Mechanisms that govern dopaminergic amacrine cell diversity

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Project Summary: Dopamine signaling in the retina is crucial for regulating circadian rhythms and circuit reconfiguration for daytime vision. Reduced dopamine signaling is associated with many visual disorders and pharmacological therapies using dopamine analogues have shown promise for treating diseases like diabetic retinopathy. The primary source of retinal dopamine is the dopaminergic amacrine cells (DACs). This project is focused on elucidating molecular mechanisms that govern the specification of these specialized amacrines. This knowledge will enable the development of novel therapeutics that harness the power of regenerative medicine to regrow repair or replace the dopaminergic amacrine cells and restore dopamine abundance to normal levels. Toward this goal, we conducted a screen to identify molecules that, when deleted, increase dopaminergic amacrine cell formation. We uncovered a candidate signaling pathway controlled by the serine- threonine kinase LKB1. When LKB1 is deleted embryonically there is an approximate doubling of the dopaminergic amacrines that persists into adulthood. Concomitantly there is an increase in dopamine when measured by High Performance Liquid Chromatography. We hypothesize that LKB1 signaling is activating developmental programs that restrict the abundance of the DAC population. Here we set out to determine whether LKB1 signaling is required intrinsically in amacrine cell precursors as well as the temporal requirement for LKB1 signaling to restrict DAC formation. We will also leverage the ability of super resolution STORM microscopy to visualize the distribution and release of dopamine in the newly generated DACs. Our second aim is to identify the downstream effectors from LKB1 that are required to restrict DAC formation. We will utilize loss of function and rescue experiments of known molecules downstream from LKB1 such as AMPK to test their contribution to DAC formation. We will also conduct single cell RNA sequencing to compare the transcriptional landscapes between DACs in control vs mutant retinas. This will allow us to find additional downstream candidates from LKB1 required for restriction of the abundance of DACs. This analysis is compelling from the basic science perspective but will also define novel molecular targets for mitigating dopamine neuron loss and restoring visual capacity. This knowledge may be useful for preventing dopaminergic neuron defects beyond the eye, such as those that occur in Parkinson’s disease.