Section I
APOE4 and the history of human disease
Apolipoprotein E, discovered in 1973, is derived from a polymorphic gene called ApoE that encodes for a signaling protein on a class of fatty proteins that primarily carry cholesterol and other molecules around the body.
We find ApoE primarily on VLDL, very low-density lipoproteins and HDL, High density lipoproteins. It primarily helps to shuttle cholesterol from the periphery of the body back to the liver. ApoE proteins are also involved in neuronal signaling, neuroinflammation and glucose metabolism making them especially critical for brain activity. This is becoming a critical part of the story for understanding dementia and neurodegeneration. (Williams T. 2020) ApoE is most famous for an isoform, APOE4/4, being highly associated with Alzheimer’s dementia in modernity. Unfortunately, as we do with many things in medicine, we have been focused on this genetic protein variant as a net negative in humans because we look at things as black and white. Either good or bad. A binary choice. Rubish!
As everyone knows, I do not believe in such things. There is no reason that genetic selection would have made this lipoprotein so prevalent if it did not serve a purpose in mammals. What is that purpose? The science is coming into light in the last 10 years and we need to understand it in order to make lifestyle decisions that can lead to longevity and a healthspan that is admirable
The full APOE protein has three isoforms that can be combined to make 5 combinations: APOE: 2/2, 2/3,3/3, 3/4, 4/4. There is a reason for this isoform variance that we are now starting to understand. It turns out that this “negative” ApoE4 isoform is incredibly valuable if you live in a region with high infectious disease burdens like equatorial Brazil, Bolivia and Congo. When the researchers began to look at these isoforms across the globe they noted a peculiar grouping of isoform 4 around the equator and no isoform 2 there. A deeper dive that we will explore today is the fact that the fourth isoform is highly protective against infectious disease burdens. We will also learn that the ApoE protein modifies the intestinal microbiome, modulates immune activity and protects the body from low level endotoxemia caused by gram negative bacterial cell wall translocation, from the likes of E. coli, into the bloodstream via the gut. This translocation is very immune stimulating and pro inflammation and believed to be at the center of the dementia story. (Freitas et. al. 2021)
Let us look at a binary that is useful. What are the pros and cons of the APOE4 carrier isoform or 4/4 status?
The pros are: increased fertility and size of the offspring, improved cognitive ability while under a significant infectious disease burden (survival advantage), improved innate immune function against enteric pathogens and reduced systemic inflammation, an increased ability to shift cholesterol away from pathogens and towards the brain, enhanced intestinal barrier and microbiome function and makeup.
The cons are: increased risk for neurodegenerative diseases like Alzheimers as we age, increased COVID19 disease burden, increased cardiovascular disease risk, increased metabolic syndrome, increased chronic inflammatory responses. (Freitas et. al. 2021)
These knowns are completely expected and wanted when contextualized to our ancestral history. What was a net benefit is now a net negative due to a mismatch of what is happening to us from an environmental perspective and especially from an infectious burden perspective. For millions of years, a primary directive for humans was survival against pathogens, predators and trying to find nourishment for survival and procreation. It is only within the last 100 years or so that this reality has flipped on its head.
