SYNOPSIS: Three isoforms of apolipoprotein E (apoE) play broad-based roles in lipid homeostasis, neuronal repair and neuronal remodeling. However these three isoforms differ in their ability to accomplish these tasks and one of them (apoE4) is a major risk factor for Alzheimer's disease (50% of all AD cases). The proposal is to identify the molecular and cellular mechanisms by which apolipoprotein E4 (apoE4) appears to impair hippocampal neurogenesis in vitro and in vivo, and to determine if apoE4-induced learning and memory deficits in mice can be rescued by stimulating endogenous hippocampal neurogenesis or by transplanting mouse or human neural stem cells (NSCs) expressing apoE3 into the hippocampus. This proposal is based on the PI’s potentially interesting preliminary results using apoE knockout (KO), and knock-in (KI) (isoforms 3 and 4) mice to study adult forebrain neurogenesis. These gain of function and loss of function studies in mice show that apoE4 modulates hippocampal neurogenesis in complex ways. On one hand, apoE4 knockout mice produce a surplus of astrocytes at the expense of neurons in the hippocampus. This observation suggests that expression of apoE4 is required for correct fate choice decisions in neural progenitor cells. On the other hand, expression of apoE4 inhibits neuronal maturation by impairing the function of GABAergic interneurons in the hilus of the hippocampus. The proposed studies could shed light on the pathogenesis of AD and establish a foundation for developing stem cell therapy for AD patients that carry the apoE4 gene, as well as possibly other brain disorders/injury.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The studies address the important question of how apoE4 is detrimental to the brain, both in AD and other brain disorders. Since apoE4 is also associated with poor clinical outcome and with earlier onset, progression, or severity of head trauma, stroke, Parkinson’s disease, MS and ALS, the proposed studies could also provide important information for the development of stem cell therapies for neurological disease other than AD. The concept is one the PI has worked on for many years and the approaches use standard techniques in mice.
The first specific aim addresses the molecular mechanisms whereby either a deficit or surplus of apoE4 can impair hippocampal neurogenesis. A number of different experiments are embedded within this first aim. These include: 1) determination of the mechanisms by which apoE deficiency inhibits neuronal, but stimulates astrocytic, differentiation of NSCs in vitro and in vivo; 2) determination of the mechanisms by which apoE4 impairs GABA-mediated neuronal maturation of adult NSCs in vitro and in vivo and 3) determination if the inhibitory effect of apoE4 on GABA-mediated neuronal maturation is specific for hippocampal NSCs or occurs also in the subventriciular zone (SVZ). Given that most of the studies are performed in vivo, it is difficult to assess whether the effect is directly related to the ApoE and GABAnergic systems. This first aim alone is complex enough that it could have been fleshed out to be the basis of an entire grant. The point was made that without establishing this first aim, the rest of the proposal might not be significant.
The second specific aim is to determine whether stimulating endogenous hippocampal neurogenesis improves learning and memory defects associated with apoE4 knock in mice. In preliminary studies, treatment of apoE4-K1 knock in mice with the GABA receptor agonist pentobarbital rescued impairment of hippocampal neurogenesis. Another way to induce hippocampal neurogenesis is via exercise. Other tactics to induce hippocampal neurogenesis include virus mediated (somewhat unproven) over expression of noggin or of neuregulin. The applicant will treat mice with all of these regimens and monitor performance by conventional methods such as the water maze test and fear conditioning.
The point was made that the preliminary study showing GABAnergic stimuli increases neuronal maturation is difficult to understand as written. Given that this was the major finding supporting the proposed studies for this aim, a reviewer would have liked the facts of this study to be more clearly conveyed. Other points made by the reviewers include the following. Pentobarbital prolongs the opening of GABA A receptor and it is not an agonist. Barbiturates also act through potent (if less well characterized) mechanisms and direct inhibition of excitatory AMPA-type glutamate receptors, resulting in a profound suppression of glutamatergic neurotransmission. Muscimol may be used instead. Also, it is hard to believe that a one time treatment with these compounds causes these dramatic changes. How many times were the animals treated and how many animals were used in this study? Also, many of the studies proposed in aim 2 (e.g., stimulation of neurogenesis by exercise and over expression of noggin or neuregulin) have nothing to do with the proposal’s hypothesis regarding the mechanism of action of apoE4 in AD pathogenesis.
