Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)_n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity.

The universally abundant polyphosphate (polyP) accelerates fibril formation of disease-related amyloids and protects against amyloid cytotoxicity. To gain insights into the mechanism(s) by which polyP exerts these effects, we focused on α-synuclein, a well-studied amyloid protein, which constitutes the major component of Lewy bodies found in Parkinson’s disease. Here, we demonstrate that polyP is unable to accelerate the rate-limiting step of α-synuclein fibril formation but effectively nucleates fibril assembly once α-synuclein oligomers are formed. Binding of polyP to α-synuclein either during fibril formation or upon fibril maturation substantially alters fibril morphology and effectively reduces the ability of α-synuclein fibrils to interact with cell membranes. The effect of polyP appears to be α-synuclein fibril specific and successfully prevents the uptake of fibrils into neuronal cells. These results suggest that altering the polyP levels in the extracellular space might be a potential therapeutic strategy to prevent the spreading of the disease.

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid–liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.

Oligomeric assemblies of amyloidogenic proteins, such as Aβ, tau, α‐synuclein, amylin, transthyretin, and TDP‐43, are increasingly recognized as key drivers of cellular dysfunction across a range of neurodegenerative and systemic disorders. However, their molecular properties remain poorly understood due to their low abundance, structural heterogeneity, and transient nature. This review outlines current methods for studying amyloid oligomers, including biophysical (NMR, cryo‐EM, HS‐AFM, mass spectrometry), computational (molecular dynamics simulations), and biological (cellular assays, organoids, and animal models) approaches. This review also covers emerging methods for detecting misfolded proteins within complex biological environments and live‐cell systems. Furthermore, we discuss recent advances that specifically address the challenges of studying oligomers, which are yielding crucial data on how these pathogenic species impair cellular homeostasis. Given the heterogeneity and transient nature of the oligomers, it is essential to utilize findings across diverse experimental platforms that yield complementary data and apply methods that also ensure reproducibility and mechanistic clarity with the goal of translating these findings into effective therapeutic strategies.

Amyloid oligomers are increasingly recognized as the major toxic contributors across protein‐misfolding disorders. In this review, we cover mechanistic evidence showing how these transient and structurally heterogeneous oligomers disrupt cellular homeostasis by: (i) permeabilizing lipid membranes and forming ion‐conducting pores; (ii) triggering endoplasmic reticulum (ER) stress and unfolded protein response (UPR), thereby compromising proteostasis via dysfunction of the ubiquitin–proteasome system (UPS) and autophagy; (iii) impairing mitochondrial function and disrupting redox balance; (iv) interfering with endosomal–lysosomal as well as axonal and synaptic trafficking; and (v) activating stress‐kinase signaling and apoptotic pathways. In relation to therapeutic intervention, we review secretase‐targeting strategies, conformation‐selective antibodies, and their mixed clinical outcomes. An in‐depth understanding of the toxic action of pathogenic oligomeric species will be critical for translating these mechanistic insights into effective therapies that comprehensively target oligomer toxicity.