Translational Research

Discover what the previously known Alzheimer’s genes can teach us about Alzheimer’s disease pathology and identify the role of the newly identified genes.

Discovery of Alzheimer’s Disease Blood Biomarkers Using Phage Display Technology

Funding year(s): 
2013
Funding to date: 
$100,000

Absence of biomarkers has posed a formidable challenge in the development of effective treatment for Alzheimer disease (AD). Blood-based biomarkers could offer advantages that allow for early AD diagnosis and are critical in monitoring efficacy in clinical studies. Proposed studies aim to identify a set of novel blood biomarkers and examine their potential application as diagnostic agents. Phage display is a powerful approach to engineer peptides or proteins for binding to targets of interest. Therefore, we will apply phage display technology to identify peptides that selectively interact with molecules in AD blood samples, not in the age matched controls. In Aim 1, we will identify potential biomarkers by screening two libraries with a diversity of approximately 2 billion peptides against AD and control blood samples. To overcome the anticipated kinetic limitations with monovalent peptides, we will polymerize them by conjugation to dendrimers combined with functional moieties including fluorescent dyes for validation studies in Aim 2. These studies will identify a set of peptides that can be potentially used as diagnostic agents for AD. Furthermore, the proposed research is highly transformative and can be widely applied for biomarker studies in other human disorders. Overall, these proposed studies address a critical unmet medical need in AD by providing large sets of new biomarkers for rapid and accurate non-invasive diagnosis of AD using innovative approaches. 

Researchers: 

The Root of Alzheimer’s Disease: Purification and Characterization of Amyloid-beta Oligomers from the Human Brain

Funding year(s): 
2013
Funding to date: 
$100,000

Large, poorly soluble aggregates of the amyloid-beta peptide form the senile plaques that are a pathological hallmark of Alzheimer’s disease, but the extent of plaque deposition correlates only moderately with dementia; for example many middle aged and elderly people have extensive plaque deposition without any signs of dementia. Instead, several types of smaller water soluble amyloid-beta oligomers have been found to be more toxic than either plaques or amyloid-beta peptide monomers. Our collaborative group has recently developed a sensitive, specific, quantitative and high-throughput assay for amyloid-beta oligomers. We propose to use this assay to facilitate purification of amyloid-beta oligomers from human brain tissue. We expect that there will be substantial complexity in the Alzheimer’s disease brain, with multiple oligomeric species having varying structural properties and toxicities. Once purified, we will use mass spectrometry to characterize the structure and cell-based toxicity assays to quantitatively assess the function of each distinct type of oligomer. The first major outcome will be identification of critical post-translational modifications, associated proteins, and conformational epitopes in amyloid-beta oligomers that could be targeted by innovative therapeutics. The second major outcome will be determining which (if any) animal models accurately reproduce the amyloid-beta oligomers found in the human brain so that candidate therapeutics targeting these oligomers can be tested appropriately in vivo. It may be that entirely new animal models will be needed. Thus, if successful, this project will facilitate an entirely new wave of preclinical and clinical therapeutic development for Alzheimer’s disease.

Vascular Regenerative Therapy for Alzheimer’s Disease

Funding year(s): 
2013
Funding to date: 
$100,000

Alzheimer’s disease (AD) is a major cause of dementia in elderly. The amyloid-β (Aβ) peptides deposit in the brain parenchyma as senile plaques and in the cerebrovasculature as cerebral amyloid angiopathy (CAA), both are hallmarks of AD pathology. Epidemiologically, cerebrovascular damages caused by diabetes mellitus or stroke increase the risk for AD. Cerebral hypoperfusion precedes cognitive decline and neurodegeneration in AD. Our recent work has also demonstrated that cerebrovasculature plays critical roles in Aβ clearance. Therefore, we aim to develop novel regenerative therapy for AD by restoring cerebral vasculature function and neural integrity through transplantation of induced pluripotent stem cell (iPSC)-derived specialty cells. Specifically, we will co-inject iPSC-derived vascular progenitor cells (VPCs) and neural stem cells (NSCs) into mouse brain to promote synergistic effects for regeneration of both cerebral vessels and neurons as neurovascular interactions play critical roles in neurogenesis and angiogenesis. Therefore, the overall goal of this proposed study is to investigate the effects of regenerative therapy through transplantation of iPSC-derived VPCs and neurospheres on Aβ clearance, amyloid pathology and cognitive function in amyloid model mice. Our innovative approach could lead to development of novel therapeutic methods to treat AD.

