Less is known about potential interactions of soluble oligomers with cell surface receptor molecules, although similar targets to the fibrillar material have been proposed. An intriguing possibility arises from recent reports that oligomeric Aβ binds to the unstructured N-terminus of cellular PrP, with initial reports suggesting that the toxic effects of small Aβ oligomers may be mediated by this interaction (Lauren et al. 2009). If true, such an association may lead to an alteration of the as yet unidentified signaling functions of PrP, or may allow PrP to act as carrier for internalization of oligomeric Aβ. In this model, once inside the cell, the prefibrillar oligomers would exert loss of function effects on internal cellular components through mechanisms not yet identified. While this observation may provide a tantalizing link between two neurodegenerative disorders, more work need to be done to confirm the

It is important to note that since Aβ exists *in vitro* and *in vivo* as a continuum of different oligomeric states, none of which are particularly stable, it is difficult to distinguish biological effects induced by one specific type of non-fibrillar oligomer. Therefore, it is entirely feasible that Aβ has significantly different physiological effects when in different oligomeric forms. Thus, it is difficult to exclude any of the putative mechanisms for involvement of Aβ

There have been conflicting reports on the toxicity of PrP(106-126) largely due to confounding effects of its ability to form amyloid oligomers as well as potentially playing a role in conversion of full-length PrP to the infectious PrPSc form (Gu et al. 2002). PrP(106- 126) has been shown to be toxic in a number of different ways. Reports initially characterized PrP(106-126) as requiring full-length PrP for toxicity in cerebral endothelial cells (Deli et al. 2000). There is also significant evidence for PrP-independent cytotoxicity,

specificity of the interaction, and to define the potential role played by PrP.

oligomers in progression of amyloid disease without further study.

for this is unclear.

effects, making them harder to confirm.

**4.2 PrP(106-126) toxicity**

possibly through disruption of calcium homeostasis (Thellung et al. 2000; Demuro et al. 2005; Ferreiro et al. 2008).

Alternatively, hydrophobic, misfolded proteins are likely to have a high propensity to associate with membranes, and membrane binding of many amyloid peptides has been described extensively (McLaurin and Chakrabartty 1996; Yip et al. 2002; Kayed et al. 2004). Once bound to the membrane surface, or inserted into the bilayer, non-fibrillar oligomers would have the potential to rearrange into channels, pores, or non-specific aggregates at the membrane surface. Any of these mechanisms are likely to cause membrane destabilization and cell death, and it has recently been demonstrated for Aβ oligomers that increased membrane conductance can occur in the absence of channel formation (Sokolov et al. 2006). Physical disruption such as the introduction of membrane defects, possibly through insertion of oligomers, or through membrane-catalyzed fibril formation, would also be sufficient to induce leakage of cell contents and ultimately lead to cell death (McLaurin and Chakrabartty 1997; Yip et al. 2002).

While the oligomer fold is distinct from that of fibrils, as determined by differential antibody reactivity, a common theme emerging from structural studies of non-fibrillar amyloid oligomers is the presence of local fibril-like structure. While it does not speak to the actual mechanism through which toxicity is exerted, this observation may suggest that small fibril-like assemblies are the key element required for cytotoxicity. A similar phenomenon has been reported by Xue *et al*. (Xue et al. 2009), who demonstrated that fragmentation of mature amyloid fibrils formed by α-synuclein, β2-microglobulin and lysozyme leads to an increase in membrane disruption and cytotoxicity (Figure 5). Likewise amyloid fibrils formed by hexapeptides gained cytotoxicity towards primary neuronal cell culture after physical disruption (Pastor et al. 2008). In both studies, it is likely that the increase in active ends allows improved interactions with cellular targets – membranes or other cell surface molecules, where they are able to rearrange to form active, toxic entities. Oligomeric species, which are known to be more conformationally flexible and less stable than their fibrillar counterparts, may act through a similar mechanism, carrying active fibril-like segments to the site of toxic activity.

#### **4.1 Aβ toxicity**

As with molecular structure, the link between oligomerization of an amyloid protein and disease progression has been explored in detail for Aβ. Several lines of evidence support a role for oligomers as the toxic entity in Alzheimer's patients. Anti-oligomer antibodies stain diffuse amyloid in human brains, showing that soluble oligomers with similar structural properties as those formed *in vitro* exist *in vivo* (Kayed et al. 2007). In addition, both injection of *in vitro* formed oligomers into brain tissue, and reinjection of small soluble Aβ oligomers isolated from diseased animals are able to induce loss of synaptic function and neuronal death (Walsh and Selkoe 2004; Cleary et al. 2005; Haass and Selkoe 2007; Walsh and Selkoe 2007). This experiment has also been performed for non-disease causing amyloid proteins, supporting a role for other amyloid oligomers as *in vivo* cytotoxins (Baglioni et al. 2006).

Aβ oligomer toxicity *in vitro* has been attributed to several distinct mechanisms, including but not limited to, membrane disruption and the direct formation of ion channels. Certainly there have been numerous reports of increased membrane conductance or leakage in the presence of Aβ oligomers ranging from small globulomers to large prefibrillar assemblies (Chimon and Ishii 2005; Yu et al. 2009), with some evidence presented to support formation of discrete ion channels of pores (Arispe 2004; Quist et al. 2005). A lot of attention has been

possibly through disruption of calcium homeostasis (Thellung et al. 2000; Demuro et al.

Alternatively, hydrophobic, misfolded proteins are likely to have a high propensity to associate with membranes, and membrane binding of many amyloid peptides has been described extensively (McLaurin and Chakrabartty 1996; Yip et al. 2002; Kayed et al. 2004). Once bound to the membrane surface, or inserted into the bilayer, non-fibrillar oligomers would have the potential to rearrange into channels, pores, or non-specific aggregates at the membrane surface. Any of these mechanisms are likely to cause membrane destabilization and cell death, and it has recently been demonstrated for Aβ oligomers that increased membrane conductance can occur in the absence of channel formation (Sokolov et al. 2006). Physical disruption such as the introduction of membrane defects, possibly through insertion of oligomers, or through membrane-catalyzed fibril formation, would also be sufficient to induce leakage of cell contents and ultimately lead to cell death (McLaurin and

While the oligomer fold is distinct from that of fibrils, as determined by differential antibody reactivity, a common theme emerging from structural studies of non-fibrillar amyloid oligomers is the presence of local fibril-like structure. While it does not speak to the actual mechanism through which toxicity is exerted, this observation may suggest that small fibril-like assemblies are the key element required for cytotoxicity. A similar phenomenon has been reported by Xue *et al*. (Xue et al. 2009), who demonstrated that fragmentation of mature amyloid fibrils formed by α-synuclein, β2-microglobulin and lysozyme leads to an increase in membrane disruption and cytotoxicity (Figure 5). Likewise amyloid fibrils formed by hexapeptides gained cytotoxicity towards primary neuronal cell culture after physical disruption (Pastor et al. 2008). In both studies, it is likely that the increase in active ends allows improved interactions with cellular targets – membranes or other cell surface molecules, where they are able to rearrange to form active, toxic entities. Oligomeric species, which are known to be more conformationally flexible and less stable than their fibrillar counterparts, may act through a similar mechanism, carrying active fibril-like segments to

As with molecular structure, the link between oligomerization of an amyloid protein and disease progression has been explored in detail for Aβ. Several lines of evidence support a role for oligomers as the toxic entity in Alzheimer's patients. Anti-oligomer antibodies stain diffuse amyloid in human brains, showing that soluble oligomers with similar structural properties as those formed *in vitro* exist *in vivo* (Kayed et al. 2007). In addition, both injection of *in vitro* formed oligomers into brain tissue, and reinjection of small soluble Aβ oligomers isolated from diseased animals are able to induce loss of synaptic function and neuronal death (Walsh and Selkoe 2004; Cleary et al. 2005; Haass and Selkoe 2007; Walsh and Selkoe 2007). This experiment has also been performed for non-disease causing amyloid proteins, supporting a role for other amyloid oligomers as *in vivo* cytotoxins (Baglioni et al. 2006). Aβ oligomer toxicity *in vitro* has been attributed to several distinct mechanisms, including but not limited to, membrane disruption and the direct formation of ion channels. Certainly there have been numerous reports of increased membrane conductance or leakage in the presence of Aβ oligomers ranging from small globulomers to large prefibrillar assemblies (Chimon and Ishii 2005; Yu et al. 2009), with some evidence presented to support formation of discrete ion channels of pores (Arispe 2004; Quist et al. 2005). A lot of attention has been

2005; Ferreiro et al. 2008).

Chakrabartty 1997; Yip et al. 2002).

the site of toxic activity.

**4.1 Aβ toxicity**

focused on the latter, and several molecular modeling studies have proposed models for the structure of a putative pore or channel – typically a picket-fence arrangement of monomers adopting the β-loop-β structure from the fibril structures (Jang et al. 2007). Alternatively, alteration of the mechanical properties of the membrane could lead to increased conductance as observed by Sokolov *et al.* (Sokolov et al. 2006), although the structural basis for this is unclear.

Several very specific proposals for Aβ oligomer toxicity have been proposed as alternatives to membrane disruption. As a group these invoke interactions with specific cell-surface targets, with subsequent alteration of normal protein activity leading to loss of neuronal function and possibly to cell death (Verdier et al. 2004; Yankner and Lu 2009). While too numerous to describe in detail here, proposed interaction partners for Aβ have included the serpin-enzyme complex receptor (SEC-R) and the insulin receptor, both of which are capable of binding to monomeric Aβ, although the physiological effects are unclear (Verdier and Penke 2004). Fibrillar Aβ, on the other hand has been shown to bind a wide array of cellsurface proteins, including the receptor for advanced glycation end products (RAGE) complex and the amyloid precursor protein (Verdier and Penke 2004), leading in some cases in increased radical formation and oxidative stress. Similarly, binding to the α-7 nicotinic receptor can mediate N-methyl-D-aspartate (NMDA) receptor activity, with broad effects on cellular metabolism (Snyder et al. 2005). Any or all of these effects may play a role in loss of synaptic function, leading to symptomatic Alzheimer's disease. Other proposed interactions, such as dysregulation of calcium channels, may be confounded by membrane disruption effects, making them harder to confirm.

Less is known about potential interactions of soluble oligomers with cell surface receptor molecules, although similar targets to the fibrillar material have been proposed. An intriguing possibility arises from recent reports that oligomeric Aβ binds to the unstructured N-terminus of cellular PrP, with initial reports suggesting that the toxic effects of small Aβ oligomers may be mediated by this interaction (Lauren et al. 2009). If true, such an association may lead to an alteration of the as yet unidentified signaling functions of PrP, or may allow PrP to act as carrier for internalization of oligomeric Aβ. In this model, once inside the cell, the prefibrillar oligomers would exert loss of function effects on internal cellular components through mechanisms not yet identified. While this observation may provide a tantalizing link between two neurodegenerative disorders, more work need to be done to confirm the specificity of the interaction, and to define the potential role played by PrP.

It is important to note that since Aβ exists *in vitro* and *in vivo* as a continuum of different oligomeric states, none of which are particularly stable, it is difficult to distinguish biological effects induced by one specific type of non-fibrillar oligomer. Therefore, it is entirely feasible that Aβ has significantly different physiological effects when in different oligomeric forms. Thus, it is difficult to exclude any of the putative mechanisms for involvement of Aβ oligomers in progression of amyloid disease without further study.

#### **4.2 PrP(106-126) toxicity**

There have been conflicting reports on the toxicity of PrP(106-126) largely due to confounding effects of its ability to form amyloid oligomers as well as potentially playing a role in conversion of full-length PrP to the infectious PrPSc form (Gu et al. 2002). PrP(106- 126) has been shown to be toxic in a number of different ways. Reports initially characterized PrP(106-126) as requiring full-length PrP for toxicity in cerebral endothelial cells (Deli et al. 2000). There is also significant evidence for PrP-independent cytotoxicity,

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 103

of PrP(106-126) have shown that it causes permeabilization of model membranes (Kayed et al. 2004; Walsh et al. 2009) and induces cytotoxicity in neuroblastoma cell cultures (Kayed et al. 2003). It is well known that PrP(106-126) interacts with phospholipid membranes even as a monomeric peptide, with lipid composition playing a role both in interaction and postbinding events. For example, PrP(106-126) has been shown to cause the aggregation of liposomes containing the ganglioside GM1 (Kurganov et al. 2004). While there is no direct link to the disruption of calcium channels or activation of the JNK-c-jun pathway, current evidence supports direct membrane interaction and disruption as a mechanism for PrP(106- 126) cytotoxicity – at least in the absence of cell surface PrP. While unlikely in the face of recent studies for Aβ oligomers that show that structural alterations of the membrane are sufficient to increase conductance without requiring channels, rearrangement of the protein in the membrane to form discrete pores or channels cannot be ruled out (Eliezer 2006;

In terms of the structure-function relationship of PrP(106-126) oligomers, the similarities between the peptide subunits within the oligomeric and fibrillar forms of this protein are reminiscent of the large oligomers of Aβ(1-40) described by Chimon *et al*. (Chimon et al. 2007). Similar to one of the hypotheses proposed for Aβ, the insertion of small fibril fragments into the membrane may directly disrupt the membrane, nucleate the assembly of membrane-disruptive amyloid fibrils, or potentially rearrange into a toroidal or barrel stave type pore. In either case, the structural data supports the possibility of a common

The toxicity associated with α-synuclein appears to be slightly different than other amyloid systems. To start with, α-synuclein is significantly larger (140 amino acids) than Aβ (40 or 42 residues), amylin (37 residues), or many other amyloidogenic proteins. Additionally, in its native state α-synuclein is intrinsically disordered, gaining α-helical structure upon binding to phospholipid membranes (Weinreb et al. 1996; Eliezer et al. 2001; Ulmer et al. 2005). In Parkinson's disease, α-synuclein forms β-sheet rich aggregates, culminating in the formation of amyloid fibrils (Lashuel et al. 2002; Lashuel et al. 2002). Not surprisingly, given its innate propensity to interact with membranes, the cytotoxic oligomers of α-synuclein have been shown to physically alter the conductance of planar bilayer membranes, presumably through the formation of pores (Kim et al. 2009). Multiple conductance levels were observed upon addition of these spherical, 15-30 nm diameter oligomers to lipid bilayers. The conductance profiles were similar to those observed for bee-venom mellitin, further supporting the formation of amyloid pores by these oligomers. Intriguingly, these poreforming α-synuclein oligomers were recognized by the A11 antibody, which is specific for prefibrillar oligomers, suggesting that there are some common structural features shared with other amyloid oligomers, and providing an additional argument in favor of direct

It is clear that despite significant advances in the past decade, the link between accumulation of amyloidogenic proteins as β-sheet rich oligomers and disease pathogenesis remains somewhat unclear. In particular, the mechanism or mechanisms through which

mechanism of action linking large spherical oligomers of Aβ and PrP(106-126).

Sokolov et al. 2006).

**4.3** α**-synuclein toxicity**

**5. Conclusion**

membrane disruption by prefibrillar oligomers.

