The paper by Lesne et al. is interesting. It would be more convincing if it had included additional controls/information relating to the Aβ oligomer.
For example, do the authors have evidence for this oligomer from in-vitro preparations of Aβ42? If not, why not? If they do, is it ThT-reactive?
Could the authors present TEM evidence of the oligomer, either generated via the transgene or from in-vitro preparations?
If, as I have assumed, the oligomer is only formed in vivo, perhaps only in transgenes, and has not been identified in in-vitro preparations, then some speculation as to why this should be so would be pertinent. It is apparently quite stable, as the authors were able to isolate it for subsequent injection into rats.
In relation to the final experiments in which the isolated oligomer was injected into rat brains, a control consisting of "the vehicle" is surely not sufficient to demonstrate activity of this particular oligomer. We are all aware that injections of Aβ cause behavioral changes in the rat. The authors could have used a positive control, for example, aggregated Aβ, to try to demonstrate that it was not simply the injection of Aβ, in any form, that produced the behavioral differences. In addition, the authors might have tried to demonstrate that the oligomer was actually present in the rat brain. Upon injection, it might have immediately aggregated or dissolved; we have no way of knowing.
The authors may be correct in their assertion that this oligomer causes the behavioral changes seen in both transgenic mice and rats, though the research as presented does not appear to do more than suggest a relationship. Given the weight afforded to research published in Nature, it is surprising that the lack of suitable controls was not commented upon in the accompanying News and Views.
Does this paper provide a new model of memory loss? No, but it advances our understanding of the basis of memory loss in a well-known transgenic mouse model of Alzheimer disease. Above all, the paper offers us a concrete biochemical entity to study and compare against other Aβ oligomer species that various groups have themselves found in recent years.
The paper fits nicely with prior studies that address the major question of what brain changes account for the deficits in memory and cognition in AD. Here is some historical context of this work: In the early 1990s, DeKosky and Scheff, 1990, as well as Robert Terry and Robert Katzman (Terry et al., 1991), showed that loss of synapses was the best correlate of the declines of memory and cognition in AD. Plaques did not correlate with memory and cognition, and tangles correlated slightly. But in these studies of the early 1990s, loss of synapses only accounted for about half the losses of memory and cognition in AD. Where might the missing 50 percent be?
In 2003, our group showed that the brains of AD patients were deficient in a protein, called dynamin 1, that is crucial to the functioning of synapses and, hence, for memory formation and information processing in the brain (Yao et al., 2003; Coleman and Yao, 2003). More specifically, dynamin 1 is a key protein in the trafficking of presynaptic vesicles that contain neurotransmitters. In 2005, Brent Kelly, Robert Vassar, and Adriana Ferreira showed that Aβ peptide caused depletion of dynamin 1, and they confirmed our major finding by showing depletion of dynamin 1 in a mouse model of AD (Kelly et al., 2005).
The current paper by Lesne et al. specifies the form of Aβ that probably was responsible for the loss of dynamin 1 described by Kelly et al. in the Tg2576 mouse model of AD, and by Yao et al. in human AD cases.
These papers all fit together when one posits that a major part of the missing 50 percent in DeKosky’s, Scheff’s, and Terry’s earlier observations lies in defective functioning of synapses that remain structurally present but are unable to function optimally due to deficient expression of dynamin 1 (and other molecules related to synaptic function), and, further, that this deficient expression of dynamin 1 is caused by a specific form of Aβ, which Lesne et al. have now identified. At present, more attention is being paid to Aβ effects on specific transmitters than on vesicle recycling. I believe the latter deserves focused exploration, as well. For one, it would be interesting to know whether Aβ effects on synaptic vesicle trafficking are selective.
One of the major questions unanswered by Lesne et al. lies in the fact that the mouse model they used contains a mutated form of the human APP molecule that is found in only a small percent of AD patients. The work of Kelly et al. apparently used the wild-type form of Aβ. Would Lesne et al. have obtained similar results with the wild-type form of Aβ?
References:
Dekosky ST, Scheff SW.
Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity.
Ann Neurol. 1990 May;27(5):457-64.
PubMed.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R.
Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment.
Ann Neurol. 1991 Oct;30(4):572-80.
PubMed.
Yao PJ, Zhu M, Pyun EI, Brooks AI, Therianos S, Meyers VE, Coleman PD.
Defects in expression of genes related to synaptic vesicle trafficking in frontal cortex of Alzheimer's disease.
Neurobiol Dis. 2003 Mar;12(2):97-109.
PubMed.
Coleman PD, Yao PJ.
Synaptic slaughter in Alzheimer's disease.
Neurobiol Aging. 2003 Dec;24(8):1023-7.
PubMed.
Kelly BL, Vassar R, Ferreira A.
Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease.
J Biol Chem. 2005 Sep 9;280(36):31746-53.
PubMed.
This study is impressive both for the breadth and detail of the experiments undertaken. Using the well-characterized Tg2576 APP transgenic mouse line, the authors searched for the appearance of an Aβ species that coincided with the first observed changes in spatial memory. Starting at 6 months, the time when cognitive changes are first apparent, the authors detected Aβ species that migrated on SDS-PAGE as nonamers and dodecamers. Aβ monomer, trimer, and hexamer were seen at earlier time points and were therefore not considered to have a deleterious effect on cognition. Indeed, comparison of spatial memory and the levels of Aβ monomer, trimer, hexamer, nonamer, and dodecamer revealed that only nonamer and dodecamer levels correlated with memory impairment.
The authenticity of these various Aβ species as discrete assemblies was confirmed using a gel filtration paradigm previously employed to fractionate cell culture-derived low-n oligomers (Walsh et al., 2005), and was combined with immunoaffinity chromatography to achieve purification of the dodecamer.
The authors then conducted the most important and compelling experiment of their study: They injected purified dodecamer into the ventricle of normal pre-trained rats and tested if the injected dodecamer could alter spatial memory. Rats given dodecamer showed a dramatic fall-off in performance; thus, the dodecamer shown to correlate with decreased cognition in Tg2576 mice was also capable of directly mediating impairment of memory in normal rats.
These studies demonstrate for the first time that a soluble, brain-derived form of Aβ can directly mediate brain dysfunction in the absence of neurodegeneration. They open up new avenues of investigation, and yet, as with all scientific advances, the Lesne study raises more questions than it answers. Going forward it will be vitally important to validate the human relevance of the Tg2576 dodecamer—is it present in human brain or CSF? Can it be detected in other animal models of AD? While there is no doubt that Aβ dodecamer present in Tg2576 brain is capable of impairing memory, it is not clear if this species also exists in human brain. Based on the novel homogenization protocol used by Lesne et al., one would predict that Aβ dodecamer should be present in the interstitial fluid and by extension should be readily detectable in CSF. To my knowledge, no such Aβ assembly has been detected in human CSF to date. Indeed, in prior studies, high-molecular-weight Aβ oligomers were not detected in human CSF, whereas Aβ monomer, dimers and trimers were consistently detected (Ida et al., 1996; Walsh et al., 2000).
Of course, the Tg2576 line is a model for AD, and, like all models, it may differ from the human condition. For instance, the authors demonstrate a time-dependent increase in "extracellular-enriched" Aβ monomer, yet in humans it is well documented that CSF Aβ falls with increasing disease severity (Nitsch et al., 1995; Andreasen et al., 1999; Lewczuk et al., 2003). Moreover, recent studies indicate that the fall in Aβ monomer (it has been previously demonstrated that the Takeda ELISA does not readily detect Aβ oligomers; see Morishima and Ihara, 1998) is due to sequestration into senile plaques (Fagan et al., 2005). Together, these results suggest that the overall economy of Aβ in the Tg2576 may be different from that of human brain, and raise the possibility that Aβ assemblies other than or in addition to Aβ dodecamer underlie the memory loss that characterizes AD.
References:
Walsh DM, Townsend M, Podlisny MB, Shankar GM, Fadeeva JV, El Agnaf O, Hartley DM, Selkoe DJ.
Certain inhibitors of synthetic amyloid beta-peptide (Abeta) fibrillogenesis block oligomerization of natural Abeta and thereby rescue long-term potentiation.
