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Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: Partial reversal by feeding acetyl-L-carnitine and/or R-alpha-lipoic acid

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Accumulation of oxidative damage to mitochondria, protein, and nucleic acid in the brain may lead to neuronal and cognitive dysfunction. The effects on cognitive function, brain mitochondrial structure, and biomarkers of oxidative damage were studied after feeding old rats two mitochondrial metabolites, acetyl-l-carnitine (ALCAR) [0.5% or 0.2% (wt/vol) in drinking water], and/or R-alpha-lipoic acid (LA) [0.2% or 0.1% (wt/wt) in diet]. Spatial memory was assessed by using the Morris water maze; temporal memory was tested by using the peak procedure (a time-discrimination procedure). Dietary supplementation with ALCAR and/or LA improved memory, the combination being the most effective for two different tests of spatial memory (P < 0.05; P < 0.01) and for temporal memory (P < 0.05). Immunohistochemical analysis showed that oxidative damage to nucleic acids (8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine) increased with age in the hippocampus, a region important for memory. Oxidative damage to nucleic acids occurred predominantly in RNA. Dietary administration of ALCAR and/or LA significantly reduced the extent of oxidized RNA, the combination being the most effective. Electron microscopic studies in the hippocampus showed that ALCAR and/or LA reversed age-associated mitochondrial structural decay. These results suggest that feeding ALCAR and LA to old rats improves performance on memory tasks by lowering oxidative damage and improving mitochondrial function.
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Memory loss in old rats is associated with brain
mitochondrial decay and RNADNA oxidation:
Partial reversal by feeding acetyl-
L-carnitine
andor
R
-
-lipoic acid
Jiankang Liu*
, Elizabeth Head
, Afshin M. Gharib*
, Wenjun Yuan*, Russell T. Ingersoll*, Tory M. Hagen
§
,
Carl W. Cotman
, and Bruce N. Ames*
†¶
*Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720;
Children’s Hospital Oakland Research Institute, 5700 Martin
Luther King, Jr., Way, Oakland, CA 94609;
Institute for Brain Aging and Dementia, University of California, Irvine, CA 92697-4540; and
§
Department of
Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University, Corvallis, OR 97331
Contributed by Bruce N. Ames, December 29, 2001
Accumulation of oxidative damage to mitochondria, protein, and
nucleic acid in the brain may lead to neuronal and cognitive
dysfunction. The effects on cognitive function, brain mitochondrial
structure, and biomarkers of oxidative damage were studied after
feeding old rats two mitochondrial metabolites, acetyl-L-carnitine
(ALCAR) [0.5% or 0.2% (wtvol) in drinking water], andor R-
-
lipoic acid (LA) [0.2% or 0.1% (wtwt) in diet]. Spatial memory was
assessed by using the Morris water maze; temporal memory was
tested by using the peak procedure (a time-discrimination proce-
dure). Dietary supplementation with ALCAR andor LA improved
memory, the combination being the most effective for two differ-
ent tests of spatial memory (P < 0.05; P < 0.01) and for temporal
memory (P < 0.05). Immunohistochemical analysis showed that
oxidative damage to nucleic acids (8-hydroxyguanosine and
8-hydroxy-2-deoxyguanosine) increased with age in the hip-
pocampus, a region important for memory. Oxidative damage to
nucleic acids occurred predominantly in RNA. Dietary administra-
tion of ALCAR andor LA significantly reduced the extent of
oxidized RNA, the combination being the most effective. Electron
microscopic studies in the hippocampus showed that ALCAR
andor LA reversed age-associated mitochondrial structural decay.
These results suggest that feeding ALCAR and LA to old rats
improves performance on memory tasks by lowering oxidative
damage and improving mitochondrial function.
M
emory, i.e., performance on memory tasks, declines with
age in animals. In the case of age-related human neuro-
degenerative diseases, such as Alzheimer’s disease (AD), the
deficit can be severe (1–4). Memory loss is accompanied but not
necessarily caused by accumulation of oxidative damage to lipids,
proteins, and nucleic acids, and by mitochondrial decay, all of
which can disrupt neuronal function (5–10).
R-
-lipoic acid (LA) is a coenzyme that is involved in carbo-
hydrate utilization necessary for the production of ATP in
mitochondria; it is reduced in mitochondria to dihydrolipoic acid
(DHLA), a potent antioxidant (11, 12). LA improves long-term
memory in aged female NMRI mice (13).
L-Carnitine is a betaine required for the transport of long-
chain fatty acids into the mitochondria for
-oxidation, ATP
production, and for the removal of excess short- and medium-
chain fatty acids (14, 15). Acetyl-
L-carnitine (ALCAR) is more
widely used than
L-carnitine in animal and clinical studies
because it enters cells and crosses the blood–brain barrier more
efficiently (16). ALCAR improves cognitive function and neu-
ronal bioenergetic mechanisms in rats with both acute and
long-term treatments (17–23).
Several clinical studies report the beneficial effects of ALCAR
or LA:ALCAR administration in a small group of patients with
AD that resulted in improved spatial orientation and short-term
memory (24, 25). LA administration in patients with AD for
approximately 1 year also resulted in mild cognitive improve-
ments and stabilization of global neuropsychological test scores
(26). Thus, as both ALCAR and LA improve mitochondrial
decay, their combination may be complementary in decreasing
oxidative damage to neurons and cognitive dysfunction.
As our understanding of the importance of mitochondrial decay
in aging advances (27–29), the importance of improving mitochon-
drial function by dietary interventions of mitochondrial metabolites
such as ALCAR or LA becomes clearer (30–33). Feeding 0.15–
0.5% ALCAR to old rats elevated the levels of carnitine in plasma
and brain to that of young rats (34) and 0.1–0.2% LA (T.M.H.,
unpublished data) was as effective in improving mitochondrial
function in the liver as the higher doses originally used (30–33). We
have examined the effects of these lower doses of ALCAR, LA, and
their combination on spatial memory by using the Morris water
maze, on temporal memory by using the peak procedure, decay in
mitochondrial structure in the hippocampus, and oxidative damage
to nucleic acids in the hippocampus and cortex.
Materials and Methods
Materials. ALCAR (hydrochloride salt) was a gift of Sigma Tau
(Pomezia, Italy), and LA was a gift of Asta Medica (Frankfurt
Main, Germany). All other chemicals were reagent grade or the
highest quality available from Sigma.
