Portia Cortes

Alzheimer’s Disease (AD) is the most common cause of dementia seen among the elderly. Familial forms of AD do exist but majority of the patients has no family history and is therefore classified as having sporadic AD (Blennow, et.al., 1994; Saunders, et.al., 1993). The hippocampus is the most medial part of the temporal lobe of the brain’s cortex that forms the floor of the lateral ventricles (Gray, 1974). Its main function together with portions of the cerebrum, is to store memories (Guyton, 1992; Tortora, 1992). It has been shown in man and experimental animals that bilateral destruction and damage to areas associated with the hippocampus results in memory and learning impairment (Hyman, et.al., 1984). Such damage would practically isolate the hippocampus leading to contextual memory defect that is a major component of the amnesia in AD (Hyman, et.al., 1994). The definite etiology of sporadic AD is still largely unknown.

Apolipoprotein is a protein moiety in it lipid-free form as designated by the prefix, "apo". It combines with lipids to form lipoproteins (Mahley, 1988; Beffert, et.al., 1998). Some of these lipoproteins are regulators for extracellular enzymatic reactions involved in lipid metabolism while others serve as ligands for cell receptors that mediate the influx of lipoprotein particles (Brown,et.al., 1986; Beffert, et.al., 1998 Mahley, 1988;). Apolipoprotein E (apoE) has been extensively studied because it plays a key role in lipid metabolism. It facilitates cholesterol transport in and out of cells (Poirier, et.al., 1993). The importance of apoE in the central nervous system (CNS) became evident with the association of the e4 allele of apoE with familial and late-onset sporadic Alzheimer’s Disease (AD)(Poirier, 1996; Saunders et.al., 1993).

In general, apoE is involved in triglyceride, phospholipid, cholesteryl ester, and cholesterol transport in and out of cells (Lehninger, 1993; Mahley, 1988). It facilitates cholesterol removal from the plasma and cerebrospinal fluid (CSF) (Beffert, et.al., 1998; Blennow, et.al., 1994; Poirier, 1994, 1996). In the peripheral nervous system (PNS), it has been shown to assist in the mobilization and redistribution of cholesterol in repair, growth, and maintenance of myelin and neuronal membranes during development and injury (Ignatius, et.al., 1985; Mahley, 1988; Poirier, 1994). Studies using the rat brain have shown that in the CNS, apoE is important in the metabolism and redistribution of cholesterol and phospholipids during myelination and membrane remodeling associated with axonal regeneration (Beffert, et.al. 1998; Ignatius, et.al., 1996; Poirier, 1994).

Apolipoprotein E polymorphism

Apolipoprotein E is a constituent of several plasma lipoprotein such as chylomicrons, very low-density lipoproteins (VLDL), and high-density lipoproteins (HDL) (Beffert, et.al., 1998; Blennow, et.al., 1994; Lehninger, 1992:675-677; Poirier, 1996). The mature form of apoE in the human plasma and CSF is a single glycosylated 36-37 kDa polypeptide containing 299 amino acids (Ignatius, et.al., 1986; Mahley, 1988; Weisgraber, 1994).

A four-exon gene located in the long arm of chromosome 19 encodes for the human apoE (Octave, 1995; Permanne, et.al., 1997; Poirier, 1996; Rubinsztein, et.al., 1994). Isoelectric focusing was used to establish the three common isoforms of human apoE gene namely e2, e3, and e4 (Octave, 1995; Poirier, 1996; Roses, 1996; Utermann, 1980). These isoforms are expressed from a single apoE genetic locus, giving rise to the three common homozygous phenotypes (e4/4, e3/3, e2/2) and three common heterozygous phenotypes (e4/3, e4/2, e3/2) (Poirier 1994, Weisgraber, 1994).

Studies have shown that e2, the isoform with cysteines at positions 112 and 158, have lower affinity for the LDL receptor (Rubinsztein, et.al., 1994). It is associated with prolonged chylomicron-remnant clearance compared to e3 and e4. The e4 (arginine at 112 and cysteine at 158) and e3 (cysteine at 112 and arginine at 158) isoforms have similar affinities for their receptors. e4 is associated with larger, less dense lipoproteins more so than e3, and is therefore associated with rapid chylomicron-remnant clearance and increased total cholesterol levels (Permanne, et.al., 1997; Rubinsztein, et.al., 1994). Studies have estimated that 60% of serum cholesterol levels in humans is genetically determined and of this, 15% is said to be due to apoE genetic variations described above (Davignon, et.al., 1988; Poirier, 1996).