J Neurosci. 2005 Mar 9;25(10):2455-62.
PubMed.
Ida N, Hartmann T, Pantel J, Schröder J, Zerfass R, Förstl H, Sandbrink R, Masters CL, Beyreuther K.
Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay.
J Biol Chem. 1996 Sep 13;271(37):22908-14.
PubMed.
Walsh DM, Tseng BP, Rydel RE, Podlisny MB, Selkoe DJ.
The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain.
Biochemistry. 2000 Sep 5;39(35):10831-9.
PubMed.
Nitsch RM, Rebeck GW, Deng M, Richardson UI, Tennis M, Schenk DB, Vigo-Pelfrey C, Lieberburg I, Wurtman RJ, Hyman BT.
Cerebrospinal fluid levels of amyloid beta-protein in Alzheimer's disease: inverse correlation with severity of dementia and effect of apolipoprotein E genotype.
Ann Neurol. 1995 Apr;37(4):512-8.
PubMed.
Andreasen N, Hesse C, Davidsson P, Minthon L, Wallin A, Winblad B, Vanderstichele H, Vanmechelen E, Blennow K.
Cerebrospinal fluid beta-amyloid(1-42) in Alzheimer disease: differences between early- and late-onset Alzheimer disease and stability during the course of disease.
Arch Neurol. 1999 Jun;56(6):673-80.
PubMed.
Lewczuk P, Esselmann H, Meyer M, Wollscheid V, Neumann M, Otto M, Maler JM, Rüther E, Kornhuber J, Wiltfang J.
The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Abeta peptide.
Rapid Commun Mass Spectrom. 2003;17(12):1291-6.
PubMed.
Morishima-Kawashima M, Ihara Y.
The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells.
Biochemistry. 1998 Nov 3;37(44):15247-53.
PubMed.
Fagan AM, Mintun MA, Mach RH, Lee SY, Dence CS, Shah AR, Larossa GN, Spinner ML, Klunk WE, Mathis CA, Dekosky ST, Morris JC, Holtzman DM.
Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans.
Ann Neurol. 2006 Mar;59(3):512-9.
PubMed.
To their credit, the authors have attempted to look for early changes in the TG 2576 mouse model, which are more likely to deal with pathogenesis than pathogenic consequences. Lesne et al. have identified an unusual, high molecular-weight component in the brains of these mice that contains Abeta determinants and is only present before amyloid deposits accumulate.
The claim that this material is necessarily all derived from extracellular spaces is questionable, since it was isolated from detergent-solubilized brain tissue. It is also not clear how much of the 56K band is made up of Abeta peptides. The authors describe an Abeta-derived peptide as representing the "core" of the material, but careful mass spec analysis should have revealed how much and what else was present in the sample. Until this is done, it is premature to declare this a special form of Abeta. I also agree that the biological activity of this material has not yet been studied adequately.
I would just like to comment on the questions/remarks that followed our article. First and foremost, I would like to point out that we did not write in the article that Aβ*56 is an assembly composed of 12 units of Aβ. We did not include any hard data that would directly demonstrate this statement. What we did mention, however, is the possibility that Aβ*56 could represent a 12-mer because of the following observations: 1) Aβ trimers are formed intracellularly and are secreted by neurons in vivo and in vitro; 2) Aβ-immunoreactive species of high molecular weights (above 20 kDa) migrate at molecular weights that match theoretical migrations for 6-mer, 9-mer, and 12-mers of Aβ1-42. It remains to be determined whether these proteins/assemblies are only composed of Aβ, but we postulated so due to the fact that trimers are predominant in vitro and in vivo and only multiples of three monomers appear to form these Aβ-immunoreactive larger structures in vivo. Further analyses are underway to confirm our hypothesis.