Researchers: 

Characterization of the pathological significance of a novel type of vascular amyloid

Funding year(s): 
2013
Funding to date: 
$100,000

The amyloid Aß peptide is deposited in at least two distinct locations in AD brain:  Parenchymal plaques and vascular amyloid deposits in the wall of arterioles, where it is associated with vascular smooth muscle cell degeneration and stroke (Congophilic amyloid angiopathy, CAA).  While CAA is commonly found in AD brain, some human mutations within the Aß domain of the amyloid precursor protein (APP) cause CAA and stroke, rather than AD indicating that these diseases can occur independently. Using a conformation-dependent monoclonal antibody, M31, we have discovered a structurally unique type of Abeta deposit that is specifically associated with vessels.  This shows that a subset of the vascular amyloid is conformationally unique and raises the hypothesis that it may be associated with a unique type of pathogenesis.  The goal of this proposal is to examine the relationship of this unique vascular amyloid to AD and CAA pathogenesis and obtain preliminary data to support an NIH application with more mechanistic and translational aims.  The results of this study may lead to the development of immunological strategies to therapeutically target CAA and image its accumulation in human brain, allowing the pre-mortem diagnosis of vascular amyloidosis and the stratification of patients for human clinical trials for both AD and CAA.  

Researchers: 

Sleep and tauopathies: Effect of an anti-tau antibody

Funding year(s): 
2013
Funding to date: 
$100,000

In neurodegenerative diseases known as the tauopathies (e.g. progressive supranuclear palsy, Alzheimer disease), there is progressive degeneration of specific brain regions that account for the symptoms and signs of each disease.  Accumulation of aggregated forms of the protein tau in structures known as neurofibrillary tangles and dystrophic neurites in these brain regions correlates well with functional decline in cognition, motor, and other functions. Cell to cell transmission of tau aggregates leading to brain dysfunction is one hypothesis which may account for spread of pathology and progressive brain dysfunction.  Our recent data, now “in press”, showing the effectiveness of certain anti-tau antibodies as a potential therapy, supports this hypothesis. One difficulty in assessing the effects of therapies for neurodegeneration in animals is the lack of a strong, quantifiable, physiologically relevant phenotype.  Here, we seek to further characterize preliminary findings that a mouse model of tauopathy (P301S Tau Tg mice) develops both decreased NREM sleep as well as a marked decline in delta power during non-REM (NREM) sleep.  In addition, we will determine whether an anti-tau antibody, HJ8.5, that we have found to strongly decrease tau pathology, will prevent this sleep phenotype when administered peripherally.

Stem Cell Consortium

Funding year(s): 
2013
Funding to date: 
$600,000

Stem cells are the least mature cells in the body. Because these cells are so immature, they can be treated with a defined cocktail of factors and, depending on which factors are used and in what sequence, those factors can cause maturation of cells along discrete cell types. With a new tool called induced pluripotent stem cells, it now is possible to take skin cells from adults and return them to this immature state. By redirecting skin cells from Alzheimer’s patients and turning them into nerve cells, we are able to study adult Alzheimer’s neurons (nerve cells) in the lab. These Alzheimer’s neurons can be studied either in a dish or by transplanting them into the brains of host mice. 

