Evidence that L1AD3, an apoptosis-inducing cyclic peptide, binds a leukemic T-cell membrane protein receptor

Human leukemic T-lymphocytes undergo extensive and rapid apoptosis in the presence of L1AD3, a small cyclic peptide derivative of cobra cardiotoxin. The first step in this process involves its binding to membranes of susceptible cells. By the use of a biotin "handle" synthetically incorporated at the N-terminus of L1AD3, we show that binding is saturable and selective: normal human peripheral blood lymphocytes do not bind this peptide. Fluorescence resonance energy transfer experiments indicate that the binding sites are separated by at least 55 A. Loss of binding occurs if membrane proteins are enzymatically degraded, suggesting that L1AD3's target is a cell-membrane surface protein receptor. Finally, crosslinking of cyclic BTNL1AD3 peptide to a leukemic T-cell membrane surface receptor, as examined using a biotin-avidin blot, indicated a molecular weight of approximately 34,400.     

A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk

Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-beta (Abeta) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1. We show that CALHM1 encodes a multipass transmembrane glycoprotein that controls cytosolic Ca(2+) concentrations and Abeta levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca(2+) conductance across the plasma membrane. Importantly, we determined that the CALHM1 P86L polymorphism (rs2986017) is significantly associated with AD in independent case-control studies of 3404 participants (allele-specific OR = 1.44, p = 2 x 10(-10)). We further found that the P86L polymorphism increases Abeta levels by interfering with CALHM1-mediated Ca(2+) permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca(2+) channel that controls Abeta levels and susceptibility to late-onset AD.     

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its beta-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1-APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.     

Two types of Alzheimer's beta-amyloid (1-40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation

The 39-42 amino acid long, amphipathic amyloid-beta peptide (Abeta) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Abeta presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Abeta(1-40) membrane assemblies for two different scenarios with potential implication in AD: Abeta peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Abeta(1-40) by stabilizing its hydrophobic transmembrane C-terminal part (residue 29-40) in an alpha-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Abeta(1-40) is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Abeta(1-40) peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide-membrane assemblies are involved in Abeta's pathophysiology with the finely balanced type of Abeta-lipid interactions against release of Abeta from neuronal membranes being overcompensated by an Abeta-membrane assembly which causes toxic beta-structured aggregates in AD. Therefore, pathological interactions of Abeta peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Abeta-membrane assemblies inherited from Abeta's origin.     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein.

Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize bt1 locus encodes an amyloplast membrane metabolite translocator protein (Sullivan, T. D., Strelow, L. I., Illingworth, C. A., Phillips, R. L., and Nelson, O. E., Jr. (1991) Plant Cell 3, 1337-1348). The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite translocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.     

The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.

Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.     

Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.     

Heterodimerization of BAK and MCL-1 activated by detergent micelles

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.     

Heavy metals toxicity: effect of cadmium ions on amyloid beta protein 1–42. Possible implications for Alzheimer’s disease

Cadmium (Cd) is an environmental contaminant, highly toxic to humans. This biologically non-essential element accumulates in the body, especially in the kidney, liver, lung and brain and can induce several toxic effects, depending on the concentration and the exposure time. Cd has been linked to Alzheimer’s disease (AD) as a probable risk factor, as it shows higher concentrations in brain tissues of AD patients than in healthy people, its implication in the formation of neurofibrillary tangles and in the aggregation process of amyloid beta peptides (AβPs). AβPs seem to have toxic properties, particularly in their aggregated state; insoluble AβP forms, such as small and large aggregates, protofibrils and fibrils, appear to be implicated in the pathogenesis of AD. In our study, we have evaluated the effect of Cd, at different concentrations, both on the AβP1–42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30 % cholesterol and on the secondary structure of AβP1–42 in aqueous environment. Cadmium is able to interact with the AβP1–42 peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both decreasing AβP1–42 channel frequency and in solution forming large and amorphous aggregates prone to precipitate. These experimental observations suggesting a toxic role for Cd strengthen the hypothesis that Cd may interact directly with AβPs and may be a risk factor in AD.     