Animals and Diet. Fischer 344 male rats were obtained from the
National Institute on Aging. Control animals were fed AIN93M
diet from Dyets (Bethlehem, PA) and MilliQ water (pH 5.2). The
rats in the experimental groups were fed either 0.5% or 0.2%
(wtvol) ALCAR in MilliQ water (pH was adjusted to 5.2 with 1
N NaOH), 0.2% or 0.1% (wtwt) LA in AIN93M diet, or a
combination of (0.5% ALCAR and 0.2% LA) or (0.2% ALCAR
and 0.1% LA). The food consumption was determined by weighing
the diet and measuring the volume of water weekly; the average
daily consumption was then calculated. The weight gain during the
course of the experiment was also measured. We did not find any
significant differences in diet, water consumption, or weight gain
between the unsupplemented old rats (13.4 0.5 gday; 18.6 1.19
mlday; body weight from 416.1 14.4 to 409.2 10.1 g mean
SE) and the old supplemented rats (For example, the ALCAR
LA group 13.1 0.4 gday; 18.4 0.9 mlday; body weight from
416.0 19.0 to 414.9 9.4 g; mean SE). All animals were
Abbreviations: ALCAR, acetyl-L-carnitine; LA, R-
-lipoic acid; oxo8dG, 8-hydroxy-2-
deoxyguanosine; oxo8G, 8-hydroxyguanosine; AD, Alzheimer’s disease.
To whom reprint requests should be addressed. E-mail: bnames@uclink4.berkeley.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
2356–2361
PNAS
February 19, 2002
vol. 99
no. 4 www.pnas.orgcgidoi10.1073pnas.261709299
acclimatized at the Northwest Animal Facilities on the University
of California at Berkeley campus for at least 2 weeks before
treatment. Rats were housed individually and provided with
ALCAR andor LA for 7 weeks. The young and old rats were 4.5
and 24.5 months old at the start of the experiment; they were more
than 7 weeks older at the time of death. Death, by approved
protocol, was with an overdose of ether.
Morris Water Maze Test of Spatial Memory. The Morris water maze
task tests spatial memory by requiring rats to find a submerged
platform in a pool of water using external visual cues (35, 36).
The time required for individual rats to find the platform and the
length of the swim path was measured by using a digital camera
and a computer system to record movement (VideoMex-V,
Columbus Instruments, Columbus, OH). Trials (4 consecutive
days, 4 trials per day) were with the same hidden platform
location, but with varied start locations. On day 5, the platform
was removed from the pool (transfer trial, 60 sec), and the time
spent at the actual site where the platform was located was
examined. On day 6, the time required to reach a visible platform
was measured to determine visual function and motor ability.
Peak Procedure Test of Temporal Memory. The peak procedure is a
modified fixed-interval schedule commonly used to study tem-
poral memory (37). Rats were tested in 18 identical boxes that
contained a light source and a speaker (for delivering light or
noise signals) and a lever that dispenses single food pellets (45
mg) when pressed (mix T101, Bioserv, Frenchtown, NJ). The
food supply of the rats was decreased to 85% of the free-feeding
amount. In this test, the animal is rewarded with one pellet only
if the lever is pressed at 40 sec from the signal. In 20% of the tests,
no food was given, and an empty trial and the signal lasted 195
sec plus a geometrically distributed duration that averaged 50
sec. The results are presented as a sum of the two types of tests.
Peak rate, which is the maximum response rate in a given trial
and reflects the rats choices of what responses to make and their
motivation, was measured.
Electron Microscopic Observations. A subset of rats from each
experimental condition was perfused transcardially with 2.5%
glutaraldehyde for 2 h. The brain was removed from the skull and
the hippocampus was postfixed in 0.1 M PBS with 1% osmium
tetroxide. The tissues were block-stained with uranyl acetate and
embedded in Epon. Sections were cut at 0.60.9-
m thick from
the block, stained with uranyl acetate and lead citrate, and
examined with a JEOL 100 CX electron microscope.
Immunohistochemical Studies. A subset of rats from each treat-
ment condition was anesthetized with ether and perfused with
4% paraformaldehyde for 1.52 h. The brain was removed and
postfixed for preparing paraffin sections. Sections of hippocam-
pus were incubated with monoclonal anti-8-hydroxy-2-
deoxyguanosine8-hydroxyguanosine (oxo8dGoxo8G; 1:2000;
QED Bioscience, San Diego) and visualized by using standard
immunocytochemical methods. Two independent analyses were
done on each rat. To determine whether DNA or RNA was
oxidatively damaged, sections were pretreated with either 10
units
l of RNase-free DNase I or 10 mgml of DNase-free
RNase (Roche Molecular Biochemicals) for 3 h prior to incu-
bation with oxo8dGoxo8G Ab (38). To quantify the extent of
oxo8Goxo8dG immunolabeling, a 525 410
m area of
staining was captured by using a 2.5 photo eyepiece, a Sony
(Tokyo) high-resolution charge-coupled device (CCD) video
camera (XC-77), and the built-in video capture capabilities of a
Macintosh 810080AV. All sections from a given region were
captured sequentially during one session and were analyzed blind
with respect to treatment condition. Subsequently, public do-
main image analysis software (
IMAGE 1.55, National Institutes of
Health) and gray-scale thresholding were used to separate
positive staining from background and to calculate the percent-
age of area occupied by oxo8Goxo8dG immunoreactivity.
Results
Spatial Memory. Rats are proficient swimmers and are motivated
to escape from water. Once animals learn where the hidden
platform is located, they can remember the location and swim
rapidly to it from any starting point. Both time taken to reach the
platform (Fig. 1) and swimming distance traveled (data not
shown) were measured and gave similar results. Fig. 1 A shows
results obtained on day 4. Young rats spent a significantly shorter
time than old rats (P 0.001) in finding the hidden platform.
ALCAR or LA seems to shorten the time in old rats, but the
differences were not significant. However, the combination
resulted in significantly shorter times (P 0.05) as compared
with old control rats. The tracks of individual rats on successive
trials and days have been shown (34).
A transfer test, in which the platform was removed, was carried
out on day 5. The time spent at the previous platform position
is a measure of search accuracy and spatial memory. Young rats
spent significantly more time at the former platform position
(P 0.001) than old rats did. The ALCAR (P 0.05) and LA
(P 0.05) significantly restored the lost procedural subcompo-
nent of spatial memory and the combination was even more
effective (P 0.01; Fig. 1B).