Normally, soluble Aβ molecule (39-43 amino acids) undergoes conformational changes in disease and is deposited in the brain as insoluble fibrils, oligomers and protofibrills. Previously it was demonstrated that Aβ neurotoxicity required insoluble fibril formation (mainly Aβ42 and to lesser degree Aβ40) (Lorenzo, 1994) and the fibrils served as inducers of neuronal apoptosis (Loo, 1993). Recently, emphasis has shifted to smaller soluble Aβ. Aβ42 dimers and trimers naturally secreted from a 7PA2 cell line were suggested to be responsible for the disruption of cognitive functions (Cleary, 2005). Importantly, intraventricular injection of such Aβ42 small oligomers inhibited long-term potentiation (LTP) in rat hippocampus and an anti-Aβ monoclonal antibody (6E10) that binds to N-terminal region of Aβ42 prevented this inhibition (Klyubin, 2005). It has also been demonstrated that passive immunization with monoclonal antibodies (NAB61), that specifically recognizes a pathologic conformation present in Aβ dimers, soluble oligomers and higher order species of Aβ, resulted in rapid improvement in spatial learning and memory (Lee, 2006). Other authors showed that 12-mer oligomers of Aβ42, also known as Aβ-derived diffusible ligands (ADDLs) increased about 70-fold in AD patient’s brains over controls (Gong, 2003; Klein, 2006). Collectively, these data suggest that the Aβ oligomers of various sizes are the most pathologic substrate responsible for disrupting neuronal functions and cognitive decline in AD.
The current paper showed that although different forms of Aβ42 are deposited in the brains of aged APP/Tg2576 mice, the memory deficits are induced in 6-month-old or older mice by that accumulated 12-mer oligomer, termed Aβ*56. This is an interesting paper that indicates that levels of soluble or insoluble Aβ do not fully correlate with behavioral changes in this mouse model of AD. Instead only levels of 9-mer (p = 0.0169; r2 = 0.4505) and 12-mer (p = 0.0014; r2 = 0.6556) oligomers showed an inverse correlation with spatial memory. These data indicates that AD therapy should target particular species of Aβ that are responsible for AD like pathology and memory deficits. Thus, if Aβ*56 is a major player in AD that is implicated in memory deficits in middle-aged Tg2576 mice, then learning about misfolding of human Aβ peptide is an important task. As we gain more knowledge of the mechanisms of assembly of Aβ peptides more potent AD therapies will be developed.
One important conclusion from this paper is that such AD therapy should be based on the prevention of accumulation of oligomers, rather than on clearing already formed toxic forms of Aβ. In other words AD therapy that can block oligomerization of Aβ should be started earlier, before the accumulation of monomers in the brains. This can be done by the prototype AD vaccine that will be able to generate antibodies that can bind all forms of Aβ and block oligomerization of the monomeric peptide. Of course such a vaccine should be used in middle-aged healthy people to prevent generation of AD-like pathology, rather than for vaccination of elderly AD patients with immunosenescence (therapeutic vaccine strategy). Such a vaccine would have to be safe and should be able to generate high titers of antibodies that can block oligomerization of β amyloid peptide, although they can be specific to any form of Aβ (monomers, oligomers, and fibrils).
Thus, the important aim of AD-immunotherapy research must be the identification of the most safe and immunogenic form of the vaccine that can generate therapeutically potent antibodies that can block oligomerization of the peptide. For example, using an epitope vaccine strategy we recently generated polyclonal antibodies specific to the N-terminal region of Aβ that can not only bind all forms of Aβ, but also delay oligomerization of Aβ42 in vitro (paper submitted). In the nearest future it will be important to test the ability of this and other prototype AD vaccines to block generation of Aβ*56 in the brains of immunized APP/Tg 2576 or other APP/Tg mice.
References:
Lorenzo A, Yankner BA.
Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12243-7.
PubMed.
Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW.
Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.
Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7951-5.
PubMed.
Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH.
Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function.
Nat Neurosci. 2005 Jan;8(1):79-84.
PubMed.
Klyubin I, Walsh DM, Lemere CA, Cullen WK, Shankar GM, Betts V, Spooner ET, Jiang L, Anwyl R, Selkoe DJ, Rowan MJ.
Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo.
Nat Med. 2005 May;11(5):556-61.
PubMed.
Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL.
Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss.
Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10417-22.
PubMed.
Comments
The paper by Lesne et al. is interesting. It would be more convincing if it had included additional controls/information relating to the Aβ oligomer.