Alzheimer's disease protein Abeta1-42 does not disrupt isolated synaptic vesicles

Synaptic vesicles are central to neurotransmission and cognition. Studies of the Alzheimer's disease (AD) associated peptide, amyloid beta (Abeta), suggest that it has the potential to non-specifically solubilize or permeabilize membranes and that it has detergent and pore-forming properties. Damage to the membrane or integrity of synaptic vesicles could compromise its function. We test the hypothesis that the intact synaptic vesicle is a direct site of attack by Abeta1-42 in AD pathology by examining the properties of individual isolated vesicles exposed to Abeta1-42. In particular, we compared the rate of leakage of dye molecules from synaptic vesicles, the rate of proton permeation across the membrane of the vesicle, and the rate of active proton transport into the vesicle interior in the presence and absence of Abeta1-42. From these experiments, we conclude that isolated synaptic vesicles are not disrupted by Abeta1-42.     

Presenilin-1, nicastrin,amyloid precursor protein,and gamma-secretase activity are co-localized in the lysosomal membrane

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion. We have recently identified nicastrin as a major lysosomal membrane protein. In the present work, we use Western blotting and immunogold electron microscopy to demonstrate that significant amounts of mature nicastrin, presenilin-1, and APP are co-localized with lysosomal associated membrane protein-1 (cAMP-1) in the outer membranes of lysosomes. Furthermore, we demonstrate that these membranes contain an acidic gamma-secretase activity, which is immunoprecipitable with an antibody to nicastrin. These experiments establish APP, nicastrin, and presenilin-1 as resident lysosomal membrane proteins and indicate that gamma-secretase is a lysosomal protease. These data reassert the importance of the lysosomal/endosomal system in the generation of Abeta and suggest a role for lysosomes in the pathophysiology of AD.     

Hemagglutinin fusion peptide mutants in model membranes: structural properties, membrane physical properties, and PEG-mediated fusion

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.     

The interactions of the N-terminal fusogenic peptide of HIV-1 gp41 with neutral phospholipids

We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1_LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the ¹³C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study. Received: 8 June 1998 / Revised version: 18 November 1998 / Accepted: 28 December 1998     

Reduced amyloid‐β degradation in early Alzheimer's disease but not in the APPswePS1dE9 and 3xTg‐AD mouse models

Alzheimer's disease (AD) is hallmarked by amyloid‐β (Aβ) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Aβ can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg‐AD mouse models. A quenched Aβ peptide that becomes fluorescent upon degradation was used to screen for Aβ‐degrading cytoplasmic peptidases cleaving the aggregation‐prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Aβ‐degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg‐AD mice. Insulin‐degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Aβ. Oligomerization of Aβ prevents its clearance by IDE. Intriguingly, the Aβ‐degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg‐AD mouse models do not show altered Aβ degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Aβ peptides are representative for human sAD.     

Cholesterol metabolism and transport in the pathogenesis of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, affecting millions of people worldwide. Apart from age, the major risk factor identified so far for the sporadic form of AD is possession of the ?4 allele of apolipoprotein E (APOE), which is also a risk factor for coronary artery disease (CAD). Other apolipoproteins known to play an important role in CAD such as apolipoprotein B are now gaining attention for their role in AD as well. AD and CAD share other risk factors, such as altered cholesterol levels, particularly high levels of low density lipoproteins together with low levels of high density lipoproteins. Statins – drugs that have been used to lower cholesterol levels in CAD, have been shown to protect against AD, although the protective mechanism(s) involved are still under debate. Enzymatic production of the beta amyloid peptide, the peptide thought to play a major role in AD pathogenesis, is affected by membrane cholesterol levels. In addition, polymorphisms in several proteins and enzymes involved in cholesterol and lipoprotein transport and metabolism have been linked to risk of AD. Taken together, these findings provide strong evidence that changes in cholesterol metabolism are intimately involved in AD pathogenic processes. This paper reviews cholesterol metabolism and transport, as well as those aspects of cholesterol metabolism that have been linked with AD.     

