- Email: [email protected]
Recent progress in Alzheimer's disease: animal models lead the way
Vol. 1, No. 2 2004
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Central nervous system
Recent progress in Alzheimer’s disease: animal models lead the way Tong Li Neuropathology Division, Department of Pathology, Johns Hopkins Medical Institutions, 720 Rutland Avenue, Room 558, Baltimore, MD 21205, USA
Alzheimer’s disease (AD) is a worldwide major public health problem. Much recent research has focused on amyloid precursor protein (APP), the secretase enzymes that cleave APP to generate Ab peptides, and the roles of these peptides in neurotoxicity. Specifically, there has been great interest in the pathogenic
Section Editors: Donald L. Price, Philip C. Wong—Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, USA In this review, Tong Li emphasizes the value of genetically engineered mouse models for studies of disease mechanisms and for development of experimental therapeutics.
role of Ab peptide oligomers and influences of apoE4 and other Ab clearance enzymes on Ab amyloidosis. Genetically engineered animal models are a central tool for studies into the disease mechanisms and for testing experimental therapeutics.
Neuropathology and biochemical features
Introduction Clinical disease Alzheimer’s disease (AD), the most common cause of the syndrome of senile dementia, is manifest by memory loss and cognitive impairments in the elderly [1]. Some patients develop psychotic symptoms, such as hallucinations and delusions. Occupational, social, and personal functions are progressively interfered with. Mental functions and activities of daily living are increasingly impaired. In the late stages, these individuals are severely compromised, bedridden and usually die of intercurrent medical illnesses. These clinical signs are the result of selective degeneration of specific populations of neurons in the central nervous system that play a crucial role in memory and cognition. Dysfunction and death of these neurons disrupt synaptic communication in these neural circuits. At present, only symptomatic treatments are available. However, AD is a disorder for which there are no E-mail address: [email protected] 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.
mechanism-based therapies. In an effort to understand the functions of some of the genes thought to play roles in AD, investigators have used transgenic or gene targeted animal models to study a variety of genes.
DOI: 10.1016/j.ddmod.2004.09.002
The pathological hallmarks of AD are neurofibrillary tangles (NFT) and neuritic plaques. The principal structural element in NFT is pared helical filaments comprised hyperphosphorylated isoforms of tau, a low molecular weight microtubuleassociated protein. The central component of plaques is Ab, an 4 kDa b-pleated sheet peptide, which is derived from amyloid precursor protein (APP). BACE1 cleaves APP to form C-terminal derivatives; subsequently, the g-secretase complex cleaves at position 40, 42, and 43 to generate Ab peptides. Ab1–42(43) appears to be the pathogenic peptide; these species are more fibrillogenic and toxic than Ab1–40 and provide a substrate for amyloid aggregation and AD neuritic plaques. The insoluble amyloid fibrils were initially regarded as the primary molecular toxic entities. However, there are disagreements over the degree of correlation between neurological deficits and amyloid plaque burden. More recent evidence suggests elevated concentrations of Ab oligomers www.drugdiscoverytoday.com
145
Drug Discovery Today: Disease Models | Central nervous system
and multimers might be more significant. In AD brain, the Ab oligomer levels can be increased up to 70-fold over control brains, suggesting that soluble oligomeric Ab ligands are intrinsic to AD pathology [2]. These putatively toxic peptide oligomers and multimers, situated in proximity to synapses, appear to interfere with synaptic functions, and, eventually lead to neuritic degeneration of terminals.
Genes involved in AD There are five principal risk factors for AD [1]: age; mutations in the presenilin 1 (PS1); presenilin 2 (PS2); APP; and the ApoE4 allele. Several familial AD (FAD) mutations linked production of increased level of total Ab or proportion of Ab42 (the more toxic Ab peptide) with the central role in the pathogenesis of familial disease. PSs are crucial components of the g-secretase complex, and mutations in PS are associated with increase in the Ab42/40 ratio. Nearly 50% of cases of early-onset familial AD are linked to the PS1 gene.
In vivo models of AD The abnormalities caused by above genes and their encoding proteins can be studied by in vitro and in vivo model systems (Table 1). Moreover, the in vivo transgenic mouse models have been the key to study the mechanisms of disease onset and progression, and to identify novel therapeutic targets.
