Current Alzheimer Research Volume 2, Number 2, 2005
Contents
ISOA Fifth Annual
Investigators Meeting “Recent Advances in Drug Discovery and Development for
Alzheimer’s Disease”
Sponsored by Elan Pharmaceuticals Inc,
The Graphic Expression, Glaxo Smith Kline Pharmaceuticals, Neurochem Inc,
Pfizer Inc.
Guest Editors: Howard M.
Fillit and Lorenzo M. Refolo
Advancing Drug Discovery for Alzheimer’s
Disease Pp.105-107
H. Fillit and L. Refolo
Development of Novel Amyloid Imaging Agents
Based Upon Thioflavin S
Pp.109-114
Jingjun Wei, Chunying
Wu, David Lankin, Anil Gulrati, Tibor Valyi-Nagy, Elizabeth Cochran, Victor W.
Pike, Alan Kozikowski, Yanming Wang
Functional Connectivity in Elderly Controls and
AD Patients Using Resting State fMRI: A Pilot Study Pp.115-116
Serge Rombouts and
Philip Scheltens
Towards Practical Cognitive Assessment for
Detection of Early Dementia: A 30-Minute Computerized Battery Discriminates as
Well as Longer Testing
Pp.117-124
Glen M. Doniger, David
M. Zucker, Avraham Schweiger, Tzvi Dwolatzky, Howard Chertkow, Howard Crystal
and Ely S. Simon
C-1073 (Mifepristone) in the Adjunctive
Treatment of Alzheimer’s Disease Pp.125-129
Charles DeBattista and
Joseph Belanoff
A Potential Role of the Curry Spice Curcumin
in Alzheimer’s Disease
Pp.131-136
John M. Ringman, Sally
A. Frautschy, Gregory M. Cole, Donna L. Masterman and Jeffrey L. Cummings
Synaptic Fatigue is More Pronounced in the
APP/PS1 Transgenic Mouse Model of Alzheimer’s Disease Pp.137-140
Hong Zhang, Bing Gong,
Shumin Liu, Mauro Fa’, Ipe Ninan, Agnieszka Staniszewski and Ottavio Arancio
Functional Central Nicotinic Acetylcholine
Receptor Antagonism by Systemic Administration of Tinuvin 770 (BTMPS) Pp.141-147
John H. Graham, Roger
L. Papke and Jerry J. Buccafusco
The Expression of Activity-Dependent
Neuroprotective Protein (ADNP) is Regulated by Brain Damage and Treatment of
Mice with the ADNP Derived Peptide, NAP, Reduces the Severity of Traumatic Head
Injury Pp.149-153
Illana Gozes, Roy Zaltzman, Janet Hauser, Douglas E. Brenneman, Esther Shohami, and Joanna M. Hill
The Molecular Basis of Memantine Action in
Alzheimer’s Disease and Other Neurologic Disorders: Low-affinity, Uncompetitive
Antagonism Pp.155-165
Stuart A. Lipton
Neurotrophin Receptor-Based Strategies for
Alzheimer’s Disease
Pp.167-169
Frank M. Longo, and Stephen M. Massa
Nitric Oxide Mimetic Molecules as Therapeutic
Agents in Alzheimer’s Disease
Pp.171-182
Gregory R.J. Thatcher, Brian M. Bennett and James N. Reynolds
Small Molecule TGF-beta Mimetics as Potential
Neuroprotective Factors
Pp.183-186
Hui Zhang, Kun Zou, Ina Tesseur, Tony Wyss-Coray
Role of Fractalkine (CX3CL1) in Regulating
Neuron-Microglia Interactions: Development of Viral-Based CX3CR1 Antagonists Pp.187-189
Wolfgang J. Streit, Christopher N. Davis and Jeffrey K. Harrison
Extracellular Superoxide Dismutase (EC-SOD)
Quenches Free Radicals and Attenuates Age-Related Cognitive Decline:
Opportunities for Novel Drug Development in Aging Pp.191-196
Edward D. Levin
Validation of the Neuroinflammation Cycle as
a Drug Discovery Target Using Integrative Chemical Biology and Lead Compound
Development with an Alzheimer’s Disease-Related Mouse Model Pp.197-205
Wenhui Hu, Hantamalala Ralay Ranaivo, Jeffrey M. Craft, Linda J. Van Eldik and D. Martin Watterson
Analysis of Genetic Polymorphisms in Acetylcholinesterase
as Reflected in Different Populations Pp.207-218
Yehudit Hasin, Nili Avidan, Dani Bercovich, Amos D. Korczyn, Israel Silman, Jacques S. Beckmann, and Joel L. Sussman
Screening for Inhibitors of Tau
Polymerization Pp.219-226
Marcus Pickhardt, Martin von Bergen, Zuzana Gazova, Antje Hascher, Jacek Bierna, Eva-Maria Mandelkow and Eckhard Mandelkow
Cytoskeletal Integrity as a Drug Target Pp.227-229
M.L. Michaelis, K. .I. Seyb and S. Ansar
Development of a High Throughput Drug
Screening Assay for the Detection of Changes in Tau Levels – Proof of Concept
with HSP90 inhibitors
Pp.231-238
Chad A. Dickey, Jason Eriksen, Adeela Kamal, Francis Burrows, Srinivas Kasibhatla, Christopher B. Eckman, Mike Hutton and Leonard Petrucelli
Lentiviral Vector-Based Models of Amyloid
Pathology: From Cells to Animals Pp.239-247
Laura Shaughnessy, Mary Beth Thomas, John Wakefield, Beth Chamblin, Ayyappan Nair, Frank Koentgen and Ram Ramabhadran
Pilot Study of the Reducing Effect on
