"Until the Last Dog(ma) Dies": Some Neuritic Dystrophy Is Reversible by Passive Immunization of PDAPP Mice
A multidisciplinary group has demonstrated that at least some neuritic dystrophy in PDAPP mice is reversible. Holtzman from Wash U, Paul from Lilly, Mathis and Klunk from Pitt, and Bacskai and Hyman from MGH contributed their considerable talent to a new paper in the current issue of The Journal of Clinical Investigation. Using the open skull method and Congo red derivative methoxy-X04 devised by the MGH and Pittsburgh groups, respectively, the team followed with serial imaging the morphology of swollen (dystrophic) neurites surrounding cortical amyloid deposits in the PDAPP mouse. Conventional wisdom would have predicted that these swellings might be permanent, but the new paper describes how passive immunization with anti-Aβ antibodies had a significant effect on partially normalizing the shapes of the processes.

The new paper builds on earlier work by the MGH group (Lombardo et al., 2003): The advance of the...  Read more

"Until the Last Dog(ma) Dies": Some Neuritic Dystrophy Is Reversible by Passive Immunization of PDAPP Mice
A multidisciplinary group has demonstrated that at least some neuritic dystrophy in PDAPP mice is reversible. Holtzman from Wash U, Paul from Lilly, Mathis and Klunk from Pitt, and Bacskai and Hyman from MGH contributed their considerable talent to a new paper in the current issue of The Journal of Clinical Investigation. Using the open skull method and Congo red derivative methoxy-X04 devised by the MGH and Pittsburgh groups, respectively, the team followed with serial imaging the morphology of swollen (dystrophic) neurites surrounding cortical amyloid deposits in the PDAPP mouse. Conventional wisdom would have predicted that these swellings might be permanent, but the new paper describes how passive immunization with anti-Aβ antibodies had a significant effect on partially normalizing the shapes of the processes.

The new paper builds on earlier work by the MGH group (Lombardo et al., 2003): The advance of the JCI paper is to study the same plaques serially during life, while the Lombardo paper relied on postmortem analysis. A key novelty is directly demonstrating a significant trend toward normalization of existing dystrophic neurites, i.e., watching the same, flagrantly abnormal neurites partially recover their normal morphology, thereby unequivocally documenting the reversibility of neuritic dystrophy.

This dovetails well with the recent report in Neuron by Oddo and colleagues (see ARF related news story) who showed that amyloid deposits and neuritic antigens were attenuated by active immunization. Janus and colleagues (Janus et al., 2000) demonstrated Aβ-dependent behavioral deficits that were reversible with active immunization, and the Holtzman paper suggests that the same may be true for passive treatment. Together, the papers portend well for the principle that Alzheimer's pathology may be treatable, even after significant dystrophy has developed.

This is important because the most sensitive means of detecting Alzheimer's today remains neuropsychological testing, implying, by definition, the existence of pathology and deficits that one would like to reverse, if possible. Interestingly, a recent PNAS paper (Lu et al., 2005) indicates that iconic memory deficits may predict Alzheimer's even before any important deficit is present or even otherwise detectable. All the usual mice-aren't-men caveats apply, of course, but these papers are very exciting because frontiers have been pushed back both in terms of early diagnosis as well as providing optimism for reversibility of pathology.

View all comments by Samuel Gandy

The authors used multiphoton microscopy, an elegant technique, to monitor the dynamics of neuritic plaques in living mice. They used the PDAPP;Thy-1:YFP transgenic mouse model, which develops plaque pathology and expresses yellow fluorescent protein in a subset of neurons. Through cranial windows, Aβ deposits were analyzed with injected methoxy-X04, and dystrophic neurites with YFP-induced fluorescence. Over a period of 72 hours, the amyloid-associated neurites remained stable. However, after application of the anti-Aβ antibody 10D5 to the cortical surface, the number and total cross-sectional area of dystrophic neuritis decreased significantly. This clearly demonstrates again the value of passive immunization to reduce extracellular plaque load and the associated neuritic pathology.

Although these results are very promising, novel transgenic mouse models teach us that extracellular amyloid plaques are not a major trigger for the dramatic neuron loss and brain atrophy. On the contrary, amyloid plaques do not correlate with the hippocampal neuron loss in the transgenic...  Read more

The authors used multiphoton microscopy, an elegant technique, to monitor the dynamics of neuritic plaques in living mice. They used the PDAPP;Thy-1:YFP transgenic mouse model, which develops plaque pathology and expresses yellow fluorescent protein in a subset of neurons. Through cranial windows, Aβ deposits were analyzed with injected methoxy-X04, and dystrophic neurites with YFP-induced fluorescence. Over a period of 72 hours, the amyloid-associated neurites remained stable. However, after application of the anti-Aβ antibody 10D5 to the cortical surface, the number and total cross-sectional area of dystrophic neuritis decreased significantly. This clearly demonstrates again the value of passive immunization to reduce extracellular plaque load and the associated neuritic pathology.

