A Novel Multi-phased Project that investigates the Glutathione Pathway by Creating Alzheimer’s model Drosophila with Pan-neuronal Over-expression of the GCLc Gene, Inducing Redox Stress through Sleep Deprivation, and Analyzing Mitochondrial Electron Transport Chain using Colorimetric Enzymatic Assays.

Lisa Michaels • Plano West Senior High School • Plano, TX •

Early Diagnosis and Treatment of Alzheimer’s:

Modulating the GCLc Gene to Mitigate Redox Stressand Mitochondrial ETC Complex Dysfunction

ALZHEIMER’S BRAIN

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Source: Alzheimer’s.org, National Center for health statisticsSource: Alzheimer’s.org, National Center for health statistics

Brain scans done with Positron Emission Tomography (PET) show how Alzheimer’s affects brain activity. The top images show a normal brain, while the right images are from a person with Alzheimer’s. The red and yellow areas in the bottom scans show Amyloid plaques, made visible with Mathis and Klunk’s traceable dye.

As Alzheimer’s disease progresses, brain tissue shrinks. However, the ventricles, chambers within the brain that contain cerebrospinal fluid, are noticeably enlarged. In the early stages of Alzheimer's disease, short-term memory begins to decline when the cells in the hippocampus degenerate.

NATIONAL SIGNIFICANCE

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MAJOR CAUSES OF DEATH - % Change 2000-08

AD has emerged as the sixth largest killer disease in the US, rapidly growing while deaths from other diseases are declining. If we make no progress in diagnosis and treatment of AD, the associated cost will rise to over a trillion dollars a year in 2050. The President recently signed into law the National Alzheimer’s Project to overcome the disease before 2025. This project contributes to this national public health goal.

Source: Alzheimer’s.org, National Center for health statisticsSource: Alzheimer’s.org, National Center for health statistics

66% Increase in Alzheimer’s during 2000-2008 as a Cause of Death!

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PREVALENT THEORY: AMYLOID CASCADE

Source: National Institutes of Health website

5Source: Alzheimer’s.org, National Center for health statisticsSource: Alzheimer’s.org, National Center for health statistics

PREVALENT THEORY II: MITOCHONDRIAL CASCADE

Balance of ROS generation and antioxidantive systems is critical for neuronal survival. An imbalance of both systems due to either excessive production of ROS (left) or reduced antioxidant defense (right) results in oxidative stress. In AD, increases in oxidative stress, mainly promoted by the interplay between the reactive species and transition metals, lead to oxidative damage that induces neuronal damage or death. Antioxidant therapies that increase endogenous antioxidant defenses or reduce oxidative sources should reduce oxidative damage, and thereby prevent or delay disease symptoms.

Amyloid Plaques Tau Tangles

OXIDATIVE STRESS

ANTIOXIDANT DEFENSE

O2

H2 O2

OH –NO –ONOO–GSSG

CATSODGSH peroxidaseGSH-S-transferaseGSH

NEURO-DEGENERATION

NEURO-PROTECTION

PREVALENT THEORY III: OXIDATIVE STRESS

The research thesis is that in vivo synthesis of Glutathione through over-expression of the GCLc gene mitigates Oxidative stress, Mitochondrial dysfunction and Amyloid toxicity, thereby slowing down the progression of AD. The Oxidative stress caused by Amyloid toxicity results in ROS, impairment of the Electron Transport Chain, further ROS generation and Mitochondrial dysfunction. Switching on the GCLc gene may protect Mitochondria from damage by improving the Redox balance (GSH/GSSG) which mitigates Mitochondrial dysfunction and the progression of Alzheimer’s.

