Biology subjects

Rizzoli, S. O.

Publications and source records attributed to Rizzoli, S. O..

At least 19 recordsLinked to original sources

bioRxiv2026

Reverse engineering Lewy bodies suggests alpha-synuclein pathology as condensatopathy

Understanding how soluble monomeric alpha-synuclein transitions into pathological alpha-synuclein (Lewy bodies) has remained a central challenge in neurodegeneration. Existing models, largely based on treating cells and animals with pre-formed alpha-synuclein fibrils cannot account for the emergence of the first inclusion without pre-existing seeds. Here, we combine in vitro reconstitutions, computational modeling and a minimal cellular system to show that phase separation of alpha-synuclein with an interacting scaffold can recapitulate Lewy body-like structures. An unbiased proteomics screen, live-cell imaging, correlative light-electron microscopy, and soft X-ray tomography show that Lewy body-like structures are associated with redistribution of actin and membrane-bound organelles, with mitochondria accumulating at their periphery in an actin-dependent manner. Structural analyses further indicate that alpha-synuclein condensates provide local environments in which the insoluble beta-sheets can form. Together, these findings support a seed-independent trajectory from soluble alpha-synuclein through condensation and progressive immobilization to Lewy body-like assemblies and, subsequently, insoluble beta-sheet fibrils. This framework offers a new mechanistic route for understanding the disease progression and promises to guide the design of innovative therapeutic interventions.

neuroscience↗

bioRxiv2026

Synapsin-1 and actin form ordered nanoscale assemblies

Liquid-liquid phase separation is a vital and ubiquitous principle of subcellular organization. Several actin-binding proteins have recently been shown to undergo liquid-liquid phase separation, forming micron-sized droplets that assemble actin into distinct network shapes. However, our knowledge of how these phase-separated proteins organize actin filaments at the nanoscale is rather limited. Here, we seek to address this knowledge gap by investigating synapsin-1 condensates through a combination of computational simulations and nanoscale imaging experiments. Our results show that while synapsin-1 condensates by themselves lack any special structural organization, the addition of actin filaments results in the generation of a regularly organized actin scaffold within the condensates. The organized scaffold forms both when actin and synapsin-1 are added simultaneously and when actin is added after the formation of synapsin condensates. Dissolving the condensates, by synapsin-1 removal, leaves the actin scaffold largely unaffected in both simulations and experiments, implying that this scaffold should guide droplet reformation when synapsin-1 is added back. Taken together, our findings suggest that the actin-rich component of the condensate retains a structural memory that promotes and guides the reassembly of synapsin-actin condensates. This structural memory may be important in the long-term maintenance of synaptic function in vivo.

biophysics↗

bioRxiv2026

Structural cell biology by mega-expansion microscopy

Structural characterization of macromolecular assemblies within intact cells remains one of the central challenges in cell biology. While cryo-electron tomography provides unparalleled structural information, its applicability is limited by sample thickness, imaging throughput, and accessibility. Fluorescence microscopy offers molecular specificity and compatibility with intact biological specimens but has so far lacked the spatial resolution required to visualize cellular ultrastructure. Here we introduce Mega-expansion microscopy (Mega-ExM), a fluorescence imaging approach that enables structural visualization of whole cells using conventional confocal microscopes. Mega-ExM combines iterative hydrogel expansion with whole-proteome NHS-dye labeling and post-expansion immunostaining to achieve tunable expansion factors of up to [~]1,500-fold while preserving ultrastructure. At expansion factors of 40-260x, Mega-ExM resolves centrioles, mitochondrial cristae, protein-dense domains within mitochondrial cristae consistent with respiratory-chain supercomplexes, the synaptonemal complex, and nuclear pore complexes (NPCs) with high fidelity. Particle averaging of [~]200x expanded NPCs yields reconstructions with a structural resolution of [~]35 [A], approaching what cryo-electron tomography has achieved for selected protein assemblies. By combining molecular specificity, large imaging volumes, and nanoscale structural resolution on conventional fluorescence microscopes, Mega-ExM establishes a broadly accessible platform for in situ structural biology.