Amyloidosis In Vivo by Three FDA Pre-Approved Drugs Via the Alzheimer’s APP
5’Untranslated
Region Pp.249-254
Stephanie Tucker, Michelle Ahl, Ashley Bush, David Westaway, Xudong Huang and Jack T. Rogers
Drug Library Screen to Identify Compounds
that Decrease Secreted Ab
from a Human Cell Line
Pp.255-259
Enakshi Chakrabarti and Jonathan D. Smith
Recent Developments on the Studies of Human
Memapsin 2 (b-Secretase) Pp.261-264
Jordan Tang, Xiangyang He , Xianping Huang and Lin Hong
An Ab
Sequestration Approach Using Non-Antibody Ab Binding Agents Pp.265-268
Yasuji Matsuoka, Li Shao, Manik Debnath, John LaFrancois, Amanda Becker, Audrey Gray, Paul Aisen, Chester Mathis, William Klunk and Karen Duff
LRP-Mediated Clearance of Ab is Inhibited by KPI-Containing Isoforms of
APP Pp.269-273
Robert D. Moir and Rudolph E. Tanzi
Abstracts
[Back
to top] Advancing Drug Discovery for Alzheimer’s Disease
H. Fillit and L.
Refolo
The abstract for this article is not available.
[Back
to top] Development
of Novel Amyloid Imaging Agents Based Upon Thioflavin S
Jingjun Wei, Chunying Wu,
David Lankin, Anil Gulrati, Tibor Valyi-Nagy, Elizabeth Cochran, Victor W.
Pike, Alan Kozikowski, Yanming Wang
To date, small-molecule amyloid-imaging agents for in vivo detection and quantitation of amyloid deposits in Alzheimer’s disease (AD) have been developed and successfully applied to human subjects. Preliminary studies have indicated that these amyloid-imaging agents were accumulated in the AD brains in a pattern that is relatively consistent with AD pathology, at least in the regions of amyloid-rich grey matter. These studies have also proven the concept that amyloid dyes, normally too hydrophilic to enter the brain, can be chemically modified to enhance brain permeability, binding affinity, as well as improve binding specificity for amyloid deposits. Related studies have suggested that structurally different agents can be developed that bind to different sites on amyloid deposits. In fact, in vivo cross-referencing studies based upon different amyloid-imaging agents may permit better characterization of AD pathology. But more importantly, novel amyloid imaging agents are required that will allow direct correlation between the results of animal models and human subjects based upon identical imaging modalities. Thus far, amyloid stains such as Congo red and thioflavin T have been extensively studied. However, another widely used amyloid dye, thioflavin S, has not been previously explored. This is in part due to the fact that thioflavin S exists as a mixture, not a pure chemical entity, albeit that the major component has been characterized. We hypothesized that neutral analogs, based upon the major component, could be developed as novel amyloid imaging agents, that exhibit complementary binding properties and pharmacokinetic profiles compatible with potential human studies.
[Back
to top] Functional Connectivity in Elderly Controls and AD Patients Using
Resting State fMRI: A Pilot Study
Serge Rombouts and
Philip Scheltens
Conventionally, Alzheimer’s disease (AD) and other dementias are diagnosed using clinical assessment, neuropsychology and also structural neuroimaging, showing neuronal degeneration starting in the hippocampal regions. However, there is an increasing need for a new method that is more sensitive to early AD identification than currently possible. A new promising technique that may be used for this is to measure local brain activation using functional magnetic resonance imaging (fMRI), since functional loss predates structural loss of brain tissue.
A new method to apply fMRI is to study connectivity between brain regions during a resting state without application of a task. Recent data suggest that connectivity within memory systems during such a resting state is associated with the level of memory function. Here we explain how we will study healthy elderly controls, patients with a mild cognitive impairment (MCI, considered to be a transitional stage between normal condition and AD), and AD patients using resting state connectivity fMRI.
If resting state connectivity is sensitive to cognitive decline, this will be of great importance for noninvasive dementia research, offering a tool to easily study functional networks in the brain without the requirement of a memory task, and perhaps offering a tool sensitive for early diagnostics.
