The Oxidative Stress Induced Mitochondrial Failure and Cellular Hypoperfusion in the Context of Alzheimer Disease: Past, Present and Future.

Gjumrakch Aliev, MD&PhD

President and CEO:

“GALLY” International Biomedical Research Consulting LLC., San Antonio, TX, USA

and

Professor of Cardiovascular, Neuropathology and Gerontology:

University of Atlanta, Atlanta, GA, USA

SCIENTIFIC PROBLEM:

Stroke and arteriosclerosis with neurological consequences such as Alzheimer disease (AD) are two leading causes of age-associated disability, dementia, and death. AD is now the sixth-leading cause of death in the United States. In the US, AD is estimated to affect 4 million people (rising steeply from <1% of the population aged 65 to 40% of those aged 90) and costs $600 billion per year, which is equivalent to the total cost of stroke, heart disease, and cancer combined. Overall, there are no effective strategies for determining and controlling this devastating disease.

Neurodegenerative disorders are characterized by a loss of cognitive function and inappropriate death of nerve cells in areas of the brain that control such functions as memory and language. The trigger for nerve cell death is unknown in AD, as well as in other neurodegenerative conditions, in which memory decline is a prominent feature.

A rapidly growing body of evidence indicates that increased oxidative stress resulting from reactive oxygen radicals is associated with the aging process and age-related degenerative disorders such as atherosclerosis, ischemia/reperfusion, arthritis, stroke, and neurodegenerative diseases. Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels they can function as signaling intermediates in the regulation of fundamental cell activities such as growth and adaptation responses. At higher concentrations, ROS can cause cell injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating blood to cells and tissue, is a major target of oxidative stress, playing a critical role in the pathophysiology of vascular diseases. Since the vascular endothelium, neurons and glia are all able to synthesize, store and release ROS and vascular active substances in response to certain stimuli, their contribution to the pathophysiology of atherosclerosis, stroke, other non-atherosclerotic cerebrovascular disease and neurodegenerative syndrome such as mild cognitive impairment (MCI) and AD is extremely important. In addition, abnormalities in cholesterol metabolism, oxidative stress and vascular lesions are important factors in the pathogenesis of late -onset forms of AD, forms of mental retardation, stroke and MCI. This idea is based on the positive correlations found between stroke, MCI, AD and cardiovascular diseases. New evidence indicates that continuous formation of free ROS induces cellular damage and decreases antioxidant defenses. Specifically, oxidative stress increases vascular endothelial permeability and promotes leukocyte adhesion, all of which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors. We theorize that the cellular and molecular mechanisms, by which cholesterol metabolism abnormalities induce the formation of large amounts of ROS, decrease endothelial barrier function via the overexpression of inducible nitric oxide synthase (iNOS) and promote leukocyte adhesion. Chronic injury stimuli has the action of inducing decompensation and or alterations in normal vascular function, which results in the development of cerebrovascular arterio- and atherosclerosis that further manifest as stroke, MCI and/or AD.

Our study compiled within this lecture highlight the most recent insights into the molecular mechanisms involved in the earliest stages of AD pathogenesis, namely that of oxidative stress-induced metabolic abnormalities, mitochondrial failure which initiates the energy crisis and vascular and cellular hypoperfusion, and lays out future potential treatment strategies based on these findings. Also discussed are the mutual pathogenic processes and antecedent biological markers shared between AD and other cardiovascular, cerebrovascular, and neurodegenerative diseases. Elucidating the commonalities between these diseases may well provide the necessary key to conquering AD.

BACKGROUND:

It is widely accepted that during neuronal energy crisis, cerebral hypometabolism and vascular hypoperfusion are major and potentially treatable contributors to the loss of function in patients with stroke as well as Alzheimer disease (AD). Based on our previous and current research, we have found that the mitochondria of the brain cellular compartments (neurons, glia, microglia and vessels wall cells) are a primary target of brain damage due to their high energy demand and susceptibility to oxidation, leading to energy failure, and resulting in cognitive impairment and memory decline. Moreover, mounting evidence indicates that targeting mitochondria with antioxidants and metabolites is a powerful treatment capable of restoring cell integrity and eliminating damage in the brain, resulting in significantly restored cognitive function and spatial memory. In addition, we have recently found an unexpected capacity for gene expression modification supplement (Aminocare A10) to retard and slow down the general aging process via gene expression modification and aid in the production of energy in the process. To achieve the goal of preventing brain dysfunction, we hypothesize that selective mitochondrial antioxidants (Acetyl -L- Carnitine and R-a-Lipoic acid (ALCAR+LA), supplement that is able to retard and slow down the general aging process via gene expression modification (Aminocare-A10) plus brain longevity substances [Aminocare Brain Longevity Forte (BLF)- as a supplement for brain aging that slow down of AD and cognitive decline] can be used as a new and more effective therapeutic approach in the treatment of stroke and AD patients.

