Regeneration of axons is an intrinsic ability of the peripheral nervous system; however, this process is slow and can be incomplete, leading to functional impairments. New therapeutic applications are necessary to improve the speed and efficacy of repair. Previously, the Butler laboratory has shown that cofilin, an actin severing protein, and its negative regulator LIM domain kinase 1 (Limk1), can be used to regulate the rate of axonal growth in mice. We are now assessing whether increasing the levels of cofilin can accelerate the axonal regeneration process after a sciatic nerve injury. This study aims to determine whether increasing cofilin activity enhances motor axon regeneration in the lower leg muscles. To investigate this, we used a Thy1::YFP reporter mouse line to reliably and precisely label motor axons. We chose viral approaches to induce the expression of a constitutively active form of cofilin in these mice using an adeno-associated virus (AAV), AAV9::cofilinS3A-mCherry. By tracking motor axons labeled with Thy1::YFP and co-expressing the mCherry-tagged transgene, we will assess regenerating markers in these axons to evaluate the extent of axonal regrowth and determine whether cofilin positively influences motor axon regeneration.

Prostate cancer (PCa) is one of the most common cancer-related causes of death in American men. Genetic variation and aging are major biological drivers of PCa. The National Comprehensive Cancer Network genetic mutation testing panel began to include variations of Homeobox B13 (HOXB13) in recent years. HOXB13 is a key transcription factor highly localized to the prostate. Studies have identified distinct heritable HOXB13 variants that contribute to increased PCa risk and early PCa onset. However, very few studies have investigated the mechanisms of early PCa onset driven by HOXB13 variants. Previous study has shown that the aging prostate contains an increasing proportion of progenitor cells that are susceptible to PCa. Interestly, our preliminary data show selected HOXB13 variants inducing progenitor-like phenotypes observed in aged prostate cells. Therefore, we hypothesize that the HOXB13 variants upregulate aging/progenitor features that make prostate cells more susceptible to cancer initiation. To test this hypothesis, we engineered non-malignant human adult prostate organoids to express selected HOXB13 variants. We found that the HOXB13 variants upregulate progenitor-like phenotypes and gene expression patterns. Additionally, the HOXB13 variants downregulate tumor suppressor TP53, suggesting that these variants make prostate cells more susceptible to cancer initiation. Uncovering the role of distinct HOXB13 variants in PCa initiation will aid the development of more targeted PCa prevention, diagnosis and treatment.

Melanoma is a type of skin cancer, yet it is one of the most lethal forms of cancer which evades current treatments. In melanoma tumor microenvironment, autophagy, a pathway through which cells sequester proteins and organelles into vesicles that fuse with lysosomes and release energy is significantly upregulated. Thus, targeting autophagy in the tumor microenvironment might be a potential therapeutic option. Our preliminary data show that loss of autophagy in the entire mouse or the tumor microenvironment results in reduced growth of melanomas. Moreover, T cell depletion results in larger tumors in autophagy-deficient mice. T cells are activated by the MHC-antigen complex on antigen presenting cells like dendritic cells (DCs). We report that autophagy ablation only in dendritic cells is enough to slow tumor growth. In vitro co-culture experiment demonstrated the number of effector CD8+ T cells cultured with autophagy-deficient DCs increases compared to wild-type. The results show that autophagy-deficient DCs are more effective at activating effector T cells than their autophagy-proficient counterparts. We conclude that autophagy-deficient DCs are better in promoting T cell activation. Autophagy-deficient DCs have the potential to be a novel method to alleviate tumor burden by promoting a heightened immune response.

CRISPR-Cas9 is a powerful genome-editing tool, but its scalability is limited by inefficient editing and labor-intensive cloning workflows. In our lab, guide RNAs (gRNAs) and repair templates are introduced via a two-step cloning process, which restricts high-throughput variant engineering. My project aims to improve CRISPR efficiency by optimizing the placement of the repair template upstream (5’) of the guide RNA. This modification could reduce cloning steps, lower costs, and enhance homology-directed repair (HDR), leading to more efficient genetic variant screening. I will construct two plasmid designs: a control with the repair template downstream of the gRNA, and an experimental version with the template positioned upstream. Using a galactose-inducible ADE2 repair system, red colony formation will serve as a phenotypic indicator of successful HDR. Yeast will be transformed and plated on galactose and glucose media to evaluate editing efficiency and cell viability. Statistical analysis of colony counts will assess whether upstream repair template placement significantly enhances HDR. Additionally, I will test co-expression of the Csy4 protein to further optimize CRISPR efficiency. By improving editing strategies in yeast, this project contributes to expanding the scale and throughput of variant engineering platforms.

Lipid droplets (LDs) store lipids in the form of triacylglycerides (TAGs). Mitochondria bound to LDs, called peridroplet mitochondria (PDM), exhibit a unique metabolic phenotype that supports LD expansion. In brown adipose tissue, PDM detaches from LD when thermogenic β-oxidation is induced. Our lab has also discovered a small molecule called SEN-110 that detaches PDM from LDs. However, there are many open questions surrounding this interaction and the induction of lipolysis needed for β-oxidation. For example, it is unknown whether PDM detachment requires lipolysis, and what the timeline for these events is. To explore this, I first determined the optimal time point when SEN-110 detaches PDM from LDs. I tested a lipase inhibitor at different concentrations and timepoints to prevent lipolysis to see its effects on PDM detachment. I am answering these questions using fixed cell confocal microscopy immunofluorescence in INS1 cells. I developed an image analysis pipeline with the software Cell Profiler. This allowed for quantification of PDM after different treatments of SEN-110 and lipase inhibitors. Preliminary results indicate that detachment happens independently of lipolysis but further exploration is required to fully validate this result. Helps us to better understand the process of thermogenesis in brown adipose tissue. Additionally, understanding the timeline of lipolysis could highlight the value of SEN-110 as an anti-obesity drug.