Going Beneath the Fold

FEATUREProtein Misfolding Transmission Electron Micrograph of Protein Filaments in Alzheimer's DiseaseCOURTESY OF HUNTINGTON POTTERHow an apolipoprotein E isoform wreaks havoc in the brain, and what we might be able to do about itBY ROBERT MAHLEY AND YADONG HUANGFor much of the 20th century, scientists have looked at brain cells from patients with Alzheimer's disease (AD), Huntington's disease, and other neurodeg

Aug 1, 2006
Robert Mahley and Yadong Huang
FEATURE
Protein Misfolding
Going Beneath the Fold
Transmission Electron Micrograph of Protein Filaments
in Alzheimer's Disease

COURTESY OF HUNTINGTON POTTER

How an apolipoprotein E isoform wreaks havoc in the brain, and what we might be able to do about it

BY ROBERT MAHLEY
AND YADONG HUANG

For much of the 20th century, scientists have looked at brain cells from patients with Alzheimer's disease (AD), Huntington's disease, and other neurodegenerative diseases and found clumps of material that are largely absent in the brains of nondemented people. The perfectly logical conclusion was that the aggregates were related to the disease process.

Through its lipid-transport functions, apoE is important in repairing and remodeling neurons. Only the liver produces more of it.

The picture is much more complicated than that, however. Various research lines have suggested that protein aggregates may result in deleterious inflammatory responses or that they arise as a response to malfunctioning cell machinery at the root of neurodegeneration. Although questions remain as to their roles in disease, with increasingly sophisticated technology, we have been able to show what these aggregates are made from and increasingly how they form.

Our groups have historically looked at the protein apolipoprotein E (apoE) and its involvement in AD.1,2 ApoE plays a fundamental role in the transport of lipids and in the maintenance and repair of neurons. A specific variant, apoE4, is associated with a wide variety of neuropathological processes and is the major known genetic risk factor for AD.1,3 Through various structural, biochemical, and cell biological studies, we've begun to untangle the role of pathogenic apoE4 in AD. We hypothesize that apoE4 works in concert with a variety of insults or so-called second hits to lead to neuropathology (see Second Hits). Understanding the structure and function of apoE and the interaction of apoE with these insults may yield new strategies for treating AD and other neuropathologies.

THE STRUCTURE OF APOE

Human apoE is a polymorphic protein 299 amino acids long.4 Its gene, on chromosome 19, encodes three alleles that differ only at two positions, 112 and 158. ApoE2, which has cysteines at both positions, occurs at a frequency of 5%-10%. ApoE3 occurs in 60%-70% of the population and has cysteine at 112 and arginine at 158. The detrimental apoE4 has arginine at both sites and occurs in 15%-20% of people.

The structural differences among apoE isoforms suggest potential therapeutic strategies.

These single amino acid sequence interchanges cause profound differences in the properties of the isoforms. ApoE4's structure confers on it two key biophysical properties - domain interaction and molten globule formation - that likely contribute to apoE4-associated neuropathology. ApoE4 is far more likely than apoE2 and apoE3 to assume this pathological conformation.

ApoE has two structural domains. The amino-terminal domain contains the region responsible for binding with members of the low-density lipoprotein receptor family (amino acids 135-150). The carboxyl-terminal domain contains the lipid-binding region (amino acids 241-270). In apoE4, which possesses Arg-112, the side chain of Arg-61 in the amino-terminal domain extends away from the helical bundle, enabling it to interact with Glu-255 in the carboxyl-terminal domain through ionic binding (see ApoE conformations). This so-called domain interaction can be abolished by mutating Arg-61 to threonine or Glu-255 to alanine.5

© 2006 ELSEVIER, LTD.
The lipid-bound apolipoprotein E: Two molecular envelopes of apoE embrace a sphere of phospholipids.

The apoE isoforms also have different stabilities (instability: apoE4 > apoE3 > apoE2), and unlike apoE3 and apoE2, apoE4 exhibits a denaturation pattern that does not fit a two-state equilibrium (native versus fully unfolded).6 ApoE4 likely forms a partially folded, reactive intermediate or molten globule, more easily than the other isoforms. This tendency of apoE4 may be important: Less stable proteins forming reactive intermediates are associated with a number of pathophysiological activities, including altered intradomain interactions, increased lipid and membrane binding, membrane disruption, translocation across membranes, and increased susceptibility to proteolysis.