The final specific aim is to determine if learning and memory defects associated with apoE4 in apoE KI mice can be rescued by transplanting neural stem cells that express apoE into the hippocampus. Mouse neural stem cells expressing human apoE3 will be cultured from the hippocampus of apoE3 knock in mice. Their human counterparts will be developed in vitro from embryonic stem cells that have been screened for the apoE genotype. This aim is the most difficult.
At a global level, reviewers struggled with several aspects of the proposal. The proposal was based on potentially interesting preliminary results. The applicant has the expertise with help of colleagues to carry out the studies. However, the innovation level is not that high, and the techniques proposed are are all standard. . Also, the behavior assay of improved learning and memory deficits may not be sensitive enough to identify positive effects. The biggest concern was that the reviewers found the proposed research diffuse, very broad and overly ambitious. They indicated that the first one or two aims could easily be turned into an entire grant proposal if better organized and described.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR:
The applicant received his M.D. and Ph.D. degrees in China. Dr Huang has been studying apoE biology since receipt of his doctorate in 1991 -- first at the University of Munster in Germany and since 1995, at the Gladstone Research Institutes, initially as a postdoctoral fellow with Mahley. Dr. Huang received numerous prizes and awards for scholarship during his postdoctoral training periods. He has risen through the ranks at the Gladstone and has been an Assistant Professor since 2005. During his time at the Gladstone, he developed mouse (and rabbit) models to study apoE3/4 function in neurodegeneration and more recently in neurogenesis (J. Neuroscience). A reviewer noted that the preliminary data to this proposal was only presented at the Society for Neuroscience meeting in 2006 and has not been published in a peer reviewed journal. Since starting his own independent laboratory in 2004, he has been senior author of seven research articles on the role of apoE4 in AD pathogenesis and on evaluating apoE4-related targets for AD drug development. These articles have appeared in well-regarded journals such as J. Biol. Chem., PNAS and J. Neuroscience. He is a Co-PI on two grants. He was invited to chair scientific sessions at two international meetings and to present at eight international or national scientific meetings. He is also an inventor on seven issued or pending patent applications.
The applicant has provided an acceptable outline for career development in the years ahead. Based on his preliminary findings, he states he has decided to redirect his research toward stem cell biology and therapy, still focusing on apoE4-related AD pathogenesis and treatment. His key goals are (1) to become an established stem cell biologist, (2) to advance stem cell therapy for neurodegenerative diseases, and (3) to train the next generation of biomedical scientists in stem cell research. He has asked two senior investigators at Gladstone – Drs. Shinya Yamanaka and Deepak Srivastava, both internationally recognized experts in stem cell research – to serve as his mentors. They will have periodic meetings with him to review and evaluate the progress of his stem cell research program and to provide suggestions and comments on future directions and plans. The milestones are realistic and achievable.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The Director of the Gladstone Institute of Neurological Disease has provided a letter documenting the institutional commitments. Some of the reviewers thought that the letter was less than clear as exactly how the institute would support the applicant’s development in stem cell research. Based on his achievements during the past 3 years, the University recently approved his accelerated promotion from Assistant Professor, Step 2 to Step 4. Dr. Huang is provided with adequate laboratory space (-800 sq. ft.), office space, and administrative support in their new research facility at UCSF’s Mission Bay campus plus shared core facility. He has all the equipment necessary to successfully complete the proposed studies. Huang is one of the participating principal investigators in the Gladstone CIRM Shared ES Cell Core Laboratory (hES Cell Biology, Physiology, Genomics and FACS sorting) and has full access to the Gladstone Neurobehavioral Core Laboratory. He also has access to a range of pertinent cores and services, including a pathogen-free animal care facility. His chairman states that Dr. Huang’s formal teaching and administrative requirements are minimal, which will allow him to dedicate most of his time to research and hands-on mentoring in the laboratory.
Over 60% of the 21 Gladstone laboratories are engaged in some aspect of stem cell research. In addition at UCSF Mission Bay, over 20 laboratories have research programs related to stem cell research or regenerative medicine, and the proposed core will provide critical services to these laboratories as well. Both Gladstone and UCSF Mission Bay are slated for significant growth in human ESC research over the next five years. Within the Gladstone Institute of Neurological Disease, along with Huang, Robert Mahley, Fen-Biao Gao and Li Gan have focused their ESC research on neurodegeneration and repair.
DISCUSSION: The reviewers all felt that the proposed research was diffuse and overly ambitious for the proposed time period and not very innovative although all acknowledged that the knock-out model was an interesting model for the study of AD. The applicant was considered good and productive but not outstanding.