Vol. 1, No. 2 2004
lines of mutant mice [4]. Although these animals have an Ab amyloidosis in the CNS, they do not fully recapitulate the pathological and clinical phenotype of AD. Classical neurofibrillary tangles and hyperphosphorylated tau are not present in the Ab amyloid mutant mice. In attempts to obtain mice with both plaques and tangles, mutant APP mice have been crossed to mice expressing the P301L tau, a mutation linked to familial frontotemporal dementia with parkinsonism (FTDP). Mice with the two mutant genes appear to have a greater number of NFT and an apparent shift in the distribution [5]. Moreover, in P301L tau mutant mice, tau pathology appears to be induced by introducing Ab fibrils into the brains. The triple-transgenic mice (3Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes progressively develop plaques and tangles [6]. Consistent with the amyloid cascade hypothesis, these mice develop extracellular Ab deposits before tangle formation. Importantly, 3Tg-AD mice exhibit deficits in synaptic plasticity, including long-term potentiation (LTP) that occurs before the appearance of extracellular Ab deposition and tangles but is associated with intracellular Ab level. Therefore, these evidences suggest a novel pathophysiologic role for Ab and provide a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be accessed.
In vivo models of ApoE and Ab degradation In vivo models of Ab amyloidosis Genetically engineered animal models have been widely used to study Ab amyloidosis features of AD. In mice, expression of APPswe or APP717 minigenes encoding FAD-linked mutations leads to an Ab amyloidosis in the CNS [3]. Levels of Ab are elevated, and diffuse Ab deposits and neuritic plaques appear in the hippocampus and cortex. The nature and levels of the expressed transgene and the specific mutation influence the severity of the pathology. Mice expressing both mutant PS1 and mutant APP develop accelerated disease. Learning deficits, problems in object recognition memory, and difficulties performing tasks assessing spatial reference and working memory have been identified in some of the
Another risk factor, ApoE4, is a glycoprotein that carries cholesterol and other lipids in the blood. At the single ApoE locus, three alleles are expressed: apoE2, apoE3, and apoE4. The risk for AD is increased by the presence of apoE4. The mechanisms whereby the apoE allele type elevates the risk for late-onset disease are not known, but might reflect differences in the abilities of apoE isoforms to bind Ab and possibly influence aggregation, deposition and/or clearance. ApoE can act as an Ab chaperone and bind to Ab, influencing the aggregates and deposits of Ab without affecting Ab synthesis. Clusterin (apolipoprotein J), similarly acts as an Ab chaperone. Previous data show that APP transgenic mice lacking either apoE or clusterin have much less amyloid burden.
Table 1. Comparison summary table In vitro models
In vivo models
Pros
Less complex system Biochemical assay for inhibitor and regulator
Animal can develop AD phenotype Be able to perform behavior studies or other complicated tests on animal models
Cons
Cultured cells cannot be used to study physiology of brain
Animal models cannot fully recapitulate human pathological phenotype Overexpression of some genes might cause unexpected effects
Best use of model
Drug screening Identification of new therapeutic targets
Identification of pathological phenotype of AD related mutants Identification of new therapeutic target
How to get access to the model
Literature
(from) Literature, (or) from company
References
[10,12]
[1,3–7,9–11,13,14,17]
146
www.drugdiscoverytoday.com
Vol. 1, No. 2 2004
Surprisingly, apoE and clusterin double knockout mice that expressed human APP with a FAD mutation (V717F) showed an increase in soluble Ab species in both the brain and CSF before Ab deposition, and an early onset and marked increase in Ab deposition [7]. These data suggest that there is a correlation between apoE and clusterin, and together, they play an important role in influencing the process of Ab deposition and in regulating the elimination of soluble Ab in the brain extracellular space. Compared with other ApoE allele, ApoE4 allele might compromise the regulation of Ab metabolism, which leads to elevated risk of late-onset disease. In addition to the study of regulation of Ab production, Ab degradation and clearance are also key factors for maintaining extracellular Ab level. Several proteases, including insulin-degrading enzyme (IDE), neprilysin (NEP), and endothelin-converting enzyme-1 and -2, have been found to be able to degrade Ab in vitro and in vivo. Several laboratories provide evidence of genetic linkage and/or allelic association of the IDE gene to late-onset AD in various populations [8]. Transgenic mice that overexpress either IDE or NEP in neurons reduce Ab levels, alleviate amyloid plaque deposition and associated pathology [9], demonstrating that chronic up-regulation of Ab-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo. However, there is no report about the side effects of the up-regulation of these enzymes. The behavioral studies of these mice models will be very helpful to address this issue.
