From localized tubulin synthesis in yeast driven by microtubule associated prions (Pub1 + Sup35) to an orthologous PTSD synaptic tag (TIA1 + GSPT2)
Abstract
PTSD affects 10-20% of military personnel involved in combat missions, and 5% of the general US population. While there has been progress in understanding the association of PTSD with stress and fear memory and in recognizing brain areas and neuronal circuits affected by traumatic events, knowledge of the underlying molecular mechanisms of PTSD and PTSD genetics is lagging. As a consequence, there is no PTSDspecific pharmacotherapy. The best treatment option is psychotherapy requiring hours of one-on-one interaction with a highly qualified specialist and this is not always available. Slow acting antidepressants approved for PTSD treatment have modest effects at most. The long-term goal of this project is to understand the molecular mechanisms that drive the continued activity of fear memory synapses in order to stimulate the development of PTSDspecific medications. Another goal is to contribute to the genotypebased prediction of the likelihood of PTSD onset after traumas. The proposed hypothesis to understand intrasynaptic molecular structures driving PTSD builds on the theory of a (synaptic tag) first proposed by F. Crick in 1984. This proposal extends the expectations for such a tag. Beyond being anchored at the synapse and capable of self-perpetuation, it should contain transcripts for intra-synaptic localized protein synthesis and the machinery for such localized protein synthesis. In previous studies the PI and collaborators identified a cytoskeleton-anchored microtubule-associated complex in yeast that contains Pub1 and Sup35. Both Pub1 and Sup35 carry prion-domains that drive their co-aggregation and association with the complex. This suggests a two-protein self-perpetuating prion-like assembly. The complex also contains components indicative of functional ribosomes. The only mRNA identified was TUB1, which suggests localized tubulin synthesis at the site of microtubule assembly. Evidence from the literature and PI’s preliminary studies suggest that mammalian neurons contain a similar complex, satisfying all proposed criteria for a synaptic tag. Indeed, the Pub1 ortholog, TIA1, and the neuron-specific Sup35 ortholog, GSPT2 (but not the ubiquitous Sup35 ortholog, GSPT1), carry prion domains and can join the yeast complex. While a neuron-specific function of GSPT2 is still unknown, several lines of evidence identify TIA1 as a likely PTSD gene. Recently, the PI identified a spontaneous mutation that promotes the assembly and function of the abovementioned yeast complex containing Pub1 and Sup35. The mutation simultaneously drives prion formation by Sup35 and stabilizes centromeric plasmids, which requires proper functioning of tubulin cytoskeleton. Whole genome sequencing followed by conventional gene cloning will be used to identify the gene containing this yeast mutation, as well as its mammalian ortholog(s). MassSpectrometry and RT RNA sequencing followed by qualitative and quantitative Westerns and RT PCRs will allow the identification of all the protein and RNA components of the complex. Forward and reverse genetic screens will elucidate the assembly and function of the complex in various growth conditions, including stress. Biochemical approaches will attempt to prove that localized protein synthesis indeed occurs on microtubules. To the best of our knowledge, this would be the first demonstration of localized protein synthesis in yeast. One of the main focuses of the project is to understand the role of the two-protein (PUB1/SUP35+) prion in this process. While currently proposed studies will use the yeast experimental system, mammalian orthologs, including TIA1 carrying PTSDrelated polymorphisms, will be employed throughout the study. The results will allow transfer of this research into neuron experimental systems and eventually aid in PTSD drug development. Importantly, based on the findings from this project, future drug screens can be initiated in an easily tractable
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 28, 2023
- Source ID
- W911NF2310122
Entities
People
- Irina Derkatch
Organizations
- Army Contracting Command
- United States Army
- University of Nevada, Reno