Although these results are very promising, novel transgenic mouse models teach us that extracellular amyloid plaques are not a major trigger for the dramatic neuron loss and brain atrophy. On the contrary, amyloid plaques do not correlate with the hippocampal neuron loss in the transgenic models (Schmitz et al., 2004; Casas et al., 2004). In both models, the increased amount of intraneuronal Aβ42 correlates best with the loss of neurons and brain tissue. These models support the idea that neuron loss and atrophy in AD is triggered by intracellular accumulation of Aβ42, and not by extracellular amyloid pathology.

It is very likely that intraneuronal Aβ accumulation leads not only to neuron loss, but also disrupts many neuronal functions, for example, axonal transport. Therefore, it will be very important to learn more about the influence of passive immunization on the potential to reduce intraneuronal Aβ levels as has been shown recently (Oddo et al., 2004). Using a triple-transgenic model (3xTg-AD) that develops plaques and tau lesions, the authors showed that Aβ immunotherapy reduces not only extracellular Aβ plaques, but also intracellular Aβ accumulation, and leads to the clearance of tau hyperphosphorylation.

Convincing evidence that passive immunization is able to rescue neuronal dysfunction and neuron loss is, however, still lacking.

References:
Blanchard V, Moussaoui S, Czech C, Touchet N, Bonici B, Planche M, Canton T, Jedidi I, Gohin M, Wirths O, Bayer TA, Langui D, Duychaerts C,Tremp G, Pradier, L (2003) Time sequence of maturation of dystrophic neurites associated with Aβ deposits in APP/PS1 transgenic mice. Exp. Neurol. 184(1):247-263. Abstract

Schmitz C, Rutten BPF, Pielen A, Schäfer S, Wirths O, Tremp G, Czech C, Blanchard V, Multhaup G, Korr H, Steinbusch HWM, Pradier L, Bayer TA (2004) Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer’s disease. American Journal of Pathol. 164:1495-1502. Abstract

Casas C, Sergeant N, Itier J-M, Blanchard V, Wirths O, van der Kolk N, Vingtdeux V, van de Steeg E, Ret G, Canton T, Drobecq H, Clark A, Bonici B, Delacourte A, Benavides J, Schmitz C, Tremp G, Bayer TA, Benoit P, Pradier L (2004) Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Aβ42 accumulation in a novel Alzheimer transgenic model. American Journal of Pathol. 165(4):1289-1300. Abstract

Oddo, S., Billings, L., Kesslak, J.P., Cribbs, D.H., and LaFerla, F.M. (2004). Abeta Immunotherapy Leads to Clearance of Early, but Not Late, Hyperphosphorylated Tau Aggregates via the Proteasome. Neuron 43, 321-332. Abstract

View all comments by Thomas Bayer

This paper by the Holtzman group supports the original work of the Bacskai/Hyman group that also showed that reduction of Aβ in the brain can induce a rapid structural recovery of existing amyloid-associated neuritic dystrophy. The originality of this new paper is that the analysis was performed in a living mouse brain as opposed to postmortem in the original study published in 2003 (Lombardo et al.). The data presented support the AD-amyloid hypothesis and, indeed, suggest that reducing Aβ (via an immunotherapy, at least) will be effective.

I would have liked to see if the same effect could be observed if the antibody is injected into the blood. Would this also reduce neurite dystrophy over the same time period? This is worthy of demonstration since, realistically, the current AD immunotherapy in development will require the injection of humanized Aβ antibodies into the bloodstream.

Moreover, although the study clearly demonstrates histological improvements (reduction of neurite dystrophy) after the antibody treatment, it...  Read more

This paper by the Holtzman group supports the original work of the Bacskai/Hyman group that also showed that reduction of Aβ in the brain can induce a rapid structural recovery of existing amyloid-associated neuritic dystrophy. The originality of this new paper is that the analysis was performed in a living mouse brain as opposed to postmortem in the original study published in 2003 (Lombardo et al.). The data presented support the AD-amyloid hypothesis and, indeed, suggest that reducing Aβ (via an immunotherapy, at least) will be effective.

I would have liked to see if the same effect could be observed if the antibody is injected into the blood. Would this also reduce neurite dystrophy over the same time period? This is worthy of demonstration since, realistically, the current AD immunotherapy in development will require the injection of humanized Aβ antibodies into the bloodstream.

Moreover, although the study clearly demonstrates histological improvements (reduction of neurite dystrophy) after the antibody treatment, it is important in the future to demonstrate if this also leads to a recovery of cognitive functions. It could also be very interesting to repeat the experiment with two kinds of antibody: one that recognizes only the monomeric form of Aβ and one that recognizes only its fibrillar form. This would answer this burning question: Shall companies develop an antibody that recognizes the fibrillar form of Aβ or its monomeric form (sink hypothesis)?