Fig. 5. The relationship between amyloid fibril length and toxicity. As the concentration of small fragments increases, increased membrane disruption and cellular toxicity are observed. As the fragments become smaller, it is proposed that they will be increasingly toxic to the cell. Reprinted with permission from Xue *et al.,* 2009. Copyright 2009 The American Chemical Society

but it is important to note that in most studies of PrP(106-126), the aggregation state of the peptide was not clearly defined, so the activity of prefibrillar oligomers is implicit rather than explicit in the results. PrP(106-126) has been shown to interact with L-type voltage sensitive calcium channels, causing apoptosis (Florio et al. 1998; Silei et al. 1999; Thellung et al. 2000). It has also been demonstrated that this peptide causes the activation of the JNK-c-Jun pathway, leading to apoptosis shortly after peptide treatment (Carimalo et al. 2005). There have been several reports of direct membrane destabilization by PrP(106-126), including the formation of ion channels (Lin et al. 1997), or alterations in membrane viscosity (Salmona et al. 1997). More recent work using well-defined prefibrillar oligomers of PrP(106-126) have shown that it causes permeabilization of model membranes (Kayed et al. 2004; Walsh et al. 2009) and induces cytotoxicity in neuroblastoma cell cultures (Kayed et al. 2003). It is well known that PrP(106-126) interacts with phospholipid membranes even as a monomeric peptide, with lipid composition playing a role both in interaction and postbinding events. For example, PrP(106-126) has been shown to cause the aggregation of liposomes containing the ganglioside GM1 (Kurganov et al. 2004). While there is no direct link to the disruption of calcium channels or activation of the JNK-c-jun pathway, current evidence supports direct membrane interaction and disruption as a mechanism for PrP(106- 126) cytotoxicity – at least in the absence of cell surface PrP. While unlikely in the face of recent studies for Aβ oligomers that show that structural alterations of the membrane are sufficient to increase conductance without requiring channels, rearrangement of the protein in the membrane to form discrete pores or channels cannot be ruled out (Eliezer 2006; Sokolov et al. 2006).

In terms of the structure-function relationship of PrP(106-126) oligomers, the similarities between the peptide subunits within the oligomeric and fibrillar forms of this protein are reminiscent of the large oligomers of Aβ(1-40) described by Chimon *et al*. (Chimon et al. 2007). Similar to one of the hypotheses proposed for Aβ, the insertion of small fibril fragments into the membrane may directly disrupt the membrane, nucleate the assembly of membrane-disruptive amyloid fibrils, or potentially rearrange into a toroidal or barrel stave type pore. In either case, the structural data supports the possibility of a common mechanism of action linking large spherical oligomers of Aβ and PrP(106-126).

#### **4.3** α**-synuclein toxicity**

102 Advanced Understanding of Neurodegenerative Diseases

Fig. 5. The relationship between amyloid fibril length and toxicity. As the concentration of small fragments increases, increased membrane disruption and cellular toxicity are observed. As the fragments become smaller, it is proposed that they will be increasingly toxic to the cell. Reprinted with permission from Xue *et al.,* 2009. Copyright 2009 The

but it is important to note that in most studies of PrP(106-126), the aggregation state of the peptide was not clearly defined, so the activity of prefibrillar oligomers is implicit rather than explicit in the results. PrP(106-126) has been shown to interact with L-type voltage sensitive calcium channels, causing apoptosis (Florio et al. 1998; Silei et al. 1999; Thellung et al. 2000). It has also been demonstrated that this peptide causes the activation of the JNK-c-Jun pathway, leading to apoptosis shortly after peptide treatment (Carimalo et al. 2005). There have been several reports of direct membrane destabilization by PrP(106-126), including the formation of ion channels (Lin et al. 1997), or alterations in membrane viscosity (Salmona et al. 1997). More recent work using well-defined prefibrillar oligomers

American Chemical Society

The toxicity associated with α-synuclein appears to be slightly different than other amyloid systems. To start with, α-synuclein is significantly larger (140 amino acids) than Aβ (40 or 42 residues), amylin (37 residues), or many other amyloidogenic proteins. Additionally, in its native state α-synuclein is intrinsically disordered, gaining α-helical structure upon binding to phospholipid membranes (Weinreb et al. 1996; Eliezer et al. 2001; Ulmer et al. 2005). In Parkinson's disease, α-synuclein forms β-sheet rich aggregates, culminating in the formation of amyloid fibrils (Lashuel et al. 2002; Lashuel et al. 2002). Not surprisingly, given its innate propensity to interact with membranes, the cytotoxic oligomers of α-synuclein have been shown to physically alter the conductance of planar bilayer membranes, presumably through the formation of pores (Kim et al. 2009). Multiple conductance levels were observed upon addition of these spherical, 15-30 nm diameter oligomers to lipid bilayers. The conductance profiles were similar to those observed for bee-venom mellitin, further supporting the formation of amyloid pores by these oligomers. Intriguingly, these poreforming α-synuclein oligomers were recognized by the A11 antibody, which is specific for prefibrillar oligomers, suggesting that there are some common structural features shared with other amyloid oligomers, and providing an additional argument in favor of direct membrane disruption by prefibrillar oligomers.

### **5. Conclusion**

It is clear that despite significant advances in the past decade, the link between accumulation of amyloidogenic proteins as β-sheet rich oligomers and disease pathogenesis remains somewhat unclear. In particular, the mechanism or mechanisms through which

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 105

Brown, D. R. (1998). Prion protein-overexpressing cells show altered response to a

Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N.,

Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J.,

Carimalo, J., Cronier, S., Petit, G., Peyrin, J. M., Boukhtouche, F., Arbez, N., Lemaigre-

Chan, J. C. Solid-State NMR Techniques for the Structural Determination of Amyloid Fibrils.

Cheng, H. M., Tsai, T. W., Huang, W. Y., Lee, H. K., Lian, H. Y., Chou, F. C., Mou, Y. and

Cheon, M., Chang, I., Mohanty, S., Luheshi, L. M., Dobson, C. M., Vendruscolo, M. and

Chimon, S. and Ishii, Y. (2005). Capturing intermediate structures of Alzheimer's beta-

*Chemical Society,* Vol. 127, No. 39, (Oct 2005), pp. 13472-3, ISSN 0002-7863. Chimon, S., Shaibat, M. A., Jones, C. R., Calero, D. C., Aizezi, B. and Ishii, Y. (2007).

 Molecular

Conway, K. A., Harper, J. D. and Lansbury, P. T., Jr. (2000). Fibrils formed in vitro from

*Topics in Current Chemistry,* Vol. 2011, (Jun pp. 1, ISSN 0340-1022.

Vol. 3, No. 9, (Sep 2007), pp. 1727-38, ISSN 1553-7358.

(Dec 2007), pp. 1157-1164, ISSN 1545-9985.

No. 48, (Nov 2007), pp. 18946-51, ISSN 1091-6490.

(Nov 1998), pp. 331-40, ISSN 0360-4012.

2311-9, ISSN 0953-816X.

ISSN 1520-4995.

ISSN 1097-6256.

6880, (Apr 2002), pp. 507-11, ISSN 0028-0836.

267, No. 1, (Jan 1992), pp. 546-54, ISSN 0021-9258.

neurotoxic prion protein peptide. *Journal of Neuroscience Research,* Vol. 54, No. 3,

Ramponi, G., Dobson, C. M. and Stefani, M. (2002). Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. *Nature,* Vol. 416, No.

Cotman, C. and Glabe, C. (1992). Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. *Journal of Biological Chemistry,* Vol.

Dubreuil, Y., Brugg, B. and Miquel, M. C. (2005). Activation of the JNK-c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection. *European Journal of Neuroscience,* Vol. 21, No. 9, (May 2005), pp.

Chan, J. C. (2011). Steric Zipper Formed by Hydrophobic Peptide Fragment of Syrian Hamster Prion Protein. *Biochemistry,* Vol. 50, No. 32, (Aug 2011), pp. 13,

Favrin, G. (2007). Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. *PLoS Computational Biology,*

amyloid, Abeta(1-40), by solid-state NMR spectroscopy. *Journal of the American*

Evidence of fibril-like beta-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's beta-amyloid. *Nature Structural and Molecular Biology,* Vol. 14, No. 12,

and Ashe, K. H. (2005). Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. *Nature Neuroscience,* Vol. 8, No. 1, (Jan 2005), pp. 79-84,

architecture of human prion protein amyloid: a parallel, in-register beta-structure. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 104,

alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. *Biochemistry,* Vol. 39, No. 10, (Mar 2000), pp. 2552-63, ISSN 0006-2960. Cooper, G. J., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B. and Reid, K. B. (1987).

Purification and characterization of a peptide from amyloid-rich pancreases of type

various non-fibrillar oligomers are capable of inducing cell death remain to be unambiguously determined. This is inextricably linked to questions of disease relevance – do all of the oligomers observed *in vitro* play a role in cell death during amyloidosis, and do they act through the same mechanisms? While the concept of a common mechanism of amyloid toxicity is attractive, given the broad range of cellular and tissue level effects reported to date, it may represent an oversimplification of a complex system.

Certainly it is possible that despite some common structural motifs and a shared ability to physically disrupt membranes, different oligomeric species formed by different amyloid proteins may exert a range of activities, all leading to the same effective endpoint – cell death and degenerative disease progression. Additional questions surround the fibril-like structures observed in most toxic amyloid oligomers described to date. Does this relate to studies showing that fragmentation of amyloid fibrils can create cytotoxic, membrane disrupting species, or are these fibril fragments acting via distinct pathways from prefibrillar oligomers? Ongoing efforts in structure determination of oligomers formed by different amyloid proteins, and in defining the molecular mechanisms for their cytotoxicity, will be required in order to address these important questions and to elucidate the link between structure and toxic function.

#### **6. References**

various non-fibrillar oligomers are capable of inducing cell death remain to be unambiguously determined. This is inextricably linked to questions of disease relevance – do all of the oligomers observed *in vitro* play a role in cell death during amyloidosis, and do they act through the same mechanisms? While the concept of a common mechanism of amyloid toxicity is attractive, given the broad range of cellular and tissue level effects

Certainly it is possible that despite some common structural motifs and a shared ability to physically disrupt membranes, different oligomeric species formed by different amyloid proteins may exert a range of activities, all leading to the same effective endpoint – cell death and degenerative disease progression. Additional questions surround the fibril-like structures observed in most toxic amyloid oligomers described to date. Does this relate to studies showing that fragmentation of amyloid fibrils can create cytotoxic, membrane disrupting species, or are these fibril fragments acting via distinct pathways from prefibrillar oligomers? Ongoing efforts in structure determination of oligomers formed by different amyloid proteins, and in defining the molecular mechanisms for their cytotoxicity, will be required in order to address these important questions and to elucidate the link

Ahmed, M., Davis, J., Aucoin, D., Sato, T., Ahuja, S., Aimoto, S., Elliott, J. I., Van Nostrand,

Arispe, N. (2004). Architecture of the Alzheimer's A beta P ion channel pore. *Journal of Membrane Biology,* Vol. 197, No. 1, (Jan 2004), pp. 33-48, ISSN 0022-2631. Baglioni, S., Casamenti, F., Bucciantini, M., Luheshi, L. M., Taddei, N., Chiti, F., Dobson, C.

Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W., Dyda, F., Reed, J. and

Baskakov, I. V., Legname, G., Baldwin, M. A., Prusiner, S. B. and Cohen, F. E. (2002).

Baskakov, I. V., Legname, G., Prusiner, S. B. and Cohen, F. E. (2001). Folding of prion protein

*Chemistry,* Vol. 277, No. 24, (Jun 2002), pp. 21140-8, ISSN 0021-9258.

*Chemistry,* Vol. 276, No. 23, (Jun 2001), pp. 19687-90, ISSN 0021-9258. Baxa, U., Wickner, R. B., Steven, A. C., Anderson, D. E., Marekov, L. N., Yau, W. M. and

W. E. and Smith, S. O. (2010). Structural conversion of neurotoxic amyloid-beta(1- 42) oligomers to fibrils. *Nature Structural and Molecular Biology,* Vol. 17, No. 5, (May

M. and Stefani, M. (2006). Prefibrillar amyloid aggregates could be generic toxins in higher organisms. *Journal of Neuroscience,* Vol. 26, No. 31, (Aug 2006), pp. 8160-7,

Tycko, R. (2000). Amyloid fibril formation by A beta 16-22, a seven-residue fragment of the Alzheimer's beta-amyloid peptide, and structural characterization by solid state NMR. *Biochemistry,* Vol. 39, No. 45, (Nov 2000), pp. 13748-59, ISSN

Pathway complexity of prion protein assembly into amyloid. *Journal of Biological*

to its native alpha-helical conformation is under kinetic control. *Journal of Biological*

Tycko, R. (2007). Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance. *Biochemistry,* Vol. 46, No. 45, (Nov

reported to date, it may represent an oversimplification of a complex system.

between structure and toxic function.

ISSN 1529-2401.

0006-2960.

2010), pp. 561-7, ISSN 1545-9985.

2007), pp. 13149-13162, ISSN 0006-2960.

**6. References**

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 107

Haass, C. and Selkoe, D. J. (2007). Soluble protein oligomers in neurodegeneration: lessons

Heise, H., Hoyer, W., Becker, S., Andronesi, O. C., Riedel, D. and Baldus, M. (2005).

Hong, D. P., Fink, A. L. and Uversky, V. N. (2008). Structural characteristics of alpha-

Huang, T. H., Yang, D. S., Plaskos, N. P., Go, S., Yip, C. M., Fraser, P. E. and Chakrabartty,

Jahn, T. R., Makin, O. S., Morris, K. L., Marshall, K. E., Tian, P., Sikorski, P. and Serpell, L. C.

Jang, H., Zheng, J. and Nussinov, R. (2007). Models of beta-amyloid ion channels in the

*Biophysical Journal,* Vol. 93, No. 6, (Sep 2007), pp. 1938-49, ISSN 0006-3495. Janson, J., Ashley, R. H., Harrison, D., McIntyre, S. and Butler, P. C. (1999). The mechanism

Jaroniec, C. P., MacPhee, C. E., Astrof, N. S., Dobson, C. M. and Griffin, R. G. (2002).

Jaroniec, C. P., MacPhee, C. E., Bajaj, V. S., McMahon, M. T., Dobson, C. M. and Griffin, R. G.

Jarrett, J. T., Berger, E. P. and Lansbury, P. T., Jr. (1993). The carboxy terminus of the beta

Jobling, M. F., Stewart, L. R., White, A. R., McLean, C., Friedhuber, A., Maher, F.,

*Biology,* Vol. 383, No. 1, (Oct 2008), pp. 214-23, ISSN 1089-8638.

Vol. 395, No. 4, (Jan 2009), pp. 717-27, ISSN 1089-8638.

No. 26, (Dec 2002), pp. 16748-53, ISSN 0027-8424.

(Oct 1999), pp. 1557-1565, ISSN 0022-3042.

No. 3, (Jan 2002), pp. 2275-86, ISSN 0021-9258.

15871-6, ISSN 0027-8424.

0022-2836.

1797.

6, ISSN 0027-8424.

4693-7, ISSN 0006-2960.