Models of potential therapeutics for AD Although the above mutant transgenic mice do not recapitulate the full phenotype of AD, they represent excellent models of Ab amyloidosis and are highly suitable for identification of therapeutic targets and for testing new treatments. Ab level can be controlled by regulating the Ab generation and the Ab clearance. Both b- and g-secretase activities represent therapeutic targets for the development of novel protease inhibitors for AD.
In vitro models of therapeutics targets Ab is generated by sequential cleavage of b- and g-secretase. In vitro assays at the cellular level have shown that both secretases can serve as therapeutic targets. BACE1, a type 1 transmembrane aspartyl protease has been identified to be the bsecretase. In vitro cultured BACE1-deficient neuronal cells could not secrete Ab peptide. PSs, Nicastrin, APH1, and PEN2 have been identified as components of the g-secretase complex [12]. Partial decreases in the level of PS1, NCT or other g-secretase components significantly reduce the secretion of Ab, suggesting that inhibiting g-secretase activity can efficiently reduce Ab secretion. However, the g-secretase complex carries out the intramembranous cleavage of APP and of many other important molecules including Notch, a
Drug Discovery Today: Disease Models | Central nervous system
receptor protein involved in crucial cell-fate decisions during development. Only in vivo studies can evaluate the therapeutic value of these secretases.
In vivo models of therapeutics targets In vivo models of the secretases as therapeutics targets
BACE1 is relatively abundant in the brain. BACE1 null mice are viable and healthy, have no obvious phenotype or pathology, and can mate successfully [10]. Significantly, BACE1deficient neurons fail to secrete Ab even when co-expressing the APPswe and mutant PS1, and mutant APP/PS gene mice lacking BACE1 do not develop Ab plaques in the brain. In a mouse model that expresses human APP-695 with the Swedish familial mutation (Tg2576), BACE1 gene deletion reduces Ab levels and prevents impairment of learning and memory and of hippocampal cholinergic dysfunction [11]. The behavioral and electrophysiological rescue of deficits in BACE1 / Tg2576+ mice correlate with a dramatic reduction of cerebral Ab40 and Ab42 levels. The results indicate that BACE1 is an excellent therapeutic target for the development of an anti-amyloidogenic therapy. The g-secretase complex carries out the intramembranous cleavage of Notch1 and other important molecules. Without this final cleavage, the Notch1 intracellular domain (NICD) is not released from the plasma membrane and does not reach the nucleus to initiate transcriptional processes essential for cell fate decisions. PS / or Nct / mice do not survive beyond the embryonic stage and show features resembling Notch1 / phenotype. To study the role of PS1 in vivo in adult mice, two groups generated conditional PS1-targeted mice lacking PS1 expression in the forebrain after embryonic development [13,14]. As expected, the absence of PS1 resulted in decreased generation of Ab. These findings suggest that g-secretase inhibitors might be useful as therapeutic agents for Ab amyloidosis. Because of the role of the g-secretase complex in Notch processing, it might be valuable to try to design therapeutics that inhibit selectively the g-secretase activity with regard to APP without allowing the activity involved in Notch1 processing. This approach is being pursued because several populations of cells, hematopoetic stem cells in particular, use Notch1 signaling for cell-fate decisions even in adults. In vivo models of other drugs
Another strategy is the use of nonsteroidal anti-inflammatory drugs (NSAIDS) as a therapeutic approach. Retrospective studies had suggested that the prevalence of AD was reduced in individuals who had used NSAIDS [15]. Ibuprofen suppresses plaque pathology and inflammation in a transgenic model of Ab amyloidosis. Initially, this outcome was thought to be attributable to the impact of NSAIDS on CNS inflammation. However, more recent results suggest that ibuprofen [16] might lower Ab42 independent of inhibition of cyclooxwww.drugdiscoverytoday.com
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Drug Discovery Today: Disease Models | Central nervous system
ygenase activity. Several, but not all, NSAIDS decrease the levels of Ab42 and increase the levels of Ab1–38, a less toxic isoform. These studies suggest that NSAIDS can impact on the pathology of the brain by reducing levels of Ab42 independent of COX activity and lead to the suggestion that other NSAIDS varieties could do this with a greater degree of specificity. Another approach has been to target copper and zinc, which are enriched in Ab deposits in the brains of individuals with AD, and have been suggested to play a role in aggregation of Ab and local H2O2 production. Thus, their removal might attenuate the deposition of Ab and its consequences. In one study [17], investigators used clioquinol, a brain-penetrating antibiotic that binds zinc and copper, to reduce the levels of these metals in the brain. Therapy, nine weeks in duration, was associated with a modest increase in soluble Ab and a reduction in Ab deposition in the brain.