[Back
to top] Towards
Practical Cognitive Assessment for Detection of Early Dementia: A 30-Minute
Computerized Battery Discriminates as Well as Longer Testing
Glen M. Doniger, David
M. Zucker, Avraham Schweiger, Tzvi Dwolatzky, Howard Chertkow, Howard Crystal
and Ely S. Simon
Early detection of cognitive decline may lead to more effective treatment. Clinical cognitive assessment is essential for early detection, but must be brief with easily interpretable results. The present study defines and evaluates a 30-minute cognitive battery consisting of a subset of tests that comprise a longer computerized battery recently validated in detecting mild cognitive impairment (MCI). Participants were from three tertiary care memory clinics and an assisted living facility (final group: N=161) with consensus diagnoses of cognitively healthy, MCI, or mild dementia. A comprehensive NeuroTrax battery evaluated memory, executive function, visual spatial perception, verbal function, information processing speed, and motor skills. Validity of a single summary measure (‘MCI Score’) designed for dementia detection and built exclusively from tests of memory, executive function, and visual spatial perception was evaluated with receiver operating characteristic (ROC) analysis. Discriminant validity (area under the curve: AUC) was at least as large for the 6-parameter MCI Score as for a 20-parameter score necessitating administration of the entire battery. Further, the MCI Score had a larger AUC with reduced variance relative to its constituent parameters. AUC for distinguishing dementia was 0.886; AUC for distinguishing cognitively healthy was 0.823. Finally, the MCI Score discriminated among all three diagnostic groups (ANOVA; F[2,150]=52.54, p<0.001). Hence a reduced NeuroTrax battery (30 minutes) with MCI Score is a useful clinical tool for summarizing cognitive data relevant to early dementia detection.
[Back
to top] C-1073
(Mifepristone) in the Adjunctive Treatment of Alzheimer’s Disease
Charles DeBattista and
Joseph Belanoff
Alzheimer’s disease is frequently associated with abnormalities in the hypothalamic pituitary adrenal axis. Elevated cortisol levels in Alzheimer’s disease may in turn be associated with a more rapid progression of the illness. In addition, elevated cortisol levels may directly contribute to cognitive deficits in Alzheimer’s disease. Mifepristone is a potent antagonist of the glucocorticoid receptor and blocks the central actions of cortisol. The purpose of this study is to determine the effects of glucocorticoid receptor blockade with mifepristone on cognition in Alzheimer’s disease.
[Back
to top] A
Potential Role of the Curry Spice Curcumin in Alzheimer’s Disease
John M. Ringman, Sally
A. Frautschy, Gregory M. Cole, Donna L. Masterman and Jeffrey L. Cummings
There is substantial in-vitro data indicating that curcumin has antioxidant, anti-inflammatory, and anti-amyloid activity. In addition, studies in animal models of Alzheimer's disease (AD) indicate a direct effect of curcumin in decreasing the amyloid pathology of AD. As the widespread use of curcumin as a food additive and relatively small short-term studies in humans suggest safety, curcumin is a promising agent in the treatment and/or prevention of AD. Nonetheless, important information regarding curcumin bioavailability, safety and tolerability, particularly in an elderly population is lacking. We are therefore performing a study of curcumin in patients with AD to gather this information in addition to data on the effect of curcumin on biomarkers of AD pathology.
[Back
to top] Synaptic
Fatigue is More Pronounced in the APP/PS1 Transgenic Mouse Model of Alzheimer’s
Disease
To search for potential mechanism that might alter synaptic transmission following Ab increase we have examined the presynaptic component of transmitter release. As parameters of synaptic transmission that might underlie presynaptic mechanisms, we have used paired-pulse facilitation (PPF), post-tetanic potentiation (PTP), and synaptic fatigue (SF) at the connection between the hippocampal Schaffer-collateral pathway and CA1 pyramidal neurons in ~5 month old double transgenic mice overexpressing the mutated form of amyloid precursor protein (APPK670N, M671L) and presenilin 1 (PS1M146V). While the presynaptic mechanisms of PPF and PTP were not compromised in the APP/PS1 mice, SF was more pronounced in the double transgenic animals. The percentage of the 40th fEPSP slope over the first during the tetanus was 18 ± 3% in APP/PS1 vs. 26 ± 2% in WT. Thus, it is likely that presynaptic mechanisms underlying SF but not PPF and PTP, may account for synaptic dysfunction in APP/PS1 mice.