GLUTATHIONE

MITOCHONDRIAL DYSFUNCTION

ROSAMYLOID TOXICITY

GCLc GENE

MITOCHONDRIAL PROTECTION

ALZHEIMER’S PROGRRESSION

Source: Thinkquest.org

GENE EXPRESSION

ANTIOXIDANT CAPACITY

PROPOSED THEORY

SUGGESTED SOLUTION: GCLc_GLUTATHIONE PATHWAY

Glutathione is the primary endogenous antioxidant. It is the body's most effective detoxifier. Stress depletes the Glutathione stores. It modulates the immune response. It assists in the regulation of the cell's vital functions, such as the synthesis and repair of DNA, the synthesis of proteins, and the activation and regulation of enzymes. As Glutathione is a large molecule the supplements are not absorbed into the body. Glutathione-rich foods include asparagus, grapefruit and peaches, garlic, whey protein. We need 250 milligrams of Glutathione a day; the average American consumes less than 35 mg!

C10H17N3O6S

Glutathione is synthesized de novo by the action of two enzymes: glutamate-cysteine ligase-catalytic subunit (GCLc) and by GSH synthase.

The determinants of Glutathione synthesis are the availability of cysteine, activity of GCLc gene and feedback regulation.

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http://max-one-cure.org/max-one/max-one-qa

REACTION 1:

GCL

Glutamate

Cysteine

ϒ-glutamylcysteine

ATP

ADP

GSHSyntha

se

Glycine

ϒ-glutamylcysteinylglyci

neGSH

ATP

ADP

REACTION 2:

RESEARCH DESIGN

GENETICMODULATION LIFESPANH1:

StressedAβ42-GCLc

LIFESPANH2:

Genetics Lab

H3:

Aβ42-GCLcINCREASED

GLUTATHIONE PRODUCTION

Aβ42-GCLcINCREASED

MITOCHONDRIALBIOENERGETICS

RESISTANCE TO REDOX STRESS

MITOCHONDRIALBIOENERGETICS ETC

ACTIVITY

INCREASED RESISTANCE TO REDOX STRESS

Home Lab

Home Lab

HYPOTHESES

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Hypothesis 1:  IF lifespans of Alzheimer’s model Aβ42 Drosophila and Aβ42-GCLc are studied, THEN the Aβ42-GCLc group will outlive the Aβ42 group of flies; all groups will exhibit shorter lifespans compared to the control group of yellow-white Drosophila melanogaster.  Hypothesis 2: IF Aβ42 and Aβ42-GCLc Drosophila are subjected to continuous oxidative stress, THEN the Aβ42-GCLc flies will be able to better resist stress, as exhibited by longer lifespan; all groups will exhibit shorter lifespans compared to the control group of yellow-white Drosophila melanogaster.

Hypothesis 3: IF the Mitochondrial Electron Transport Chain of Aβ42 and  Aβ42-GCLc groups of Drosophila are analyzed, THEN Aβ42-GCLc will exhibit more enzymatic activity in the mitochondrial complexes compared to Aβ42; all groups will exhibit lower enzyme activity compared to the control group of yellow-white Drosophila melanogaster.

MATERIALS

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12University Lab: ETC studiesUniversity Lab: ETC studies

Oscillator For inducing Oxidative StressOscillator For inducing Oxidative Stress

Home Lab: Lifespan and Oxidative stress Experiments

Source: All Charts and Photographs by the Researcher

TRANSGENIC DROSOPHILA

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• Drosophila melanogaster is an ideal animal model widely used in human longevity and disease studies for several reasons. About 75% of the known human disease genes have a recognizable match in the genetic code of fruit flies and 50% of fly protein sequences have human analogues (Culliton, 2000; Reiter et al, 2001; UCSD, 2001). It has a relatively short life cycle, is easily reared in the laboratory, and the fecund females produce large populations that make statistical studies easy and reliable.

• Drosophila melanogaster is ideal for genetic intervention studies in neurodegenerative diseases. Several genes have been identified that are preferentially expressed inneurons, and have been used to drive transgene expression in tissues. In this research, elav-GAL4 technique for pan-neuronal expression was used in the Aβ42-GCLc Drosophila.