“In populations with frequent exposure to diverse pathogens and who eat energy-restricted diets, high lipid levels would be important to boost innate responses against these pathogens. Such benefits would disappear in post-industrialized populations most exposed to high-calorie Western diets and without the energy delivery needed to fight heavy burdens of recurrent pathogens, thus causing chronic systemic inflammation and lifelong predisposition to aging-related chronic diseases.” (Freitas et. al. 2021)
ApoE has the ability to modulate immune activity as well as increase cholesterol usage to make new synapses which will be discussed later. It specifically has the ability to take degraded bacterial cell wall debris called lipopolysaccharide out of the blood stream and return it to the liver for clearance. This is super important in an infectious world. This protein’s function can be the difference between death or life when one has bacterial sepsis, a blood infection. Multiple translational animal studies have shown that ApoE knockout mice are much more susceptible to infections than ApoE containing mice. We know that an ApoE4 carrier has an increased survival rate when infected with a serious bacterium that kills. Thus, it leaves us thinking that this genetic variant has continued to exist as procreation has occurred before the gene causes damage at the advanced age that they are doing so. The genetic advantage outweighs the long term consequence. At a species level, to be able to procreate effectively and live into the 50s and 60s is perfectly fine. Not so much for us individually that are aging however. (Yassine et. al. 2020)
If we look at this ApoE gene from another perspective, there is growing evidence that intestinal dysbiosis or unbalanced bacteria that reside within our intestines are causing increased pathogenic gram-negative rod bacterial translocation from the intestine into the systemic circulation/blood stream and eventually making their way to the brain. These bacteria and bacterial cell wall debris are intensely immune stimulating if they cross the blood brain barrier and enter the brain tissue. This is a major area of study as this innate immune stimulation, if occurring in the brain, is very dysfunctional.
Let us look in the brain where things get super interesting. ApoE is the major cholesterol transporter in the brain leading to beneficial functions in the brain. Cholesterol is critical for synapse formation, migration, myelin insulation and maturation as well as the regulation of signal transduction through its function as a component of the cell membrane. 20% of the body’s cholesterol is in the brain. (Van Vliet P. 2012) Thus, ApoE is a major player in the movement of another major player, cholesterol.
Let us look deeper at cholesterol first: Biologically, cholesterol is a very expensive molecule to produce as it costs 36 moles of ATP to generate. Therefore, it would not make sense to waste it when it is not needed in an area. The body would want to recirculate it to other body sites that are in need, especially when energy levels are low or during fasting/famine. Cholesterol is a molecule that is a critical piece of the following vital cellular structures: steroid hormones, all cell membranes throughout the body (especially the brain), bile acids and vitamin D as well as the brain actions as stated. It is essential and necessary for human survival. We can obtain cholesterol through our diet as a poorly absorbed cholesterol ester when we consume animal products. Cholesterol is also naturally produced in all mammalian cells throughout the body but primarily in the liver in response to low cholesterol levels in the bloodstream. However, this process to make cholesterol requires energy as ATP and the fed state. Thus, the primary source of all cholesterol in the body is not dietary cholesterol as it makes up less than 25% of our cholesterol volume. Plants do not have cholesterol, therefore consuming them has no effect on levels. They have a special plant type sterol called a phyto-sterol which humans do not use. Cholesterol cannot move freely around the blood stream; therefore, it has to be transported around the body in lipoproteins like the LDL or low-density lipoprotein. I like to think of these fat proteins as cholesterol carrying "cars." These cars are going to be critical to understanding heart and neurodegenerative disease as they travel around our circulation highways with the intent of delivering the cholesterol package to the needed cell membrane, hormone or bile acid.
The lipoprotein cars are combinations of fat and protein that aggregate together to carry triglycerides and cholesterol. These lipoproteins have the major job of transporting energy as fats called triglycerides around the body to tissues. The cholesterol carrying issue is a secondary event. There are many different types of cars and they are named by the volume of triglycerides that are carried on board. The large lipoproteins are called chylomicrons, buses, and as they lose triglycerides at different cells they shrink and concentrate more cholesterol and become low density lipoproteins, cars. The LDL protein cars are called the bad lipoprotein/cholesterol. Again, I find it hard to believe that anything in the body is inherently bad or good! In no way does it make evolutionary sense for God to put a bad thing in our body.
However, the volume of any lipoprotein particle could convert it to an unbalanced state which makes it unhealthy. That I believe. I think that this is truth.
These lipoproteins have signaling molecules attached to them. Apolipoprotein A is found on all high density lipoproteins. Apolipoprotein B is found on low, very low and intermediate density molecules. Apolipoprotein E can be on both lipoprotein types. These signaling molecules are how the function and movement of the lipoproteins takes shape. The proinflammatory isoforms like Apo E4, Apo (a), Apo C3 and others are all protective in an infectious burdened environment, but pro dementia and heart disease in a western food based low infectious burdened environment. This is the main fascinating twist on the cardiovascular story. No mistakes here people!
