Parkinson’s disease (PD) is a neurodegenerative movement disorder that affects 1 in 100 people over the age of 60, one million people in the US and six million worldwide. Patients show a resting tremor, slowness of movement (bradykinesia), postural instability and rigidity. Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). There are actually two groups of midbrain dopaminergic (DA) neurons, and only one, those in the substantia nigra (SN) are highly susceptible to degeneration in Parkinson’s patients. There is a relative sparing of the second group and these are called ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside in different regions of the adult ventral midbrain and importantly, they deliver dopamine to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine.
A major therapeutic strategy for Parkinsons’ patients is to produce DA neurons from human embryonic stem cells for use in transplantation therapy. However early human trials were disappointing, since a number of patients with grafts of human fetal neurons developed additional, highly undesirable motor dyskinesias. Why this occurred is not known, but one possibility is that the transplant mixture, which contained both SN and VTA DA neurons, provided too much or unregulated amounts of DA (from the VTA neurons), overloading or confusing the target region in the brain that usually receives dopamine from SN neurons in small, regular quantities. Future human trials will likely utilize DA neurons that have been made from human embryonic stem cells (hES). Since stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to specifically produce SN neurons and not VTA neurons from stem cells for use in transplantation. However, although we can produce dopaminergic neurons from hES cells, to date the scientific community cannot distinguish SN from VTA neurons outside of their normal brain environment and therefore has no ability to produce one selectively and not the other. We do know, however, that these two populations of neurons normally form connections with different regions in the brain, and we propose to use this fact to identify molecular markers that distinguish SN from VTA neurons and to determine optimal conditions for the differentiation of hES to SN DA neurons, at the expense of VTA DA neurons. Our studies have the potential to significantly impact transplantation therapy by enabling the production of SN over VTA neurons from hES cells, and to generate hypotheses about molecules that might be useful for coaxing SN DA neurons to form appropriate connections within the transplanted brain.
The goal of our work is to further optimize our ability to turn undifferentiated human stem cells into differentiated neurons that the brain can use as replacement for neurons damaged by disease. We focus on Parkinson’s disease, a neurodegenerative disease that afflicts 4-6 million people worldwide in all geographical locations, but which is more common in rural farm communities compared to urban areas, a criteria important for California's large farming population. In Parkinson’s patients, a small, well-defined subset of neurons, the midbrain dopaminergic neurons have died, and one therapeutic strategy is to transplant healthy replacement neurons to the patient. Our work will further our understanding of the biology of these neurons in normal animals. This will allow us to refine the process of turning human embryonic stem cells onto biologically active dopaminergic neurons that can be used in transplantation therapy. Our work will be of benefit to all Parkinson's patients including afflicted Californians. Further, this project will utilize California goods and services whenever possible.
A hallmark of Parkinson's disease (PD) is the death and degeneration of midbrain dopaminergic (DA) neurons in the substantia nigra (SN) and a relative sparing of DA neurons in the ventral tegmental area (VTA). This proposal seeks to identify molecular markers that distinguish SN from VTA neurons and to determine optimal conditions for the differentiation of human embryonic stem cells (hESCs) to SN DA neurons, at the expense of VTA DA neurons. In Aim 1, the applicants propose to test the hypothesis that axon guidance molecules may be used as molecular markers for identification of specific subpopulations of DA neurons. In Aim 2, the applicants plan to determine whether anatomically distinct subgroups of DA neurons respond differently to axon guidance ligands. Finally, in Aim 3 the applicants will use the information obtained in Aims 1 and 2 to develop protocols for the preferential generation of SN DA neurons from hESC.
In terms of impact, reviewers agreed that the proposal is highly innovative and addresses a major unsolved problem in stem cell-based therapies for PD. Reviewers found the fundamental idea of this proposal to be very intriguing and sophisticated. Since SN neurons have different functional properties than VTA neurons, reviewers supported the hypothesis that dyskinesias, observed in some PD patients, may have resulted from transplanting a mix of the two sub-populations of DA neurons rather than only those that degenerate in the disease. Consequently, generation of pure populations of SN DA neurons may provide cells that have the required functional capacity to cure the disease without causing such dyskinesias. This concept has great appeal experimentally and has been posited by others. Furthermore, since no molecular markers have yet been identified to distinguish SN from VTA neurons, a prerequisite for developing protocols to produce those neurons from stem cells, reviewers considered the proposal highly relevant and logically developed, and appreciated that the proposed identification of such markers and optimization of hESC differentiation protocols is based on a fundamental understanding of the biology of the developing brain.
Reviewers found the proposal to be very well written, and its feasibility to be supported by a sound experimental plan and outstanding preliminary data. Although the studies in Aim 1 were considered technically very demanding, all the proposed studies fall within the applicant's skill set. Despite the observation that Aim 3’s success depends on the progress attained in Aims 1 and 2, reviewers believed the risk/benefit ratio of this study was very favorable. Reviewers expressed confidence that what is proposed will be accomplished and is likely to yield significant results.
The principal investigator (PI) is a distinguished developmental neuroscientist, and his/her qualifications to carry out the work described here are outstanding. The collaborators are highly qualified as well, and the scientific environment for developmental neurobiology is exceptional. A reviewer mentioned that this is the PI’s first endeavor in disease-related research, but given the PI’s immense credentials, this foray is promising as the PI brings fresh ideas and expertise into an important area.
In summary, this application centers on the goal to identify markers of different DA neuron sub-populations, and to use that information to develop protocols for the generation of pure preparations of SN DA neurons from hESC for transplantation purposes in PD. Reviewers found this proposal to be exceptional as it addresses a critical problem, has a sound approach, assembles a well qualified team of researchers, and contains a cogent experimental design.