Prions cause a set of neurodegenerative diseases collectively called transmissible spongiform encephalopathies (TSEs) in humans and other mammals, which are characterized by rapid neurodegeneration. Yeast prions can be used as a trackable system due to their similar features to mammalian prions, such as the protein only hypothesis. While many aspects of prion pathology have been identified, the molecular basis of prion structure and propagation is still elusive. Here we report a comprehensive structural study of the amyloid fibrils formed by yeast prion protein Mot3. Using site-directed spin labeling, we created a total of 14 spin-labeled Mot3 variants, followed by structural studies using electron paramagnetic resonance (EPR) spectroscopy. Strong intermolecular spin-spin interactions were found between residue positions of 40 and 120, suggesting that the parallel in-register β-sheet region, or amyloid core, of Mot3 may consist of the region of 40-120. To further characterize the core, we performed truncation mutations at the C-terminal region and studied morphological differences of the Mot3 aggregates using transmission electron microscopy (TEM). We found that the deletion of the last 95 residues yielded fibrils and oligomers in the same truncation mutant. These results suggest that non-core residues may affect the aggregation pathway without affecting the formation of the final aggregation product. This work will identify key structures which may inform drug design and uncover principles of amyloid fibril formation.

Metabolic dysfunction and insulin resistance underpin heightened type 2 diabetes and cardiovascular disease risk in women during the menopausal transition. These changes in metabolic health coincide with impairments in estrogen receptor alpha (ERa) action. Polymerase γ (POLG/Polg1) expression is reduced in many cell types lacking Esr1/ERa, and Polg1 is a novel ERa target gene. Esr1 deletion produces marked dysregulation of mitochondrial morphology and metabolism, and Polg1 is the only known mammalian mtDNA polymerase involved in the replication of the mitochondrial genome. Since Esr1 deletion from macrophages is shown to heighten susceptibility to insulin resistance and cardiometabolic disease, our current study tested whether a myeloid-specific Polg1 deletion recapitulated the same metabolic disease phenotypes. We employed the Lox-Cre approach to generate mice with a myeloid-specific deletion of Polg1 using Cre recombinase driven by the LysM promoter. Our findings show a novel sex-biased link between mitochondrial dysfunction, inflammation, and metabolic dysfunction in the context of high fat diet (HFD) feeding. Myeloid-specific Polg1 deficiency promoted inflammatory signaling and insulin resistance in gonadal white adipose tissue (gWAT), the liver, and skeletal muscle despite a blunting of adipose weight gain in the context of HFD feeding. Our preliminary findings reinforce the notion that mitochondrial function in the immune compartment exerts important regulatory control over metabolic health especially in females.

Intellectual disability (ID) syndromes are neurodevelopmental disorders often caused by genetic abnormalities that impair cognitive and adaptive functioning. Previous research suggests that neuronal senescence mediated by activation of the p53 pathway contributes to the pathology of Down and Rett Syndromes. Specifically in Rett Syndrome, prior studies found that the neuronal senescence phenotype driven by elevated DNA damage can be ameliorated by restoring levels of PARP1, a DNA repair enzyme, via NAD stimulation. This shared phenotype has sparked interest to elucidate if senescence caused by elevated DNA damage is a common etiology across other genetic ID syndromes. Using a disease-in-a-dish model, neural progenitor cells and neurons were generated from patient-derived induced pluripotent stem cells to recapitulate Down, Bohring-Opitz (BOS), and Arboleda-Tham Syndromes (ARTHS). Molecular profiling using β-galactosidase staining, comet assays, and immunofluorescence, showed elevated senescence in Down Syndrome, while ARTHS and BOS lines exhibit more variable stress responses. Similar to Rett Syndrome, ELISA-based chemiluminescent assays showed that all disease lines except BOS exhibited reduced PARP1 baseline activity in neurons. This study characterizes key cellular stress markers associated with each ID, providing insights into the varying molecular mechanisms underlying their etiologies and offering new ID therapeutic avenues if a shared stress phenotype is observed.

Axon caliber (cross-sectional diameter) is critical to neuronal function because it directly influences the speed at which neurons send messages. Abnormal axon caliber leads to diseases like Giant Axonal Neuropathy. Despite the essential role of axon caliber in neurons, most research assumes that caliber is static in individual cells. There has been little investigation into axon caliber variation. Previously, the Sagasti lab demonstrated that average caliber varies within peripheral axon arbors of skin-innervating sensory neurons. Specifically, primary axons tend to be thicker than higher order branches. Moreover, every location measured was dynamic, such that each axon branch thickens and narrows over time. The mechanisms regulating these axon caliber variations remain poorly understood. Neurofilaments are known to contribute to cell-wide caliber, however, it remains unclear whether they contribute to caliber variation between branches or caliber dynamics over time. To determine how the abundance of neurofilaments affects axon caliber, we will utilize a previously established Zebrafish gene knockout model with a deletion of neflb, a neurofilament subunit that is necessary for polymer assembly. Caliber measurements will be taken from mutant and wildtype time lapse images and subsequently compared to determine how neurofilament depletion impacts caliber dynamics. As caliber is critical to nervous system function, these data could provide a foundation for more accurate assessments of neural circuit transmission speeds.