eBriefing

Targeting Metals in Alzheimer's and Other Neurodegenerative Diseases

Targeting Metals in Alzheimer's and Other Neurodegenerative Diseases
Reported by
Jennifer Cable

Posted January 17, 2013

Presented By

Overview

As life expectancy increases, the prevalence of age-related diseases will also increase. Efforts to develop therapies for age-related neurodegenerative diseases have yet to succeed in impacting disease pathogenesis; current treatments target only the symptoms, not the causes. To develop disease-modifying agents, some researchers aim to identify common features of neurodegenerative diseases that might provide clues to potential drug targets. On November 29, 2012, researchers investigating Alzheimer's, Parkinson's, and Huntington's diseases met at the New York Academy of Sciences to discuss one such feature—the effect of metals in neurodegenerative diseases—and to highlight clinical achievements using metal-binding compounds to influence disease progression. The symposium, Targeting Metals in Alzheimer's and Other Neurodegenerative Diseases, was presented by the Academy's Brain Dysfunction Discussion Group.


Presentations available from:

Robert A. Cherny, PhD (The Florey Institute of Neuroscience and Mental Health, Australia)
Steven M. Hersch, MD, PhD (Massachusetts General Hospital; Harvard Medical School)
Rudolph Tanzi, PhD (Massachusetts General Hospital; Harvard Medical School)
Daniel Tardiff, PhD (Whitehead Institute for Biomedical Research)
Panel moderator: George Zavoico, PhD (MLV)


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Transition Metals and Neurodegenerative Disease: An Opportunity for Therapeutic Intervention


Robert A. Cherny (Florey Institute of Neuroscience and Mental Health, Australia)
  • 00:01
    1. Introduction; Copper, zinc, and glutamatergic neurotransmission
  • 03:12
    2. Metal dyshomeostasis and oxidative stress; Beta-amyloid accumulation
  • 09:51
    3. PBT2 mouse study
  • 16:58
    4. Anti-oligomer effects; Metal dependent modulation of signaling pathways; PBT2 mechanism of action
  • 26:43
    5. Huntington's Disease mouse study
  • 29:26
    6. A neuroprotection approach to Parkinson's Disease therapy; PBT434
  • 43:36
    7. Summary, acknowledgements, and conclusion
  • 45:25
    8. Q and A sessio

Transition Metals and the Pathogenesis of Treatment of Huntington's Disease


Steven M. Hersch (Massachusetts General Hospital and Harvard Medical School)
  • 00:01
    1. Introduction and overview
  • 06:55
    2. The relevance of transition metals; Increase in paramagnetic brain metals
  • 13:55
    3. Oxidative stress markers; Systemic metal dyshomeostasis
  • 18:56
    4. Copper interaction with Huntingtin; The formation of toxic htt oligomers
  • 31:33
    5. Posttranslational modifications; The Reach2HD trial
  • 33:11
    6. Summary, acknowledgements, and conclusion
  • 36:05
    7. Q and A sessio

Panel Discussion


Moderator: George Zavoico (MLV)
  • 00:01
    1. Alleviation of endocytosis; A-beta clearance
  • 07:15
    2. Manipulation of nitrogen and hydroxl; Fenton chemistry; Amyloid aggregation
  • 15:36
    3. Synaptic zinc; Executive function metrics
  • 21:27
    4. Peripheral metal homeostasis; Diversity profiling; PBT2 and zinc levels
  • 27:30
    5. Other hypotheses; Clinical trials; Conclusio

The Metal Hypothesis of Alzheimer's Disease


Rudolph Tanzi (Massachusetts General Hospital and Harvard Medical School)
  • 00:01
    1. Introduction and overview; APP processing
  • 08:39
    2. Known AD genes; Functional grouping; Excess amyloid and inflammation
  • 14:55
    3. The amyloid hypothesis; Phase 3 failures and current studies
  • 19:47
    4. The metal hypothesis; PBT2
  • 29:06
    5. Executive dysfunction; Imaging and Huntingon's phase II trials
  • 32:00
    6. Metal chaperones; Summary, acknowledgements, and conclusion
  • 34:50
    7. Q and A sessio

Targeting Metal Homeostasis Protects Against the Toxicity Caused by Neurodegenerative Disease Proteins in Yeast


Daniel Tardiff (Whitehead Institute for Biomedical Research)
  • 00:01
    1. Introduction
  • 05:07
    2. Phenotypic screening of yeast; Models of proteotoxicity; TDP-43
  • 13:06
    3. Creating a yeast A-beta toxicity model; CQ rescue modulation
  • 24:25
    4. Steady state A-beta levels in yeast; Early events in A-beta pathogenesis
  • 30:13
    5. Other metal-related compounds rescue A-beta toxicity
  • 32:19
    6. Summary, acknowledgements, and conclusion
  • 36:10
    7. Q and A sessio

Introduction


George Zavoico (MLV)

Journal Articles

Information on the metal hypothesis of neurodegenerative diseases

Crouch PJ, Barnham KJ. Therapeutic redistribution of metal ions to treat Alzheimer's disease. Acc Chem Res. 2012;45(9):1604-1611.