For example, do the authors have evidence for this oligomer from in-vitro preparations of Aβ42? If not, why not? If they do, is it ThT-reactive?
Could the authors present TEM evidence of the oligomer, either generated via the transgene or from in-vitro preparations?
If, as I have assumed, the oligomer is only formed in vivo, perhaps only in transgenes, and has not been identified in in-vitro preparations, then some speculation as to why this should be so would be pertinent. It is apparently quite stable, as the authors were able to isolate it for subsequent injection into rats.
In relation to the final experiments in which the isolated oligomer was injected into rat brains, a control consisting of "the vehicle" is surely not sufficient to demonstrate activity of this particular oligomer. We are all aware that injections of Aβ cause behavioral changes in the rat. The authors could have used a positive control, for example, aggregated Aβ, to try to demonstrate that it was not simply the injection of Aβ, in any form, that produced the behavioral differences. In addition, the authors might have tried to demonstrate that the oligomer was actually present in the rat brain. Upon injection, it might have immediately aggregated or dissolved; we have no way of knowing.
The authors may be correct in their assertion that this oligomer causes the behavioral changes seen in both transgenic mice and rats, though the research as presented does not appear to do more than suggest a relationship. Given the weight afforded to research published in Nature, it is surprising that the lack of suitable controls was not commented upon in the accompanying News and Views.
View all comments by Chris ExleyBanner Research Institute
Does this paper provide a new model of memory loss? No, but it advances our understanding of the basis of memory loss in a well-known transgenic mouse model of Alzheimer disease. Above all, the paper offers us a concrete biochemical entity to study and compare against other Aβ oligomer species that various groups have themselves found in recent years.
The paper fits nicely with prior studies that address the major question of what brain changes account for the deficits in memory and cognition in AD. Here is some historical context of this work: In the early 1990s, DeKosky and Scheff, 1990, as well as Robert Terry and Robert Katzman (Terry et al., 1991), showed that loss of synapses was the best correlate of the declines of memory and cognition in AD. Plaques did not correlate with memory and cognition, and tangles correlated slightly. But in these studies of the early 1990s, loss of synapses only accounted for about half the losses of memory and cognition in AD. Where might the missing 50 percent be?
In 2003, our group showed that the brains of AD patients were deficient in a protein, called dynamin 1, that is crucial to the functioning of synapses and, hence, for memory formation and information processing in the brain (Yao et al., 2003; Coleman and Yao, 2003). More specifically, dynamin 1 is a key protein in the trafficking of presynaptic vesicles that contain neurotransmitters. In 2005, Brent Kelly, Robert Vassar, and Adriana Ferreira showed that Aβ peptide caused depletion of dynamin 1, and they confirmed our major finding by showing depletion of dynamin 1 in a mouse model of AD (Kelly et al., 2005).
The current paper by Lesne et al. specifies the form of Aβ that probably was responsible for the loss of dynamin 1 described by Kelly et al. in the Tg2576 mouse model of AD, and by Yao et al. in human AD cases.
These papers all fit together when one posits that a major part of the missing 50 percent in DeKosky’s, Scheff’s, and Terry’s earlier observations lies in defective functioning of synapses that remain structurally present but are unable to function optimally due to deficient expression of dynamin 1 (and other molecules related to synaptic function), and, further, that this deficient expression of dynamin 1 is caused by a specific form of Aβ, which Lesne et al. have now identified. At present, more attention is being paid to Aβ effects on specific transmitters than on vesicle recycling. I believe the latter deserves focused exploration, as well. For one, it would be interesting to know whether Aβ effects on synaptic vesicle trafficking are selective.
One of the major questions unanswered by Lesne et al. lies in the fact that the mouse model they used contains a mutated form of the human APP molecule that is found in only a small percent of AD patients. The work of Kelly et al. apparently used the wild-type form of Aβ. Would Lesne et al. have obtained similar results with the wild-type form of Aβ?
References:
Dekosky ST, Scheff SW. Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity. Ann Neurol. 1990 May;27(5):457-64. PubMed.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol. 1991 Oct;30(4):572-80. PubMed.
