Prion-Like Propagation of Protein Misfolding and Aggregation in Amyotrophic Lateral Sclerosis

Abstract

The discovery that prion protein can misfold into a pathological conformation that encodes structural information capable of both propagation and inducing severe neuropathology has revolutionized our understanding of neurodegenerative disease. Many neurodegenerative diseases with a protein misfolding component are now classified as "prion-like" owing to the propagation of both symptoms and protein aggregation pathology in affected individuals. The neuromuscular disorder amyotrophic lateral sclerosis (ALS) is characterized by protein inclusions formed by either TAR DNA-binding protein of 43 kDa (TDP-43), Cu/Zn superoxide dismutase (SOD1), or fused in sarcoma (FUS), in both upper and lower motor neurons. Evidence from in vitro, cell culture, and in vivo studies has provided strong evidence to support the involvement of a prion-like mechanism in ALS. In this article, we review the evidence suggesting that prion-like propagation of protein aggregation is a primary pathomechanism in ALS, focusing on the key proteins and genes involved in disease (TDP-43, SOD1, FUS, and C9orf72). In each case, we discuss the evidence ranging from biophysical studies to in vivo examinations of prion-like spreading. We suggest that the idiopathic nature of ALS may stem from its prion-like nature and that elucidation of the specific propagating protein assemblies is paramount to developing effective therapies.

Keywords: amyotrophic lateral sclerosis; prion; protein aggregation; protein misfolding; proteostasis.

Figure 1

A model of superoxide dismutase-1 (SOD1) aggregation and prion-like propagation. (A) The native folding (green background) and off-folding pathways (red background) of SOD1. When undergoing native folding, SOD1 is initially in a metal-free intermediate state [E, E(SH)] which is primed for Zn binding. Following Zn binding, the copper chaperone for SOD1 (CCS) heterodimerizes with a SOD1 monomer to implant Cu and facilitate the oxidation of the disulfide bond between Cys57 and Cys146 to form a mature SOD1 monomer [Cu, Zn(SS)]. Subsequent dimerization of two mature monomers gives the fully folded SOD1 enzyme. In the off-folding pathway (red background) amyotrophic lateral sclerosis (ALS)-associated mutations have the ability to push the folding back towards the nascent intermediates (red arrows), which are more aggregation prone than mature states. Misfolded SOD1 is capable of recruiting nascent SOD1 states to form oligomers. Oligomers can grow in size to form larger aggregates that may have strain-like properties. Strains that are prone to fragment are the most likely to propagate to adjacent cells and seed naïve SOD1. (B) Graphic representation of the SOD1 primary structure with important features. SOD1 is composed of 8 beta-strands and contains two major loop regions. The Zn-binding loop is responsible for coordination of Zn and, to a lesser extent, Cu. The electrostatic loop is responsible for guidance of superoxide substrate to the active site of the enzyme. A disulfide bond is formed between Cys57 and Cys146 in the mature protein. Red dots below the graphic represent the location and number of ALS-associated mutations that have been identified to date within SOD1. Coloured bars represent potential cores or contributing regions of the protein to the formation of aggregate strains from corresponding colour coded publications.

Figure 2

A model of the aggregation and prion-like propagation of TAR DNA-binding protein of 43 kDa (TDP-43) in ALS. (A) TDP-43 is composed of four domains which include the ubiquitin-like N-terminal domain (NTD), tandem RNA-recognition motifs (RRM), and a C-terminal low complexity domain (LCD). The NTD contains the nuclear localization signal (NLS) of TDP-43, whereas the nuclear export signal (NES) is in RRM2. The pathological 35 kDa (TDP-35) and 25 kDa (TDP-25) are formed via aberrant caspase-mediated cleavage at the NLS and in RRM2 respectively. The blue bar below RRM2 represents an amyloidogenic segment. The LCD is a prion-like domain due to it being enriched for G/N/Q/S residues. It also contains a helix-turn-helix motif which is important for association with other TDP-43 LCDs. Red dots below both the main TDP-43 graphic and the LCD expansion represent ALS-associated mutations. Colored bars represent experimentally identified regions of the protein that are capable of forming amyloid-like structures. Bar colors correspond to the publications listed on the right. (B) TDP-43 is a primarily nuclear-localized protein, however, it shuttles between the nucleus and cytoplasm. Impairment of nucleocytoplasmic transport (top) can lead to the cytoplasmic concentration of TDP-43 increasing, leading to liquid demixing to form biomolecular condensates distinct from stress granules. Prolonged residency of TDP-43 in these condensates can lead to the formation of aggregates. Aberrant cleavage (middle) leads to the formation of the highly aggregation-prone TDP-35 and TDP-25 pathological fragments. These fragments, and even full-length TDP-43 can aberrantly self-associate to form oligomers that may grow to become larger aggregates. Also, TDP-43 is known to inhabit stress granules (bottom). Given prolonged stress or genetic mutations, stress granules can persist for longer than necessary and can fail to disassemble, which leads to the formation of aggregates. Each of these possible pathways may also result in strains of TDP-43 aggregates that can propagate from cell-to-cell and seed further aggregation.

Figure 3

Mechanisms by which prion-like ALS-associated protein aggregates may transfer between cells. (Top left) Once formed, misfolded monomers and soluble oligomers can potentially exit the cell through diffusion and enter nearby cells either through diffusion or receptor-mediated endocytosis. (Top right) Cells that are dead or dying can potentially release larger insoluble aggregates that can be taken up into nearby naïve cells through micropinocytosis. (Bottom left) Misfolded and oligomeric proteins are capable of being loaded into either microvesicles or exosomes for transport to nearby cells. (Bottom right) Misfolded monomers or soluble oligomers are capable of being transported across axon terminals to naïve cells.