biophysics↗

bioRxiv2025

Brain-derived synaptic vesicles have an intrinsic ability to sequester tubulin

The presence and function of microtubules within the synaptic bouton has long been under investigation. In recent years evidence has accumulated that connects the synaptic vesicle cluster to the local dynamics of microtubule ends. Nonetheless, one question remains open, namely whether the vesicles influence the availability of tubulin within the synaptic compartment. An analysis of previously published live imaging experiments indicates that tubulin is strongly enriched in the synaptic vesicle cluster. To analyze the vesicle-tubulin interaction directly, we isolated vesicles from the mouse brain and imaged them together with fluorescent tubulin in vitro. We found that soluble tubulin is collected by synaptic vesicles in physiological buffers, resulting in the formation of tubulin-rich regions (TRRs) on the respective vesicle clusters. We conclude that the synaptic vesicle cluster is indeed able to recruit soluble tubulin.

neuroscience↗

bioRxiv2025

Nanobinders for Synaptotagmin 1 enable the analysis of synapticvesicle dynamics in rodent and human models.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

neuroscience↗

bioRxiv2025

Glutamatergic synaptic resilience to overexpressed human alpha-synuclein

Alpha synuclein (aSyn) is an abundant protein that, in the brain, is concentrated in neuronal presynaptic boutons and associates with synaptic vesicles. aSyn is strongly linked with Parkinsons disease (PD) due to its accumulation in pathognomonic inclusions in neuronal cells, including in glutamatergic neurons. While increased expression of wild-type (WT) aSyn due to multiplications of the SNCA gene, or the expression of mutant forms of aSyn, can cause familial forms of PD, it is still unclear whether increased levels of aSyn alone are sufficient to impair synaptic function. Previous studies have used experimental systems that do not allow systematic characterisation of presynaptic physiology. To address this gap in research, we used lentiviral vectors to overexpress human aSyn (haSyn) in continental and autaptic glutamatergic neurons from rodent hippocampus and systematically analysed their presynaptic function. Virally-transduced neurons exhibited levels of expression of haSyn that mimic those associated with triplications of the SNCA gene in PD patients (2-fold increase in total aSyn), as determined using quantitative immunofluorescence imaging and immunoblots. Neuronal toxicity, neuronal morphology, and SNAP-25, a presynaptic protein, were not altered in continental cultures. Finally, a systematic characterization of autaptic neurons expressing haSyn exhibited no significant difference in any parameter of synaptic function, including basal properties of evoked and spontaneous neurotransmitter release, and synaptic plasticity compared to neurons infected with a control virus. These results indicate that rodent glutamatergic neurons are resilient to aSyn overexpression. In conclusion, our findings suggest neurotoxicity associated with aSyn overexpression is not universal, and that a deeper understanding of aSyn biology and pathobiology is necessary.

neuroscience↗

bioRxiv2024

SynapseNet: Deep Learning for Automatic Synapse Reconstruction

Electron microscopy is an important technique for the study of synaptic morphology and its relation to synaptic function. The data analysis for this task requires the segmentation of the relevant synaptic structures, such as synaptic vesicles, active zones, mitochondria, presynaptic densities, synaptic ribbons, and synaptic compartments. Previous studies were predominantly based on manual segmentation, which is very time-consuming and prevented the systematic analysis of large datasets. Here, we introduce SynapseNet, a tool for the automatic segmentation and analysis of synapses in electron micrographs. It can reliably segment synaptic vesicles and other synaptic structures in a wide range of electron microscopy approaches, thanks to a large annotated dataset, which we assembled, and domain adaptation functionality we developed. We demonstrated its capability for (semi-)automatic biological analysis in two applications and made it available as an easy-to-use tool to enable novel data-driven insights into synapse organization and function.

bioinformatics↗

bioRxiv2024

PHluorin-conjugated secondary nanobodies as a tool for measuring synaptic vesicle exo- and endocytosis

Neuronal communication relies on synaptic vesicle recycling, which has long been investigated by live imaging approaches. Synapto-pHluorins, genetically encoded reporters that incorporate a pH-sensitive variant of GFP within the lumen of the synaptic vesicle, have been especially popular. However, they require genetic manipulation, implying that a tool combining their excellent reporter properties with the ease of use of classical immunolabeling would be desirable. We introduce this tool here, relying on primary antibodies against the luminal domain of synaptotagmin 1, decorated with secondary single-domain antibodies (nanobodies) carrying a pHluorin moiety. The application of the antibodies and nanobodies to cultured neurons results in labeling their recycling vesicles, without the need for any additional manipulations. The labeled vesicles respond to stimulation, in the expected fashion, and the pHluorin signals enable the quantification of both exo- and endocytosis. We conclude that pHluorin-conjugated secondary nanobodies are a convenient tool for the analysis of vesicle recycling.

