This study examines for the first time the qualitative and quantitative pattern of mitochondria in hippocampal neurons affected by age and demonstrates the restoration of mitochondrial ultrastructure by the dietary supplementation of ALCAR + LA. Perhaps even more important is the observation that dietary supplementation stimulated mitochondrial proliferation in young animals, suggesting early interventions are important in fighting age-related decline.
An accumulating body of evidence strongly suggests that mitochondrial decay plays a key role in aging and age-associated neurodegenerative disease, such as AD [14
]. Mutant mitochondrial DNA is implicated in impairing cognitive functioning [32
] and cellular energy production, disrupting the normal connections between brain cells and increasing the generation of oxidants [33
]. Further, beta-amyloid-binding alcohol dehydrogenase is a direct molecular link from amyloid β to mitochondrial toxicity [34
]. A recent study of extending mouse lifespan with overexpression of catalase in mitochondria provides strong support to the oxidative mitochondrial decay theory of aging [35
] and age-associated diseases, including mice that mimic human AD [17
]. However, no detailed quantitative electron microscopic study examines rat brain mitochondrial changes with age.
In the present study, the data indicate a significant decrease in the number and percentage of intact mitochondria and a significant increase in the number and percentage of damaged mitochondria (mitochondria with broken cristae, vacuoles and vacuolar lipofuscin) in aged hippocampal neurons. Neuronal damage is characterized by an increase in the number of vacuolar lipofuscin structures as well as the number of electron-dense and giant mitochondria, lysosomes, lipofuscin deposits, mitochondrial matrix oedema, internal and external membrane breaks and a decrease in the density of microtubules per neurons [36
It is important to note that the tissues in our present study were fixed with paraformaldehyde. Moreover, previous studies also demonstrate the consistency of optimal tissue preservation by this technique and the consequent EM images [17
]. In addition, our unpublished data from these experiments show that mitochondrial DNA overproliferation and/or deletion were dependent on mitochondrial structural damage seen in hippocampal neurons. Vibratome sections from each brain were used for the in situ
hybridization study to compare the spectrum of mitochondrial structural and DNA damage and/or prevention by using selective mitochondrial antioxidants, consequently greatly reducing the risk of potential artifacts.
Feeding ALCAR + LA ameliorated the age-associated neuronal damage evident by marked decreases of vacuoles and lipofuscin as well as decreases in partially and completely damaged mitochondria. In our preliminary study using in situ hybridization of mitochondrial DNA, we have found that the main source (almost 100%) of lipofuscin formation appeared to be mitochondria because ‘young’ lipofuscin contains mtDNA positive signals. Well-developed granular and agranular endoplasmic reticulum and free ribosomes (or polysomes) are present in the matrix of cell bodies, which were often comparable to the ultrastructure of hippocampal neurons in young rat brains. In addition, ALCAR + LA dietary supplementation also increased the percentage of area covered by intact mitochondria not only in old groups but also in young treated animals, suggesting a much more significant effect of the ALCAR + LA dietary supplementation can be achieved if treatment is started at an early age.
Our observations support recent meta-analysis of 21 double blind clinical trials of ALCAR in the treatment of mild cognitive impairment and mild AD that showed modest but significant efficacy of ALCAR [37
]. A meta-analysis of four clinical trials of LA for treatment of neuropathic deficits in diabetes showed modest efficacy [37
]. In addition, immune function decline with age has been extensively documented in human beings [38
] and in rodents [40
]. The deleterious effects of oxidants on immune cells such as T and B lymphocytes were ameliorated by ALCAR treatment in aged animals [42
]. Moreover, Franceschi and colleagues demonstrated that ALCAR treatment increased phyto-haemagglutinin-induced peripheral blood lymphocyte proliferation in young and old subjects [44
] and LA, as an antioxidant, may have protective effects on immune cells leading to improved immune system functioning in general.
