Aging, cumulative DNA Aging, cumulative DNA damage, and deficits in damage, and deficits in

genomic repair:genomic repair:

A story of micronutrients and A story of micronutrients and lifelong neural integritylifelong neural integrity

The accrual of DNA damage leads The accrual of DNA damage leads to progressive aging.to progressive aging.

• DNA damage is ubiquitous. DNA damage is ubiquitous. • Our cells are well-equipped with DNA repair enzymes.Our cells are well-equipped with DNA repair enzymes.

• However, DNA damage can occur at such high levels However, DNA damage can occur at such high levels that the cell’s capacity for DNA repair is overwhelmed.that the cell’s capacity for DNA repair is overwhelmed.

• As DNA damage accumulates over time, our DNA repair As DNA damage accumulates over time, our DNA repair mechanisms become increasingly inefficient.mechanisms become increasingly inefficient.

Ionizing radiation and oxidative stress Ionizing radiation and oxidative stress can cause single- and can cause single- and

double- stranded breaks in DNA.double- stranded breaks in DNA.

• Ionizing radiation kills cells by inducing double-stranded DNA breaks. Ionizing radiation kills cells by inducing double-stranded DNA breaks. Double stranded breaks are the most lethal form of DNA damage: one Double stranded breaks are the most lethal form of DNA damage: one unrepaired DSB is sufficient to kill a cell.unrepaired DSB is sufficient to kill a cell.

• ROS destroy DNA by creating single-stranded breaks; when two single-ROS destroy DNA by creating single-stranded breaks; when two single-stranded breaks on opposite strands are within 10 to 20 base pairs of stranded breaks on opposite strands are within 10 to 20 base pairs of each other, a DSB occurs.each other, a DSB occurs.

The constitutively expressed nuclear protein The constitutively expressed nuclear protein poly(ADP-ribose) polymerase-1 becomes poly(ADP-ribose) polymerase-1 becomes immediately activated upon DNA strand immediately activated upon DNA strand

breakage.breakage.

PARP-1 catalyzes the cleavage of NADPARP-1 catalyzes the cleavage of NAD++ into ADP-ribose into ADP-ribose and nicotinamide.and nicotinamide.

Mechanism of action of PARP-1:Mechanism of action of PARP-1:

2 PARP-1 molecules bind to the damaged DNA at the site of the nick. ADP-2 PARP-1 molecules bind to the damaged DNA at the site of the nick. ADP-ribose polymers (pink) are synthesized and attached to associated proteins. ribose polymers (pink) are synthesized and attached to associated proteins. These polymers create a negative charge, purportedly changing the These polymers create a negative charge, purportedly changing the conformation of the DNA molecule, making it more accessible for repair conformation of the DNA molecule, making it more accessible for repair enzymes. The negatively charged protein-(ADP-ribose) polymers dissociate enzymes. The negatively charged protein-(ADP-ribose) polymers dissociate from the negatively-charged DNA molecule. The green molecule is a from the negatively-charged DNA molecule. The green molecule is a histone, which, when dissociated, gives the DNA molecule a more relaxed histone, which, when dissociated, gives the DNA molecule a more relaxed and accessible shape.and accessible shape.

Cellular response to DNA damage fluctuates Cellular response to DNA damage fluctuates according to the severity of the insult:according to the severity of the insult:

• Under physiological Under physiological conditions, PARP-1 plays conditions, PARP-1 plays a critical role in DNA base a critical role in DNA base excision repair excision repair machinery. machinery.

• Also, degradation of the Also, degradation of the ADP-ribose polymer may ADP-ribose polymer may provide a source of ATP provide a source of ATP for ligation of DNA nicks.for ligation of DNA nicks.

• End result: DNA repair End result: DNA repair and genomic stability.and genomic stability.

• Severe DNA damage can Severe DNA damage can activate PARP-1 500-fold:activate PARP-1 500-fold:

• Extremely high levels of Extremely high levels of PARP-1 activity will PARP-1 activity will deplete the cell of NAD+ deplete the cell of NAD+ and ATP.and ATP.

• This activates p53, This activates p53, triggering a cascade of triggering a cascade of cellular events leading to cellular events leading to apoptosis.apoptosis.

• End result: The cell dies.End result: The cell dies.