Finally, the fact that the reduction of neuritic dystrophy can only be seen on the side ipsilateral to 10D5 application (when applied to 17-18-month-old PDAPP mice) suggests that the effect won't be observed unless there is a massive amount of antibody present.

Altogether, this is an important paper because it shows clearly that ongoing axonal or dendritic damage by Aβ seems to be, in part, reversible.

View all comments by Frank Bernier

This paper by Brendza et al. elegantly confirms and extends previous studies by this group and others. It uses living PDAPP:Thy-1:YFP transgenic mice and multiphoton microscopy to show that passive immunization with anti-Aβ antibodies not only is able to reduce or eliminate cortical deposits of Aβ, but also the associated dystrophic neurites. These are novel and important studies, as they imply that the therapeutic effects of passive immunization may extend beyond fibrillar or aggregated Aβ deposits themselves to pathological dystrophic processes that are a prominent feature of Alzheimer disease (AD) brain pathology.

This study is significant, as dystrophic processes are well-recognized but poorly understood components of AD brain pathology despite having been described nearly 20 years ago as sites of tau accumulation (e.g., Ihara, 1988). In addition, many studies have shown that dystrophic neurites also contain other elements including fragments of APP flanking the Aβ domain and neurofilament proteins (e.g.,   Read more

This paper by Brendza et al. elegantly confirms and extends previous studies by this group and others. It uses living PDAPP:Thy-1:YFP transgenic mice and multiphoton microscopy to show that passive immunization with anti-Aβ antibodies not only is able to reduce or eliminate cortical deposits of Aβ, but also the associated dystrophic neurites. These are novel and important studies, as they imply that the therapeutic effects of passive immunization may extend beyond fibrillar or aggregated Aβ deposits themselves to pathological dystrophic processes that are a prominent feature of Alzheimer disease (AD) brain pathology.

This study is significant, as dystrophic processes are well-recognized but poorly understood components of AD brain pathology despite having been described nearly 20 years ago as sites of tau accumulation (e.g., Ihara, 1988). In addition, many studies have shown that dystrophic neurites also contain other elements including fragments of APP flanking the Aβ domain and neurofilament proteins (e.g., Arai et al., 1990). Although dystrophic neurites are the locus of more than 95 percent of immunohistochemically ascertainable, pathological tau amyloid as measured morphometrically (Mitchell et al., 2000), they are lesions with a complex composition and uncertain etiology. It is likely that they contribute to cognitive impairments in AD, although how they do this is incompletely understood.

Thus, Brendza et al. point the way to render these enigmatic lesions more tractable to experimental investigation. It will be interesting to see if they or other investigators are able to take further steps toward elucidating the nature and pathological significance of dystrophic neurites with the elegant methods used in their current study. A better understanding of these issues will help clarify the extent to which dystrophic neurites should become a deliberate focus of therapeutic intervention in AD.

Several of the investigators of the present paper also have shown recently how passive immunotherapy with anti-Aβ antibodies exacerbates cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice (Racke, 2005). It would be important to know if the methods used by Brendza et al. could be exploited to gain insight into mechanisms underlying this complication of Aβ immunotherapy. .

View all comments by John Trojanowski

Thank you for offering such a variety of papers by people who are spending their lives looking for answers.

PS: Footnotes for lay persons would help.

View all comments by Elizabeth Petersen

Brendza and colleagues demonstrate that administration of anti-Aβ antibodies into APP-transgenic mouse brain results in recovery of dystrophic neuritis surrounding Aβ plaques. The authors employed multiphoton microscopy to detect dystrophic neuritis labeled by transgene-derived YFP. This detection method allows in-vivo observation of Aβ plaques and dystrophic neuritis in living mice, although it is not fully non-invasive, as it requires cranial surgery to make a small window on a cranial bone.

The effect of the antibody administration is not very large, but is statistically significant. The authors’ observation indicates that Aβ removal leads to recovery of neuritic dystrophy and that the processes involved in dystrophic neuritis are reversible until they reach a certain point. The results shown by Brendza and colleagues thus provide additional support for therapeutic strategies targeting Aβ.

There remains, however, a primary question whether the formation of dystrophic neurites is a main pathway causing cognitive dysfunction in the pathological cascade of Alzheimer...  Read more

Brendza and colleagues demonstrate that administration of anti-Aβ antibodies into APP-transgenic mouse brain results in recovery of dystrophic neuritis surrounding Aβ plaques. The authors employed multiphoton microscopy to detect dystrophic neuritis labeled by transgene-derived YFP. This detection method allows in-vivo observation of Aβ plaques and dystrophic neuritis in living mice, although it is not fully non-invasive, as it requires cranial surgery to make a small window on a cranial bone.