Part of this reason rests with the intestinal gut bacterial changes that are occurring in the modern environment of highly processed foods laden with flour, sugar and trans or saturated fats that feed that gram-negative rods and bacteria growing their volume and number triggering the lipoprotein system to activate and clear the translocated lipopolysaccharides in the blood stream. There is also evidence that mental stress, toxin exposure, antibiotic use and other common events are similarly disrupting the biodiversity of the microbiome of the gut and intestinal permeability. The chronic leaky gut may facilitate gut-to-blood increased pathogenic bacterial translocation and systemic inflammation. Lipopolysaccharides (LPS) may induce innate immune neutrophil and monocyte activation and the subsequent release of myeloperoxidase, a bactericidal enzyme. This enzyme and other chemicals released may injure brain capillary endothelial cells leading to blood-brain barrier disruption with an end result being apathway toward inflammation and neurodegeneration. (Freitas et. al. 2021) Again, if these lipoprotein cells are pathologically active in the heart vessel, we develop coronary vascular disease. If these lipoprotein cells are pathologically active in the brain, they can trigger neurodegeneration.
Let us recap: We now know that these metaphorical cars can bind to and eliminate viruses, bacterial cell wall debris and parasites throughout the body as a first line of defense, innate immunity. The gut microbiome harbors trillions of good and bad bacteria and their respective cell wall components like LPS/lipopolysaccharides as stated. We know from the amazing work of Patrice Cani and Alessio Fasano that the bacterial cell wall debris from a dysfunctional intestinal microbiome can translocate into the bloodstream and circulate around the body causing inflammation, called low level endotoxemia. (Amar J. et. al. 2008) (Cani P. et. al. 2008) The lipoprotein cars have binding sites, "trunks", to grab the LPS and other pathogenic bacterial endotoxins and transport them back to the liver for excretion in bile and therefore your stool. (Harris et. al. 1990) This is a very advantageous mechanism for survival, if you are in a highly infectious environment like a third world country. (Read et. al. 1993)
Let us look at this infection fighting reality in study. Let us say that we have two sets of rats and infect them both with a deadly dose of a bacteria called E. coli, a gram negative rod, while simultaneously giving one group an infusion of these protective lipoproteins. In the end, you will have most of the lipoprotein infused group relaxed and alive while the other group has died from the infection. (Harris et. al. 1993) It turns out that HDL cholesterol gets used up trying to clear the acute inflammation and infection. As the HDL level drops, LDL cholesterol and triglyceride levels rise as the next line of defense. As the infection clears, we would expect the HDL and LDL levels to flip flop back to normal. This appears to happen and is an amazing reality.
Now that we have reviewed lipoproteins in general, back to signaling molecule Apolipoprotein E4 and the brain story. If ApoE is important in cholesterol efflux and the production of synapses, then it would follow that defects in ApoE activity could worsen synapse formation and neurological function over time. Specifically, ApoE is made by astrocytes with receptors for ApoE on the neurons. It is a process by which increased ApoE molecules will increase the transport and usage of cholesterol in the brain. It is also known that a blockade of the ApoE receptor decreases cholesterol drop off and synaptogenesis making it a question mark in the benefit of Apo E isoforms for brain function. It appears that the ApoE2 isoform has the most activity here whereas ApoE4 has the least. ApoE2 and ApoE3 preferentially interact with high-density lipoproteins. ApoE4 preferentially interacts with triglyceride-enriched lipoproteins like very low-density lipoproteins. Another distinction is that ApoE2 has the lowest binding affinity for low-density lipoprotein receptors while ApoE4 has the highest. (Williams T. 2020) In the periphery, i.e. not in the brain, the ApoE 2/3 isoforms are carrying cholesterol efficiently back to the liver for recycling.
