AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, with over 100 identified downstream targets throughout the cell. In response to cellular stress, including energetic stress, AMPK is activated via binding of AMP and phosphorylation by upstream kinases, including liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). We and others have found that the activation of AMPK in response to cellular stress has distinct subcellular mechanisms, indicating compartmentalized regulation of AMPK signaling. Although oxidative stress is known to stimulate AMPK activity, how AMPK is spatially regulated by oxidative stress is underexplored. Using a single-fluorophore excitation-ratiometric AMPK activity reporter (ExRai AMPKAR), we find that oxidative stress induced by hydrogen peroxide (H2O2) results in AMPK activity with distinct spatiotemporal dynamics. We found that across all locations measured, phosphorylation of AMPK by LKB1 is required for AMPK activity. Using a multi-‘omics approach, we discover that in response to oxidative stress, AMPK mediates significant metabolic and gene expression changes. These findings support a role for AMPK in regulating adaptive responses to oxidative stress. Altogether, this work provides new insights into how the subcellular environment influences localized AMPK activity, and identifies how AMPK regulates the cellular response to oxidative stress.

Cancer is characterized by excessive cell division, causing tumor initiation and progression. In eukaryotes, somatic cells divide by mitosis, requiring major structural changes. Microtubules, the largest cytoskeletal filaments, support phenotypic changes during mitosis through polymerization and depolymerization. Such dynamic rearrangements are also mediated by numerous enzyme complexes; namely, the heterodimer katanin severs microtubules via catalytic subunits KATNA1 (A1, p60), KATNAL1 (AL1), and KATNAL2 (AL2), and regulatory subunits KATNB1 (B1, p80) and KATNBL1 (BL1). Previous studies have shown that katanin regulates mitotic spindle length and premitotic severing in Xenopus and Chlamydomonas, respectively. Additionally, A1 knockout in mouse neuronal progenitors produces lethal phenotypes indicative of defective mitosis. However, the role of katanin in human cell division remains uncertain. This project examines katanin subunit involvement in human mitosis through CRISPR-Cas9 knockdown. Retinal pigment epithelial (RPE1) cell lines expressing unique guide RNA sequences were generated to produce single-subunit knockdowns of A1, AL1, and B1. Additionally, double subunit knockdowns (A1/B1, A1/AL1, AL1/B1) were established to assess potential functional redundancies or cooperativity. Immunofluorescent imaging showed elongated mitotic spindles, implicating katanin in division. Analyses of such defects will further characterize the impact of katanin in mitosis, possibly revealing targetable pathways for novel cancer therapies.

Infertility affects 17.5% of the global adult population and continues to burden society due to scientific ambiguity. Central to this ambiguity is the limited understanding of oogenesis, or egg development. Proteomics is a powerful tool for probing cellular mechanisms, revealing protein expression patterns, and dynamics across developmental stages. We use Drosophila melanogaster, a genetically tractable model, to study egg development. However, proteomic studies in Drosophila oogenesis are limited by sample preparation challenges. Bulk ovary sampling obscures stage-specific signals due to the ovary’s heterogeneous structure. Separating thousands of egg chambers to produce stage-specific samples is not feasible. Single-cell proteomics and Data-Independent Acquisition (DIA) will help us overcome these limitations. Single-egg-chamber sampling reduces dissection time, while DIA enhances protein identification, enabling richer profiles from smaller samples. To perform DIA, a spectral library must first be created to define elution windows. This requires high-fractionation Data-Dependent Acquisition (DDA) data. I have worked to optimize a medium-scale sample preparation method, SP3, significantly enhancing protein retention and increasing potential proteome coverage in Drosophila. I am also developing protocols for hydrophilic interaction liquid chromatography (HILIC) to improve the quality of the spectral library.

Neurodegenerative diseases (NDs) involve progressive loss of neuronal structure and function, yet their molecular mechanisms remain incompletely understood. A key challenge is tracking disease-relevant molecular changes in living neurons over time. Human induced pluripotent stem cell (iPSC)-derived neurons provide a platform to model these processes. In this study, we used NGN2-induced excitatory neurons (iNeurons) to examine how a pathogenic tau mutation influences neuronal development, maturation, and degeneration at the proteomic level. We compared wild-type neurons with neurons harboring a CRISPR knock-in V337M mutation in the MAPT gene, associated with familial frontotemporal dementia (FTD-Tau). Proteomic profiling was performed across six time points spanning differentiation and aging. Across all samples, we identified 9,294 proteins. Clustering revealed early divergence between mutant and wild-type neurons, with the largest differences around day 14, suggesting altered early maturation. By day 28, both genotypes converged toward a mature proteomic state, after which progressive differences emerged during later stages of neuronal aging and degeneration. In mutant neurons, proteins related to mitochondrial function and energy metabolism were increasingly upregulated, while those involved in RNA processing and spliceosomal regulation were downregulated. Together, these results highlight the value of time-resolved proteomics and support iNeuron models for studying tauopathy mechanisms and identifying candidate biomarkers.

Primary Hyperoxaluria Type 1 (PH1) is a metabolic disorder caused by autosomal recessive mutations, culminating in End Stage Renal Disease (ESRD). Point mutations in the protein alanine-glyoxylate aminotransferase (AGT) result in mislocalization of a metabolically active protein, typically from peroxisomes to mitochondria, or aggregation in other cellular compartments. Mislocalization disrupts AGT’s detoxification role, causing oxalate buildup and kidney stones characteristic of ESRD. Current treatments, like kidney stone removal, dialysis, and renal allografts, do not fully attenuate PH1, and mortality from complications remains high, especially in younger patients. This project aims to ameliorate PH1 by using small molecules to relocate mutant AGT from mitochondria to peroxisomes and restore function. We developed a human liver model using a HepG2 FlpIn cell line expressing mutant AGT P11L-G170R for high-throughput screening. AGT is fused to a C-terminal GFP, while the remaining GFP1-10 is targeted to peroxisomes. Upon small molecule treatment, successful protein return to peroxisomes is confirmed by green puncta upon GFP reconstitution. After screening ~55,000 compounds, we discovered nine capable of rescuing mutant AGT and producing phenotypes comparable to wildtype cells. Our work highlights the potential of mitochondrial import inhibitors for treating diseases of similar pathology, with our high-throughput screening protocol offering a promising approach to identifying drug targets and understanding disease mechanisms.