Heat Shock Protein Expression in Drosophila melanogaster as a Model System of Glial Tauopathy
Student name: Jaasiel Alvarez
Neurodegenerative diseases, defined as proteinopathies, contain filamentous protein aggregates that are associated with lesions in the brain. One class of neurodegenerative diseases includes tauopathies, which are characterized by aggregates of the microtubule-associated protein tau, in both neuronal and glial cells. Protein chaperones are vital for maintaining proteostasis, thus allowing proteins to function as needed. Specifically, heat shock proteins (HSP) play an essential role in sustaining proteostasis, supporting protein folding and preventing degradation that leads to misfolding, aggregation. However, Hsps are unable to regulate tau proteotoxicity, which we hypothesize to be due to dysregulation of the chaperone network.
In order to examine the effects of tauopathies on the broad HSP response in the brain, we employed Drosophila melanogaster as a model organism which exhibits disease associated genes that are the equivalent to at least 70% of those in humans. Through the use of the GAL4/UAS system we overexpressed the human wild type tau isoform 0N4R in the flies and aged female groups to Day 10 (Tau D10) and Day 30 (Tau D30) at 25°C to allow GAL4 to remain in a DNA-binding permissive conformation. Tau D10 and Tau D30 female fly groups, along with their accompanying control groups (Control D10 and Control D30), after aging were each divided into two groups. One group was exposed to a 37°C heat shock (HS) and the other group was exposed to no heat (NH) and kept at 25°C. Drosophila heads from all groups were processed via homogenization in TRIzol, chloroform extraction, RNA extraction, purification and were all subjected to qRT-PCR in order to synthesize cDNA. Interestingly, each Hsp had a unique heat shock response and there was no consistent up or downregulation across the different Hsps. Our data were consistent with previous studies where s constitutive Hsps did not undergo large changes in expression, except for early (D10) tau expression. Notable results include: no difference in the expression levels of Hsp26 across tau expression and age, whereas Hsp70 exhibited significant changes in expression due to both the presence of tau and aging variables. Furthermore, we analyzed basal expression levels across age, and tau vs control and found that in Hsp27 there was extremely low basal expression for Tau D10 flies. Global Hsp responses are significant as the network of chaperone interactions are involved in proteostasis. Therefore identifying the cause and cascade of tau’s self-aggregation is imperative to developing therapeutic measures to target the chaperone network and ultimately these aggregation diseases.

Developing a robust system for genetic detection of RNA-binding protein
Student name: Linda Wang
Suxuan Wang, Chandra M. Gravel and Katherine E. Berry
RNA-binding proteins play important roles in gene regulation across all domains of life. In order to detect and characterize bacterial RNA-protein interactions in a native cellular environment, our laboratory has developed a bacterial three-hybrid (B3H) assay that connects the strength of RNA-protein interactions inside of Escherichia coli cells to the activity of a reporter gene. In this B3H assay, plasmids encoding two hybrid proteins (DNA-RNA “adapter” and RNAP- bound “prey”) and one hybrid RNA (“bait”) are transformed into E. coli reporter cells and the strength of the RNA-protein interaction is linked to the transcriptional output of a reporter gene. With this system, interacting RNAs and proteins do not need to be purified and their interactions can be assessed inside living bacterial cells. Since its inception, our B3H assay has been used exclusively with a lacZ reporter, encoding β-galactosidase. While β-gal assays and blue/white screens are effective for testing the effects of site-directed mutants, screening small libraries, several exciting potential assay applications would require alternate reporter genes and detection methods: for example, screening of large libraries to uncover rare interaction partners, mapping mutational fitness landscapes of RNA-protein interactions, and directed evolution to generate novel RNA- binding proteins with new properties.
In order to expand the applications of the B3H assay to higher-throughput screening and selection strategies, this study aims to develop reporters with both dual-fluorescence reporters and the selectable reporter gene tetA. We have designed and cloned dual-fluorescent reporters (using sYFP2 and mKate2) to quantitatively report on the real-time protein-RNA interactions in vivo and provide an intrinsic negative control. These initial constructs show promising results in the detection of protein-protein interactions using a related two-hybrid assay. Ongoing work aims to optimize the genetic background of these reporters and benchmark them using a variety of fluorescence detection methods against established B3H interactions between ProQ and Hfq and their respective RNA ligands. The ability to utilize fluorescence-activated cell sorting (FACS) or selectable markers (e.g. tetA) to examine larger library sizes will be an important advance in expanding the applications of our B3H assay, and will support our ongoing goals of searching for new RNA-protein interactors and identifying protein and RNA variants with desirable molecular phenotypes.