Throughout life and increasing with age, neurons must be remodeled and repaired to maintain synaptodendritic connections. Through its lipid transport function, apoE is an important factor in these processes. ApoE is expressed in a variety of cell types in the central nervous system including astrocytes and some neurons. Only the liver produces more of it. ApoE also exists in the cerebrospinal fluid as small, high-density lipoprotein-like particles or phospholipid disks that deliver lipids, including cholesterol, to sites of injury for cell repair. ApoE3 and apoE2 seem more effective in the normal maintenance and repair of cells than apoE4 due to differences in their structure and biophysical properties.


APOE AND AD

Forty percent to as many as 80% of patients with AD possess at least one apoE4 allele. ApoE3 seems to be neutral, and apoE2 somewhat protective. Although apoE4 is strongly linked to AD pathology, its mode of action is unknown. Several mechanisms have been proposed. Through interactions with the plaque-forming amyloid b peptide, apoE4 may increase Ab deposition in plaques and impair its clearance. However, apoE may act through other pathways that may or may not involve Ab (see Many roles). Insights into the role of apoE in neuropathology have come from studies of human patients and transgenic mice expressing human apoE3 or apoE4 in neurons or astrocytes. Several mechanisms have been suggested, including the regulation of Ab production, the effects of lysosomal leakage and apoptosis, and the actions of toxic apoE fragments.1,2,7

Second hits: We hypothesize that apoE4 acts in concert with a variety of cellular insults or so-called second hits (genetic, metabolic, and environmental) that lead to poor neuronal repair/remodeling and neuropathology.

Ab has been implicated in AD primarily by its association with plaques, a pathological hallmark of AD.8 Levels of Ab can be affected by decreasing Ab clearance or increasing Ab deposition. ApoE4 may influence both processes adversely.

In cultured Neuro-2a cells, we showed that Ab-induced lysosomal leakage and apoptotic cell death are much greater with apoE4 than apoE3.9 ApoE4 is more unstable and thus more likely to form a reactive intermediate (molten globule) at the acidic pH of late endosomes or lysosomes and may destabilize membranes. The activity of apoE3 was not affected by neutralizing the lysosomal pH.10


A DISRUPTIVE LITTLE PROTEIN

Neurons synthesize apoE in response to injury.11 But, when apoE is synthesized by neurons, the 27 carboxyl-terminal amino acids can be lopped off the end by a neuron-specific, chymotrypsin-like serine protease.12-14 Because of its unique conformation (domain interaction), apoE4 is much more susceptible than apoE3 to this proteolysis. Interestingly, mutating Arg-61 to threonine or Glu-255 to alanine, which eliminates domain interaction, reduces the susceptibility of apoE4 to proteolysis.

Top: Courtesy of Karl H. Weisgraber, Gladstone Institute of Neurological Disease
Middle: ©2006 Am Soc Biochem Molec Biol
Bottom Two: ©2005 National Academy Of Sciences
ApoE conformations: Lipid-free apoE has a four-helix bundle structure of the N-terminal domain as determined by X-ray crystallography. The series of helices depicted for the C-terminal domain is based on structure prediction.

Lipid-bound apoE forms a molecular envelope. In apoE4, Arg-112 orients the side chain of Arg-61 into the aqueous environment, where it can interact with Glu-255, resulting in interaction between the amino- and carboxyl-terminal domains.

In apoE3, Arg-61 is not available to interact with residues in the carboxyl-terminal domain, resulting in a different overall conformation. Small molecules that interact with apoE4 in the region of Arg-61 might disrupt the domain interaction, converting apoE4 to an apoE3-like molecule.

Not all fragments are toxic. Only those generated by the apoE-cleaving enzyme cause neuropathology. Mutating four conserved amino acids in the lipid-binding region or three conserved positively charged amino acids in the receptor-binding region abolished the neurotoxicity. Furthermore, fragments lacking the receptor-binding region and the lipid-binding region are not neurotoxic.15

These apoE4 fragments have pathophysiological relevance. Notably, similar carboxyl-terminal-truncated fragments are found in the brains of patients with AD and in transgenic mice expressing apoE4 in neurons.12,14 In mice, accumulation of the fragments peaks at 6 to 7 months of age, just about the time that neurodegenerative changes and significant deficits in learning and memory occur.14,16

Expression of toxic fragments in neurons has several effects: translocation of the fragments into the cytosol, and accumulation of the fragments in filamentous cytoplasmic structures (phosphorylated tau and neurofibrillary tangle-like structures) and in mitochondria, and ultimately neurotoxicity.

