.The resulting cybrids then enable one to determine
whether any observed defects in oxidative phospho-
rylation are attributable to alterations in the patient's
mtDNA, since the patient's mitochondria now func-
tion in the presence of a di?erent nuclear back-
ground.Neurocbrillary tangle bearing
neurons showed increased immunostaining with anti-
bodies to advanced glycation end products, hemeox-
ygenase-1, malondialdehyde, 4-hydroxynonenal, pro-
tein carbonyl groups, carbonylated neuroclaments,
and 3-nitrotyrosine [13,32^38].Previous studies showed that impairment of
cytochrome oxidase in vitro leads to an increase in
C-terminal fragments of the amyloid precursor pro-
tein, which contain the L-amyloid peptide [39], and a
decrease in non-amyloidogenic processing of the
amyloid precursor protein [40].Phosphorus magnetic resonance spectroscopy
has demonstrated abnormalities in either phospho-
creatine (PCr) or inorganic phosphate (Pi) in Alz-
heimer's disease patients as compared with elderly
controls [11,12].Familial
autosomal dominant Alzheimer's disease is associ-
ated with point mutations in the amyloid precursor
protein as well as in novel proteins entitled preseni-
lin-1 and presenilin-2.Studies utilizing posi-
tron emission tomography consistently show reduced
glucose metabolism in temporoparietal regions of
Alzheimer's disease patients, and this appears to oc-
cur quite early in the disease course [4].Recent stud-
ies have demonstrated that this occurs in patients at
risk for Alzheimer's disease [5], and there appeared
to be reduced glucose utilization in asymptomatic
patients who are homozygous for the Apo O4 allele,
a known risk factor for sporadic Alzheimer's disease
[6].An increase in intra-
cellular L-amyloid 1^42 was found after exposure of
cultured guinea pig neurons to hydrogen peroxide,
and oxidative stress increased L-amyloid in mamma-
lian lens tissue [41,42].Reduced
ATP generation also leads to activation of ERK1
M.F. Beal / Biochimica et Biophysica Acta 1366 (1998) 211^223 213
and ERK2 kinases which phosphorylate tau proteins
into a paired helical clament-like state similar to that
in ADAlthough prior biochemical studies suggested that
there were decreases in cytochrome oxidase activity
in Alzheimer's disease platelets and cerebral tissue, it
was unclear whether this was a primary or secondary
e?ect of the disease process.The cytochrome oxidase activity shows
a reduction in catalytic activity yet normal amounts
of cytochrome aa3 , suggesting that reduced complex
IV activity is a consequence of abnormal catalytic
activity rather than decreased enzyme levels [16].Ethidium bromide is con-
centrated within mitochondria and preferentially in-
M.F. Beal / Biochimica et Biophysica Acta 1366 (1998) 211^223212
hibits mtDNA replication in comparison to nuclear
DNA replication.The Alzheimer's disease
cybrids show elevated basal cytosolic calcium con-
centrations as well as enhanced sensitivity to inosi-
tol-1,4,5-triphosphate mediated calcium release.A sporadic inheritance pattern with familial as-
sociation and evidence for maternal transmission are
characteristic features of known mitochondrial genet-
ic diseases.Recent studies using the cybrid technique to dem-
onstrated that the cytochrome oxidase defects in Alz-
heimer's disease appear to be encoded on mtDNA
[20,21].Point mutations were found in the cyto-
chrome oxidase-1 and cytochrome oxidase-2 mtDNA
encoded subunits of cytochrome oxidase, however
further work needs to be done to exclude the possi-
bility that these mutations are not present in nuclear
pseudogenes.The study of Smith and colleagues
demonstrated a reduction in PCr/Pi ratio in the fron-
tal cortex of Alzheimer's disease patients [13].Studies of post-
mortem cerebral tissue of Alzheimer's disease
patients concrmed reduced cytochrome oxidase ac-
tivity [17,18].Cell lines from a
variety of sources can be depleted of mitochondrial
DNA (mtDNA) by exposing them to low concentra-
tions of ethidium bromide.Nuclear pseudogenes are mitochondrial
DNA sequences which are randomly incorporated
into the nuclear genome by unclear mechanisms,
but which exist for much of the mitochondrial ge-
nome.Consistent with
this possibility we found a 3-fold increase in 8-hy-
droxy-2-deoxyguanosine content of mtDNA in AD
postmortem tissue as compared to age-matched con-
trols [28].Positron emission tomography studies also show
increased oxygen utilization in comparison with glu-
cose utilization in Alzheimer's disease patients [7].Familial Alzheimer's disease
accounts for approximately 5% of all cases.