[Back
to top] Functional
Central Nicotinic Acetylcholine Receptor Antagonism by Systemic Administration
of Tinuvin 770 (BTMPS)
John H. Graham, Roger
L. Papke and Jerry J. Buccafusco
Tinuvin 770 (BTMPS) is a non-competitive, use-dependent antagonist of nicotinic acetylcholine receptors (nAChRs). The drug is highly lipid soluble and as such it has the potential to act within the brain. Presently the ganglionic blocking drug mecamylamine is used almost exclusively to block central nAChRs upon peripheral administration. These experiments were designed to confirm the nAChR antagonism effectiveness of BTMPS in both peripheral (ganglionic stimulation) and central (locomotor activity and thermal nociceptive sensitivity) nicotinic system in vivo. BTMPS inhibited the expression of the pressor response produced by i.v. injection of the ganglionic stimulant DMPP in anesthetized rats. The inhibition dose-response profile appeared to be biphasic with the maximal inhibition occurring after administration of the 0.48 mg/kg dose of BTMPS. In rats acclimated to the test apparatus, nicotine increased different measures of locomotor activity, particularly at the 0.75 mg/kg dose. BTMPS pretreatment significantly inhibited the nicotine-induced increase in motor behaviors, again with a biphasic dose-response relationship. Lastly, nicotine elicited an antinociceptive response in rats (hot plate test). BTMPS almost completely blocked the antinociceptive responses to 1 and 1.5 mg/kg nicotine. On its own, BTMPS failed to decrease blood pressure and to decrease the nociceptive threshold. The drug also generally failed to alter locomotor activity. The use-dependent aspect of BTMPS-induced inhibition of nAChRs was evident in the drug’s greater effectiveness in the presence of the highest doses of nicotine. Therefore, BTMPS can be considered as an alternative to or as a confirmatory drug for mecamylamine when inhibition of central nicotinic receptors is required.
[Back
to top] The
Expression of Activity-Dependent Neuroprotective Protein (ADNP) is Regulated by
Brain Damage and Treatment of Mice with the ADNP Derived Peptide, NAP, Reduces
the Severity of Traumatic Head Injury
Illana Gozes, Roy Zaltzman, Janet Hauser, Douglas E. Brenneman, Esther Shohami, and Joanna M. Hill
NAP is a short octapeptide sequence (single letter code, NAPVSIPQ) that protects neurons against a wide variety of insults. The NAP sequence was identified by peptide structure/function scanning of activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation. To further evaluate the in vivo efficacy of NAP neuroprotection we used a mouse model of head trauma; a condition that presents a risk factor for the development of Alzheimer's disease in injured patients. In the mouse model, NAP treatment (prophylactic or curative) indicated improvement in longitudinal clinical, biochemical and anatomical outcomes. Furthermore, closed head injury was associated with a delayed increase in the expression of the immune cell surface glycoprotein Mac-1 (CD11B antigen) at the injury site that was decreased in NAP-treated mice. Additional experiments with Mac-1-deficient mice suggested partial protection against death related to severe head injury. NAP protection in Mac-1-deficient mice against adverse clinical outcome was concomitant with the time period when increases in Mac-1 transcripts were observed in the Mac-1 expressing mice (~four weeks after the injury). The expression of ADNP (the NAP parent protein) was also increased at the injured brain site four weeks after the traumatic event, only in Mac-1 expressing mice. Here, using immunocytochemistry, we localized the increase in ADNP to microglia and astrocyte-like cells. The increase in ADNP in injured brains is now suggested to be a part of an endogenous compensatory mechanism and NAP treatment provides an additional protection. Toxicology studies suggest NAP as safe for further clinical development.
[Back
to top] The
Molecular Basis of Memantine Action in Alzheimer’s Disease and Other Neurologic
Disorders: Low-affinity, Uncompetitive Antagonism
Stuart A. Lipton
In western countries, Alzheimer’s disease (AD) is the most common form of dementia. In fact, if left uncurbed, the economic cost of caring for AD patients could consume the entire gross national product of the U.S.A. by the middle of this century. Until recently, the only available drugs for this condition were cholinergic treatments, which symptomatically enhance cognitive state to some degree, but they were not neuroprotective. In fact, many potential neuroprotective drugs tested in clinical trials failed because they were poorly tolerated. However, after our discovery of its clinically-tolerated mechanism of action, one neuroprotective drug, memantine, was recently approved by the European Union and the U.S. Food and Drug Administration (FDA) for the treatment of Alzheimer’s disease. Recent phase 3 clinical trials have shown that memantine is effective in the treatment of both mild and moderate-to-severe Alzheimer’s disease and possibly vascular dementia (multi-infarct dementia). Here we review the molecular mechanism of memantine’s action and also the basis for the drug’s use in these neurological diseases, which are mediated at least in part by excitotoxicity. Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. Excitotoxic neuronal cell death is mediated in part by overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors, which results in excessive Ca2+ influx through the receptor’s associated ion channel. Physiological NMDA receptor activity, however, is also essential for normal neuronal function. This means that potential neuroprotective agents that block virtually all NMDA receptor activity will very likely have unacceptable clinical side effects. For this reason many previous NMDA receptor antagonists have disappointingly failed advanced clinical trials for a number of neurodegenerative disorders. In contrast, studies in our laboratory have shown that the adamantane derivative, memantine, preferentially blocks excessive NMDA receptor activity without disrupting normal activity. Memantine does this through its action as an uncompetitive, low-affinity, open-channel blocker; it enters the receptor-associated ion channel preferentially when it is excessively open, and, most importantly, its off-rate is relatively fast so that it does not substantially accumulate in the channel to interfere with normal synaptic transmission. Clinical use has corroborated the prediction that memantine is thus well tolerated. Besides Alzheimer’s disease, memantine is currently in trials for additional neurological disorders, including other forms of dementia, depression, glaucoma, and severe neuropathic pain. A series of second-generation memantine derivatives are currently in development and may prove to have even greater neuroprotective properties than memantine. These second-generation drugs take advantage of the fact that the NMDA receptor has other modulatory sites in addition to its ion channel that potentially could also be used for safe but effective clinical intervention.