A clearly visible platform was used to measure deficits in vision,
motivation, motor strength, or coordination on day 6 of the training
cycle. The platform protruded 1 cm above the surface of the water.
Young rats required less time to find the visible platform than the
old animals (Fig. 1C). All three supplementation groups showed
improvement, but only the combination treatment group reached
statistical significance (Fig. 1C).
Fig. 1. Morris water maze test in relation to age and treatment. (A) Time on
day 4 taken to find the hidden platform. (B) Time spent at the former platform
position in the transfer test. (C) Time to find the visible platform. Data are
mean SEM of 9 rats in young and old, 5 in LA (0.1%), and 6 in ALCAR (0.2%)
and ALCAR LA groups. Higher doses, 0.2% LA andor 0.5% ALCAR, showed
similar results (data not shown). Statistical differences were examined with
two-tailed Student t test.
***
, P 0.001 vs. young rats; #, P 0.05 and ##, P
0.01 vs. old rats.
Liu et al. PNAS
February 19, 2002
vol. 99
no. 4
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NEUROBIOLOGY
Temporal Memory. The response rate to a sound (Fig 2A) and to
a light (Fig. 2B) signal is the same, indicating that the rats
responded similarly to both signals. Results from the last 10 days
of testing were used, where responses had reached asymptotic
levels.
Peak rate (Fig. 2 C and D) of young animals was significantly
higher than that of all other groups: young compared with old
(P 0.001); young compared with old ALCAR (P 0.004);
young compared with old LA (P 0.043); and young
compared with old ALCAR LA (P 0.046). Although
ALCAR does not show any significant effect (comparing the
old ALCAR group to the old control rats), LA seems to
slightly increase peak rate. The old ALCAR LA treatment
showed a more significant increase (P 0.033) in peak rate in
old animals than treatment with LA alone.
Ultrastructural Observations of Neuronal Mitochondria. Electron
microscope observations of hippocampal neuronal mitochondria
indicate that structural abnormalities develop with age. Com-
pared with young rats, old rats showed some disruption and loss
of cristae in about half of the mitochondria in the dentate gyrus
area, indicating structural decay. Animals treated with 0.5%
ALCAR andor 0.2% LA showed less structural disruption and
loss of cristae. In addition, old rats had more lipofuscin in the
cytoplasm of granule cells of the dentate gyrus, and the com-
bined treatment rats also seemed to have less lipofuscin. How-
ever, these results were obtained from one or two animals per
group. Clearly, further quantitative studies with more animals
and more fields are needed to confirm these observations.
Oxidative Damage to Nucleic Acids. Various regions of the brain
were stained with an Ab that recognizes oxidized DNA or RNA
Fig. 2. Peak procedure test related to age and treatment. Response rate functions plotted separately for sound-signaled trials (A) and light-signaled trials (B)
obtained during the last 10 days of the test. (C) Peak rate over the 20 days of peak procedure testing. Each data point averages 2 days of testing. (D) Peak rate
of the last 10 days averaged. Data are mean SEM of 6 in young, 7 in old, 4 in LA (0.2%), and 5 in ALCAR (0.5%) and ALCAR LA groups. Treatment with lower
doses, 0.1% LA andor 0.2% ALCAR, showed similar results (data not shown). Statistical differences were examined with two-tailed Student t test.
*
, P 0.05
and
***
, P 0.001 vs. young rats; #, P 0.05 vs. old unsupplemented rats.
2358
www.pnas.orgcgidoi10.1073pnas.261709299 Liu et al.
(oxo8dG or oxo8G; ref. 39). Fig. 3A shows representative images
of the CA1 region of the hippocampus. Fig. 3C shows that old
rats without treatment showed significantly higher immunore-
activity than young rats in areas CA1, CA4, cerebral cortex, and
in the white matter. Both ALCAR and LA reduced immunore-
activity, but only LA showed a significant effect in the CA4
region. The combination showed a significant effect on lowering
immunoreactivity in CA1, CA3, CA4, and dentate granule cells
in old rats.
Fig. 3B illustrates that pretreatment of sections, including area
CA1 with RNase but not DNase, virtually eliminated the im-
munoreactivity, indicating that the predominant damage to
neuronal nucleic acids is to RNA (oxo8G). In CA1, RNase
pretreatment reduced the immunoreactivity by 92%, whereas
DNase, rather than reducing, enhanced (168%) the immuno-
reactivity (Fig. 3B).
Discussion
Old rats have increased mitochondrial dysfunction and oxidative
damage, which is associated with cognitive deficits in both spatial
and temporal memory. Spatial memory relies on intact hip-
pocampal function. Temporal memory may also be associated
Fig. 3. Immunostaining relative to age and treatment for oxidized nucleic acids in neurons. (A) Representative photographs of oxo8G immunoreactivity in area
CA1 of the hippocampus and adjacent white matter, from individual rats selected from young, old, old ALCAR (0.5%), old LA (0.2%), and old ALCAR
(0.5%) LA (0.2%) groups. (B) CA1 sections pretreated with either DNase or RNase before incubation with Ab. (C) Extent of immunoreactivity to oxo8G in the
hippocampus [CA1, CA3, CA4, dentate gyrus (DG), cerebral cortex (CX), and white matter (WM) in rat brain]. [Bar 50
m.] Values are mean SEM of 5 animals
for young and old groups, 3 for old ALCAR and old LA groups, and 2 for the old ALCAR LA group. The MannWhitney U test was used to compare values.
*
, P 0.005 vs. young rats; #, P 0.05 vs. old control rats.
Liu et al. PNAS
February 19, 2002
vol. 99
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NEUROBIOLOGY
with the hippocampus, although it may be more closely associ-
ated with the striatum and cerebellum. The dietary administra-
tion of a combination of ALCAR and LA to old rats improves
mitochondrial function in liver (40). The purpose of this study
was to determine whether it also improves cognition.
Spatial memory was assayed in the Morris water maze. The
Morris water maze has been used extensively to measure cog-
nitive deficits in spatial memory in lesion studies (4147) and in
aging (4852). Old rats showed decreased spatial memory
compared with young rats; ALCAR andor LA restored some of
this function, the combination being more effective than each
compound alone. We also observed significant age effects in the
transfer test, which measures search accuracy and is considered
a procedural (habitual) subcomponent of spatial memory (36).