GOAL:

We have determined the cellular and subcellular features of vascular lesions and mitochondria i n brain vascular wall cells as well as neurons from human AD brain biopsies, human short postmortem brain tissues, rat model of 2 vessel occlusion (2-VO), yeast artificial chromosome (YAC), and C57B6/SJL transgenic positive (Tg+) mice overexpressing amyloid beta precursor protein (AßPP). We expand our models towards the E4 isoform of apolipoprotein E (ApoE) which is involved in cardiovascular and cerebrovascular disorders and is the most prevalent risk factor for late onset of sporadic AD. Moreover, ApoE4 transgenic (Tg+) mice are appropriate models for studying the pathogenesis and preclinical treatment of ApoE-related cognitive deficits associated with late onset and sporadic AD. We have also determined the potential therapeutic effects of using a combination of a selective mitochondrial antioxidant plus A10 and BLF both in combination with our recently developed brain exercise program in patients with stroke and AD (moderate and severe AD symptoms).

METHODS:

In situ hybridization, using mitochondrial DNA (mtDNA) probes for human wild type, 5kb deleted and mouse mtDNA, was performed in conjunction with immunocytochemistry using antibodies against AßPP,

8-hydroxyguanosine, all three isoforms of nitric oxide synthase (neuronal, inducible and endothelial NOS), GRK-2 and cytochrome c oxidase. We have also measured age-dependent effects of the human ApoE4 on cerebral blood flow (CBF) using ApoE4 transgenic mice compared to age-matched wild-type (WT) mice by use of [14C] iodoantipyrene autoradiography. Spatial memory and temporal memory tests

were also employed to determine the potential protective effects of ALCAR+LA as a selective mitochondrial antioxidants treatment. Our animal study applies the vascular dementia paradigm to ApoE4

Tg+ mice in order to analyze the effects of the selective mitochondrial antioxidants ALCAR+LA on cerebral blood flow (CBF), neuropathology, brain and vessel ultrastructural abnormalities and behavior. Moreover, these studies can apply to the brain hypometabolism and mitochondrial failure paradigm to stroke and AD patients in order to analyze cognitive function in patients who receive ALCAR+LA, Omega-3-6-9 Fish, Flax, Borage oil as well as Coenzyme Q-10 and A10+BLF, along with diet changes in combination with our recently developed brain activation program (a home-based protocol involving mild physical exercise and cognitive training). The average age of the patients was 72. The patients were evaluated at baseline and in 5 years post treatment.

RESULTS:

A significant higher degree of mitochondrial damage was found in neurons and cerebrovascular cell walls in AD and in animal models used when compared to age-matched controls and non-treated subjects. These abnormalities coexist with the over expression of GRK-2, AßPP and inducible NOS immunoreactivity in these cells, and closely related to amyloid deposition in the same regions. They were also characterized by the presence of large, lipid-laden vacuoles in the cell body of severely damaged neurons and cytoplasmic matrix of the vascular endothelium. In situ hybridization revealed deleted mtDNA positive signals in the damaged mitochondria of neurons, vascular endothelium and perivascular cells. Moreover, brain microvessels with atherosclerotic lesions revealed endothelium and perivascular cells, which stained positively and in clusters when probed with wild and deleted mtDNA probes. These mtDNA deletions were associated with increased amounts of immunoreactive GRK-2, AßPP, 8OHG, and COX in the same cellular and subcellular compartments. Moreover, GRK overexpression appeared to be a selective hallmark for mitochondrial damage at the earlier but not late stages of neuronal and other brain cellular compartment lesions. ApoE4 associated factors reduced the CBF gradually and created brain hypoperfusion when compared to the WT and the differences in CBF were greatest as animals aged from 6 weeks to 12 months. Transmission electron microscopy (TEM) with colloidal gold immunocytochemistry and in situ hybridization using human and mouse DNA probes showed structural damage and mitochondrial DNA overproliferation and/or deletion in the young and aged microvessels endothelium of ApoE4 animals, extending to the cytoplasm of perivascular cells, perivascular nerve terminals, hippocampal neurons, and glial cells. These blood flow changes associates with severe structural lesions in young and aged microvessels endothelium of ApoE4 animals extended to the cytoplasmic matrix of perivascular cells, perivascular nerve terminals and hippocampal neurons and glial cells in the damaged regions of the brain. Moreover, mitochondrial structural alterations coexist with mitochondrial DNA overproliferation and/or deletion in all brain cellular compartments. Most likely, further development of these alterations can lead t o blood brain barrier (BBB) failure and breakage during the development of AD. In contrary to this observation, the animals that received selective mitochondrial antioxidants (ALCAR+LA) treatment showed an absence of any cellular or subcellular abnormality in brain cellular compartments. Spatial and temporal memory tests showed a trend in improving cognitive function in ApoE4 Tg+ mice that were fed with the selective mitochondrial antioxidants (ALCAR+LA).

