Alzheimer’s Disease

Role of Amyloid β in Alzheimer's

Alzheimer’s is a complex neurodegenerative disease that accounts for 60-70% of dementia.
Alzheimer’s Disease (AD) was first described in 1907 by the German psychiatrist Alois Alzheimer. He did a histopathological study of his patient Auguste, who was suffering from dementia. Dr. Alzheimer found two types of legions in her cerebral cortex, senile plaques composed of amyloid beta (Aβ) protein and neurofibrillary tangles composed of Tau protein. Both are linked to the pathogenesis of Alzheimer’s.
Aβ is an abnormal fibrous, extracellular proteinaceous deposit. Tangles are Intracellular deposits of defective tau protein.

What is the cause of Alzheimer’s disease?

It is widely believed that there are several factors contributing to Alzheimer’s, or as a consequence of Alzheimer’s, including Aβ rich plaque, Tau rich tangles, Apolipoprotein (ApoE) variants, inflammation, and others.

Bricks are to a house like proteins are to you, me, and all life on planet earth. There is a protein for everything, including the color of your hair, eyes, muscles. The 20 amino acids are the building blocks of proteins. Broadly, large amino acid chains are proteins, and smaller ones are peptides. Proteins fold and unfold much like a ribbon.
The surfaces of neurons have a protein called Amyloid Precursor Protein (APP) that is sectioned by enzymes to free up Aβ protein, which is then cleared by the body. In Alzheimer’s patients, there is an imbalance whereby Aβ protein is not regulated and builds up abnormally into insoluble fibrils depositing as senile plaques.
The dynamics of protein folding and unfolding and in particular, it is believed, mis-folding play a key role in vitro fibril formation. Several factors including polypeptide charge, sequence, hydrophobicity, secondary structure, among others, contribute to peptides forming amyloid.
Senile plaque deposit around neurons and shut down neural activity. If neurons cannot fire, memory is lost and dementia sets in.
The path way for tau and tangles is different. Tau makes up the framework for microtubules within a neuron. When tau is hyperphosphorylated it breaks down, causing the microtubules to break down that eventually damage neurons.

In healthy individuals, Aβ protein is regulated and cleared by the body.
In AD patients, Aβ protein is not cleared and it builds up from monomers to dimers to oligomers and continues to build abnormally into insoluble fibrils depositing as senile plaques.
The creation of these mature insoluble fibrils involves many intermediaries: dimers, trimers, tetramers (collectively called oligomers) that are soluble, and others like Aβ derived diffusible plaque and protofibrils that are insoluble.

The amyloid fibrils that are deposited extracellularly are thought to have a stable β-sheet structure.
Our in vivo research shows that dose dependent Hyalolex can reduce Aβ aggregation and inhibit hyperphosphorylation of tau protein.

Role of IGC-AD1 on Aβ aggregation

Of the two types of legions found in the brains of AD patients, extracellular senile plaques are composed of Aβ proteins. In this study, IGC-AD1 is shown to inhibit the aggregation of Aβ monomers into oligomers, which deposit as senile plaque.

Aβ peptide monomers aggregate into toxic, prefibrillar oligomers (dimers, trimers, tetramers, oligomers), the key pathogenic event in the onset of Alzheimer’s.

Aβ oligomers directly cause synaptic dysfunction and neuronal death, both of which are implicated in the initiation and progression of Alzheimer’s1&2.

Aβ plaques trigger pathological events such as oxidative damage and inflammation, which lead to the progression of AD1&2

Dr. Chuanhai Cao’s demonstrates that IGC-AD1 inhibits Aβ peptide aggregation in a dose-dependent manner by increasing monomer Aβ levels as measured by an immunoblotting technique.

1. Brain2008; 131: 2414–24.

2.Prog Neurobiol 2009; 87: 181–94.