[Back
to top] Neurotrophin
Receptor-Based Strategies for Alzheimer’s Disease
Frank M. Longo, and Stephen M. Massa
The traditional perspective of applying neurotrophins in the context of Alzheimer’s disease is based on the premise that neurotrophins are capable of upregulating cholinergic function and of rendering neurons less vulnerable to certain processes causing degeneration. Factors limiting the therapeutic application of neurotrophin proteins include their poor pharmacological properties and their pleiotropic effects mediated by interaction with Trk, p75NTR and sortilin receptors. Recent studies suggesting and that pro-forms of neurotrophins accumulating in Alzheimer’s and other pathological states cause cell death, that p75NTR modulates amyloid beta- and injury-induced neurodegeneration and that small molecules can be created that bind specifically to individual neurotrophin receptors point to novel strategies by which neurotrophin receptors might be targeted in Alzheimer’s and other neuropathological states.
[Back
to top] Nitric
Oxide Mimetic Molecules as Therapeutic Agents in Alzheimer’s Disease
Gregory R.J. Thatcher, Brian M. Bennett and James N. Reynolds
Nitric oxide is multifunctional messenger molecule in the brain, playing important roles including in learning and memory and in regulating the expression of trophic factors that may be reduced with aging. Small molecules that mimic the biological activity of NO, NO mimetics, will bypass cholinergic receptor activation and are anticipated to provide multiple pathways of treating and circumventing dementia in Alzheimer’s disease. Activation of soluble guanylyl cyclase and cGMP formation in the brain represents one element of effective neuroprotective pathways mediated by NO. Substantial evidence suggests that NO mimetics may display cGMP-dependent and cGMP-independent activity and may operate via multiple biochemical signaling pathways, both to ensure the survival of neurons subjected to stress and also to provide cognition-enabling pathways to circumvent dementia. GT 1061 is an NO mimetic compound currently in clinical trials for Alzheimer’s. A survey of current research indicates that NO mimetics will provide a combined neuroprotective and cognition-enabling approach to anti-neurodegenerative therapy.
[Back
to top] Small
Molecule TGF-beta Mimetics as Potential Neuroprotective Factors
Hui Zhang, Kun Zou, Ina Tesseur, Tony Wyss-Coray
Neurodegenerative and dementing illnesses are becoming an increasing social and economical burden as the number of older people continues to grow in industrialized countries. Current knowledge of the processes leading to these diseases is still limited, and very few effective treatments are available. Because neurodegeneration is associated with an activation of injury and innate immune responses in the brain, drugs that could mimic the beneficial aspects of this response are potential therapeutic candidates. The cytokine transforming growth factor (TGF)-b1 is an organizer of the brain’s response to injury and is known to be neuroprotective. Previous studies from our lab also showed that TGF-b1 can reduce accumulation of b-amyloid peptide (Ab), which appears to be central to Alzheimer’s disease (AD) pathogenesis, and we therefore initiated a search for small molecule chemical compounds that could mimic this effect. We report here the identification of several such TGF-b mimetics detected in an in vitro screen of a library with 5000 chemically diverse compounds. If active in vivo, these mimetics could be developed into candidates for the treatment of neurodegeneration.
[Back
to top] Role
of Fractalkine (CX3CL1) in Regulating Neuron-Microglia Interactions:
Development of Viral-Based CX3CR1 Antagonists
Wolfgang J. Streit, Christopher N. Davis and Jeffrey K. Harrison
Blocking the effects of fractalkine therapeutically may regulate microglia cell activation and provide neuroprotection in the AD brain. A human herpesvirus 8-encoded chemokine, termed vMIP-II is a non-selective chemokine receptor antagonist (binding multiple chemokine receptors, including CX3CR1). By comparing vMIP-II and FKN, we have generated molecules that selectively antagonize CX3CR1 activation. The results from these studies will guide future development of therapeutic agents designed to modulate microglial activation with the goal of preventing or slowing the progression of AD.
