1. Aging, cumulative DNAAging, cumulative DNA
damage, and deficits indamage, and deficits in
genomic repair:genomic repair:
A story of micronutrients andA story of micronutrients and
lifelong neural integritylifelong neural integrity

2. The accrual of DNA damage leadsThe 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 levelsHowever, 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 repairAs DNA damage accumulates over time, our DNA repair
mechanisms become increasingly inefficient.mechanisms become increasingly inefficient.

3. Ionizing radiation and oxidative stressIonizing radiation and oxidative stress
can cause single- andcan cause single- and
double- stranded breaks in DNA.double- stranded breaks in DNA.
• Ionizing radiation kills cells by inducing double-strandedIonizing radiation kills cells by inducing double-stranded
DNA breaks. Double stranded breaks are the mostDNA breaks. Double stranded breaks are the most
lethal form of DNA damage: one unrepaired DSB islethal form of DNA damage: one unrepaired DSB is
sufficient to kill a cell.sufficient to kill a cell.
• ROS destroy DNA by creating single-stranded breaks;ROS destroy DNA by creating single-stranded breaks;
when two single-stranded breaks on opposite strandswhen two single-stranded breaks on opposite strands
are within 10 to 20 base pairs of each other, a DSBare within 10 to 20 base pairs of each other, a DSB
occurs.occurs.

4. The constitutively expressed nuclear proteinThe constitutively expressed nuclear protein
poly(ADP-ribose) polymerase-1 becomespoly(ADP-ribose) polymerase-1 becomes
immediately activated upon DNA strandimmediately activated upon DNA strand
breakage.breakage.
PARP-1 catalyzes the cleavage of NADPARP-1 catalyzes the cleavage of NAD++
into ADP-riboseinto ADP-ribose
and nicotinamide.and nicotinamide.

5. 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 theThese polymers create a negative charge, purportedly changing the
conformation of the DNA molecule, making it more accessible for repairconformation of the DNA molecule, making it more accessible for repair
enzymes. The negatively charged protein-(ADP-ribose) polymers dissociateenzymes. The negatively charged protein-(ADP-ribose) polymers dissociate
from the negatively-charged DNA molecule. The green molecule is afrom the negatively-charged DNA molecule. The green molecule is a
histone, which, when dissociated, gives the DNA molecule a more relaxedhistone, which, when dissociated, gives the DNA molecule a more relaxed
and accessible shape.and accessible shape.

6. Cellular response to DNA damage fluctuatesCellular response to DNA damage fluctuates
according to the severity of the insult:according to the severity of the insult:
• Under physiologicalUnder physiological
conditions, PARP-1 playsconditions, PARP-1 plays
a critical role in DNA basea critical role in DNA base
excision repairexcision repair
machinery.machinery.
• Also, degradation of theAlso, degradation of the
ADP-ribose polymer mayADP-ribose polymer may
provide a source of ATPprovide a source of ATP
for ligation of DNA nicks.for ligation of DNA nicks.
• End result: DNA repairEnd result: DNA repair
and genomic stability.and genomic stability.
• Severe DNA damage canSevere DNA damage can
activate PARP-1 500-fold:activate PARP-1 500-fold:
• Extremely high levels ofExtremely high levels of
PARP-1 activity willPARP-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 oftriggering a cascade of
cellular events leading tocellular events leading to
apoptosis.apoptosis.
• End result: The cell dies.End result: The cell dies.

7. Severe DNA damageSevere DNA damage
presents a huge problempresents a huge problem
for non-proliferating cellsfor non-proliferating cells
such as neurons:such as neurons:
It makes sense that a lifetime ofIt makes sense that a lifetime of
unrepaired DNA damage can leadunrepaired DNA damage can lead
to neurodegeneration.to neurodegeneration.

8. Is there anything we can do toIs there anything we can do to
assist our cells in repairing DNAassist our cells in repairing DNA
damage?damage?

9. Folic Acid Deficiency andFolic Acid Deficiency and
Homocysteine Impair DNA Repair inHomocysteine Impair DNA Repair in
Hippocampal Neurons and SensitizeHippocampal Neurons and Sensitize
Them to Amyloid Toxicity inThem to Amyloid Toxicity in
Experimental Models of Alzheimer’sExperimental 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

10. Overexpression of PARP-1 hasOverexpression of PARP-1 has
been detected in the brains ofbeen detected in the brains of
Alzheimer’s disease patients.Alzheimer’s disease patients.
This suggests that the overactivation ofThis suggests that the overactivation of
PARP and the associated exhaustion ofPARP and the associated exhaustion of
cellular energy sources due to thecellular energy sources due to the
depletion of NADdepletion of NAD++
lead to the extensivelead to the extensive
neuron death seen in AD.neuron death seen in AD.

11. 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.

12. Folic acid deficiency induces DNAFolic 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 thefactor in the synthesis of dTMP. Low dTMP yields the
DNA polymerase-mediated dUTP misincorporation intoDNA polymerase-mediated dUTP misincorporation into
DNA.DNA.
2. DNA hypomethylation: folate deficiency translates into2. DNA hypomethylation: folate deficiency translates into
fewer methyl groups available to covalently attach tofewer methyl groups available to covalently attach to
DNA for gene regulation.DNA for gene regulation.
3. Impairment of DNA repair: uracil is excised from DNA3. Impairment of DNA repair: uracil is excised from DNA
through base excision repair, resulting in nicks. Thethrough 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.

