Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell without sample translation. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometers resolution, fluorescence-guided localized molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platform's capabilities by visualizing imipramine-induced transverse-tubule (TT) remodeling in live cardiomyocytes, revealing pronounced detubulation of internal TT invaginations while surface TT opening characteristics remain largely preserved, and capturing high-speed correlative volumetric images of clathrin-mediated endocytosis in Cos-7 cells. Additionally, we show precision delivery of fluorescent cargos, including dextrans and α-synuclein, into mammalian cells and diatoms, alongside localized stiffness mapping to evaluate mechanical responses of mammalian cells. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

Abstract Soluble Aβ oligomers are regarded as major neurotoxic agents in Alzheimer’s disease. Several monoclonal antibodies have been developed to target Aβ oligomers, but most of them show limited specificity binding also to monomers and fibrils. To generate an antibody with high specificity for the oligomers, we aimed to increase the efficiency and sensitivity of a human VH–derived Aβ-oligomer-specific single domain antibody, called DesAb-O. We engineered a dimeric DesAb-O variant, DiDesAb-O, which showed significantly higher binding affinity for Aβ oligomers as compared to the monomeric sdAb. DiDesAb-O selectively detected Aβ42 oligomers not only in vitro and in cultured cells using synthetic preparations, but also in the cerebrospinal fluid from Alzheimer’s patients. Moreover, it inhibited the binding of these toxic species to cellular membranes and neutralized their neurotoxicity both in cells and in patient-derived cerebrospinal fluid at lower concentrations compared to DesAb-O. These results indicate that rational dimerization of single-domain antibodies can substantially enhance target engagement and functional efficacy, providing a promising strategy for the development of improved diagnostic and therapeutic molecules for Alzheimer’s disease.

Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents in Alzheimer’s disease (AD). In the last few years, Aprile and co-workers designed and purified a single-domain antibody (sdAb), called DesAb-O, with high specificity for Aβ 1-42 oligomeric conformers. Recently, Cascella and co-workers showed that DesAb-O can selectively detect synthetic Aβ 1-42 oligomers both in vitro and in cultured cells, neutralizing their associated neuronal dysfunction. DesAb-O can also identify Aβ 1-42 oligomers in the cerebrospinal fluid (CSF) of AD patients, with respect to healthy individuals, preventing cell dysfunction induced by the administration of CSFs to neuronal cells. Given the extraodinary potentialities of this nanobody, we design a dimeric-structure of DesAb-O, with the aim to increase its avidity and affinity for toxic Aβ 1-42 oligomers. We designed the dimeric-DesAb-O structure by linking two DesAb-O monomeric domains with a flexible linker region (GGGGS) 3. Once expressed and purified the protein, we characterised its molecular weight by mass spectrometry and its secondary structure by Circular Dichroism. Then, we performed an aggregation assay to monitor its ability to interfere with the Aβ 1-42 aggregation process and a Real-Time based ELISA assay to study its binding for Aβ 1-42 oligomers. The Dimeric-DesAb-O is able to interfere with the Aβ 1-42 aggregation process to a greater extent than DesAb-O. Furthermore, the dimeric structure of DesAb-O showed a higher specificity and affinity for Aβ 1-42 oligomers compared to DesAb-O. The Dimeric-DesAb-O appears to be a promising tool for the future development of sdAbs-based immunodiagnostic tests for the early diagnosis of AD.

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platform’s capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos—including dextrans and α-synuclein—into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology. ### Competing Interest Statement AS is a shareholder in ICAPPIC, Ltd., a company commercialising nanopipette-based instrumentation. EPSRC, EP/W012219/1, EP/W015005/1, EP/X034968/1 Imperial College London and the China Scholarship Council (CSC) Scholarship, File No. 202309370006 Early Career Pathway Award of the CRUK International Alliance for Cancer Early Detection, EDDAPA-2024/100006 British Heart Foundation, RG/F/22/110081 Alzheimer's Society, Dementia Research Leader Fellowship AS-DRL-24-012 UKRI Future Leaders Fellowship, MR/S033947/1, MR/Y003616/1 Alzheimer's Research UK, Major Project Grant ARUK-PG2019B-020 UK Natural Environment Research Council, NE/V01451X/2