Kaden D, Bush AI, Danzeisen R, Bayer TA, Multhaup G. Disturbed copper bioavailability in Alzheimer's disease. Int J Alzheimers Dis. 2011;345614.

Kenche VB, Barnham KJ. Alzheimer's disease & metals: therapeutic opportunities. Br J Pharmacol. 2011;163(2):211-219.

General information on metal chaperones

Sampson EL, Jenagaratnam L, McShane R. Metal protein attenuating compounds for the treatment of Alzheimer's dementia. Cochrane Database Syst Rev. 2012;16(5):CD005380.

Adlard PA, Bush AI. Metal chaperones: a holistic approach to the treatment of Alzheimer's disease. Front Psychiatry. 2012;3:15.

Preclinical characterization of PBT2

Crouch PJ, Sawa MS, Hung LW, et al. The Alzheimer's therapeutic PBT2 promotes amyloid-β degradation and GSK3 phosphorylation via a metal chaperone activity. J Neurochem. 2011;119(1):220-230.

Adlard PA, Cherny RA, Finkelstein DI, et al. Rapid restoration of cognition in Alzheimer's transgenic mice with 8-hydroxyquinoline analogs is associated with decreased interstitial Aβ. Neuron. 2008;59(1):43-55.

Clinical trials with PBT2

Faux NG, Ritchie CW, Gunn A, et al. PBT2 rapidly improves cognition in Alzheimer's Disease: additional phase II analyses. J Alzheimers Dis. 2010;20(2):509-516.

Lannfelt L, Blennow K, Zetterberg H, et al. Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomized, placebo-controlled trial. Lancet Neurol. 2008;7(9):779-786.

Modeling neurodegenerative diseases in yeast

Tardiff DF, Tucci ML, Caldwell KA, Caldwell GA, Lindquist S. Different 8-hydroxyquinolines protect models of TDP-43 protein, α-synuclein, and polyglutamine proteotoxicity through distinct mechanisms. J Biol Chem. 2012;287(6):4107-4120.

Su LJ, Auluck PK, Outeiro TF, et al. Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models. Dis Model Mech. 2010;3(3-4):194-208.

Targeting transition metals in Huntington's Disease

Rosas HD, Reuter M, Doros G, et al. A tale of two factors: what determines the rate of progression in Huntington's disease? A longitudinal MRI study. Mov Disord. 2011;26(9):1691-1697.

Rosas HD, Chen YI, Doros G, et al. Alterations in brain transition metals in Huntington disease: an evolving and intricate story. Arch Neurol. 2012;69(7):887-893.

Fox JH, Kama JA, Lieberman G, et al. Mechanisms of copper ion mediated Huntington's disease progression. PLoS One. 2007;2(3):e334.

Fox JH, Connor T, Stiles M, et al. Cysteine oxidation with N-terminal mutant huntingtin promotes oligomerization and delays clearance of soluble protein. J Biol Chem. 2011;286(20):18320-18330.

Nguyen T, Hamby A, Massa SM. Clioquinol down-regulates mutant huntingtin expression in vitro and mitigates pathology in a Huntington's disease mouse model. Proc Natl Acad Sci USA. 2005;102(33):11840-11845.


Patient Advocacy and Educational Organizations

Alzheimer's Foundation of America

Alzheimer's Association

The Michael J. Fox Foundation

Parkinson's Disease Foundation

Huntington's Disease Society of America

Hereditary Disease Foundation

Organizers

Rudolph Tanzi, PhD

Massachusetts General Hospital and Harvard Medical School
e-mail | website | publications

Rudolph Tanzi is the Joseph P. and Rose F. Kennedy Professor of Neurology at Harvard University and the director of the Genetics and Aging Research Unit at Massachusetts General Hospital. Tanzi has been investigating the genetics of neurological disease since the 1980's, when he participated in the first study to use genetic markers to find a disease gene (Huntington's disease). Tanzi co-discovered the three familial early-onset Alzheimer's disease (FAD) genes and several other neurological-disease genes, including the gene responsible for Wilson's disease. As leader of the Cure Alzheimer's Fund Alzheimer's Genome Project, Tanzi has carried out multiple genome-wide association studies of thousands of Alzheimer's families, leading to the identification of novel AD candidate genes and the first two rare mutations causing late-onset AD in the ADAM10 gene. His research on the role of zinc and copper in AD has led recently to successful clinical trials. Tanzi serves on dozens of editorial and scientific advisory boards and as chair of the Cure Alzheimer's Fund Research Consortium. He has received numerous awards, including the two highest awards for Alzheimer's disease research: the Metropolitan Life Award and the Potamkin Prize. He co-authored the popular trade book Decoding Darkness: The Search for the Genetic Causes of Alzheimer's Disease and the recent New York Times bestseller Super Brain.