The in-vitro studies, further, indicate that IGC-AD1 at low non-toxic concentrations has an effect on reducing the Aβ1-42 dimers and tetramers in N2a AβPPswe cells as determined by a reduction in Thioflavin T (a marker of Aβ aggregation (1)) binding using a fluorescence assay.

J Alzheimers Dis. 2014;42(3):973-84.

Dr. Cao shows that in vitro repeated exposure over 48 hours to IGC-AD1 is not toxic to cells as determined by MTT assay that shows the number of viable cells present remains constant despite the exposure.

MTT assay is a colorimetric assay that measures cell metabolic activity. In general, increased MTT activity indicates damage to the cell. The graph shows that at the certain doses of IGC-AD1, MTT is not increased. The conclusion is that there is no neuro-toxicity.

Collectively these studies support the following in an animal model of Alzheimer’s:

IGC-AD1 modulates Aβ production
IGC-AD1 inhibits Aβ aggregation
IGC-AD1 is not toxic to neurons

Role of IGC-AD1 on Tau Protein

Of the two types of legions found in the brains of AD patients, intracellular neurofibrillary tangles (NFTs) are composed of hyperphosphorylated tau protein. This study shows that IGC-AD1 can inhibit glycogen synthase kinase-3β (GSK-3β) a major kinase (catalyst) in the phosphorylation of tau. Curtailing abnormal hyperphosphorylation of tau, which leads to NFTs, is an accepted strategy for addressing AD.

Tau protein is Microtubule Associated Proteins (MAPs) that stabilize microtubules within a neuron. Tau stabilizes microtubules under physiological conditions and regulates axonal stability and cell morphology. (Physiol. Rev. 2004;84(2):361)

“Tau is able to impact synaptic activity in several ways: studies show tau interacting directly with post-synaptic signaling complexes, regulating glutamatergic receptor content in dendritic spines, and influencing targeting and function of synaptic mitochondria.” (Amy Pooler, et al. A role for tau at the synapse in AD pathogenesis. Neuropharmacology, volume 76, 1/2014, 1-8).

In Alzheimer’s, tau is abnormally phosphorylated leading to disassociation of tau from microtubule-associated protein, leading to the destabilization of microtubule-associated protein complexes.

The free soluble hyperphosphorylated tau protein then aggregates into soluble and insoluble intracellular aggregates known as neurofibrillary tangles (NFTs), a hallmark of Alzheimer’s. NFTs are formed from paired helical filaments composed of hyperphosphorylated tau.

Role of tau hyperphosphorylation in NFTs

The phosphorylation of tau in Alzheimer’s is 3 to 4 times that of normal brain. (Kopke E, et al. J. Biol. Chem 1993;268:24374– 24384. PubMed: 8226987)

“Phosphorylation of tau negatively impacts its ability to promote microtubule assembly. (Lindwall G, Cole RD. J. Biol. Chem 1984;259:5301–5305. PubMed: 6425287)

As tau hyperphosphorylate and aggregate, the microtubules within a neuron disintegrate resulting in neurodegeneration and NFTs. Tau appears to spread across synapsis from neuron to neuron, much like a prion. (Li Liu, et al. (2012), the Trans-Synaptic spread of tau pathology in vivo. PLOS ONE 7(2))

“Recent studies have suggested that abnormal hyperphosphorylation of tau in the brain plays a vital role in the molecular pathogenesis of AD and in neurodegeneration”. (C.-X. Gong and K. Iqbal. Hyperphosphorylation of Microtubule-Associated Protein Tau: A Promising Therapeutic Target for Alzheimer Disease. Curr Med Chem. 2008;15(23): 2321–2328.)

GSK3β is an enzyme that enables (kinase) phosphorylation. It promotes the transfer of phosphate groups to specific substrates.