Neurotoxicity correlates with the ability of the fragments to enter the cytosol, where they interact with the cytoskeleton or mitochondria,15 but how apoE4 escapes the secretory pathway and enters the cytosol isn't clear. ApoE4 could be translocated through the ribosome-membrane junction during protein synthesis, undergo cleavage, and then enter the cytosol. Or, apoE4 or the apoE4 fragment within the endoplasmic reticulum/Golgi compartment may assume a conformation conducive to membrane translocation. Molten globules can easily assume conformations that might promote translocation across membranes.

Peptides that penetrate cell membranes have common features. The process relies on the interaction of positively charged amino acids with negatively charged membranes. Hydrophobic amino acids may contribute to the binding and enhance protein translocation. ApoE contains both of these structural elements.

40% to as many as 80% of patients with Alzheimer's disease possess at least one apoE4 allele.

Once in the cytoplasm, apoE could interfere with a number of important cell processes. For example, tau is a critical element of the cytoskeleton and the major component of the intracellular neurofibrillary tangles that are also a pathological hallmark of AD. The carboxyl-terminal-truncated fragments of apoE stimulate tau phosphorylation and the formation of neurofibrillary tangle-like inclusions in nerve cells in transgenic mice.13,14 Thus, the greater sensitivity of apoE4 to the protease may be manifested in a greater degree of damage to the cell cytoskeleton.

Mitochondria are important for normal neuronal structure and function. Patients with AD, especially those with apoE4, show mitochondrial dysfunction.17 Exactly how apoE4 disrupts mitochondrial function is unknown, but there are some interesting clues. For example, mitochondria play a critical role in synaptogenesis, and apoE4 expression in mice results in a significant loss of synaptodendritic connections within the brain.18

ApoE fragments may also induce the mitochondrial-apoptotic pathway, disrupt mitochondrial regulation of energy and glucose metabolism in neurons, or disrupt mitochondrial trafficking, resulting in failure to deliver these organelles to appropriate sites in neurons and causing energy depletion and disruption of calcium homeostasis. The disruption of microtubules in cultured neurons by apoE4 may accentuate the mitochondrial dysfunction, or the mitochondrial dysfunction may in part cause the cytoskeletal abnormality observed with apoE4. The association of apoE4 with hyperphosphorylation of tau and the occurrence of neurofibrillary tangle-like structures in neurons could result in an abnormal distribution of mitochondria in neurons.1

How apoE fragments come to be associated with the mitochondria is unknown. The fragments may bind directly to lipids in the mitochondria. Alternatively, the fragments may interact with cytoplasmic factors (chaperones) that target the apoE to the import channels, or after proteolytic cleavage in the endoplasmic reticulum they might diffuse through the endoplasmic reticulum membranes to access the mitochondria. For example, apoE binds to the a- and b-subunits of mitochondrial F1-ATPase, an enzyme involved in ATP synthesis.19 We localized the mitochondrial-binding region to the lipid-binding region of apoE.15

NEW THERAPEUTIC APPROACHES

Understanding the nature of proteinopathies has transformed the search for potential therapies. The target is no longer necessarily an attack on protein inclusions. In some cases, attacking the protein deposits may increase the concentration of toxic molecules and do more harm than good. The goal now may be to mitigate the results of a protein's structure or conformation.

©2006 NATIONAL ACADEMY OF SCIENCES
Many roles: ApoE is synthesized by astrocytes, activated microglia, and some neurons, and is redistributed among several cell types to repair damaged cells.

Three potential detrimental roles played by apoE4 include: 1) enhanced Ab production, 2) potentiation of Ab-induced lysosomal leakage and apoptosis, and 3) enhanced neuron-specific proteolysis resulting in translocation of neurotoxic apoE4 fragments into the cytosol, where they are associated with cytoskeletal disruption and mitochondrial dysfunction.

ApoE4 represents an especially attractive therapeutic target. Domain interaction mediates several neuropathological effects of apoE4, including increases in Ab production, potentiation of Ab-induced lysosomal leakage and apoptosis, and enhanced proteolytic cleavage in neurons.

Knowledge of apoE4's activity suggests several therapeutic approaches. Production of the toxic apoE fragments could be prevented by an inhibitor of the protease. The interaction of the apoE4 fragments with the mitochondria could be blocked by an inhibitor. Drugs that increase the number or activity of neuronal mitochondria might have value. In fact, AD patients treated with rosiglitazone maleate, an insulin sensitizer and mitochondrial stimulator, appear to have improved cognition.20

The structural and functional differences between the apoE isoforms suggest another therapeutic strategy: Make apoE4 behave more like apoE3. If domain interaction could be disrupted, it might mitigate apoE4's detrimental effects. Inhibiting apoE4 domain interaction with small-molecule "structure correctors" represents a new therapeutic strategy.

