng) is used as positive control. D, Reversal of spontaneous mtDNA damage by mitochondrial hOGG1 results in increased neurogenesis. Top, mtDNA integrity in cells from differentiation day 1 and 5 (D1 and D5, respectively) was determined as in Figure 1B. Numbers are average with SD from three independent experiments. Bottom, Spontaneous differentiation pattern in wt and ogg1^−/− cells and ogg1^−/− cells expressing mtOGG1, determined as in Figure 2D. E, Induced mtDNA damage in NSCs increases astrogliosis. Top, NSCs were treated with menadione and mtDNA integrity was examined as in D, after (1 h) and 4 h after treatment. Bottom, Different NSCs treated with menadione and recovered for 4 h as above, were cultivated in differentiation medium and the differentiation pattern determined after 1 d, as in Figure 2D. Results shown are experiments from two independent cultures. *p < 0.05, **p < 0.01.