GSK3β is the most important tau kinase, along with GSK3α and MAPK13, and is implicated in the hyper-phosphorylation of tau, memory impairment, the increased production of Aβ, and other inflammatory responses. (Journal of Neurochemistry: 2008 Mar; 104(6): 1433–1439)

GSK3 is known to reduce acetylcholine synthesis, and is a key mediator of apoptosis (cell death) and thereby might directly contribute to neuronal loss in AD. (BMC Cell Biology: 2001;2:12)

Inhibiting GSK3 activity reverses some of the pathological effects of over-expression of mutated APP and tau in models of AD. (Proc Natl Acad Sci U S A. 2005 May 10; 102(19):6990-5)

Certain low doses of IGC-AD1 reduce the level of GSK3β in N2a AβPPswe cells as measured by immunoblotting studies.

The studies conducted by Dr. Chuanhai Cao show that based on dosage IGC-AD1 reduces the expression of GSK3β by as much as 53% to 62%.

As shown in the slides above, reducing GSK3β activity can beneficially effect some of the pathological effects of the mutated APP and decrease hyperphosphorylation of tau.

The study below directly measures the impact on tau phosphorylation.

The in vitro study directly demonstrates that dosage dependent IGC-AD1 reduces the phosphorylation of tau protein.

The study is based on measuring the density of deposits at various concentrations of IGC-AD1.

These studies demonstrate that IGC-AD1 can be beneficial in treating Alzheimer’s as it can

(i) inhibit the buildup of tau by decreasing the production of GSK3β levels by as much as 53% to 62%, and

(ii) inhibit the hyperphosphorylation of tau protein.

Effect of IGC-AD1 on mitochondrial function

Mitochondria mediate cell energy and cell death. The mitochondrial cascade hypothesis puts mitochondria at the apex of AD. Our study shows that IGC-AD1 can increase mitochondrial function.

Mitochondrial dysfunction has been linked to Alzheimer’s and may be a trigger that starts a cascade leading to the disease. (Paula I. Moreira, et al. Mitochondrial dysfunction is a trigger of AD pathophysiology, Biochinica et Biaphysica Acta 10/21/2009)

Growing evidence suggest that elevated amyloid-β (Aβ) levels contribute to mitochondrial abnormalities. (Dong-Hyung Cho, et al. S-Nitrosylation of Drp1 Mediates β-Amyloid-Related Mitochondrial Fission and Neuronal injury. Science 03 April 2009 vol 324.)

Overproduction of APP and Aβ may disturb the dynamics of mitochondrial fusion/fission, impair mitochondrial transport, disrupt the electron transfer chain, increase reactive oxygen species (ROS) production and alter mitochondrial function.

The mitochondrial cascade hypothesis for sporadic AD:

Mitochondrial function declines with age and a threshold is reached that sets of the histopathology observed in Alzheimer’s.

Aβ production is elevated-leading to Aβ aggregation-leading to extracellular senile plaques.

Further, increased Aβ leads-to mitochondrial injury that leads-to hyperphosphorylation of tau that leads to-NFTs.

((1) Russell Swerdlow, Kansas University Medical College May 1, 2017)

In-vitro studies done with IGC-AD1, by Dr. Chuanhai Cao, show that mitochondria isolated N2a AβPPswe cells which were exposed to 36 hours to either THC or melatonin (a potent antioxidant) or caffeine or both had the effect on enhancing mitochondrial function.

Role of Amyloid β in Alzheimer's

Alzheimer’s is a complex neurodegenerative disease that accounts for 60-70% of dementia.

Aβ is an abnormal fibrous, extracellular proteinaceous deposit. Tangles are Intracellular deposits of defective tau protein.

It is widely believed that there are several factors contributing to Alzheimer’s, or as a consequence of Alzheimer’s, including Aβ rich plaque, Tau rich tangles, Apolipoprotein (ApoE) variants, inflammation, and others.

The dynamics of protein folding and unfolding and in particular, it is believed, mis-folding play a key role in vitro fibril formation. Several factors including polypeptide charge, sequence, hydrophobicity, secondary structure, among others, contribute to peptides forming amyloid.

Senile plaque deposit around neurons and shut down neural activity. If neurons cannot fire, memory is lost and dementia sets in.