neuroscience↗

bioRxiv2024

Calcium-triggered (de)ubiquitination events in synapses

Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by calcium-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on [~]2,000 proteins, the majority of which participate in SV recycling processes. Several proteins showed significant changes in ubiquitination in response to calcium influx, with the most pronounced changes in CaMKII and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKII mutant lacking the ubiquitination target site (K291) and analysed it both in neurons and non-neuronal cells. K291 ubiquitination influences CaMKII activity and synaptic function by modulating its autophosphorylation at a functionally important site (T286). We suggest that ubiquitination in response to synaptic activity is an important regulator of synaptic function.

neuroscience↗

bioRxiv2024

MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity

Lewy pathology, consisting of Lewy bodies and Lewy neurites, is the pathological hallmark of synucle-inopathies such as Parkinsons disease and dementia with Lewy bodies, but it is generally thought to represent late-stage pathological changes. In contrast, -synuclein oligomers are regarded as early-stage pathology, likely involved in disease progression and cellular toxicity. Oligomers, however, are not de-tected by standard immunohistochemistry but require specific detection techniques such as the proxim-ity ligation assay (PLA). Here, we describe the MJF-14 PLA, a new PLA towards aggregated -synuclein with unprecedented specificity, attained by the utilization of aggregate conformation-specific -synu-clein antibody MJFR-14-6-4-2 (hereafter MJF-14). Signal in the assay directly correlates with -synuclein aggregation in SH-SY5Y cells, as treatment with aggregation inhibitor ASI1D significantly lowers PLA sig-nal. In human cortical neurons, MJF-14 PLA detects pre-formed fibril-induced aggregation, especially prominent when using stealth PFFs invisible to the MJF-14 antibody. Co-labelling of MJF-14 PLA and pS129--synuclein immunofluorescence in post-mortem dementia with Lewy bodies cases showed that while the MJF-14 PLA reveals extensive non-inclusion pathology, it is not sensitive towards Lewy bodies. In Parkinsons disease brain, direct comparison of PLA and IHC with the MJF-14 antibody, combined with machine learning-based quantification, showed striking -synuclein pathology preceding the formation of conventional Lewy pathology. The majority of the PLA-revealed non-inclusion pathology was found in the neuropil, including some clearly located in the presynaptic terminals. With this work, we introduce an improved -synuclein aggregate PLA to uncover abundant non-inclusion pathology, which deserves future validation with multiple brain bank resources and in different synucleinopathies.

neuroscience↗

bioRxiv2023

Doubling the resolution of single-molecule localization microscopy with image scanning microscopy

In this study, we integrate a single-photon detector array into a Confocal Laser Scanning Microscope (CLSM), enabling the combination of Fluorescence Life-time Single-Molecule Localization Microscopy (FL-SMLM) with Image Scanning Microscopy (ISM). This unique combination delivers a two-fold improvement in lateral localization accuracy for SMLM while maintaining its simplicity. More-over, the addition of lifetime information from our CLSM eliminates chromatic aberration, particularly crucial for achieving few-nanometer resolution in SMLM. Our approach, named Fluorescence Lifetime ISM-SMLM (FL-iSMLM), is demon-strated through direct Stochastic Optical Reconstruction Microscopy (dSTORM) and DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence lifetime multiplexing capabilities.

biophysics↗

bioRxiv2022

Age-dependent structural reorganization of utricular ribbon synapses

In mammals, spatial orientation is synaptically-encoded by sensory hair cells of the vestibular labyrinth. Vestibular hair cells (VHCs) harbor synaptic ribbons at their presynaptic active zones (AZs), which play a critical role in molecular scaffolding and facilitate synaptic release and vesicular replenishment. With advancing age, the prevalence of vestibular deficits increases; yet, a direct link to the functional decline of VHC ribbon synapses remains to be demonstrated. To address this issue, we investigated the effects of aging on the ultrastructure of the ribbon-type AZs in murine utricles using various electron microscopic techniques and combined them with confocal and super-resolution light microscopy as well as metabolic imaging up to one year of age. In older animals, we detected predominantly in type I VHCs the formation of floating ribbon clusters. Our findings suggest that VHC ribbon-type AZs undergo dramatic structural alterations upon aging.