[Back
to top] Extracellular
Superoxide Dismutase (EC-SOD) Quenches Free Radicals and Attenuates Age-Related
Cognitive Decline: Opportunities for Novel Drug Development in Aging
Edward D. Levin
[Back
to top] Validation
of the Neuroinflammation Cycle as a Drug Discovery Target Using Integrative
Chemical Biology and Lead Compound Development with an Alzheimer’s
Disease-Related Mouse Model
Wenhui Hu, Hantamalala Ralay Ranaivo, Jeffrey M. Craft, Linda J. Van Eldik and D. Martin Watterson
The neuroinflammation cycle has been proposed as a potential therapeutic target in the development of new approaches to altering Alzheimer’s disease (AD) progression. However, the efficacy and toxicological profile of compounds that focus only on classical NSAID targets have been disappointing to date. Therefore, we recently initiated an unbiased, integrative chemical biology approach that used a hierarchal set of cell-based screens, followed by efficacy analysis in a new AD-relevant animal model that more closely resembles human pathology endpoints in terms of neuroinflammation and neuronal loss. The prior investigations provided a proof of concept that targeting the neuroinflammation cycle may be a viable drug discovery approach for AD. However, recent informatics analyses of the high attrition rate in drug development have identified the need for starting drug development with lead compounds that are well below cut off values in computed molecular properties in order to facilitate late stage medicinal chemistry refinement to improve in vivo functions. We describe here how we are leveraging our novel, unbiased, integrative chemical biology approach for the rapid discovery of potential lead compounds for AD drug discovery. Specifically, we show that orally bioavailable compounds with the desired physical properties and in vivo functions can be identified in focused synthetic libraries composed of chemical diversifications of the inactive but privileged pyridazine molecular fragment.
[Back
to top] Analysis
of Genetic Polymorphisms in Acetylcholinesterase as Reflected in Different
Populations
Yehudit Hasin, Nili Avidan, Dani Bercovich, Amos D. Korczyn, Israel Silman, Jacques S. Beckmann, and Joel L. Sussman
Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. In addition, it was implicated in amyloid plaque formation, a hallmark of Alzheimer’s disease (AD), and most of the drugs used in AD treatment are AChE inhibitors. Thus ACHE is an obvious candidate gene for pharmacogenetic study of AD treatment. However, AChE is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goals of this study were to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene, and to reveal their population specific architecture. To this end, the genomic coding sequences for AChE of 96 unrelated control individuals from three distinct ethnic groups, African Americans, Ashkenazi Jews and Israeli Arabs, were analyzed. Thirteen ACHE SNPs were identified, ten of which are newly described, and five of which should produce amino-acid substitutions (Arg34Gln, Gly57Arg, Glu344Gly, His353Asn and Pro592Arg). Population frequencies of 11 of the 13 SNPs were established in four different populations, African Americans, Ashkenazi Jews, Sephardic Jews and Israeli Arabs; 17 haplotypes and 5 ethno-specific alleles were identified, and a cladogram of ACHE haplotypes was constructed. Among the SNPs resulting in an amino-acid substitution, three are within the mature protein, mapping on its external surface; they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes – such as adverse drug responses to AChE inhibitors employed in treatment of AD patients and hypersensitivity to pesticides.
[Back
to top] Screening
for Inhibitors of Tau Polymerization
Marcus Pickhardt, Martin von Bergen, Zuzana Gazova, Antje Hascher, Jacek Bierna, Eva-Maria Mandelkow and Eckhard Mandelkow
The histopathological diagnosis of Alzheimer’s disease relies on two kinds of proteinaceous aggregates: the extracellular plaques built from filaments of the Ab-peptide and the intracellular tangles consisting of tau polymerized into Paired Helical Filaments (PHFs). The order of aggregation events is still under debate, but it is well accepted that tau-related changes have an important impact on the viability of neurons. In neurons, early morphological changes are seen in axons which begin to loose and retract synapses. This process is accompanied by an increase of aggregated tau protein. Thus the prevention of tau aggregation seems to be a valuable target for therapy of Alzheimer’s disease. Here we present a screening procedure by which we identified inhibitors of tau polymerization. In the primary screen we used a thioflavin-S based assay which detects PHF formation in solution. These initial hits were further analyzed for their capacity to depolymerize preformed PHFs. These results were confirmed by several secondary assays (tryptophan fluorescence, pelleting, filter trapping and electron microscopy). By this approach it is possible to identify small molecule compounds which prevent or reverse the aggregation of tau and thereby might improve the viability of neurons in a therapeutic approach.
