Alzheimer’s disease (AD) and Huntington’s disease (HD) neurodegenerative diseases involve loss of neurons in certain brain regions. Replacement of neurons in these diseases by stem cells differentiated into functional neurotransmitter phenotypes has high potential to provide stem cell therapy for these diseases.
The nervous system utilizes integrated actions of multiple neurotransmitters that mediate communication among neurons. It is, therefore, critical to identify factors that promote differentiation of human stem cells into systems of neurotransmitters present in normal neurons. However, most studies have examined functions of only one neurotransmitter at a time, rather the groups of transmitters that function together. It is,, therefore, important to assess factors that differentiate human stem cells into profiles of neurotransmitters that represent the normal neurons. Analyses of ‘systems of neurotransmitters’ requires advanced technology in mass spectrometry for determining profiles of neurotransmitters. With the strong and long-standing efforts of the Hook laboratory to investigate peptide neurotransmitters, we have implemented state-of-the-art peptidomic technology for high throughput LC-MS/MS mass spectrometry to identify neurotransmitters. This new state-of-the-art technology will be used for this project.
Therefore, the goal of this project will be to evaluate agents that transform human stem cells into neurons containing the ‘system of neurotransmitters’ present in the normal condition of brain regions affected in AD and HD. The specific aims will (1) test growth factors and related differentiating agents to induce human stem cells into neurotransmitter phenotypes of brain regions affected in AD and HD, and (2) compare the ‘system of neurotransmitters’ in normal human hippocampus and striatum with that in differentiated stem cells to guide differentiating conditions to generate the normal ‘system of neurotransmitters’ in selected human brain regions. Such differentiated cells may provide benefit for cell therapy to improve the health of patients affected with neurodegenerative diseases.
Statement of Benefit to California:
Numerous citizens in California are affected by devastating neurodegenerative diseases, including Alzheimer’s, Huntington’s, and Parkinsons’ disease. The unique opportunity for human stem cell therapy in California provides special research that can potentially improve health for California citizens affected by these neurodegenerative diseases.
The focus of this project to utilize novel peptidomic technology to understand the systems of neurotransmitters utilized in brain function is critical to guiding research to differential human stem cells into functional brain neurons. Achievement of the goals of this project is key to providing California citizens with the hope of new medical treatments using stem cells to relieve affected patients of the detrimental neurodegeneration in brain diseases.
SYNOPSIS: Dr. Vivian Hook from UCSD proposes to evaluate differentiating agents the transform hESC to neurons. These include growth factors, comparison of neurotransmitters. Dr. Hook is a well-known scientist who has published over 120 papers on neurochemistry of Alzheimer's disease and Huntington's disease. Specific factors planned for evaluation include neurotrophic factors NGF, BDNF, NT-3, PACAP, forskolin. The cells will be evaluated by the neurotransmitter systems they express in co-culture with PA6 cells, reported to facilitate neurotransmitter expression by cells. They will compare neurotransmitter systems expressed by hESC-differentiated neurons and actual differentiated neurons obtained from the brain.
SIGNIFICANCE AND INNOVATION: This is not particularly innovative nor original.
The recent advances in embryonic stem cell research raise high hopes for their future use in curing serious human conditions such as Alzheimer’s and Huntington’s diseases, both involving neuronal loss in certain brain regions. As has been demonstrated, it is becoming feasible to culture, under the right set of circumstances, the embryonic stem cells into neuronal cells, but how will they become functional to allow stem cell therapy?
To secure communication among neurons, most if not all “biochemical circuits” (including many neurotransmitters) must be in place. Precise measurements of many neurotransmitters under such circumstances are not yet at a point where they can inform studies wiith stem cells aimed at validating the derivation of functional cells for therapy. To her credit, Dr. Vivian Hook, an experienced neuropharmacologist, wishes to address this extremely complex set of neurobiological problems through the analysis of neurotransmitter profiles (“system of neurotransmitters”) and the use of differentiating agents. Besides the usual neurotransmitters such as catecholamines, there are many peptides representing largely still a terra incognita of the field. Consequently, such inquiries should be encouraged and supported.
As far as innovation is concerned, this proposal represents a fairly routine approach of acquiring stem cells in certain stages, analyzing their extracts for the numerous low-molecular weight constituents, and seeing eventually if “differentiating agents” change the substance profiles. The substance profiles will also be compared with those extracted from the brain tissue (hippocampus and striatum) of humans from “standard” sources, representing the sites of Alzheimer’s and Huntington’s diseases. It is commendable that the P.I. wishes to address the issue of profiles rather than individual neurotransmitters, one at the time. This, however, opens the proposal to a criticism that P.I. is ignoring perhaps the vast problems associated with peptide analysis in complex mixtures. While this is a worthwhile undertaking, this project is likely to move slowly without a collaboration involving stem cell specialists and an expert bioanalytical chemist.
STRENGTHS: The P.I. has assembled in her laboratory a research staff with considerable expertise, ranging from biology to mass spectrometry, to bioinformatics. Her plans addressing the whole profiles of neurotransmitters are a plus of the overall plan, as are the plans for comprehensive analyses of normal human brain tissues against the extracts from stem cells under differentiating conditions. The P.I. is a very experienced and productive investigator in the Alzheimer's and Huntington's disease fields. The P.I.'s laboratory has all the equipment and facilities to carry out the proposed studies.
Testing growth factors and differentiating agents to induce the desirable changes during cell culturing is viewed positively as a good strategy, and having a laboratory of this experience and capability characterize the neurons produced from hESC would be an advantage.
The project has relevance to improving human conditions in serious CNS diseases. It is uniquely suited for this California-based initiative, which is not easily fundable by federal grants.
WEAKNESSES: While this is clearly a multidisciplinary research project, the P.I. has decided to contain it solely in her laboratory. Consequently, the plans involving stem cell preparations are a bit sketchy. While the recently acquired LC/MS-MS system is state-of-the-art in the category of small instruments, it is not clear that the P.I. appreciates the difficulties associated with the MS analysis of trace levels of peptides in complex biological mixtures. The equipment and facilities appear adequate for this research. Dr. Steven Bark, a research scientist on the project has previous experience in proteomics (at Scripps), but his publication record is modest. This mulidisciplinary project would benefit from the participation of of expert collaborators in the areas of stem cell research and bioanalytics.
Additionally, profiling all small molecule neurotransmitters simultaneously is highly unlikely, as they are typically present in very different concentrations. How will be the “normal levels” of neurotransmitters established? On what criteria will be the “families” of peptide neurotransmitters established?
The proposal lacked clarity regarding what has already been done and tried by other laboratories.
DISCUSSION: The PI is an experienced neuropharmacologist but has little experience with hESC, and many similar studies are likely to exist in the literature already. This proposal was not well developed, and the go-it-alone strategy without expertise was not good. The quantitative neuropeptide analysis is an unknown, and the PI needs help to do it and much time. There was a general lack of appreciation for the technical problems that may be encountered. Many investigators are studying these factors and how they affect cells; the P.I. probably doesn't fully comprehend many of the challenges that are ahead.