Together the Cure Alzheimer’s Fund Stem Cell Consortium team—Drs. Scott Noggle, Kevin Eggan, Sam Gandy, Doo Kim, Rudy Tanzi, Tamir Ben-Hur and Marc Tessier-Lavigne—will develop, study and maintain Alzheimer’s neurons that will be used to screen for new drugs. This “Stem Cell Bank” can be used by these and other researchers around the world to advance drug screening much more rapidly. The first targets for such screening will be drugs that already have been proven safe in humans. Other targets will include compounds developed specifically for interruption of Alzheimer’s pathology. Most excitingly, new drugs will be based on new clues that will arise only from the study of these human Alzheimer’s neurons.

A. Specific Aims

Genetic approaches have provided major insights into the molecular pathogenesis of Alzheimer’s disease (AD). However, only about 3 percent of all AD is due to genetic mutations in either amyloid precursor protein (APP), or presenilin 1 or 2 (PSEN1, PSEN2). About 25 to 33 percent of all AD is associated with a polymorphism in the apolipoprotein E (APOE) gene, yet there is little consensus surrounding the molecular pathway(s) leading from APOEε4 alleles to an enhanced risk for AD. A particular promise for the recent success in differentiating skin fibroblasts into phenotypes of brain neurons provides an unprecedented and unequaled cell system for exploring AD pathogenesis in both familial and sporadic AD. We propose to generate a human in vitro model using induced pluripotent stem (iPS) cells, in which the genetic and developmental aspects of familial and sporadic AD can be studied more accurately and therapeutic targets can be identified for subsequent drug discovery. The cell-type-specificity of key AD risk molecules (e.g., APOE and astrocytes) dictates that the complete modeling of the AD brain in culture will require the generation of neurons and glia and the study of these cells in mixed cultures. Ultimately, we will transplant these neurons into mouse brain in order to study their molecular and physiological properties in vivo.

Specific Aim 1: Drs. Noggle and Eggan will generate iPS cells and neurons from skin fibroblasts from subjects with familial and sporadic AD. We already have succeeded in generating differentiated neurons from fibroblasts from subjects with PSEN1 mutations. We have demonstrated that differentiation of these neurons leads to their acquisition of an obvious standard molecular phenotype; i.e., a shift in the Aβ42/40 ratio). The initial essential standardization of these neurons will include, for each PSEN1 mutation, the exploration of intra-individual and inter-individual variability in the Aβ42/40 phenotype within patients, affected and unaffected family members, and across different families that carry either the identical mutation or across different PSEN1 mutations. Inasmuch as possible, priority will be given to the naturally occurring prevalence of PSEN1 mutations, although practical issues in acquiring skin fibroblasts also will be a factor. Once we have completed this survey of intra-individual vs. intra-mutation/inter-individual, and inter-mutation variability, we will expand our array of iPS cell lines to include patients with pathologically proven sporadic AD with segregation of analyses according to homozygosity or heterozygosity for APOEε3 or APOEε4 alleles. A longer-term goal will be the generation of glia and mixed cell cultures.

Specific Aim 2: Dr. Gandy will perform molecular, biochemical and functional characterization of AD iPS cell lines. We have defined a culture system for AD iPS cell-derived neurons that includes the essential Aβ42/40 phenotype. We now will proceed to establish the content of AD-related molecules in these iPS cells while seeking to establish the cell biological basis for the Aβ42/40 phenotype. This will include an assessment of the autophagic pathway. We will use this model system to define survival kinetics and molecular responses of AD iPS cells to apoptotic stresses, including neurotrophic factor withdrawal and addition of NGF or pro-NGF.

Specific Aim 3: Drs. Noggle, Eggan and Gandy will identify transcriptional and proteomic profiles of familial and sporadic AD iPS cells. Our primary goal in this aim is to establish a baseline molecular characterization of forebrain neural cells derived from the panel of iPS cell lines specified above. Informatic analysis of these profiles will be performed in order to identify possible AD-related networks, as recently defined by Geschwind and colleagues. We will examine how in vitro cellular and molecular phenotypes in telencephalic neural cells derived from patient iPS cells vary and are similar across individuals and mutations related to either familial or sporadic AD. 