Vol. 8, No. 2, (Feb 2007), pp. 101-12, ISSN 1471-0072.

potentially neurotoxic transmembrane PrP. *Journal of Biological Chemistry,* Vol. 277,

from the Alzheimer's amyloid beta-peptide. *Nature Reviews Molecular Cell Biology,*

Molecular-level secondary structure, polymorphism, and dynamics of full-length alpha-synuclein fibrils studied by solid-state NMR. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 102, No. 44, (Nov 2005), pp.

synuclein oligomers stabilized by the flavonoid baicalein. *Journal of Molecular*

A. (2000). Structural studies of soluble oligomers of the Alzheimer beta-amyloid peptide. *Journal of Molecular Biology,* Vol. 297, No. 1, (Mar 2000), pp. 73-87, ISSN

(2009). The common architecture of cross-beta amyloid. *Journal of Molecular Biology,*

membrane suggest that channel formation in the bilayer is a dynamic process.

of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. *Diabetes,* Vol. 48, No. 3, (Mar 1999), pp. 491-8, ISSN 0012-

Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 99,

(2004). High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 101, No. 3, (Jan 2004), pp. 711-

amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. *Biochemistry,* Vol. 32, No. 18, (May 1993), pp.

Beyreuther, K., Masters, C. L., Barrow, C. J., Collins, S. J. and Cappai, R. (1999). The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106-126. *Journal of Neurochemistry,* Vol. 73, No. 4,

2 diabetic patients. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 84, No. 23, (Dec 1987), pp. 8628-32, ISSN 0027-8424.

Deli, M. A., Sakaguchi, S., Nakaoke, R., Abraham, C. S., Takahata, H., Kopacek, J.,

Demuro, A., Mina, E., Kayed, R., Milton, S. C., Parker, I. and Glabe, C. G. (2005). Calcium

Eanes, E. D. and Glenner, G. G. (1968). X-ray diffraction studies on amyloid filaments.

Eliezer, D. (2006). Amyloid ion channels: a porous argument or a thin excuse? *The Journal of General Physiology,* Vol. 128, No. 6, (Dec 2006), pp. 631-3, ISSN 0022-1295. Eliezer, D., Kutluay, E., Bussell, R., Jr. and Browne, G. (2001). Conformational properties of

Fandrich, M., Meinhardt, J. and Grigorieff, N. (2009). Structural polymorphism of Alzheimer

Ferreiro, E., Oliveira, C. R. and Pereira, C. M. (2008). The release of calcium from the

Florio, T., Thellung, S., Amico, C., Robello, M., Salmona, M., Bugiani, O., Tagliavini, F.,

Forloni, G., Angeretti, N., Chiesa, R., Monzani, E., Salmona, M., Bugiani, O. and Tagliavini,

Geddes, A. J., Parker, K. D., Atkins, E. D. and Beighton, E. (1968). "Cross-beta" conformation

Glenner, G. G. and Wong, C. W. (1984). Alzheimer's disease: initial report of the purification

Govaerts, C., Wille, H., Prusiner, S. B. and Cohen, F. E. (2004). Evidence for assembly of

Gu, Y., Fujioka, H., Mishra, R. S., Li, R. and Singh, N. (2002). Prion peptide 106-126

Vol. 307, No. 4, (Apr 2001), pp. 1061-73, ISSN 0022-2836.

*of America,* Vol. 84, No. 23, (Dec 1987), pp. 8628-32, ISSN 0027-8424.

2000), pp. 3931-6, ISSN 0959-4965.

2005), pp. 17294-300, ISSN 0021-9258.

ISSN 0022-1554.

1933-6896.

0022-2836.

0006-291X.

ISSN 0027-8424.

pp. 331-42, ISSN 1095-953X.

1998), pp. 341-52, ISSN 0360-4012.

(Apr 1993), pp. 543-6, ISSN 0028-0836.

2 diabetic patients. *Proceedings of the National Academy of Sciences of the United States*

Shigematsu, K., Katamine, S. and Niwa, M. (2000). PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C). *Neuroreport,* Vol. 11, No. 17, (Nov

dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. *Journal of Biological Chemistry,* Vol. 280, No. 17, (Apr

*Journal of Histochemistry & Cytochemistry,* Vol. 16, No. 11, (Nov 1968), pp. 673-7,

alpha-synuclein in its free and lipid-associated states. *Journal of Molecular Biology,*

Abeta and other amyloid fibrils. *Prion,* Vol. 3, No. 2, (Apr 2009), pp. 89-93, ISSN

endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway. *Neurobiology of Disease,* Vol. 30, No. 3, (Jun 2008),

Forloni, G. and Schettini, G. (1998). Prion protein fragment 106-126 induces apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in the GH3 cell line. *Journal of Neuroscience Research,* Vol. 54, No. 3, (Nov

F. (1993). Neurotoxicity of a prion protein fragment. *Nature,* Vol. 362, No. 6420,

in proteins. *Journal of Molecular Biology,* Vol. 32, No. 2, (Mar 1968), pp. 343-58, ISSN

and characterization of a novel cerebrovascular amyloid protein. *Biochemical and Biophysical Research Communications,* Vol. 120, No. 3, (May 1984), pp. 885-90, ISSN

prions with left-handed beta-helices into trimers. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 101, No. 22, (Jun 2004), pp. 8342-7,

modulates the aggregation of cellular prion protein and induces the synthesis of

potentially neurotoxic transmembrane PrP. *Journal of Biological Chemistry,* Vol. 277, No. 3, (Jan 2002), pp. 2275-86, ISSN 0021-9258.

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 109

Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowak, R. J., Walz, T. and Lansbury, P. T.,

Lauren, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W. and Strittmatter, S. M. (2009).

Lee, S. W., Mou, Y., Lin, S. Y., Chou, F. C., Tseng, W. H., Chen, C. H., Lu, C. Y., Yu, S. S. and

Lin, H., Bhatia, R. and Lal, R. (2001). Amyloid beta protein forms ion channels: implications

Lin, M. C., Mirzabekov, T. and Kagan, B. L. (1997). Channel formation by a neurotoxic prion

Losic, D., Martin, L. L., Mechler, A., Aguilar, M. I. and Small, D. H. (2006). High resolution

*Structural Biology,* Vol. 155, No. 1, (Jul 2006), pp. 104-10, ISSN 1047-8477. Luca, S., Yau, W. M., Leapman, R. and Tycko, R. (2007). Peptide conformation and

*Biochemistry,* Vol. 46, No. 47, (Nov 2007), pp. 13505-22, ISSN 0006-2960. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H., Schubert, D. and

Madine, J., Jack, E., Stockley, P. G., Radford, S. E., Serpell, L. C. and Middleton, D. A. (2008).

McLaurin, J. and Chakrabartty, A. (1996). Membrane disruption by Alzheimer beta-amyloid

McLaurin, J. and Chakrabartty, A. (1997). Characterization of the interactions of Alzheimer

*Biochemistry,* Vol. 245, No. 2, (Apr 1997), pp. 355-63, ISSN 0014-2956.

322, No. 5, (Oct 2002), pp. 1089-102, ISSN 0022-2836.

2008), pp. 1142-54, ISSN 1089-8638.

2001), pp. 2433-44, ISSN 1530-6860.

2005), pp. 17342-7, ISSN 0027-8424.

No. 43, (Oct 1996), pp. 26482-9, ISSN 0021-9258.

ISSN 0021-9258.

1520-5126.

9258.

Jr. (2002). Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils. *Journal of Molecular Biology,* Vol.

Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. *Nature,* Vol. 457, No. 7233, (Feb 2009), pp. 1128-32, ISSN 1476-4687. Laurents, D. V., Gorman, P. M., Guo, M., Rico, M., Chakrabartty, A. and Bruix, M. (2005).

Alzheimer's Abeta40 studied by NMR at low pH reveals that sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) binds and promotes beta-ball oligomerization. *Journal of Biological Chemistry,* Vol. 280, No. 5, (Feb 2005), pp. 3675-85, ISSN 0021-

Chan, J. C. (2008). Steric zipper of the amyloid fibrils formed by residues 109-122 of the Syrian hamster prion protein. *Journal of Molecular Biology,* Vol. 378, No. 5, (May

for Alzheimer's disease pathophysiology. *FASEB Journal,* Vol. 15, No. 13, (Nov

protein fragment. *Journal of Biological Chemistry,* Vol. 272, No. 1, (Jan 1997), pp. 44-7,

scanning tunnelling microscopy of the beta-amyloid protein (Abeta1-40) of Alzheimer's disease suggests a novel mechanism of oligomer assembly. *Journal of*

supramolecular organization in amylin fibrils: constraints from solid-state NMR.

Riek, R. (2005). 3D structure of Alzheimer's amyloid-beta(1-42) fibrils. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 102, No. 48, (Nov

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction. *Journal of the American Chemical Society,* Vol. 130, No. 45, (Nov 2008), pp. 14990-5001, ISSN

peptides mediated through specific binding to either phospholipids or gangliosides. Implications for neurotoxicity. *Journal of Biological Chemistry,* Vol. 271,

beta-amyloid peptides with phospholipid membranes. *European Journal of*

Kayed, R., Head, E., Sarsoza, F., Saing, T., Cotman, C. W., Necula, M., Margol, L., Wu, J.,

Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W. and

Kayed, R., Pensalfini, A., Margol, L., Sokolov, Y., Sarsoza, F., Head, E., Hall, J. and Glabe, C.

Kayed, R., Sokolov, Y., Edmonds, B., McIntire, T. M., Milton, S. C., Hall, J. E. and Glabe, C.

Kim, H. Y., Cho, M. K., Kumar, A., Maier, E., Siebenhaar, C., Becker, S., Fernandez, C. O.,

Kumar-Singh, S., Theuns, J., Van Broeck, B., Pirici, D., Vennekens, K., Corsmit, E., Cruts, M.,

Kurganov, B., Doh, M. and Arispe, N. (2004). Aggregation of liposomes induced by the toxic

Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., Morgan,

Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T. and Lansbury, P. T., Jr. (2002).

418, No. 6895, (Jul 2002), pp. 291, ISSN 0028-0836.

*Chemical Society,* Vol. 4, (Nov 2009), pp. 4, ISSN 1520-5126.

1326.

486-9, ISSN 1095-9203.

4230-7, ISSN 0021-9258.

2010), pp. 19808-13, ISSN 1091-6490.

2006), pp. 686-95, ISSN 1098-1004.

6448-53, ISSN 0027-8424.

0196-9781.

Breydo, L., Thompson, J. L., Rasool, S., Gurlo, T., Butler, P. and Glabe, C. G. (2007). Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. *Molecular Neurodegeneration,* Vol. 2, No. 2, (Sept 2007), pp. 18, ISSN 1750-

Glabe, C. G. (2003). Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. *Science,* Vol. 300, No. 5618, (Apr 2003), pp.

(2009). Annular protofibrils are a structurally and functionally distinct type of amyloid oligomer. *Journal of Biological Chemistry,* Vol. 284, No. 7, (Feb 2009), pp.

G. (2004). Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. *Journal of Biological Chemistry,* Vol. 279, No. 45, (Nov 2004), pp. 46363-6, ISSN 0021-9258. Khan, M. Q., Sweeting, B., Mulligan, V. K., Arslan, P. E., Cashman, N. R., Pai, E. F. and

Chakrabartty, A. (2010). Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 107, No. 46, (Nov

Lashuel, H. A., Benz, R., Lange, A. and Zweckstetter, M. (2009). Structural Properties of Pore-Forming Oligomers of alpha-Synuclein. *Journal of the American*

Dermaut, B., Wang, R. and Van Broeckhoven, C. (2006). Mean age-of-onset of familial alzheimer disease caused by presenilin mutations correlates with both increased Abeta42 and decreased Abeta40. *Human Mutation,* Vol. 27, No. 7, (Jul

peptides Alzheimer's Abs, human amylin and prion(106-126): facilitation by membrane-bound GM1 ganglioside. *Peptides,* Vol. 25, No. 2, 2004), pp. 217-232, ISSN

T. E., Rozovsky, I., Trommer, B., Viola, K. L., Wals, P., Zhang, C., Finch, C. E., Krafft, G. A. and Klein, W. L. (1998). Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 95, No. 11, (May 26 1998), pp.

Neurodegenerative disease: amyloid pores from pathogenic mutations. *Nature,* Vol.

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 111

Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J. and

Salmona, M., Forloni, G., Diomede, L., Algeri, M., De Gioia, L., Angeretti, N., Giaccone, G.,

Salmona, M., Malesani, P., DeGioia, L., Gorla, S., Bruschi, M., Molinari, A., Della Vedova, F.,

Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I.,

Schmidt, M., Sachse, C., Richter, W., Xu, C., Fandrich, M. and Grigorieff, N. (2009).

Shewmaker, F., Wickner, R. B. and Tycko, R. (2006). Amyloid of the prion domain of Sup35p

Silei, V., Fabrizi, C., Venturini, G., Salmona, M., Bugiani, O., Tagliavini, F. and Lauro, G. M.

Silveira, J. R., Raymond, G. J., Hughson, A. G., Race, R. E., Sim, V. L., Hayes, S. F. and

Simoneau, S., Rezaei, H., Sales, N., Kaiser-Schulz, G., Lefebvre-Roque, M., Vidal, C.,

Vol. 194, No. 3, (Aug 1993), pp. 1380-6, ISSN 0006-291X.

*Research,* Vol. 818, No. 1, 1999), pp. 168-170, ISSN 0006-8993.

*Pathogens,* Vol. 3, No. 8, (Aug 2007), pp. e125, ISSN 1553-7374.

No. 7056, (Sep 2005), pp. 257-61, ISSN 1476-4687.

*of America,* Vol. 102, No. 30, (Jul 2005), pp. 10427-32, ISSN 0027-8424. Sachse, C., Fandrich, M. and Grigorieff, N. (2008). Paired beta-sheet structure of an Abeta(1-

7462-6, ISSN 1091-6490.

1476-4687.

0027-8424.

Vol. 4, No. 1, 1997), pp. 47-57, ISSN 0969-9961.

1999), pp. 207-214, ISSN 0264-6021.

Lal, R. (2005). Amyloid ion channels: a common structural link for proteinmisfolding disease. *Proceedings of the National Academy of Sciences of the United States*

40) amyloid fibril revealed by electron microscopy. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 105, No. 21, (May 2008), pp.