The effect of the antibody administration is not very large, but is statistically significant. The authors’ observation indicates that Aβ removal leads to recovery of neuritic dystrophy and that the processes involved in dystrophic neuritis are reversible until they reach a certain point. The results shown by Brendza and colleagues thus provide additional support for therapeutic strategies targeting Aβ.

There remains, however, a primary question whether the formation of dystrophic neurites is a main pathway causing cognitive dysfunction in the pathological cascade of Alzheimer disease development, since it is generally accepted that tauopathy rather than Aβ amyloidosis is more closely associated with the symptoms.

One technical point to note is that the authors administered the anti-Aβ antibodies directly into the cranial windows. This protocol probably does not mimic the processes evoked by Aβ vaccination, active or passive, because intravenously administered anti-Aβ antibodies do not seem to penetrate into brain parenchyma.

In any case, removal of Aβ deposition in the early stages of the disease development before tauopathy and neurodegeneration proceeds to an irreversible extent will certainly stop this tragic disease from rising. Thus, the key words for combating Alzheimer disease in a pragmatic manner are “presymptomatic diagnosis” and “preventive medication.” For this purpose, we need to identify the primary cause of Aβ deposition in sporadic Alzheimer disease development.

View all comments by Takaomi Saido

This very nice study confirms the finding of Matthias Jucker and colleagues [1] that anti-Aβ antibody treatment can provoke CAA-related hemorrhage, in particular, antibodies that bind to deposited rather than soluble Aβ. The mechanism remains unclear; a logical possibility is that the same mechanisms that clear Aβ deposits can also "punch holes" in the amyloid-laden vessel wall.

For now, immunization-related hemorrhage remains a phenomenon of transgenic mice rather than human disease. Hemorrhagic stroke was not reported in the Elan-Wyeth vaccine studies [2], and the microhemorrhages seen on pathological examination of these brains [3,4] appear related to the underlying CAA rather than the vaccine itself. Human CAA may instead respond to inflammation by vascular dysfunction and reversible white matter changes [5]. Clearly, much remains to be learned about the role of CAA in determining the safety and efficacy of immune-based therapies for AD.

References:
1. Pfeifer M, Boncristiano S, Bondolfi L, Stalder A, Deller T, Staufenbiel M, Mathews PM, Jucker M. Cerebral...  Read more

This very nice study confirms the finding of Matthias Jucker and colleagues [1] that anti-Aβ antibody treatment can provoke CAA-related hemorrhage, in particular, antibodies that bind to deposited rather than soluble Aβ. The mechanism remains unclear; a logical possibility is that the same mechanisms that clear Aβ deposits can also "punch holes" in the amyloid-laden vessel wall.

For now, immunization-related hemorrhage remains a phenomenon of transgenic mice rather than human disease. Hemorrhagic stroke was not reported in the Elan-Wyeth vaccine studies [2], and the microhemorrhages seen on pathological examination of these brains [3,4] appear related to the underlying CAA rather than the vaccine itself. Human CAA may instead respond to inflammation by vascular dysfunction and reversible white matter changes [5]. Clearly, much remains to be learned about the role of CAA in determining the safety and efficacy of immune-based therapies for AD.

References:
1. Pfeifer M, Boncristiano S, Bondolfi L, Stalder A, Deller T, Staufenbiel M, Mathews PM, Jucker M. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science. 2002;298:1379. Abstract

2. Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, Jouanny P, Dubois B, Eisner L, Flitman S, Michel BF, Boada M, Frank A, Hock C. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 2003;61:46-54. Abstract

3. Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat. Med. 2003;9:448-452. Abstract

4. Ferrer I, Boada Rovira M, Sanchez Guerra ML, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol. 2004;14:11-20. Abstract

5. Eng JA, Frosch MP, Choi K, Rebeck GW, Greenberg SM. Clinical Manifestations of Cerebral Amyloid Angiopathy-Related Inflammation. Ann. Neurol. 2004;55:250-256. Abstract

View all comments by Steven Greenberg

This article points out an important consideration with respect to passive immunotherapy against Aβ and its possible application to Alzheimer patients, namely, the increased risk for vascular hemorrhage with N-terminal-specific antibodies. Equally important, Racke et al. demonstrate that a mid-domain anti-Aβ antibody, which fails to decorate amyloid deposits in brain, does not increase this hemorrhage risk.

Coupled with the observation of Pfeifer et al. (2002) using N-terminal-specific antibodies in old APP23 mice, and our recent observations with C-terminal antibodies in old Tg2576 mice (Wilcock et al., 2004), this may be a common sequel of antibody-mediated amyloid removal. Our work also found 90 percent reduction in parenchymal, but fourfold elevation of vascular amyloid deposits with antibody therapy, implying a redistribution of the material.