• A yeast transgene for the transcription factor GAL4 (elav) is inserted in an arbitrary location in the Drosophila genome. There, the expression of GAL4 is controlled by flanking Drosophila enhancers (GCLc) and suppressors that normally regulate a Drosophila gene in the neighborhood. The resulting expression pattern of GAL4 might include certain cells and tissues. As GAL4 is itself a transcription factor, it can drive the expression of other genes that are placed downstream of a DNA sequence that binds GAL4. Hence the combination of GAL4 driver and effector gene is a way to target the effector to tissues that happen to express GAL4 in a particular driver line.

Neuronal network of Natural (A) and Transgenic (B) Drosophila visualized by immuno-staining technique .Neuronal network of Natural (A) and Transgenic (B) Drosophila visualized by immuno-staining technique .

http://sanpatricio.co.uk/Innexins/pg2%20dev.php

METHODS 1

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SUBJECT PREPARATIONSUBJECT PREPARATION

1. Breeding: Male and female natural yellow-white Drosophila melanogaster and the transgenic GAL4-GCLc flies were obtained from the Southern Methodist University Biology lab in Dallas. Alzheimer model Aβ42 (elav-GAL4 ; UAS Aβ42/cyo) Drosophila melanogaster flies were obtained from Thomas Jefferson Universities in Philadelphia to be used as the parent population.

2. The Alzheimer’s/GCLc crosses were made by cross breeding virgin female Azheimer elav-Aβ42 flies with male GAL4-GCLc flies. Multiple culture bottles were maintained till a large sample subject population was obtained

http://www.nature.com/nrg/journal/v3/n3/box/nrg751_BX2.html

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The dominating role of the mouse in modeling Alzheimer's disease has been challenged by Drosophila melanogaster. Its well organized brain permits the study of complex behaviors such as learning and memory. Transgenic flies have been established that express human Aβ42 in the nervous system using the UAS-GAL4 system. These flies developed age-dependent short-term memory impairment and neurodegeneration. Transgenic flies with overexpression of Abeta42 peptides in the nervous system results in phenotypes associated with neuronal degeneration in a dose- and age-dependent manner. It is these peptides that accumulate in human disease and are thought to be the initiating factor in Alzheimer's disease. The flies exhibit a clear phenotype from a few days of age, including reduced locomotor function, impaired olfactory memory and shortened lifespan. Therapeutic agents that interfere with the generation of toxic aggregates of beta-amyloid peptides have been shown to rescue the flies.

ALZHEIMER’S MODEL DROSOPHILA

Spatial targeting of transgene expression in Drosophila. GAL4/UAS system. Driver lines expressing the transcriptional activator GAL4 in a tissue-specific fashion are crossed to UAS-lines with genomic inserts of a target gene fused to five GAL4-binding sites arrayed in tandem (5 × UAS) (shown here as UAS-Aβ42). Source: Adapted from Development, Brand and Perrimon, 118: 401-415, 1993

METHODS – Phase 2

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EXPERIMENTSEXPERIMENTS

PROCEDURE

1) Breeding: Male and female yellow-white Drosophila, Alzheimer model “c155-elav/elav GAL4; UAS-Aβ42/cyo” Drosophila and GCLc-GAL4 Drosophila melanogaster parent flies were obtained from the SMU biology Department and Thomas Jefferson Universities. A total of about 1000 subject Drosophila were used for this research study.

2) Mating: They were bred at the home lab in Drosophila glass culture bottles with Carolina blue medium 424 formula, activated yeast and water. The Alzheimer’s/GCLc transgenic fly crosses were made in the home lab by breeding virgin female Alzheimer’s elav-GAL4;UAS-Aβ42 flies with male GCLc-GAL4 flies.

3) Anesthetizing: Once the larvae hatched, they were immediately isolated, anesthetized by a CO2 anesthetizer, and divided on an ice pack by gender. Only male flies were used as the subject population.

