Lakhasly

.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.

. Alzheimer's disease
Alzheimer's disease (AD) is the most common of
the neurodegenerative diseases. The most important
risk factor is advancing age [1]. The illness occurs in
both a familial form which is autosomal dominant
inherited and an apparently sporadic illness. 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. Familial Alzheimer's disease
accounts for approximately 5% of all cases. The re-
maining apparently sporadic cases of Alzheimer's
disease show an increased risk in families of 2.5^3-
fold. A sporadic inheritance pattern with familial as-
sociation and evidence for maternal transmission are
characteristic features of known mitochondrial genet-
ic diseases. Some evidence has suggested that there is
maternal inheritance in Alzheimer's disease [2,3].
These studies showed an increase in female to male
ratio in the parental generation of Alzheimer's dis-
ease probands. In a group of families in which there
were both an a°ected parent and at least two a°ected
siblings the ratio of mothers to fathers in the paren-
tal generation was 9:1 [2].
There is substantial evidence implicating metabolic
defects in Alzheimer's disease. 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]. Positron emission tomography studies also show
increased oxygen utilization in comparison with glu-
cose utilization in Alzheimer's disease patients [7].
This latter observation has been con¢rmed with di-
rect measurements in arterial and jugular venous
samples [8,9]. Prior work also demonstrated abnor-
mal glucose metabolism in brain biopsy specimens
[10]. 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]. The study of Smith and colleagues
demonstrated a reduction in PCr/Pi ratio in the fron-
tal cortex of Alzheimer's disease patients [13].
Initial studies which suggested that there were de-
fects in cytochrome oxidase in Alzheimer's disease
were done in platelets. Parker and colleagues re-
ported signi¢cant decreases in cytochrome oxidase
activity in Alzheimer's disease platelets as compared
to normal controls [14]. This work was disputed uti-
lizing less puri¢ed platelets preparations but was con-
¢rmed in a follow-up study [15,16]. Studies of post-
mortem cerebral tissue of Alzheimer's disease
patients con¢rmed reduced cytochrome oxidase ac-
tivity [17,18]. 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].
Although 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. A novel technique to
investigate the role mitochondrial defects is to utilize
cybrid technology, which was pioneered by King and
Attardi [19]. This technique involves the transfer of
mitochondria from living patients or cell lines to mi-
tochondria de¢cient cells (b0 cells). Cell lines from a
variety of sources can be depleted of mitochondrial
DNA (mtDNA) by exposing them to low concentra-
tions of ethidium bromide. 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. Exposed cells lose their mtDNA
and assume an anaerobic phenotype. Cybrids are
cells formed by fusing mitochondria from platelets
or other tissues into the b0 cells. Since the b0 cells
are auxotrophic for uridine and pyruvate, any cells
which are not transformed are then eliminated by
removing uridine and pyruvate from the medium.
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.
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]. It was shown that cytochrome oxidase de-
fects can be transferred from Alzheimer's disease
platelets into cybrids. Furthermore the ensuing
cybrid cell lines show markedly increased free radical
production. 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. 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.
The consequences of cytochrome oxidase defects in
cybrid cell lines on intracellular calcium bu°ering
have been determined [22]. 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. They
also show slower recovery from the increased calci-
um levels. These ¢ndings are consistent with prior
observations in AD ¢broblasts [23^25]. They are
also consistent with the ¢nding of decreased calcium
uptake in mitochondria from AD ¢broblasts [26].
Impaired calcium bu°ering is also known to occur
in ¢broblasts of patients with a known mitochondrial
disorder, MELAS syndrome [27].
Alzheimer's disease cybrid cell lines are associated
with increased free radical production. One therefore
might expect that there would be evidence for in-
creased free radical damage in Alzheimer's disease
postmortem tissue. Mitochondrial DNA may be
preferentially vulnerable since it is located close to
the inner mitochondrial membrane. 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]. In other studies there have been reports of
increased tissue concentrations of malondialdehyde
and protein carbonyl groups [29,30]. Furthermore
novel spin trapping techniques demonstrated in-
creased oxidative damage to both lipids and proteins
[31]. Immunocytochemical studies have demon-
strated that there is evidence for oxidative damage
at the cellular level. Neuro¢brillary 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 neuro¢laments,
and 3-nitrotyrosine [13,32^38]. Interestingly these
antibodies show increased staining in cell bodies
and neurites rather than in senile plaques, suggesting
an intracellular source of free radicals.
Mitochondrial dysfunction may be linked to the
other neuropathological hallmarks of Alzheimer's
disease including senile plaques and neuro¢brillary
tangles. 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]. 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]. An increase in intracellular L-
amyloid was also observed in cultured astrocytes
from Down syndrome patients in which there is in-
creased free radical production [43]. Oxidative dam-
age may lead to cross-linking and impaired solubility
of L-amyloid [44,45].
Oxidative injury has also been shown to lead to
intermolecular cross links in covalent bonds which
could contribute to the generation of paired helical
¢laments [46]. Oxidation of critical cysteine residues
seems to be associated with the aggregation of tau
proteins into paired helical ¢laments [47]. 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 ¢lament-like state similar to that
in AD