Tagliavini, F. and Bugiani, O. (1997). A neurotoxic and gliotrophic fragment of the prion protein increases plasma membrane microviscosity. *Neurobiology of Disease,*

Pedrotti, B., Marrari, M. A., Awan, T., Bugiani, O., Forloni, G. and Tagliavini, F. (1999). Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126. *Biochemical Journal,* Vol. 342, (Aug

Thompson, M. J., Balbirnie, M., Wiltzius, J. J., McFarlane, H. T., Madsen, A. O., Riekel, C. and Eisenberg, D. (2007). Atomic structures of amyloid cross-beta spines reveal varied steric zippers. *Nature,* Vol. 447, No. 7143, (May 2007), pp. 453-7, ISSN

Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) amyloid fibrils reveals similar protofilament structures. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 106, No. 47, (Nov 2009), pp. 19813-8, ISSN 1091-6490. Selvaggini, C., De Gioia, L., Cantu, L., Ghibaudi, E., Diomede, L., Passerini, F., Forloni, G.,

Bugiani, O., Tagliavini, F. and Salmona, M. (1993). Molecular characteristics of a protease-resistant, amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein. *Biochemical and Biophysical Research Communications,*

has an in-register parallel beta-sheet structure. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 103, No. 52, (Dec 2006), pp. 19754-9, ISSN

(1999). Activation of microglial cells by PrP and beta-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels. *Brain*

Caughey, B. (2005). The most infectious prion protein particles. *Nature,* Vol. 437,

Fournier, J. G., Comte, J., Wopfner, F., Grosclaude, J., Schatzl, H. and Lasmezas, C. I. (2007). In vitro and in vivo neurotoxicity of prion protein oligomers. *PLoS*

Miller, Y., Ma, B., Tsai, C. J. and Nussinov, R. Hollow core of Alzheimer's Abeta42 amyloid

Naito, A., Kamihira, M., Inoue, R. and Saito, H. (2004). Structural diversity of amyloid fibril

Necula, M., Kayed, R., Milton, S. and Glabe, C. G. (2007). Small molecule inhibitors of

Nielsen, J. T., Bjerring, M., Jeppesen, M. D., Pedersen, R. O., Pedersen, J. M., Hein, K. L.,

Olofsson, A., Lindhagen-Persson, M., Sauer-Eriksson, A. E. and Ohman, A. (2007). Amide

Ono, K., Condron, M. M. and Teplow, D. B. (2009). Structure-neurotoxicity relationships of

Paravastu, A. K., Petkova, A. T. and Tycko, R. (2006). Polymorphic fibril formation by

Pastor, M. T., Kummerer, N., Schubert, V., Esteras-Chopo, A., Dotti, C. G., Lopez de la Paz,

Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin, O. N., Leapman, R. D., Delaglio, F. and

Petkova, A. T., Leapman, R. D., Guo, Z., Yau, W. M., Mattson, M. P. and Tycko, R. (2005).

fibrils. *Science,* Vol. 307, No. 5707, (Jan 2005), pp. 262-5, ISSN 1095-9203. Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. *Science,* Vol.

216, No. 4542, (Apr 1982), pp. 136-44, ISSN 0036-8075.

*Journal,* Vol. 404, No. 1, (May 2007), pp. 63-70, ISSN 1470-8728.

33, ISSN 1091-6490.

ISSN 0749-1581.

2007), pp. 10311-24, ISSN 0021-9258.

pp. 2118-21, ISSN 1521-3773.

5386-93, ISSN 1520-6882.

ISSN 0027-8424.

No. 12, (Jun 2006), pp. 4618-29, ISSN 0006-3495.

2008), pp. 695-707, ISSN 1089-8638.

observed by cryoEM is relevant at physiological pH. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 107, No. 32, (Aug pp. 14128-

formed in human calcitonin as revealed by site-directed 13C solid-state NMR spectroscopy. *Magnetic Resonance in Chemistry,* Vol. 42, No. 2, (Feb 2004), pp. 247-57,

aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. *Journal of Biological Chemistry,* Vol. 282, No. 14, (Apr

Vosegaard, T., Skrydstrup, T., Otzen, D. E. and Nielsen, N. C. (2009). Unique identification of supramolecular structures in amyloid fibrils by solid-state NMR spectroscopy. *Angewandte Chemie International Edition,* Vol. 48, No. 12, (Mar 2009),

solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloidbeta(1-42) form different fibrillar structures under identical conditions. *Biochemical*

amyloid beta-protein oligomers. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 106, No. 35, (Sep 2009), pp. 14745-50, ISSN 1091-6490. Pan, J., Han, J., Borchers, C. H. and Konermann, L. (2011). Conformer-specific hydrogen

exchange analysis of abeta(1-42) oligomers by top-down electron capture dissociation mass spectrometry. *Analytical Chemistry,* Vol. 83, No. 13, (Jul 2011), pp.

residues 10-40 of the Alzheimer's beta-amyloid peptide. *Biophysical Journal,* Vol. 90,

M. and Serrano, L. (2008). Amyloid toxicity is independent of polypeptide sequence, length and chirality. *Journal of Molecular Biology,* Vol. 375, No. 3, (Jan

Tycko, R. (2002). A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 99, No. 26, (Dec 2002), pp. 16742-7,

Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid

Structure-Toxicity Relationships of Amyloid Peptide Oligomers 113

Walsh, D. M. and Selkoe, D. J. (2004). Oligomers on the brain: the emerging role of soluble

Walsh, D. M. and Selkoe, D. J. (2007). A beta oligomers - a decade of discovery. *Journal of Neurochemistry,* Vol. 101, No. 5, (Jun 2007), pp. 1172-84, ISSN 0022-3042. Walsh, P., Neudecker, P. and Sharpe, S. (2010). Structural Properties and Dynamic Behavior

Walsh, P., Simonetti, K. and Sharpe, S. (2009). Core structure of amyloid fibrils formed by

Walsh, P., Yau, J., Simonetti, K. and Sharpe, S. (2009). Morphology and Secondary Structure

Wasmer, C., Lange, A., Van Melckebeke, H., Siemer, A. B., Riek, R. and Meier, B. H. (2008).

Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. and Lansbury, P. T., Jr. (1996). NACP,

*Biochemistry,* Vol. 35, No. 43, (Oct 1996), pp. 13709-15, ISSN 0006-2960. Williams, A. D., Sega, M., Chen, M., Kheterpal, I., Geva, M., Berthelier, V., Kaleta, D. T.,

*Chemistry,* Vol. 284, No. 49, (Dec 2009), pp. 34272-82, ISSN 1083-351X. Yankner, B. A. and Lu, T. (2009). Amyloid beta-protein toxicity and the pathogenesis of

Yip, C. M., Darabie, A. A. and McLaurin, J. (2002). Abeta42-peptide assembly on lipid

Yu, L., Edalji, R., Harlan, J. E., Holzman, T. F., Lopez, A. P., Labkovsky, B., Hillen, H.,

Yu, X., Wang, Q. and Zheng, J. (2010). Structural determination of Abeta25-35 micelles by

Yu, X. and Zheng, J. (2011). Polymorphic Structures of Alzheimer's beta-Amyloid Globulomers. *PLoS One,* Vol. 6, No. 6, (Jun 2011), pp. e20575, ISSN 1932-6203. Zhang, A., Qi, W., Good, T. A. and Fernandez, E. J. (2009). Structural differences between

No. 9, (Feb 2009), pp. 1870-1877, ISSN 1520-4995.

*Society,* Vol. 132, No. 22, (Jun 2010), pp. 7684-7695, ISSN 0002-7863.

48, No. 25, (Jun 2009), pp. 5779-5781, ISSN 0969-2126.

(Jun 2004), pp. 213-28, ISSN 0929-8665.

pp. 417-426, ISSN 0969-2126 (Print).

9203.

4755-9, ISSN 0021-9258.

666-74, ISSN 1542-0086.

0022-2836.

protein aggregates in neurodegeneration. *Protein & Peptide Letters,* Vol. 11, No. 3,

of Nonfibrillar Oligomers Formed by PrP(106-126). *Journal of the American Chemical*

residues 106-126 of the human prion protein. *Structure,* Vol. 17, No. 3, (Mar 2009),

of Stable β-Oligomers Formed by Amyloid Peptide PrP(106-126). *Biochemistry,* Vol.

Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core. *Science,* Vol. 319, No. 5869, (Mar 2008), pp. 1523-6, ISSN 1095-

a protein implicated in Alzheimer's disease and learning, is natively unfolded.

Cook, K. D. and Wetzel, R. (2005). Structural properties of Abeta protofibrils stabilized by a small molecule. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 102, No. 20, (May 2005), pp. 7115-20, ISSN 0027-8424. Xue, W. F., Hellewell, A. L., Gosal, W. S., Homans, S. W., Hewitt, E. W. and Radford, S. E.

(2009). Fibril fragmentation enhances amyloid cytotoxicity. *Journal of Biological*

Alzheimer disease. *Journal of Biological Chemistry,* Vol. 284, No. 8, (Feb 2009), pp.

bilayers. *Journal of Molecular Biology,* Vol. 318, No. 1, (Apr 2002), pp. 97-107, ISSN

Barghorn, S., Ebert, U., Richardson, P. L., Miesbauer, L., Solomon, L., Bartley, D., Walter, K., Johnson, R. W., Hajduk, P. J. and Olejniczak, E. T. (2009). Structural Characterization of a Soluble Amyloid beta-Peptide Oligomer. *Biochemistry,* Vol. 48,

molecular dynamics simulations. *Biophysical Journal,* Vol. 99, No. 2, (Jul 2010), pp.

Abeta(1-40) intermediate oligomers and fibrils elucidated by proteolytic

Snyder, E. M., Nong, Y., Almeida, C. G., Paul, S., Moran, T., Choi, E. Y., Nairn, A. C., Salter,

Sokolov, Y., Kozak, J. A., Kayed, R., Chanturiya, A., Glabe, C. and Hall, J. E. (2006). Soluble

Sokolowski, F., Modler, A. J., Masuch, R., Zirwer, D., Baier, M., Lutsch, G., Moss, D. A., Gast,

Thellung, S., Florio, T., Villa, V., Corsaro, A., Arena, S., Amico, C., Robello, M., Salmona, M.,

Tycko, R. and Ishii, Y. (2003). Constraints on supramolecular structure in amyloid fibrils

Ulmer, T. S., Bax, A., Cole, N. B. and Nussbaum, R. L. (2005). Structure and dynamics of

van der Wel, P. C., Lewandowski, J. R. and Griffin, R. G. (2007). Solid-state NMR study of

van der Wel, P. C., Lewandowski, J. R. and Griffin, R. G. (2007). Structural characterization

Verdier, Y. and Penke, B. (2004). Binding sites of amyloid beta-peptide in cell plasma

Verdier, Y., Zarandi, M. and Penke, B. (2004). Amyloid beta-peptide interactions with

Vilar, M., Chou, H. T., Luhrs, T., Maji, S. K., Riek-Loher, D., Verel, R., Manning, G.,

(Aug 2005), pp. 1051-8, ISSN 1097-6256.

Vol. 62, (May 2011), pp. 279-99, ISSN 0066-426X.

10, (Mar 2005), pp. 9595-603, ISSN 0021-9258.

No. 44, (Nov 2007), pp. 9457-69, ISSN 1520-4995.

*Science,* Vol. 5, No. 1, (Feb 2004), pp. 19-31, ISSN 1389-2037.

2007), pp. 5117-30, ISSN 0002-7863.

2008), pp. 8637-42, ISSN 1091-6490.

839-40, ISSN 0028-0836.

6606-7, ISSN 0002-7863.

ISSN 1075-2617.

M. W., Lombroso, P. J., Gouras, G. K. and Greengard, P. (2005). Regulation of NMDA receptor trafficking by amyloid-beta. *Nature Neuroscience,* Vol. 8, No. 8,

amyloid oligomers increase bilayer conductance by altering dielectric structure. *Journal of General Physiology,* Vol. 128, No. 6, (Dec 2006), pp. 637-47, ISSN 0022-1295.

K. and Naumann, D. (2003). Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein. *Journal of Biological Chemistry,* Vol. 278, No. 42, (Oct 2003), pp. 40481-92, ISSN 0021-9258. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. and Goedert, M.

(1997). Alpha-synuclein in Lewy bodies. *Nature,* Vol. 388, No. 6645, (Aug 1997), pp.

Forloni, G., Bugiani, O., Tagliavini, F. and Schettini, G. (2000). Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106-126. *Neurobiology of Disease,* Vol. 7, No. 4, (Aug 2000), pp. 299-309, ISSN 0969-9961. Tycko, R. (2011). Solid-state NMR studies of amyloid fibril structure. *Annu Rev Phys Chem,*

from two-dimensional solid-state NMR spectroscopy with uniform isotopic labeling. *Journal of the American Chemical Society,* Vol. 125, No. 22, (Jun 2003), pp.

micelle-bound human alpha-synuclein. *Journal of Biological Chemistry,* Vol. 280, No.

amyloid nanocrystals and fibrils formed by the peptide GNNQQNY from yeast prion protein Sup35p. *Journal of the American Chemical Society,* Vol. 129, No. 16, (Apr

of GNNQQNY amyloid fibrils by magic angle spinning NMR. *Biochemistry,* Vol. 49,

membrane and implications for Alzheimer's disease. *Current Protein & Peptide*

neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease. *Journal of Peptide Science,* Vol. 10, No. 5, (May 2004), pp. 229-48,

Stahlberg, H. and Riek, R. (2008). The fold of alpha-synuclein fibrils. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 105, No. 25, (Jun

**5**

*Japan*

**Disruption of Calcium Homeostasis**

**Channel Formation by** β **Amyloid Protein**

Alzheimer's disease (AD) is a severe senile type of dementia that affects a large number of elderly people. It is estimated that 5.4 million people in the worldwide are affected by this disease in 2011 and this number continues to grow yearly. AD is characterized by profound memory loss and severe cognitive decline. The pathological hallmarks of AD are the presence of numerous extracellular deposits termed senile plaques and intraneuronal neurofibrillary tangles (NFTs) (Selkoe, 1991). The selective loss of synapses and neurons is observed in the brain of patients, and this decrease in the number of synapses is strongly correlated with memory impairment (Terry et al., 1991). The major component of NFTs is the phosphorylated tau protein, while β-amyloid protein (AβP) is the major component of

Although the pathological cause of AD has not yet strictly been elucidated, numerous biochemical, toxicological, cell biological, and genetic studies have supported the "amyloid cascade hypothesis", which suggests that the neurotoxicity and synaptotoxicity (synaptic degeneration) caused by AβP play a central role in AD (Hardy & selkoe, 2002). Moreover, oligomerization and conformational changes in AβP are important for its neurodegenerative

There is considerable interest regarding the mechanism by which AβPs cause neurodegeneration. AβPs reportedly cause numerous adverse effects on neuronal survival, e.g., reactive oxygen species (ROS) production, cytokine induction, endoplasmic reticulum (ER) stresses induction, and the abnormal increase in intracellular Ca2+ levels ([Ca2+]i) (Small et al., 2001). These adverse effects are complex and may be interwoven. Among them, the disruption of Ca2+ homeostasis could be the primary event in the pathogenesis of AD, since Ca2+ ions play critical roles in the function and structure of neurons and other cells. Increasing evidence suggests the implications of Ca2+ dyshomeostasis in the pathogenesis of

AD (Green and LaFerla, 2008; Demuro et al., 2010).

**1. Introduction**

senile plaques.

capacity.

**in Alzheimer's Disease: Role of**

Susumu Ohkawara1 and Midori Negishi-Kato2

*2Institute of Industrial Science (IIS), The University of Tokyo,*

Masahiro Kawahara1, Hironari Koyama1,

*1Department of Analytical Chemistry, School of Pharmaceutical Sciences,*

*Kyushu University of Health and Welfare,*

fragmentation and hydrogen/deuterium exchange. *Biophysical Journal,* Vol. 96, No. 3, (Feb 2009), pp. 1091-104, ISSN 1542-0086.

## **Disruption of Calcium Homeostasis in Alzheimer's Disease: Role of Channel Formation by** β **Amyloid Protein**

Masahiro Kawahara1, Hironari Koyama1, Susumu Ohkawara1 and Midori Negishi-Kato2 *1Department of Analytical Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 2Institute of Industrial Science (IIS), The University of Tokyo, Japan*

#### **1. Introduction**

114 Advanced Understanding of Neurodegenerative Diseases

Zhang, R., Hu, X., Khant, H., Ludtke, S. J., Chiu, W., Schmid, M. F., Frieden, C. and Lee, J. M.

