ApoE gene and Alzheimer's disease (2)

There are three common variants of ApoE gene, which are E2, E3 and E4

There is only one nucleotide difference between them

Type E3 is the most common, accounting for 65-70%, while type E4 accounts for 15-20%, and type E2 accounts for 5-10%

Type E3 is considered as normal type

Compared with type E3, type E4 significantly increases the risk of Alzheimer's disease, while type E2 slightly decreases the risk

What happened first in terms of molecular biology and protein structure? There are two important functional regions in ApoE protein molecule: LDL receptor binding domain and lipid binding domain

The receptor binding region is located in the middle of the protein, rich in positively charged basic amino acids, which is conducive to binding with negatively charged cell surface receptors

The fat binding region is located in the tail (C-terminal) of protein, which is rich in hydrophobic amino acids

The E4 and E2 variants of ApoE gene showed amino acid changes in two positions: E4 variant changed the position 130 from Cys to Arg, while E2 variant changed the position 176 from Arg to Cys

In other words, the normal E3 protein apoE3 is 130cys, 176arg; the high-risk E4 protein apoE4 is 130arg, 176arg; and the low-risk E2 protein apoe2 is 130cys, 176cy

What are the functional changes caused by these amino acid changes? First, simply from the amino acid level, we can see that apoE4 added a positively charged amino acid Arg at position 130, while apoe2 lost a positively charged amino acid Arg at position 176

Interestingly, sites 130 and 176 are located on both sides of the receptor binding region of apoE protein, respectively

The core region of apoE receptor binding is located at 152-168, which is rich in positively charged basic amino acids for binding to receptors rich in negatively charged amino acids

Apoe2 protein lost a positively charged amino acid near the receptor binding region

The results showed that the binding ability of apoe2 and its LDLR receptor on hepatocytes decreased greatly, only 2% of that of normal apoE3

However, apoE4 protein increases a positively charged amino acid slightly away from the receptor binding region, and the binding ability of apoE4 to the receptor is slightly higher than that of normal apoE3

Therefore, if the ability of binding to the receptor is used to evaluate the functional change, apoE3 is normal, apoe2 is severely inactivated, and apoE4 is weakly inactivated

In the population, we have observed that people with E2 / E2 homozygous mutation are prone to a disease called type III hyperlipidemia

The important feature of this disease is that chylomicrons and other lipoprotein residues in the blood can not be removed by the liver cells, so it is also called residual metabolic disease

Further observation also found that the loss of positive charge base acid variation in the receptor binding region of several other apoE genes will also lead to familial type III hyperlipidemia, which further confirmed that the loss of positive charge in this region will systematically damage the binding energy of ApoE and its receptor

The binding ability of apoE to receptor is apoE4 > apoE3 > apoe2

From the point of view of receptor binding ability, it is difficult to explain why the E4 variation that only slightly increases receptor binding ability can significantly increase the risk of Alzheimer's disease, while the E2 variation that loses 90% of receptor binding ability can only slightly reduce the risk

Therefore, we conclude that the pathway of apoE binding to receptor may not be the main pathogenesis of Alzheimer's disease

Secondly, from the perspective of the change of three-dimensional structure of protein, we can see that the variation of E4 will cause new salt bond between the regions of apoE4 protein

The introduction of a positively charged amino acid Arg at position 130 will lead to a special new salt bond, which just locks the lipid binding region of apoE protein tail and makes this region lose flexibility, thus affecting the binding energy and selectivity between this region and lipid molecules

See schematic diagram

The experiment of replacing amino acids on apoE gene one by one showed that only the position of E4 mutation could change the structure of lipid binding region through salt bond

It is suggested that the conformational change of lipid binding region caused by E4 mutation is unique and special, and the change of amino acids in other positions is almost impossible to obtain similar conformational change

In real life, no other mutations in the ApoE gene increase the risk of Alzheimer's disease

Figure 6: three dimensional structure model of apoE protein

The lipid binding region of apoE4 tail is locked by salt bond, while the tail of apoE3 is relatively free, which is more compatible with cholesterol and phospholipid

Now let's see what functional changes might be caused by locking the tail lipid binding region due to E4 variation

The experimental data showed that the lipid content of apoE4 was 2-3 times less than that of normal apoE3

However, although the binding ability of apoe2 to its receptor was greatly reduced, its lipid content was not affected, which was similar to that of normal apoE3

In addition to the amount of fat, apoE4 preferred to be compatible with triglycerides, but decreased with cholesterol and phospholipids

Apoe2 and apoE3 are more compatible with cholesterol and phospholipids

From the perspective of lipoproteins' lipid content, apoe4apoe3 > > apoe2, it can be inferred that the stronger the binding ability of apoE to receptor, the stronger the ability of preventing soluble a β from clearing the brain through LRP1 channel, and vice versa

The evidence supporting the inhibition of soluble a β clearance by apoE also comes from the mouse gene knockout experiment (apoE knockout mouse)

When ApoE gene was removed from the Alzheimer's model mice, the amyloid plaques in the brain decreased significantly

In addition to the LRP1 channel, another LDL receptor related protein 2 (LRP2) expressed on the blood-brain barrier can transport insoluble a β binding to APOJ lipoproteins