ApoE expression in response to neuronal injury.

Large amounts of lipids and cholesterol are released from degenerating membrane and myelin following neuronal cell loss and termination of afferent connections to vital nerve centers

(deafferentation). Astrocytes in the CNS and macrophages in the PNS respond via the synthesis and release of apoE that scavenges cholesterol and phospholipids from cellular and myelin debris (Figure 1)(Brown and Goldstein, 1986; Beffert, 1998; Ignatius, et.al., 1986; Poirier, 1996). Most of the cholesterol generated during this process is stored in the astrocytes or the macrophages in the form cholesteryl ester (CE) for later use in PNS regeneration and CNS reinnervation. Astrocytes are one of the neuroglial cells in the nervous system that assist normal brain physiology by assisting in the metabolism and brain development.

During this degradation process, the activity of 3,3-hydroxymethylglutaryl-Coenzyme A (HMG-CoA) reductase, an important enzyme regulator in the mevalonate pathway of cholesterol synthesis, is much reduced in the astrocytes and

Figure 1: This diagram shows that the astroglia participate in brain development and support by degrading damaged nerve terminals. The non-esterified free cholesterol (FC) may be used in the formation of ApoE complexes (E) or converted into cholesterol esters (CE)for storage. ApoE may be directed to the ventricles of the brain and eventually to the general circulation. Some ApoE may enter the neurons via LDL receptors where they are broken down into components one of which is cholesterol. Cholesterol is used by the cell in the repair and development of dendrites, nerve processes important for the proliferation of synapses. It was proposed that HMG CoA reductase decreases when cholesterol levels from ApoE increases. (Diagram redrawn from Poirier, 1996, and Beffert, et. al. 1998).

neurons of the CNS, and in the macrophages of the PNS (Brown, et.al., 1973; Goldstein, et.al., 1990). This suggests a receptor-mediated down regulation of cholesterol synthesis due to lipoprotein degradation (Poirier, 1996). Since apoE is the main apolipoprotein produced and secreted in the CNS parenchyma, its role in lipid transport and regulation of cholesterol homeostasis in the CNS cannot be overlooked (Beffert, et.al., 1998; Pitas, 1987; Poirier, 1994, 1996).

Neurons in the CNS have not been known to readily regenerate. However, recent studies in rats have demonstrated that in response to entorhinal cortex lesions (ECL), the hippocampus has the ability to induce the proliferation of presynaptic extensions from terminals of undamaged neurons to compensate for the loss of input (Mathews, et.al., 1976; Poirier, 1996;). The entorhinal cortex is made up of layers of gray mater that sends cortical input to the hippocampus.

Lesions in this area was shown to cause a 60% loss in synaptic input to the granular cell layer of the hippocampus; but within a few months, lost input was replaced with the formation of new synapses (synaptogenesis) (Mathews, et.al., 1976; Poirier, 1996).

After damage to nerve cells, astrocytes engulf the degenerating presynaptic terminals and axons (see Figure 1). During this process of catabolism, lipids are produced which are stored and later utilized for membrane synthesis of precursors used in the formation of neuronal sprouts and reorganization of dendrites and axons. Part of this mechanism is the increased production of cholesterol-phospholipid-apoE complex that gets secreted to the ventricles and the general circulation or targeted to other sites in the CNS (Figure 1) (Beffert, 1998; Ignatius, et.al., 1986; Poirier, 1996). In response to the initial reinnervation process, there is an increase in the expression of LDL receptors in neurons undergoing remodeling (Brown and Goldstein, 1986; Poirier, 1996). As mentioned earlier, this is accompanied by the reduction in the hippocampal HMG-CoA reductase activity, a regulator in the pathway for cholesterol synthesis in the neurons (Figure 1).

After binding of the apoE complex with the LDL-receptor, the apoE-cholesterol-LDL receptor complex is internalized and degraded with the release of cholesterol into the neuronal cytoplasm. Free cholesterol is then transported to the different layers of the hippocampus for membrane and synapse formation.