Amyloid beta ion channel: 3D structure and relevance to amyloid channel paradigm

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Aβ) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Aβ specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Aβ oligomers (∼ > 60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Aβ oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Aβ channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.     

Molecular mechanism of antimicrobial peptides: the origin of cooperativity

Based on very extensive studies on four peptides (alamethicin, melittin, magainin and protegrin), we propose a mechanism to explain the cooperativity exhibited by the activities of antimicrobial peptides, namely, a non-linear concentration dependence characterized by a threshold and a rapid rise to saturation as the concentration exceeds the threshold. We first review the structural basis of the mechanism. Experiments showed that peptide binding to lipid bilayers creates two distinct states depending on the bound-peptide to lipid ratio P/L. For P/L below a threshold P/L*, all of the peptide molecules are in the S state that has the following characteristics: (1) there are no pores in the membrane, (2) the axes of helical peptides are oriented parallel to the plane of membrane, and (3) the peptide causes membrane thinning in proportion to P/L. As P/L increases above P/L*, essentially all of the excessive peptide molecules occupy the I state that has the following characteristics: (1) transmembrane pores are detected in the membrane, (2) the axes of helical peptides are perpendicular to the plane of membrane, (3) the membrane thickness remains constant for P/L> or =P/L*. The free energy based on these two states agrees with the data quantitatively. The free energy also explains why lipids of positive curvature (lysoPC) facilitate and lipids of negative curvature (PE) inhibit pore formation.     

CALHM1 P86L polymorphism modulates CSF Aβ levels in cognitively healthy individuals at risk for Alzheimer's disease

The calcium homeostasis modulator 1 (CALHM1) gene codes for a novel cerebral calcium channel controlling intracellular calcium homeostasis and amyloid-β (Aβ) peptide metabolism, a key event in the etiology of Alzheimer's disease (AD). The P86L polymorphism in CALHM1 (rs2986017) initially was proposed to impair CALHM1 functionally and to lead to an increase in Aβ accumulation in vitro in cell lines. Recently, it was reported that CALHM1 P86L also may influence Aβ metabolism in vivo by increasing Aβ levels in human cerebrospinal fluid (CSF). Although the role of CALHM1 in AD risk remains uncertain, concordant data have now emerged showing that CALHM1 P86L is associated with an earlier age at onset of AD. Here, we have analyzed the association of CALHM1 P86L with CSF Aβ in samples from 203 AD cases and 46 young cognitively healthy individuals with a positive family history of AD. We failed to detect an association between the CALHM1 polymorphism and CSF Aβ levels in AD patients. Our data, however, revealed a significant association of CALHM1 P86L with elevated CSF Aβ42 and Aβ40 in the normal cohort at risk for AD. This work shows that CALHM1 modulates CSF Aβ levels in presymptomatic individuals, strengthening the notion that CALHM1 is involved in AD pathogenesis. These data further demonstrate the utility of endophenotype-based approaches focusing on CSF biomarkers for the identification or validation of risk factors for AD.     

The role of membrane trafficking in the processing of amyloid precursor protein and production of amyloid peptides in Alzheimer's disease

Alzheimer's disease (AD) is characterized by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and the release of inflammatory mediators by innate immune responses. β-Amyloid peptide (Aβ) is derived from sequential processing of the amyloid precursor protein (APP) by membrane-bound proteases, namely the β-secretase, BACE1, and γ-secretase. Membrane trafficking plays a key role in the regulation of APP processing as both APP and the processing secretases traffic along distinct pathways. Genome wide sequencing studies have identified several AD susceptibility genes which regulate membrane trafficking events. To understand the pathogenesis of AD it is critical that the cell biology of APP and Aβ production in neurons is well defined. This review discusses recent advances in unravelling the membrane trafficking events associated with the production of Aβ, and how AD susceptible alleles may perturb the sorting and transport of APP and BACE1. Mechanisms whereby inflammation may influence APP processing are also considered.