Biology subjects

Aprile, F. A.

Publications and source records attributed to Aprile, F. A..

12 recordsLinked to original sources

bioRxiv2026

A Molecular Rotor-Based Platform for Dissecting Protein Phase Separation and Aggregation

Biomolecular condensates formed via phase separation play critical roles in regulating cellular processes, and their dysregulation is increasingly associated with neurodegeneration. A key challenge in understanding condensate pathology lies in distinguishing dynamic condensates from neurotoxic solid aggregates, the formation of which can, in some systems, occur within existing condensates. Yet current detection techniques often lack the sensitivity and resolution to directly monitor these assemblies and their transitions in dynamic and complex environments. Here, we combined fluorescent probes, based on molecular rotors, with fluorescence lifetime imaging microscopy to quantify changes in protein dynamics during phase separation-associated aggregate formation. Molecular rotors respond to local viscosity changes, which in our study allowed discrimination between fluid condensates and rigid amyloids based on rotors' fluorescence lifetime. Using -synuclein, a hallmark protein in Parkinson's disease, as a model system, we demonstrate real-time visualization of phase separation and condensate maturation within a unified experimental framework. Our method preserves protein integrity and provides quantitative, spatially resolved insights into structural transitions. This technology bridges a critical gap in the study of pathological protein aggregation and offers a powerful platform for mechanistic studies, biomarker discovery, and drug discovery in neurodegenerative disease research.

biophysics↗

bioRxiv2025

Tracking gene expression of single mitochondria in live neurons using nanotweezers

Neurons are highly polarised cells that depend on mitochondria for energy and signalling homeostasis. Importantly, energy and signalling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyse mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction a individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with mitochondrial gene expression tracking and mtDNA profiling. By tracking mitochondrial gene expression in the same neurons over time, we reveal a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to -synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides precise, dynamic measurements of mitochondrial composition and gene expression in vivo at single-organelle resolution, enabling mechanistic studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.

neuroscience↗

bioRxiv2025

Engineering a Dimeric Single-Domain Antibody for Improved Detection and neutralization of Amyloid-β oligomers

Soluble A{beta} oligomers are regarded as major neurotoxic agents in Alzheimers disease. Several monoclonal antibodies have been developed to target A{beta} 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 humanized A{beta}-oligomer-specific single domain antibody, called DesAb-O. We engineered a dimeric DesAb-O variant, DiDesAb-O, which showed a significantly improved binding properties for A{beta} oligomers as compared to the monomeric sdAb. Furthermore, DiDesAb-O detected A{beta}42 oligomers in cells, prevented their binding to cell membranes and the A{beta}42 oligomers-induced neurotoxicity from both synthetic A{beta}42 samples and cerebrospinal fluid of Alzheimers patients at lower concentrations compared to DesAb-O. Overall, our findings indicate that the rational engineering of dimeric sdAb variants is an effective strategy to improve their binding properties offering new opportunities for the development of clinical molecules in the early diagnosis and cure of Alzheimers disease. Significance StatementProtein aggregates, characterized by high structural heterogeneity, are central to numerous neurodegenerative diseases, yet the development of aggregate-specific molecular probes and therapeutic agents remains a significant challenge. We present a novel approach to enhance the binding of single-domain antibodies by engineering multivalent, specifically dimeric, molecules to exploit the avidity effect. As a proof of concept, we developed a dimeric version of a previously studied single-domain antibody named DesAb-O, which exhibits enhanced binding sensitivity and specificity to toxic amyloid-{beta} oligomers and greater efficacy in inhibiting their toxicity. These findings provide a robust foundation for creating next-generation antibody fragments with enhanced binding to heterogeneous protein aggregates, opening new avenues for innovative diagnostic tools and therapeutic strategies in neurodegenerative disease research and treatment.

biochemistry↗

bioRxiv2025

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

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 platforms 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 -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.

biophysics↗

bioRxiv2024

Molecular rotors provide insight into the mechanism of formation and conversion of α-synuclein aggregates

-Synuclein is an intrinsically disordered protein that forms amyloids in Parkinsons disease. Currently, detection methods predominantly report on the formation of mature amyloids but have poor sensitivity to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitor -synuclein oligomer formation using the fluorescence lifetime of molecular rotors. We detect oligomer formation and conversion into amyloids for wild type and two -synuclein variants; the pathological mutant A30P and {Delta}P -synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30P -synuclein shows a similar rate of oligomer formation compared to wild type -synuclein, whereas {Delta}P -synuclein shows delayed oligomer formation. Additionally, both variants demonstrate a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism of -synuclein aggregation which is key to understanding the pathology of Parkinsons disease.

biochemistry↗

bioRxiv2024

The N-terminal Region of alpha-Synuclein Controls Amyloid Nucleation at the Condensate Interface