cell biology↗

bioRxiv2022

Heat denaturation enables multicolor X10-STED microscopy at single-digit nanometer resolution

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the 10-fold expansion of the samples through high-temperature homogenization (X10ht). The resulting gels exhibited relatively high fluorescence intensity, enabling the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm. X10ht offers a more thorough homogenization than previous X10 protocols based on enzymatic digestion, and thereby enables the expansion of thick samples. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.

bioengineering↗

bioRxiv2022

Protein nanobarcodes enable single-step multiplexed fluorescence imaging

Multiplexed cellular imaging typically relies on the sequential application of detection probes, such as antibodies or DNA barcodes, which is complex and time-consuming. To address this, we developed here protein nanobarcodes, composed of combinations of epitopes recognized by specific sets of nanobodies. The nanobarcodes are read in a single imaging step, relying on nanobodies conjugated to distinct fluorophores, which enables a precise analysis of large numbers of protein combinations.

cell biology↗

bioRxiv2021

Super-resolution microscopy informs on the molecular architecture of alpha-synuclein inclusions in model systems and in the human brain

Lewy bodies (LBs) and Lewy neurites are pathological hallmarks of Parkinsons disease and other progressive neurodegenerative disorders known as Lewy body diseases (LBD). These proteinaceous deposits are immunopositive for alpha-synuclein (aSyn) and several other proteins, as neurofilament components. The structural organization and composition of aSyn inclusions is still unclear and needs to be addressed in greater detail, as this may open novel avenues for our understanding of the disease-relevant pathological events. In this study, we investigated the molecular architecture of aSyn inclusions, both in cell models and in human brain tissue, using state-of-art super resolution X10 Expansion microscopy (ExM). This approach physically expands specimens embedded into a swellable gel, preserving their biological information. Then, the specimen can be analyzed using standard epifluorescence microscopes, thereby obtaining nanoscale information. The combination of different cell models, mouse and human brain tissue enabled us to distinguish different types aSyn assemblies (e.g. ring shape or tubular structures), and a conserved pattern of aSyn inclusions surrounded/encaged by intermediate filament proteins. Overall, X10 ExM enabled us to gain insight into the architecture and biology of aSyn inclusions and constitutes a powerful tool in the quest to understanding underlying disease mechanisms in synucleinopathies.

neuroscience↗

bioRxiv2021

Human brain organoids assemble functionally integrated bilateral optic vesicles

During embryogenesis, optic vesicles develop from the diencephalon via a complex process of organogenesis. Using iPSC-derived human brain organoids, we attempted to simplify the complexities and demonstrate the formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day thirty, brain organoids could assemble optic vesicles, which progressively develop as visible structures within sixty days. These optic vesicle-containing brain organoids (OVB-Organoids) constitute a developing optic vesicles cellular components, including the primitive cornea and lens-like cells, developing photoreceptors, retinal pigment epithelia, axon-like projections, and electrically active neuronal networks. Besides, OVB-Organoids also display synapsin-1, CTIP-positive, myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photoreceptor activity of OVB-Organoids, and light sensitivities could be reset after a transient photo bleach blinding. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow conducting interorgan interaction studies within a single organoid.

developmental biology↗

bioRxiv2020

Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R

The brain extracellular matrix (ECM) assembles around neurons and synapses, and is thought to change only rarely, through proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules. We found that a key ECM protein, Tenascin-R, is frequently endocytosed, and later resurfaces, preferentially near synapses. The TNR molecules complete this cycle within [~]3 days, in an activity-dependent fashion.

neuroscience↗

bioRxiv2020

Local translation in synaptic mitochondria influences synaptic transmission

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system, and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we addressed this by devising an imaging approach to analyze mitochondrial translation, by following the incorporation of clickable non-canonical amino acids. We applied this method to multiple cell types, including hippocampal neurons, where we found ample evidence for mitochondrial translation in both dendrites and axons. Translation levels were surprisingly heterogeneous, were typically stronger in axons, and were independent of their distance from the cell soma, where mitochondria presumably descent from. Presynaptic mitochondrial translation correlated with local synaptic activity, and blocking mitochondria translation reduced synaptic function. Overall, these findings demonstrate that mitochondrial gene expression in neurons is intimately linked to neuronal function.

biochemistry↗