Optimization of guidance response in human embryonic stem cell derived midbrain dopaminergic neurons in development and disease
Statement of Benefit to California:
SYNOPSIS: In order to facilitate the survival, integration, and axonal innervation of grafted dopamine neurons in the Parkinson's disease (PD) brain, it is important to understand the molecular milieu of the degenerating host brain before evaluating which cell is best to restore the dopaminergic nigrostriatal axis. This proposal focuses on two aspects of this molecular milieu: axonal guidance molecules and neuroinflammatory molecules. hES-derived DA neurons are looked at, and compared with ventral mesencephalon dopamine neurons, for the role of differentiation state in vitro and response to axon guidance (e.g. ephrins) and inflammatory (interlukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (Infγ) molecules. Three aims are proposed that 1. Confirm the roles, on hES-derived dopamine neurons, of certain guidance molecules established in previous funded studies on mouse cells; 2. Determine the influences of inflammation on DA neuron axon guidance; chemokines and immune effector. SIGNIFICANCE AND INNOVATION: This is a very innovative grant that will address the roles of axon guidance and neuroinflammation molecules in getting cells to respond to guidance cues. A co-culture method previously used by the PI, will now be used with hESC aggregates, and COS cells secreting guidance molecules. The idea is innovative and should yield novel and important findings on factors that direct guidance of DA axons. STRENGTHS: This proposal combines expertise from two well established and respected groups to study axon guidance and inflammatory molecules in a co-culture bioassay with hESC derived DA neuron precursors. A key strength is the inclusion of Mary Hynes, a senior researcher in the PI's lab, who is a world expert in dopaminergic neurons. She will be directing the proposed studies. The goal of this work is highly meritorious - to understand the potential neurodegenerative disease inflammatory environment of the PD brain, and shed light on how this environment influences guidance and connectivity for future studies that exploit genetic and drug-based approaches for maximizing stem cell based neuron replacement strategies. WEAKNESSES: A potential weakness is which dopaminergic neuron will one get from hESC differentiation. There are many different sub-types of dopaminergic neurons. Only a specific sub-type is lost in PD. Therefore it is critical to focus on ensuring that the differentiation protocol specifically targets this sub-type, and not just any neuron that makes tyrosine hydroxylase. This kind of differentiation has not yet been perfected and the applicant knows this and recognizes the challenge. Additionally, some of the previous studies showed effects of these molecules on midbrain DA neuron topography, so it is not certain how axons will be the only elements affected by these molecules. Also, there is little discussed with regard to diverse outcomes from studying “immune effector cells” on a heterogeneous population of hES-derived cells in these cultures. DISCUSSION: This proposal aims to investigate the guidance response in hESC-derived midbrain dopaminergic neurons in development and disease. The PI proposes to look at axon guidance molecules and the inflammatory response. Two issues were raised regarding the potential effects of these molecules, first with respect to axons being the only affected elements, and second with respect to the diverse outcome that may arise from treating a heterogeneous cell population. The PI should be able to overcome the latter. One suggestion was that the protocol of Ericson, Perlman and Studer should be used rather than the older protocols cited.