Project Overview

TDP-43, SOD1, and FUS proteinopathies in vivo: interrogating prion-like mechanisms and drug rescue in zebrafish and ALS hiPSC-derived neuron xenotransplant models

RATIONALE: Proteins of ALS-associated gene variants in TDP-43, SOD1, and FUS exhibit misfolding and prion-like properties under certain conditions. Whether these prion-like properties occur in a complex central nervous system (CNS), and are a contributor to disease, have been challenging to interrogate in in vivo models. Protein misfolding and spread from cell to cell would be attractive therapeutic targets to slow or stop disease spread. Recent in vitro studies suggest misfolding of one protein type leads to misfolding of other types, which requires assessment in vivo. The zebrafish model is ideal for these questions; it is a facile genetic model, has rapid external development and a CNS that is well-studied at the cell, electrophysiologic, and behavioural levels, and is amenable to xenotransplantation, a unique method I propose to utilize with ALS patient human induced pluripotent stem cells (hiPSC).

HYPOTHESIS: I hypothesize that TDP-43, SOD1, and FUS misfolded proteins have capacity to spread in a prion-like fashion in vivo, and thus halting their spread is a viable therapeutic target. I also hypothesize that expression of mutant forms of TDP-43 or of FUS leads to misfolding of wildtype (WT) SOD1 protein, based on previous in vitro literature.

AIMS: 1) I will test if expression of TDP-43, SOD1, and/or FUS misfolded proteins leads to misfolding of WT equivalents in vivo. Co-expression of tagged WT and disease variants of each of human TDP-43, SOD1, or FUS will be achieved using two techniques: mRNA injection and transgene expression. Tracking of protein aggregation will include live fluorescent imaging and immunocytochemistry. 2) I will test if expression of one protein type leads to misfolding of another, e.g. TDP-43 expression leading to SOD1 misfolding. In addition to the experimental approaches described in (1), I will create zebrafish genetic chimeras via xenotransplantation to study non-cell autonomous effects, e.g. protein misfolding in neighbouring cells, protein spread from cell to cell. To interrogate these properties in human cells, I will transplant hiPSC-derived neural progenitors from fALS and sALS patients into zebrafish embryos and assess protein misfolding and spread. 3) I will characterize the zebrafish phenotypes of disease variants of TDP-43, SOD1, and FUS in parallel, by quantifying motor neuron and neuromuscular junction structure, electrophysiology, and swim performance. These assays will be used as measurable outputs for testing drugs that target protein misfolding and/or spread. Zebrafish transplanted with sALS and fALS hiPSC-derived neurons will also be used in drug screening, modelling “bench to bedside” preclinical trials.

SIGNIFICANCE: Understanding the prion-like properties of these major proteins will have implications for designing future therapeutic strategies in the heterogeneous ALS population, including for sporadic disease which has no known disease mutations. Creating a model that incorporates hiPSC-derived neurons into an in vivo CNS holds significant promise for future studies on sALS, and pre-clinical drug screening including for under-represented populations.”

Principal Investigator

Michele DuVal , University of Alberta

Partners and Donors

ALS Society of Canada