TDP-43 protein is an RNA-binding protein linked to amyotrophic lateral sclerosis, frontotemporal dementia, and Alzheimer disease. While normally a protein that shuttles between the nucleus and cytoplasm, TDP-43 has recently been found also in extracellular vesicles. These are an important medium for cell-cell communication that allows the transfer of lipids, proteins, and genetic material among cells. An increasing concern in neurodegenerative diseases, however, is the possibility that extracellular vesicles can also provide an effective way to spread misfolded proteins that could "infect" other cells according to a "prion-like" mechanism. To characterize the interaction of TDP-43 with lipid membranes, we carried out a systematic biophysical study using a TDP-43 fragment lacking the first 84 N-terminal residues, called M85, and synthetic model phospholipid membranes. We utilized standard techniques, such as fluorescence and microscopy, complemented by neutron reflectivity measurements. Our results show that lipid charge affects the modality by which M85 interacts with membranes: a higher negative charge induces the protein to bind to the bilayer surface, promoting protein aggregation and decreasing lipid bilayer damage that this interaction causes. Thus, we speculate that the M85-lipid membrane interaction could play an important and previously undefined role in TDP-43-related neurodegenerative diseases.

Introducing exogenous biomolecules into individual cells with precise control over space, time, and dosage is crucial for both fundamental and applied biological research. Glass nanopipettes have long been employed to deliver biomolecules into individual cells; yet, their reliance on the electrical charge of the target molecule and the need for penetrating the cellular membrane pose significant limitations. We demonstrate that voltage pulses applied through a glass nanopipette in proximity to the cell membrane induce localized electroporation and generate directional flow, enabling controlled delivery of both charged and neutral biomolecules into subcellular compartments, e.g., the nucleus, without the need for penetrating the cellular membrane. This approach minimizes cell damage and preserves cell viability, even after multiple rounds of injection. Our findings will serve as a reference for the design of novel nanopipette methods, contributing to the newly established field of spatiotemporal analysis of live cells.

α-Synuclein condensates can mature into amyloid fibrils, demonstrating a link between phase separation and amyloid aggregation. However, the mechanisms driving this maturation are not fully understood, particularly in the context of pathological post-translational modifications that modulate α-synuclein amyloid aggregation. Although often studied in isolation, condensates appear to interact with surfaces in vitro and in the cell. Notably, the N-terminus of α-synuclein is implicated in membrane binding and may influence condensate-surface interactions. Here, we developed a microscopy-based protocol to investigate how N-terminal truncation affects α-synuclein condensate formation, surface wetting, and maturation. We found that N-terminal truncation enhances condensate wettability and accelerates maturation. Conversely, perturbing condensate-surface interactions reduces condensate wettability and delays maturation. These results suggest that enhanced wettability promotes condensate maturation, likely by increasing condensate surface-to-volume ratios. Our findings reveal distinct mechanistic roles for the N-terminus of α-synuclein and highlight condensate wettability and interfacial dynamics as key modulators of aggregate formation. The transition of α-synuclein from biomolecular condensates to amyloid fibrils is a key process in neurodegeneration, yet the mechanisms underlying this transition remain unclear. Here, the authors use microscopy-based quantitative imaging to show that the early N-terminus decreases surface wettability and delays maturation, implicating condensate interface interactions in toxic aggregate formation.

α-Synuclein can self-assemble into amyloid fibrils in neurodegenerative diseases, including Parkinson’s disease. Recent studies show α-synuclein can undergo liquid-liquid phase separation, forming biomolecular condensates. The interconnection between α-synuclein amyloid formation and liquid-liquid phase separation is evident as these condensates can mature into solid gel-like condensates containing amyloid fibrils. Among the many α-synuclein post-translational modifications, N-terminally truncated fragments have been found in aggregates isolated from Parkinson’s disease affected brains and are known to affect the in vitro aggregation kinetics. Here, we show that physiologically relevant N-terminal truncation of residues 1-4 significantly enhances α-synuclein aggregation. Combining amyloid aggregation and liquid-liquid phase separation studies, we determined that this truncation increases the propensity of α-synuclein to nucleate at the interface, specifically at the fibril surface, on lipid vesicles and at the interface of biomolecular condensates. Our results enhance the understanding of α-synuclein pathology and offer new targets for therapeutic treatment. ### Competing Interest Statement The authors have declared no competing interest. * IDP : intrinsically disordered protein; α-syn, α-synuclein PD : Parkinson’s disease NAC : non-amyloid-β component PTMs : post-translational modifications LLPS : liquid-liquid phase separation PEG : poly-ethylene glycol PLK : poly-L-lysine FL : full length; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; E. coli, Escherichia coli SDS-PAGE : sodium dodecyl sulphate–polyacrylamide gel electrophoresis PBS : phosphate buffered saline UV-Vis : Ultraviolet– visible PCR : polymerase chain reaction ESI-MS : electrospray ionization mass spectrometry DMPS : 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) PB : 20 mM phosphate buffer ThT : Thioflavin T DLS : dynamic light scattering DIC : differential interference contrast GA3 : General Analysis 3 TEM : transmission electron microscopy.