Yao PJ, Zhu M, Pyun EI, Brooks AI, Therianos S, Meyers VE, Coleman PD. Defects in expression of genes related to synaptic vesicle trafficking in frontal cortex of Alzheimer's disease. Neurobiol Dis. 2003 Mar;12(2):97-109. PubMed.
Coleman PD, Yao PJ. Synaptic slaughter in Alzheimer's disease. Neurobiol Aging. 2003 Dec;24(8):1023-7. PubMed.
Kelly BL, Vassar R, Ferreira A. Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease. J Biol Chem. 2005 Sep 9;280(36):31746-53. PubMed.
View all comments by Paul ColemanBrigham & Women's Hospital
This study is impressive both for the breadth and detail of the experiments undertaken. Using the well-characterized Tg2576 APP transgenic mouse line, the authors searched for the appearance of an Aβ species that coincided with the first observed changes in spatial memory. Starting at 6 months, the time when cognitive changes are first apparent, the authors detected Aβ species that migrated on SDS-PAGE as nonamers and dodecamers. Aβ monomer, trimer, and hexamer were seen at earlier time points and were therefore not considered to have a deleterious effect on cognition. Indeed, comparison of spatial memory and the levels of Aβ monomer, trimer, hexamer, nonamer, and dodecamer revealed that only nonamer and dodecamer levels correlated with memory impairment.
The authenticity of these various Aβ species as discrete assemblies was confirmed using a gel filtration paradigm previously employed to fractionate cell culture-derived low-n oligomers (Walsh et al., 2005), and was combined with immunoaffinity chromatography to achieve purification of the dodecamer.
The authors then conducted the most important and compelling experiment of their study: They injected purified dodecamer into the ventricle of normal pre-trained rats and tested if the injected dodecamer could alter spatial memory. Rats given dodecamer showed a dramatic fall-off in performance; thus, the dodecamer shown to correlate with decreased cognition in Tg2576 mice was also capable of directly mediating impairment of memory in normal rats.
These studies demonstrate for the first time that a soluble, brain-derived form of Aβ can directly mediate brain dysfunction in the absence of neurodegeneration. They open up new avenues of investigation, and yet, as with all scientific advances, the Lesne study raises more questions than it answers. Going forward it will be vitally important to validate the human relevance of the Tg2576 dodecamer—is it present in human brain or CSF? Can it be detected in other animal models of AD? While there is no doubt that Aβ dodecamer present in Tg2576 brain is capable of impairing memory, it is not clear if this species also exists in human brain. Based on the novel homogenization protocol used by Lesne et al., one would predict that Aβ dodecamer should be present in the interstitial fluid and by extension should be readily detectable in CSF. To my knowledge, no such Aβ assembly has been detected in human CSF to date. Indeed, in prior studies, high-molecular-weight Aβ oligomers were not detected in human CSF, whereas Aβ monomer, dimers and trimers were consistently detected (Ida et al., 1996; Walsh et al., 2000).
Of course, the Tg2576 line is a model for AD, and, like all models, it may differ from the human condition. For instance, the authors demonstrate a time-dependent increase in "extracellular-enriched" Aβ monomer, yet in humans it is well documented that CSF Aβ falls with increasing disease severity (Nitsch et al., 1995; Andreasen et al., 1999; Lewczuk et al., 2003). Moreover, recent studies indicate that the fall in Aβ monomer (it has been previously demonstrated that the Takeda ELISA does not readily detect Aβ oligomers; see Morishima and Ihara, 1998) is due to sequestration into senile plaques (Fagan et al., 2005). Together, these results suggest that the overall economy of Aβ in the Tg2576 may be different from that of human brain, and raise the possibility that Aβ assemblies other than or in addition to Aβ dodecamer underlie the memory loss that characterizes AD.
References:
Walsh DM, Townsend M, Podlisny MB, Shankar GM, Fadeeva JV, El Agnaf O, Hartley DM, Selkoe DJ. Certain inhibitors of synthetic amyloid beta-peptide (Abeta) fibrillogenesis block oligomerization of natural Abeta and thereby rescue long-term potentiation. J Neurosci. 2005 Mar 9;25(10):2455-62. PubMed.