4) Grouping: The research subject flies were divided into 100 flies each in four vials of about 25 each. There were 3 types of flies consisting of Male Yellow White, Male Aβ42 Alzheimer’s and Male Aβ42/GCLc flies. There were three study groups in each type of fly, one group was used for natural lifespan, one for redox stress lifespan and one for the mitochondrial ETC assays.

5) Redox Stress: The 24 hr Redox stress was achieved by constant oscillation, noise and light in each group. The non stressed flies were in an insect culture incubator with no movement, natural noise and light.

1) Life Span: The 40 vials of 25 flies each were changed every other day. This consists of making the right food for every vial (Carolina 424 blue formula with dry activated yeast and water) and transferring the flies from one vial to the other and the number of flies that died each time was recorded. This was continued until the last fly died in each vial. The average 50%survival life spans for each group of flies in each group (in each of the 4 vials) were recorded, tabulated and graphed.

7) The ETC subject group of flies were collected and frozen, 15 each time, at four weeks for further testing at the genetics laboratory

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The Mitochondrial Electron Transport Chain (ETC) colorimetric enzymatic assays and analysis was performed at a Human Genetics Lab in a Medical College. HOMOGENIZING:For each group of Drosophila studied, (YW, Aβ42, and Aβ42-GCLc) 15 flies each were frozen at four weeks of their lifespan. They were taken on dry ice to the Baylor Medical Genetics Lab to do the Electron Transport Chain Complex Enzymatic Assays using the Spectrophotometer.Flies were homogenized and Drosophila lysate transferred to eppendorph tubes.Whole fly lysate was spun at 2500rpms for 10 minutes and supernatant containing mitochondria was separately placed into new tubes. STANDARDIZING:Bradford protein reagent was used to measure diluted concentrations of Drosophila lysate described above.BSA was used to create a standard curve, to which each sample was measured against. Each Drosophila lysate was standardized to 1ug/ul. SET-UP:The activities of complex II (succinate dehydrogenase), total and rotenone sensitive complex I+III (NADH: Cytochrome C Oxido reductase), and complex IV (cytochrome c oxidase), and CS (Citrate Synthase) was measured using appropriate electron acceptors/donors according to published procedures. Each assay was performed in duplicate. COLORIMETRIC ASSAY:ETC enzymes will be assayed at 30 °C using a temperature-controlled spectrophotometer; Ultraspec 6300 pro, Biochrom Ltd., (Cambridge, England).The increase or decrease in the absorbance of cytochrome C at 550 nm will be measured for complexes I+III, II+III, and complex IV. For complex II, the reduction of 2,6-dichloroindophenol (DCIP) at 600 nm was measured.Citrate Synthase (CS) was used as a marker for mitochondrial content and was measured by reduction of Ellman's reagent at 412 nm. Enzyme activities are expressed as nmol/min/mg protein.Data was recorded, graphed, tested for statistical significance using the two tailed T test, analyzed and conclusions drawn.

(Complete protocol in research folder)Sources: Kirby M. Biochemical assays of respiratory chain complex activity. Methods in Cell Biology. 2007;80:93-119. Medja F. Development and implementation of standardized spectrophotometric assays for clinical diagnosis. Mitochondrion. 2009 Sept; 9(5); 331-339.