ALCAR andor LA significantly restored performance in this
test, the combination being more effective (P 0.01) and not
significantly different from that of young rats (Fig. 1B). Schenk
and Morris (36) have shown that after a retrohippocampal
lesion, the procedural component of spatial memory can be
partially recovered after training. We also observed significant
age effects on the latencies of animals in finding a visible
platform, which is a control procedure used to detect sensory
motor deficits or motivational differences that impair water
maze performance. The dietary interventions have similar ef-
fects on the visible platform test as those observed during the
hidden platform tests (Fig. 1C).
Many physiological changes occur with age and can have
major consequences on cognitive performance (53). We ob-
served age and treatment effects on several noncognitive factors,
such as motivation and locomotor activity, which can potentially
contribute to the cognitive results. The age-associated decline in
the Morris water maze test, therefore, should not be considered
solely a test of cognition, but also as revealing a general decline
in other systems as a result of aging. Old animals are known to
be less sensitive to pain and possibly to temperature, which may
affect their motivation to find the hidden platform. ALCAR and
LA reduce mitochondrial dysfunction in peripheral systems (31,
33, 54, 55), including sensory systems such as hearing (56).
Therefore, improvements shown here in test performances
attributable to ALCAR, LA, or their combination, including the
visible platform test and ambulatory activity (see ref. 40), suggest
that reversing mitochondrial decay might reverse age-associated
declines in nervous, cardiovascular, visual, and auditory systems,
as well as general effects on motivation and physical strength.
Temporal memory, as assayed by the peak procedure, mea-
sures the function of the internal clock, learning processes,
attention, and exploratory behavior. The combination of LA
with ALCAR showed a significant improvement on peak rate
(P 0.05). The peak procedure is a time-discrimination proce-
dure, which resembles a discrete-trials fixed-interval schedule
with catch trials; it has been used to study the timing abilities of
animals (37). Several studies have shown that old rats have
deficits in time perception (5761). One advantage of the peak
procedure is that it allows for comparison of performance by
using different types of signals and sensory modalities. The
similarity of performance with light and sound signals suggests
that the deficits are the results of deficits in cognition as rats of
different ages do not differ in their sensitivity to light and sound
at the two levels of light and sound used in this study. Peak rate
reflects changes in a response learning mechanism. Old rats had
lower peak rates, suggesting that old animals have difficulty
learning the relevant response. The combination of ALCAR and
LA seems to have a complementary effect on improving the peak
procedure performance.
Not all of the old rats tested had cognitive deficits; this resulted
in a large SD and the need for larger numbers of rats to achieve
statistical significance. In future experiments it would be useful
to separate cognitively impaired from unimpaired old rats to
show more pronounced effects in old rats that receive treatment
(23, 52).
The current study also has tested the hypothesis that cognitive
improvements in response to ALCAR andor LA interventions
are linked to reductions in oxidative damage in old brain. To
measure oxidative damage to nucleic acids, we used an Ab that
detects both oxidized DNA and RNA (39). RNase pretreatment
decreased immunoreactivity extensively, whereas DNase had a
smaller effect. This result suggests that the oxidized nucleic acid
in the aged rat brain is predominantly RNA, which is consistent
with studies in human brains with AD (38). It is clear that more
than 90% immunoreactivity is from RNA, suggesting that RNA
oxidation is a significant biomarker of aging in rat brain. The
mechanism of the DNase enhancement of immunoreactivity
remains unclear; the digestion of DNA may have unmasked
binding sites allowing greater access of the mAb to the RNA.
Cytoplasmic punctate staining is consistent with either cellular
Rna or mtDNARNA. RNA being the predominant oxidized
nucleic acid is consistent with the lack of staining of nuclear
DNA. The type of RNA oxidized and its subcellular localization
remain to be determined, particularly with respect to mitochon-
dria, the most likely oxidant target and the one that is improved
by ALCAR andor LA. RNA oxidation increased significantly
as a function of age in rats in areas CA1 and CA4 in the
hippocampus, in cortical neurons, and in white matter in the
frontoparietal cortex. Feeding old rats LA significantly reduced
the levels of oxidized RNA in CA4. The combination of ALCAR
and LA was effective in significantly reducing oxidative RNA
damage in neurons in CA1, CA3, CA4, and dentate gyrus of the
hippocampus to levels not significantly different from young
animals.
Poorer performance on memory tasks by old rats could
involve, in part, oxidative damage to RNA, with errors in
translation (62) compromising protein synthesis critical for the
formation of new memories (63, 64). Although oxidative damage
to RNA has been shown to be more extensive than damage to
DNA in urine and plasma (39), oxidized RNA has not been a
focus of interest as an oxidative damage marker for brain aging
or cognition, except in some patients with AD sample studies.
Neuronal RNA oxidation is a prominent feature of vulnerable
neurons in AD, Downs syndrome, and Parkinsons disease, all
of which are diseases associated with severe cognitive deficits
(38, 65, 66). Neuronal RNA oxidation may thus contribute to
memory decline and serve as a sensitive marker for intervention
studies. However, oxidant-induced enzyme dysfunction is also an
important contributor to neuronal decay and aging (6769).
The improving effects on performance on memory tasks by
ALCAR andor LA on hippocampal mitochondria are sup-
ported by morphological observations. There seems to be a loss
of mitochondrial cristae with age. Evidence that ALCAR re-
versed this loss with a dose-dependent response has been
presented (34). Similar to ALCAR, LA also reduced age-
dependent cristae loss in the dentate granule cells of the
hippocampus. Because ALCAR alone showed a virtually com-
plete reversal of the cristae loss, we cannot say whether the
combination has an improving effect or not, but it produced at
least as large a reduction as the ALCAR or LA alone.
The loss of memory with age seems to be caused in good part
by oxidative mitochondrial decay in neurons. (i) The effective-
ness of the mitochondrial metabolites ALCAR and LA suggests
that mitochondrial decay is involved. (ii) The oxidation of
RNADNA in neurons is likely to be mitochondrial (70). (iii)
Neuronal mitochondria show structural decay with age.
The cognition-improving effect of ALCAR may also be
caused in part by the donation of an acetyl group for the synthesis
of the neurotransmitter acetylcholine through choline acetyl-
transferase and carnitine acetyltransferase (17, 71, 72). Low
acetylcholine levels in certain brain regions are associated with
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www.pnas.orgcgidoi10.1073pnas.261709299 Liu et al.
age-related cognitive dysfunction, including AD (73). Because of
the profound effects of calorie restriction, we have compared
dietary intakes carefully and have found no significant differ-
ences in food and water consumption or in body weight (see
Materials and Methods).