[Back
to top] Cytoskeletal
Integrity as a Drug Target
M.L. Michaelis, K. .I. Seyb and S. Ansar
The cytoskeleton provides structural integrity and determines localization of proteins and organelles throughout the cell. The focus on structure and transport has overshadowed the role this ubiquitous network plays in cell signaling cascades, though it participates in transduction of signals from the plasma membrane to the nucleus. Clearly the discovery that neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) brain are made up of the microtubule (MT)-associated protein t and evidence that the toxic amyloid peptides in AD can lead to t hyper-phosphorylation and cytoskeletal dystrophy support the assertion that disruption of the MT network is an early signaling event in neurodegenerative cascades. Thus we have been testing the hypothesis that drugs that can moderate such signals through interactions with MTs would protect neurons against Ab toxicity. Drugs targeted to MTs are currently used as anti-cancer agents, due to their blockade of cell proliferation and induction of cell death. However, we and others have now found that low concentrations of compounds that help stabilize MTs do indeed protect post-mitotic neurons challenged with various toxic stimuli. Therefore we propose that the cytoskeletal network actually serves as a sensor for the overall state of the neurons and a first-line transducer of stress signals. Drugs that can moderate initiation of such early signaling events do protect against disruption of the cytoskeleton and neuritic dystrophy in neuronal cell cultures. In vivo proof-of-concept studies in animal models will require the development of agents that can protect cytoskeletal integrity and also cross the blood brain barrier.
[Back
to top] Development
of a High Throughput Drug Screening Assay for the Detection of Changes in Tau
Levels – Proof of Concept with HSP90
inhibitors
Chad A. Dickey, Jason Eriksen, Adeela Kamal, Francis Burrows, Srinivas Kasibhatla, Christopher B. Eckman, Mike Hutton and Leonard Petrucelli
Therapeutic development for Alzheimer’s disease has largely focused on the removal of beta amyloid because of its suggested role in the primary agent in initiating the disease process. However, with the recent discovery of mutations that result as pathologic buildup of tau in the absence of amyloid pathology, tau is beginning to be recognized as a potential target for drug discovery. We have developed a high-throughput drug screening method that allows for direct intracellular quantitation of tau protein species, enabling the fast, reliable detection of these changes. We have identified a family of small, blood brain barrier penetrant heat shock protein 90 inhibitors that significantly reduce tau protein levels in vitro. Western blot analysis demonstrated a clear inverse correlation between the tau levels and the increase in HSP27, HSP40 and HSP90. Modifications to this assay will further allow the specific analysis of pathologically relevant species. Using this assay, we have demonstrated that a class of HSP90 inhibitors is able to significantly lower intracellular tau levels most likely through induction of a heat shock response.
[Back
to top] Lentiviral
Vector-Based Models of Amyloid Pathology: From Cells to Animals
Laura Shaughnessy, Mary Beth Thomas, John Wakefield, Beth Chamblin, Ayyappan Nair, Frank Koentgen and Ram Ramabhadran
Lentiviral vectors are efficient tools for the introduction of genes into a wide range of established and primary cells in vitro, ex vivo, and in vivo, and also permit efficient transgenesis in a wide range of mammalian species. Our goals have been to apply the broad capabilities of the lentiviral vector system to AD research. Using a set of vectors expressing APP and PS1 genes, we demonstrated the efficiency and fidelity of the system for in vitro biochemical analyses of genes and pathways involved in plaque deposition. These analyses were performed in cell lines and in primary neuronal cultures, which have previously been difficult to use. The methods and tools described here are applicable to the study of effects of other genes and gene combinations on APP processing, including suppression of gene activity by delivering shRNAs. We have attempted to create local plaque pathology by stereotactic injection of APP and PS1 expressing vectors into mouse brains for use as a rapid model for plaque pathology that can be used in a broad range of mammals. No amyloid or preamyloid pathology has been detected over a six-month period; the possible reasons are discussed. Lastly, we have used the vectors to create transgenic rats expressing mutant APP and mutant PS1 and have obtained the first set of positive pups with more expected. The results presented here demonstrate the utility of Lentiviral vector-based approaches to the study of AD and other neurodegenerative diseases.
[Back
to top] Pilot
Study of the Reducing Effect on Amyloidosis In Vivo by Three FDA
Pre-Approved Drugs Via the Alzheimer’s APP 5’Untranslated Region
Stephanie Tucker, Michelle Ahl, Ashley Bush, David Westaway, Xudong Huang and Jack T. Rogers
A pilot study was conducted employing a well known mouse model for Alzheimer’s disease to evaluate the anti-amyloid efficacy of three FDA pre-approved drugs. Paroxetine (SSRI and APP 5’UTR directed lead compound), N-acetyl cysteine (antioxidant), and erythromycin (macrolide antibiotic) were provided to the drinking water of TgCRND8 mice for three months. This report provides data that measured the steady-state levels of amyloid Ab-40 and Ab-42 Ab as pmol Ab per gram of mouse brain cortex in drug treated and placebo animals. The relative levels of Ab peptide levels were reduced after exposure of mice to paroxetine (N=5), NAC (N=7), and erythromycin (N=7) relative to matched placebo counterparts. These results demonstrated proof-of concept for a strategy to further screen the APP 5’UTR target to identify novel drugs that exhibit anti-amyloid efficacy in vivo. These data also demonstrated a statistically significant anti-amyloid trend for paroxetine, NAC and erythromycin. The potential for conducting further studies with these compounds using larger cohorts of TgCRND8 mice is discussed.