In silico models of AD Currently, in silico models are not available for AD. AD is an extremely complicated neuronal degeneration disease, our knowledge of which is still premature. The causes of the disease are not fully understood and the disease onset is not clearly defined. The disease progression, which we know little about, can last for decades. Therefore, generating an in silico model is currently practically impossible. However, new techniques will provide more information about AD. The computational approach will be a valuable tool for AD research in the future.
Vol. 1, No. 2 2004
AD [20]. Specifically, some areas of cortex with very few plaques, contained a relatively high density of tangles in neuropil and vascular amyloid deposits, but there was paucity of plaque-associated dystrophic neurites and astrocytic clusters. Ab immunoreactivity was associated with microglia. A Tcell predominant meninogpencephalilits was identified. Investigators have continued to pursue the passive immunization approach and attempts to make antigens that do not stimulate T-cell mediated immunologic attacks.
Conclusions Investigations of genetically engineered (transgenic or gene targeted) mice have reproduced some of the features of AD, have provided important new information about the disease mechanisms, have disclosed participants in pathogenic pathways, and allowed identification of new therapeutic targets. Moreover, the models are being used to test novel treatments. These lines of research have made extremely good progress over the past few years, and we anticipate that further discoveries will lead to the design of more promising therapies that can be tested in models of this terrible disease of the elderly.
Acknowledgements The author wishes to thank Donald Price and Philip Wong for their helpful comments. This work is supported by National Institution of Neurological Disorder and Stroke (NS45150), the CART (Coins for Alzheimer’s Research Trust) Fund, The Alder Foundation.
From mouse to human? A variety of other treatments have been tested in mouse models. However, to illustrate the challenges of extrapolating outcomes in mice to trials with humans, it is useful to briefly discuss recent problems with Ab immunotherapy. In both prevention and treatment trials, both Ab immunization (with Freund’s adjuvant) and passive transfer of Ab antibodies reduce levels of Ab and plaque burden in mutant APP transgenic mice. Efficacy seems to be related to antibody titer. The mechanisms of enhanced clearance are not certain, but two not mutually exclusive hypotheses have been suggested: first, a small amount of Ab antibody reaches the brain, binds to Ab peptides, promotes the disassembly of fibrils, and via the Fc antibody domain, attracts activated microglia that remove Ab [18]; and second, serum antibodies serve as a sink to draw the amyloid peptides from the brain into the circulation, thus changing the equilibrium of Ab in different compartments and promoting removal from the brain [19]. Although phase 1 trials with Ab peptide and adjuvant were not associated with any adverse events, phase 2 trials of this strategy have been suspended because of severe adverse reactions (encephalomyelitis) in a subset of patients. One of these patients died and autopsy showed several unusual features in comparison with unimmunized cases of 148
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References 1 Price, D.L. and Sisodia, S.S. (1998) Mutant genes in familial Alzheimer’s disease and transgenic models. Annu. Rev. Neurosci. 21, 479–505 2 Gong, Y. et al. (2003) Alzheimer’s disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss 538. Proc. Natl. Acad. Sci. USA 100, 10417–10422 3 Wong, P.C. et al. (2002) Genetically engineered mouse models of neurodegenerative diseases. Nat. Neurosci. 5, 633–639 4 Chen, G. et al. (2000) A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer’s disease. Nature 408, 975–979 5 Lewis, J. et al. (2001) Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293, 1487–1491 6 Oddo, S. et al. (2003) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39, 409–421 7 DeMattos, R.B. et al. (2004) ApoE and clusterin cooperatively suppress Abeta levels and deposition. Evidence that ApoE regulates extracellular Abeta metabolism in vivo. Neuron 41, 193–202 8 Ertekin-Taner, N. et al. (2004) Genetic variants in a haplotype block spanning IDE are significantly associated with plasma Abeta42 levels and risk for Alzheimer disease. Hum. Mutat. 23, 334–342 9 Leissring, M.A. et al. (2003) Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40, 1087–1097 10 Cai, H. et al. (2001) BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat. Neurosci. 4, 233–234 11 Ohno, M. et al. (2004) BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer’s disease. Neuron 41, 27–33