Ida N, Hartmann T, Pantel J, Schröder J, Zerfass R, Förstl H, Sandbrink R, Masters CL, Beyreuther K. Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay. J Biol Chem. 1996 Sep 13;271(37):22908-14. PubMed.
Walsh DM, Tseng BP, Rydel RE, Podlisny MB, Selkoe DJ. The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry. 2000 Sep 5;39(35):10831-9. PubMed.
Nitsch RM, Rebeck GW, Deng M, Richardson UI, Tennis M, Schenk DB, Vigo-Pelfrey C, Lieberburg I, Wurtman RJ, Hyman BT. Cerebrospinal fluid levels of amyloid beta-protein in Alzheimer's disease: inverse correlation with severity of dementia and effect of apolipoprotein E genotype. Ann Neurol. 1995 Apr;37(4):512-8. PubMed.
Andreasen N, Hesse C, Davidsson P, Minthon L, Wallin A, Winblad B, Vanderstichele H, Vanmechelen E, Blennow K. Cerebrospinal fluid beta-amyloid(1-42) in Alzheimer disease: differences between early- and late-onset Alzheimer disease and stability during the course of disease. Arch Neurol. 1999 Jun;56(6):673-80. PubMed.
Lewczuk P, Esselmann H, Meyer M, Wollscheid V, Neumann M, Otto M, Maler JM, Rüther E, Kornhuber J, Wiltfang J. The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Abeta peptide. Rapid Commun Mass Spectrom. 2003;17(12):1291-6. PubMed.
Morishima-Kawashima M, Ihara Y. The presence of amyloid beta-protein in the detergent-insoluble membrane compartment of human neuroblastoma cells. Biochemistry. 1998 Nov 3;37(44):15247-53. PubMed.
Fagan AM, Mintun MA, Mach RH, Lee SY, Dence CS, Shah AR, Larossa GN, Spinner ML, Klunk WE, Mathis CA, Dekosky ST, Morris JC, Holtzman DM. Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans. Ann Neurol. 2006 Mar;59(3):512-9. PubMed.
View all comments by Dominic WalshYale University School of Medicine
To their credit, the authors have attempted to look for early changes in the TG 2576 mouse model, which are more likely to deal with pathogenesis than pathogenic consequences. Lesne et al. have identified an unusual, high molecular-weight component in the brains of these mice that contains Abeta determinants and is only present before amyloid deposits accumulate.
The claim that this material is necessarily all derived from extracellular spaces is questionable, since it was isolated from detergent-solubilized brain tissue. It is also not clear how much of the 56K band is made up of Abeta peptides. The authors describe an Abeta-derived peptide as representing the "core" of the material, but careful mass spec analysis should have revealed how much and what else was present in the sample. Until this is done, it is premature to declare this a special form of Abeta. I also agree that the biological activity of this material has not yet been studied adequately.
View all comments by Vincent MarchesiUniversity of Minnesota
I would just like to comment on the questions/remarks that followed our article. First and foremost, I would like to point out that we did not write in the article that Aβ*56 is an assembly composed of 12 units of Aβ. We did not include any hard data that would directly demonstrate this statement. What we did mention, however, is the possibility that Aβ*56 could represent a 12-mer because of the following observations: 1) Aβ trimers are formed intracellularly and are secreted by neurons in vivo and in vitro; 2) Aβ-immunoreactive species of high molecular weights (above 20 kDa) migrate at molecular weights that match theoretical migrations for 6-mer, 9-mer, and 12-mers of Aβ1-42. It remains to be determined whether these proteins/assemblies are only composed of Aβ, but we postulated so due to the fact that trimers are predominant in vitro and in vivo and only multiples of three monomers appear to form these Aβ-immunoreactive larger structures in vivo. Further analyses are underway to confirm our hypothesis.