In conclusion, feeding old rats ALCAR andor LA improved
performance on memory tasks, reduced brain mitochondrial
structure decay, and reduced oxidative damage in the brain. The
combination of ALCAR and LA showed a greater effect than
ALCAR or LA alone. These results suggest that feeding a
combination of mitochondrial metabolites to old animals may
prevent mitochondrial decay in neurons and restore cognitive
dysfunction. These results also suggest that consumption of high
levels of mitochondrial metabolites may be an efficient inter-
vention in humans for delaying brain aging and age-associated
neurodegenerative diseases.
We are indebted to Seth Roberts for his stimulation and advice in using
the peak procedure; Judith Campisi, John Nides, and Seth Roberts for
critical reading of the manuscript; and the Electron Microscope Lab at
the University of California at Berkeley for the electron microscopic
studies. We thank M. Nistor at the Institute for Brain, Aging, and
Dementia for technical assistance. This work was supported by grants
from the Ellison Foundation, the National Institute on Aging, the
Wheeler Fund of the Dean of Biology, and the National Institute of
Environmental Health Sciences Center Grant ES01896 (to B.N.A.), and
by National Institute of Aging Grant AG12694 (to C.W.C.).
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Liu et al. PNAS
February 19, 2002
vol. 99
no. 4
2361
NEUROBIOLOGY
... It has been scientifically evidenced that exogenously supplied ALA cannot be used as a booster of mitochondrial metabolism, but it exhibits a unique set of biochemical interactions with a potential pharmacotherapeutic value against numerous pathophysiological factors. ALA is known as a detoxifying agent and antidiabetic drug, and has been used to ameliorate age-related cardiovascular, cognitive, and neuromuscular deficits as a modulator of various inflammatory signaling pathways [39][40][41]. As already mentioned in the introduction to this paper, ALA demonstrates antioxidant activity [42]. ...
... Cell Physiol Biochem 2022;56(S1): [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] ...
Article
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Background/aims: The aim of the present study was to investigate whether α-lipoic acid (ALA) could reverse/prevent high fat diet (HFD) -induced salivary gland dysfunction and oxidative damage in the salivary glands of rats, and strengthen their antioxidant defense. Methods: The enzymatic and non-enzymatic antioxidants as well as their redox status, oxidative damage products and salivary flow rate were investigated in the parotid (PG) and submandibular (SMG) glands of Wistar rats exposed to a high-fat diet and then supplemented with ALA for a period of 4 weeks. The rats in the study were divided into 4 groups of 10 animals each: C (control), HFD, C + ALA, HFD + ALA. Results: The HFD + ALA group in comparison to the HFD group showed normalization of the activity of antioxidant enzymes to the levels observed in the C group only in the case of the SMG. Additionally, ALA supplementation was more effective in reducing the value of oxidative damage products in the PG compared to the SMG. ALA supplementation in the HFD group was not able to restore the disturbed total antioxidant capacity (TAC) of the salivary glands to the level observed in the C group. In the group of HFD + ALA rats, both unstimulated and stimulated salivation and the protein concentration in the SMG did not differ significantly from the parameters recorded in the group fed with HFD. Conclusion: ALA supplementation by rats fed the HFD diet prevents/reverses oxidative damage in the PG to a greater extent than in the SMG and is unable to completely restore disturbed TAC to the levels seen in C rats. Moreover, we observed that ALA supplementation did not improve the impaired secretory function of the salivary glands.
... The administration of ALA has been shown to reduce lipid peroxidation and increase the activity of antioxidant molecules in different areas of the brain of experimental animals [10,11]. In elder rats, ALA has been demonstrated to revert the effects of age-induced oxidative damage in the prefrontal cortex and hippocampi [12]. In addition, ALA has been suggested to improve memory by increasing the activity of choline acetyltransferase (ChAT), a crucial step in the biosynthesis of acetylcholine, in the hippocampi of treated rats [13]. ...
Article
Full-text available
Background: Memory disorders are common among elder people, and nonclinical cognitive decline is commonly experienced with age. Preclinical investigations have explored the possible role of alpha-lipoic acid (ALA), a known antioxidant compound abundant in vegetables and animal tissues, in reducing oxidative stress in the aging brain and preventing cognitive decline. However, clinical evidence is limited, and the few existing results are contrasting. In addition, while most of the existing trials have been focused on the effects of ALA administration in Alzheimer’s disease (AD) or other types of dementia, studies evaluating its effects on nonclinical elder population are still missing. Methods: In the present open-label, pilot study, fifteen elder patients (mean age: 84.5 ± 5.77) received ALA at a daily dose of 600 mg/day for 12 weeks. General cognitive function, executive function, and mood symptom assessment were carried out at baseline and at the endpoint. Results: Overall, ALA administration was generally well-tolerated (only one dropout due to gastrointestinal side effects). However, no statistically significant effects either on cognitive function, executive function, or mood were found. Conclusions: Despite several limitations, our study found no evidence of positive effects on cognition and mood after ALA administration in elder people without the diagnosis of AD or cognitive impairment. Further clinical trials are needed to better investigate ALA effectiveness on cognition and mood in elder subjects.
... Later, o 8 G was confirmed to occur in cytoplasmic RNAs by observing retained 8-oxoguanine immunostaining in DNase I-treated brains of patients with Alzheimer's disease (AD) 129 , which is associated with increasing ROS. o 8 G was also identified in the brains of patients with Parkinson's disease 130 and aged mouse brains 131 , wherein the quantity of o 8 G correlated with memory loss and mitochondrial decay, which could be partially reversed by antioxidant treatment 132 . Based on these observations, the oxidative modification of cytoplasmic RNAs, which may include mRNA, rRNA, tRNA, and miRNA, has been proposed to function in redox-related disease phenotypes, especially in the case of neurodegenerative disorders. ...
Article
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In pathophysiology, reactive oxygen species control diverse cellular phenotypes by oxidizing biomolecules. Among these, the guanine base in nucleic acids is the most vulnerable to producing 8-oxoguanine, which can pair with adenine. Because of this feature, 8-oxoguanine in DNA (8-oxo-dG) induces a G > T (C > A) mutation in cancers, which can be deleterious and thus actively repaired by DNA repair pathways. 8-Oxoguanine in RNA (o8G) causes problems in aberrant quality and translational fidelity, thereby it is subjected to the RNA decay pathway. In addition to oxidative damage, 8-oxo-dG serves as an epigenetic modification that affects transcriptional regulatory elements and other epigenetic modifications. With the ability of o8G•A in base pairing, o8G alters structural and functional RNA-RNA interactions, enabling redirection of posttranscriptional regulation. Here, we address the production, regulation, and function of 8-oxo-dG and o8G under oxidative stress. Primarily, we focus on the epigenetic and epitranscriptional roles of 8-oxoguanine, which highlights the significance of oxidative modification in redox-mediated control of gene expression.