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12 Francis, R. et al. (2002) aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev. Cell 3, 85–97 13 Feng, R. et al. (2001) Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32, 911–926 14 Yu, H. et al. (2001) APP processing and synaptic plasticity in presenilin-1 conditional knockout mice. Neuron 31, 713–726 15 Lim, G.P. et al. (2000) Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer’s disease. J. Neurosci. 20, 5709– 5714 16 Weggen, S. et al. (2001) A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414, 212–216
Drug Discovery Today: Disease Models | Central nervous system
17 Cherny, R.A. et al. (2001) Treatment with a copper–zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron 30, 665–676 18 Schenk, D. et al. (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400, 173–177 19 DeMattos, R.B. et al. (2001) 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. USA 98, 8850– 8855 20 Nicoll, J.A. et al. (2003) Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat. Med. 9, 448–452
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149
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Central nervous system
Recent progress in Alzheimer’s disease: animal models lead the way Tong Li Neuropathology Division, Department of Pathology, Johns Hopkins Medical Institutions, 720 Rutland Avenue, Room 558, Baltimore, MD 21205, USA
Alzheimer’s disease (AD) is a worldwide major public health problem. Much recent research has focused on amyloid precursor protein (APP), the secretase enzymes that cleave APP to generate Ab peptides, and the roles of these peptides in neurotoxicity. Specifically, there has been great interest in the pathogenic
Section Editors: Donald L. Price, Philip C. Wong—Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, USA In this review, Tong Li emphasizes the value of genetically engineered mouse models for studies of disease mechanisms and for development of experimental therapeutics.
role of Ab peptide oligomers and influences of apoE4 and other Ab clearance enzymes on Ab amyloidosis. Genetically engineered animal models are a central tool for studies into the disease mechanisms and for testing experimental therapeutics.
Neuropathology and biochemical features
Introduction Clinical disease Alzheimer’s disease (AD), the most common cause of the syndrome of senile dementia, is manifest by memory loss and cognitive impairments in the elderly [1]. Some patients develop psychotic symptoms, such as hallucinations and delusions. Occupational, social, and personal functions are progressively interfered with. Mental functions and activities of daily living are increasingly impaired. In the late stages, these individuals are severely compromised, bedridden and usually die of intercurrent medical illnesses. These clinical signs are the result of selective degeneration of specific populations of neurons in the central nervous system that play a crucial role in memory and cognition. Dysfunction and death of these neurons disrupt synaptic communication in these neural circuits. At present, only symptomatic treatments are available. However, AD is a disorder for which there are no E-mail address: [email protected] 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.
mechanism-based therapies. In an effort to understand the functions of some of the genes thought to play roles in AD, investigators have used transgenic or gene targeted animal models to study a variety of genes.
DOI: 10.1016/j.ddmod.2004.09.002
The pathological hallmarks of AD are neurofibrillary tangles (NFT) and neuritic plaques. The principal structural element in NFT is pared helical filaments comprised hyperphosphorylated isoforms of tau, a low molecular weight microtubuleassociated protein. The central component of plaques is Ab, an 4 kDa b-pleated sheet peptide, which is derived from amyloid precursor protein (APP). BACE1 cleaves APP to form C-terminal derivatives; subsequently, the g-secretase complex cleaves at position 40, 42, and 43 to generate Ab peptides. Ab1–42(43) appears to be the pathogenic peptide; these species are more fibrillogenic and toxic than Ab1–40 and provide a substrate for amyloid aggregation and AD neuritic plaques. The insoluble amyloid fibrils were initially regarded as the primary molecular toxic entities. However, there are disagreements over the degree of correlation between neurological deficits and amyloid plaque burden. More recent evidence suggests elevated concentrations of Ab oligomers www.drugdiscoverytoday.com
145
Drug Discovery Today: Disease Models | Central nervous system
and multimers might be more significant. In AD brain, the Ab oligomer levels can be increased up to 70-fold over control brains, suggesting that soluble oligomeric Ab ligands are intrinsic to AD pathology [2]. These putatively toxic peptide oligomers and multimers, situated in proximity to synapses, appear to interfere with synaptic functions, and, eventually lead to neuritic degeneration of terminals.