Sexually Dimorphic and Age-Dependent Heat Shock Protein Induction in a Drosophila melanogaster Model of Glial Tauopathy
Student Presenter: Margot Whitmore
Project Advisor: Dr. Kathryn McMenimen
Tauopathies are a family of neurodegenerative diseases which includes Alzheimer’s disease, frontotemporal lobe dementia, and progressive supranuclear palsy. This subclass of diseases is characterized by the aggregation of hyperphosphorylated tau in neurons and glia. Heat shock chaperone proteins (HSPs) are able to help maintain proteostasis by helping to monitor protein synthesis, folding, trafficking, assembly of quaternary structures, turnover, and clearance of toxic aggregates1 . However, these protective mechanisms are insufficient when responding to tau proteotoxicity.
Further complicating the matter, many neurodegenerative diseases present sexual dimorphisms in their onset, progression, and severity. Additionally, studies have shown that certain HSPs can become overwhelmed by stress as the system ages which in turn causes a decline in the global defense mechanism2 . As a result, this study seeks to uncover how glial tau aggregation, age, and sex concurrently impact the chaperone system by performing a holistic screening of HSP induction in a Drosophila melanogaster model.
Using the repo-GAL4, UAS system, the human tau isoform 0N4R was expressed in a subset of D. melanogaster glia. Multiple experimental variables were examined including; glial tau aggregation, age, sex, and exposure to heat shock were varied to determine the effects of these variables on HSP response. mRNA was extracted from the central nervous system of these samples via TRIzol homogenization and isopropanol precipitation, and all samples were subjected to qRT-PCR. Finally, relative fold expression and basal expression were determined to approximate the protein response. Current results highlight each individual heat shock chaperone’s unique contribution to the proteostasis machinery, as responses vary greatly across the HSP family. Furthermore, in female flies, the age-dependency of the HSP response was emphasized by the significant decline in Hsp70 and Hsp27 induction in day 30 flies compared to d10 flies. Ongoing experiments are looking to elaborate sex-specific responses; however, preliminary data suggest that female flies exhibit a more pronounced generalized upregulation of HSPs in response to heat stress.

Title: Exploring the Relationship Between Calcium and Dysmenorrhea: Understanding the Role of Nutritional Intervention
Student: Valentina Shrum
Dysmenorrhea, or menstrual pain, affects a large number of women worldwide. While there are various treatments available, there is limited research on the potential role of calcium consumption in alleviating menstrual pain. This research proposal aims to investigate the relationship between calcium consumption and dysmenorrhea in women of reproductive age. The study will involve a cross-sectional survey of women aged 18-45 years old, who will be asked to provide information on their dietary habits, including calcium intake, and their experience of menstrual pain. Data will be collected using a self-administered questionnaire and analyzed using statistical software. The proposed research will contribute to our understanding of the potential benefits of calcium consumption for the management of dysmenorrhea. If the findings demonstrate a significant association between calcium intake and reduced menstrual pain, this could inform the development of new strategies for managing dysmenorrhea. Additionally, this study will raise awareness about the importance of a balanced diet in managing menstrual pain and may encourage individuals to consider their calcium intake when addressing menstrual pain. Overall, this research proposal aims to improve our understanding of the potential role of calcium consumption in the management of dysmenorrhea, and could ultimately lead to the development of new treatment approaches for menstrual pain.