L-Carnitine
Memory loss in old rats is associated with brain
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 RΑ-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.
Memory, i.e., performance on
memory tasks, declines with age in animals. In the case of age-related
human neurodegenerative diseases, such as Alzheimer's disease (AD),
the deficit can be severe. 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.
RΑ-lipoic acid (LA) is a coenzyme that is involved in
carbohydrate utilization necessary for the production of ATP in
mitochondria; it is reduced in mitochondria to dihydrolipoic acid
(DHLA), a potent antioxidant. LA improves long-term memory in
aged female NMRI mice.
L-Carnitine is a betaine required for the transport of
long-chain fatty acids into the mitochondria for Β-oxidation, ATP
production, and for the removal of excess short- and medium-chain fatty
acids. 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. ALCAR improves cognitive function and neuronal
bioenergetic mechanisms in rats with both acute and long-term
treatments.
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. LA administration in patients with AD for approximately 1 year
also resulted in mild cognitive improvements and stabilization of
global neuropsychological test scores. 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, the importance of improving mitochondrial function by dietary interventions of mitochondrial metabolites such as
ALCAR or LA becomes clearer. Feeding 0.15-0.5% ALCAR to old
rats elevated the levels of carnitine in plasma and brain to that of
young rats 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. 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 & 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% (wt/vol) ALCAR in MilliQ water (pH was
adjusted to 5.2 with 1 N NaOH), 0.2% or 0.1% (wt/wt) 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 g/day; 18.6 ± 1.19 ml/day; 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 g/day; 18.4 ± 0.9 ml/day; body weight from 416.0 ± 19.0 to 414.9 ± 9.4 g; mean ± SE). All animals were 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 and/or 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. 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 temporal memory. 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 0.6-0.9-µm 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 treatment condition was anesthetized with
ether and perfused with 4% paraformaldehyde for 1.5-2 h. The brain
was removed and postfixed for preparing paraffin sections. Sections of
hippocampus were incubated with monoclonal anti-8-hydroxy-2'-deoxyguanosine/8-hydroxyguanosine (oxo8dG/oxo8G; 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/µl of
RNase-free DNase I or 10 mg/ml of DNase-free RNase (Roche Molecular
Biochemicals) for 3 h prior to incubation with oxo8dG/oxo8G Ab. To quantify the extent of oxo8G/oxo8dG immunolabeling, a
525 × 410 µm 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 8100/80AV. All sections from a given region were
captured sequentially during one session and were analyzed blind with
respect to treatment condition. Subsequently, public domain 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 percentage of area
occupied by oxo8G/oxo8dG 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 and swimming distance traveled (data
not shown) were measured and gave similar results. 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.
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
subcomponent of spatial memory and the combination was even more
effective.
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. All three supplementation groups
showed improvement, but only the combination treatment group reached
statistical significance.
Temporal Memory.
The response rate to a sound and to a light 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 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. Compared 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 and/or 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 combined treatment rats also seemed to have less
lipofuscin. However, 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 (oxo8dG or oxo8G;). Fig. 3A shows representative images
of the CA1 region of the hippocampus. Fig. 3C shows that old
rats without treatment showed significantly higher immunoreactivity
than young rats in areas CA1, CA4, cerebral cortex, and in the white
matter. Both ALCAR and LA reduced immunoreactivity, 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
immunoreactivity, 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 immunoreactivity.
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 hippocampal function.
Temporal memory may also be associated with the hippocampus, although
it may be more closely associated with the striatum and cerebellum. The
dietary administration of a combination of ALCAR and LA to old rats
improves mitochondrial function in liver. 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 cognitive deficits in spatial
memory in lesion studies and in aging. Old rats
showed decreased spatial memory compared with young rats; ALCAR
and/or 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.
ALCAR and/or LA significantly restored performance in this test, the
combination being more effective (P < 0.01) and not
significantly different from that of young rats.
Schenk and Morris 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 effects on the visible platform test as
those observed during the hidden platform tests.
Many physiological changes occur with age and can have major
consequences on cognitive performance. We observed 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 including sensory systems such as hearing. Therefore, improvements shown here in test performances
attributable to ALCAR, LA, or their combination, including the visible
platform test and ambulatory activity, 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, measures 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 procedure, which resembles a
discrete-trials fixed-interval schedule with catch trials; it has been
used to study the timing abilities of animals. Several studies have shown that old rats have deficits in time perception. 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.
The current study also has tested the hypothesis that cognitive
improvements in response to ALCAR and/or 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. 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. 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
mtDNA/RNA. 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 mitochondria, the most likely oxidant
target and the one that is improved by ALCAR and/or 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 compromising
protein synthesis critical for the formation of new memories.
Although oxidative damage to RNA has been shown to be more extensive
than damage to DNA in urine and plasma, 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, Down's
syndrome, and Parkinson's disease, all of which are diseases
associated with severe cognitive deficits. 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.
The improving effects on performance on memory tasks by ALCAR and/or
LA on hippocampal mitochondria are supported by morphological observations. There seems to be a loss of mitochondrial cristae with
age. Evidence that ALCAR reversed this loss with a dose-dependent response has been presented. Similar to ALCAR, LA also reduced
age-dependent cristae loss in the dentate granule cells of the
hippocampus. Because ALCAR alone showed a virtually complete 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
effectiveness of the mitochondrial metabolites ALCAR and LA suggests
that mitochondrial decay is involved. (ii) The oxidation of
RNA/DNA in neurons is likely to be mitochondrial.
(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 acetyltransferase and
carnitine acetyltransferase. Low acetylcholine levels in
certain brain regions are associated with age-related cognitive
dysfunction, including AD. Because of the profound effects of
calorie restriction, we have compared dietary intakes carefully and
have found no significant differences in food and water consumption or
in body weight (see Materials and Methods).
In conclusion, feeding old rats ALCAR and/or 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 intervention in humans
for delaying brain aging and age-associated neurodegenerative diseases.
Credits
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;
§ Department of Biochemistry and Biophysics, Linus Pauling
Institute, Oregon State University, Corvallis, OR 97331