George Zavoico, PhD

MLV
e-mail | website | publications

George Zavoico is managing director of research and a senior equity research analyst at MLV & Co., a boutique investment bank and institutional broker-dealer based in New York. He has over six years of experience as a life sciences analyst, writing on publicly traded equities. Prior to MLV, he was an equity analyst with Westport Capital Markets and Cantor Fitzgerald. Before that, Zavoico established his own consulting company serving the biotech and pharmaceutical industries. He also wrote extensively on healthcare and the biotech and pharmaceutical industries for periodicals targeting the general public and industry executives. Zavoico began his career as a senior research scientist at Bristol-Myers Squibb Co., moving on to management positions at Alexion Pharmaceuticals, Inc. and T Cell Sciences, Inc. (now Celldex Therapeutics, Inc.). He holds a PhD in physiology from the University of Virginia and held postdoctoral positions at the University of Connecticut Health Sciences Center, Brigham and Women's Hospital, and Harvard Medical School.

Jennifer Henry, PhD

The New York Academy of Sciences
e-mail

Jennifer Henry is the director of Life Sciences at the New York Academy of Sciences. Henry joined the Academy in 2009, before which she was a publishing manager in the Academic Journals division at Nature Publishing Group. She also has eight years of direct editorial experience as editor of Functional Plant Biology for CSIRO Publishing in Australia. She received her PhD in plant molecular biology from the University of Melbourne, specializing in the genetic engineering of transgenic crops. As director of Life Sciences, she is responsible for developing scientific symposia across a range of life sciences, including biochemical pharmacology, neuroscience, systems biology, genome integrity, infectious diseases, and microbiology. She also generates alliances with organizations interested in developing programmatic content.


Speakers

Robert A. Cherny, PhD

The Florey Institute of Neuroscience and Mental Health, Australia
e-mail | website | publications

Robert A. Cherny is an associate professor and principal research fellow in the Oxidation Biology Laboratory at the Florey Institute of Neuroscience and Mental Health at the University of Melbourne, Australia. He has published extensively on the neurobiology of metals and its relevance to neuodegenerative disease, with a focus on translational medicine. He is also head of research at Prana Biotechnology, Ltd., a pharmaceutical company based in Melbourne that specializes in the development of novel drugs for neurodegenerative diseases.

Steven M. Hersch, MD, PhD

Massachusetts General Hospital, Harvard Medical School
e-mail | website | publications

Steven M. Hersch directs the New England Center of Excellence for Huntington's Disease and the Laboratory of Neurodegeneration and Neurotherapeutics at Massachusetts General Hospital. His research areas have included the synaptic organization and molecular pharmacology of the cerebral cortex and basal ganglia and basic, translational, and clinical research related to HD. His laboratory studies the pathogenesis of HD, with a focus on identifying and validating potential neuroprotective treatments and biomarkers using genetic HD mouse models and human clinical and postmortem samples. Hersch has been a principal investigator, steering committee member, and site investigator for many observational and therapeutic studies in HD patients. He is the principal investigator of the first prevention trial in presymptomatic HD and of a global phase III study examining high dose creatine in early symptomatic HD. He is leading a collaborative NIH-supported program to develop neuroimaging and protein, small molecule, and genomic biomarkers of HD. He is the co-chair of the Huntington Study Group, an international consortium of academic investigators at almost 100 leading institutions, devoted to HD clinical research.

Rudolph Tanzi, PhD

Massachusetts General Hospital, Harvard Medical School
e-mail | website | publications

Daniel Tardiff, PhD

Whitehead Institute for Biomedical Research
e-mail | website | publications

Daniel Tardiff holds a PhD in molecular and cell biology from Brandeis University, where he worked in the lab of Michael Rosbash, a pioneer in the study of basic gene expression work in yeast and Circadian rhythms in fruit flies. He studied new applications of chromatin immunoprecipitation to study cotranscriptional pre-mRNA splicing in budding yeast and developed an in vivo crosslinking approach to affinity purify large, labile complexes that typically do not survive affinity purification. He completed a postdoctoral program with Susan Lindquist at the Whitehead Institute for Biomedical Research. He has worked on several yeast models that recapitulate the basic protein misfolding problems associated with PD, ALS, FTLD, and AD. Most of his efforts have focused on high-throughput screens for small molecules that rescue the cell death associated with toxic expression of disease proteins.