... Uszkodzenia oksydacyjne w mitochondriach, białkach i DNA w mózgu mogą prowadzić do zaburzeń neuronalnych i poznawczych. Eksperymenty przeprowadzone na szczurach wykazały, że α-LA i acetylo-L-karnityna przywracają sprawność pamięciową [76,77]. Dlatego też suplementy oparte na tych związkach zalicza się do substancji przeciwstarzeniowych i wykazujących korzystny wpływ na długość i jakość życia. ...
Article
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Kwas α-liponowy (α-LA) jest naturalnym antyoksydantem występującym w żywności w postaci lipolizyny. Zarówno α-LA, jak i jego zredukowana postać DHLA (kwas dihydroliponowy) hamują produkcję reaktywnych form tlenu (ROS) i redukują szkodliwe ich działanie, mają zdolność chelatowania jonów metali przejściowych oraz regeneracji innych przeciwutleniaczy. Oprócz efektu antyoksydacyjnego wykazują także działanie przeciwzapalne. Stres oksydacyjny i stan zapalny odgrywają kluczową rolę w patogenezie miażdżycy i chorób sercowo-naczyniowych. α-LA obniża poziom lipidów we krwi, zapobiega oksydacyjnym modyfikacjom cząsteczki LDL i hamuje tworzenie blaszki miażdżycowej. Jądrowy czynnik transkrypcyjny kappa B (NF-κB) przyczynia się do rozwoju miażdżycy poprzez aktywację genów prozapalnych, natomiast α-LA hamuje odpowiedź zapalną indukowaną przez NF-κB poprzez redukcję stresu oksydacyjnego, hamowanie ekspresji cząsteczek adhezyjnych oraz proliferacji i migracji komórek mięśni gładkich naczyń. Ponadto, suplementacja α-LA może obniżać ciśnienie krwi. W pracy dokonano przeglądu badań dotyczących roli α-LA w patogenezie miażdżycy i chorób sercowo-naczyniowych.
... Because of its antioxidant action, exogenous ALA is utilized as a dietary supplement in significant areas like Europe, the United States, and Japan (Takahashi H, Bungo Y, 2011). Additionally, it has been demonstrated in rats that ALA lessens oxidative stress damage, improves mitochondrial performance, and treats memory impairment by reversing the structural degeneration of the mitochondria that comes with aging (Liu J, Head E, 2002). Multiple sclerosis and diabetic polyneuropathy are only a couple of the neurological disorders that are frequently treated using an antioxidant called alpha-lipoic acid (ALA).Through eating, it enters the body and crosses the blood-brain barrier ( Biewenga GP,1997).It is safe at therapeutic dosages ( Shay KP, Moreau RF,2009). ...
Article
Full-text available
Background: Alpha lipoic acid has both hydrophilic and hydrophobic characteristics and is abundantly distributed in cellular membranes and the cytoplasm. It is among the top cell- protective antioxidants. Material and methods : The present work investigated the possible therapeutic effects of alpha lipoic acid in a male rat model of brain aging induced by D-galactose. Four equal-sized groups of 40 male rats were randomly assigned: G1, the control group, G2, and G3, which each received daily doses of 200 mg/kg of D-gal for 30 days. Alpha lipoic acid was given orally for 30 days to the G4 D-gal + alpha lipoic acid group at 200 mg/kg bw, IP. daily with 100 mg/kg. thirty days of IP. Glutamate is deposited in the brain, according to research on behavioral alterations and brain glutamate. Indicators of oxidative stress are increased Our Results show that whereas brain glutamate deposition declines in the D-gal model of aging, the Forced Swimming Test (FST) and Morris Water Maze Test considerably rise (MWM).
... (2) hydroxyl radical formation, ROS production, and neuroinflammation; (3) impaired amyloid plaque formation; (4) decreased glutathione expression; and (5) impaired neurotransmitter levels [23]. Several cell and animal studies [24][25][26] have demonstrated beneficial effects and even some smaller human intervention studies showed promising results [27][28][29]. ...
Article
Full-text available
Introduction: Alpha lipoic acid (ALA) is a sulphur-containing organic compound, derived from octanoic acid, and an important cofactor for mitochondrial respiratory enzymes. It has strong antioxidant properties that improve mitochondrial function. We investigated if ALA improves mitochondrial dysfunction in a cellular model of Alzheimer's disease (AD). Methods: SH-SY5Y-APP695 cells were used as a model for an early stage of AD. Vector-transfected SH-SY5Y-MOCK cells served as controls. Using these cells, we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, and citrate synthase activity (CS) in cells treated with ALA. Cells were treated for 24 h with different concentrations of ALA and with or without the complex I inhibitor rotenone. Results: Incubation with ALA showed a significant increase in ATP levels in both SH-SY5Y-APP695 and SH-SY5Y-MOCK cells. MMP levels were elevated in SH-SY5Y-MOCK cells, treatment with rotenone showed a reduction in MMP, which could be partly alleviated after incubation with ALA in SH-SY5Y-MOCK cells. ALA treatment showed significant differences in respiration chain complex activities in SH-SY5Y-MOCK cells. Citrate synthase activity was unaffected. ROS levels were significantly lower in both cell lines treated with ALA. Conclusions: ALA increased the activity of the different complexes of the respiratory chain, and consequently enhanced the MMP, leading to increased ATP levels indicating improved mitochondrial function. ALA only marginally protects from additional rotenone-induced mitochondrial stress.
... Instead, a focus mainly on the cellular actions of orally supplied, nonprotein-bound LA will be presented. Pertinent clinical benefits of LA will also be discussed in light of this molecular mechanism (Liu et al., 2002, Shay et al., 2009).Therefore, this study investigated the modulating and antioxidant activity of lipoic acid on renal toxicity induced by cyclosporine A in male albino rats. ...