The Mitochondrial Electron Transport Chain (ETC) colorimetric enzymatic assays and analysis was performed at a Human Genetics Lab in a Medical College. HOMOGENIZING:For each group of Drosophila studied, (YW, Aβ42, and Aβ42-GCLc) 15 flies each were frozen at four weeks of their lifespan. They were taken on dry ice to the Baylor Medical Genetics Lab to do the Electron Transport Chain Complex Enzymatic Assays using the Spectrophotometer.Flies were homogenized and Drosophila lysate transferred to eppendorph tubes.Whole fly lysate was spun at 2500rpms for 10 minutes and supernatant containing mitochondria was separately placed into new tubes. STANDARDIZING:Bradford protein reagent was used to measure diluted concentrations of Drosophila lysate described above.BSA was used to create a standard curve, to which each sample was measured against. Each Drosophila lysate was standardized to 1ug/ul. SET-UP:The activities of complex II (succinate dehydrogenase), total and rotenone sensitive complex I+III (NADH: Cytochrome C Oxido reductase), and complex IV (cytochrome c oxidase), and CS (Citrate Synthase) was measured using appropriate electron acceptors/donors according to published procedures. Each assay was performed in duplicate. COLORIMETRIC ASSAY:ETC enzymes will be assayed at 30 °C using a temperature-controlled spectrophotometer; Ultraspec 6300 pro, Biochrom Ltd., (Cambridge, England).The increase or decrease in the absorbance of cytochrome C at 550 nm will be measured for complexes I+III, II+III, and complex IV. For complex II, the reduction of 2,6-dichloroindophenol (DCIP) at 600 nm was measured.Citrate Synthase (CS) was used as a marker for mitochondrial content and was measured by reduction of Ellman's reagent at 412 nm. Enzyme activities are expressed as nmol/min/mg protein.Data was recorded, graphed, tested for statistical significance using the two tailed T test, analyzed and conclusions drawn.

(Complete protocol in research folder)Sources: Kirby M. Biochemical assays of respiratory chain complex activity. Methods in Cell Biology. 2007;80:93-119. Medja F. Development and implementation of standardized spectrophotometric assays for clinical diagnosis. Mitochondrion. 2009 Sept; 9(5); 331-339.

METHODS – Phase 3

RESULTS: HYPOTHESIS 1

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Results: There was a dramatic decrease in lifespan as hypothesized. The increase was 40% in the Aβ42 Alzheimer model flies compared to the natural yellow whites. GCLc overexpression in the Aβ42-GCLc flies increased lifespan by 28%!

Charts conceived and created by researcher

* Statistically Significant at 99% level; using two-tailed t-tests; SE shown on the bars.

*

*

*

+28%

-40%

T

RESULTS: HYPOTHESIS 2

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Results: As hypothesized, stress was extremely significant in the Alzheimer model flies. Stress decreased average lifespan from 25 days to 11 days! But the lifespan of the Aβ42-GCLc flies was 26 days (136% increase compared to Aβ42) showing that increased Glutathione can mitigate redox stress especially in Alzheimer’s.

Charts conceived and created by researcher

**

*

*

+136%

-62%

T* Statistically Significant at 99% level; using two-tailed t-tests; SE shown on the bars.

RESULTS: HYPOTHESIS 3 Hypothesis 3: As hypothesized Aβ42-GCLc exhibited more activity in mitochondrial complexes I+III, and IV compared to Aβ42. As hypothesized Aβ42-GCLc exhibited dramatically more activity in mitochondrial complex IV compared to Aβ42. The GCLc activity in the Alzheimer flies restored and even increased complex IV activity compared to the control group of yellow-white.

Original ETC Assay readings in nmol/min/mg protein; converted to percentages to adjust for Citrate Synthase variations

Charts conceived and created by researcher

+63%

-34%

+116%-50%

* Two trials; Each trial done in duplicate; SD shown on bars

RESULTS: HYPOTHESIS 3 Hypothesis 3: As hypothesized Aβ42-GCLc exhibited more activity in mitochondrial complexes II and II+III compared to Aβ42. Complex II and II+III Aβ42-GCLc exhibited lower enzyme activity compared to the yellow-white Drosophila melanogaster.

Charts conceived and created by researcher

Original ETC Assay readings in nmol/min/mg protein; converted to percentages to adjust for Citrate Synthase variations

+141%

-68%

T

T

T

+151%

-69%

T

T

T

* Two trials; Each trial done in duplicate; SD shown on bars

CONCLUSIONS

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Hypothesis 1: STRONGLY SUPPORTED. The Alzheimer flies had a 40% decrease in lifespan compared to the Yellow Whites. But it was found that the Aβ42-GCLc group had a 28% increased lifespan compared to the Aβ42 group! The increased average lifespan was found significant at the 99% confidence level.