Zheng, J., Jang, H., Ma, B. and Nussinov, R. (2008). Annular structures as intermediates in

Zhu, M., Rajamani, S., Kaylor, J., Han, S., Zhou, F. and Fink, A. L. (2004). The flavonoid

3, (Feb 2009), pp. 1091-104, ISSN 1542-0086.

No. 22, (Jun 2008), pp. 6856-65, ISSN 1520-6106.

9258.

fragmentation and hydrogen/deuterium exchange. *Biophysical Journal,* Vol. 96, No.

(2009). Interprotofilament interactions between Alzheimer's Abeta1-42 peptides in amyloid fibrils revealed by cryoEM. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 106, No. 12, (Mar 2009), pp. 4653-8, ISSN 1091-6490.

fibril formation of Alzheimer Abeta17-42. *Journal of Physical Chemistry B,* Vol. 112,

baicalein inhibits fibrillation of alpha-synuclein and disaggregates existing fibrils. *Journal of Biological Chemistry,* Vol. 279, No. 26, (Jun 2004), pp. 26846-57, ISSN 0021-

> Alzheimer's disease (AD) is a severe senile type of dementia that affects a large number of elderly people. It is estimated that 5.4 million people in the worldwide are affected by this disease in 2011 and this number continues to grow yearly. AD is characterized by profound memory loss and severe cognitive decline. The pathological hallmarks of AD are the presence of numerous extracellular deposits termed senile plaques and intraneuronal neurofibrillary tangles (NFTs) (Selkoe, 1991). The selective loss of synapses and neurons is observed in the brain of patients, and this decrease in the number of synapses is strongly correlated with memory impairment (Terry et al., 1991). The major component of NFTs is the phosphorylated tau protein, while β-amyloid protein (AβP) is the major component of senile plaques.

> Although the pathological cause of AD has not yet strictly been elucidated, numerous biochemical, toxicological, cell biological, and genetic studies have supported the "amyloid cascade hypothesis", which suggests that the neurotoxicity and synaptotoxicity (synaptic degeneration) caused by AβP play a central role in AD (Hardy & selkoe, 2002). Moreover, oligomerization and conformational changes in AβP are important for its neurodegenerative capacity.

> There is considerable interest regarding the mechanism by which AβPs cause neurodegeneration. AβPs reportedly cause numerous adverse effects on neuronal survival, e.g., reactive oxygen species (ROS) production, cytokine induction, endoplasmic reticulum (ER) stresses induction, and the abnormal increase in intracellular Ca2+ levels ([Ca2+]i) (Small et al., 2001). These adverse effects are complex and may be interwoven. Among them, the disruption of Ca2+ homeostasis could be the primary event in the pathogenesis of AD, since Ca2+ ions play critical roles in the function and structure of neurons and other cells. Increasing evidence suggests the implications of Ca2+ dyshomeostasis in the pathogenesis of AD (Green and LaFerla, 2008; Demuro et al., 2010).

Disruption of Calcium Homeostasis in Alzheimer's Disease:

impairment observed in AD patients.

AßP

• randam coil • soluble • non-toxic

Fig. 1. Oligomerization of AβP

monomers and fibrils are rather nontoxic.

• aging (incubation) • metals (Al, Cu, Zn, *etc.*)

Acceleratory factors

• oxidation • Mutation • racemization *etc.*

• rifampicin • curcumine • polyphenol • aspirin

Inhibitory factors

• ß-sheet breaker peptide *etc.*

Role of Channel Formation by β Amyloid Protein 117

Recent studies on the identified AβP species further strengthened and refined the amyloid cascade hypothesis. Approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy (AFM) have demonstrated that there are several stable types of oligomers: naturally occurring soluble oligomers (dimers or trimers), AßP-derived diffusible ligands (ADDLs), AβP globulomers, and protofibrils. Walsh et al. found the existence of SDS-stable oligomers in the conditioned medium of cultured cells transfected with the human APP gene. The intracerebral administration of these SDS-stable low-molecularweight oligomers (dimers, trimers, or tetramers) inhibited long-term potentiation (LTP), which is a form of synaptic information storage that is a well-known paradigm for the mechanisms underlying memory formation (Walsh and Selkoe, 2007). They also demonstrated that LTP was not blocked by AβP monomers or larger aggregates. Natural AβP oligomers derived from the cerebrospinal fluid of AD patients induced the loss of dendritic spines and synapses, and also blocked the oligomers in the conditioned medium. Klein and the colleagues reported that ADDLs obtained by sedimentation with clusterin are highly toxic to cultured neurons. They also reported that ADDLs inhibited LTP and exhibited adverse effects on synaptic plasticity e.g., decreased spine density, abnormal spine morphology, and decreased levels of synaptic proteins (Lacor et al., 2007). Tomiyama et al. found a new AβP variant (AβP E42Δ) that exhibited enhanced oligomerization but no fibrillization (Tomiyama et al., 2008). This unique variant AβP decreased synaptophysin immunostaining and blocked LTP. Since synaptic plasticity is crucial for the process of memory formation and is involved in the early stages of AD, these lines of evidence indicate that the synaptic impairment induced by AβP oligomers is the primary event in the memory

*monomer Soluble oligomer Amyloid fibril*

• ß-sheet • soluble • toxic

Toxicity

AβP monomers exhibit random-coil structures. However, during aging or in the presence of some acceleratory factors, AβP self-aggregates and forms several types of oligomers (SDSsoluble oligomers, ADDLS, globulomers, protofibrils, *etc.*) and finally forms insoluble aggregates, which are termed amyloid fibrils. Oligomeric soluble AβPs are toxic, although

*protofibril ADDLs*

*globulomer etc.*

• ß-sheet • insoluble • less or non-toxic

Several possible mechanisms account for AβP-induced Ca2+ dyshomeostasis, e.g., its interaction with endogenous Ca2+-permeable ion channels, disruption of membrane integrity, and the formation of Ca2+ permeable channels (pores) by the direct incorporation of AβP into membranes. Here, we focus on the 'amyloid channel hypothesis' (Arispe et al., 2007; Lin et al. 2001; Kawahara, 2011b), namely, that the direct incorporation of AβPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels may be the primary event in AßP neurotoxicity.

In this chapter, we review the current understanding of the link between Ca2+ homeostasis and AD pathogenesis based on the amyloid channel hypothesis the characteristics of AβPinduced neurotoxicity and synaptotoxicity caused by Ca2+ dyshomeostasis *via* amyloid channels. We also discuss the possible development of new drugs for the treatment and prevention of AD by attenuating amyloid channels and inhibiting AβP-induced Ca2+ dyshomeostasis.

### **2. Amyloid cascade hypothesis**

AβP is a small peptide with 39–43 amino acid residues. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP) by the cleavage of its N-terminal by β-secretase (BACE), followed by the intra-membrane cleavage of its C-terminal by γ-secretase. There are several species of AβP, such as AβP(1–40), the first 40 amino acid residues, or AβP(1–42), which are generated by the different cleavage processes in its C-terminal domain. Genetic studies of early-onset cases of familial AD indicated that APP mutations and the consequent changes in AβP metabolism are associated with AD (Goate et al., 1991). Moreover, mutations in the presenilin genes also account for the majority of cases of early-onset familial AD (Sherrington et al., 1995). Presenilins have been identified as one of γ -secretase proteins, and their mutations also influence the production of AβP and its neurotoxicity (Selkoe and Wolfe, 2007).

Yankner *et al.* reported that AβP(1–40) caused the death of cultured rat hippocampal neurons and the neurodegeneration in the brains of experimental animals (Yankner et al., 1991). A smaller fragment of AβP (AβP (25–35)) or a longer variant (AβP (1–42)) were also reported to cause neuronal death. AβP is a hydrophobic peptide with an intrinsic tendency to self-assemble to form sodium dodecyl sulfate (SDS)-stable oligomers. In an aqueous solution, freshly prepared and dissolved AβP exists as a monomeric protein with a random coil structure. However, following incubation at 37۫ºC for several days (*aging*), AβPs form aggregates (oligomers) with β-pleated sheet structures, and finally form insoluble aggregates, termed amyloid fibrils (Fig. 1). Pike et al. revealed that *aged* AβP(1–40) peptides were considerably more toxic to cultured neurons than *fresh* (freshly prepared just before the experiment) AβP(1–40) (Pike et al, 1991). The ß-sheet content of AßP solutions, as determined by circular dichroism (CD) spectroscopy, correlates with its neurotoxicity (Simmons et al., 1994). Jarrett and Lansbury demonstrated that the longer peptide variant, AβP(1–42), polymerizes much quicker than AβP(1–40) (Jarrett and Lansbury, 1993). AβP(1– 42) enhances the aggregation of AβP(1–40) and functions as a "seed" for amyloid fibrils. AβP (1–42) is more abundant in the brains of AD patients than in age-matched controls. The point mutations of APP are located near the γ-secretase cleavage-site and influences the ratio of AβP(1–40) and AβP(1–42). Mutations of APP and the presenilin genes increase the production of AβP (1–42) in the transfected cell lines.

Several possible mechanisms account for AβP-induced Ca2+ dyshomeostasis, e.g., its interaction with endogenous Ca2+-permeable ion channels, disruption of membrane integrity, and the formation of Ca2+ permeable channels (pores) by the direct incorporation of AβP into membranes. Here, we focus on the 'amyloid channel hypothesis' (Arispe et al., 2007; Lin et al. 2001; Kawahara, 2011b), namely, that the direct incorporation of AβPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels may be the

In this chapter, we review the current understanding of the link between Ca2+ homeostasis and AD pathogenesis based on the amyloid channel hypothesis the characteristics of AβPinduced neurotoxicity and synaptotoxicity caused by Ca2+ dyshomeostasis *via* amyloid channels. We also discuss the possible development of new drugs for the treatment and prevention of AD by attenuating amyloid channels and inhibiting AβP-induced Ca2+

AβP is a small peptide with 39–43 amino acid residues. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP) by the cleavage of its N-terminal by β-secretase (BACE), followed by the intra-membrane cleavage of its C-terminal by γ-secretase. There are several species of AβP, such as AβP(1–40), the first 40 amino acid residues, or AβP(1–42), which are generated by the different cleavage processes in its C-terminal domain. Genetic studies of early-onset cases of familial AD indicated that APP mutations and the consequent changes in AβP metabolism are associated with AD (Goate et al., 1991). Moreover, mutations in the presenilin genes also account for the majority of cases of early-onset familial AD (Sherrington et al., 1995). Presenilins have been identified as one of γ -secretase proteins, and their mutations also influence the production

Yankner *et al.* reported that AβP(1–40) caused the death of cultured rat hippocampal neurons and the neurodegeneration in the brains of experimental animals (Yankner et al., 1991). A smaller fragment of AβP (AβP (25–35)) or a longer variant (AβP (1–42)) were also reported to cause neuronal death. AβP is a hydrophobic peptide with an intrinsic tendency to self-assemble to form sodium dodecyl sulfate (SDS)-stable oligomers. In an aqueous solution, freshly prepared and dissolved AβP exists as a monomeric protein with a random coil structure. However, following incubation at 37۫ºC for several days (*aging*), AβPs form aggregates (oligomers) with β-pleated sheet structures, and finally form insoluble aggregates, termed amyloid fibrils (Fig. 1). Pike et al. revealed that *aged* AβP(1–40) peptides were considerably more toxic to cultured neurons than *fresh* (freshly prepared just before the experiment) AβP(1–40) (Pike et al, 1991). The ß-sheet content of AßP solutions, as determined by circular dichroism (CD) spectroscopy, correlates with its neurotoxicity (Simmons et al., 1994). Jarrett and Lansbury demonstrated that the longer peptide variant, AβP(1–42), polymerizes much quicker than AβP(1–40) (Jarrett and Lansbury, 1993). AβP(1– 42) enhances the aggregation of AβP(1–40) and functions as a "seed" for amyloid fibrils. AβP (1–42) is more abundant in the brains of AD patients than in age-matched controls. The point mutations of APP are located near the γ-secretase cleavage-site and influences the ratio of AβP(1–40) and AβP(1–42). Mutations of APP and the presenilin genes increase the

primary event in AßP neurotoxicity.

**2. Amyloid cascade hypothesis**

of AβP and its neurotoxicity (Selkoe and Wolfe, 2007).

production of AβP (1–42) in the transfected cell lines.

dyshomeostasis.

Recent studies on the identified AβP species further strengthened and refined the amyloid cascade hypothesis. Approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy (AFM) have demonstrated that there are several stable types of oligomers: naturally occurring soluble oligomers (dimers or trimers), AßP-derived diffusible ligands (ADDLs), AβP globulomers, and protofibrils. Walsh et al. found the existence of SDS-stable oligomers in the conditioned medium of cultured cells transfected with the human APP gene. The intracerebral administration of these SDS-stable low-molecularweight oligomers (dimers, trimers, or tetramers) inhibited long-term potentiation (LTP), which is a form of synaptic information storage that is a well-known paradigm for the mechanisms underlying memory formation (Walsh and Selkoe, 2007). They also demonstrated that LTP was not blocked by AβP monomers or larger aggregates. Natural AβP oligomers derived from the cerebrospinal fluid of AD patients induced the loss of dendritic spines and synapses, and also blocked the oligomers in the conditioned medium. Klein and the colleagues reported that ADDLs obtained by sedimentation with clusterin are highly toxic to cultured neurons. They also reported that ADDLs inhibited LTP and exhibited adverse effects on synaptic plasticity e.g., decreased spine density, abnormal spine morphology, and decreased levels of synaptic proteins (Lacor et al., 2007). Tomiyama et al. found a new AβP variant (AβP E42Δ) that exhibited enhanced oligomerization but no fibrillization (Tomiyama et al., 2008). This unique variant AβP decreased synaptophysin immunostaining and blocked LTP. Since synaptic plasticity is crucial for the process of memory formation and is involved in the early stages of AD, these lines of evidence indicate that the synaptic impairment induced by AβP oligomers is the primary event in the memory impairment observed in AD patients.

Fig. 1. Oligomerization of AβP

AβP monomers exhibit random-coil structures. However, during aging or in the presence of some acceleratory factors, AβP self-aggregates and forms several types of oligomers (SDSsoluble oligomers, ADDLS, globulomers, protofibrils, *etc.*) and finally forms insoluble aggregates, which are termed amyloid fibrils. Oligomeric soluble AβPs are toxic, although monomers and fibrils are rather nontoxic.

Disruption of Calcium Homeostasis in Alzheimer's Disease:

NMDA-R AMPA-R Ach-R

AßP

VDCC

presenilin

can participate in the abnormal increase in [Ca2+]i in neurons

**4. Formation of Ca2+ permeable pores by AßP**

the ER, and mitochondrial Ca2+ signaling (Querfurth and LaFerla, 2010).