The path way for tau and tangles is different. Tau makes up the framework for microtubules within a neuron. When tau is hyperphosphorylated it breaks down, causing the microtubules to break down that eventually damage neurons.

In healthy individuals, Aβ protein is regulated and cleared by the body.

In AD patients, Aβ protein is not cleared and it builds up from monomers to dimers to oligomers and continues to build abnormally into insoluble fibrils depositing as senile plaques.

The creation of these mature insoluble fibrils involves many intermediaries: dimers, trimers, tetramers (collectively called oligomers) that are soluble, and others like Aβ derived diffusible plaque and protofibrils that are insoluble.

The amyloid fibrils that are deposited extracellularly are thought to have a stable β-sheet structure.

Our in vivo research shows that dose dependent Hyalolex can reduce Aβ aggregation and inhibit hyperphosphorylation of tau protein.

Role of IGC-AD1 on Aβ aggregation

Of the two types of legions found in the brains of AD patients, extracellular senile plaques are composed of Aβ proteins. In this study, IGC-AD1 is shown to inhibit the aggregation of Aβ monomers into oligomers, which deposit as senile plaque.

Aβ peptide monomers aggregate into toxic, prefibrillar oligomers (dimers, trimers, tetramers, oligomers), the key pathogenic event in the onset of Alzheimer’s.

Aβ oligomers directly cause synaptic dysfunction and neuronal death, both of which are implicated in the initiation and progression of Alzheimer’s1&2.

Aβ plaques trigger pathological events such as oxidative damage and inflammation, which lead to the progression of AD1&2

Dr. Chuanhai Cao’s demonstrates that IGC-AD1 inhibits Aβ peptide aggregation in a dose-dependent manner by increasing monomer Aβ levels as measured by an immunoblotting technique.

The in-vitro studies, further, indicate that IGC-AD1 at low non-toxic concentrations has an effect on reducing the Aβ1-42 dimers and tetramers in N2a AβPPswe cells as determined by a reduction in Thioflavin T (a marker of Aβ aggregation (1)) binding using a fluorescence assay.

Dr. Cao shows that in vitro repeated exposure over 48 hours to IGC-AD1 is not toxic to cells as determined by MTT assay that shows the number of viable cells present remains constant despite the exposure.

MTT assay is a colorimetric assay that measures cell metabolic activity. In general, increased MTT activity indicates damage to the cell. The graph shows that at the certain doses of IGC-AD1, MTT is not increased. The conclusion is that there is no neuro-toxicity.

Collectively these studies support the following in an animal model of Alzheimer’s:

IGC-AD1 modulates Aβ production

IGC-AD1 inhibits Aβ aggregation

IGC-AD1 is not toxic to neurons

Role of IGC-AD1 on Tau Protein

Tau protein is Microtubule Associated Proteins (MAPs) that stabilize microtubules within a neuron. Tau stabilizes microtubules under physiological conditions and regulates axonal stability and cell morphology. (Physiol. Rev. 2004;84(2):361)

In Alzheimer’s, tau is abnormally phosphorylated leading to disassociation of tau from microtubule-associated protein, leading to the destabilization of microtubule-associated protein complexes.

The free soluble hyperphosphorylated tau protein then aggregates into soluble and insoluble intracellular aggregates known as neurofibrillary tangles (NFTs), a hallmark of Alzheimer’s. NFTs are formed from paired helical filaments composed of hyperphosphorylated tau.

The phosphorylation of tau in Alzheimer’s is 3 to 4 times that of normal brain. (Kopke E, et al. J. Biol. Chem 1993;268:24374– 24384. PubMed: 8226987)

“Phosphorylation of tau negatively impacts its ability to promote microtubule assembly. (Lindwall G, Cole RD. J. Biol. Chem 1984;259:5301–5305. PubMed: 6425287)

As tau hyperphosphorylate and aggregate, the microtubules within a neuron disintegrate resulting in neurodegeneration and NFTs. Tau appears to spread across synapsis from neuron to neuron, much like a prion. (Li Liu, et al. (2012), the Trans-Synaptic spread of tau pathology in vivo. PLOS ONE 7(2))

GSK3β is an enzyme that enables (kinase) phosphorylation. It promotes the transfer of phosphate groups to specific substrates.

GSK3β is the most important tau kinase, along with GSK3α and MAPK13, and is implicated in the hyper-phosphorylation of tau, memory impairment, the increased production of Aβ, and other inflammatory responses. (Journal of Neurochemistry: 2008 Mar; 104(6): 1433–1439)

GSK3 is known to reduce acetylcholine synthesis, and is a key mediator of apoptosis (cell death) and thereby might directly contribute to neuronal loss in AD. (BMC Cell Biology: 2001;2:12)

Inhibiting GSK3 activity reverses some of the pathological effects of over-expression of mutated APP and tau in models of AD. (Proc Natl Acad Sci U S A. 2005 May 10; 102(19):6990-5)

Certain low doses of IGC-AD1 reduce the level of GSK3β in N2a AβPPswe cells as measured by immunoblotting studies.

The studies conducted by Dr. Chuanhai Cao show that based on dosage IGC-AD1 reduces the expression of GSK3β by as much as 53% to 62%.

As shown in the slides above, reducing GSK3β activity can beneficially effect some of the pathological effects of the mutated APP and decrease hyperphosphorylation of tau.

The study below directly measures the impact on tau phosphorylation.

The in vitro study directly demonstrates that dosage dependent IGC-AD1 reduces the phosphorylation of tau protein.

The study is based on measuring the density of deposits at various concentrations of IGC-AD1.

These studies demonstrate that IGC-AD1 can be beneficial in treating Alzheimer’s as it can

(i) inhibit the buildup of tau by decreasing the production of GSK3β levels by as much as 53% to 62%, and

(ii) inhibit the hyperphosphorylation of tau protein.

Effect of IGC-AD1 on mitochondrial function

Mitochondria mediate cell energy and cell death. The mitochondrial cascade hypothesis puts mitochondria at the apex of AD. Our study shows that IGC-AD1 can increase mitochondrial function.

Mitochondrial dysfunction has been linked to Alzheimer’s and may be a trigger that starts a cascade leading to the disease. (Paula I. Moreira, et al. Mitochondrial dysfunction is a trigger of AD pathophysiology, Biochinica et Biaphysica Acta 10/21/2009)

Growing evidence suggest that elevated amyloid-β (Aβ) levels contribute to mitochondrial abnormalities. (Dong-Hyung Cho, et al. S-Nitrosylation of Drp1 Mediates β-Amyloid-Related Mitochondrial Fission and Neuronal injury. Science 03 April 2009 vol 324.)

Overproduction of APP and Aβ may disturb the dynamics of mitochondrial fusion/fission, impair mitochondrial transport, disrupt the electron transfer chain, increase reactive oxygen species (ROS) production and alter mitochondrial function.

The mitochondrial cascade hypothesis for sporadic AD:

Mitochondrial function declines with age and a threshold is reached that sets of the histopathology observed in Alzheimer’s.

Aβ production is elevated-leading to Aβ aggregation-leading to extracellular senile plaques.

Further, increased Aβ leads-to mitochondrial injury that leads-to hyperphosphorylation of tau that leads to-NFTs.

((1) Russell Swerdlow, Kansas University Medical College May 1, 2017)

In-vitro studies done with IGC-AD1, by Dr. Chuanhai Cao, show that mitochondria isolated N2a AβPPswe cells which were exposed to 36 hours to either THC or melatonin (a potent antioxidant) or caffeine or both had the effect on enhancing mitochondrial function.

IGC-AD1 can enhance mitochondrial function between 30% and 60% at the basal and in the presence of FCCP (FCCP disrupts ATP synthesis).