Jennifer Cable

Jennifer Cable resides in New York City, where she experiments with different methods and outlets to communicate science. She enjoys bringing science to scientists and nonscientists alike. She writes for Nature Structural and Molecular Biology, Bitesize Bio, Under the Microscope, and the Nature New York blog. She received a PhD from the University of North Carolina at Chapel Hill for her research in investigating the structure/function relationship of proteins.

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Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) disproportionately affect elderly populations, inflicting a considerable physical, emotional, and economic burden. Approximately 5.4 million Americans suffer from AD, and another one million suffer from PD. In 2009, AD and PD were the 6th and 14th most common causes of death in the United States, respectively. Since the number of people aged 65 and older is expected to reach 20% of the U.S. population by 2050, these numbers are expected to increase. Indeed, recent data indicate that this is already occurring. From 2000 to 2008, deaths related to HIV and several types of cancer decreased by as much as 30%, but deaths related to AD increased by 66%. The associated cost of neurodegenerative diseases, estimated at $200 billion for AD and $25 billion for PD, will also increase. It is clear that new approaches are needed; these must not only offer symptomatic relief but also target the mechanisms behind disease progression.

While the percentages of deaths caused by many other diseases have fallen, the percentage of deaths due to Alzheimer's disease is rising. (Image courtesy of Rudolph Tanzi)

To develop new approaches, researchers are examining the commonalities between different neurodegenerative disorders. AD, PD, and HD are characterized by an accumulation of misfolded proteins in the brain. When two or more of these proteins come together, they form higher-order oligomers, such as dimers, trimers, and tetramers, which can cause neuron death and a decline in brain function. Extracellular oligomers of the peptide Aβ form the notorious plaques for which AD is so well known. The protein α-synuclein accumulates within neurons in PD, eventually leading to cell death. Mutations in the huntingtin protein cause it to misfold and oligomerize in HD. While a considerable amount of research has been directed at targeting oligomers and preventing their toxic effects, available medications are palliative: they alleviate symptoms without modifying the course of disease.

One approach is to target a common feature of all three diseases. Researchers at the symposium explored this possibility in a discussion of the "metal hypothesis of neurodegenerative disease." This posits that metals such as iron, copper, and zinc are implicated in the pathogenesis of some neurodegenerative diseases and that agents targeting these metals can slow or potentially reverse the course of disease.

Several neurodegenerative diseases result from abnormal protein aggregation in the brain. Metals have been implicated in the pathogenesis of AD, HD, and PD and may play a role in other neurodegenerative diseases. (Image courtesy of Rudolph Tanzi)

Normal brain function is heavily dependent on metals: copper and zinc are released into the synaptic cleft during neurotransmission and are essential for the activity of several enzymes, such as superoxide dismutase.

The metal hypothesis of neurodegenerative diseases is based on two observations. First, metals promote protein oligomerization. The presence of zinc or copper is required for oligomerization of Aβ in AD, and copper promotes oligomerization of huntingtin in HD. Second, several studies demonstrate that metal homeostasis is disrupted in neurodegenerative diseases. In AD mouse models, brain imaging studies show an increased concentration of zinc; in HD, iron accumulation correlates with disease progression; and in PD, there is an increased concentration of iron in the substantia nigra, the area of the brain most affected by the disease.

Speakers at the symposium highlighted the roles metals play in neurodegenerative diseases; presented preclinical and clinical data on a metal-binding agent, PBT2, in AD and HD; and discussed ongoing studies of a similar compound, PBT434, in PD. Rudolph Tanzi from Massachusetts General Hospital and Harvard Medical School described how copper catalyzes Aβ aggregation in AD and discussed phase IIa data on PBT2. Robert Cherny from the Florey Institute of Neuroscience and Mental Health presented preclinical data from PBT2 trials in mouse models of AD and HD, as well as preclinical data on PBT434 in mouse models of PD. Steven Hersch from Massachusetts General Hospital and Harvard Medical School showed how copper and iron play important yet distinct roles in the pathogenesis of HD. Daniel Tardiff from the Whitehead Institute for Biomedical Research demonstrated how yeast models of neurodegenerative diseases can be used in drug discovery.

