Parkinson's disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
The mechanisms of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in seven genes, including ?-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
Parkinson's disease (PD) is the second leading neurodegenerative disease with no current cure available. Compared to other states, California is the highest in the incidence of this particular disease. First, California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the US (Cal Pesticide use reporting system). A commonly used herbicide, paraquat, has been shown to induce parkinsonism in both animals and human. Other pesticides are also proposed as potential causative agents for PD. Studies have shown increased PD-caused mortality in agricultural pesticide-use counties in comparison to those non-use counties in California. Second, California has the largest Hispanic population. Studies suggest that incidence of PD is the highest among Hispanics (Van Den Eeden et al, American Journal of Epidemiology, Vol 157, pages 1015-1022, 2003). Thus, finding effective treatments of PD will significantly benefit citizens in California.
This proposal seeks to create human cell-based models of Parkinson’s disease (PD) by deriving human embryonic stem cells (hESCs) from affected embryos and by creating induced pluripotent stem (iPS) cells from patient fibroblasts. PD is the most common neurodegenerative movement disorder and human cell-based models would provide new tools for investigating the cellular and molecular pathology of this disease as well as support the discovery of new therapeutics. The principal investigator (PI) has access to preimplantation genetic diagnosis (PGD) embryos and fibroblasts from families with well-defined mutations in two PD genes. Once pluripotent stem cells are derived, the PI intends to differentiate these cells into dopaminergic neurons, and to examine the effect of the mutations on cell survival in response to chemicals known to induce PD in humans.
Reviewers agreed that PD cell lines would be a valuable resource, and that the experimental design to generate the cell lines is realistic. However, the reviewers identified several weaknesses in the proposed analysis. The major drawback of the proposal is the lack of a well laid out experimental plan in Aim 3 to investigate the cellular and molecular pathology of PD neurons. The only outcome measure to be analyzed is cell death. However, a measure of cellular dysfunction is necessary to better model the disease phenotype. Furthermore, reviewers criticized the lack of experience of the PI with deriving hES or iPS cells, and expressed concern that the highest percentage of cells differentiating into dopaminergic neurons reported by the applicant is lower than that reported by other laboratories, and is not sufficient for biochemical studies. Finally, a reviewer was concerned about the use of iPS cells for the analysis of PD mechanisms, since the effect of multiple viral insertions may influence that analysis.
The responsiveness to the RFA was considered appropriate. The applicant proposes to assess pluripotency and the generation of mutation-bearing pluripotent human cell lines from patients with specific diseases would seem to be one of the most exciting applications of the re-programming technology.
Programmatic Discussion: A motion was made to recommend that this application be moved to Tier 1 – Recommended for Funding. A reviewer noted that a main reason why this application had a relatively low score was due to the proposed use of cell death as the only endpoint to investigate the pathology of PD neurons. However, reviewers agreed that despite this weakness, the application was very strong. A panelist asked whether embryos carrying the PD mutations had already been identified. This was affirmed by a reviewer who emphasized that the applicant has access to genetically well-defined material, an important advantage of the present proposal. The motion to move this application to Tier 1 carried.
Reviewer One Comments
PD lines would be a valuable resource, but two issues need to be considered. First, will these really be better than mouse lines with the same mutations? The authors argue that human cells will better model the disease, but you might argue that, with their much shorter life span, mouse cells might be better for looking at the mechanisms of neurodegeneration. While I do accept the need for human cells, it would have been nice to have seen this addressed. Second, while the use of the PGD embryos is a major strength of the application and a novel feature, if it does not work then re-programming is the likely way forward. What would be the effect on the cells of the multiple viral insertions? Would this affect their analysis and utility for looking at mechanisms of PD?
The design to generate the cell lines is realistic, but there are two weaknesses in the analysis. First, the only outcome measure to be analyzed is cell death. A measure of dysfunction might also be valuable to better model disease activity. Second, the optimum level of differentiation into dopaminergic neurons is apparently 30% (and even this is not shown in the preliminary data). This is, as the authors acknowledge, not enough for biochemical studies. Other laboratories have reported much higher percentages in recent years, and different methods could be explored. Further issues regarding feasibility are:
1) The lack of any re-programming evidence in the preliminary data,
2) The rather modest publication record of the PI (although there are two good 2006 papers on PD mechanisms),
3) Issues of consent for the PGD embryos. Are there really going to be no problems associated with this?
Regarding resources, it is not made clear why three postdoctoral assistants are required, or what their respective roles will be.
Responsiveness to RFA:
This is fine. The applicants do propose to assess pluripotency and the generation of mutation-bearing pluripotent human cell lines from patients with specific diseases would seem to me to be one of the most exciting applications of the re-programming technology.
Reviewer Two Comments
This proposal seeks to create human cell-based models of Parkinson’s disease (PD) by deriving hES cells from affected embryos and by creating induced pluripotent stem (iPS) cells from patient fibroblasts. PD is the most common neurodegenerative movement disorder and human cell-based models would provide new tools for the understanding the cellular and molecular pathology of this disease as well as perhaps help discover new therapeutics.
This proposal is clear, focused and well designed. Given the preliminary data, previously published results, and expertise of the group the proposed experiments are likely to be successful. The major drawback of the proposal is the lack of a well laid out experimental plan (Aim 3) to investigate the cellular and molecular pathology of PD neurons. An additional minor concern is the lack of experience of the principal PI in deriving hES or iPS cells, which means it would be necessary to rely on collaborators’ expertise.
Responsiveness to RFA:
The proposal is highly responsive to the RFA.