Specific Aim 4: Dr. Kim will generate human neural progenitor (NP) cells overexpressing AD genes with familial mutations. We will establish AD cell models based on human NP cell lines established from fetal brains, embryonic stem (ES) and iPS cells. These NP cells will be transfected with the constructs designed to overexpress human APP with KM670/671NL (Swedish) and V717I (London) mutations (APPSweLon) and/or presenilin 1 with Delta E9 (PS1dE9) familial AD mutation. To enhance the AD pathology, we also will co-express APP and PSEN1 constructs with multiple familial AD mutations. Plasmids and lentiviral expression vectors will be used for the transient and/or stable expression of the select AD genes. The NP cells will be differentiated into neurons in vitro and the expression of neuron/astrocyte/oligodendrocyte markers will be measured. AD pathological markers will be analyzed by ELISA, immunohistochemistry and Western blot as summarized in Diagram 1. In these cells, we will also test the effects of γ-secretase inhibitors/modulators on AD pathology, including Aβ accumulation.

Specific Aim 5: Drs. Tanzi and Kim will analyze pathological changes of AD NP cells in vivo. In this aim, we will establish a method to analyze AD pathology of AD NP cell models in adult mouse brains. Using AD NP cell models that are developed in Aims 1, 3 and 4, we will test whether these

cells can differentiate and develop AD pathology in vivo. AD NP and the control cells will be engrafted into hippocampal/cortical regions of mouse brains. In addition to young and aged wild-type mice, young Tg2576 mice that would show high concentration of soluble brain Aβ species will be used. These models would enhance AD pathology of the engrafted AD NP cells. We will analyze pathogenic AD markers, including Aβ42/40 levels, amyloid plaque load, synaptic dysfunction and neurodegenerative changes in one to six months after the NP cell injections (Diagram 1).

Specific Aim 6: Dr. Ben-Hur will identify pathologic functional properties of human AD cells that affect their bilateral interactions with brain environment. Neural precursors in the neurogenic niches of the adult brain have neurotrophic properties that are important for maintaining the physiologic homeostasis in the normal adult brain. We will test the hypothesis that AD pathogenesis is related in part to either abnormal trophic homeostatic support by neurogenic niches, and/or that AD neurons are deficient in their response to environmental support. To that end, we will use in vitro co-culture systems and transplantation experiments into adult mouse brains to examine how AD NP cells affect neurogenesis and neuronal fate in normal and pathological conditions. Reciprocally, we will compare how AD neurons (vs. normal neurons) survive and connect in the brain environment.

Specific Aim 7: Dr. Tessier-Lavigne will derive PSEN1-mutant neurons in two distinct ways, i.e., from induced pluripotent stem cells (iPSCs) or directly from fibroblasts by trans-differentiation. His lab then will characterize the epigenetic signatures of these neurons and determine whether the two reprogramming techniques yield phenotypically similar neurons or if one set more closely resembles adult, aged neurons from diseased patients.

Stem cell funding $600,000

Please note: The $600,000 'funding to date' includes $100,000 given independently to the Harvard Stem Cell Institute for this project plus $100,000 given in 2012 to the Rockefeller University for related research.
 

 

The roles of Eps homology domain (EHD) proteins and synaptic activity in axon transport of the Alzheimer’s β-secretase BACE1 in the brain

Funding year(s): 
2012 to 2013
Funding to date: 
$200,000

The membrane-bound aspartic protease 13-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the 13-secretase enzyme that generates the first cleavage in the formation of the 13-amyloid (AI3) peptide from APP (1). Thus, BACE1 is a prime therapeutic target for Alzheimer's disease (AD). However, BACE1 inhibitors with drug-like properties that cross the blood-brain barrier (BBB) have proven difficult to develop. Although the first BBB-penetrant BACE1 inhibitors are currently entering clinical trials in humans, we are still years away from knowing whether any will be successful in treating or preventing AD. Meanwhile, it is of paramount importance to study the cell biology of BACE1 to fully elucidate its mechanism of action in Al3 generation, for deep understanding of factors that regulate BACE1 trafficking and access to APP substrate in neurons of the brain may uncover novel, effective, and practical AD therapeutic targets.

