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.

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

Researchers: 
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.
 

BACE1 transcytosis in Alzheimer’s disease pathogenesis

Researchers: 
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.
 

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.
 

Antibody Signature of Alzheimer’s Disease: Promise of an Early Diagnostic Test

Funding year(s): 
2012
Funding to date: 
$100,000

A physician can’t cure what he can’t diagnose. The diagnosis of Alzheimer's disease is based on the exclusion of several neurological syndromes, rather than directly testing for the disease of interest. This can be an inaccurate exercise in up to 20% of the cases. Promising biomarkers are being developed, such as the cerebrospinal fluid profile of beta amyloid and tau proteins, as well as amyloid imaging with positron-emission tomography. However, these tests are not universally available and have some disadvantages, including the need for a spinal tap or the injection of radioactive material. A plasma biomarker capable of identifying asymptomatic individuals developing Alzheimer's-type pathology is needed, as they are ideal targets for intervention (i.e., amyloid-binding therapies) to prevent dementia or delay its onset. PSEN-1 mutations cause a predictable onset of mild cognitive dysfunction by age 40, followed by frank dementia a few years later. If characteristic biomarkers accompany different disease stages, these patterns could guide clinicians in the future to decide when to pursue more elaborate tests such a spinal tap and PET scans.  Immunosignaturing, a technology that employs antibody binding to a random-peptide microarray, is capable of generating profiles that distinguish transgenic mice engineered with familial Alzheimer’s disease mutations (APPswe and PSEN1-dE9) from non-transgenic littermates. The signature is distinguishable in transgenic mice as early as 2 months of age and intensifies as animals grow older. Immunosignatures can also distinguish individuals with non-genetic Alzheimer’s disease from non-demented elderly controls. In this project, we will evaluate whether late-stage Alzheimer’s disease patients with presenilin-1 (PSEN-1) mutations have a different signature as compared to young non-demented PSEN-1 carriers. In addition, we will assess the differences between the signature of demented patients with PSEN-1 mutations and elderly Alzheimer’s disease patients without PSEN-1 mutations. We will also investigate whether age-matched individuals without the mutation can be distinguished from asymptomatic carriers. Finally, we will determine if patients with different PSEN-1mutations born and raised in different continents (North America and South America) have similar signatures. This will be a collaborative project between 4 institutions: Arizona State University (Tempe, AZ), Banner Alzheimer’s Institute (Phoenix, AZ), Universidad de Antioquia (Medellin, Columbia) and UCLA Medical Center.

iPS-derived and trans-differentiated human neurons as models to study Alzheimer’s disease

Funding year(s): 
2012
Funding to date: 
$100,000

Recent groundbreaking work in stem cell biology has made it possible to reprogram non-neuronal cells obtained from Alzheimer’s diseased patients into neurons. For the first time, the research community has the means to study diseased human neurons from Alzheimer’s patients. These models have already yielded novel insights into the disease. However, different reprogramming techniques and various sources of cell material have been used to generate these models, and it is currently unclear whether one approach provides an advantage over the other (in terms of phenotype robustness and disease-relevance). Here, we propose to derive PSEN1-mutant neurons in two distinct ways, i.e., from induced pluripotent stem cells (iPSCs) or directly from fibroblasts by trans-differentiation. We will then characterize the epigenetic signatures of these neurons and determine if the two reprogramming techniques yield phenotypically similar neurons or if one set more closely resembles adult, aged neurons from diseased patients.

The Putative Role of Red Blood Cell CR1 levels in Amyloid Beta Clearance and Alzheimer’s Disease Pathogenesis.

Researchers: 
Funding year(s): 
2012
Funding to date: 
$100,000

The immune system uses complement proteins and receptors to “coat and clear” pathogens and proteins from the body. Complement Receptor 1 (CR1/CD35) is found on the surface of red blood cells in humans and helps shuttle cellular debris to the liver for degradation. Recently, specific genetic variations, called polymorphisms, in the CR1 gene were found to be associated with an increased risk of late-onset Alzheimer’s disease. We hypothesize that people with AD-risk CR1polymorphisms have low levels of CR1 protein on their red blood cells and therefore, are less efficient at clearing amyloid-â protein (Aâ) throughout life, gradually leading to Aâ aggregation and deposition in the brain. To test this hypothesis, we will examine Aâ and CR1 in archived human brain and measure the amount of CR1 molecules on red blood cells in individuals with and without AD-risk CR1polymorphisms.