Genes involved in AD There are five principal risk factors for AD [1]: age; mutations in the presenilin 1 (PS1); presenilin 2 (PS2); APP; and the ApoE4 allele. Several familial AD (FAD) mutations linked production of increased level of total Ab or proportion of Ab42 (the more toxic Ab peptide) with the central role in the pathogenesis of familial disease. PSs are crucial components of the g-secretase complex, and mutations in PS are associated with increase in the Ab42/40 ratio. Nearly 50% of cases of early-onset familial AD are linked to the PS1 gene.
In vivo models of AD The abnormalities caused by above genes and their encoding proteins can be studied by in vitro and in vivo model systems (Table 1). Moreover, the in vivo transgenic mouse models have been the key to study the mechanisms of disease onset and progression, and to identify novel therapeutic targets.
Vol. 1, No. 2 2004
lines of mutant mice [4]. Although these animals have an Ab amyloidosis in the CNS, they do not fully recapitulate the pathological and clinical phenotype of AD. Classical neurofibrillary tangles and hyperphosphorylated tau are not present in the Ab amyloid mutant mice. In attempts to obtain mice with both plaques and tangles, mutant APP mice have been crossed to mice expressing the P301L tau, a mutation linked to familial frontotemporal dementia with parkinsonism (FTDP). Mice with the two mutant genes appear to have a greater number of NFT and an apparent shift in the distribution [5]. Moreover, in P301L tau mutant mice, tau pathology appears to be induced by introducing Ab fibrils into the brains. The triple-transgenic mice (3Tg-AD) harboring PS1M146V, APPSwe, and tauP301L transgenes progressively develop plaques and tangles [6]. Consistent with the amyloid cascade hypothesis, these mice develop extracellular Ab deposits before tangle formation. Importantly, 3Tg-AD mice exhibit deficits in synaptic plasticity, including long-term potentiation (LTP) that occurs before the appearance of extracellular Ab deposition and tangles but is associated with intracellular Ab level. Therefore, these evidences suggest a novel pathophysiologic role for Ab and provide a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be accessed.
In vivo models of ApoE and Ab degradation In vivo models of Ab amyloidosis Genetically engineered animal models have been widely used to study Ab amyloidosis features of AD. In mice, expression of APPswe or APP717 minigenes encoding FAD-linked mutations leads to an Ab amyloidosis in the CNS [3]. Levels of Ab are elevated, and diffuse Ab deposits and neuritic plaques appear in the hippocampus and cortex. The nature and levels of the expressed transgene and the specific mutation influence the severity of the pathology. Mice expressing both mutant PS1 and mutant APP develop accelerated disease. Learning deficits, problems in object recognition memory, and difficulties performing tasks assessing spatial reference and working memory have been identified in some of the
Another risk factor, ApoE4, is a glycoprotein that carries cholesterol and other lipids in the blood. At the single ApoE locus, three alleles are expressed: apoE2, apoE3, and apoE4. The risk for AD is increased by the presence of apoE4. The mechanisms whereby the apoE allele type elevates the risk for late-onset disease are not known, but might reflect differences in the abilities of apoE isoforms to bind Ab and possibly influence aggregation, deposition and/or clearance. ApoE can act as an Ab chaperone and bind to Ab, influencing the aggregates and deposits of Ab without affecting Ab synthesis. Clusterin (apolipoprotein J), similarly acts as an Ab chaperone. Previous data show that APP transgenic mice lacking either apoE or clusterin have much less amyloid burden.
Table 1. Comparison summary table In vitro models
In vivo models
Pros
Less complex system Biochemical assay for inhibitor and regulator
Animal can develop AD phenotype Be able to perform behavior studies or other complicated tests on animal models
Cons
Cultured cells cannot be used to study physiology of brain
Animal models cannot fully recapitulate human pathological phenotype Overexpression of some genes might cause unexpected effects
Best use of model
Drug screening Identification of new therapeutic targets
Identification of pathological phenotype of AD related mutants Identification of new therapeutic target
How to get access to the model
Literature
(from) Literature, (or) from company
References
[10,12]
[1,3–7,9–11,13,14,17]
146
www.drugdiscoverytoday.com
Vol. 1, No. 2 2004
Surprisingly, apoE and clusterin double knockout mice that expressed human APP with a FAD mutation (V717F) showed an increase in soluble Ab species in both the brain and CSF before Ab deposition, and an early onset and marked increase in Ab deposition [7]. These data suggest that there is a correlation between apoE and clusterin, and together, they play an important role in influencing the process of Ab deposition and in regulating the elimination of soluble Ab in the brain extracellular space. Compared with other ApoE allele, ApoE4 allele might compromise the regulation of Ab metabolism, which leads to elevated risk of late-onset disease. In addition to the study of regulation of Ab production, Ab degradation and clearance are also key factors for maintaining extracellular Ab level. Several proteases, including insulin-degrading enzyme (IDE), neprilysin (NEP), and endothelin-converting enzyme-1 and -2, have been found to be able to degrade Ab in vitro and in vivo. Several laboratories provide evidence of genetic linkage and/or allelic association of the IDE gene to late-onset AD in various populations [8]. Transgenic mice that overexpress either IDE or NEP in neurons reduce Ab levels, alleviate amyloid plaque deposition and associated pathology [9], demonstrating that chronic up-regulation of Ab-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo. However, there is no report about the side effects of the up-regulation of these enzymes. The behavioral studies of these mice models will be very helpful to address this issue.
