Imaging Downstream Intracellular Signals Activated by Axon Guidance Cues in ES-derived Dopaminergic Neurons

Lay Summary

Can Cellular Imaging Provide Pivotal Information for Improving Stem Cell Therapy in Parkinson’s Disease?

Researchers will use “fluorescence imaging biosensors” to visualize biochemical signals within neurons derived from embryonic stem cells to test whether these neurons respond appropriately to molecular guidance cues.  The guidance cues to be tested regulate the orientation the axons (communication cables) of developing and regenerating neurons, which controls neural network formation, necessary for stem cell therapy to benefit patients with Parkinson’s disease.

Parkinson’s disease progressively depletes neurons that use the transmitter dopamine to communicate with one anther.  While the drug L-DOPA is effective in controlling symptoms for a long period in most patients, eventually the drug’s effectiveness is severely compromised.  Researchers have been experimenting with stem cell therapy to determine whether the stem cell-derived neurons can effectively be reincorporated into the correct neural networks. To do so, stem cell-derived neurons need to respond to environmental factors that attract of repel outgrowth, to guide axon extension in the correct direction. Improperly activated signals can cause mis-wiring of the axons.

The researchers hypothesize that modulation of specific signaling pathways must occur with precise spatial and temporal control, as occurs in developing neurons.  They will use fluorescence imaging to determine whether two key biochemical signals are activated normally within dopaminergic neurons derived from stem cells.  If improper activation of one or both signals occurs, this information will help to identify the proteins that are necessary to effectively guide the axons of stem cell-derived neurons to their appropriate targets in Parkinson’s disease.

Significance:  This research provides a new direction for correcting defects in cultured stem cells that might be used to treat Parkinson’s disease.

Abstract

Imaging Downstream Intracellular Signals Activated by Axon Guidance Cues in ES-derived Dopaminergic Neurons

Parkinson's disease (PD) is a progressively debilitating neurological disorder that results from the loss of dopaminergic (DA) neurons of the ventral midbrain.  One promising treatment of PD patients is stem cell therapy, which requires the integration of new DA neurons into existing circuits.  For cell therapy to succeed, new DA neurons derived from embryonic stem cells (ESC) must respond appropriately to extracellular guidance information.  Two extracellular factors that guide fetal DA neurons are Netrin and Slit.  However, a recent study using dopaminergic neurons derived from embryonic stem cells found that these neurons do not respond to chemotropic gradients of Netrin or Slit.  Netrin and Slit are important chemotropic factors that promote attractive and repulsive turning, respectively.  Interestingly, Netrin and Slit regulate axon outgrowth by bi-directionally modulating tyrosine phosphorylation and Cdc42 activity (Netrin increases and Slit decreases).  Here we will test whether these two fundamental downstream signals of Netrin and Slit are locally modulated in primary neurons and DA neurons derived from ESCs.

To complete this study, we will use novel fluorescence biosensors to visualize in live developing neurons tyrosine phosphorylation and Cdc42 activity.  Our published and preliminary data demonstrate that we can measure dynamic changes in Src kinase-dependent tyrosine phosphorylation and Cdc42 activity with high spatial and temporal precision in living cells.  In Aim 1, we will examine whether localized changes in tyrosine phosphorylation and Cdc42 activity correlate with chemotropic turning of growth cones of primary embryonic neurons in gradients of Netrin and Slit. In Aim 2, we will perform similar experiments using dopaminergic neurons derived from mouse embryonic stem cells.   This research will establish techniques to visualize the temporal and spatial dynamics of key downstream signals of both attractive and repulsive guidance cues and test whether these signals are locally generated in neurons that are necessary for the functional recovery of patients with Parkinson's disease.