Rescue of Neurodegenerative Models in Drosophila melanogaster and Conservation of Metabolic and Degradative Regulation by Heparan Sulfate in Mammalian Cells
Open Access
- Author:
- Schultheis, Nicholas
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 30, 2024
- Committee Members:
- Lorraine Santy, Major Field Member
Scott Selleck, Chair & Dissertation Advisor
Zhi-Chun Lai, Major Field Member
Claire Thomas, Major Field Member
Santhosh Girirajan, Program Head/Chair
Amie Boal, Outside Unit & Field Member - Keywords:
- Heparan Sulfate
Autophagy
Alzheimer's Disease
Parkinson's Disease
Amyotrophic Lateral Sclerosis
Metabolism
Mitochondria
Cholesterol Biosynthesis
Growth Factor Signaling
MPS IIIa - Abstract:
- Alzheimer’s Disease (AD) is characterized by protein aggregates, mitochondrial dysfunction, autophagy inhibition, and lipid accumulation. Other neurodegenerative diseases such as Parkinson’s Disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are characterized by protein aggregations and mitochondrial disruption. Despite extensive characterization, AD and other neurodegenerative disorders retain unclear etiology and treatment is poor. The cellular hallmarks of these neurodegenerative diseases are abrogated under typical conditions through autophagy, a process of cellular self-eating and renewal that degrades cellular organelles and macromolecules into their constituent parts for further use. An increase in autophagy is an attractive means of treatment for neurodegenerative disorders. Heparan Sulfate (HS) is a glycosaminoglycan added post-translationally to a set of proteins referred to as Heparan Sulfate Proteoglycans (HSPGs). HS operates as a co-receptor or signal modifier for a range of growth factors that affect signaling pathways that inhibit autophagy. Previous work has shown that a decrease in HS biosynthetic enzymes increases autophagy in Drosophila. The present work demonstrates the rescue of characteristics of models of PD and ALS in flies through a decrease in the expression of HS biosynthetic enzymes. The flight muscles of flies modeling PD demonstrated less ubiquitin upon introduction of a mutant allele of HS biosynthetic enzymes, indicating an increased capacity for flies to remove cellular components marked for degradation. Flies modeling ALS had a lower rate of lethality exiting the pupal stage of life upon knockdown of a HS biosynthetic enzyme. Reinforcing the capacity of increases in autophagy to ameliorate neurodegenerative diseases, treatment of mice modeling mucopolysaccharidoses IIIa (MPSIIIa) with a drug that increases autophagy resulted in the morphological rescue of its brown adipose tissue (BAT). The present work also characterizes and rescues a model of AD in flies through downregulation of HS biosynthetic enzymes. Neurological and metabolic disruptions were analyzed through confocal microscopy and TEM analysis. Neuronal death, disruptions in lipid morphology, and disruptions in mitochondrial morphology were all reversed in the AD model in flies upon knockdown of HS biosynthetic enzymes. RNA sequencing analysis revealed a set of genes involved in solute transport concomitantly rescued with the cellular abnormalities, strengthening the association between AD and metabolic disruption. The present work serves to demonstrate the conservation of the regulation of autophagy, mitochondrial volume and efficacy, and lipid volume by HS. Confocal microscopy demonstrated increased autophagy in one cell line and increased mitochondrial volume and decreased neutral lipid droplet presence across multiple cell lines upon knockout or knockdown of HS biosynthetic enzymes. A mitochondrial stress test demonstrated an increase in oxygen consumption rate across three cell lines upon a decrease in HS biosynthetic enzymes, indicating mitochondrial efficacy. RNA sequencing analysis of Hep3B cells revealed a broad shift in gene expression associated with increases in mitochondrial biogenesis and energy metabolism. Furthermore, the RNA sequencing analysis demonstrated that knockout of an HS biosynthetic enzyme significantly altered expression of AD-associated genes. The present work contains preliminary research into the drug bosutinib, a tyrosine kinase inhibitor that inhibits pathways upon which HS acts. So far, the research is indicative that bosutinib may rescue cellular disturbances in flies modeling AD, but further investigation is required.