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Library Screen to Identify Compounds that Decrease Secreted Ab from a Human Cell Line
Enakshi Chakrabarti and Jonathan D. Smith
In order to discover compounds that may be useful in the prevention of Alzheimer disease, a drug library was screened for drugs that could decrease amyloid b peptide (Ab) accumulation secreted from a human CNS-derived cell line. Of the 2160 compounds subjected to a primary screen, 16, or 0.74%, reduced Ab accumulation by at least 40%. Seven of these compounds were confirmed to be effective in a secondary screen using each compound at a dose of 5 mM. Three of these seven compounds were more effective than the others at reducing Ab levels in a tertiary screen, and led to 68 to 85% reductions in total Ab, at the 25 mM dosage. The effects of these three drugs on secreted Ab40, Ab42, and sAPPa were also determined. Thus, we have identified lead compounds that may be useful for subsequent studies to determine the mechanism of action of each drug, as well as for pre-clinical studies to determine whether each of these drugs is safe and effective in vivo.
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Developments on the Studies of Human Memapsin 2 (b-Secretase)
Jordan Tang, Xiangyang He , Xianping Huang and Lin Hong
Several specific topics on recent findings in the studies of memapsin 2 (b-secretase) are discussed in this article. New structural evidence on memapsin 2 has pointed to the structural basis of flap opening. Such a mechanism is thought to participate in the substrate sequence selection during the cellular function. Another potentially important area is the intracellular transport of memapsin 2. Here we discuss the involvement of APP in memapsin 2 endocytosis and the GGA proteins in recycling the protease back to the cell surface. These mechanisms may be connected with the cellular regulation of neuronal activity through the regulation of memapsin 2 activity and Ab production.
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Ab Sequestration Approach Using Non-Antibody Ab Binding Agents
Yasuji Matsuoka, Li Shao, Manik Debnath, John LaFrancois, Amanda Becker, Audrey Gray, Paul Aisen, Chester Mathis, William Klunk and Karen Duff
Amyloid beta (Abeta) has been considered as a primary cause of Alzheimer’s disease (AD), and Abeta lowering approaches have been tested. Active immunization against Abeta is one of several promising Abeta-lowering approaches. Two mechanisms have been proposed: enhancement of microglial phagocytosis and Abeta sequestration (also called “peripheral sink”). We hypothesized that Abeta sequestration without immune modulation is sufficient to reduce the brain Abeta load and have demonstrated effective sequestration with Abeta binding agents that do not stimulate an immune reaction. Recent reports from other groups showed two other non-immune related Abeta binding agents, which have no structural relation to compounds we previously tested, reduced brain Abeta after peripheral administration. Congo red is a chemically synthesized small molecule that has binding affinity to Abeta. In the present study, we tested three Congo red derivatives in Abeta plaque-forming mice at an early pathological stage. Unfortunately, peripheral administration for three weeks did not substantially alter brain Abeta load. Optimized Abeta binding agents with high affinity to soluble Abeta are necessary for the sequestration approach.
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Clearance of Ab is Inhibited by
KPI-Containing Isoforms of APP
Robert D. Moir and Rudolph E. Tanzi
The pathogenesis of Alzheimer’s disease (AD) involves the abnormal accumulation and deposition of b-amyloid in cerebral blood vessels and in the brain parenchyma. Critical in modulating b-amyloid deposition in brain is the flux of Ab across the blood brain barrier. The low-density lipoprotein receptor-related protein (LRP), is a large endocytic receptor that mediates the efflux of Ab out of brain and into the periphery. The first step in the LRP-mediated clearance of Ab involves the formation of a complex between Ab and the LRP ligands apolipoprotein E (apoE) or a2‑macroglobulin (a2M). The Ab/chaperone complexes then bind to LRP via binding sites on apoE or a2M. The efflux of Ab/chaperone complexes out of the neuropil and into the periphery may be attenuated by LRP-ligands that compete with apoE or a2M for LRP binding. LRP is also the cell surface receptor for Kunitz Protease Inhibitor (KPI) containing isoforms of Ab’s parent protein, the amyloid protein precursor (APP). Protein and mRNA levels of KPI-containing APP isoforms (APP‑KPI) are elevated in AD brain and are associated with increased Ab production. In this study we show that soluble non-amyloidogenic APP-KPI can also inhibit the uptake of Ab/ a2M in a cell culture model of LRP mediated Ab clearance. Clearance of Ab/apoE complexes was not inhibited by APP-KPI. Our findings are consistent with studies showing that apoE and a2M have discrete binding sites on LRP. Most significantly, our data suggests that the elevated levels of APP-KPI in AD brain may attenuated the clearance of Ab, the proteins own amyloidogenic catabolic product.