Ca2+

ER

Role of Channel Formation by β Amyloid Protein 119

Ca2+

Amyloid channel

AßP AßP

mitochondoria

Ca2+ ROS

Ca2+

Neuronal death

AβP reportedly binds to *N*-methyl D-aspartate (NMDA)-, and α-amino-3-hydroxy-5 methylisoxazole-4-propionic acid (AMPA) -type glutamate receptors (Parameshwaran et al., 2008), and nicotinic ACh receptors (Parri and Dineley, 2010). All of these receptors contain Ca2+ channels and regulate [Ca2+]i during membrane depolarization. AβP also influences voltage-gated Ca2+ channels and the inositol triphosphate (IP3) receptor. The influx of Ca2+ from the ER is regulated by ryanodine-type Ca2+ channels, the sarcoplasmic reticulum Ca-ATPase (SERCA), and presenilins (Green et al., 2008). Presenilins are found in the ER membrane, and are involved in capacitative Ca2+ entry, ER Ca2+ signaling, Ca2+ leakage from

Our and other numerous studies have demonstrated that AβPs are directly incorporated into the surfaces of cellular membrane and create unregulated cytotoxic pore-like channels. In 1993, Arispe et al. first demonstrated that AβP(1–40) directly incorporates into artificial lipid bilayer membranes and forms cation-selective ion channels (Arispe et al., 1993a, 1993b). These channels termed amyloid channels were revealed to be giant multilevel pores that can facilitate the transport of large amounts of Ca2+. Their activity was blocked by Zn2+ ions, which are abundant in the brain (Arispe et al., 1996). Electrophysiological studies have revealed that other neurotoxic peptide fragments of AβP including AβP(25–35) and

Fig. 2. AβP-induced calcium dyshomeostasis in neurons. It is possible that AßP-induced Ca2+ dyshomeostasis by (i) its interactions with endogenous Ca2+-permeable ion channels, (ii) disruption of membrane integrity, and (iii) formation of Ca2+ permeable channels (pores) by the direct membrane incorporation of AβP. AβP can directly cause membrane disruption, or AβP-induced ROS can impair membrane structure. Presenilins in the ER or mitochondria

AβP is secreted into the cerebrospinal fluid (CSF) of young individuals as well as in aged or dementia patients (Fukuyama et al., 2000). Therefore, factors that accelerate or inhibit AβP oligomerization may play essential roles in the pathogenesis of AD. Several factors such as peptide concentration, the pH or composition of solvents, and temperature can influence the oligomerization processes. In addition, oxidations, mutations, and racemization of AβP enhance its oligomerization. Substances that can influence the oligomerization processes, e.g., cholesterol or its oxidation products, transthyretin, rifampicin, curcumin, aspirin, docosahexaenoic acid (DHA) and peptides such as the β-sheet breaker peptide, reportedly inhibit AβP oligomerization *in vitr*o.

Among these factors, Al is of particular interest considering its epidemiological link with AD (Kawahara and Kato-Negishi, 2011). Al3+ has strong positive charges and a relatively small ionic radius in comparison to other metal ions; thus, Al3+ firmly binds to metalbinding amino acids (e.g. histidine, tyrosine, arginine *etc.*) or phosphorylated amino acids and acts as a cross-linker. Owing to this property, Al can cause the aggregation of various proteins and induce the conformational changes. Exposure to Al causes the accumulation of AβP in cultured neurons and in the brains of experimental animals and humans (Pratico et al., 2002; Exley and Esiri, 2006).

#### **3. Alzheimer's Disease and calcium homeostasis**

Ca2+ is required for various normal brain functions and is a component of key enzymes such as kinases, phosphatases, and proteases, it is highly possible that Ca2+ dyshomeostasis could be the earliest adverse event among AβP-induced various adverse effects such as ROS production, cytokine induction, endoplasmic reticulum (ER) stresses induction. Once neuronal Ca2+ homeostasis is disrupted and [Ca2+]i is changed, various apoptotic pathways, such as Ca2+-activated neutral protease (calpain) and caspase, are activated, leading to neuronal death. The disruption of Ca2+ homeostasis can trigger membrane disruption, the formation of free radicals, and the induction of other adverse effects, which are often observed after exposure to AβP. It is widely recognized that an increase in [Ca2+]i induces changes in the number and morphology of synapses and spines, and that an imbalances of Ca2+ in synapses directly influences neuronal activity and synaptic impairment. Considering that APP localizes in synapses and that AβP is secreted from APP into synaptic clefts by neuronal excitation, the adverse effects caused by AβP-induced Ca2+ dyshomeostasis can occur in synaptic compartments and induce synaptotoxicity. Therefore, the influx of Ca2+ is tightly controlled and [Ca2+]i levels are strictly regulated by various mechanisms including voltage-dependent Ca2+ channels (VDCC), and neurotransmitter receptors e.g., glutamate (Glu) and acetylcholine (ACh) (Zorumski and Thio, 1992). Moreover, the ER and mitochondoria represent the major intracellular stores of Ca2+ (Green and LaFerla, 2008; Leuner et al., 2007).

An increasing amount of data indicates that exposure to AβP causes an abnormal increase in [Ca2+]i in intoxicated neurons. There is considerable interest regarding the mechanism by which AβPs interact with neurons and disrupts Ca2+ homeostasis (Demuro et al., 2010). There are several possible mechanisms that account for AβP-induced Ca2+ dyshomeostasis, e.g., interaction with endogenous Ca2+-permeable ion channels, disruption of membrane integrity, and formation of Ca2+-permeable channels (pores) by the direct membrane incorporation of AβP (Fig.6). It is possible that AβP directly binds to membranes and causes their disruption, or that AβP-induced ROS impairs membrane structures.

AβP is secreted into the cerebrospinal fluid (CSF) of young individuals as well as in aged or dementia patients (Fukuyama et al., 2000). Therefore, factors that accelerate or inhibit AβP oligomerization may play essential roles in the pathogenesis of AD. Several factors such as peptide concentration, the pH or composition of solvents, and temperature can influence the oligomerization processes. In addition, oxidations, mutations, and racemization of AβP enhance its oligomerization. Substances that can influence the oligomerization processes, e.g., cholesterol or its oxidation products, transthyretin, rifampicin, curcumin, aspirin, docosahexaenoic acid (DHA) and peptides such as the β-sheet breaker peptide, reportedly

Among these factors, Al is of particular interest considering its epidemiological link with AD (Kawahara and Kato-Negishi, 2011). Al3+ has strong positive charges and a relatively small ionic radius in comparison to other metal ions; thus, Al3+ firmly binds to metalbinding amino acids (e.g. histidine, tyrosine, arginine *etc.*) or phosphorylated amino acids and acts as a cross-linker. Owing to this property, Al can cause the aggregation of various proteins and induce the conformational changes. Exposure to Al causes the accumulation of AβP in cultured neurons and in the brains of experimental animals and humans (Pratico et

Ca2+ is required for various normal brain functions and is a component of key enzymes such as kinases, phosphatases, and proteases, it is highly possible that Ca2+ dyshomeostasis could be the earliest adverse event among AβP-induced various adverse effects such as ROS production, cytokine induction, endoplasmic reticulum (ER) stresses induction. Once neuronal Ca2+ homeostasis is disrupted and [Ca2+]i is changed, various apoptotic pathways, such as Ca2+-activated neutral protease (calpain) and caspase, are activated, leading to neuronal death. The disruption of Ca2+ homeostasis can trigger membrane disruption, the formation of free radicals, and the induction of other adverse effects, which are often observed after exposure to AβP. It is widely recognized that an increase in [Ca2+]i induces changes in the number and morphology of synapses and spines, and that an imbalances of Ca2+ in synapses directly influences neuronal activity and synaptic impairment. Considering that APP localizes in synapses and that AβP is secreted from APP into synaptic clefts by neuronal excitation, the adverse effects caused by AβP-induced Ca2+ dyshomeostasis can occur in synaptic compartments and induce synaptotoxicity. Therefore, the influx of Ca2+ is tightly controlled and [Ca2+]i levels are strictly regulated by various mechanisms including voltage-dependent Ca2+ channels (VDCC), and neurotransmitter receptors e.g., glutamate (Glu) and acetylcholine (ACh) (Zorumski and Thio, 1992). Moreover, the ER and mitochondoria represent the major intracellular stores of Ca2+ (Green and LaFerla, 2008;

An increasing amount of data indicates that exposure to AβP causes an abnormal increase in [Ca2+]i in intoxicated neurons. There is considerable interest regarding the mechanism by which AβPs interact with neurons and disrupts Ca2+ homeostasis (Demuro et al., 2010). There are several possible mechanisms that account for AβP-induced Ca2+ dyshomeostasis, e.g., interaction with endogenous Ca2+-permeable ion channels, disruption of membrane integrity, and formation of Ca2+-permeable channels (pores) by the direct membrane incorporation of AβP (Fig.6). It is possible that AβP directly binds to membranes and causes

their disruption, or that AβP-induced ROS impairs membrane structures.

inhibit AβP oligomerization *in vitr*o.

al., 2002; Exley and Esiri, 2006).

Leuner et al., 2007).

**3. Alzheimer's Disease and calcium homeostasis**

Fig. 2. AβP-induced calcium dyshomeostasis in neurons. It is possible that AßP-induced Ca2+ dyshomeostasis by (i) its interactions with endogenous Ca2+-permeable ion channels, (ii) disruption of membrane integrity, and (iii) formation of Ca2+ permeable channels (pores) by the direct membrane incorporation of AβP. AβP can directly cause membrane disruption, or AβP-induced ROS can impair membrane structure. Presenilins in the ER or mitochondria can participate in the abnormal increase in [Ca2+]i in neurons

AβP reportedly binds to *N*-methyl D-aspartate (NMDA)-, and α-amino-3-hydroxy-5 methylisoxazole-4-propionic acid (AMPA) -type glutamate receptors (Parameshwaran et al., 2008), and nicotinic ACh receptors (Parri and Dineley, 2010). All of these receptors contain Ca2+ channels and regulate [Ca2+]i during membrane depolarization. AβP also influences voltage-gated Ca2+ channels and the inositol triphosphate (IP3) receptor. The influx of Ca2+ from the ER is regulated by ryanodine-type Ca2+ channels, the sarcoplasmic reticulum Ca-ATPase (SERCA), and presenilins (Green et al., 2008). Presenilins are found in the ER membrane, and are involved in capacitative Ca2+ entry, ER Ca2+ signaling, Ca2+ leakage from the ER, and mitochondrial Ca2+ signaling (Querfurth and LaFerla, 2010).

### **4. Formation of Ca2+ permeable pores by AßP**

Our and other numerous studies have demonstrated that AβPs are directly incorporated into the surfaces of cellular membrane and create unregulated cytotoxic pore-like channels. In 1993, Arispe et al. first demonstrated that AβP(1–40) directly incorporates into artificial lipid bilayer membranes and forms cation-selective ion channels (Arispe et al., 1993a, 1993b). These channels termed amyloid channels were revealed to be giant multilevel pores that can facilitate the transport of large amounts of Ca2+. Their activity was blocked by Zn2+ ions, which are abundant in the brain (Arispe et al., 1996). Electrophysiological studies have revealed that other neurotoxic peptide fragments of AβP including AβP(25–35) and

Disruption of Calcium Homeostasis in Alzheimer's Disease:

quantitatively analyze Ca2+ influx.

membranes, ultimately resulting in neuronal death.

al., 2010).

Role of Channel Formation by β Amyloid Protein 121

resolution multi-site video imaging system with Ca2+-sensitive fluorescent dye (fura-2) (Kawahara et al., 2000; Kawahara & Kuroda, 2001). We also observed AβP-induced abnormal increases in [Ca2+]i in primary cultured rat hippocampal neurons (Kato-Negishi & Kawahara, 2008). Shown in figure 3 are temporal changes of [Ca2+]i in GT1-7 cells before and after exposure to AβP(1-40) and related peptides. Although a marked increase in [Ca2+]i was caused by AβP(1–40) (*line (a)*), AβP(1–42) (*line (c)*), and AβP(25–35) (*line (e)*), no remarkable changes were induced by AβP(40–1), control peptide with no toxicity (*line (b)*). There is controversy over whether the AβP-induced [Ca2+]i changes occur through receptormediated pathways or amyloid channels formed by direct incorporation of AβP. To clarify the precise characterization of the AβP-induced [Ca2+]i changes, we performed detailed and quantitative analysis of the AβP-induced Ca2+ influx using a high-resolution multi-site video imaging system. This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i in more than 50 neurons. There are 5 major pieces of evidence supporting the hypothesis that AßP-induced [Ca2+]i changes occur through amyloid channels. *First*, the AβP-induced [Ca2+]i rise was highly heterogeneous among genetically identical GT1-7 cells. Even in the same field of view, exposure to the same peptide solution produced differential changes in the [Ca2+]i levels (Fig. 4A). Considering the heterogeneity, we compared the peak increase in [Ca2+]i (∆[Ca2+]i) induced by AβPs, and its latency (the lag between the [Ca2+]i increase and the time of AβP addition) in each cell to

Although AβP (1–40) induced an increase in [Ca2+]i levels either instantly or after some delay, the magnitude and latency differed. In addition, some adjacent cells still did not exhibit any responses. *Second*, the average ∆[Ca2+]i was increased and the average latency was shortened in a dose-dependent manner (Fig. 4B and C). These features are considerably similar to those observed in relation to toxic peptide channels formed on membranes. *Third*, the AβP-induced increase in [Ca2+]i was not influenced by the addition of a Na+ channel blocker (tetrodotoxin), a Ca2+ channel blocker (nifedipine), a glutamate receptor antagonist (D-APV), or a γ-aminobutyric acid (GABA) antagonist (bicuculline). However, antibodies to AβP remarkably inhibited the [Ca2+]i increase resulting from AβP (Kawahara, 2004). *Fourth*, D-AβP(1–40), AβP(1–40) composed of D-amino acid residues, also caused the elevation of [Ca2+]i in a manner similar to AβP(1–40) (see Fig. 3, *line (d)*). *Fifth*, the vulnerability of primary cultured rat hippocampal neurons to AβP was changed during the culture period, despite that the expression and the function of neurotransmitter are not changed (Kato-Negishi and Kawahara, 2008). On the basis of these lines of evidence, we conclude that AβP causes the disruption of Ca2+ homeostasis *via* the formation of amyloid channels in

Pore formation-induced cytotoxicity is commonly observed in our biological system, particularly in the presence of certain toxins and venoms including the α-toxin of *Staphylococcus aureus*, magainin 2, a 26-residue antimicrobial peptide obtained from *Xenopus laevis*, melitin, a bee venom composed of 28 amino acids, or antibiotics such as amphotericin and gramicidin (Bechinger, 1997). In this respect, AβPs may share the similar toxicity mechanism as various antimicrobial or antifungal peptides that exhibit pore-forming ability and cell toxicity. Indeed, Soscia et al. demonstrated that AβP exerts antimicrobial activity against 8 common and clinically relevant microorganisms (Soscia et

AβP(1–42) form Ca2+-permeable pores in artificial lipid bilayers. The C-terminal fragment of APP (CT105) including AβP also formed ion channels in Xenopus oocytes (Fraser et al., 1996).