Severe DNA damage Severe DNA damage presents a huge problem presents a huge problem for non-proliferating cells for non-proliferating cells

such as neurons:such as neurons:It makes sense that a lifetime of It makes sense that a lifetime of

unrepaired DNA damage can lead unrepaired DNA damage can lead to neurodegeneration.to neurodegeneration.

Is there anything we can do to Is there anything we can do to assist our cells in repairing DNA assist our cells in repairing DNA

damage?damage?

Folic Acid Deficiency and Folic Acid Deficiency and Homocysteine Impair DNA Repair in Homocysteine Impair DNA Repair in Hippocampal Neurons and Sensitize Hippocampal Neurons and Sensitize

Them to Amyloid Toxicity in Them to Amyloid Toxicity in Experimental Models of Alzheimer’s Experimental Models of Alzheimer’s

DiseaseDisease

Inna I. Kruman, T.S. Kumaravel, Althaf Lohani, Ward A. Inna I. Kruman, T.S. Kumaravel, Althaf Lohani, Ward A. Pedersen, Roy G. Cutler, Yuri Kruman, Norman Haughey, Pedersen, Roy G. Cutler, Yuri Kruman, Norman Haughey,

Jaewon Lee, Michele Evans, and Mark P. MattsonJaewon Lee, Michele Evans, and Mark P. Mattson

The Journal of Neuroscience, March 1, 2002, 22(5): 1752-The Journal of Neuroscience, March 1, 2002, 22(5): 1752-17621762

Overexpression of PARP-1 has Overexpression of PARP-1 has been detected in the brains of been detected in the brains of Alzheimer’s disease patients.Alzheimer’s disease patients.

This suggests that the overactivation of This suggests that the overactivation of PARP and the associated exhaustion of PARP and the associated exhaustion of cellular energy sources due to the cellular energy sources due to the depletion of NADdepletion of NAD++ lead to the extensive lead to the extensive neuron death seen in AD.neuron death seen in AD.

The chief risk factor for AD is advanced age.The chief risk factor for AD is advanced age.

Top left: healthy 20-year old; middle: normal 80-Top left: healthy 20-year old; middle: normal 80-year-old; right: AD patient. year-old; right: AD patient.

Folic acid deficiency induces DNA Folic acid deficiency induces DNA damage in cells via several pathways:damage in cells via several pathways:

1. The promotion of uracil misincorporation: folate is a co-1. The promotion of uracil misincorporation: folate is a co-factor in the synthesis of dTMP. Low dTMP yields the factor in the synthesis of dTMP. Low dTMP yields the DNA polymerase-mediated dUTP misincorporation into DNA polymerase-mediated dUTP misincorporation into DNA.DNA.

2. DNA hypomethylation: folate deficiency translates into 2. DNA hypomethylation: folate deficiency translates into fewer methyl groups available to covalently attach to fewer methyl groups available to covalently attach to DNA for gene regulation. DNA for gene regulation.

3. Impairment of DNA repair: uracil is excised from DNA 3. Impairment of DNA repair: uracil is excised from DNA through base excision repair, resulting in nicks. The through base excision repair, resulting in nicks. The formation of two opposing nicks leads to deletions, formation of two opposing nicks leads to deletions, duplications, chromosome breaks.duplications, chromosome breaks.

Folate coenzymes are critical for Folate coenzymes are critical for DNA integrity:DNA integrity:

Hypothesis: Folic acid deficiency Hypothesis: Folic acid deficiency and homocysteine sensitize and homocysteine sensitize

neurons to Aneurons to Aββ-induced death.-induced death.

• Increased DNA damage has been found in Increased DNA damage has been found in the neurons of AD patientsthe neurons of AD patients

• Non-neuronal cells from AD patients Non-neuronal cells from AD patients display defective DNA repairdisplay defective DNA repair

• Folic acid deficiency and high cellular Folic acid deficiency and high cellular levels of homocysteine impair DNA repair levels of homocysteine impair DNA repair in non-neuronal cellsin non-neuronal cells

MethodsMethods

• Hippocampal and Hippocampal and cortical cell culturescortical cell cultures

• Included neurons and Included neurons and gliaglia

• Control: complete Control: complete mediummedium

• Methyl-donor deficient Methyl-donor deficient medium lacked folic medium lacked folic acid and methionineacid and methionine

• APP transgenic APP transgenic mouse models are mouse models are homozygous for the homozygous for the “Swedish mutation”. “Swedish mutation”. Cells that express this Cells that express this mutant sequence mutant sequence secrete approximately secrete approximately 6-8 times more A6-8 times more Aββ peptide than wild-type peptide than wild-type cells.cells.