Models of potential therapeutics for AD Although the above mutant transgenic mice do not recapitulate the full phenotype of AD, they represent excellent models of Ab amyloidosis and are highly suitable for identification of therapeutic targets and for testing new treatments. Ab level can be controlled by regulating the Ab generation and the Ab clearance. Both b- and g-secretase activities represent therapeutic targets for the development of novel protease inhibitors for AD.
In vitro models of therapeutics targets Ab is generated by sequential cleavage of b- and g-secretase. In vitro assays at the cellular level have shown that both secretases can serve as therapeutic targets. BACE1, a type 1 transmembrane aspartyl protease has been identified to be the bsecretase. In vitro cultured BACE1-deficient neuronal cells could not secrete Ab peptide. PSs, Nicastrin, APH1, and PEN2 have been identified as components of the g-secretase complex [12]. Partial decreases in the level of PS1, NCT or other g-secretase components significantly reduce the secretion of Ab, suggesting that inhibiting g-secretase activity can efficiently reduce Ab secretion. However, the g-secretase complex carries out the intramembranous cleavage of APP and of many other important molecules including Notch, a
Drug Discovery Today: Disease Models | Central nervous system
receptor protein involved in crucial cell-fate decisions during development. Only in vivo studies can evaluate the therapeutic value of these secretases.
In vivo models of therapeutics targets In vivo models of the secretases as therapeutics targets
BACE1 is relatively abundant in the brain. BACE1 null mice are viable and healthy, have no obvious phenotype or pathology, and can mate successfully [10]. Significantly, BACE1deficient neurons fail to secrete Ab even when co-expressing the APPswe and mutant PS1, and mutant APP/PS gene mice lacking BACE1 do not develop Ab plaques in the brain. In a mouse model that expresses human APP-695 with the Swedish familial mutation (Tg2576), BACE1 gene deletion reduces Ab levels and prevents impairment of learning and memory and of hippocampal cholinergic dysfunction [11]. The behavioral and electrophysiological rescue of deficits in BACE1 / Tg2576+ mice correlate with a dramatic reduction of cerebral Ab40 and Ab42 levels. The results indicate that BACE1 is an excellent therapeutic target for the development of an anti-amyloidogenic therapy. The g-secretase complex carries out the intramembranous cleavage of Notch1 and other important molecules. Without this final cleavage, the Notch1 intracellular domain (NICD) is not released from the plasma membrane and does not reach the nucleus to initiate transcriptional processes essential for cell fate decisions. PS / or Nct / mice do not survive beyond the embryonic stage and show features resembling Notch1 / phenotype. To study the role of PS1 in vivo in adult mice, two groups generated conditional PS1-targeted mice lacking PS1 expression in the forebrain after embryonic development [13,14]. As expected, the absence of PS1 resulted in decreased generation of Ab. These findings suggest that g-secretase inhibitors might be useful as therapeutic agents for Ab amyloidosis. Because of the role of the g-secretase complex in Notch processing, it might be valuable to try to design therapeutics that inhibit selectively the g-secretase activity with regard to APP without allowing the activity involved in Notch1 processing. This approach is being pursued because several populations of cells, hematopoetic stem cells in particular, use Notch1 signaling for cell-fate decisions even in adults. In vivo models of other drugs
Another strategy is the use of nonsteroidal anti-inflammatory drugs (NSAIDS) as a therapeutic approach. Retrospective studies had suggested that the prevalence of AD was reduced in individuals who had used NSAIDS [15]. Ibuprofen suppresses plaque pathology and inflammation in a transgenic model of Ab amyloidosis. Initially, this outcome was thought to be attributable to the impact of NSAIDS on CNS inflammation. However, more recent results suggest that ibuprofen [16] might lower Ab42 independent of inhibition of cyclooxwww.drugdiscoverytoday.com
147
Drug Discovery Today: Disease Models | Central nervous system
ygenase activity. Several, but not all, NSAIDS decrease the levels of Ab42 and increase the levels of Ab1–38, a less toxic isoform. These studies suggest that NSAIDS can impact on the pathology of the brain by reducing levels of Ab42 independent of COX activity and lead to the suggestion that other NSAIDS varieties could do this with a greater degree of specificity. Another approach has been to target copper and zinc, which are enriched in Ab deposits in the brains of individuals with AD, and have been suggested to play a role in aggregation of Ab and local H2O2 production. Thus, their removal might attenuate the deposition of Ab and its consequences. In one study [17], investigators used clioquinol, a brain-penetrating antibiotic that binds zinc and copper, to reduce the levels of these metals in the brain. Therapy, nine weeks in duration, was associated with a modest increase in soluble Ab and a reduction in Ab deposition in the brain.