As proof of this concept, we have identified several candidate molecules that do exactly that. We used the DOCK screening program to identify 65 small-molecule candidates, and in vitro assays to test their effectiveness. Nine inhibited preferential binding of apoE4 to emulsion particles. Two decreased Ab production induced by apoE4 to levels similar to those of apoE3.1,21 Other small molecules prevented the Ab-induced lysosomal leakage and apoptosis mediated by apoE4.

As more evidence is uncovered, the concept of neurodegenerative diseases as proteinopathies has become accepted. ApoE isoforms and AD are an excellent example of this new paradigm. The exciting aspect of these structural differences is that they suggest multiple therapeutic strategies that can be easily tested. Since apoE4 is involved in not only AD but also in other neurodegenerative disorders such as Parkinson disease and multiple sclerosis, these potential therapeutic strategies might prove beneficial for those diseases as well.

Robert Mahley (rmahley@the-scientist.com) is president of the J. David Gladstone Institutes.
Yadong Huang is an assistant investigator at the Gladstone Institute of Neurological Disease.

References
1. R.W. Mahley et al., "Apolipoprotein E4: A causative factor and therapeutic target in neuropathology, including Alzheimer's disease," Proc Natl Acad Sci, 103:5644-51, April 11, 2006.
2. Y. Huang et al., "Apolipoprotein E. Diversity of cellular origins, structural and biophysical properties, and effects in Alzheimer's disease," J Mol Neurosci, 23:189-204, 2004.
3. E.H. Corder et al., "Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families," Science, 261:921-3, 1993.
4. R.W. Mahley, "Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology," Science, 240:622-30, 1988.
5. L.-M. Dong et al., "Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms," J Biol Chem, 269:22358-65, 1994.
6. J.A. Morrow et al., "Apolipoprotein E4 forms a molten globule: A potential basis for its association with disease," J Biol Chem, 277:50380-5, 2002.
7. Y. Huang, "Apolipoprotein E and Alzheimer disease," Neurology, 66 (Suppl 1):S79-S85, 2006.
8. D.J. Selkoe, "Clearing the brain's amyloid cobwebs," Neuron, 32:177-80, 2001.
9. Z.-S. Ji et al., "Apolipoprotein E4 potentiates amyloid b peptide-induced lysosomal leakage and apoptosis in neuronal cells," J Biol Chem, 277:21821-8, 2002.
10. Z.-S. Ji et al., "Reactivity of apolipoprotein E4 and amyloid b peptide: Lysosomal stability and neurodegeneration," J Biol Chem, 281:2683-92, 2006.
11. Q. Xu et al., "Profile and regulation of apolipoprotein E (apoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the apoE locus," J Neurosci, 26:4985-94, May 10, 2006.
12. Y. Huang et al., "Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons," Proc Natl Acad Sci, 98:8838-43, 2001.
13. F.M. Harris et al., "Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice," Proc Natl Acad Sci, 100:10966-71, 2003.
14. W.J. Brecht et al., "Neuron-specific apolipoprotein E4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice," J Neurosci, 24:2527-34, 2004.
15. S. Chang et al., "Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity," Proc Natl Acad Sci, 102:18694-9, 2005.
16. J. Raber et al., "Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: Increased susceptibility of females," Proc Natl Acad Sci, 95:10914-9, 1998.
17. G.E. Gibson et al., "Mitochondrial damage in Alzheimer's disease varies with apolipoprotein E genotype," Ann Neurol, 48:297-303, 2000.
18. M. Buttini et al., "Expression of human apolipoprotein E3 or E4 in the brains of ApoE-/- mice: Isoform-specific effects on neurodegeneration," J Neurosci, 19:4867-80, 1999.
19. R.W. Mahley et al., "Chylomicron remnant metabolism. Role of hepatic lipoprotein receptors in mediating uptake," Arteriosclerosis, 9:I-14-I-18, 1989.
20. M.E. Risner et al., "Efficacy of rosiglitazone in a genetically defined population with mild-to-moderate Alzheimer's disease," Pharmacogenomics J, (in press), 2006.
21. S. Ye et al., "Apolipoprotein (apo) E4 enhances amyloid b peptide production in cultured neuronal cells: ApoE structure as a potential therapeutic target," Proc Natl Acad Sci, 102:18700-5, 2005.