Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder affecting millions worldwide. Impaired mitophagy, the process by which damaged mitochondria are removed, has been implicated in AD, though the downstream mechanisms connecting mitophagy dysfunction to disease progression remain poorly understood. In this study, we investigated whether cytosolic double-stranded DNA (dsDNA) and/or mitochondrial DNA (mtDNA) may contribute to AD progression through an inflammatory response. Using a Drosophila AD model, we found accumulation of dsDNA, but not mtDNA, in the cytosol, which is associated with increased inflammatory responses and the development of AD phenotypes. Notably, upregulation of DNaseII, a dsDNA-removing enzyme, significantly improved the health of AD flies, enhancing olfactory memory, increasing activity, improving climbing ability, and extending lifespan. This rescue effect was independent of amyloid-beta (Aβ42) accumulation. These results suggest that cytosolic dsDNA accumulation contributes to AD progression, and that its removal may offer a therapeutic target, though the exact role of mitophagy, cytosolic DNA, and immune responses in this pathway remains to be fully elucidated.

RNA interference (RNAi) is a highly conserved gene-silencing mechanism that is found in a wide range of eukaryotic organisms. Unlike many organisms, Caenorhabditis elegans has a highly efficient RNAi system that allows gene silencing to spread throughout the organism. Leveraging this trait, we aim to understand how natural genetic variations influence RNAi response. More specifically, we examined differences in RNAi sensitivity across multiple C. elegans strains in a large bulk assay and selected strains with contrasting RNAi responses to investigate the genetic factors underlying their differences. We identified JU1793 as an outlier strain that exhibited resistance to RNAi targeting both pos-1, a gene essential for embryogenesis, and mig-6, which is involved in cell development. To investigate the genetic basis of this resistance, we crossed JU1793 to two moderately RNAi-sensitive strains, AB1 and JU2466. Segregant populations were subsequently exposed to pos-1 RNAi and mig-6, revealing a shared QTL on the right arm of Chromosome III. This overlap of the QTL likely suggests a general RNAi response rather than a gene-specific effect. Our work continues to fine-map the region by constructing Near Isogenic Lines (NILs) and CRISPR-Cas9 mediated gene knockouts. This work seeks to further our understanding of how natural genetic variation shapes quantitative traits and ultimately contributes to how complex traits arise within natural populations.

The widespread use of the CRISPR/Cas9 gene-editing tool necessitates the evaluation of its efficiency in inducing gene disruption, particularly for genes for which no antibodies are available to validate knockout efficiency. The Tarling-Vallim Laboratory routinely employs liver-directed delivery of adeno-associated viral (AAV) CRISPR to investigate genes involved in lipid metabolism. We and others have found that, in addition to the insertion and deletion of nucleotides (INDELs), sequences from the AAV vector can also integrate at the target cut sites. Using DNA-based techniques, we have developed a protocol consisting of Sanger PCR and a novel qPCR-based method that quantifies both INDEL and AAV integration events in murine livers treated with AAV-CRISPR. I have completed this editing analysis pipeline for over a dozen AAV-CRISPRs targeting genes involved in the bile acid and lipid metabolism pathways. Because the liver is comprised of hepatocytes (targeted by AAV), and non-hepatocyte cells (not targeted by AAV), efficient editing was characterized by a 50-70% combined INDEL and AAV integration. These new methods improve the assessment of genomic disruption by AAV-CRISPR in the liver and will lay the groundwork for improved CRISPR editing design in the future.

Chromatinopathies are a subset of rare developmental disorders caused by germline mutations in genes that regulate the epigenome. In 2017, germline pathogenic variants were identified in epigenetic “reader” BRPF1, resulting in a neurodevelopmental syndrome termed Intellectual Developmental Disorder with Dysmorphic Facies and Ptosis (IDDDFP). BRPF1 is part of a complex with the lysine acetyltransferases KAT6A/B to facilitate histone H3 acetylation. While mutations in BRPF1 result in a primarily neurodevelopmental syndrome, mutations in KAT6A and KAT6B result in developmental syndromes that affect multiple organ systems (e.g., neurological, gastrointestinal, and cardiovascular), suggesting some divergence in disease mechanisms. To identify novel protein interactors of BRPF1, we performed immunoprecipitation-mass spectrometry (IP-MS) to bind BRPF1 and associated proteins in an immortalized schwann cell line. IP-MS confirmed enrichment of BRPF1 (log2FC 1.16, p-adjusted 0.000179), and identified known interactors such as KAT6A (log2FC 1.010, p-adjusted 0.000434) and KAT6B (log2FC 0.761, p-adjusted 0.000668), demonstrating the validity of this approach. Our results indicate that BRPF1 interacts with RNA binding proteins to regulate both RNA transcription and pre-mRNA processing. Further, our work highlights potential BRPF1 interactions with key chromatin remodeling proteins, including SMARCA5 and EZH2. Ultimately, this work has identified novel BRPF1 protein interactors and further defined its role in gene regulation.

Neurodegenerative diseases of the retina involve irreversible degradation of photoreceptors and retinal ganglion cells, and currently available treatments are only capable of slowing down the progression of degeneration. Although neurogenesis does not occur in the mature mammalian retinas, Müller glia in fish and amphibians can serve as endogenous stem cells to generate new neurons to repair the retinas after injury. This project seeks to stimulate Müller glial cells’ self-repair capability in the retina of mice, a model system to study retinal degeneration and repair. Mpc2 is one subunit of the mitochondrial pyruvate carrier (Mpc) complex required to transport pyruvate into the mitochondria. With either Mpc1 or Mpc2 inactivated, the cell is unable to transport and use pyruvate to perform oxidative phosphorylation. We hypothesize that deletion of the Mpc2 gene in retinal Müller glial cells can modify the metabolic status of mouse Müller glial cells to become more similar to that of progenitor cells, thus potentiating their endogenous repair capabilities in order to replace degenerated photoreceptors and retinal ganglion cells in the mammalian retina. This investigation utilizes inducible Cre-loxP gene knockout of Mpc2 in both wild type and retinal degeneration mutant (rds) backgrounds. While literature suggests Müller glia are very sensitive to changes in their microenvironment, knockout of Mpc2 has not significantly altered retina morphology. More data analysis must be done to confirm these initial findings.