Biology subjects

Lam, J. Y. L.

Publications and source records attributed to Lam, J. Y. L..

7 recordsLinked to original sources

bioRxiv2025

Super-resolution microscopy of alpha-synuclein aggregates in brain samples indicates a subset of cells have disrupted protein homeostasis

Nanoscopic aggregates of alpha-synuclein have been observed in Parkinsons disease (PD). However, the processes that occur in-vivo leading to the formation of these small aggregates are not well understood. We used ultra-sensitive single-molecule methods including SIMOA and super-resolution microscopy to quantify and characterise alpha-synuclein aggregates harvested from human brain samples alongside the Line 61 mouse model using different tissue processing methods. While aggregate numbers did not differ between PD and control samples, larger aggregates were detected in PD brain samples. Moreover, different sub-populations of aggregates were obtained by different extraction methods, with diffusible and membrane-bound aggregates producing a more pronounced difference between disease and control samples. Our data suggests that alpha-synuclein aggregates slowly in the brain, leading to formation of larger aggregates in a sub-set of cells.

neuroscience↗

bioRxiv2025

Small tau aggregates exhibit disease-specific molecular profiles across tauopathies

Tauopathies are neurodegenerative diseases marked by pathological tau aggregation. While disease-specific folds of insoluble tau filaments have been established, it remains unclear whether the smaller, earlier species also differ across tauopathies. Here, we characterise these small tau aggregates from post-mortem brain of individuals with Alzheimers disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration, Picks disease, and healthy controls. Using two complementary single-molecule assays, we confirm that small tau aggregates vary in abundance, morphology, and post-translational modifications. AD features specific long, fibrillar-shaped aggregates enriched in phospho-epitopes, while PSP aggregates are shorter, round, and selectively phosphorylated at serine-356, a site we identify as correlating with markers of inflammation and apoptosis. Aggregate properties co-vary with cellular stress signatures and align with disease-specific seeding profiles, suggesting distinct pathological mechanisms. These findings suggest that small tau aggregates are not a shared intermediate, but instead encode disease-specific mechanisms, with potential as both biomarkers and therapeutic targets. Key pointsSmall tau aggregates show disease-specific signatures across tauopathies, differing in abundance, morphology, and post-translational modifications. Tau aggregates in AD show enhanced phosphorylation density and structural heterogeneity, including a distinct population of long fibrillar species detectable in the soluble fraction. Alzheimers disease is characterised by specific long, fibrillar-shaped tau aggregates enriched in disease-relevant phospho-epitopes. PSP features round pSer356-positive aggregates that correlate with apoptotic and inflammatory markers, suggesting a distinct mechanism of toxicity. Isoform-specific biosensor assays reveal divergent seeding behaviour: CBD shows strong 4R seeding, while PSP lacks seeding activity. Features of small aggregates co-vary with distinct patterns of gliosis and cell stress, suggesting disease-specific mechanisms of tau-mediated toxicity.

neuroscience↗

bioRxiv2024

Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab

Monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab have been developed for treatment of Alzheimers disease. Here, we have used single-molecule detection techniques and super-resolution imaging to characterise the binding of these antibodies to beta-amyloid aggregates including human post-mortem brain samples. Lecanemab is the best antibody in terms of binding to the small-soluble beta-amyloid aggregates, affinity, aggregate coating, and the ability to bind to post-translationally modified species, explaining its therapeutic success.

neuroscience↗

bioRxiv2024

SynPull: a novel method for studying neurodegeneration-related aggregates in synaptosomes using super-resolution microscopy