Speakers

Rudolph Tanzi

Massachusetts General Hospital and Harvard Medical School

Robert A. Cherny

The Florey Institute of Neuroscience and Mental Health, Australia

Highlights

Metal homeostasis in the brain is disrupted in Alzheimer's disease.

Metals such as zinc and copper are essential for Aβ aggregation and toxicity.

Therapeutic agents that remove metals from Aβ aggregates may be effective in treating Alzheimer's disease.

The role of metals in the pathogenesis of Alzheimer's disease

The primary culprit in AD pathogenesis is the peptide Aβ. Aβ is formed as a result of aberrant processing of the plasma membrane protein amyloid precursor protein (APP). APP is normally cleaved by the proteases α- and γ-secretase to create peptides that perform functions such as regulating transcription in the nucleus. Aβ is formed if, instead, APP is cleaved by β-secretase. Monomeric Aβ is not pathogenic and can usually be removed from the brain by apolipoprotein E. However, when these processes are not functioning properly, Aβ can oligomerize into higher-order species which form the amyloid fibrils and plaques that characterize AD.

Rudolph Tanzi of Massachusetts General Hospital and Harvard Medical School showed that Aβ oligomerization is dependent on the presence of either zinc or copper. When copper binds to Aβ, it forms oxidative crosslinks between monomers. The resulting crosslinked species are very difficult for the brain to clear and eventually cause neuron death. Copper binding to Aβ can also generate free radicals, which cause oxidative damage. When amyloid plaques sequester zinc and copper, preventing these metals from performing their normal functions in the brain, it is unclear whether the resulting decrease in the bioavailable pool of metals is high enough to cause any detrimental effect. However, metal dyshomeostasis has been observed in a mouse model of AD. Targeting the interaction between Aβ and metals, therefore, appears to be a mechanism for therapeutic intervention that could both normalize metal homeostasis and reduce the levels of toxic Aβ oligomers in the brain.

The metal hypothesis of Alzheimer's disease. Copper and zinc can bind to Aβ, promoting Aβ oligomerization, fibril formation, and oxidative stress. (Image courtesy of Rudolph Tanzi)

Preclinical studies of PBT2 in Alzheimer's disease

Robert A. Cherny of the Florey Institute of Neuroscience and Mental Health presented preclinical results for PBT2, which targets the interaction between Aβ and metals. PBT2 is a metal-binding compound that binds to copper and zinc with an intermediate affinity; this is essential to its function, as it has a high enough affinity to remove copper and zinc from Aβ oligomers but cannot compete with physiological metal-binding proteins. When PBT2 binds to metal, it undergoes a conformational change that enables the complex to cross the plasma membrane; thus, PBT2 shuttles metal from amyloid fibrils and delivers it to neurons. The results are twofold: without metal, amyloid fibrils dissolve into monomers that can be cleared from the brain via normal processes; and metal becomes available to perform its normal functions, thus restoring metal homeostasis.

The mechanism of action of PBT2 in Alzheimer's disease. PBT2 removes zinc and copper from Aβ and delivers it to neurons. This dissolves Aβ fibrils into monomers, which can then be cleared from the brain, and restores metal homeostasis. These processes promote several neuroprotective effects. (Image courtesy of Robert Cherny)

Cherny presented several promising results from preclinical studies of PBT2 in a transgenic mouse model of AD. Consistent with its metal-binding properties, PBT2 reduced the levels of soluble and insoluble Aβ plaques. Mice treated with PBT2 also showed improved cognitive function. In a test of spatial memory, untreated mice were unable to find a submerged platform in a pool of water, whereas PBT2-treated mice performed as well as healthy mice and found the platform quite easily. PBT2 treatment also increased synaptophysin in the brain, suggesting that the drug promotes neuron remodeling. Furthermore, several markers involved in memory and neuron plasticity increased, including calcium/calmodulin-dependent protein kinase II (CAMKII), N-methyl-D-aspartate receptor (NMDAR) 1 and 2a, and tyrosine-related kinase B (TrkB). PBT2 treatment in wild-type mice did not result in any of the changes in cognitive function or biomarkers observed in transgenic mice, suggesting that PBT2 acts on a specific AD-related defect.

Clinical studies of PBT2 in Alzheimer's disease

Tanzi presented data from a 12-week phase IIa clinical study of PBT2 in 78 patients with mild AD. Results mirrored the mouse studies. Patients treated with PBT2 had significantly less Aβ in their cerebral spinal fluid and significant improvements in executive function (cognitive abilities like reasoning, adaptability, self-monitoring, and multitasking). Executive function is often used to assess whether patients are able to care for themselves or should enter a nursing home.

