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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons that appears to spread through the neuroaxis in a spatiotemporally restricted manner. Misfolded Cu/Zn superoxide dismutase (SOD1) has been detected in all ALS patients, despite SOD1 mutations accounting for only 2% of total cases, while the presence of inclusions containing pathological TAR-DNA binding protein-43 (TDP-43) represent a hallmark of all non-SOD1/FUS familial ALS. We previously reported that TDP-43 and FUS can trigger misfolding of human wild-type SOD1 (HuWtSOD1) in living cells, however the mechanisms and consequences are unknown. Here, we used immunocytochemistry, immunoprecipitation and cell viability studies to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. Furthermore, we developed several chimeric SOD1-GFP proteins that we validated to aggregate in the presence of pathological SOD1 or TDP-43 seed. We used this assay, along with immunofluorescence, live-cell microscopy and flow cytometry studies, to show that intermolecular conversion of SOD1 by pathological TDP-43 is mediated by tryptophan residues in both proteins. Furthermore, we used the reporter proteins to show that human spinal cord extracts prepared from familial, but not sporadic, ALS patients can trigger SOD1 aggregation in cultured cells. Finally, we used this system to show that small molecules, akin to 5-fluorouridine, can block this intermolecular kindling of SOD1 aggregation, and demonstrated that our assay can be used as a high-throughput tool for screening drugs against induced SOD1 aggregation. Altogether, our studies indicate that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of tryptophan-dependent propagated SOD1 misfolding. Furthermore, it is key to recognize that elucidation of the pathogenic role of a simple structural motif in ALS may provide a framework for understanding other neurodegenerative diseases in which propagated protein misfolding is shown to occur.
Protein misfolding diseases represent a large burden to human health for which only symptomatictreatment is generally available. These diseases, such as Creutzfeldt-Jakob disease, amyotrophiclateral sclerosis, and the systemic amyloidoses, are characterized by conversion of globular, nativelyfoldedproteins into pathologic β-sheet rich protein aggregates deposited in affected tissues. Understandingthe thermodynamic and kinetic details of protein misfolding on a molecular level dependson accurately appraising the free energies of the folded, partially unfolded intermediate,and misfolded protein conformers. There are multiple energetic and entropic contributions to thetotal free energy, including nonpolar, electrostatic, solvation, and configurational terms. To accuratelyassess the electrostatic contribution, a method to calculate the spatially-varying dielectricconstant in a protein/water system was developed using a generalization of Kirkwood Frohlich theoryalong with brief all-atom molecular dynamics simulations. This method was combined withpreviously validated models for nonpolar solvation and configurational entropy in an algorithm tocalculate the free energy change on partial unfolding of contiguous protein subsequences. Resultswere compared with those from a minimal, topologically-based Gō model and direct calculationof free energies by steered all-atom molecular dynamics simulations. This algorithm was appliedto understand the early steps in the misfolding mechanism for β₂-microglobulin, prion protein,and superoxide dismutase 1 (SOD1). It was hypothesized that SOD1 misfolding may follow atemplate-directed mechanism like that discovered previously for prion protein, so misfolding ofSOD1 was induced in cell culture by transfection with mutant SOD1 constructs and observed tostably propagate intracellularly and intercellularly much like an infectious prion. A defined minimalassay with recombinant SOD protein demonstrated the sufficiency of mutant SOD1 aloneto trigger wtSOD1 misfolding, reminiscent of the “protein-only” hypothesis of prion spread. Finally,protein misfolding as a feature of disease may extend beyond neurodegeneration and amyloidformation to cancer, in which derangement of protein folding quality control may lead to antibodyrecognizablemisfolded protein present selectively on cancer cell surfaces. The evidence for thishypothesis and possible therapeutic targets are discussed as a future direction.
Master's Student Supervision (2010 - 2018)
The presence of Amyloid-β (Aβ) aggregates in the brain is a hallmark of Alzheimer’s disease. However, the heterogeneity of these assemblies and their highly dynamic nature have rendered them challenging to target. It is known that the smaller aggregates are toxic to neurons and show a potential to stimulate a glia-mediated pro-inflammatory response in the brain. We first sought to further characterize the pathway of Aβ aggregation by targeting different stages of assembly with an oligomer-specific antibody (anti-cSNK) and the sugars trehalose and glucosamine. Incubation of Aβ₄₂ with the anti-cSNK antibody and glucosamine lengthened the nucleation phase and diminished the maximum β-sheet signal after 72 hrs, as tracked with a Thioflavin T fluorescence aggregation assay. Incubation with trehalose only affected maximum β-sheet signal. The analysis of samples collected at different timepoints with TEM and immunoblotting revealed a delay in Aβ₄₂ aggregation into insoluble species in presence of the two sugars and the anti-cSNK antibody. In conclusion, we lend further support to the hypothesis that AβOs are part of the fibril formation pathway. To better understand the role that different Aβ soluble aggregates play in microglia activation, we used stabilized Aβ oligomers consisting primarily of tetramers. Upon exposure to AβOs at different concentrations (ranging from picomolar to nanomolar), NO and TNF-α levels in addition to cell morphology were used as indicators of cell activation after 24 hrs. Cell viability was tracked at various time points with different cytotoxicity assays. While the Aβ tetramers did not cause significant NO and TNF-α release, they did induce significant decrease in the signal detected by the MTT assay, usually used as an indirect measure of cell viability. These experiments were conducted in both primary and immortalized murine microglial cells in vitro, yielding consistent results across both systems. However, other measures of cell viability did not reflect the same results, suggesting that the AβOs used in this experiment have an alternative effect on the cells.Although our data do not support our initial hypothesis, they generate a new line of inquiry with regards to the effects of Aβ tetramers on microglia.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.