Along the lines of recycling, the ApoE4 isoform is less efficiently recycled by the LDL receptors in the liver leading to enhanced innate immunity but also with a metabolic drawback. “Reduced recycling of apoE4 affects its cellular energy source preferences. ApoE complexes with the Insulin receptor (IR) and reduced apoE recycling trap the IR in the endosomes away from the cell surface. This reduction in IR surface expression causes reduced utilization of glucose to generate ATP and promotes fatty acid oxidation. Neuronal cell lines expressing APOE2 have more hexokinase, a critical enzyme of glycolysis, which yields a more efficient production of energy from glucose. Neuronal cell lines expressing APOE4, on the other hand, have lower hexokinase activity. Also, human APOE2 expressing immortalized astrocytes have a 2.5-fold greater glucose uptake, while APOE4 astrocytes have half the glucose uptake capacity of APOE3.” “Reduced recycling of apoE4 also affects cellular cholesterol metabolism. ApoE4 traps ATP binding cassette 1 or ABCA1 in endosomes away from the cell surface. Reduced ABCA1 activity results in lower cholesterol efflux to HDL and redistributes cholesterol to cell membranes. In macrophages, greater membrane cholesterol is associated with activated TLR4 signaling, which, in turn, induces NFkB and inflammatory gene responses. A greater distribution of cholesterol to the neuronal plasma membrane promotes BACE1 expression and APP processing to produce more Aβ-amyloid peptide. In microglia and astrocytes, less cholesterol efflux reduces Aβ degradation. “(Yassine et. al. 2020)
Nutrition appears to be heavily involved in the outcomes of modern ApoE4 carriers. As ApoE is expressed on fat cells throughout the body, it would follow that whatever drives up adiposity will have a major role in ApoE4 biology. Here we fall back again on flour, sugar, trans and high volume animal fats as root causes of dysfunction in modernity. You can think of it as a replete diet from a calorie perspective, but the brain will be starved of glucose sugar due to the ApoE 4 biology causeing intracerebral insulin resistance. This is such a paradox of human biology of modernity. Overweight individuals with peripheral insulin resistance will have peripheral cellular starvation while the ApoE4 carrier can have this reality occurring in the brain at the overweight or normal weight state. One complete antagonist to this reality on both ends of the spectrum is exercise. Exercise is the ultimate fighter of this ApoE4 carrier biology.
This is some incredible science emerging whereby the innate immunology of the ApoE4 carrier has enhanced infection fighting skills peripherally and, in the brain, but at the cost of the ability to prevent neurodegeneration with age in modernity. Researchers have demonstrated that ApoE4 can trap ABCA1 endosomes causing lower cholesterol transport and an increase in intracellular and plasma membrane cholesterol content which affects amyloid processing and increases inflammasome activation. Inflammasome activation and toll like receptor signaling are enhanced which is amazing for the ability to survive infection, but they are not great now. The reduced recycling of ABCA1, the discussed neuronal insulin resistance, and other mechanisms to be discovered help us understand the accelerated brain aging phenotype observed over the past 50 years. (Yassine et. al. 2020)
ApoE4 carriers are known to preferentially oxidize fatty acids for neuronal fuel in the brain due to the mild insulin resistance as stated already. The work of Dale Bredesen at the Buck Institute has noted an improvement in cognition in study in individuals with neurodegenerative issues. Thus, the type and volume of fat is going to be the future study locale.
This is just my first deep look at ApoE4 biology and the positive and negative aspects of it in context of modernity. Much more to come in the coming years. At this point, I think that it is safe to say that refined sugar and flour are net negatives for neurodegeneration for ApoE4 carriers especially, but for all of us in general. Poor quality fats from corn fed cows, pigs and other CAFU animals are a net negative based on inflammatory risk.
Round one for ApoE in the books.
Have a great week,
Dr. M
Williams Molecular Neurodegenration