The repression of cholesterol synthesis via the HMG-CoA pathway is essential. This allows for the salvage and reutilization of cholesterol from the damaged terminals through the apoE complex-LDL receptor pathway. ApoE deficient (knockout) mice have been used by a group of investigators to demonstrate the importance of the apoE complex-LDL receptor pathway (Masliah, et.al., 1995). They reported that the knockout mice displayed a significant loss of synapses and marked disruption in dendritic cytoskeleton in the neurons with age. They also failed to induce compensatory synaptogenesis in response to entorhinal cortex lesions (ECL) (Masliah, et.al., 1995). In another study, it was found that the same kind of knockout mice exhibited normal PNS regeneration in the sciatic nerve (Popko, et.al., 1993). This supports what has already been proven that CNS relies mainly on apoE, whereas the PNS has other lipoproteins; apoB, apoA1, apoD, available to compensate for the loss of apoE function for the maintenance of lipid homeostasis (Pitas, et.al., 1987; Poirier, 1996). These combined results establish the role of apoE in the process of nerve innervation following deafferentation and cell loss due to either experimental lesion or to normal aging process (Poirier, 1996).

Genetic mapping and positional cloning technology has linked apolipoprotein E e4 allele to familial and sporadic late-onset AD (Arendt, et.al., 1997; Beffert, et.al., 1998; Corder, et.al., 1995; Poirier, et.al., 1993, 1996; Roses, 1996; Yasuda, et.al., 1993). Several laboratories have confirmed this all over the world including the USA, Canada, France, Japan, Germany, and Italy. A gene dosage effect on risk and age of onset was observed in both familial and sporadic AD cases (Arendt, et.al., 1998; Corder, et.al., 1995 Poirier, 1996;). The independent studies by Arendt and Corder found that individuals who inherited two e4 alleles had an increased risk and earlier age of onset than individuals with only one e4 allele. Individuals with only one allele had earlier disease onset compared to individuals with no e4 allele. These results show that AD is not an autosomal dominant condition. Instead, the effect of e4 is dose-related (Roses, 1996). Studies have shown that circulating concentration of apoE was markedly reduced among individuals carrying apoE e4 allele compared to non-e4 subjects (Utermann, et.al. 1985). This suggests that apoE expression may be compromised among AD individuals carrying the allele, leading to a disruption in their lipid homeostasis. The presence of the apoE genotype has so far been the most important genetic risk factors for AD found by different researchers in the field (Arendt, et.al.,1998; Corder, et.al. 1995; Geffert, et.al., 1998; Golabek, et.al., 1996; Payami; Poirier, 1996; Roses, 1996; Schmechel, et.al., 1993; Yasuda, et.al., 1998).

The association of apoE e4 allele and AD holds true for both sexes, but there are reports that suggests a higher frequency of e4 alleles in women compared to men (Poirier, 1993). Corder and colleagues reported that nearly 100% of women aged 85, and with one dose of e4, were affected with AD compared to only 50% of men, but they did not consider this significant due to the small sample size (Corder, et.al., 1995). Another study found an increased risk for women with one e4 allele compared to men (Payami, et.al., 1974) Others have concluded that the higher susceptibility of women could be due to independent factors such as estrogen (Beffert, et.al., 1998; Farrer, et.al., 1997).

Two major neuro-pathological features of AD include generalized deposition of amyloid ß (Aß) in the form of senile plaques and aggregation of abnormally phosphorylated tau protein into neurofibrillary tangles (Castano, et.al., 1995; Ii, 1995; Octave, 1995; Permanne, et.al., 1997).