-Synuclein self-assembles into amyloid fibrils during neurodegeneration. The protein can also self-assemble via liquid-liquid phase separation to form biomolecular condensates. The link between these processes is evident, as -synuclein condensates can mature into amyloids. However, the mechanisms driving this maturation remain largely unknown, particularly when incorporating pathological post-translational modifications known to affect -synuclein self-assembly in the absence of LLPS, such as N-terminal truncation. Moreover, condensates are primarily studied as isolated entities; however, it is increasingly evident that they interact with various cellular components and surfaces. Here, we developed a microscopy-based quantitative real-time imaging protocol to investigate how N-terminal truncation influences -synuclein condensate formation, well surface wetting, and maturation. We found that increasing -synuclein truncation, which reduces N-terminal hydrophobicity, inhibits condensate sedimentation, enhances surface wettability, and accelerates maturation. Additionally, by increasing well surface hydrophobicity we decreased -synuclein condensate wettability, delaying their maturation. Thus, we propose that enhanced wettability, which increases the condensate surface-to-volume ratio, promotes -synuclein nucleation at the condensate-bulk solution interface, thereby accelerating maturation. Our results reveal distinct mechanistic roles for -synuclein N-terminal residues and indicate that condensate wetting on cellular surfaces, such as synaptic vesicles, may drive toxic aggregate formation during neurodegeneration.

biochemistry↗

bioRxiv2022

Amyloid-β Oligomers Serve as Nucleation Sites for α-Synuclein Aggregation

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-{beta} peptide or the protein -synuclein are traditionally considered hallmarks of Alzheimers and Parkinsons 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-{beta} can induce the aggregation of -synuclein. Fibrils formed under these conditions are solely composed of -synuclein to which amyloid-{beta} 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 -synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-{beta}.

biochemistry↗

bioRxiv2022

A Facile Method to Produce N-Terminally Truncated α-Synuclein

-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinsons disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of -synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of -synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of -synuclein play an important role in the formation of the amyloids.

biochemistry↗

bioRxiv2022

A Chemical Mutagenesis Approach to Insert Post-Translational Modifications in Aggregation-Prone Proteins

Neurodegenerative diseases are a class of disorders linked to the formation in the nervous system of fibrillar protein aggregates called amyloids. This aggregation process is affected by a variety of post-translational modifications, whose specific mechanisms are not fully understood yet. Emerging chemical mutagenesis technology is currently striving to address the challenge of introducing protein post-translational modifications, while maintaining proteins stable and soluble during the modification reaction. Several amyloidogenic proteins are highly aggregation-prone, and current modification procedures lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic proteins soluble during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of the serine 26 and the acetylation of the lysine 28 of the 40-residue long variant of amyloid-{beta} peptide, whose aggregation is linked to Alzheimers disease.

biochemistry↗

bioRxiv2020

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

While aggregation-prone proteins are known to accelerate ageing and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A and SERF2 have recently been identified as cellular modifiers of such cytotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with specific patterns of negatively charged and hydrophobic, aromatic amino acids. The absence of such patterns, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In a protein aggregation model in the nematode C. elegans, protein aggregation was suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that charge interactions are required for MOAG-4 and SERF2 to promote aggregation. Such charged interactions might accelerate the primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our finding that negatively charged segments are overrepresented in amyloid-forming proteins suggests that inhibition of charge interactions deserves exploration as a strategy to target age-related protein toxicity. Significance StatementHow aging causes relatively common diseases such as Alzheimers and Parkinsons is still a mystery. Since toxic structural changes in proteins are likely to be responsible, we investigated biological mechanisms that could drive such changes. We made use of a modifying factor called SERF2, which accelerates structural changes and aggregation of several disease-related proteins. Through a peptide-binding screen, we found that SERF2 acts on negatively charged protein regions. The abundance of such regions in the disease-related proteins explains why SERF has its effect. Removing positive charge in SERF was sufficient to suppress protein aggregation in models for disease. We propose that blocking charge-interactions with SERF or other modifiers could serve as a general approach to treat age-related protein toxicity.

molecular biology↗

bioRxiv2019

Small molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer’s disease

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that has been shown to modify the properties of their monomeric states. Here, we identify a small molecule capable of binding and sequestering the amyloid-{beta} peptide (A{beta}) in its monomeric, soluble state. Our analysis reveals that this compound interacts with A{beta} and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterise this interaction using biophysical experiments and integrative structural ensemble determination methods. We thus observe that this small molecule has the remarkable effect of increasing the conformational entropy of monomeric A{beta} while decreasing its hydrophobic surface area. We then show that this small molecule rescues a Caenorhabditis elegans model of A{beta}-associated toxicity in a manner consistent with the mechanism of action identified from the in silico and in vitro studies. These results provide an illustration of the strategy of targeting the monomeric states of disordered proteins with small molecules to alter their behaviour for therapeutic purposes.

biophysics↗

bioRxiv2019

ADSoluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression

Soluble aggregates of amyloid-{beta} (A{beta}) have been associated with neuronal and synaptic loss in Alzheimers disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we studied the soluble A{beta} aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls. We found that these aggregates vary structurally and in their mechanisms of toxicity. More small aggregates of A{beta} that can cause membrane permeabilization already found in MCI; in established AD, the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells. These results suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

neuroscience↗