A number of important questions need to be addressed. First, given the observation of Hock...  Read more

This article points out an important consideration with respect to passive immunotherapy against Aβ and its possible application to Alzheimer patients, namely, the increased risk for vascular hemorrhage with N-terminal-specific antibodies. Equally important, Racke et al. demonstrate that a mid-domain anti-Aβ antibody, which fails to decorate amyloid deposits in brain, does not increase this hemorrhage risk.

Coupled with the observation of Pfeifer et al. (2002) using N-terminal-specific antibodies in old APP23 mice, and our recent observations with C-terminal antibodies in old Tg2576 mice (Wilcock et al., 2004), this may be a common sequel of antibody-mediated amyloid removal. Our work also found 90 percent reduction in parenchymal, but fourfold elevation of vascular amyloid deposits with antibody therapy, implying a redistribution of the material.

A number of important questions need to be addressed. First, given the observation of Hock et al., (2003), that the only patients in the truncated Elan trial with a cognitive benefit had antibodies that decorated brain amyloid deposits, it may be that antibodies binding soluble Aβ, such as m266, may not benefit cognition. However, if the risk of hemorrhage is increased with antibodies against deposited Aβ, that will cause a serious dilemma regarding any form of Aβ immunotherapy.

A second issue regards the possibility that the increased microhemorrhage may apply to any therapy clearing Aβ. If our observation that vascular amyloid increases generalizes to other agents clearing preexisting plaques (e.g., plaque busters, zinc chelators), then these agents may have the same consequences.

A third question is if the problem is primarily due to the rapid rate of amyloid removal by these agents. The dose of antibody used by Racke et al. was 3-5 times higher than that used by others before (50 mg/kg). If the problem is saturation of efflux mechanisms at the vessels and resultant buildup of vascular amyloid, then a lower dose and more protracted exposure may reduce the amyloid loads without a concomitant rise in vascular deposits and increased risk of hemorrhage. Interestingly, one feature of the Dutch mutation in the Aβ peptide is a dramatic reduction in vascular efflux (Monro et al., 2002). While some of these issues are tractable in mouse models, others can only be addressed by carefully controlled trials in AD patients. Such trials are already in progress. Hopefully, detailed work in mouse models educated by the results of human trials will permit development of safe and effective Aβ immunotherapy.

View all comments by Dave Morgan

The study by Racke and colleagues is the third report showing cerebral bleeding in different AD mouse models following passive immunization with monoclonal antibodies with high affinity for Aβ plaques and congophilic angiopathy (Pfeifer et al., 2002; Wilcock et al., 2004b; Racke et al., 2005). This effect was not observed by other investigators employing another anti-Aβ antibody administered intracerebroventricularly in the Tg2576 model (Chauhan and Siegel, 2003). Also, microhemorrhages have not been reported following active immunizations, although it is unlikely that this has been assessed in most studies. We have recently sampled mouse brain sections from Tg2576 mice immunized with our Aβ derivatives (Sigurdsson et al., 2001; Sigurdsson et al., 2004), and we have yet to detect any microhemorrhages, although many of the animals immunized with K6Aβ1-30 had high anti-Aβ titer (Sigurdsson et al., unpublished observation).

Racke and colleagues did not assess amyloid burden or behavior (Racke et al., 2005), whereas Pfeifer’s study resulted in a modest reduction in plaque burden...  Read more

The study by Racke and colleagues is the third report showing cerebral bleeding in different AD mouse models following passive immunization with monoclonal antibodies with high affinity for Aβ plaques and congophilic angiopathy (Pfeifer et al., 2002; Wilcock et al., 2004b; Racke et al., 2005). This effect was not observed by other investigators employing another anti-Aβ antibody administered intracerebroventricularly in the Tg2576 model (Chauhan and Siegel, 2003). Also, microhemorrhages have not been reported following active immunizations, although it is unlikely that this has been assessed in most studies. We have recently sampled mouse brain sections from Tg2576 mice immunized with our Aβ derivatives (Sigurdsson et al., 2001; Sigurdsson et al., 2004), and we have yet to detect any microhemorrhages, although many of the animals immunized with K6Aβ1-30 had high anti-Aβ titer (Sigurdsson et al., unpublished observation).

Racke and colleagues did not assess amyloid burden or behavior (Racke et al., 2005), whereas Pfeifer’s study resulted in a modest reduction in plaque burden (Pfeifer et al., 2002). Also, Wilcock et al. observed that while causing microhemorrhages, their antibody treatment improved cognition, reduced plaque burden, but increased congophilic angiopathy (Wilcock et al., 2004b). That increase may be related to plaque clearance via the vasculature. In the three autopsy studies from the AN-1796 trial, vascular amyloid remained while plaques appeared to have been cleared in certain brain regions (Nicoll et al., 2003; Ferrer et al., 2004; Masliah et al., 2005). Eli Lilly’s antibody, 266, which binds soluble Aβ but does not recognize plaques and did not produce bleeding, has previously been shown to reduce plaque burden (DeMattos et al., 2001) and also to improve cognition acutely without affecting amyloid burden (Dodart et al., 2002). Elan was not able to replicate the effect of 266 on amyloid burden (Seubert et al., 2003), questioning its long-term efficacy.

