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.