AD patients with the e4 allele were found to have an increased formation of senile plaques compared to AD patients without the e4 allele (Corder, 1995). Correlation of senile plaque density among patients with varying apoE genotypes showed that e4 patients demonstrated a signe4 allele were found to have an increased formatien of senile plaques compared to AD patients without the e4 allele (Corder, 1995). Correlation of senile plaque density among patients with varying apoE genotypes showed that e4 patients demonstratedn apoE can induce in vitro formation of amyloid plaques (Castano, et.al., 1995). Briefly, peptides amyloid b (1-40) with Glu in position 22, Ab (1-40Q) with Gln in position 22 at concentrations of 300 m M, were synthesized by solid phase procedures. Human apoE was purchased from a reagent company. Ultrafiltered human cerebrospinal fluid was also used in the study to mimic physiological ionic environment. The apoE was prepared at0.7 mg/mL in 0.1 M Tris/HCl buffer at pH 7.4. The peptides were prepared in 0.1% trifluroacetic acid/50% acetonitrile. In the experiment the peptide and ApoE or CSF was incubated at room temperature and then added to 50mM glycine (pH 9)/2uM thioflavine-T (Sigma) in a final volume of 2 ml. Fluorescence was measured at excitation 435 nm and emission 485 nm in a fluorescent spectrophotometer. This thioflavin-T fluorescence assay is used to measure the degree of fibril formation. Results from the fluorimetric data showed that Ab (1-40) had a fluorescent signal at 485 nm and the signal increased 2-fold with Ab (1-40Q). Freshly prepared Ab (1-40) suspended only in buffer did not show specific Th-T fluorescence at 485 nm. According to Castano and colleagues, this shift in the Th-T fluorescence is related to the formation of b -pleated secondary structure of aggregated Ab (1-40). It showed that the substituted peptide formed amyloid at a much faster rate and a higher mass of peptide aggregated than the unsubstituted peptide.

This study illustrates in vitro how the binding of apoE to amyloid ß proteins may slow down the soluble amyloid ß clearance from the brain or CS space turning it into amyloid plaque deposits. It supports the hypothesis that apoE acts as a pathological chaperone promoting conformational change of the normally ß-pleated sheet amyloid into amyloid fibers (Castano, et.al., 1995; Mori, et.al., 1994).

Another feature of AD neuropathology is the presence of intraneuronal neurofibrillary tangles. A correlation between neurofibrillary tangle formation and apolipoprotein E allele has not been reported. However, it was observed that neurofibrillary tangle formation often follows senile plaque formation (Smith, et.al., 1995).

ApoE plays a pivotal role in the proper function of the cholinergic system, which relies heavily on the availability of lipids to synthesize acetylcholine (Ach), in the neurons (Beffert, et.al., 1998). Two components of brain cell membrane; phosphatidyl choline (PC) and phosphatidylethanolamine (PE) have been shown to serve as donors of choline, a rate-limiting precursor for Ach (Beffert, et.al.,1998; Blesztajn, et.al., 1987). It was suggested by Poirier that the low levels of apoE in the hippocampus, temporal cortex, and CSF of AD patients with the e4 allele may compromise cholesterol and phospholipid transport in the CNS, which in turn restricts cholinergic neurotransmission (Poirier, 1994, 1995). The loss of neurons in the nucleus basalis of Meynert, a major source of cholinergic innervation for the cerebral cortex, and the concomitant loss of activity of choline acetyl transferase (the enzyme responsible for Ach synthesis) are significant neurochemical hallmarks of AD (Poirier, et.al., 1995; Soininen, et.al., 1995; Whitehouse, et.al., 1982). Studies have shown that brain levels of choline are decreased by up to 40-50% in the frontal and parietal cortices of AD patients. As a consequence, acetylcholine production is significantly reduced among AD patients carrying the e4 allele (Beffert, et.al., 1998; Poirier, et.al., 1995; Soininen, et.al., 1995).

This decade has seen some major advances in the search for the biochemical and pathological mechanisms that may explain the signs and lesions involved in AD. Although not all people with the ApoE e 4 allele will eventually develop AD, a significant majority of familial and sporadic cases of AD do have one or two copies of the gene. The genetic expression of the gene may act alone in the pathophysiology of the disease when it leads to a malfunctioning lipid and cholesterol biosynthesis. As a consequence of decreased cholesterol in the brain, synaptic integrity is compromised leading to clinical signs of memory loss or dementia. With impaired phosphatidylcholine synthesis, there is decreased production of choline, which leads to a decrease in the levels of the neurotransmitter acethylcholine.

The expression of apoE e 4 allele may also act in tandem with other processes as in the role of apoE in the formation of senile plaques and neurofibrillary tangles. Although it is widely accepted that e 4 allele is clearly associated with increased risk for developing AD, the exact mechanism by which the e 4 allele operates is still under investigation. Treatment plans have been investigated using steroidal and nonsteroidal antiinflammatory drugs since studies have shown that their use tend to ameliorate the symptoms of AD (Breitner, 1996). This is an area with hopeful potential since it is paving the way for in-depth clinical studies to search for proof of definitive efficacy.

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