Microtubule Deficit in Glaucoma
Glaucoma is one of the leading causes of blindness worldwide, in which the retinal ganglion cells and the axons (i.e., the retinal nerve fibers) are gradually lost. At the time of diagnosis, nearly half of the retinal ganglion cells are irreversibly lost. The pathogenic mechanism is unknown, but evidence suggests that microtubules in the retinal nerve fibers are compromised in the process. Our hypothesis is that microtubule disruption is reversible and occurs before the loss of cells (‘microtubule hypothesis of glaucoma’). To test the notion, we developed a novel retinal imaging based on second-harmonic generation (SHG) from microtubules (Lim et al., Opt. Lett. 2012), whose unique properties allow measurement of new aspects of the cytoskeleton, for example, molecular conformation (Sharoukhov et al., J. Mod. Opt. 2016). From SHG imaging of the DBA/2 retina, a mouse model of glaucoma, we found compelling supports for the microtubule hypothesis: Microtubule deficit occurs earlier than the loss of the retinal ganglion cell axons, and shares a common pathogenic insult as the loss of the retinal nerve fibers (Sharoukhov et al., IOVS 2018). Unraveling the role of microtubule deficit in the progressive cell death could offer tremendous opportunities for diagnosis and neuroprotection against glaucoma.
Deciphering Transcription with Live Retina/Brain Imaging
Transcription, the first step in gene expression, is intricately regulated to enables specific cells to respond appropriately to the organism’s activities. Despite rapid advances in RNA sequencing, we still lack the knowledge of how genes are expressed in a cell-type-specific and activity-induced manner. It is a fundamental question for understanding the retina and brain, where diverse types of cells are organized in a hierarchy best to react to the sensory stimuli and cognition. Also, dysfunctional transcription regulations are known to underlie many neurological disorders. To decipher cell-type-specific and activity-induced transcription, we are developing novel methods to listen in the process in situ, as it occurs in the native tissue where the definition of cell types is preserved. It is based on the MS2 tagging (pioneered by R.H. Singer at Albert Einstein College of Medicine), where multiple copies of MS2 binding sequence (24xMS2) are inserted in the gene of interest and then the RNA stem loops are recognized by MS2 capsid protein tagged with green fluorescent protein (MCP-GFP). The intense fluorescence from multiple MCP-GFPs allows detecting single RNA molecules and, ultimately, the regulation of transcription process. In collaboration with the Singer Lab, we demonstrated the first proof of principle of visualizing nascent transcripts in live mouse by two-photon microscopy (Park et al., Science 2014, Nwokafor et al., Methods 2019). Now we are innovating the next-generation live RNA imaging to learn more about controlling gene expression in the retina/brain, which could guide gene therapy.