Hypothesis 2: STRONGLY SUPPORTED. The Alzheimer flies had a 62% decrease in lifespan when stressed. But the Aβ42-GCLc group under stress had an increase of 136% in lifespan compared to the stressed Alzheimer’s group! The increased average lifespan was found significant at the 99% confidence level.

Hypothesis 3: SUPPORTED. The Mitochondrial activities in all four complexes tested were significantly lower in the Alzheimer flies compared to the yw, reduced by 34-69%. But in each complex tested, the Aβ42-GCLc flies enzyme activity increased 63-151% compared to the Alzheimer’s flies! In complex I+III and IV, the enzyme activities in Aβ42-GCLc flies were even higher than the YW flies, suggesting increased protection for mtDNA.

The research findings underscore the importance of an integrated model on Alzheimer’s and the key role of antioxidant genes, like GCLc in redox stress management. The dramatic change in lifespan and mitochondrial function when Glutathione was increased in the Alzheimer’s flies (Aβ42-GCLc), opens new research avenues in the field of antioxidants in Alzheimer’s as preventive and therapeutic agents.

AD therapies designed to reduce Aβ42 thus far have had very limited clinical benefits. This research identifies alternative therapeutic targets. Since it is increasingly accepted that mitochondria play an important role in the late-onset forms Alzheimer’s, this could potentially advance our understanding especially of sporadic, late-onset AD, making mitochondria an ideal diagnostic and therapeutic target. This is consistent with the Mitochondrial Cascade Hypothesis.

This study also shows that novel animal models such as Aβ42-GCLc are valuable tools in investigating AD diagnostics and therapies.

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SCIENTIFIC INSIGHTSThis study offers support for an integrated approach toward Alzheimer’s. An improper mitochondrial complex function leads to a decreased mitochondrial membrane potential of the organelle impairing ATP formation. Increased ROS levels act at multiple levels to impair mitochondrial function, and increase Amyloid accumulation.

Also the critical role of mitochondria in the early pathogenesis of AD may make them attractive as a preferential target for treatment strategies. In view of the increasing interest in mitochondrial protection as a treatment strategy in dementia, besides strategies with regard to the treatment and/or removal of both Aβ and tau pathology, the findings of a substantial protection of mitochondria against Aβ-induced dysfunction deserve further attention.

This project underscores efforts to develop therapies aimed at modulating GSH levels so as to modulate disease risk and progression.  Increasing the neuronal GSH level, whether endogenously or exogenously, would prevent the progression of Alzheimer’s by protecting against oxidative stress. It is unclear whether exogenous GSH supplements are clinically effective, whereas genetic pathways inducing GSH synthesis might be an alternative strategy against neuro-degenerative diseases.

APPLICATIONSThis research has medical implications for diagnosis, treatment and prognosis of Alzheimer’s. Diagnostic tools can be developed to map mitochondrial function and test enzymatic assay levels as a biomarker for susceptibility to disease. Biotechnology research may be developed and targeted at the 15 million Americans who are at risk of Alzheimer’s.

①This research confirmed the importance of redox stress on health and longevity. Antioxidant genes like GCLc may be able to mitigate some of the negative effects of stress on health and longevity, especially in people with neurodegenerative diseases of aging.

②People with Alzheimer’s with decreased Glutathione levels may have a decreased innate capacity to withstand stress. Over-expressing the GCLc genes through epigenetic intervention may be a potential Biotechnology application for the future.

③Physicians may want to test serum Glutathione levels in patients who suffer from stress, sleep deprivation and Alzheimer’s. As Glutathione oxidizes quickly outside the body, blood tests may not reveal the antioxidant deficiency; this research suggests that genetic testing may produce more reliable results.

④We need 250 milligrams of Glutathione a day; the average American consumes less than 35 mg! Over the counter, Glutathione supplements could substitute the 60 million prescriptions every year for sleep disorders and may be a valuable supplement in delaying Alzheimer’s disease.