View all comments by Sylvain LesneThe Institute for Molecular Medicine
Normally, soluble Aβ molecule (39-43 amino acids) undergoes conformational changes in disease and is deposited in the brain as insoluble fibrils, oligomers and protofibrills. Previously it was demonstrated that Aβ neurotoxicity required insoluble fibril formation (mainly Aβ42 and to lesser degree Aβ40) (Lorenzo, 1994) and the fibrils served as inducers of neuronal apoptosis (Loo, 1993). Recently, emphasis has shifted to smaller soluble Aβ. Aβ42 dimers and trimers naturally secreted from a 7PA2 cell line were suggested to be responsible for the disruption of cognitive functions (Cleary, 2005). Importantly, intraventricular injection of such Aβ42 small oligomers inhibited long-term potentiation (LTP) in rat hippocampus and an anti-Aβ monoclonal antibody (6E10) that binds to N-terminal region of Aβ42 prevented this inhibition (Klyubin, 2005). It has also been demonstrated that passive immunization with monoclonal antibodies (NAB61), that specifically recognizes a pathologic conformation present in Aβ dimers, soluble oligomers and higher order species of Aβ, resulted in rapid improvement in spatial learning and memory (Lee, 2006). Other authors showed that 12-mer oligomers of Aβ42, also known as Aβ-derived diffusible ligands (ADDLs) increased about 70-fold in AD patient’s brains over controls (Gong, 2003; Klein, 2006). Collectively, these data suggest that the Aβ oligomers of various sizes are the most pathologic substrate responsible for disrupting neuronal functions and cognitive decline in AD.
The current paper showed that although different forms of Aβ42 are deposited in the brains of aged APP/Tg2576 mice, the memory deficits are induced in 6-month-old or older mice by that accumulated 12-mer oligomer, termed Aβ*56. This is an interesting paper that indicates that levels of soluble or insoluble Aβ do not fully correlate with behavioral changes in this mouse model of AD. Instead only levels of 9-mer (p = 0.0169; r2 = 0.4505) and 12-mer (p = 0.0014; r2 = 0.6556) oligomers showed an inverse correlation with spatial memory. These data indicates that AD therapy should target particular species of Aβ that are responsible for AD like pathology and memory deficits. Thus, if Aβ*56 is a major player in AD that is implicated in memory deficits in middle-aged Tg2576 mice, then learning about misfolding of human Aβ peptide is an important task. As we gain more knowledge of the mechanisms of assembly of Aβ peptides more potent AD therapies will be developed.
One important conclusion from this paper is that such AD therapy should be based on the prevention of accumulation of oligomers, rather than on clearing already formed toxic forms of Aβ. In other words AD therapy that can block oligomerization of Aβ should be started earlier, before the accumulation of monomers in the brains. This can be done by the prototype AD vaccine that will be able to generate antibodies that can bind all forms of Aβ and block oligomerization of the monomeric peptide. Of course such a vaccine should be used in middle-aged healthy people to prevent generation of AD-like pathology, rather than for vaccination of elderly AD patients with immunosenescence (therapeutic vaccine strategy). Such a vaccine would have to be safe and should be able to generate high titers of antibodies that can block oligomerization of β amyloid peptide, although they can be specific to any form of Aβ (monomers, oligomers, and fibrils).
Thus, the important aim of AD-immunotherapy research must be the identification of the most safe and immunogenic form of the vaccine that can generate therapeutically potent antibodies that can block oligomerization of the peptide. For example, using an epitope vaccine strategy we recently generated polyclonal antibodies specific to the N-terminal region of Aβ that can not only bind all forms of Aβ, but also delay oligomerization of Aβ42 in vitro (paper submitted). In the nearest future it will be important to test the ability of this and other prototype AD vaccines to block generation of Aβ*56 in the brains of immunized APP/Tg 2576 or other APP/Tg mice.
References:
Lorenzo A, Yankner BA. Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12243-7. PubMed.
Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW. Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7951-5. PubMed.
Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005 Jan;8(1):79-84. PubMed.
Klyubin I, Walsh DM, Lemere CA, Cullen WK, Shankar GM, Betts V, Spooner ET, Jiang L, Anwyl R, Selkoe DJ, Rowan MJ. Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo. Nat Med. 2005 May;11(5):556-61. PubMed.
Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL. Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10417-22. PubMed.
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