Parkinson’s disease is caused by the death of neurons in the substantia nigra. These neurons extend projections, called axons, to another region of the brain called the corpus striatum. Here, they release dopamine, an essential chemical required for smooth and coordinated movement. Loss of 80% of the dopamine producing cells leads to the symptoms of Parkinson’s disease which include muscle rigidity, tremor, and uncoordinated movement. Current therapy involves supplementing the dwindling dopamine production with drugs, but this therapy loses effectiveness over time. Surgical options also exist, such as neurostimulation from battery-operated implants, but surgical complications occur in addition to problems associated with implanted hardware such as broken or dislocated wires. Successful long-term treatment has been attained for a subset of patients using fetal midbrain tissue grafts that reconstitute the axonal pathway, but general use of this therapy has been limited by requirements for fresh human fetal tissue. Consequently, there are clear advantages to using sources of dopaminergic neurons for transplantation, such as human embryonic stem cells (hESC), that can be perpetually grown in tissue culture Great strides toward treatment have recently been made in our ability to reliably differentiate hESCs into dopaminergic neurons, but ultimately, therapeutic success will require reconstituting the precise neuronal connections with engrafted hESC-derived dopaminergic neurons. In the nervous system, neurons and their axons make appropriate connections by following cues present in the extracellular environment. Studies on dopaminergic neurons have shown that they are guided by two families of environmental cues called Netrins and Slits. In this application, we propose to investigate the response of hESC-derived dopaminergic neurons to the Netrin and Slit cues. Neurons respond to cues based on the expression of receptors on their cell surface. Consequently, in Aim I, we propose to determine the profile of receptors expressed on dopaminergic neurons. Studies on mouse embryonic stem cells suggest that at least one receptor for each family of cue will be expressed by hESCs. In Aim II, we propose to evaluate how hESCs respond to Netrin and Slit cues, using an assay in which aggregates of hESC-derived dopaminergic neurons are placed in a 3-dimensional matrix near a point source of Netrin and Slit. We will determine how axons of these dopaminergic neurons respond to cues by recording the migration behavior of the axons, toward or away from, the point source. If the dopaminergic neurons do not respond, we have previously identified methods to stimulate the neurons to generate a response. Finally in Aim III, we propose to observe the response of hESC-derived dopaminergic neurons to Netrins and Slits that are present in adult brains by transplanting the neurons into mice and evaluating the response of their axons one and four weeks post-transplantation.
Statement of Benefit to California:
The citizens of California voted to support research on potential stem cell therapies that can be used to treat serious medical conditions that cripple millions of Americans. Parkinson’s disease is one of these conditions. The experiments proposed in this application tackle key issues that must be resolved before neurons derived from human embryonic stem cells can be successfully used to treat this neural disorder. Recently, researchers have developed ways to reliably differentiate human embryonic stem cells into the type of neurons, dopaminergic, that degenerate in patients with Parkinson’s disease. This application addresses the next step required for the development of successful therapeutic strategies. These strategies will require that transplanted dopaminergic neurons respond robustly and appropriately to environmental cues in the patient’s brain so that they supply the crucial chemical, dopamine, to the correct target. Parkinson’s disease is one of the illnesses in which a stem cell-based therapy is within grasp. An early success in using human stem cells to treat a devastating illness will be inspirational to California citizens.
SYNOPSIS OF PROPOSAL: Dr. Lindsay Hinck from UC Santa Cruz proposes to study netrin and slit receptor expression in dopaminergic cells grown from hESC, to assess the response of the cells grown in 3D collagen gells containing point sources of netrin and slit, and assess the behavior of differentiated hESC cells implanted into immunocompromised mice. INNOVATION AND SIGNIFICANCE: This is an innovative and original proposal. While most researchers have selected dopaminergic cells for transplantation into animal models (and human cases) of Parkinson's disease, they have seldom considered other factors that might account for the success or failure of the grafts. In these experiments, the investigator has proposed that two well-known guidance molecules, Netrin and Slit, are important for the successful use of dopaminergic neuronal transplants. They will study Netrin and Slit receptor expression in cells differentiated from hESC line, assess their response to these two guidance molecules in vitro, and then test the hypothesis in vivo. The project may lead to advances in understanding progress of and developing treatments for neurodegenerative diseases such as Parkinson's Disease. The approach has been used for various types of neurons previously. It may be interesting to learn if Netrins and Slits also affect hESC-derived DA neurons. The proposal is moderately innovative in that this approach has never before been applied to hESC lines, and is potentially significant. STRENGTHS OF PROPOSAL: The proposed research addresses an important problem: the selection of hESC derived dopaminergic cells for transplantation for treating Parkinson's disease. Results are expected to lead to advances in understanding dopaminergic neuron axonal guidance and the role of Netrins and Slits as cues. The proposed experiments are clear, focused, and feasible with likely successful outcomes. The PI's laboratory is in a unique position to answer the question. He is an experienced and productive investigator (10 years experience in field), who trained as a postdoctoral fellow with Marc Tessier-Lavigne (1994-1998) where the applicant and his colleagues cloned the netrin receptor and developed numerous reagents to study the receptors, including antibodies against the receptors and cues, stable cell lines expressing netrins and slits, blocking reagents (ectodomain constructs), and a viral system to transfect neurons. He has experience with performing all the proposed assays. WEAKNESSES OF PROPOSAL; The proposed research has no clear hypothesis, is not feasible, as presented and is NIH fundable. The research design is not well described and not well thought through. There is a lack of details. For example, the PI proposes to use SSEA as a marker. Which SSEA? There are several of them and not every one is a marker for the same hESC state. The PI proposes to transplant terminally differentiated neurons - this may lead to cell death upon transplantation and/or a very low yield of cells surviving the procedure. A large amount of DA neurons will be required - will need high yield and relatively pure populations of DA neurons. The PI does not describe how the DA neurons will be recognized. The PI and team do not have any direct hESC experience, nor do they have a collaborator experienced in hESC culture. There is only a graduate student on the team who is currently scheduled to be trained in a hands-on hESC course in Wisconsin. The PI allocates only 11% of her time and states that she will be responsible for scientific, financial, and administrative tasks for this project. This may not be enough commitment. DISCUSSION: To do the type of study proposed, one would need a high yield differentiation method for differentiating hESC to dopinergic neurons. There is no high yield dopaminergic neuron differentiation protocol currently. Also, applicant does not state how cultures will be characterized. It was mentioned that transplantation of dopaminergic neurons can be problematic as terminally differentiated neurons do not transplant well. The investigator was regarded as experienced but new to hESC field, exactly the kind of researcher the RFA was hoping to attract. There was some question about what is the review panel's philosophy regarding the value of testing any given morphogenic molecule on hESC. A point was raised that simply calling something a dopaminergic neuron is misleading as there are many types of dopaminergic neurons - that making TH is not sufficient to make a neuron a 'true' dopaminergic neuron, noting that olfactory bulb neurons are dopaminergic - but are not the same dopaninergic neurons as those that are lost in Parkinson's Disease.