13. Folate coenzymes are critical forFolate coenzymes are critical for
DNA integrity:DNA integrity:

14. Hypothesis: Folic acid deficiencyHypothesis: Folic acid deficiency
and homocysteine sensitizeand homocysteine sensitize
neurons to Aneurons to Aββ-induced death.-induced death.
• Increased DNA damage has been foundIncreased DNA damage has been found
in the neurons of AD patientsin the neurons of AD patients
• Non-neuronal cells from AD patientsNon-neuronal cells from AD patients
display defective DNA repairdisplay defective DNA repair
• Folic acid deficiency and high cellularFolic acid deficiency and high cellular
levels of homocysteine impair DNA repairlevels of homocysteine impair DNA repair
in non-neuronal cellsin non-neuronal cells

15. MethodsMethods
• Hippocampal andHippocampal and
cortical cell culturescortical cell cultures
• Included neurons andIncluded neurons and
gliaglia
• Control: completeControl: complete
mediummedium
• Methyl-donor deficientMethyl-donor deficient
medium lacked folicmedium lacked folic
acid and methionineacid and methionine
• APP transgenicAPP transgenic
mouse models aremouse models are
homozygous for thehomozygous for the
“Swedish mutation”.“Swedish mutation”.
Cells that express thisCells that express this
mutant sequencemutant sequence
secrete approximatelysecrete approximately
6-8 times more A6-8 times more Aββ
peptide than wild-typepeptide than wild-type
cells.cells.

16. Hippocampal neurons subjected to folateHippocampal neurons subjected to folate
deficiency and homocysteine exhibitdeficiency and homocysteine exhibit
increased vulnerability to Aincreased vulnerability to Aββ-induced death.-induced death.
 80% of the neurons maintained in methyl-donor80% 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 mediumOnly 10% of the neurons in complete medium
died within 72 hours.died within 72 hours.

17. Folic acid deficiency increasesFolic acid deficiency increases
homocysteine levels and sensitizeshomocysteine levels and sensitizes
hippocampal neurons to death in APPhippocampal neurons to death in APP
mutant transgenic mice.mutant transgenic mice.

18.  AmyloidAmyloid ββ--
peptide levelspeptide levels
are unchangedare unchanged
in APP mutantin APP mutant
mice maintainedmice maintained
on a folic acid-on a folic acid-
deficient dietdeficient diet

19. Folic acid deficiency and homocysteineFolic acid deficiency and homocysteine
enhance Aenhance Aββ-induced DNA damage in-induced DNA damage in
cultured hippocampal neurons andcultured hippocampal neurons and
APP mutant miceAPP mutant mice

20. The Olive Tail Moment is aThe Olive Tail Moment is a
measure of DNA damage inmeasure of DNA damage in
a cell.a cell.
OTM = (amount of DNA in tail) xOTM = (amount of DNA in tail) x
(distance between head and tail(distance between head and tail
regions)regions)

21. Folic acid deficiency promotes uracilFolic acid deficiency promotes uracil
misincorporation and impairs DNA repairmisincorporation and impairs DNA repair
under conditions of exposure to Aunder conditions of exposure to Aββ
• Neurons incubated inNeurons incubated in
methyl-donor deficientmethyl-donor deficient
medium experiencedmedium experienced
a significant increasea significant increase
in uracilin uracil
misincorporationmisincorporation
(p<0.01).(p<0.01).

22. DiscussionDiscussion
• The combination of low folic acid intake and theThe combination of low folic acid intake and the
consequent hyperhomocysteinemia might put agingconsequent hyperhomocysteinemia might put aging
individuals at increased risk for developing Alzheimer’sindividuals at increased risk for developing Alzheimer’s
disease.disease.
• The CA3 neurons of the hippocampus may beThe CA3 neurons of the hippocampus may be
selectively vulnerable to DNA damage, since noselectively 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 hyperhomocysteinemiaFolic acid deficiency and hyperhomocysteinemia
accelerate the accumulation of DNA damage that isaccelerate the accumulation of DNA damage that is
promoted by age-related increases in oxidative stresspromoted 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.

23. Folic Acid deficiency is quiteFolic Acid deficiency is quite
common in humans.common in humans.
• Approximately 10% of the U.S. populationApproximately 10% of the U.S. population
is folate-deficient.is folate-deficient.
• Folate deficiency is much more prevalentFolate deficiency is much more prevalent
in developing countries. Worldwide, therein developing countries. Worldwide, there
are about 200,000 severe birth defects perare about 200,000 severe birth defects per
year resulting from folate-deficient diets.year resulting from folate-deficient diets.
• Reduced intestinal absorption of folic acidReduced intestinal absorption of folic acid
in the elderly increases folate-deficiency inin the elderly increases folate-deficiency in
this population to almost 50%.this population to almost 50%.

24. Sources of folic acidSources of folic acid
• Most cereals and breads are fortified withMost 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 leafyNatural sources of folic acid include leafy
greens and citrus.greens and citrus.
• Most supplements contain 400 µg of folicMost supplements contain 400 µg of folic
acid.acid.
• Less than one gram per day of folic acid isLess than one gram per day of folic acid is
considered non-toxic.considered non-toxic.

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