So which type of antibody do you pick for costly clinical development? The Zurich AN-1792 trial indicated that AD patients who developed antibodies against amyloid plaques showed less cognitive decline than did other patients. However, even if 266 may potentially be less efficacious than antibodies binding to Aβ aggregates, it seems appropriate to start therapy with that antibody if it appears to be safer. Although, if 266 needs to be given more often than other monoclonals to improve cognition, it may be more likely to elicit an anti-idiotypic response with subsequent vasculitis and glomerulonephritis. If that is ineffective, then antibodies with high affinity for Aβ fibrils could be tested.

What about the isotype? IgG2a recognizing amino acids 3 to 7 of Aβ has been shown to promote plaque clearance after intraperitoneal injection, but IgG1 or IgG2b against the same epitope were not effective (Bussiere et al., 2004). However, different anti-Aβ IgG1 has been shown to result in plaque clearance when given intracranially (Wilcock et al., 2003; Lombardo et al., 2003) or intraperitoneally (Pfeifer et al., 2002; Wilcock et al., 2004a). Preferably, all these issues should have been sorted out in animal studies by direct comparison of these different types of antibodies, but clinical trials have already started on at least one of these approaches (AAB-001 by Elan/Wyeth), which is understandable because no effective therapy exists.

In addition to monoclonals, clinical trials of ivIg therapy are ongoing. This preparation contains pooled human immunoglobulins including autoantibodies against Aβ. If those trials will improve cognition in AD patients, future studies will have to determine if that effect was caused by the known antiinflammatory effect of ivIg and/or by the autoantibodies against Aβ.

Alternatively, active immunization with Aβ derivatives may be the way to go. This approach should produce several isotypes of antibodies against different epitopes and perhaps conformations, as well. Our Aβ derivative vaccination approaches have all improved cognition while eliciting different antibody responses and having various effects on amyloid burden (Scholtzova et al., 2002; Sigurdsson et al., 2004), indicating that a modest immune response is sufficient to improve cognition in AD mouse models. Prior active and passive immunization studies have also observed cognitive improvements without obvious correlation with certain Aβ measurements (Janus et al., 2000; Morgan et al., 2000; Dodart et al., 2002; Kotilinek et al., 2002).

It is fair to say at this point that low molecular weight species of Aβ, including oligomers and perhaps monomers, are likely to affect cognition. However, although extensive plaque deposition is a defense mechanism to isolate excess Aβ that cannot be cleared, it is likely that it will eventually affect neuronal connectivity, and plaque-associated glial activation should also have some toxic effects. Any immunotherapy should, therefore, at least slow the progression of plaque deposition.

All these different approaches are still worth pursuing. Because a self-antigen is being targeted, the immunotherapy may have to be individually tailored as a regular drug treatment. For example, an Aβ-derived immunogen may be chosen based on haplotype screening to provide first a T cell-independent IgM response with antibodies recognizing different epitopes and conformations of Aβ, which may prove to be more efficacious than targeting a single entity. If ineffective, a different Aβ derivative could then be administered that would be predicted to elicit a slightly stronger immune response. This process could then be continued until cognition improves.

References:

1. Bussiere T, Bard F, Barbour R, Grajeda H, Guido T, Khan K, Schenk D, Games D, Seubert P, Buttini M. Morphological characterization of Thioflavin-S-positive amyloid plaques in transgenic Alzheimer mice and effect of passive Abeta immunotherapy on their clearance. Am J Pathol. 2004 Sep;165(3):987-95. Abstract

2. Chauhan NB, Siegel GJ. Intracerebroventricular passive immunization with anti-Abeta antibody in Tg2576. J Neurosci Res. 2003 Oct 1;74(1):142-7. Abstract

3. DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8850-5. Epub 2001 Jul 03. Abstract

4. Dodart JC, Bales KR, Gannon KS, Greene SJ, DeMattos RB, Mathis C, DeLong CA, Wu S, Wu X, Holtzman DM, Paul SM. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci. 2002 May;5(5):452-7. Abstract

5. Ferrer I, Boada RM, Sanchez Guerra ML, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol. 2004 Jan;14(1):11-20. Abstract

