Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease

Abstract

Recent epidemiological and clinical data suggest that persons with low folic acid levels and elevated homocysteine levels are at increased risk of Alzheimer's disease (AD), but the underlying mechanism is unknown. We tested the hypothesis that impaired one-carbon metabolism resulting from folic acid deficiency and high homocysteine levels promotes accumulation of DNA damage and sensitizes neurons to amyloid beta-peptide (Abeta) toxicity. Incubation of hippocampal cultures in folic acid-deficient medium or in the presence of methotrexate (an inhibitor of folic acid metabolism) or homocysteine induced cell death and rendered neurons vulnerable to death induced by Abeta. Methyl donor deficiency caused uracil misincorporation and DNA damage and greatly potentiated Abeta toxicity as the result of reduced repair of Abeta-induced oxidative modification of DNA bases. When maintained on a folic acid-deficient diet, amyloid precursor protein (APP) mutant transgenic mice, but not wild-type mice, exhibited increased cellular DNA damage and hippocampal neurodegeneration. Levels of Abeta were unchanged in the brains of folate-deficient APP mutant mice. Our data suggest that folic acid deficiency and homocysteine impair DNA repair in neurons, which sensitizes them to oxidative damage induced by Abeta.

Figures

Fig. 1.

Methyl donor deficiency induces death of hippocampal neurons and potentiates Aβ toxicity. a, Phase-contrast micrographs showing hippocampal neurons in a control culture and a culture that had been maintained for 48 hr in medium lacking methionine and folic acid. b, Cultures were exposed to control medium, medium lacking l-methionine and folic acid (M/F), medium containing 5 μm Aβ1-42 (Aβ), or medium containing a combination of M/F plus Aβ; neuronal survival was quantified at the indicated time points. Neuronal survival is expressed as a percentage of the initial number of neurons present before experimental treatment (see Materials and Methods). Values are the mean and SD of determinations made in six cultures. c, Cultures were incubated for 48 hr in control medium, medium lacking methionine and folic acid (M/F), medium lacking folate (FA), medium lacking methionine but containing folic acid (M), or medium containing 20 μm methotrexate (Methotr). Neuron survival was quantified (mean and SD; n = 6). *p < 0.001 compared with control; **p < 0.01 compared with M/F;#p < 0.05 and##p < 0.01 compared with control (ANOVA with Scheffe's post hoc tests).d, Cultures were exposed for 48 hr to saline (Control), M/F-deficient medium, 5 μm Aβ1-42 (Aβ), a combination of M/F-deficient medium plus 5 μm Aβ1-42 (Aβ+M/F), 250 μm homocysteine (Hom), or a combination of homocysteine plus Aβ (Aβ+Hom). Neuron survival was quantified (mean and SD;n = 6). *p < 0.01, **p < 0.001 compared with control;#p < 0.01 compared with M/F deficiency and with Aβ; ##p < 0.01 compared with homocysteine and with Aβ (ANOVA with Scheffe's post hoc tests).

Fig. 2.

A methyl donor-deficient diet induces hyperhomocysteinemia and promotes neuronal degeneration in APP mutant mice. a, Levels of homocysteine in serum samples from wild-type (WT) and APP mutant mice that had been maintained for 3 months on the normal control diet or the experimental folic acid-deficient diet were quantified. Values are the mean and SD (n = 8). *p < 0.0001 compared with corresponding control value (ANOVA with Scheffe's post hoc tests). b, Micrographs showing cresyl violet-stained sections of hippocampus (region CA3) from wild-type and APP mutant mice maintained for 3 months on either the control diet or the folic acid-deficient diet (APP diet; WT diet). The micrographs at thebottom show high magnification of the lower limb of CA3.c, Numerical densities of neurons in regions CA3 and CA1 of hippocampus were quantified in the brains of wild-type and APP mutant mice maintained for 3 months on either control or folic acid-deficient diets. Values are the mean and SD (n= 8). *p < 0.01 compared with APP mutant or wild-type mice maintained on the control diet and compared with wild-type mice on the experimental diet (ANOVA with Scheffe'spost hoc tests).

Fig. 3.

