Folic Acid Deficiency and Homocysteine Impair DNA Repair in Hippocampal Neurons and Sensitize Them to Amyloid Toxicity in Experimental Models of Alzheimer's Disease

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).

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.

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.

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.