Synaptic dysfunction is one of the primary hallmarks of both Alzheimers and Parkinsons disease, leading to cognitive and behavioural decline. While alpha-synuclein, beta-amyloid, and tau are involved in the physiological functioning of synapses, their pathological aggregation has been linked to synaptic dysfunction. However, the methodology for studying the small (sub-diffraction limit) and soluble aggregates -often called oligomers, formed by these proteins is limited. Here we describe SynPull, a novel method combining single-molecule pulldown, super-resolution microscopy, and advanced computational analyses, in order to reliably study the quantity and morphology of the oligomeric alpha-synuclein, beta-amyloid, and AT8-positive tau aggregates in synaptosomes harvested from post-mortem human brain samples and mouse models. Using SynPull, we show that AT8-positive tau is the predominant aggregate type in AD, with significantly more aggregates compared to the control samples, yet the aggregate size does not differ between disease and control samples. Meanwhile, the relatively smaller amount of alpha-synuclein and beta-amyloid aggregates found in the synapses are larger than the extra-synaptic ones. Collectively, these results show the utility of SynPull to study pathological aggregates in dementia, which can help further understand the disease mechanisms causing synaptic dysfunction. O_FIG O_LINKSMALLFIG WIDTH=140 HEIGHT=200 SRC="FIGDIR/small/609517v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@d53d06org.highwire.dtl.DTLVardef@1995971org.highwire.dtl.DTLVardef@bf3edaorg.highwire.dtl.DTLVardef@1b75ca5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO Human post-mortem orbitofrontal cortex samples from subjects with neuropathological diagnosis of Alzheimers and Parkinsons disease, as well as age-matched controls cut into [~]300 mg sections, and MI2, APPNL-G-F, P301S, and C57Bl/6J mouse brains were first homogenised in synaptosome buffer using a Dounce homogeniser and then filtered and centrifuged to separate nuclei and organelles from the synaptic fragments. Then, the isolated synaptosomes were incubated on the SiMPull surface with anti-neurexin antibody overnight, followed by fixation and permeabilisation. Imaging antibodies against beta-amyloid, alpha-synuclein, and AT8-positive tau were added to the samples and dSTORM imaging was performed to super-resolve the aggregates. C_FIG

neuroscience↗

bioRxiv2023

Characterization and super-resolution imaging of small tau aggregates in human samples.

Hyperphosphorylation and aggregation of the microtubule binding protein tau plays a key role in the development of Alzheimers disease. While the structure of the filamentous aggregates formed in humans has recently been determined to atomic resolution, there is far less information available about the smaller aggregate precursors, thought to be the most neurotoxic. To address this gap, we have developed a single molecule pull-down (SiMPull) able to detect tau aggregates in clinically relevant human samples. This method enables the detection and characterisation of individual tau aggregates, as opposed to averaged features obtained from traditional bulk techniques. We report the number, size and shape of individual aggregates measured via super-resolution microscopy, revealing disease-specific differences in tau aggregate morphology. By adapting the assay to simultaneously detect multiple phosphorylation sites in individual aggregates, we were also able to derive compositional profiles for pathological modifications present in individual aggregates. We demonstrate that tau aggregates in Alzheimers disease are significantly more likely to contain both the AT8 and T181 pathological phosphorylation markers, rather than only one. Together, tau SiMPull identified distinct subpopulations of large, modified tau aggregates that were invisible to traditional methodologies. These morphological and compositional differences distinguish samples taken from disease cohorts, offering to illuminate underlying disease mechanisms, and providing a foundation for novel diagnostic strategies.

biochemistry↗

bioRxiv2021

AN ECONOMIC, SQUARE-SHAPED FLAT-FIELD ILLUMINATION MODULE FOR TIRF-BASED SUPER-RESOLUTION MICROSCOPY

Super-resolution (SR) microscopy allows complex biological assemblies to be observed with remarkable resolution. However, the presence of uneven Gaussian-shaped illumination hinders its use in quantitative imaging or high-throughput assays. Methods developed to circumvent this problem are often expensive, hard-to-implement, or not applicable to total internal reflection fluorescence (TIRF) imaging. We herein demonstrate a cost-effective method to overcome these challenges using a small square-core multimodal optical fibre as the coupler. We characterise our method with synthetic, recombinant and cellular systems imaged under TIRF and highly inclined and laminated optical sheet (HILO) illuminations to demonstrate its ability to produce highly uniform images under all conditions.

biophysics↗

bioRxiv2021

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-β in Alzheimer's disease

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimers Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-{beta} (A{beta}) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with A{beta} in the early stages of aggregation and then fall away as fibrillation happens. ApoE-A{beta} co-aggregates account for [~]50% of the mass of soluble A{beta} aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of A{beta}. Selectively removing non-lipidated apoE4-A{beta} co-aggregates enhances clearance of toxic A{beta} by glial cells, and reduces inflammation and membrane damage, demonstrating a clear path to AD therapeutics.

neuroscience↗