Cerebral organoids with chromosome 21 trisomy secrete Alzheimer's disease-related soluble aggregates detectable by single-molecule-fluorescence and super-resolution microscopy.

Understanding the role of small, soluble aggregates of beta-amyloid (Aß) and tau in Alzheimer's disease (AD) is of great importance for the rational design of preventative therapies. Here we report a set of methods for the detection, quantification, and characterisation of soluble aggregates in conditioned media of cerebral organoids derived from human iPSCs with trisomy 21, thus containing an extra copy of the amyloid precursor protein (APP) gene. We detected soluble beta-amyloid (Aß) and tau aggregates secreted by cerebral organoids from both control and the isogenic trisomy 21 (T21) genotype. We developed a novel method to normalise measurements to the number of live neurons within organoid-conditioned media based on glucose consumption. Thus normalised, T21 organoids produced 2.5-fold more Aß aggregates with a higher proportion of larger (300-2000 nm2) and more fibrillary-shaped aggregates than controls, along with 1.3-fold more soluble phosphorylated tau (pTau) aggregates, increased inflammasome ASC-specks, and a higher level of oxidative stress inducing thioredoxin-interacting protein (TXNIP). Importantly, all this was detectable prior to the appearance of histological amyloid plaques or intraneuronal tau-pathology in organoid slices, demonstrating the feasibility to model the initial pathogenic mechanisms for AD in-vitro using cells from live genetically pre-disposed donors before the onset of clinical disease. Then, using different iPSC clones generated from the same donor at different times in two independent experiments, we tested the reproducibility of findings in organoids. While there were differences in rates of disease progression between the experiments, the disease mechanisms were conserved. Overall, our results show that it is possible to non-invasively follow the development of pathology in organoid models of AD over time, by monitoring changes in the aggregates and proteins in the conditioned media, and open possibilities to study the time-course of the key pathogenic processes taking place.

Single-molecule characterization of salivary protein aggregates from Parkinson's disease patients: a pilot study.

Saliva is a convenient and accessible biofluid that has potential as a future diagnostic tool for Parkinson's disease. Candidate diagnostic tests for Parkinson's disease to date have predominantly focused on measurements of α-synuclein in CSF, but there is a need for accurate tests utilizing more easily accessible sample types. Prior studies utilizing saliva have used bulk measurements of salivary α-synuclein to provide diagnostic insight. Aggregate structure may influence the contribution of α-synuclein to disease pathology. Single-molecule approaches can characterize the structure of individual aggregates present in the biofluid and may, therefore, provide greater insight than bulk measurements. We have employed an antibody-based single-molecule pulldown assay to quantify salivary α-synuclein and amyloid-ß peptide aggregate numbers and subsequently super-resolved captured aggregates using direct Stochastic Optical Reconstruction Microscopy to describe their morphological features. We show that the salivary α-synuclein aggregate/amyloid-ß aggregate ratio is increased almost 2-fold in patients with Parkinson's disease (n = 20) compared with controls (n = 20, P < 0.05). Morphological information also provides insight, with saliva from patients with Parkinson's disease containing a greater proportion of larger and more fibrillar amyloid-ß aggregates than control saliva (P < 0.05). Furthermore, the combination of count and morphology data provides greater diagnostic value than either measure alone, distinguishing between patients with Parkinson's disease (n = 17) and controls (n = 18) with a high degree of accuracy (area under the curve = 0.87, P < 0.001) and a larger dynamic range. We, therefore, demonstrate for the first time the application of highly sensitive single-molecule imaging techniques to saliva. In addition, we show that aggregates present within saliva retain relevant structural information, further expanding the potential utility of saliva-based diagnostic methods.

Improved Imaging Surface for Quantitative Single-Molecule Microscopy.

Preventing nonspecific binding is essential for sensitive surface-based quantitative single-molecule microscopy. Here we report a much-simplified RainX-F127 (RF-127) surface with improved passivation. This surface achieves up to 100-fold less nonspecific binding from protein aggregates compared to commonly used polyethylene glycol (PEG) surfaces. The method is compatible with common single-molecule techniques including single-molecule pull-down (SiMPull), super-resolution imaging, antibody-binding screening and single exosome visualization. This method is also able to specifically detect alpha-synuclein (α-syn) and tau aggregates from a wide range of biofluids including human serum, brain extracts, cerebrospinal fluid (CSF) and saliva. The simplicity of this method further allows the functionalization of microplates for robot-assisted high-throughput single-molecule experiments. Overall, this simple but improved surface offers a versatile platform for quantitative single-molecule microscopy without the need for specialized equipment or personnel.