Article
Full-text available
Cyclosporine A (CsA) is the most widely used immunosuppressive drug for preventing graft rejection and autoimmune disease. However, the therapeutic treatment induces several side effects such as nephrotoxicity, cardiotoxicity and hepatotoxicity. This study aimed to assess the protective role of lipoic acid (LA) on kidney toxicity of male albino rats induced by cyclosporine (CsA). Forty adult male rats were allocated into four groups: Group (I) served as a control group. Group (II); received treatments orally with CsA (25 mg/kg b.w.), daily for 3 weeks. Group III: (Recovery CsA group): treated orally with CsA (25 mg/kg b.w.), daily for 3 weeks, then recovered for another 3 weeks. Group IV (LA and CsA group): received LA (100 mg/kg b. w.) orally 1 h before treatment by CsA (25 mg/kg b. w.) daily for 3 weeks. The results indicated that treatment of CsA caused a significant elevation in the concentrations of serum urea, creatinine, and uric acid which indicate injury to the kidney function. Renal malondialdehyde (MDA) concentration was markedly increased reflecting increased lipid peroxidation, whereas, reduced glutathione (GSH) and superoxide dismutase (SOD) were significantly decreased. On the other hand, LA plus CsA dose-dependently inhibited activities of serum urea, creatinine, and uric acid. The administration of LA plus CsA exhibited significant reduction in lipid peroxidation while GSH content and SOD activity were enhanced significantly which reflect an improvement in renal toxicity. In conclusion, the results indicated a negative role of CsA on kidney function and oxidative stress in induction toxicity, suggested Thus, Lipoic acid play a positive role on toxicity of kidney induced by cyclosporine A.
... In addition, carnitine-fed rats showed improved brain function [79]. Carnitine also affects cognitive function in the human brain. ...
Article
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Although facial nerve palsy is not a life-threatening disease, facial asymmetry affects interpersonal relationships, causes psychological stress, and devastates human life. The treatment and rehabilitation of facial paralysis has many socio-economic costs. Therefore, in cases of facial paralysis, it is necessary to identify the cause and provide the best treatment. However, until now, complete recovery has been difficult regardless of the treatment used in cases of complete paralysis of unknown cause and cutting injury of the facial nerve due to disease or accident. Therefore, this article aims to contribute to the future treatment of facial paralysis by reviewing studies on drugs that aid in nerve regeneration after peripheral nerve damage.
... In vivo, ALA is reduced to dihydro-LA (DHLA), which neutralizes ROS, chelates metal ions and promotes regeneration of endogenous antioxidants such as glutathione (GSH), vitamin E, and C [18]. Moreover, it is reported to inhibit the release of pro-inflammatory cytokines as well as improve cognitive function and reduce brain-oxidative stress and helped in the treatment of neurodegenerative diseases [19,20]. Moreover, it has been reported that ALA affected the glucose and lipid metabolism [21], regulated the appetite [22], and combated complications of diabetic origin [23] and myocardial and cerebral reperfusion injuries [24]. ...
Article
Full-text available
Background Reserpine is a monoamine depletory drug cause oxidative damage and used to induce depression-like features in rodent model. Paroxetine is an antidepressant drug that exerts its effects by inhibiting dopaminergic neurons although it may exert much pathological damage. Alpha-lipoic acid (ALA) is an endogenous antioxidant co-factor of important enzymatic complexes. The present study was aimed to elucidate the possible protective effect of ALA in the improvement of the deleterious cerebral cortex injury after reserpine and paroxetine treatment. Forty adult male albino rats were equally divided into 5 groups. Group I served as control group orally treated with saline solution all the experiment period. Group II animals orally treated with ALA (200 mg/kg/day) for six weeks. The induction of depression-like features occurred when the rest of animals were intraperitoneally treated with 25 mg/kg of reserpine once daily for consecutive 14 day. Then these animals were divided into; Group III (reserpine group) animals in this group were sacrificed on 15th day. Group IV; reserpine-treated animals were treated with paroxetine (20 mg/kg) daily for 6 weeks. Group V, animals in this group were received paroxetine and ALA daily for 6 weeks. Results Reserpine-treated rats showed disorganized layers of cerebral cortex with degenerative, apoptotic and necrotic changes. Ultrastructure changes include both pyramidal and granule cells with severe degenerative, necrotic and apoptotic features. The nuclei appeared pyknotic; irregular with chromatin condensation as well as the cytoplasm of these cells contained many degenerated organelles. In addition, a significant increase in total oxidative stress and decrease in total antioxidant capacity, norepinephrine, dopamine and serotonin levels were recorded. The same treatment showed significant decrease in proliferating cell nuclear antigen (PCNA) expression and significant increase in caspase-3 expression in the granule and pyramidal cells. After paroxetine-treatment these parameters were more or less similar to those observed in reserpine-treated ones. While an obvious improvement was appeared when animal treated with both paroxetine and ALA and; all parameters restored its normal features. Conclusions This study concluded that; ALA treatment attenuated the cerebral injury induced by reserpine and improved the effects of paroxetine in rats due to its anti-inflammatory, anti-apoptotic and antioxidant activities.
Article
This study was designed to explore the effects of valproic acid (VPA) on spatial and passive avoidance learning and memory as well as to assess the protective effects of L-Carnitine (LC) against VPA-induced memory deficit in the rat. Male Wistar rats received VPA (300 mg/kg/daily by i.p. injection), or LC (50 mg/kg/ daily by i.p. injection), or co-treatment with VPA and LC for 28 days. Following 28 days, Elevated Plus-Maze (EPM), Morris Water Maze (MWM), and Passive Avoidance Learning (PAL) tasks were used to evaluate the anxiety-like behavior and spatial and passive learning and memory, respectively. Our results showed that VPA has no effect on memory acquisition (in both MWM and PAL) but induced reference memory impairment. We demonstrated that treatment with LC partially ameliorated the impairment in the retrieval of reference memory and passive avoidance learning. Moreover, VPA increased anxiety-like behavior, which was partially reversed by the administration of LC. In conclusion, these results show that LC is effective in counteracting the anxiety-like behavior and reference memory impairment caused by VPA. Therefore, LC may serve as a possible therapeutic agent for VPA-induced memory change.