Hippocampal neurons subjected to folate Hippocampal neurons subjected to folate deficiency and homocysteine exhibit deficiency and homocysteine exhibit

increased vulnerability to Aincreased vulnerability to Aββ-induced death.-induced death.

80% of the neurons maintained in methyl-donor 80% of the neurons maintained in methyl-donor deficient medium died within 72 hours.deficient medium died within 72 hours.

Only 10% of the neurons in complete medium Only 10% of the neurons in complete medium died within 72 hours.died within 72 hours.

Folic acid deficiency increases Folic acid deficiency increases homocysteine levels and sensitizes homocysteine levels and sensitizes

hippocampal neurons to death in APP hippocampal neurons to death in APP mutant transgenic mice.mutant transgenic mice.

Amyloid Amyloid ββ--peptide levels peptide levels are unchanged are unchanged in APP mutant in APP mutant mice maintained mice maintained on a folic acid-on a folic acid-deficient dietdeficient diet

Folic acid deficiency and homocysteine Folic acid deficiency and homocysteine enhance Aenhance Aββ-induced DNA damage in -induced DNA damage in

cultured hippocampal neurons and cultured hippocampal neurons and APP mutant miceAPP mutant mice

The Olive Tail Moment is a The Olive Tail Moment is a measure of DNA damage in measure of DNA damage in

a cell. a cell.

OTM = (amount of DNA in tail) x OTM = (amount of DNA in tail) x (distance between head and tail (distance between head and tail

regions)regions)

Folic acid deficiency promotes uracil Folic acid deficiency promotes uracil misincorporation and impairs DNA repair misincorporation and impairs DNA repair

under conditions of exposure to Aunder conditions of exposure to Aββ

• Neurons incubated in Neurons incubated in methyl-donor deficient methyl-donor deficient medium experienced medium experienced a significant increase a significant increase in uracil in uracil misincorporation misincorporation (p<0.01).(p<0.01).

DiscussionDiscussion

• The combination of low folic acid intake and the The combination of low folic acid intake and the consequent hyperhomocysteinemia might put aging consequent hyperhomocysteinemia might put aging individuals at increased risk for developing Alzheimer’s individuals at increased risk for developing Alzheimer’s disease.disease.

• The CA3 neurons of the hippocampus may be The CA3 neurons of the hippocampus may be selectively vulnerable to DNA damage, since no selectively vulnerable to DNA damage, since no significant damage was seen in the CA1 region.significant damage was seen in the CA1 region.

• Folic acid deficiency and hyperhomocysteinemia Folic acid deficiency and hyperhomocysteinemia accelerate the accumulation of DNA damage that is accelerate the accumulation of DNA damage that is promoted by age-related increases in oxidative stress promoted by age-related increases in oxidative stress and Aand Aββ..

• Dietary supplementation may reduce the risk of AD.Dietary supplementation may reduce the risk of AD.

Folic Acid deficiency is quite Folic Acid deficiency is quite common in humans.common in humans.

• Approximately 10% of the U.S. population Approximately 10% of the U.S. population is folate-deficient.is folate-deficient.

• Folate deficiency is much more prevalent Folate deficiency is much more prevalent in developing countries. Worldwide, there in developing countries. Worldwide, there are about 200,000 severe birth defects per are about 200,000 severe birth defects per year resulting from folate-deficient diets.year resulting from folate-deficient diets.

• Reduced intestinal absorption of folic acid Reduced intestinal absorption of folic acid in the elderly increases folate-deficiency in in the elderly increases folate-deficiency in this population to almost 50%.this population to almost 50%.

Sources of folic acidSources of folic acid

• Most cereals and breads are fortified with Most cereals and breads are fortified with 200 µg of folic acid per serving.200 µg of folic acid per serving.

• Natural sources of folic acid include leafy Natural sources of folic acid include leafy greens and citrus.greens and citrus.

• Most supplements contain 400 µg of folic Most supplements contain 400 µg of folic acid.acid.

• Less than one gram per day of folic acid is Less than one gram per day of folic acid is considered non-toxic.considered non-toxic.

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