Results from a phase IIa study of PBT2 in patients with mild Alzheimer's disease. Treatment with PBT2 resulted in significant decreases in Aβ (left) and significant increases in executive function (right) compared to placebo. (Image courtesy of Rudolph Tanzi)

In previous AD trials, treatment has simply slowed cognitive decline; therefore, an improvement in executive function is quite unique. However, many studies assess the effect of drugs on memory, not executive function. As Tanzi pointed out, loss of executive function, heralded by symptoms like depression, apathy, and personality changes, occurs before memory loss; by the time patients exhibit memory loss it may be too late to alter the course of disease. Therefore, clinical trials may have more success if patients are treated before memory loss occurs, using executive function as a clinical endpoint to assess drug efficacy.

A second phase II trial is underway to monitor the effect of PBT2 on amyloid plaques and cognition. Results are expected in the second half of 2013.

Speaker

Daniel Tardiff

Whitehead Institute for Biomedical Research

Highlights

Yeast models of neurodegenerative diseases can elucidate the mechanisms of toxicity of protein aggregation.

Several drug hits identified in yeast models are metal-binding compounds.

Metal-binding compounds show varying activities in different disease models, suggesting different mechanisms.

Yeast as a model of neurodegenerative diseases

How is it possible to model neurodegenerative diseases in yeast? At first glance, yeast may seem a poor replica of the human brain. However, as Daniel Tardiff of the Whitehead Institute for Biomedical Research explained, several attributes of yeast make it a useful model for non-biased drug screens.

Many important cellular processes are conserved between yeast and humans. On a practical note, yeast is easy to work with. It grows well in the lab and is inexpensive to maintain. Furthermore, the yeast genome is very well understood and is amenable to many genetic techniques—genes can easily be knocked out or overexpressed, and other analyses, such as proteomics and lipidomics, are easily accessible. Performing drug screens in yeast can often filter out drugs that are grossly toxic to living cells, while also selecting for compounds that are able to traverse the plasma membrane, features that cannot be tested in in vitro assays. After being identified in yeast models, potential drugs can be validated in higher-order neuronal models, such as C. elegans and Drosophila melanogaster, and in neuronal cell culture models.

Tardiff uses yeast models of neurodegenerative diseases to better understand how protein aggregation leads to cell toxicity. He described several models, all expressing a key protein involved in a particular disease: TDP-43 (amyotrophic lateral sclerosis [ALS]), α-synuclein (PD), huntingtin (HD), and Aβ (AD). Expression of these proteins is toxic to yeast. Tardiff screens a library of over 100 000 potential drugs to find compounds that are able to rescue yeast survival. Some of the strongest drug hits in these screens are analogs of 8-hydroxyquinoline, with the same basic structure as PBT2. Despite this shared basic structure, the compounds have distinct activities; while one may be active in yeast models of ALS, HD, and PD, another may only be active in ALS.

Tardiff gave further details about one drug hit, identified in yeast expressing Aβ. Aβ toxicity in yeast results from an accumulation of oligomers between the plasma membrane and the cell wall. Aβ expression caused a defect in endocytosis, although it is unclear whether this defect is specific to yeast or mimics any aspect of AD pathology in humans.

Tardiff identified a metal-binding molecule known as clioquinol that rescued Aβ toxicity in yeast and C. elegans. Consistent with clioquinol's metal-binding properties, adding copper to yeast abolished the ability of clioquinol to rescue Aβ toxicity. Interestingly, the effects of iron and zinc were less straightforward: iron attenuated the effect of clioquinol, whereas zinc had no effect. Tardiff found that several genes involved in copper and iron transport are upregulated by clioquinol. The genetic profile of yeast exposed to clioquinol was similar to the response to a copper/iron deficiency.

In yeast expressing Aβ, clioquinol reverses many of the cellular events associated with early Alzheimer's disease: it destabilizes Aβ fibrils, restores endocytosis, and alters metal homeostasis. (Image courtesy of Daniel Tardiff)

In addition to rescuing survival, clioquinol also decreased the level of Aβ in yeast, presumably by removing metal from Aβ oligomers and thus enabling monomers to be degraded. Clioquinol also rescued the endocytosis defect observed in yeast expressing Aβ and affected copper/iron homeostasis, although the mechanism of these effects is unclear.

Speakers

Steven M. Hersch

Massachusetts General Hospital and Harvard Medical School

Robert Cherny

The Florey Institute of Neuroscience and Mental Health, Australia

Highlights

Copper and iron serve important yet distinct functions in the pathogenesis of Huntington's disease.

The metal-binding compound PBT2 improves motor function and survival in mouse models.