6. Janus C, Pearson J, McLaurin J, Mathews PM, Jiang Y, Schmidt SD, Chishti MA, Horne P, Heslin D, French J, Mount HT, Nixon RA, Mercken M, Bergeron C, Fraser PE, George-Hyslop P, Westaway D. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):979-82. Abstract

7. Kotilinek LA, Bacskai B, Westerman M, Kawarabayashi T, Younkin L, Hyman BT, Younkin S, Ashe KH. Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J Neurosci. 2002 Aug 1;22(15):6331-5. Abstract

8. Lombardo JA, Stern EA, McLellan ME, Kajdasz ST, Hickey GA, Bacskai BJ, Hyman BT. Amyloid-beta antibody treatment leads to rapid normalization of plaque-induced neuritic alterations. J Neurosci. 2003 Nov 26;23(34):10879-83. Abstract

9. Masliah E, Hansen L, Adame A, Crews L, Bard F, Lee C, Seubert P, Games D, Kirby L, Schenk D. Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease. Neurology. 2005 Jan 11;64(1):129-31. Abstract

10. Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, Connor K, Hatcher J, Hope C, Gordon M, Arendash GW. A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):982-5. Erratum in: Nature 2001 Aug 9;412(6847):660. Abstract

11. Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 2003 Apr;9(4):448-52. Epub 2003 Mar 17. Abstract

12. Pfeifer M, Boncristiano S, Bondolfi L, Stalder A, Deller T, Staufenbiel M, Mathews PM, Jucker M. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science. 2002 Nov 15;298(5597):1379. No abstract available. Abstract

13. Racke MM, Boone LI, Hepburn DL, Parsadainian M, Bryan MT, Ness DK, Piroozi KS, Jordan WH, Brown DD, Hoffman WP, Holtzman DM, Bales KR, Gitter BD, May PC, Paul SM, DeMattos RB. Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta. J Neurosci. 2005 Jan 19;25(3):629-36. Abstract

14. Scholtzova H, Wisniewski T, Ahlawat S, Watanabe M, Quartermain D, Frangione B, and Sigurdsson EM. Safety of potential vaccines for Alzheimer's disease. Society for Neuroscience Abstracts, 227.1.2002.

15. Seubert P, Games D, Khan K, Buttini M, Bard F, Guido T, Grajeda H, Barbour R, Nguyen M, Kling K, Vasquez N, Schenk D, Hagen M, and Eldridge J. Comparative efficacy of different immunotherapeutic approaches in reducing AD-like neuropathology. Society for Neuroscience Abstracts, 133.3, 2003.

16. Sigurdsson EM, Knudsen E, Asuni A, Fitzer-Attas C, Sage D, Quartermain D, Goni F, Frangione B, Wisniewski T. An attenuated immune response is sufficient to enhance cognition in an Alzheimer's disease mouse model immunized with amyloid-beta derivatives. J Neurosci. 2004 Jul 14;24(28):6277-82. Abstract

17. Sigurdsson EM, Scholtzova H, Mehta PD, Frangione B, Wisniewski T. Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice. Am J Pathol. 2001 Aug;159(2):439-47. Abstract

18. Wilcock DM, DiCarlo G, Henderson D, Jackson J, Clarke K, Ugen KE, Gordon MN, Morgan D. Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent of and associated with microglial activation. J Neurosci. 2003 May 1;23(9):3745-51. Abstract

19. Wilcock DM, Rojiani A, Rosenthal A, Levkowitz G, Subbarao S, Alamed J, Wilson D, Wilson N, Freeman MJ, Gordon MN, Morgan D. (2004a) Passive amyloid immunotherapy clears amyloid and transiently activates microglia in a transgenic mouse model of amyloid deposition. J Neurosci. 2004 Jul 7;24(27):6144-51. Abstract

20. Wilcock DM, Rojiani A, Rosenthal A, Subbarao S, Freeman MJ, Gordon MN, Morgan D. (2004b) Passive immunotherapy against Abeta in aged APP-transgenic mice reverses cognitive deficits and depletes parenchymal amyloid deposits in spite of increased vascular amyloid and microhemorrhage. J Neuroinflammation. 2004 Dec 08;1(1):24. Abstract

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Are conformation-dependent antibodies the key to a safe and effective AD immunotherapy?
Although immunotherapy for AD has become very attractive and its effectiveness is being applauded, there are lingering safety concerns as scientists continue to work to develop an effective and safe therapy. The recent paper by Racke et al. [1] confirms and extends the results from Mathias Jucker’s group [2] that indicate that passive immunization results in an increase in cerebral hemorrhage in addition to reducing amyloid deposition. Some scientists criticized these results because of the lack of control antibodies, and it was proposed that the cerebral hemorrhage observed in response to passive immunization was related to the particular mouse model used (APP23) rather than the actual passive immunization.

