Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.

Since early oligomeric intermediates in amyloid assembly are often transient and difficult to distinguish, characterize and quantify, the mechanistic basis of the initiation of spontaneous amyloid growth is often opaque. We describe here an approach to the analysis of the Aβ aggregation mechanism that uses Aβ-polyglutamine hybrid peptides designed to retard amyloid maturation and an adjusted thioflavin intensity scale that reveals structural features of aggregation intermediates. The results support an aggregation initiation mechanism for Aβ-polyQ hybrids, and by extension for full-length Aβ peptides, in which a modular Aβ C-terminal segment mediates rapid, non-nucleated formation of α-helical oligomers. The resulting high local concentration of tethered amyloidogenic segments within these α-oligomers facilitates transition to a β-oligomer population that, via further remodelling and/or elongation steps, ultimately generates mature amyloid. Consistent with this mechanism, an engineered Aβ C-terminal fragment delays aggregation onset by Aβ-polyglutamine peptides and redirects assembly of Aβ42 fibrils.

Objective To explore the role of alpha‐synuclein (αSyn) oligomers and neurofilament light chain (NfL) in cerebrospinal fluid (CSF) of patients with pure autonomic failure (PAF) as markers of future phenoconversion to multiple system atrophy (MSA). Methods Well‐characterized patients with PAF (n = 32) were enrolled between June 2016 and February 2019 at Mayo Clinic Rochester and followed prospectively with annual visits to determine future phenoconversion to MSA, Parkinson's disease (PD), or dementia with Lewy bodies (DLB). ELISA was utilized to measure NfL and protein misfolding cyclic amplification (PMCA) to detect αSyn oligomers in CSF collected at baseline. Results Patients were followed for a median of 3.9 years. Five patients converted to MSA, 2 to PD, and 2 to DLB. NfL at baseline was elevated only in patients who later developed MSA, perfectly separating those from future PD and DLB converters as well as non‐converters. ASyn‐PMCA was positive in all but two cases (94%). The PMCA reaction was markedly different in five samples with maximum fluorescence and reaction kinetics previously described in MSA patients; all of these patients later developed MSA. Interpretation αSyn‐PMCA is almost invariably positive in the CSF of patients with PAF establishing this condition as α‐synucleinopathy. Both NfL and the magnitude and reaction kinetics of αSyn PMCA faithfully predict which PAF patients will eventually phenoconvert to MSA. This finding has important implications not only for prognostication, but also for future trials of disease modifying therapies, allowing for differentiation of MSA from Lewy body synucleinopathies before motor symptoms develop. ANN NEUROL 2021;89:1212–1220

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. Light chain (AL)2 amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. LC proteins are comprised of two distinct domains: the variable (V L ) and constant (C L ) domains (also called LC full-length (FL) protein to differentiate with the V L domain). In patients with AL amyloidosis, the LC aggregate and deposit in vital organs, causing organ failure and death (1). The factors governing deposition in individual tissues are unknown. Patients with cardiac AL amyloidosis have the worst prognosis, with a median survival of less than a year (2, 3).The finding that the V L was the primary component of amyloid fibrils influenced previous biophysical studies (4,5). Recent proteomic studies have demonstrated that amyloid deposits are likely heterogeneous in nature and can be formed by FL, V L , C L , or mixtures of all types of LC fragments (6 -8). Thermodynamic studies proposed a stabilizing role for the 3C L domain in the stability and a modulating effect on fibril formation (9). Recently, our laboratory has demonstrated that the C L domain modulates the amyloid formation reaction but has no effect on the stability of the protein (10).Soluble monoclonal LC, isolated from patients with amyloidosis, can impair rat cardiomyocyte function (11) and induce apoptotic events in mouse cardiomyocytes (12, 13). Also, urinederived LC can be internalized into primary rat cardiac fibroblasts (14) and primary human renal mesangial cells (15) through a pinocytic pathway (16) or via receptor, clathrin-mediated mechanisms, respectively (15).Within the amyloid field, it is widely accepted that oligomeric species are potentially more toxic than mature fibrils (17-20). However, toxicity associated with amyloid fibrils may also be pathologically relevant. Engel et al. (21) described a mechanism in which growth of islet amyloid associated polypeptides fibrils is re...

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.