⑤As Glutathione is not absorbed well into the body, Glutathione precursors such as N-acetyl-cysteine (NAC), Selenium and Vitamin C may be taken. Glutathione-rich foods such as asparagus, mangoes, eggs, garlic and whey protein would also help.

⑥Future studies may explore genetic intervention in the brain to treat Alzheimer’s. Increasing Glutathione (GSH) levels in the brain by exploring new molecular targets and transcription factors may be a focus area for such research.

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BIOMARKERS FOR AD > CURRENT - PROPOSED

1MILD COGNITIVE IMPAIRMENT:Risk Factors

Age, Genetics, metabolic, traumatic

Plasma Biomarkers: ApoE genotype status; Plasma Abeta Biomarkers

Mitochondrial Dysfunction using ETC Enzymatic Assays

Complex I+III: NADH-Cytochrome C ReductaseComplex II: Succinate- CoQ ReductaseComplex II+III: Ubiquinol - Cytochrome C ReductaseComplex IV: Cytochrome C Oxidase

2MILD AD: Early Molecular Mechanisms

Beta Amyloid Cleaving Enzyme (BACE1) activity; Brain Abeta; Tau

Cerebrospinal Fluid Biomarkers such as T-tau, P-tau and Abeta42

3MODERATE AD: Neuro-degeneration Biomarkers

Tau, Neurofibrillary Tangles, Volume loss

Cerebrospinal Fluid Biomarkers such as T-tau, P-tau and Abeta42

4SEVERE AD: Neuro-degeneration Biomarkers

Tau, Neurofibrillary Tangles, Volume loss

Cerebrospinal Fluid Biomarkers such as T-tau, P-tau and Abeta42

AD DIAGNOSIS: CURRENT VS. PROPOSED

Source: Adapted from Hampel, H et al. (2011), Biomarkers for Alzheimer’s disease therapeutic trials, Progress in Neurobiology 95 (2011) 579–593.

THERAPEUTIC TARGETS FOR AD > CURRENT - PROPOSED

1MITOCHONDRIAL DYSFUNCTION MitoQ; CoQ10; Creatine; Idebenone, SS31

GCLc-Glutathione Pathway targeted on Mitochondria:

• Gene Regulation

• Epigenetics

• Gene Therapy

2OXIDATIVE STRESS

Vitamin E; Vitamin C; Melatonin; Pycnogenol; SOD2

3AMYLOIDTOXICITY

Anti-Abeta – Rifampicin; 8-hidroxy-3R-methyl-2R, Type IV collagen; Danuomycin; Fullerine; Tannic acid; Savianolic acid

4 OTHER

Hormone therapy using Estrogen; Gene therapy using Lentiviral vectors; Stem cell transplantation; Neurosteroids; NMDA receptor antagonists – Memantine; Prednisone.

Source: Tarawneh R and Galvin JE (2010), Potential Future Neuroprotective Therapies For Neurodegnerative Disorders, Clinical Geriatric Medicine, February; 26 (1): 125-147.

Bolognesi ML, Matera R, Minarini A et al. (2009) Alzheimer’s Disease: New Approaches to Drug Discovery. Current Opinions in Chemical Biology 2009; 13:303.

AD TREATMENT: CURRENT VS. PROPOSED

James Michael Luchak, Baylor Genetics Laboratory.

Professor Lee-Jun Wong, Dept of Molecular Medicine.

Professor Koichi Iijima, Dept of Molecular Biology.

Professor. William Orr, Chairman of the Dept of Biology.

REFERENCES & REFERENCES & ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

This research was made possible with the access to the Genetics Labs at three research institutions.

The project idea, research design and methodology are original and independent, and not a part of any ongoing projects at any of these institutions.

A patent application has been filed by the researcher with USPTO on the novel animal model created for the project, and the diagnostic tool proposed for Alzheimer's Disease based on the findings from this research.

I thankfully acknowledge the guidance of the following mentors in this research project: