Chapter 1 Part 3 How are Neurofibertangles formed in the brain ?

How are NFTs formed in the brain?

NFTs are insoluble tangles of soluble phosphoproteins called tau.  Because these tangles start forming inside neurons they are called neurofibrillary tangles or NFTs. The appearance in the brain of these NFTs as “tombstones” or “ghosts” is one of the hallmarks of AD. NFT formation requires two things:

·         An above normal amount of phosphoryl groups (PO₃^2-) coated on the tau proteins. This phosphoryl coated tau protein is called hyper-phosphorylated tau.

·         Metal ions that facilitate the formation and pairing of hyper-phosphorylated tau to form paired helical filaments (PHFt) called NFTs. 

Lesions in the brain caused by NFTs are better correlated with cognitive decline in AD than intracellular Aβ oligomer and extracellular Aβ plaque formation¹⁹.  

Does aluminum cause NFTs or does aluminum complex with NFTs after they form?

Some metal cations inhibit numerous key enzymes in the brain.  One of these key enzymes is PP2A that keeps tau from being covered with too many phosphoryl (PO₃^2-) groups (i.e. over-phosphorylated).  When PP2A is inhibited by zinc²⁰, mercury²¹, or aluminum²² ions the result is over-phosphorylation of tau leading to PHFt formation, neuronal death, and ultimately neurofibrillary tangles in place of former neurons.  Of the three cations that are known to inhibit PP2A, zinc is a neurotransmitter that is required by our brains and zinc and mercury are not on the list of suspects in the case of strange death as they are not contained in alum. Therefore Watson, zinc and mercury are “red-herrings” and this leaves aluminum in alum on the suspect list.

In 1988 aluminum chloride added to rat brain cells resulted in NFTs that were “distinct” immunochemically from human Alzheimer NFTs²³.  This resulted in more controversy but was resolved in 1998 when aluminum chloride was injected into the cerebrospinal fluid in the brains of New Zealand white rabbits²⁴.    The resulting NFTs were immunochemically identical to NFTs found in the brains of AD patients. In 1992 it was found that aluminum stimulates the interaction between filaments of hyper-phosphorylated tau. This interaction results in paired helical filaments of tau (PHFt) and NFT formation²⁵. 

Is aluminum linked to NFTs in neurons?

In 1973 levels of aluminum were found to be higher than normal in some regions of the brains of Alzheimer patients²⁶. This finding remained controversial until 1980 when a combination scanning electron microscope and x-ray spectrometer analysis showed there was aluminum in neurons with NFTs in the brains of both Alzheimer and elderly non-Alzheimer patients and no aluminum in adjacent neurons without NFTs²⁷.  Since NFTs are a hallmark of AD, this finding was the first to link aluminum to AD at the neuronal level.  Even more disturbing was the fact that aluminum and NFTs are in the neurons of the elderly in general not just those with an AD diagnosis.  These findings are consistent with research that finds aluminum in the brains of the elderly in general^28,29. 

Aluminum is a non-essential cation in our brains and an unwanted intruder.  It is obvious Watson, since aluminum promotes the formation of paired helical filaments of tau and NFTs, NFTs are at best only a secondary cause or symptom and not the primary cause of Alzheimer’s.

But Watson was looking skeptical and asked: “Since aluminum is looking more and more like the culprit in the case of strange death, why do people with the apoE4 gene have a 50% higher chance of getting AD than people without this gene?”

Holmes replied: Carriers of the apoE4 gene have lived with higher levels of Aβ peptides, oligomers, and plaque for thousands of years and not had AD.  There must be an environmental factor that facilitates the formation of Aβ peptides, oligomers, or plaque to cause AD”.    

Watson asked: “Could this environmental factor be aluminum?”

Yes Watson aluminum is a likely environmental casual factor of AD. Metal cations such as aluminum, copper, zinc, and iron are known to complex with Aβ peptides to form Aβ oligomers^16,17.  However, only aluminum complexes of Aβ oligomers are neurotoxic¹⁶.  Aluminum makes the degree of neurotoxicity worse because it has the unique property of “freezing” Aβ peptides in the oligomeric state, resulting in high concentrations of Aβ oligomers¹⁶. When these Aβ oligomers are complexed with aluminum they cause excess calcium to diffuse into neurons which can ultimately kill them.  Therefore aluminum can cause AD both with and without the apoE4 gene but is 50% more likely to cause AD in carriers of the apoE4 gene. This is due to the carriers having higher levels of Aβ peptides in their brains making it possible for aluminum to freeze more Aβ peptides in the neurotoxic oligomeric state.

Aluminum acts in five ways to increase the accumulation of Aβ oligomers in the brain putting carriers of the apoE4 gene at greater risk of getting AD:

·         Aluminum freezes Aβ peptides in the neurotoxic oligomeric state¹⁶.  

·         Aluminum lowers gene expression of neprilysin, an enzyme that is the rate limiting step in Aβ peptide and oligomer degradation^30,31.

·         Aluminum lowers gene expression of the LDL receptor LRP1, required for Aβ peptide clearance and the importation of cholesterol to neurons^30,32.

·         Aluminum increases gene expression of BACE1, the β-secretase enzyme that cleaves the amyloid precursor protein (APP) to the precursor of Aβ peptides^30,31.

·         Aluminum increases gene expression for the production of APP, the precursor of Aβ peptides³¹.

Those with the apoE4 gene are also more likely to get AD due to head trauma or aluminum accumulation leading to an ischemic event, such as stroke.  Ischemia occurs when blood flow is temporarily suspended to some regions of the brain. Sporadic AD is believed to be associated in some cases with an ischemic event³³.  Post mortem analysis of the brains of those with AD has shown that 30% have evidence of an ischemic event³³.  Shortly after an ischemic event there is increased genetic expression of an apoE protein, such as apoE4³³.  Also there is both an increased genetic expression of amyloid precursor protein (APP) and the enzyme β-secretase (BACE1) that cleaves APP to the precursor of Aβ peptides³³.  Those with the apoE4 gene have slower Aβ peptide clearance than normal.  Because of this, after an ischemic event there is even less clearance than normal in the brains of those with the apoE4 gene and more production of Aβ peptides and Aβ oligomers. 

Accumulation of aluminum in the brain increases the toxicity of Aβ oligomers, the amount of aluminum-complexed Aβ oligomers, and the likelihood of an ischemic event (see Chapter 2).   Aluminum is a likely environmental causal factor for both toxic Aβ oligomers and ischemic events and puts people with the apoE4 gene at greater risk than normal for AD.

Watson, this is the second crux of the case. Since Aβ oligomers are only a facilitating factor for AD they can be removed from our list of suspects.

Watson looked alarmed for the first time and asked:  “Is there aluminum in my brain that is ‘freezing’ neurotoxic Aβ peptides in the oligomeric state?”  Holmes replied “I am afraid that we are all suffering from some frozen neurotoxic Aβ oligomers. But luckily we can control the amount of aluminum we ingest, absorb, and excrete. As you have sensed Watson, the sum total of this research has now reached a tipping point”.

Aluminum is the only remaining suspect for causing the case of strange death.  But before we can be certain that aluminum is the culprit we need to find if aluminum has the means and motive to cause AD in the case of strange death.  

Chapter 1 Part 2 Is there a genetic defect that could be responsible for AD ?

Is there a genetic defect that could be responsible for AD?  Yes Watson, there are two types of AD: familial and sporadic. Familial AD is believed to have a genetic link but accounts for only 1-2% of AD cases⁹.  Sporadic AD accounts for the remaining 98-99% of AD cases and after extensive searching only the apoE4 gene has been found to increase the risk for sporadic AD⁹.  Carriers of the apoE4 gene, about 20% of the population, have a 60% chance over age 80 of having AD versus a 10% overall risk of AD over age 80 in the general population.  Therefore the difference in risk for AD between carriers and non-carriers of this gene is 50%. In 2002 there were 9 million people in the U.S. who were 80 years of age or older¹⁰.  A 50% chance of AD in the 20% of those over 80 with the apoE4 gene corresponds to 10% of 9 million people or 0.9 million cases of AD.  There were 2.7 million AD cases in the U.S. in 2002¹¹.  This means approximately 1/3 (33%) of the sporadic AD cases in the U.S. could be due to genetics involving the apoE4 gene. 

Carriers of the apoE4 gene are more vulnerable to AD because they have higher than normal levels of Aβ peptides that can result in higher levels of Aβ oligomers and Aβ plaques. ApoE proteins are chaperones that complex with Aβ peptides and facilitate there transfer across the blood-brain-barrier and out the brain.  Carriers of the apoE4 gene may be more vulnerable to AD because they produce an Aβ peptide chaperone molecule apolipoprotein E4 (apoE4).  This chaperone works with a slow receptor that slows Aβ peptide clearance from the brain¹².  Also carriers of the apoE4 gene produce have less apoE protein in their serum and this also slows Aβ peptide clearance from the brain⁹. Therefore carriers of the apoE4 gene have higher than normal levels of Aβ peptides in their brains that can result in higher levels of Aβ oligomers and plaques in their brains.

Is there any other indication that AD is caused by a genetic defect? In 1987 L.E. Nee, et al. reported a clinical and family study of 22 twin pairs in which one or both twins had AD.    In approximately 40% of the cases, AD affects both twins showing that about 40% of cases in the U.S. could be due to genetics (i.e. the apoE4 gene) ¹³.  Within experimental errors this 40% agrees with the 33% derived from the math based upon the frequency of the apoE4 gene in the U.S. population. 

So as you can see Watson with the data agreeing from a risk analysis and a study of twin pairs we can conclude that greater than 50% of AD is not related to genetics and more than likely caused by environmental factors.  But before turning our back on genetics, could it be possible there is a gene other than the apoe4 gene responsible for AD? 

Could any dominant gene or genes spread through the population and cause a modern disease? If a dominant gene is inherited from either parent it is always expressed in that parent’s children. For a recessive gene to be passed on, both parents must have it, so dominant genes spread more quickly through a population than recessive genes.    

As we now know Watson, Alzheimer’s disease is a modern disease first observed 110 years ago in a person 56 years old at the time of death. Since Alzheimer’s is a modern disease, a mutation, such as the apoE4 gene, that could cause Alzheimer’s would have had to occur approximately 180 years ago. A human generation lasted, on average, 30 years during the last 200 years¹⁴.  A period of 180 years corresponds to 6 generations.  But how many generations did this gene have to spread through the population by the time people were born who are currently over 80 years old? The 9 million people in the U.S. over 80 were born after approximately 3 of those 6 generations.  Therefore the gene carrying the mutation would have had only 3 generations to spread through the population before those people currently over 80 were born.

Watson asked: “How many people can potentially carry a dominant gene three generations after the single mutation that created the gene?” Holmes answered: “If there was a single mutant and every one of the three succeeding generations had both a dominant gene and eight children, there would be only 512 carriers of the gene worldwide after three generations.  As already mentioned there are 9 million people over age 80 in the U.S. and 20% (1.8 million) of them are carriers of the apo4 gene. The calculated 512 carriers are not even close to the estimated 1.8 million carriers of this gene in the U.S. population over age 80.  So a mutation 180 years ago of the apo4 gene, or for that matter any gene, could not be the cause of AD”. 

At this point Holmes turned and noticed that Watson was madly calculating 8 x 8 x 8 = 512 on a sheet of scratch paper and looking confused.  Watson looked up and asked: “So can a modern disease as common as AD be due to a recent mutation?”  Holmes replied: “No, it takes thousands of years to spread even a dominant mutation to a significant percentage of the population”.

Since AD is a modern disease and not an ancient disease we now know three important facts from which we can draw a conclusion:

·         Because of its prevalence the apoE4 gene as it exists today is an ancient gene not a modern gene

·         Twenty percent of humans who have the apoE4gene have been living with higher than normal levels of Aβ peptides in their brains for thousands of years and did not get AD.

·         Only one third of those with AD in the U.S. have the apoE4 gene.

This is the first crux of the case Watson! Because the apoE4 gene is ancient it alone can’t be the cause of a modern disease like AD. Since the apoE4 gene causes higher than normal levels of Aβ peptides, a good percentage of people have lived with these high levels of Aβ peptides for thousands of years and not gotten AD. Therefore excessive levels of these Aβ peptides can’t cause a modern disease like AD.  

Watson looked startled and said: “Amazing Holmes this proves that AD is not caused by the ancient apoE4 gene or a recent genetic mutation or even higher than normal levels of Aβ peptides in the brain. So if not genetics and Aβ peptides, then what is the cause of AD?”  

 Is AD caused by high levels of Aβ plaque?

Watson noted: “Aβ plaque is a hallmark of AD and it is found in higher than normal levels in the brain of the case of strange death.  This puts Aβ plaque on the list of suspects. But is there any evidence that Aβ plaque is harmful to the brain?” Holmes replied: “No, AD-related neuron loss and dementia are mediated by Aβ oligomers, not Aβ plaque⁵³⁶.  This is because Aβ oligomers inhibit neuronal viability ten times more that Aβ plaque and forty times more than Aβ peptides¹⁵.  Soluble Aβ oligomers (e.g. clusters of soluble Aβ peptides) are converted in the brain to insoluble Aβ plaque¹⁶.  Metals, such as aluminum, facilitate the conversion of Aβ peptides to Aβ oligomers and then to Aβ plaque^16,17.   Aβ oligomers have been isolated from the cerebral cortex and cerebrospinal fluid in concentrations six times higher than normal in the brains of AD patients¹⁸”. 

Watson then suggested: “Since these findings suggest that Aβ oligomers rather than Aβ plaque might be a cause of AD, we should add Aβ oligomers to our list of suspects. After all it would appear that Aβ oligomers had to be present in order to make Aβ plaque observed in the brain of the case of strange death.” 

Holmes replied: “Excellent deduction Watson, our new list of suspects now includes:

·         NFTs

·         Aβ oligomers

·         Aluminum”

Chapter 1 Part 1 What Causes Alzheimer's ? The Case of the Cloaked Assassin

Chapter 1 – Alzheimer’s Disease

“… anatomically it provided a result which departed from all previously known disease pathology.”  L. Alzheimer 1907¹

What Causes Alzheimer’s Disease?

The book begins as I did, searching for the cause of Alzheimer’s. I initiated this search when simultaneously my mother and a friend’s mother began having panic attacks due to short term memory failure at age 85. The progression of short term memory loss had been slow and steady and was just beginning to impact their daily living skills.  Routine things they had done on a daily basis, such as grocery shopping, balancing the checkbook, and sewing, had become impossible tasks. I hoped that learning what was known about the cause of Alzheimer’s would allow me to help these women slow the progression of the disease and possibly prevent further erosion of their memories.  

I quickly discovered that finding the cause versus symptom of a disease like Alzheimer’s is a tricky business.  It takes a sleuth with the knowledge and cunning of Sherlock Holmes.  Being a chemist I have always had a fascination with Mr. Holmes, probably because he was portrayed as a chemist by Sir Arthur Conan Doyle. Young Stamford said Holmes was a “first-class chemist” when he first introduced Watson to Holmes in “A Study in Scarlet”.  In order to understand my search for a cause of Alzheimer’s (AD) please follow Mr. Holmes and Doctor Watson and read “The Case of the Cloaked Assassin”.

The Case of the Cloaked Assassin

Watson we may have a killer loose on the streets!  I just read on the internet in the Journal of Medical Case Reports 8:41 (2014) of the strange death of a 58 year old man with no prior medical history who was diagnosed with early-onset AD.  Ten years prior to his diagnosis he began working on a daily basis handling alum dust in order to develop a new insulation for the nuclear and space industry.  He was working with minimal respiratory protection.  After six years performing this work he began suffering from memory loss and depression.  In 2011, after his death due to AD at age 66, the frontal lobe of his brain was analyzed for aluminum.  It contained approximately 3mcg (3 micrograms) of aluminum per gram of dried brain tissue (3mcg/gr. dry wt.)².  This is more than three times higher than the median concentration of aluminum (e.g. 0.87mcg/gr. dry wt.) found after death in the frontal lobes of people 60-70 years old³.  In addition, an abundance of beta-amyloid (Aβ) plaque and a profusion of neurofibrillary tangles (NFTs), hallmarks of AD, were found in his frontal cortex. 

So Watson what is coincidence and what caused AD in this case of strange death?  There are four possibilities:

·         The victim was genetically predisposed toward AD

·         Aβ plaques were the cause of AD

·         NFTs were the cause of AD

·         Alum was the cause of AD

The first question – what are Aβ plaques and NFTs? These plaques and NFTs are insoluble protein molecules that are three dimensional aggregates of smaller soluble molecules called peptides and oligomers that are comprised of amino acids.  An amino acid is a molecule with at least one each of an amine and acid.

Amyloid beta (Aβ) plaques are made from Aβ oligomers that are aggregates of Aβ peptides that are in turn fragments enzymatically cleaved from a larger protein called amyloid precursor protein (APP). 

NFTs are filaments of a phosphoprotein called tau that stabilizes structural elements in neurons called microtubules. Tau has 79 potential sites for phosphorylation. Thirty of these sites are usually phosphorylated in normal tau proteins giving them an overcoat of phosphorus, oxygen and hydrogen. The tau in NFTs is more highly phosphorylated than normal and is called hyper-phosphorylated tau.      

The second question - what is in alum?  Alum is a combination of aluminum and sulfate ions making a salt. In some cases alum salts also include other ions such as in ammonium alum (a.k.a. ammonium aluminum sulphate), and potassium alum (a.k.a. potassium aluminum sulphate, potash alum). 

After hearing this definition of alum as being a salt of aluminium sulphate, Doctor Watson became agitated and said: “Wait just a minute Holmes, as a medical doctor I believe aluminium should not be on the list of suspects for AD!” Watson pointed out that according to the Alzheimer’s Association “… studies have failed to confirm any role for aluminum in causing Alzheimer’s”⁴. Holmes turned to Watson and replied: I applaud you, your profession, and the Alzheimer’s Association for their dedication to caring for and supporting those with illnesses and their mission to eliminate Alzheimer’s disease through the advancement of research and the promotion of brain health. But historically there have been examples when the medical profession has not been sufficiently open-minded to explore all the potential causes of a disease.

One such example of a missed opportunity to save lives was the prevention of pellagra. Pellagra results in dementia, painful disfigurement, and death and is now known to be caused by a lack of niacin in the diet.  Niacin is concentrated in the outer-most layer of the endosperm and germ layers of the corn kernel that are removed by milling. In 1901 the Beall Degerminator was patented that could mill the corn removing the outer endosperm and germ layers from the kernels for improved storage. In the southern part of the U.S. between 1906 and 1940 mechanical corn milling resulted in 3 million people sickened by pellagra and more than 100,000 deaths due to pellagra. In 1913 Casmir Funk wrote an article suggested that the new procedure for corn milling was causing pellagra, but he was ignored⁵. The medical profession in the U.S. firmly believed a toxin in rancid unripe corn was the cause of pellagra⁶. It took a number of years after the connection between niacin deficiency and pellagra was discovered for the medical profession to finally abandon their toxin theory.  So as you can see Watson we should remain open-minded and leave aluminium on the list of suspects for AD so we do not repeat any missed opportunities to save lives.

Watson, who was still pondering this dilemma regarding aluminium as a suspect, responded: “I am all for saving lives and I know that aluminum is a proven to be toxic to neurons but I have read and believe the following:”

·         Aluminum compounds are inert or insoluble preventing entry into the body.

·         Any aluminum that does get into the body is immediately excreted.

·         Any aluminum that is not excreted is deposited in biologically inert stores such as bone.

Dear Watson we may find comfort in believing this dogma but these are untrue myths that give us a false sense of security.  Aluminum can cloak itself in a variety of chemical disguises and enter the body through the gut, skin, lung, and nose.  Acidification of aluminum compounds in the environment by acid rain and in the gut by stomach acid, solubilizes these compounds and allows aluminum the freedom to change its chemical disguise. Once in the body some of the disguised aluminum finds its way to the brain and accumulates in our brain during our lifetime.   I know these facts are unpleasant to deal with, but facing reality is better than keeping our heads in the sand.  So Watson let’s proceed ahead with aluminum on the suspect list and see where this investigation takes us.  

 The third question that begs an answer is how old is Alzheimer’s disease?  Is AD an ancient disease that happened to finally get a diagnosis or is it a modern disease?  At age 35 in 1901 Doctor Alois Alzheimer began observing a patient named Auguste Deter in a Frankfort Germany asylum.  She had strange behavioral symptoms and had lost her short term memory.  After she died in 1906 her brain was autopsied by Dr. Alzheimer. Using special stains he found amyloid plaques and neurofibrillary tangles. So let’s ask the doctor himself if AD is a modern disease.  After an exchange of letters, Holmes had the answer. In a translation of Dr. Alzheimer’s words:

“The case presented even in the clinic such a different picture, that it could not be categorized under known disease headings, and also anatomically it provided a result which departed from all previously known disease pathology”¹.

Doctor Alzheimer believed AD is a modern disease but did anyone else share his opinion?  The mental health of older people living between 1886 and 1889 is described in the monograph “Old Age” published in Cambridge England in 1889⁷.  The monograph summarizes the results of British general practitioners studying the mental health of their oldest patients during the mid-1880s.  The study group was 900 subjects who were 80 or older including 74 centenarians.  The monograph’s author concludes that dementia:

 “… was witnessed only in two of our centenarians … indeed the brain held out as well or better than other organs” 

This 3% rate of dementia among centenarians in the mid-1880s can be compared with a study 111 years later.  In 2000 a study was done of people in three Dutch towns with populations greater than 250,000.  Of the 17 centenarians found in this more recent study 15 had dementia for an 88% rate of dementia.  The other 2 centenarians could not be examined⁸. 

From Doctor Alzheimer’s comments in 1907 and comparing these two studies of dementia among centenarians done 111 years apart, it is obvious Watson that AD is a modern disease that is between 110 and 180 years old. So now Watson let us look at the first suspect: genetics.