LRP2 gene and clu gene encoding APOJ are risk genes of Alzheimer's disease

In addition to clearing a β through the blood-brain barrier, there are also a part of a β, especially insoluble a β, which may be transported into the cell through binding with APOE lipoproteins, and then transported into the cell through binding with lipoproteins receptor on the cell surface, and then degraded in the lysosomes within the cell

LDLR and LRP1 are expressed in astrocytes, microglia and nerve cells in the brain, which can be combined with APOE

In addition, microglia, as the immune cells in the brain, can also internalize amyloid plaques into the cells for digestion through non-specific endocytosis mechanism (phagocytosis)

ABCA7 and TREM2 genes related to endocytosis are also strongly associated with the risk of Alzheimer's disease

Figure 8: apoE lipoprotein particles secreted by astrocytes meet with a β short peptide secreted by nerve cells in extracellular matrix

Previously, we analyzed the changes of protein three-dimensional conformation caused by E4 variation

The variation of E4 changed the flexibility of the lipid binding region in the tail of the protein, which resulted in the poor binding of apoE4 with polar cholesterol molecules and phospholipids, resulting in a significant decrease in the lipid content of apoE4 lipoproteins

We can imagine that astrocytes synthesize apoE lipoproteins and secrete them out of the cell, and they are mixed with a β short peptide secreted by nerve cells and Co located in the intercellular substance

The lipid molecules on apoE lipoproteins, especially cholesterol, may be affinity with the hydrophobic amino acids exposed on the surface of a β short peptide, so as to break the balance point of self polymerization of a β short peptide and affect the amyloid Protein formation

Just like in the crystallization process when the solution concentration reaches the critical point, there is still a need for seed to trigger crystallization

If cholesterol molecules on apoE lipoproteins can interfere with the formation of "nucleation" in a β polymerization, even small changes in lipoproteins may significantly change the final production of a β polymerization

Let's first review that apoE is mainly synthesized by astrocytes, and HDL like lipoproteins are formed together with cholesterol and phospholipids and other oil molecules synthesized in astrocytes, and secreted to the outside of cells

Its main physiological function is considered to transport cholesterol and phospholipids for nerve cells, which are used for the maintenance and expansion of nerve cells

Cholesterol and phospholipid are polar oils, just like detergents, which can emulsify and fuse molecules with hydrophobic groups

It is not hard to imagine that the higher the APOE lipoproteins contain, the larger the capacity of emulsification fusion a β, and vice versa

In this way, we can explain why apoE4 has poor ability to dissolve the formation of "crystal nucleus" of a β polymerization because of its low fat content and small capacity of a β fusion

Therefore, a β is more likely to aggregate into amyloid plaques in the brain of people with E4 mutation

The experimental evidence shows that the addition of lipolized apoE in saturated a β solution can greatly delay the self polymerization of a β

Here, the role of apoE lipoproteins is to slow down the formation of polymeric nuclei

More experiments have shown that cholesterol molecules, rather than phospholipid molecules, can effectively block the polymerization of an amyloid protein called amylin in the pancreas

And lanosterol, the precursor of cholesterol, can effectively dissolve the insoluble proteins in cataract

These insoluble proteins, including a β amyloid, are all polymers formed by β lamellar structure

An important evidence supporting the APOE fat content hypothesis comes from the ABCA1 gene

The lipid level of apoE is mainly regulated by ABCA1 gene

ABCA1 is a kind of molecular pump, whose function is to guide cholesterol in cells to HDL like lipoproteins containing apoE or APOJ

Activation of ABCA1 expression can significantly increase the lipid content of apoE lipoproteins

The expression of ABCA1 gene is regulated by retinoid receptor x (RXR)

Retinoic acid is a derivative of vitamin A

both synthetic RXR antagonists and activated vitamin A can effectively activate the expression of ABCA1 gene

In the experiment of feeding the Alzheimer's model mice with RXR agonists, it was found that the amyloid plaques in the brain of the mice would start to be removed in large quantities a few hours after feeding RXR antagonists

In addition, the removal of amyloid protein induced by ABCA1 depends on the expression of ApoE gene, which does not play a role in ApoE gene knockout mice

Why is such an effective RXR agonist not used to treat people with Alzheimer's disease? Unfortunately, when RXR agonist was used in human clinical experiment, although the removal of amyloid plaques was also observed, but the agonist in a very short period of time caused extreme elevation of blood lipid in patients, so this human clinical experiment could not be stopped

Several other agonist drugs that can activate the expression of ABCA1 have the same results in Alzheimer's mice

ABCA1 gene mutation is closely related to Alzheimer's disease

It has been found that the inactivated mutation of ABCA1 gene increases the risk of Alzheimer's disease by 2-3 times, while the inactivated mutation of ABCA1 can reduce the risk

In addition to the above apoE amyloid hypothesis, many experiments have shown that the E4 mutation of ApoE gene increases the activity and inflammatory response of glial cells responsible for immune function in the brain

Other evidence suggests that E4 expressing neurons produce more a β 42, which is easier to aggregate

Moreover, the nerve cells with E4 are more sensitive to amyloid accumulation and more tolerant to apoptosis and necrosis

Interestingly, if people with E4 variant have concussion or brain injury during exercise, they will take longer to recover than those with normal E3 variant

reference: Bell RD, Sagare AP, Friedman AE, Zlokovic BV, et al

Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system

J Cereb Blood Flow Metab

2007 May;27(5):909-18

PMID: 17077814

Boehm-Cagan A, Bar