Ion channel hypothesis for Alzheimer amyloid peptide neurotoxicity

1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the AβP peptide in amyloid plaques. 2. The AβP[1–40] peptide is
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  Cellular and Molecular Neurobiology Vol. 15 No. 5. 1995 Ion Channel Hypothesis for lzheimer myloid Peptide Neurotoxicity Harvey B. Pollard, 1' Nelson Arispe, ~ and Eduardo Rojas ~ Received January 5 1995; accepted February 20 1995 KEY WORDS: Alzheimer s Disease: calcium channels: amyloid. SUMMARY 1. Alzheimer s disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the A/3P peptide in amyloid plaques. 2. The A/3P[1-40] peptide is derived by proteolytic processing from a much larger amyloid precursor protein (APP), and has been circumstantially identified as the toxic principle causing cell damage in the disease. 4. The A/3P[1-40] peptide is able to form quite characteristic calcium channels in planar lipid bilayers. These channels have conductances in the nS range, and can dissipate ion gradients quickly. The peptide can also cause equivalent cation conductances in cells. 5. We suggest that amyloid channel blocking agents might be therapeutically useful in Alzheimer s Disease, and have constructed molecular models of the channels to aid in the design of such compounds. INTRODUCTION Alzheimer s disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. It is characterized pathologically by extracellular amyloid plaques, intraneuronal neurofibrillary tangles, and vascular and neuronal damage (Masters et al. 1985; Neve et al. 1990: Selkoe, 1991; Hardy and Higgins, 1992). The major component of brain amyloid is a ~ Laboratory of Cell Biology and Genetics, NIDDSK, National Institutes of Health, Bethesda. MD 20892. 2 To whom correspondence should be addressed at Laboratorw of Cell Biology and Genetics, Building 8, Room 402, National Institutes of Health, Bethesda, MD -50892. 513 0272.4340/95tlOOO.0513507.50to ~ 1995 Plenum Pubhshmg Corporation  514 Pollard Arispe and Rojas 38-42 residue peptide, termed amyloid /3 protein (Al3P or/3A/4) (Glenner and Wong, 1984; Masters et al. 1985; Kang et al. 1987), which is a proteolytic product of the widely distributed amyloid precursor protein (APP7s~) defined by a locus on chromosome 21 (Goldgaber et al. 1987; Tanzi et al. 1987; Goate et al. 1991). A/3P has been circumstantially identified as the toxic principle causing cell damage in the disease, although the mechanism of this toxicity has been a matter of contention (e.g., Price et al. 1992). Amyloid peptides do not appear to interact with target cells via receptors, since no binding sites have been detected. Indeed, direct interaction of the peptide with the target cell membrane has been most often observed (Mattson et al. 1993). In addition, increases in intracellular calcium have been noted to follow addition of amyloid peptide to the target cells (Mattson et al. 1992), indicating that toxicity might involve disordering of intracellular calcium homeostasis. The toxicity of amyloid peptide has not been ameliorated by u-type calcium channel blockers (Whitson and Appel, 1995), further indicating some specificity in the actions of amyloid peptides on calcium metabolism. Finally, amyloid peptide toxicity has been shown to be associated with the formation of free radicals in target cells (Behl et al. 1994), and the characteristic antiparallel t3-sheet conformation of the amyloid peptide has been implicated in the toxic process (Schubert et al. 1995). The centrality of calcium, even in the latter processes, is manifest by the fact that elevation of intracellular calcium in neuronal cells is closely associated with the generation of free radicals (Olanow and Arendash, 1994). It was for these reasons that we have given particular emphasis to our srcinal observation that the amyloid peptide (ACP[1-40]) was able to form quite characteristic calcium channels in planar lipid bilayers (Arispe et al. 1993a,b). This observation led us to propose that the toxic action of amyloid peptide on target cells in the brain might be due to