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Mitochondrial function and ambulatory activity were monitored after feeding old rats acetyl-l-carnitine (ALCAR). Young (3–5 mo) and old (22–28 mo) rats were given a 1.5% (wt/vol) solution of ALCAR in their drinking water for 1 mo, were sacrificed, and their liver parenchymal cells were isolated. ALCAR supplementation significantly reverses the age-associated decline of mitochondrial membrane potential, as assessed by rhodamine 123 staining. Cardiolipin, which declines significantly with age, is also restored. ALCAR increases cellular oxygen consumption, which declines with age, to the level of young rats. However, the oxidant production per oxygen consumed, as measured by 2′,7′-dichlorofluorescin fluorescence levels, is ≈30% higher than in untreated old rats. Cellular glutathione and ascorbate levels were nearly 30% and 50% lower, respectively, in cells from ALCAR-supplemented old rats than in untreated old rats, further indicating that ALCAR supplementation might increase oxidative stress. Ambulatory activity in young and old rats was quantified as a general measure of metabolic activity. Ambulatory activity, defined as mean total distance traveled, in old rats is almost 3-fold lower than in young animals. ALCAR supplementation increases ambulatory activity significantly in both young and old rats, with the increase being larger in old rats. Thus, ALCAR supplementation to old rats markedly reverses the age-associated decline in many indices of mitochondrial function and general metabolic activity, but may increase oxidative stress.
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Aging is the accumulation of changes that increase the risk of death. The major contributors after age 28 years are the endogenous chemical reactions that, collectively, produce aging changes that exponentially increase the chances for disease and death with age. These reactions constitute the 'inborn aging process.' This process is the major risk factor for disease and death of the 98% to 99% of cohorts still alive at age 28 in developed countries, where living conditions are now near optimum. The Free Radical Theory of aging (FRTA) and, simultaneously, the discovery of the ubiquitous, important involvement of endogenous free radical reactions in the metabolism of biologic systems, arose in 1954 from a consideration aging phenomena from the premise that a single common process, modifiable by genetic and environmental factors, was responsible for the aging and death of all living things. The FRTA postulates that the single common process is the initiation of free radical reactions. These reactions, however initiated, could be responsible for the progressive deterioration of biologic systems with time because of their inherent ability to produce random change. The theory was extended in 1972 with the suggestion that the life span was largely determined by the rate of free radical damage to the mitochondria. The FRTA suggests the possibility that measures to decrease the rate of initiation and/or the chain length of free radical reactions may, at least in some cases, decrease the rate of reactions that produce aging changes without significantly depressing those involved in maintenance and function. Many studies support this possibility. Applications of the FRTA have been fruitful. For example, it is a useful guide to efforts to increase the life span, and it provides plausible explanations for the aging phenomenon (e.g., the association of disease with age as well as insight into pathogenesis; the gender gap; the association between events in early life and late onset disease; and the shortening of telomeres with cell division). Further, it is reasonable to expect on the basis of animal and epidemiologic studies that the increasing population-wide use of antioxidant supplements and ingestion of foods high in antioxidant capacity over the past 40 years have helped to increase the functional life span of the population by contributing significantly to the decline in 'free radical' diseases, to increases in the fraction of elderly, and to the decline in chronic disability in this group.
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In Alzheimer and Parkinson diseases, oxidative alterations, affecting lipids, proteins, and DNA, have been described. Using an in situ approach to identify 8-hydroxyguanosine, an oxidized nucleoside, we recently identified RNA as a major target of oxidation in Alzheimer and Parkinson diseases as well as Down syndrome, where premature Alzheimer-like neuropathology is invariably found. RNA oxidation is localized to the neuronal populations potentially affected in these diseases. Together with the known mitochondrial dysfunction in Alzheimer and Parkinson diseases, the cytoplasmic predominance of neuronal 8-hydroxyguanosine supports mitochondria as the most likely source of reactive oxygen responsible for RNA oxidation. The consequence of oxidatively damaged RNA is not fully understood; however, the potential of oxidized RNA to cause errors in translation indicates a metabolic abnormality in neurodegenerative diseases.
Article
: The oxidative modification of proteins by reactive species, especially reactive oxygen species, is implicated in the etiology or progression of a panoply of disorders and diseases. These reactive species form through a large number of physiological and non-physiological reactions. An increase in the rate of their production or a decrease in their rate of scavenging will increase the oxidative modification of cellular molecules, including proteins. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation, and a decrease in the efficiency of proteolysis will cause an increase in the cellular content of oxidatively modified proteins. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, most notably during aging. Accumulation of modified proteins disrupts cellular function either by loss of catalytic and structural integrity or by interruption of regulatory pathways.
Article
The aging laboratory animal is recognized as a suitable experimental model for the investigation on drugs potentially able to retard the age-dependent decline in cognitive functions. There is robust evidence that levocarnitine acetyl (ALCAR), the acetyl derivative of carnitine, when administered chronically, prevents some age-related deficits of the central nervous system, mainly at the hippocampal level. On the basis of this evidence and because learning of active avoidance was demonstrated to become impaired with age, we decided to investigate the effect of ALCAR in rats. For statistical evaluation of results, the Cluster Analysis technique was chosen. This procedure pointed out the great heterogeneity of the old population and allowed the classification of the animals into homogeneous groups according to their response pattern. The effect of ALCAR was evident in the higher number of treated old animals yielding escape responses, indicating that ALCAR can preserve, at least partially, learning and memory from the natural decay occurring with age.
Article
Although protein synthesis inhibition has been shown to affect long-term memory in a wide variety of animal species, cases have been reported in which protein synthesis inhibition failed to affect memory consolidation [S. Wittstock, R. Menzel, Color learning and memory in honey bees are not affected by protein synthesis inhibition, Behav. Neural Biol., 62 (1994) 224–229.]. Most findings argue that the critical time for protein synthesis is during or immediately after training. However, other reports show a second time window, hours after training, where protein synthesis inhibition can cause amnesia [F.M. Freeman, S.P.R. Rose, A.B. Scholey, Two time windows of anisomycin-induced amnesia for passive avoidance training in the day-old chick, Neurobiol. Learn. Mem., 63 (1995) 291–295.][G. Grecksch, H. Matthies, Two sensitive periods for the amnesic effect of anisomycin, Pharmacol. Biochem. Behav., 12 (1980) 663–665.]. In this study, we addressed two questions: (1) Is protein synthesis essential for spatial memory? and (2) At what injection time window(s) will protein synthesis inhibition cause spatial memory amnesia? We report that bilateral intraventricular microinjection of anisomycin (Ani) impairs consolidation of long-term memory, in the hippocampal-dependent Morris water maze spatial memory task. Memory was impaired in a dose-dependent manner without affecting short-term memory. Spatial memory was affected only if Ani was injected 20 min before performing the task and not in any other time window before or after the behavioral test. The inhibition did not affect pre-existing memories or the capability to memorize once the effect of the inhibition diminished.