Durell et al. proposed a 3-D structural model of amyloid channels obtained from a computer simulation of the secondary structure of AβP(1–40) in membranes that showed the aggregation of 5- to 8-mers to form pore-like structures on the membranes (Durell et al., 1994). Jang et al. established a model for amyloid channels on membranes and observed the pentameric AβP forms pores (Jang et al, 2009). Strodel et al. proposed a model of AβP(1–42) pores which consist of tetrameric and hexameric β-sheet subunits from the observations in NMR (Strodel et al., 2010). The dimension, shape, and subunit organization of these models were in good agreement with the morphological observations using high resolution AFM that demonstrated that AβPs form pore-like structures on mica plates or on reconstituted membranes (Lal et al., 2007, Jang et al., 2010). Furthermore, the presence of pore-like structures of AßPs *in vivo* was demonstrated in the neuronal cell membrane of the brains of AD patients and of AD-model mice. Using high resolution transmission electron microscopy, Inoue observed in situ AßP pores in the neuronal cell membrane in AD brains (Inoue, 2008). Kayed *et al*. reported that the annular protofibrils (APFs) of AßP exhibit ringshaped and pore-like structures (Kayerd, et al., 2009). The age-dependent accumulation of APFs was observed on the membranes of AD model mice (APP transgenic mice; APP23) (Kokubo et al., 2009).

It is important to determine whether AβPs form channels in neuronal cell membranes in addition to artificial lipid bilayers. To address this issue, we employed membrane patches from a neuroblastoma cell line (GT1-7 cells), and found that AβP(1–40) formed amyloid channels on GT1-7 cell membranes (Kawahara et al., 1997). GT1-7 cells (immortalized hypothalamic neurons) are derived from murine hypothalamic neurons by site-directed tumorigenesis and exhibit several neuronal characteristics such as the extension of neurites and the expression of neuron-specific proteins and receptors (Mellon et al., 1990). The features of amyloid channels formed on GT1-7 membranes were considerably similar to those observed in artificial lipid bilayers; cation-selective, multilevel, voltage-independent, and long-lasting; channel activity was inhibited by the addition of Zn2+, and recovered by the addition of zinc chelator *o*-phenanthroline. Moreover, Sepulveda et al. revealed that AβP(1-40) formed perforations on membranes excised from hippocampal neurons and induced currents (Sepulveda et al., 2010). The effect of AβP was similar to that of gramicidin and amphotericin which are commonly used to perforate neuron membranes.

Furthermore, we have revealed that AβP directly caused the disruption of liposomal membrane vesicles by observing the release of fluorescent dye (Kawahara et al., 2011b). These results are consistent with the findings that AßP causes membrane disruption, increases membrane permeability, causes hemolysis, and changes membrane fluidity (Eckert et al., 2005).

These results strongly support the hypothetical idea termed amyloid channel hypothesis namely, that the direct incorporation of AβPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels may be the primary event in AßP neurotoxicity.

### **5. Disruption of calcium homeostasis by amyloid channels**

In order to test the validity of the amyloid channel hypothesis more precisely, we examined whether AβP alters the [Ca2+]i levels of GT1-7 cells under the same conditions, using a high-

AβP(1–42) form Ca2+-permeable pores in artificial lipid bilayers. The C-terminal fragment of APP (CT105) including AβP also formed ion channels in Xenopus oocytes (Fraser et al.,

Durell et al. proposed a 3-D structural model of amyloid channels obtained from a computer simulation of the secondary structure of AβP(1–40) in membranes that showed the aggregation of 5- to 8-mers to form pore-like structures on the membranes (Durell et al., 1994). Jang et al. established a model for amyloid channels on membranes and observed the pentameric AβP forms pores (Jang et al, 2009). Strodel et al. proposed a model of AβP(1–42) pores which consist of tetrameric and hexameric β-sheet subunits from the observations in NMR (Strodel et al., 2010). The dimension, shape, and subunit organization of these models were in good agreement with the morphological observations using high resolution AFM that demonstrated that AβPs form pore-like structures on mica plates or on reconstituted membranes (Lal et al., 2007, Jang et al., 2010). Furthermore, the presence of pore-like structures of AßPs *in vivo* was demonstrated in the neuronal cell membrane of the brains of AD patients and of AD-model mice. Using high resolution transmission electron microscopy, Inoue observed in situ AßP pores in the neuronal cell membrane in AD brains (Inoue, 2008). Kayed *et al*. reported that the annular protofibrils (APFs) of AßP exhibit ringshaped and pore-like structures (Kayerd, et al., 2009). The age-dependent accumulation of APFs was observed on the membranes of AD model mice (APP transgenic mice; APP23)

It is important to determine whether AβPs form channels in neuronal cell membranes in addition to artificial lipid bilayers. To address this issue, we employed membrane patches from a neuroblastoma cell line (GT1-7 cells), and found that AβP(1–40) formed amyloid channels on GT1-7 cell membranes (Kawahara et al., 1997). GT1-7 cells (immortalized hypothalamic neurons) are derived from murine hypothalamic neurons by site-directed tumorigenesis and exhibit several neuronal characteristics such as the extension of neurites and the expression of neuron-specific proteins and receptors (Mellon et al., 1990). The features of amyloid channels formed on GT1-7 membranes were considerably similar to those observed in artificial lipid bilayers; cation-selective, multilevel, voltage-independent, and long-lasting; channel activity was inhibited by the addition of Zn2+, and recovered by the addition of zinc chelator *o*-phenanthroline. Moreover, Sepulveda et al. revealed that AβP(1-40) formed perforations on membranes excised from hippocampal neurons and induced currents (Sepulveda et al., 2010). The effect of AβP was similar to that of gramicidin

Furthermore, we have revealed that AβP directly caused the disruption of liposomal membrane vesicles by observing the release of fluorescent dye (Kawahara et al., 2011b). These results are consistent with the findings that AßP causes membrane disruption, increases membrane permeability, causes hemolysis, and changes membrane fluidity

These results strongly support the hypothetical idea termed amyloid channel hypothesis namely, that the direct incorporation of AβPs and the subsequent imbalances of Ca2+ and other ions through amyloid channels may be the primary event in AßP neurotoxicity.

In order to test the validity of the amyloid channel hypothesis more precisely, we examined whether AβP alters the [Ca2+]i levels of GT1-7 cells under the same conditions, using a high-

and amphotericin which are commonly used to perforate neuron membranes.

**5. Disruption of calcium homeostasis by amyloid channels**

1996).

(Kokubo et al., 2009).

(Eckert et al., 2005).

resolution multi-site video imaging system with Ca2+-sensitive fluorescent dye (fura-2) (Kawahara et al., 2000; Kawahara & Kuroda, 2001). We also observed AβP-induced abnormal increases in [Ca2+]i in primary cultured rat hippocampal neurons (Kato-Negishi & Kawahara, 2008). Shown in figure 3 are temporal changes of [Ca2+]i in GT1-7 cells before and after exposure to AβP(1-40) and related peptides. Although a marked increase in [Ca2+]i was caused by AβP(1–40) (*line (a)*), AβP(1–42) (*line (c)*), and AβP(25–35) (*line (e)*), no remarkable changes were induced by AβP(40–1), control peptide with no toxicity (*line (b)*). There is controversy over whether the AβP-induced [Ca2+]i changes occur through receptormediated pathways or amyloid channels formed by direct incorporation of AβP. To clarify the precise characterization of the AβP-induced [Ca2+]i changes, we performed detailed and quantitative analysis of the AβP-induced Ca2+ influx using a high-resolution multi-site video imaging system. This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i in more than 50 neurons. There are 5 major pieces of evidence supporting the hypothesis that AßP-induced [Ca2+]i changes occur through amyloid channels. *First*, the AβP-induced [Ca2+]i rise was highly heterogeneous among genetically identical GT1-7 cells. Even in the same field of view, exposure to the same peptide solution produced differential changes in the [Ca2+]i levels (Fig. 4A). Considering the heterogeneity, we compared the peak increase in [Ca2+]i (∆[Ca2+]i) induced by AβPs, and its latency (the lag between the [Ca2+]i increase and the time of AβP addition) in each cell to quantitatively analyze Ca2+ influx.

Although AβP (1–40) induced an increase in [Ca2+]i levels either instantly or after some delay, the magnitude and latency differed. In addition, some adjacent cells still did not exhibit any responses. *Second*, the average ∆[Ca2+]i was increased and the average latency was shortened in a dose-dependent manner (Fig. 4B and C). These features are considerably similar to those observed in relation to toxic peptide channels formed on membranes. *Third*, the AβP-induced increase in [Ca2+]i was not influenced by the addition of a Na+ channel blocker (tetrodotoxin), a Ca2+ channel blocker (nifedipine), a glutamate receptor antagonist (D-APV), or a γ-aminobutyric acid (GABA) antagonist (bicuculline). However, antibodies to AβP remarkably inhibited the [Ca2+]i increase resulting from AβP (Kawahara, 2004). *Fourth*, D-AβP(1–40), AβP(1–40) composed of D-amino acid residues, also caused the elevation of [Ca2+]i in a manner similar to AβP(1–40) (see Fig. 3, *line (d)*). *Fifth*, the vulnerability of primary cultured rat hippocampal neurons to AβP was changed during the culture period, despite that the expression and the function of neurotransmitter are not changed (Kato-Negishi and Kawahara, 2008). On the basis of these lines of evidence, we conclude that AβP causes the disruption of Ca2+ homeostasis *via* the formation of amyloid channels in membranes, ultimately resulting in neuronal death.

Pore formation-induced cytotoxicity is commonly observed in our biological system, particularly in the presence of certain toxins and venoms including the α-toxin of *Staphylococcus aureus*, magainin 2, a 26-residue antimicrobial peptide obtained from *Xenopus laevis*, melitin, a bee venom composed of 28 amino acids, or antibiotics such as amphotericin and gramicidin (Bechinger, 1997). In this respect, AβPs may share the similar toxicity mechanism as various antimicrobial or antifungal peptides that exhibit pore-forming ability and cell toxicity. Indeed, Soscia et al. demonstrated that AβP exerts antimicrobial activity against 8 common and clinically relevant microorganisms (Soscia et al., 2010).

Disruption of Calcium Homeostasis in Alzheimer's Disease:

Exposure to AßP

latency

(C) and the latency (D) after exposure to AßP (1–40)

∆[Ca2+]i

Fig. 4. (A) Heterogeneity of AβP-induced changes in [Ca2+]i Temporal changes of 50 randomly chosen GT1-7 cells in the same field of view before and after the exposure to AβP(1–40). The arrow indicates the time of peptide addition. B~C: The peak increase in [Ca2+]i (∆[Ca2+]i) in each cell and the latency after exposure to AβP (1–40) were analyzed (B) in more than 50 cultured neurons in a single field of view (360×420 µm3) (mean ± S.E.M., n=300). Typical responses of [Ca2+]i in cultured neurons following exposure to various concentrations of AβP (1–40) (2.5~10 µM). The peak increase in [Ca2+]i (∆[Ca2+]i) in each cell

A

Role of Channel Formation by β Amyloid Protein 123

We have also demonstrated that these amyloidogenic peptides including PrP106–126 (a peptide fragment of prion protein), human amylin, NAC, or antimicrobial peptide magainin2 also caused an elevation in the [Ca2+]i levels similar to that induced by AβPs (Kawahara et al., 2000; Kawahara, 2004). However, rat amylin and a peptide a randomized PrP106–126 sequence (scrambled PrP106–126) did not induce any [Ca2+]i changes (Fig. 3). We have also demonstrated that PrP106-126 forms β-sheet structures and exhibits neurotoxicity as well as AβP (Kawahara et al., 2011a). In addition, oligomeric α-synuclein causes neuronal death *via* the Ca2+ influx (Danzer et al., 2007). Considering these results

B

0

40

80

∆[Ca2+]I (nM)

Latency (sec)

C

0

20

40

60

80

100

120

120

160

∆[Ca2+]i

Latency

AßP[1-40] (μM)

AßP[1-40] (μM)

02468 10 12

0 2 4 6 8 10 12

Fig. 3. Characteristics of the elevations in [Ca2+]i induced by AβP and other amyloidogenic proteins. Typical time course of [Ca2+]i at 2 min prior to and at 3 min after the application of the peptide is depicted. (a) AßP(1-40); (b) AßP(40-1); (c) AßP(1-42); (d) D-AßP(1-40); (e) PrP106-126; (f) scramble PrP106-126; (g) human amylin; (h) rat amylin; (i) NAC; and (j) magainin 2. All peptides were at 10 µM. The arrow indicates the time of peptide addition

Recently, a new concept of "conformational disease," had been proposed, suggesting that the conformation of disease-related proteins (amyloidogenic proteins) is an important determinant of their toxicity, and consequently, the disease development (Carrell and Lomas, 1997). The conformational diseases includes prion diseases, triplet-repeat diseases, e.g., Huntington's disease, Parkinson's disease and other neurodegenerative diseases that can be categorized under dementia with Lewy bodies (DLB). Increasing evidence indicates that most of these disease-related amyloidogenic proteins or their peptide fragments are directly incorporated into membranes to form ion channels as well as AβP (Lashuel and Lansbury, 2002; Kawahara et al., 2011b). It was also demonstrated that AßP (1–40), αsynuclein, amylin, ABri, or other amyloidogenic peptides morphologically similar common ion channel-like structures and elicit single channel currents using AFM, CD, gel electrophoresis, and electrophysiological recordings (Quist et al., 2005; Lal et al., 2007).

122 Advanced Understanding of Neurodegenerative Diseases

Fig. 3. Characteristics of the elevations in [Ca2+]i induced by AβP and other amyloidogenic proteins. Typical time course of [Ca2+]i at 2 min prior to and at 3 min after the application of

the peptide is depicted. (a) AßP(1-40); (b) AßP(40-1); (c) AßP(1-42); (d) D-AßP(1-40); (e) PrP106-126; (f) scramble PrP106-126; (g) human amylin; (h) rat amylin; (i) NAC; and (j) magainin 2. All peptides were at 10 µM. The arrow indicates the time of peptide addition Recently, a new concept of "conformational disease," had been proposed, suggesting that the conformation of disease-related proteins (amyloidogenic proteins) is an important determinant of their toxicity, and consequently, the disease development (Carrell and Lomas, 1997). The conformational diseases includes prion diseases, triplet-repeat diseases, e.g., Huntington's disease, Parkinson's disease and other neurodegenerative diseases that can be categorized under dementia with Lewy bodies (DLB). Increasing evidence indicates that most of these disease-related amyloidogenic proteins or their peptide fragments are directly incorporated into membranes to form ion channels as well as AβP (Lashuel and Lansbury, 2002; Kawahara et al., 2011b). It was also demonstrated that AßP (1–40), αsynuclein, amylin, ABri, or other amyloidogenic peptides morphologically similar common ion channel-like structures and elicit single channel currents using AFM, CD, gel electrophoresis, and electrophysiological recordings (Quist et al., 2005; Lal et al., 2007).