Background Amyloid-β 42 (Aβ 42 ) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer’s disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aβ 42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aβ 42 oligomers. Methods We investigated the ability of DesAb-O to selectively detect preformed Aβ 42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. Results DesAb-O was found to selectively detect synthetic Aβ 42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aβ 42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. Conclusions Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

Extracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein are the main hallmarks of Alzheimer’s disease (AD). Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents. The heterogeneous structures and the transient nature of these oligomers make their isolation and characterization very challenging. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early diagnosis and therapy for AD. Performing an in vitro and in vivo screening of different sdAbs, we selected those targeting Aβ 42 oligomers or fibrils with high specificity using dot-blot, ELISA assay and the super resolution stimulated emission depletion (STED) microscopy. The potential of sdAbs was also investigated in preventing Aβ 42 oligomer-induced cytotoxicity in cultured neuronal cells. Then, the sdAbs were used to selectively detect Aβ 42 species in the cerebrospinal fluid (CSF) of AD patients and control subjects and to neutralize their associated harmful effects following cell exposure to CSF samples. sdAbs can selectively detect Aβ 42 oligomers or fibrils both in vitro and in cultured cells, preventing the neurotoxicity induced by small oligomers. Moreover, sdAbs can significantly identify Aβ 42 oligomers in the CSF of AD patients counteracting their induced toxicity in our cell models. All these data provide a solid foundation for the development of sdAbs-based immunodiagnostic tools that can selectively detect toxic aggregates in human body fluids for an early differential diagnosis of protein deposition diseases, such as AD. Furthermore, our results contribute to the improvement of the current therapeutic approaches against AD. The study was supported by Airalzh (AGYR 2020 to R.C.).

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC) n in C9orf72 . Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC) n , which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

Aging is the main risk factor for Alzheimer’s disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-β (Aβ) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc −/− ). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aβ in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aβ levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aβ and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aβ accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aβ accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.

Parkinson's Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identified an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fibrils. In addition, aggregation experiments show that Gal3 affects spatial propagation and the stability of pre-formed αSyn fibrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD.

The presence of extracellular amyloid β (Aβ) plaques in the brain parenchyma and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein is the main hallmark of Alzheimer’s disease (AD). Aβ aggregation consists of a complex chain of nucleation events producing soluble oligomer intermediates, which are the major neurotoxic agents. The transient nature of these oligomers makes very challenge their isolation and characterization. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early accurate diagnosis and therapy for AD. An in vitro and in vivo screening of different sdAbs was performed, selecting those targeting Aβ 42 oligomers with high specificity using immunoassays and the super resolution stimulated emission depletion (STED) microscopy. The ability of sdAbs to prevent Aβ 42 -induced cytotoxicity in neuronal cells was investigated. Then, the sdAbs were used to detect and quantify the levels of Aβ 42 assemblies in the cerebrospinal fluid (CSF) of AD patients and controls. SdAbs can detect neurotoxic Aβ assemblies and prevent the associated neuronal dysfunction in cell models. Furthermore, sdAbs significantly discriminated Aβ aggregates in the CSF. All these data provide solid grounds for the understanding of the molecular basis of oligomers toxicity, thus establishing a compelling structure-toxicity relationship. Moreover, sdAbs-based approaches hold a great potential as they allow detection in complex mixtures such as a biological sample for an early differential diagnosis of AD, also providing therapeutic benefits. The study was supported by Airalzh (AGYR 2020 to R.C.).

An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-β peptide or the protein α-synuclein are traditionally considered hallmarks of Alzheimer's and Parkinson's diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-β can induce the aggregation of α-synuclein. Fibrils formed under these conditions are solely composed of α-synuclein to which amyloid-β can be found associated but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of α-synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-β.