Our clinical results showed that patients who received ALCAR+LA, Omega-3-6-9 Fish, Flax, Borage oil as well as Coenzyme Q-10 and A10+BLF, along with diet changes in combination with our recently developed brain activation program exhibited the maximum cognitive improvement at the end of 60 months of treatment. The maximum cognitive improvement was seen with the combined treatment in MMSE, attention, memory, naming, construction, clock drawing, verbal fluency, and Ruff Frontal Fluency tests. By the end of 12 months of treatment, significant improvement was observed, especially in attention, construction and clock drawing, when patients received the combined treatment. In addition,

this group also showed that in all categories there were no signs of a decrease and/or decline below the base line for the entire period of treatment (5 years).

CONCLUSION:

Our conclusion is that for the first time we were able to demonstrate the potential pharmacologic modulation of brain hypometabolism and therefore the cognitive improvements by using combination of selective mitochondrial antioxidants/metabolites, a gene expression modification substance (A10) and supplement for brain aging (BLF) with diet changes and brain exercise training. This represents a completely new and more effective strategy to treat stroke, Alzheimer and/or other types of dementia. Moreover, further increase in the examination of the ultrastructural degeneration caused by aging, especially under cardio- and cerebrovascular disease complications, is likely to contribute to our understanding of neurodegenerative etiology and will indicate a new avenue for the development of novel prophylactic and treatment strategies by offering selective mitochondrial antioxidants like ALCAR+LA and gene expression modification substrate (A10) and brain aging supplement (BLF) to the stroke, AD and/or other demented patients.

LEARNING OBJECTIVE: Project 1:

(1) Determine the effect of aging in animals genetically lacking LDL receptor and ApoE gene as a model of CBH on regional cerebral blood flow (rCBF), and functional state of the hippocampal endothelium and neighboring neuronal tissue.

(2) Investigate the effect of additional cholesterol supplementation (1, 2, and 3 months after feeding) and chronic vessel occlusion (2vessel occlusion, 2-VO) in an animal with genetic dyslipidaemia or ApoE gene lacking on rCBF, amyloid production and activities of NOS isoforms and EC markers.

(3) Evaluate the effect of selective pharmacological inhibition of iNOS, nNOS, and eNOS after cholesterol supplementation with additional 2-vessel occlusion at 1, 2, and 3 months before and after the administration of the hydroxymethylglutaryl coenzyme A reductase inhibitors atorvastatin and simvastatin, of the NOS donor S-nitroso-N-acetylpenicillamine (SNAP), the angiotensin II type 1- receptor blocker candesartan, A10 and BLF on hippocampal Aβ accumulation and on oxidative stress markers and rCBF.

Project 2:

(1) To determine how nicotine exposure and hypoxia/reperfusion independently affect CBF and Spatial Memory in ApoE transgenic (Tg+) mice before any amyloid deposition and/or any pathology is accountable.

(2) Investigate the cellular, subcellular and biochemical pattern of the changes in NOS, ET -1, and nAChR

activity in the brain of the mouse overexpressing ApoE after the nicotine or hypoxia/reperfusion exposure.

(3) Determine the biochemical, cellular, and subcellular features of the pattern on the oxidative stress markers, RNA oxidation, mitochondrial abnormalities (mtDNA overproliferation and/or deletion, mitochondrial enzyme activity, and structural changes) and nAChR receptors activity in ApoE Tg+ mice brain after nicotine treatment or hypoxia/reperfusion exposure.

(4) Determine the functional, biochemical, and structural features of the changes in NOS, ET -1, nAChR activity, mitochondrial pathology, and oxidative stress markers when ApoE Tg+ mice are exposed to chronic nicotine treatment or hypoxia/reperfusion with additional drug treatment (A10+BLF).

Acknowledgements

Supported by grants from the Philip Morris USA Research Managements, “GALLY” International Biomedical Research Institute Inc., and Alzheimer Association. I am very grateful to Mr. Russell Pool and Ms. Galina Alieva for their critiques and editorial work.

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* Equal Contribution.

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