We have also demonstrated that these amyloidogenic peptides including PrP106–126 (a peptide fragment of prion protein), human amylin, NAC, or antimicrobial peptide magainin2 also caused an elevation in the [Ca2+]i levels similar to that induced by AβPs (Kawahara et al., 2000; Kawahara, 2004). However, rat amylin and a peptide a randomized PrP106–126 sequence (scrambled PrP106–126) did not induce any [Ca2+]i changes (Fig. 3). We have also demonstrated that PrP106-126 forms β-sheet structures and exhibits neurotoxicity as well as AβP (Kawahara et al., 2011a). In addition, oligomeric α-synuclein causes neuronal death *via* the Ca2+ influx (Danzer et al., 2007). Considering these results

Fig. 4. (A) Heterogeneity of AβP-induced changes in [Ca2+]i Temporal changes of 50 randomly chosen GT1-7 cells in the same field of view before and after the exposure to AβP(1–40). The arrow indicates the time of peptide addition. B~C: The peak increase in [Ca2+]i (∆[Ca2+]i) in each cell and the latency after exposure to AβP (1–40) were analyzed (B) in more than 50 cultured neurons in a single field of view (360×420 µm3) (mean ± S.E.M., n=300). Typical responses of [Ca2+]i in cultured neurons following exposure to various concentrations of AβP (1–40) (2.5~10 µM). The peak increase in [Ca2+]i (∆[Ca2+]i) in each cell (C) and the latency (D) after exposure to AßP (1–40)

Disruption of Calcium Homeostasis in Alzheimer's Disease:

treatment (Diaz et al., 2009, Arispe et al., 2010).

neurotoxicity (Malchiodi-Albedi et al., 2010).

AβP-induced increase in [Ca2+]i .

compounds.

Role of Channel Formation by β Amyloid Protein 125

Here, we focused on substances that inhibit the formation of amyloid channels. As discussed, the elevation of [Ca2+]i by its permeation through amyloid channels is considered to be the primary event of AβP-induced neurotoxicity; therefore, such compounds could

Inorganic cations such as Zn2+ inhibit the current induced by amyloid channels. Zn is abundant in presynaptic terminal vesicles and is secreted into synaptic clefts following neuronal excitation. Considering that Zn binds to the His residues of AβP, Arispe et al. found that His-related peptide derivatives, such as His-His, effectively inhibit the current through amyloid channels, attenuate AβP-induced [Ca2+]i changes, and protect neurons from AβP toxicity (Arispe et al., 2008). They also developed new compounds with pyrimidine structures that inhibit the amyloid channels and investigated its efficacy for

We focused substances which modify the membrane properties and inhibit amyloid channels. It is widely accepted that the composition of membrane lipids strongly influences the direct incorporation of peptides into membranes and consequent channel formation (Bechinger and Lohner, 2006; Simakova & Arispe, 2007). In particular, electrostatic interactions between peptides and lipids, namely, the net charges of the membrane surface, and membrane fluidity play crucial roles in the affinity for peptides (Seelig et al., 1994). Several AβP residues (e.g., Arg5, Lys16, and Lys28 residues) have a positive charge at a neutral pH; thus, AβP has an affinity for negatively charged phospholipids, such as phosphatidylserine (PS) or phoshatidylglycerol (PG), but not for neutral phospholipids such as phosphatidylcholine (PC). The formation of ß-sheet structures by AβP (1–40) was increased after the addition of PG. Meanwhile, substances that decrease membrane fluidity are known to enhance membrane stiffness and influence pore formation by toxins (Tomita et al., 1992). Gangliosides also contribute to the net charge of the outer membrane surface and to the binding to AβPs. Micro-circumstances on the membranes, such as rafts, containing gangliosides and cholesterol, may provide suitable locations that facilitate this process (Matsuzaki et al., 2010). Indeed, disruption of raft protected neurons from AβPs-induced

Thus, we have developed a screening system for compounds that influence membrane properties by observing the AβP-induced influx of Ca2+. Among tested, we demonstrated that several lipophilic substances such as phloretin, cholesterol, 17β-estradiol, 17α-estradiol, and neurosteroids including dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and pregnenolone, significantly inhibit AβP-induced [Ca2+]i elevation (Kawahara and Kuroda, 2001, Kato-Negishi and Kawahara, 2008). Figure 5 exhibits the structures of these

Phloretin, a plant-derived flavonoid, decreases the membrane potential and inhibits the electrostatic interaction between AβP and membrane lipids (Hertel et al., 1997). Cholesterol decreases membrane fluidity and inhibits channel formation by peptides such as α-toxin, gramicidine, amylin, and AβP. The pretreatment with phloretin or cholesterol significantly inhibited the AßP-induced increase in [Ca2+]i in cultured neurons. Meanwhile, 6-ketocholestanol, which increases the membrane dipole potential, did not influence the

Furthermore, 17β-estradiol, a female hormone, is neuroprotective and affects membrane fluidity (Schwartz et al., 1996). Considering that both of 17β-estradiol and 17α-estradiol inhibit the AβP-induced [Ca2+]i elevation, this inhibition may depend on their membrane modifying effects (Whiting et al, 2000). All of these compounds inhibit AβP neurotoxicity.

serve as the foundation for the new and effective drugs with fewer adverse effects.

together, it is plausible that these disease-related amyloidogenic proteins share similarities in channel formation and the disruption of Ca2+ homeostasis as well as β-sheet formation and cytotoxicity. Table 1 summarizes the common properties of these proteins.

n.d.: not determined

Table 1. Characteristics of amyloidogenic proteins and the related peptides

## **6. Possible candidates for the treatment of AD**

The search for protective agents against AβP-induced neurotoxicity is of great importance. Substances that prevent the oligomerization of AβP such as rifampicin, curcumin, aspirin, DHA can be potential candidates against AβP neurotoxicity (Fig. 2). Reduction of AβP production using BACE or γ-secretase inhibitors is reportedly effective for the treatment of AD. In addition, even though AβP vaccines had been associated with adverse effects, they may be considered as a potential treatment alternative. Conversely, trace metals such as Al, Zn, and Cu enhance AβP oligomerization; thus, chelation therapy with clioquinol, deferoxamine, or silicates has been proposed to be effective in the treatment of AD (Lannfelt et al., 2008; Exley, 2007).

together, it is plausible that these disease-related amyloidogenic proteins share similarities in channel formation and the disruption of Ca2+ homeostasis as well as β-sheet formation

– Parkinson's disease

The search for protective agents against AβP-induced neurotoxicity is of great importance. Substances that prevent the oligomerization of AβP such as rifampicin, curcumin, aspirin, DHA can be potential candidates against AβP neurotoxicity (Fig. 2). Reduction of AβP production using BACE or γ-secretase inhibitors is reportedly effective for the treatment of AD. In addition, even though AβP vaccines had been associated with adverse effects, they may be considered as a potential treatment alternative. Conversely, trace metals such as Al, Zn, and Cu enhance AβP oligomerization; thus, chelation therapy with clioquinol, deferoxamine, or silicates has been proposed to be effective in the treatment of AD (Lannfelt

ß-sheet formation

+

–

+

+

+

–

EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV + + + + +

QQQQQQQQ------------------- + + + n.d. n.d.

ASNCPAIRHPGNKPAVGTLICSRTVKKNIIGGN + + + + n.d.

CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP + + + + +

+

–

Cytotoxicity Channel

+

–

+

+

+

–

+

–

formation

+

–

+

+

+

–

+

–

Morphol ogical pores

+

n.d.

+

n.d.

n.d. +

+ +

–

[Ca2+]i rise

+

–

+

+

and cytotoxicity. Table 1 summarizes the common properties of these proteins.

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL

VVGGVMLGIIAGKNSGVDEAFFVLKQHHVEYGS

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL

KCNTATCATQRLANFLVHSSNNFGAILSSTNVGS

KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGS

Table 1. Characteristics of amyloidogenic proteins and the related peptides

Disease Amyloidogenic protein or its fragment peptide and the primary sequence

> MVGGVV *AßP(40-1)*

DHRFEAD *AßP(25-35)*  DVGSNKGAII *AßP(1-42)*

MVGGVVIA

*Scramble PrP106-126*

KTNMKHMAGAAAAGAVVGGLG

NGAKALMGGHGATKVMVGAAA

*NAC* ( α *fragment of* α*-synuclein )*

**6. Possible candidates for the treatment of AD**

Prion disease *PrP106-126 (prion protein fragment)*

α*-synuclein*

*ABri35*

NTY *Rat amylin*

NTY

Calcitonin

Alzheimer's disease *AßP(1-40)*

(DLB; diseases with Lewy bodies)

Familial British dementia

Medullary carcinoma of the thyroid

n.d.: not determined

et al., 2008; Exley, 2007).

Triplet-repeat disease *Polyglutamine*

Diabetes mellitus *Human amylin*

Here, we focused on substances that inhibit the formation of amyloid channels. As discussed, the elevation of [Ca2+]i by its permeation through amyloid channels is considered to be the primary event of AβP-induced neurotoxicity; therefore, such compounds could serve as the foundation for the new and effective drugs with fewer adverse effects.

Inorganic cations such as Zn2+ inhibit the current induced by amyloid channels. Zn is abundant in presynaptic terminal vesicles and is secreted into synaptic clefts following neuronal excitation. Considering that Zn binds to the His residues of AβP, Arispe et al. found that His-related peptide derivatives, such as His-His, effectively inhibit the current through amyloid channels, attenuate AβP-induced [Ca2+]i changes, and protect neurons from AβP toxicity (Arispe et al., 2008). They also developed new compounds with pyrimidine structures that inhibit the amyloid channels and investigated its efficacy for treatment (Diaz et al., 2009, Arispe et al., 2010).

We focused substances which modify the membrane properties and inhibit amyloid channels. It is widely accepted that the composition of membrane lipids strongly influences the direct incorporation of peptides into membranes and consequent channel formation (Bechinger and Lohner, 2006; Simakova & Arispe, 2007). In particular, electrostatic interactions between peptides and lipids, namely, the net charges of the membrane surface, and membrane fluidity play crucial roles in the affinity for peptides (Seelig et al., 1994). Several AβP residues (e.g., Arg5, Lys16, and Lys28 residues) have a positive charge at a neutral pH; thus, AβP has an affinity for negatively charged phospholipids, such as phosphatidylserine (PS) or phoshatidylglycerol (PG), but not for neutral phospholipids such as phosphatidylcholine (PC). The formation of ß-sheet structures by AβP (1–40) was increased after the addition of PG. Meanwhile, substances that decrease membrane fluidity are known to enhance membrane stiffness and influence pore formation by toxins (Tomita et al., 1992). Gangliosides also contribute to the net charge of the outer membrane surface and to the binding to AβPs. Micro-circumstances on the membranes, such as rafts, containing gangliosides and cholesterol, may provide suitable locations that facilitate this process (Matsuzaki et al., 2010). Indeed, disruption of raft protected neurons from AβPs-induced neurotoxicity (Malchiodi-Albedi et al., 2010).

Thus, we have developed a screening system for compounds that influence membrane properties by observing the AβP-induced influx of Ca2+. Among tested, we demonstrated that several lipophilic substances such as phloretin, cholesterol, 17β-estradiol, 17α-estradiol, and neurosteroids including dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and pregnenolone, significantly inhibit AβP-induced [Ca2+]i elevation (Kawahara and Kuroda, 2001, Kato-Negishi and Kawahara, 2008). Figure 5 exhibits the structures of these compounds.

Phloretin, a plant-derived flavonoid, decreases the membrane potential and inhibits the electrostatic interaction between AβP and membrane lipids (Hertel et al., 1997). Cholesterol decreases membrane fluidity and inhibits channel formation by peptides such as α-toxin, gramicidine, amylin, and AβP. The pretreatment with phloretin or cholesterol significantly inhibited the AßP-induced increase in [Ca2+]i in cultured neurons. Meanwhile, 6-ketocholestanol, which increases the membrane dipole potential, did not influence the AβP-induced increase in [Ca2+]i .

Furthermore, 17β-estradiol, a female hormone, is neuroprotective and affects membrane fluidity (Schwartz et al., 1996). Considering that both of 17β-estradiol and 17α-estradiol inhibit the AβP-induced [Ca2+]i elevation, this inhibition may depend on their membrane modifying effects (Whiting et al, 2000). All of these compounds inhibit AβP neurotoxicity.

6-ketocholestanol(6KC) 17-ß estradiol

Disruption of Calcium Homeostasis in Alzheimer's Disease:

pathogenesis of AD.

**7. Amyloid channel hypothesis**

facilitate the retention of AβP in the brain.

is important in the pathogenesis of AD.

neurodegeneration is initiated.

Role of Channel Formation by β Amyloid Protein 127

aging hormones, and are widely used as supplements to improve the impaired cognitive functions of the elderly (Huppert et al., 2000). Considering that plasma DHEA-S levels are reduced in healthy individuals in an age-dependent manner and in AD patients (Hillen et al., 2000; Aldred and Mecocci, 2010), neurosteroids may have an important role in the

Considering the results of our study together with those of the others, we propose the

AβPs are normally secreted following the cleavage of APP in the synaptic compartment into the cerebrospinal fluid. The secreted AβPs are usually degraded proteolytically within a short period. However, the upregulation of AβP secretion from APP, or an increased ratio of AβP(1–42) to AβP(1–40), which are influenced by APP or presenilin gene mutations, may

As descried previously, the net charge of the outer membrane surface may be a determinant when secreted AβPs bind to cellular membranes. The distribution of phospholipids on cellular membranes is usually asymmetrical; neutral lipids such as PC usually exist on the outer surface of plasma membranes, whereas negatively charged phospholipids such as PS exist on the inner membrane surface. When this asymmetric distribution is disrupted by apoptotic conditions or aging, AβPs can bind to the membrane surfaces (Fig. 6, step (A)). After incorporation into the membrane, the conformation of AβPs change and the accumulated AβPs aggregate on the membranes. The ratio of cholesterol to phospholipids in the membrane may alter membrane fluidity, thereby affecting the process from step (A) to (B). Considering that apoE, its phenotype is a risk factor of AD, is a cholesterol-binding protein, the relationship between cholesterol and AD

Finally, aggregated AβP oligomers form ion channels (Fig. 6 (C)), leading to the various neurodegenerative processes. Once AβP channels are formed in neuronal membranes, the homeostasis of Ca2+ and other ions is disrupted. Unlike endogenous Ca2+ channels, these AβP channels are not regulated by the usual channel blockers; thus, once formed on the membrane, a continuous flow of Ca2+ is initiated. However, Zn2+ ions, which are secreted into the synaptic cleft in a neuronal activity-dependent manner, inhibit AβP-induced Ca2+ entry, and thus have a protective function in AD. Disruption of Ca2+ homeostasis triggers several apoptotic pathways and promotes numerous degenerative processes, including free radical formation and tau phosphorylation, thereby accelerating neuronal death. Presenilins can influence Ca2+ homeostasis through the disturbances of the capacitive Ca2+ entry or other pathways, and may influence these pathways. Free radicals also induce membrane disruption, by which further amplifies unregulated Ca2+ influx. The disruption of Ca2+ homeostasis influences the production and processing of APP. Thus, a vicious cycle of

The velocity of channel formation will be regulated by the binding of AßP on membranes and its concentration, considering that *in vitro* aging can enhance the neurotoxicity of AβP and natural oligomers (dimmers or trimers) are more toxic as compared to monomers, it is provable that AßP oligomerization *in vitro* accelerates the velocity from (A) to (B), and enhances the formation of tetrameric or hexameric pores on membranes. These oligomers might easily form tetrameric or hexameric pores and exhibit neurotoxicity. Indeed, O'Nuallain

following hypothetical scheme of AβP-induced neurodegeneration (Fig. 6).

Phloretin (PH) Cholesterol (Chol)

pregnenolone

Fig. 5. Chemical structures of substances which influence AβP-induced [Ca2+]i elevation