Dietary folic acid deficiency does not affect levels of Aβ in the brains of APP mutant mice. a, Levels of Aβ1-40 and Aβ1-42 were quantified by ELISA analysis in cerebral cortex of APP mutant mice that had been maintained on either a control diet or a folate-deficient diet (Exp. Diet). Values are the mean and SE of determinations (6 mice per group).b, Levels of Aβ, expressed as a ratio of Aβ1-42/Aβ1-40 in brain tissue from APP mutant mice that had been maintained for 3 months on folic acid-deficient or control diets. Values are the mean and SE of measurements made in samples from six mice on each diet. c, Micrographs showing Aβ immunoreactivity (brown) in sections of hippocampus from APP mutant transgenic mice that had been maintained for 3 months on a folic acid-deficient diet (APP diet) or a control diet. Note the absence of plaques in mice on either diet.

Fig. 4.

Amyloid β-peptide enhances DNA damage in neurons under conditions of methyl donor deficiency. a, Comet assay analysis of nuclear DNA in neurons in a control culture and a culture that had been exposed for 8 hr in methionine/folic acid-deficient medium. b, DNA damage was quantified by comet analysis in hippocampal neurons after the indicated time periods of incubation in methionine/folic acid-deficient medium. Values are the mean and SD of determinations made in six cultures; *p < 0.01. c, DNA damage was quantified in neurons that had been exposed for 4 hr to the indicated treatments (5 μm Aβ1-42; 250 μm homocysteine). Values are the mean and SD (n = 6). **p < 0.01 compared with control. #p < 0.05,##p < 0.01 compared with Aβ alone (ANOVA with Scheffe's post hoc test). d, DNA damage was quantified by comet assay in hippocampal tissue samples from wild-type and APP mutant mice that had been maintained for 3 months on either the control diet or the methyl donor-deficient experimental diet. Values are the mean and SD (n = 8). *p < 0.01 compared with the value for APP mutant mice on the control diet and with WT mice on either diet.

Fig. 5.

Impaired DNA repair and increased accumulation of oxidatively modified bases in neurons exposed to Aβ under methyl donor-deficient conditions. a, Cultured neurons were exposed for 5 hr to the indicated treatments (5 μm Aβ1-42; 250 μm homocysteine). Then the cultures either were processed for analysis of DNA damage (Treatment) or were incubated for an additional 12 hr in the absence of treatments (Recovery) and then processed for analysis of DNA damage. Values are the mean and SD (n = 6). *p < 0.01 compared with the corresponding treatment value. b, Neurons were exposed for 5 hr to the indicated treatments and then subjected to comet analysis of DNA damage, using FPG treatment as described in Materials and Methods. Values are the mean and SD (n = 6). *p < 0.01, **p < 0.001 compared with the corresponding buffer value. c, Cultured neurons were exposed for 5 hr to the indicated treatments (5 μm Aβ1-42). Cultures either were processed for analysis of DNA damage (Treatment) or were incubated for an additional 12 hr in the absence of treatments (Recovery) and subjected to comet analysis of DNA damage by FPG treatment. Values are the mean and SD (n = 6). *p < 0.01 compared with the corresponding treatment value. d, Micrographs showing 8-oxoguanine immunoreactivity in cultured hippocampal neurons subjected to the indicated treatments. Cultures were exposed to the indicated treatments for 5 hr and either were analyzed at that time (treatment) or were allowed to recover for 12 hr in the absence of treatment (recovery). Control, Exposure of 5 hr to buffer; Aβ, 5 μmAβ1-42; M/F def., methionine- and folic acid-deficient medium.

Fig. 6.

Methyl donor deficiency-induced neuronal death involves misincorporation of uracil into DNA. a, Neurons were exposed for 5 hr to saline (Control), methionine/folic acid-deficient medium (M/F), or 250 μm homocysteine (Hom) and then were subjected to comet analysis of DNA damage via UDG treatment. Next the nuclei were subjected to comet analysis of DNA damage. Values are the mean and SD (n = 6). *p < 0.01, **p < 0.001 compared with control value and with the corresponding buffer value. b, Cultures were exposed for 48 hr to saline (Control), methionine/folic acid-deficient medium (M/F), 15 μm thymidine plus 30 μm hypoxanthine (Th+H), or the combination of methionine/folic acid-deficient medium plus Th+H. Neuron survival was quantified; the values are the mean and SD (n = 6). **p < 0.01 compared with control;#p < 0.05 compared with the methionine/folic acid-deficient value.