The current proposal aims to elucidate the roles of Eps homology domain (EHD) proteins and synaptic activity in BACE1 axon transport in the brain and is linked to the application of our collaborator Dr. Gopal Thinakaran (U. Chicago) to determine the function of EHD proteins in Al3 production and amyloid deposition in vivo. EHD proteins regulate dynamic BACE1 axon transport in primary hippocampal neurons in vitro (manuscript in preparation). In addition, synaptic activity controls Al3 generation in vivo (2). Here, we will investigate the dependence of BACE1 axon transport on EHD function and synaptic activity in the hippocampus, a brain region critical for memory formation that is severely affected in AD. We hypothesize that inhibition or stimulation of EHD protein function or synaptic activity will decrease or increase hippocampal BACE1 axon transport, respectively. Our Specific Aims are: 1) Determine whether EHD proteins regulate dynamic BACE1 axon transport in ex vivo hippocampal slice cultures, and 2) Determine whether synaptic activity regulates dynamic BACE1 axon transport in ex vivo hippocampal slice cultures in an EHD-dependent manner. Our studies together with those of Dr. Thinakaran's proposal will increase our understanding of Aj3 production in the brain and may reveal new therapeutic strategies for AD.
 

Researchers: 

BACE1 transcytosis in Alzheimer’s disease pathogenesis

Funding year(s): 
2012 to 2013
Funding to date: 
$200,000

Many lines of evidence suggest that beta-amyloid peptides cause neuronal damage and affect fundamental memory processes early in the course of Alzheimer's disease (AD). Two membrane-associated enzymes, namely betasecretase (BACEl) and gamma-secretase are responsible for beta-amyloid production. Understanding the details regarding the cellular and molecular mechanisms involved in beta-amyloid production in neurons is a topic of central importance in molecular AD research. Many investigators have studied the membrane transport of amyloid precursor protein in cultured cell lines and neurons in order to ascertain where in neurons this protein is processed by BACEl and gamma-secretase. There is a general agreement in the field that amyloid precursor protein is transported along the nerve fibers (called axons) and is proteolytically converted into beta-amyloid near axon terminals, termed presynaptic sites. BACEl has been found in neuronal dendrites and axons (the two types of neuronal projections). How BACE1 is transported in axons is not clearly understood. Recent findings from our lab suggest that BACEl is transported in membrane organelles called recycling endosomes in neurons cultured from embryonic mouse hippocampus. Moreover, we found evidence for a highly polarized transport of BACEl from the cell surface of dendrites towards axons (a process termed transcytosis). The goal of this proposal is to characterize the functional significance of polarized BACE1 transport in neurons. Specifically, we propose to interfere with BACEl transcytosis in cultured hippocampal neurons and in brains of transgenic mice to test our hypothesis that this process contributes to neuronal beta-amyloid production and deposition.

Our proposal is timely, unique and highly innovative because BACEl transcytosis in recycling endosomes has never been described. Our proposal is also highly significant because we employ both in vitro and in vivo models to investigate the molecular and cellular mechanisms involved in neuronal BACE1 trafficking that is functionally important for Af3production. This is a novel and exciting area of research, and we feel that our investigation will uncover significant insights on cellular and molecular mechanisms that are relevant to AD pathogenesis.
 