Clinical trials are underway to assess the effect of PBT2 in patients with Huntington's disease.

The pathogeny of Huntington's disease

According to the Huntington's Disease Society of America, 250 000 Americans suffer from or are at risk for HD, a slowly progressing inherited neurological disorder often characterized by impaired mobility, depression, mood swings, and clumsiness. Most patients die within 15 to 20 years of experiencing symptoms and there are no treatments that affect disease progression.

The pathogeny of Huntington's disease. The huntingtin protein is cleaved near the N-terminus, resulting in a peptide containing the poly-glutamine region. The peptide can form toxic oligomers, which have many detrimental effects on the cell. (Image courtesy of Steven M. Hersch)

HD is an autosomal dominant genetic disease caused by a series of CAG repeats in the huntingtin gene. This mutation results in a string of glutamine residues in the N-terminus of the huntingtin protein. In general, the more CAG repeats present in the gene, the longer the string of glutamine residues (known as the poly-glutamine region) in the protein and the earlier the disease manifests. Although the physiological role of huntingtin is not well understood, it is clearly important as it is necessary for survival in animal models. Huntingtin interacts with hundreds of proteins and may act as a scaffold, bringing proteins together to perform their functions. In Huntington's disease, the protein is cleaved near the N-terminus, releasing a peptide that contains the abnormal poly-glutamine region. This peptide can form toxic oligomers within the cytoplasm and nucleus, with many detrimental effects including oxidative damage, metabolic changes, mitochondrial dysfunction, and metal dyshomeostasis.

A tale of two metals

Steven M. Hersch of Massachusetts General Hospital and Harvard Medical School discussed the distinct roles iron and copper play in HD pathology. In HD, iron accumulates in the brain and its concentration increases as the disease progresses. Genes involved in iron absorption, such as hepcidin and divalent metal transporter 1 (DMT1), are upregulated, partially explaining the increased iron levels. Elevated iron levels correlate with increased oxidative damage in the brain, suggesting at least one harmful effect of iron.

The connection between copper and HD is quite different. Hersch showed that copper binds directly to the huntingtin fragment containing the poly-glutamine region. He narrowed the binding site to two histidine residues and showed that copper binding induces huntingtin oligomerization by catalyzing the formation of a disulfide bond between two huntingtin monomers. Hersch speculated that blocking copper binding could prevent the toxic effects of huntingtin oligomerization. The metal-binding compound clioquinol, also described by Tardiff for its ability to rescue Aβ toxicity in yeast, interfered with huntingtin oligomerization in mice and inhibited neuron death, resulting not only in improved motor function but also in increased overall survival.

Copper plays a role in HD pathogenesis. Mutant huntingtin protein is cleaved near the N-terminus, resulting in a peptide that contains the poly-glutamine region. While monomeric peptide is non-toxic and can generally be cleared from the brain, interaction with copper catalyzes the formation of a disulfide bond between two monomers, which promotes huntingtin aggregation and disease progression. (Image courtesy of Steven M. Hersch)

Preclinical studies of PBT2 in Huntington's disease

In addition to studies in AD, PBT2 is also under investigation in HD due to its copper-binding properties. Cherny showed that treatment with PBT2 improved motor function in mouse models of HD. Mice with the huntingtin mutation exhibit a distinct clasping of their hind limbs when held upside down, whereas wild-type mice splay their hind limbs. Treatment with PBT2 ameliorated this limb-clasping phenotype, which is used as a surrogate for motor function. Furthermore, similar to the effect of clioquinol in yeast, PBT2 increased median lifespan by 26%.

Clinical studies of PBT2 in Huntington's disease

Based on the promising preclinical results of PBT2 in mice, a phase II study by the Huntington Study Group, REACH2HD, will investigate the effects of PBT2 in HD patients. Results are expected near the end of 2013. In addition to monitoring cognitive changes, the study will assess metal levels in the brain, oxidative damage, and huntingtin oligomerization in the blood.

Speaker

Robert A. Cherny

The Florey Institute of Neuroscience and Mental Health, Australia

Highlights

Metal dyshomeostasis may play a role in Parkinson's disease, as indicated by the high levels of iron in the substantia nigra of patients with Parkinson's disease.

The metal-binding compound PBT434 improved several characteristics of Parkinson's disease in a transgenic mouse model and is currently being investigated in preclinical toxicology assessments.

Targeting iron in Parkinson's disease

Parkinson's disease is characterized by progressive impairment of motor function. The shaking and tremors commonly associated with PD are due to loss of dopaminergic neurons in the substantia nigra. Current therapies rely on replacing dopamine; however, over 70% of dopaminergic neurons are lost by the time symptoms appear, limiting the efficacy of dopamine-based treatments. Therefore, a strategy that prevents neuron damage is needed.