To date, three different groups using three different transgenic mouse models and different antibodies showed that passive immunization itself caused increased cerebral hemorrhage. Interestingly, all of the antibodies associated with increased cerebral hemorrhage bind...  Read more

Are conformation-dependent antibodies the key to a safe and effective AD immunotherapy?
Although immunotherapy for AD has become very attractive and its effectiveness is being applauded, there are lingering safety concerns as scientists continue to work to develop an effective and safe therapy. The recent paper by Racke et al. [1] confirms and extends the results from Mathias Jucker’s group [2] that indicate that passive immunization results in an increase in cerebral hemorrhage in addition to reducing amyloid deposition. Some scientists criticized these results because of the lack of control antibodies, and it was proposed that the cerebral hemorrhage observed in response to passive immunization was related to the particular mouse model used (APP23) rather than the actual passive immunization.

To date, three different groups using three different transgenic mouse models and different antibodies showed that passive immunization itself caused increased cerebral hemorrhage. Interestingly, all of the antibodies associated with increased cerebral hemorrhage bind plaques at either the N-terminus [1,2] or C-terminus [2], while antibodies against the middle region of the Aβ sequence, which do not bind plaques, do not increase hemorrhage. The mid-region antibodies can be considered conformation-dependent in the sense that their epitope is buried within the insoluble fibril structure and is exposed in the soluble monomer. Importantly, in fibrils, both the N-terminus (1-13) and C-terminus (39-42) are exposed at the surface of the fibril, while the middle region of the sequence is buried [3,4]. Therefore, m266 and 4G8 (which bind at approximately the same region, residues 13-28), which appear to be conformation-specific (their epitopes are buried within fibrils), may be the solution, since they would not bind amyloid fibril deposits.

The results of Racke et al. also indicate that in order for an active vaccine to be both safe and effective, it would have to lead to the production of antibodies that do not bind to insoluble amyloid deposits. Conformation-dependent antibodies may provide a solution to this problem [5,6]. Vaccination with a molecular mimic of amyloid oligomers gives rise to conformation-dependent antibodies that are specific for amyloid oligomers, which represent an intermediate in the fibril formation pathway and may be the primary toxic species of amyloids [7]. This oligomer-specific antibody does not bind extensively to insoluble amyloid deposits in AD brain; rather, it recognizes a much more restricted subset of soluble oligomers [7], suggesting that it would not cause problems with cerebral hemorrhage. In addition, this antibody neutralizes the toxicity of amyloid oligomers, which may represent a therapeutic benefit beyond removing amyloid deposits. Another attractive feature of conformation-dependent antibodies, such as the oligomer-specific antibody, is that they recognize an epitope that is specifically associated with pathogenesis. They do not recognize the normal, native protein structure. This suggests that anti-oligomer antibodies would be less likely to cause autoimmune complications.

Overall, the results of this paper make it appear increasingly likely that antibodies that bind to amyloid deposits (whether passive or active) may be more likely to have problems with cerebral hemorrhage than are other antibodies. The solution may be in conformation-dependent antibodies that specifically target restricted epitopes. It is still too early to judge the therapeutic benefits of conformation-specific antibodies. At this point, we think these antibodies represent an attractive opportunity to develop an effective immunotherapy for AD with a lower risk of hemorrhage or inflammatory complications.

References:
[1] Racke MM, Boone LI, Hepburn DL, Parsadainian M, Bryan MT, Ness DK, Piroozi KS, Jordan WH, Brown DD, Hoffman WP, Holtzman DM, Bales KR, Gitter BD, May PC, Paul SM, DeMattos RB. Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid beta. J Neurosci. 2005 Jan 19;25(3):629-36. Abstract

[2] Pfeifer M, Boncristiano S, Bondolfi L, Stalder A, Deller T, Staufenbiel M, Mathews PM, Jucker M. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science. 2002 Nov 15;298(5597):1379. No abstract available. Abstract

[3] Torok M, Milton S, Kayed R, Wu P, McIntire T, Glabe CG, Langen R. Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling. J Biol Chem. 2002 Oct 25;277(43):40810-5. Epub 2002 Aug 13. Abstract

[4] Kheterpal I, Williams A, Murphy C, Bledsoe B, Wetzel R. Structural features of the Abeta amyloid fibril elucidated by limited proteolysis. Biochemistry. 2001 Oct 2;40(39):11757-67. Abstract

[5] Dumoulin M, Dobson CM. Probing the origins, diagnosis and treatment of amyloid diseases using antibodies. Biochimie. 2004 Sep-Oct;86(9-10):589-600. Abstract

[6] Glabe CG. Conformation-dependent antibodies target diseases of protein misfolding. Trends Biochem Sci. 2004 Oct;29(10):542-7. Review. Abstract

[7] Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE, Glabe CG. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. J Biol Chem. 2004 Nov 5;279(45):46363-6. Epub 2004 Sep 21. Abstract

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