The role of PICALM in vascular clearance of amyloid-β

Funding year(s): 
2012
Funding to date: 
$100,000

PICALM, the gene encoding phosphatidylinositol binding clathrin assembly (picalm) protein, plays a key role in endocytosis, a process which regulates the function of cell receptors and synaptic transmission. PICALM is one of the most highly validated Alzheimer’s disease (AD) risk factors. Its role in AD, however, is unknown. A recent genome-wide screen for modifiers of amyloid-b peptide (Aβ) toxicity in yeast has identified the key role of the yeast homologue of PICALM. This study has shown that PICALM efficiently controls Aβ toxicity in yeast, nematode models and mammalian neurons by regulating endocytosis-dependent cell vulnerability to Aβ. Our preliminary data in human and mouse brain show that picalm protein is most abundantly expressed in blood vessels which have been shown to provide a major pathway for Aβ removal from brain into the bloodstream. Therefore, picalm in brain endothelium is ideally situated to participate in Aβ clearance from brain. Interestingly, our pilot data also show significantly reduced picalm expression in brain vessels in AD. Previous findings have established that low density lipoprotein receptor (LRP) in brain endothelium mediates vascular clearance of Aβ from brain via transport across the brain capillary endothelium, a site of the blood-brain barrier (BBB) in vivo. Our preliminary data using human brain endothelial cells show that PICALM is required for rapid endothelial internalization of Aβ after its initial binding to LRP. The current proposal will determine the role of PICALM in regulating internalization and transcellular transport (transcytosis) of LRP-bound Aβ across the endothelial cell wall of the BBB in vitro and in vivo. To test our hypotheses we will use a human model of the BBB and a mouse model of Aβ clearance, both developed in our laboratory. In collaboration with Dr Tanzi we will study the effects of novel PICALM mutations on amyloid-β vascular clearance once the sequence of these functional variants/mutations becomes available. The proposed studies will represent a novel advance in our understanding of the molecular regulation of CNS Aβ homeostasis and will demonstrate a pivotal role of PICALM in controlling brain Aβ.

General Anesthetics and Alzheimer’s Disease

Funding year(s): 
2012
Funding to date: 
$100,000

The goal of this project is to test the hypothesis that desflurane is a safer anesthetic than isoflurane for AD patients in order to find safer anesthetics that won’t worsen AD symptoms.

Age is one of the most important risk factors for Alzheimer’s Disease (AD), with an incidence of 6.8 percent in people older than 65 years. One-third of all anesthetics are administered to people older than 65. Therefore, it is inevitable that many older patients who present to anesthesiologists will have AD. Just as the anesthesia specialty became intimately involved with the management of coronary artery disease (CAD), it is time for the anesthesiology specialty to develop guidelines for safer anesthesia care for AD patients. As the first step of these efforts, Dr. Zhongcong Xie and his fellow researchers will set out to identify anesthetics that will exacerbate the AD pathology, such as neuronal death, increases of Abeta levels, learning/memory impairment and synapse loss.

In their preliminary studies, they found the inhalation anesthetic isoflurane, but not desflurane, can induce cell death and increase Abeta levels in the cultured cells. In this application they will repeat these experiments in the mice having AD pathology (Aim #1) and in real human AD patients (Aim #3). In addition, they will study the up-stream mechanism of the anesthetics-induced cell death and increases in Abeta levels (Aim #2). The hypothesis they will test is desflurane is a safer anesthetic than isoflurane for both AD patients and normal patients. The anticipated results from the proposed studies will finally help them find safer anesthetics, which may not worsen the AD symptoms (e.g., learning/memory impairment). These efforts are consistent with the goals of Cure Alzheimer’s Fund research grant program in identifying the risk factors of AD and in finding the prospects and strategies for the prevention of AD, which, ultimately, will help AD patients.