It has long been known that iron levels are increased in the substantia nigra in PD. Iron can cause oxidative damage and plays a role in α-synuclein oligomerization, which is implicated in PD. Iron levels are currently used to monitor disease progression, but work is underway to develop therapies that directly target iron.

Cherny described studies of PBT434, a compound that maintains the metal-binding motif of PBT2 but has a different backbone structure. PBT434 inhibited the production of iron-mediated reactive oxygen species in vitro. In a mouse model of PD in which the substantia nigra was selectively damaged by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), PBT434 treatment reduced neuron death in the substantia nigra, improved motor function, reduced oxidative stress, decreased levels of α-synuclein, and reduced iron levels. PBT434 treatment had similar effects in a transgenic mouse model of PD. It is currently undergoing preclinical toxicology assessments funded by the Michael J. Fox Foundation.

The interplay between iron, dopamine, and α-synuclein in Parkinson's disease. (Image courtesy of Robert A. Cherny)

Panelists

Robert A. Cherny

The Florey Institute of Neuroscience and Mental Health, Australia

Steven M. Hersch

Massachusetts General Hospital and Harvard Medical School

Rudolph Tanzi

Massachusetts General Hospital and Harvard Medical School

Daniel Tardiff

Whitehead Institute for Biomedical Research

George Zavoico

Moderator

MLV

Highlights

Metal-binding compounds like PBT2 and PBT434 likely act by preventing protein aggregation and normalizing metal dyshomeostasis. The relative contribution of each of these mechanisms is still unclear.

Future trials of neurodegenerative diseases may require new trial designs and clinical endpoints to assess the efficacy of potential therapies.

Which came first...

The panel discussion delved further into the two mechanisms by which drugs like PTB2 appear to function in neurodegenerative diseases: normalizing metal dyshomeostasis and clearing protein oligomers. Which of these is the primary mechanism is still up for discussion. Much remains unknown about the effects of metal dyshomeostasis in AD and whether it plays a role in pathology. However, it is clear that amyloid fibrils have a detrimental effect on cognition. The panelists acknowledged that amyloid aggregation and metal dyshomeostasis may develop differently depending on the cause of AD. Patients with a genetic risk for AD are predisposed to amyloid aggregation and may develop metal dyshomeostasis as a secondary event due to aging. In sporadic cases of AD, however, metal dyshomeostasis and amyloid aggregation may develop concurrently over time.

Ionophore vs. chelator

Several audience members expressed concern that agents such as PBT2 might remove metals from the body and cause metal deficiency syndromes like anemia. The speakers clearly specified that PBT2 is not a metal chelator, a type of compound that irreversibly binds to and removes metals from the body, but an ionophore, a type of compound that delivers metals to cells. Although long-term results are needed to fully understand the safety profile of compounds like PBT2, preclinical and clinical analyses do not suggest metal depletion.

Because PBT2 has an intermediate metal-binding affinity, it is able to remove metals from protein aggregates and to deliver them to neurons. This mechanism is not expected to deplete metal concentrations in the body, but long-term safety data are needed. (Image courtesy of Rudolph Tanzi)

How can cognition be measured?

The phase IIa study of PBT2 assessed cognition by measuring executive function. In contrast, many phase III studies on AD use tools such as the Alzheimer's Disease Assessment Scale-Cognitive Subscale and the Clinical Dementia Rating Sum of Boxes, which measure memory, orientation, language, and other cognitive functions. Several audience members questioned whether the metrics used in phase III trials should monitor earlier symptoms instead. Executive function as well as biomarkers of disease progression may be candidates for phase III endpoints in the future, but these need to be validated and approved by the FDA.

The role of metals in Alzheimer's disease

Does the sequestration of metals by amyloid fibrils contribute to disease pathogenesis?

Are the effects of metal ionophores like PBT2 primarily due to the release of metals from Aβ or does metal homeostasis also play a role?

What are the best endpoints to monitor changes in cognition in clinical trials?

The role of metals in Huntington's disease

Is there any interplay between copper-mediated oligomerization of huntingtin and the poly-glutamine region?

Does PBT2 have a direct effect on iron in Huntington's disease or are its effects primarily mediated by copper binding?

Will clinical studies of PBT2 reinforce the promising results seen in preclinical analyses?

The role of metals in Parkinson's disease

What is the mechanism behind metal dyshomeostasis in Parkinson's disease?

Will metal-binding agents like PBT434 prove to be safe and effective in clinical trials of Parkinson's disease?