In silico models of AD Currently, in silico models are not available for AD. AD is an extremely complicated neuronal degeneration disease, our knowledge of which is still premature. The causes of the disease are not fully understood and the disease onset is not clearly defined. The disease progression, which we know little about, can last for decades. Therefore, generating an in silico model is currently practically impossible. However, new techniques will provide more information about AD. The computational approach will be a valuable tool for AD research in the future.
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AD [20]. Specifically, some areas of cortex with very few plaques, contained a relatively high density of tangles in neuropil and vascular amyloid deposits, but there was paucity of plaque-associated dystrophic neurites and astrocytic clusters. Ab immunoreactivity was associated with microglia. A Tcell predominant meninogpencephalilits was identified. Investigators have continued to pursue the passive immunization approach and attempts to make antigens that do not stimulate T-cell mediated immunologic attacks.
Conclusions Investigations of genetically engineered (transgenic or gene targeted) mice have reproduced some of the features of AD, have provided important new information about the disease mechanisms, have disclosed participants in pathogenic pathways, and allowed identification of new therapeutic targets. Moreover, the models are being used to test novel treatments. These lines of research have made extremely good progress over the past few years, and we anticipate that further discoveries will lead to the design of more promising therapies that can be tested in models of this terrible disease of the elderly.
Acknowledgements The author wishes to thank Donald Price and Philip Wong for their helpful comments. This work is supported by National Institution of Neurological Disorder and Stroke (NS45150), the CART (Coins for Alzheimer’s Research Trust) Fund, The Alder Foundation.
From mouse to human? A variety of other treatments have been tested in mouse models. However, to illustrate the challenges of extrapolating outcomes in mice to trials with humans, it is useful to briefly discuss recent problems with Ab immunotherapy. In both prevention and treatment trials, both Ab immunization (with Freund’s adjuvant) and passive transfer of Ab antibodies reduce levels of Ab and plaque burden in mutant APP transgenic mice. Efficacy seems to be related to antibody titer. The mechanisms of enhanced clearance are not certain, but two not mutually exclusive hypotheses have been suggested: first, a small amount of Ab antibody reaches the brain, binds to Ab peptides, promotes the disassembly of fibrils, and via the Fc antibody domain, attracts activated microglia that remove Ab [18]; and second, serum antibodies serve as a sink to draw the amyloid peptides from the brain into the circulation, thus changing the equilibrium of Ab in different compartments and promoting removal from the brain [19]. Although phase 1 trials with Ab peptide and adjuvant were not associated with any adverse events, phase 2 trials of this strategy have been suspended because of severe adverse reactions (encephalomyelitis) in a subset of patients. One of these patients died and autopsy showed several unusual features in comparison with unimmunized cases of 148
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12 Francis, R. et al. (2002) aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev. Cell 3, 85–97 13 Feng, R. et al. (2001) Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32, 911–926 14 Yu, H. et al. (2001) APP processing and synaptic plasticity in presenilin-1 conditional knockout mice. Neuron 31, 713–726 15 Lim, G.P. et al. (2000) Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer’s disease. J. Neurosci. 20, 5709– 5714 16 Weggen, S. et al. (2001) A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414, 212–216
Drug Discovery Today: Disease Models | Central nervous system
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