A recent study by McMackin and coworkers in a double-blind crossover study showed the effect of combined ALCAR + LA on vasodilator function and blood pressure in patients with coronary artery disease [45
]. Active treatment increased brachial artery diameter and had a significant effect on individuals with either elevated blood pressure or metabolic syndrome [45
]. Recent studies report protective effects of LA on endothelial function and the reduction of markers of inflammation in metabolic syndrome and in patients with diabetic syndrome, albuminuria, and symptomatic diabetic polyneuropathy as well as in aging rats. [27
]. Metabolic function as a possible key factor in the mitochondrial decay seen in aging and micronutrient deficiency has been discussed [49
]. Future studies are needed to determine in more detail the potential protective effect of ALCAR + LA treatment in different experimental models such as stroke and cerebrovascular disease.
We previously reported preliminary qualitative electron microscopic observations of age-associated mitochondrial morphological decay and the positive effects of ALCAR and/or LA in old rat brain [22
]. 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 preservation of cristae and less structural disruption compared to controls. Formation or accumulation of lipofuscin granules in the cytoplasmic matrix of hippocampal neurons and other AD brain cell types [15
] and rodent brain samples demonstrating AD-like pathology [13
] appears to be a feature of mitochondrial damage associated with age and especially disease. As a major source of reactive oxygen species, mitochondria are particularly vulnerable to oxidative stress [15
]. A recent morphometric study found a significant reduction in intact mitochondria in different cellular compartments of AD and AD-like rodent brain [13
] and other cells (e.g.
fibroblasts) obtained from patients with AD [53
]. In addition, old rats had more lipofuscin in the cytoplasm of granular cells of the dentate gyrus, and the combined-treatment rats appeared to have less lipofuscin accumulation.
In the present study, we replicated our previous experiment with more animals for quantitative analysis of mitochondria in the hippocampus. The morphological observations clearly show that old rat brain is characterized by age-associated increases in the number of electron-dense and giant mitochondria, mitochondrial oedema, external membrane breaks, lysosomes, lipofuscin formation and decreases in the density of microtubules. Golgi, granular and agranular endoplasmic reticulum and microvessels structures display abnormalities. Treatment with ALCAR + LA for 3 months ameliorated the age-associated neuronal damage and increased proliferation of intact mitochondria in hippocampal neurons in the young animals.
Mitochondria are essential to the functions of neurons because their limited glycolytic capacity makes them highly dependent on aerobic oxidative phosphorylation. We and others demonstrated that oxidative stress is one of the earliest events in susceptible neurons and vascular endothelium in AD [13
] and mitochondrial dysfunction is implicated in the associated increased oxidative stress. Among other mitochondrial abnormalities, we found that the levels of mitochondrial DNA and cytochrome oxidase-1 in susceptible neurons and vascular endothelial cells are significantly increased compared with those of age-matched controls, even though the number of mitochondria per neuron is significantly decreased [15
]. The present results confirm our previous qualitative observation in aged rats and provide not only quantitative morphological confirmation of mitochondrial decay, but also suggest that the ameliorating effect of ALCAR + LA on age-associated activity and memory decline [20
] is a result of repairing mitochondrial structure, thereby restoring mitochondrial function in aged animal brain. A preventive effect of ALCAR + LA supplementation was seen in all brain cellular compartments including microvascular systems, indicating a systemic effect of the treatment.
Our results demonstrate for the first time that ultrastructural morphometric analysis provides criteria for documenting mitochondrial damage that increases with aging. Treatment with ALCAR + LA restores intact mitochondrial morphology normally degraded with age. Moreover, quantitative electron microscopic observation for the first time demonstrated that with ALCAR + LA supplementation neuronal mitochondria from young treated animals show better intact morphology compared to young non-treated controls. Further investigation on the effect of ALCAR + LA supplementation by using electron microscopic quantitative/qualitative study will provide new information for the current model of aging as well as neurodegenerative diseases such as AD where mitochondrial damage and energy failure appear to be a primary step in the development of cognitive impairment or brain pathology such as amyloid beta deposition, a hallmark for AD. In conclusion, old rats showed morphological mitochondrial decay and dietary supplementation with ALCAR + LA restored mitochondrial morphology and prevented decay. Further, a key observation of this study is the proliferation of intact mitochondria in young treated rat brain neurons. These results support the role of mitochondrial decay as an important factor in aging. Further, dietary supplementation with selective mitochondrial antioxidants and metabolites such as ALCAR and LA may be an effective strategy for delaying aging, as well as neurodegeneration such as AD, where mitochondrial damage appears to be a primary target before the development of any amyloid deposition or cognitive impairment.