these channels. We further suggested that amyloid channel blocking agents might be therapeutically useful, and have constructed molecular models of the amyloid channels to aid in the design of such compounds (Durrell et al. 1994). In the remainder of this article we will summarize the data leading to this conclusion, and mention studies supporting the concept that similar channels may indeed be formed in intact cells. AMYLOID PRECURSOR PROTEIN APP) AND THE Af~P[1-40] PEPTIDE The A/3P[1-40] peptide is derived by proteolytic processing from a much larger amyloid precursor protein (APP). The mRNA for APP is said to represent as much as 2.5 of the total mRNA in the brain, and its synthesis is regulated by physical and chemical damage (Siman et al. 1989; Kawarabayashi et al. 1991; Roberts et aL 1991; Heurteaux et al. 1993). As shown in Fig. 1, this precursor is a glycosylated integral membrane protein, which is synthesized from a single copy gene in three alternatively spliced forms: APP 695, 751, and 770 (Ponte et aL 1988; Tanzi et aI. 1988; Kitaguchi et at. 1988). The larger 751 and 770 forms of  Amyloid Peptide Calcium Channels 515 A extracellular membrane Lysine-16 ') Amyloid Beta Protein[I-40] . cytoplasmic I ' ,','~ C I B H 2N'D I A2 E3-F4 R5 Hs'D7 S8-Gg Ylo'E1 l V12 H13 H 14 Q 15 Kls'L17- V t 8 F19 F20 A21 E22 D23 V24 G25 S 26 N27 K2s G 29 A30 131 132 G 33 L34 M 35 V36 G37 G38 V39. V40 OH. Fig. 1. Structure of the amyloid precursor protein APP). A) The alterna- tively spliced forms are shown numbered. The Kunitz sequence is in the alternative spliced forms. B) The sequence of the A/3P[l-40] domain is shown. The internally numbered sequence correlates with the amyloid domain in part A. K~ is lysine-16 in part A. APP contain an exon-7-defined Kunitz type serine proteinase inhibitor ( KPI') sequence (Ponte et aL 1988; Tanzi et al. 1988; Kitaguchi et al. 1988), and a large soluble fragment of APP, containing the Kunitz sequence, is eventually released from cells. This fragment is the previously described protease inhibitor protease nexin II (Hardy and Allsop, 1991). The remaining short segment of trans- membrane and cytosolic sequence is subsequently processed into the toxic amyloid /3 peptide (A/3P, /3A/4). The 40 residue peptide [1-40] is the most common, but [1-41] and [1-42] peptides also occur with specific anatomical distributions. Prior to the major cleavage step, APP is also processed within the cell by sequential glycosylation and other post-translational modifications reactions. Consequently, using antibodies to the distal N-terminal domain, three bands of APP are routinely seen by Western analysis (e.g., Sambamurti et aL 1992a,b). In PC12 cells, for example, a core glycosylated form of endogenous APP appears in the endoplasmic reticulum as a 100 kDa species. This protein is then further glycosylated and sulfated to a mature 140kDa form in the trans-golgi network (TGN). Finally, after reaching the TGN, the mature form is proteolyzed to release the above-mentioned 120kDa soluble fragment. However, only the mature 140kDa and immature 110kDa forms can be detected by antibodies against the proximal C-terminal or transmembrane domains (Setkoe et aL t988; Sambamurti et al. 1992a,b). Similar results have been reported for endogenous APP in secretory vesicles  5 6 Pollard, Arispe, and Rojas such as alpha granules in platelets (Van Nostrand et al., 1990) and chromaffin granules (Vassilacopoulou et al., 1995), and for recombinant AAP in CHO cells (Caporaso et al., 1994). Cleavage to form the soluble APP species is not a lysosomal process (Sambamurti et al., 1992a,b), and actually has been shown to occur within the chromaffin granule (Vassilacopoulou et al., 1995). In rat hippocampal slices, electrical stimulation causes the release of soluble forms of the APP lacking the APP carboxy terminus (Nitsch et al., 1993). Depending on where the major proteotytic secretase step occurs on te APP, the remainder of the protein left in the membrane is further processed to form either a non-toxic 3 kDa species, or a longer, toxic 4kDa species (A/3P, or /3A/4 ) (Haas and Selkoe, 1993). This processing step may occur in lysosomes (Cole et al., 1989; Caporaso et al., 1992; Golde et al., 1992), although there is evidence that the 4 kDa A/3P may not occur by normal processing (Sisodia er al., 1990). The sequence of the toxic At3P[1-40] product, numbered internally as in Fig. 1, is as follows: H2N-D1-A2-E3-F4-Rs-H6 DT-Ss-G9-Ylo-E1 l V12-H13-H~4-Q15 K~6-L17-V18-Flg- F20-A21-E22-D23-V24-G25-S26 N27 K28-G29-A30-I31-I32-G333 L34M35-V36-G37 G38-V39-V40-OH. The most frequent cleavage occurs between lysine16 (KI6) and leucine 17 (L17), leaving the sequence [1-16] attached to the secreted soluble APP. The protease has been termed the a-secretase, although the hydrolysis position on the APP is not sequence specific (Maruyama et al., 1991). Following further processing of the cytosolic domain, the resulting 3 kDa sequence [17-40] is either retained in the membrane or released, but is not toxic. Historically, it was presumed that patients with Alzheimer's Disease suffered because of aberrant proteolysis by a 13-secretase just before the N-terminal aspartic acid residue (viz. D1). This generates the toxic AI3P[1-40] peptide, which is either retained in the membrane or released extracellularly. However, it is now known that the toxic A/3P[1-40] peptide also occurs naturally in the CSF of non-affected controls, as well as Alzheimer's disease patients (Gold et al., 1992; Shoji et al., 1992; Haas et aL, 1992; Seubert et al., 1993; Busciglio et aL, 1993). The interpretation of this result is still evolving. Perhaps there are no real controls in the case of Alzheimer's disease, since the incidence increases with age. Or, perhaps target cells may express some prodromal property that renders them uniquely susceptible to attack by the toxic A/3P[1-40] peptide (Pollard et al., 1994a). It is well known that the potency of the A/3P[1-40] is cell specific, and from this perspective the A/3P[1-40] may act by delivering a coup de grace to a susceptible cell, leaving otherwise robust cells intact. CATION CHANNEL ACTIVITY OF AI3P[1 40] We noted that the first 16 residues of the toxic A~P[1-40] peptide had the remarkable property of alternating charged or neutral residues with hydrophobic residues. This was consistent with the amphipathic beta sheet structure occurring  Amyloid Peptide Calcium Channels 517 in this region, and the relationship between such structures and ion channels as disparate as porin/VDAC and shaker K Guy and Conti, 1990; Durrell and Guy, 1992) led us to consider the possibility that the A/3P[1-40] might also have ion channel properties. To test this hypothesis, liposomes containing A/3P were added to the cis chamber of a POPE/PS bilayer separating symmetric solutions of CsC12 Srispe et al. 1993a). We had anticipated that the A/3P[1-40] might conduct calcium, and we picked Cs+ for our first analysis because many calcium channels efficiently conduct this ion. The channels formed were multiconductance in character Pollard et al. 1994), with the most frequent species having a slope conductance of 206 pS. We next performed a classical ion gradient experiment to decide which charge the amyloid channel preferred to conduct. As shown in Fig. 2A upper traces), in a symmetric KC1 system, no conductance occurred at 0 mV driving force. However, in an asymmetric KC1 system positive current was observed at A" 20 Io " o,,F7 o 'Jli F10 5 pa 20 10 o o mvF 7 ~ 7 j I l C .... I10 i~ I 10 s pA 5 J ° mV  5 10 Fig. 2. The A/3P[1-40] channel is cation selective. A) In the upper trace, a symmetric system of KCI is used to measure the current conducted by A/3P[1- 40] channels between 20 and -20 mV. In the lower trace the KC1 solution on the trans side is increased and the experiment repeated. B) In symmetrical KCI the slope conductance was 325 pS, while in the asymmetrical KCI system 60 mM KCI vs. 40mMKC1) the slope conductance was 346pS. However, the reversal potential moved in the posi- tive direction. Thus the system is selective for K ~ over C1- from Arispe et aL 1993).
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