Researchers: 

Aβ Oligomers and the Pathogenic Spread of Tau Aggregation: Implications for Alzheimer’s Disease Mechanism and Treatment

Funding year(s): 
2012 to 2013
Funding to date: 
$251,000

The goal of this project is to conduct a series of experiments designed to elucidate the role of Abeta and exosomes (vesicles involved in “cell-to-cell signaling”) in the transfer of Tau clumps from nerve cell to nerve cell.

Two proteins are known to be critically involved in Alzheimer’s disease: Abeta and Tau. Both are prone to “self-associate,” such that in the Alzheimer brain clumps of Abeta, known as amyloid plaques, are found in the spaces in between nerve cells and clumps of Tau, known as neurofibrillary tangles, are found within nerve cells. Until recently it was assumed that Abeta had to form plaques to be toxic; however, it is now clear that smaller, mobile clumps of Abeta (referred to as oligomers) are also damaging. When Dr. Walsh’s lab isolated an oligomer from a human brain composed of just two Abeta molecules (referred to as Abeta dimer) and injected it into rats, it caused amnesia. Studies also show that lowering Tau levels can protect nerve cells against the toxic effects of Abeta oligomers. These data indicate that Abeta oligomers cause changes in Tau that harm brain cells. In parallel, evidence has emerged that clumps of Tau can be passed from one nerve cell to another. Indeed this process may explain why neurofibrillary tangles appear to spread through the brain as the disease progresses.

Thus understanding how Tau pathology is “transmitted” and, if Abeta is involved, should identify novel targets for therapeutic intervention. For instance, if Abeta is found to cause the release of Tau via small membranous vesicles known as exosomes, it should be possible to prevent either the release of Tau-containing exosomes or their uptake by unaffected recipient cells. If this is possible, drugs designed to prevent the spread of Tau pathology should halt further cognitive deterioration. Accordingly, this project will include a series of experiments designed to elucidate the role of Abeta and exosomes in the transfer of Tau clumps from nerve cell to nerve cell.

Potential for Host Cytotoxicity from Microbially-derived Abeta Oligomers

Funding year(s): 
2009
Funding to date: 
$250,000

Alzheimer’s disease (AD) is the most common form of dementia in the elderly afflicting over 20 million people worldwide. Two decades of findings from cell biology, genetic, neuropathological, biochemical and animal studies overwhelmingly point to the β-amyloid peptide (Aβ) as the key protein in the disease’s pathology (see review by Hardy and Selkoe, 20001). Aβ appears to be a soluble component of normal brain. However, in AD brain the peptide accumulates as β-amyloid, an insoluble semi-crystalline deposit that is the hallmark of the disease pathology. The most pathologically important forms of Aβ appear to be oligomers that are intermediates between insoluble β-amyloid and normal soluble monomeric species. Mounting evidence suggests that these soluble, low molecular weight oligomeric forms of Aβ are the critical cytotoxic species mediating neuronal death in AD. Of particular interest are soluble cross-linked β-amyloid protein species (CAPS) containing between 2 and 12 cross-linked Aβ subunits. CAPS, particularly dimeric2,3 forms, are highly neurotoxic. CAPS are also abundant in vivo with dimeric species alone comprising as much as 40% of the total Aβ pool in late stage AD brain. While the mechanism of Aβ cytotoxicity remains contentious, evidence is accumulating that membrane permiabilization plays a key role in the pathological activity of the peptide. In this study we propose to focus on role of Aβ oligomerization in the Aβ-mediated disruption of lipid bilayers.

The planned experiments will use many of the methods and techniques we have
developed in our previous CAF-funded project. Experiments will test our own CAPS
preparations as well as material from collaborators, including the “prion”-like Aβ oligomers generated in Dr Charles Glabe's laboratory at the University of California-Irvine. Immunochemical, chromatographic and electron microscopic techniques will be used to characterize Aβ oligomers. Characterization experiments will include immuno studies using conformation-dependent antibodies developed by Dr. Glabe’s laboratory. Antimicrobial activities will be tested using published assays previously employed in our study that identified Aβ as an AMP.