Biology subjects

Zacco, E.

Publications and source records attributed to Zacco, E..

6 recordsLinked to original sources

bioRxiv2024

The role of RNA in the nanoscale organization of alpha-synuclein phase separation

The cellular accumulation of alpha-synuclein (aS) aggregates is a hallmark of several neurodegenerative diseases. Recent studies suggest that the aberrant transition of monomeric aS into solid-like aggregates may occur through an intermediate liquid-like state, where the protein partitions between dense and dilute phases. Although aS is not typically recognized as an RNA-binding protein, it can bind RNA under aggregation conditions, but its impact on aS liquid-like phases remains unexplored. Employing a combination of fluorescence spectroscopy techniques, we investigated aS dynamics in both phases in the presence of RNA. Our analysis revealed the formation of nanoclusters involved in initiating phase separation and uncovered heterogeneity within the dense phase, discovering that aS molecules exist in two distinct mobility states. Additionally, we demonstrated that RNA induces morphological changes and promotes the liquid-to-solid transition of aS dense phase. These findings underscore the active role of RNA in modulating aS phase transitions.

biophysics↗

bioRxiv2024

Amygdala TDP-43 pathology is associated with behavioural dysfunction and ferritin accumulation in amyotrophic lateral sclerosis.

BackgroundCognitive and behavioural symptoms associated with amyotrophic lateral sclerosis and frontotemporal spectrum disorders (ALSFTSD) are thought to be driven, at least in part, by the pathological accumulation of TDP-43. MethodsHere we examine post-mortem tissue from six brain regions associated with cognitive and behavioural symptoms in a cohort of 30 people with sporadic ALS (sALS), a proportion of which underwent standardized neuropsychological behavioural assessment as part of the Edinburgh Cognitive ALS Screen (ECAS). ResultsOverall, the behavioural screen performed as part of the ECAS predicted accumulation of pathological phosphorylated TDP-43 (pTDP-43) with 100% specificity and 86% sensitivity in behaviour-associated brain regions. Notably, of these regions, pathology in the amygdala was the most predictive correlate of behavioural dysfunction in sALS. In the amygdala of sALS patients, we show variation in morphology, cell type predominance, and severity of pTDP-43 pathology. Further, we demonstrate that the presence and severity of intra-neuronal pTDP-43 pathology, but not astroglial pathology, or phosphorylated Tau pathology, is associated with behavioural dysfunction. Cases were also evaluated using a TDP-43 aptamer (TDP-43APT), which revealed that pathology was not only associated with behavioural symptoms, but also with ferritin levels, a measure of brain iron. ConclusionsIntra-neuronal pTDP-43 and cytoplasmic TDP-43APT pathology in the amygdala is associated with behavioural symptoms in sALS. TDP-43APT staining intensity is also associated with increased ferritin, regardless of behavioural phenotype, suggesting that ferritin increases may occur upstream of clinical manifestation, in line with early TDP-43APT pathology, representing a potential region-specific imaging biomarker of early disease in ALS. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSThe amygdala is a key brain region in regulating behavior and emotional cognition and has been shown recently, through imaging studies, to be affected in ALS and FTD patients. What this study addsHere we examine the underlying pathology driving the association between the amygdala and behavioural symptoms in sporadic ALS demonstrating that region specific TDP-43 pathology and brain iron accumulation could represent potential early biomarkers of dysfunction. How this study might affect research, practice, or policyThe correlation between early TDP-43 pathology (detected by RNA aptamer) and increased ferritin (brain iron accumulation) occurring upstream of clinical manifestation represents a potential, region-specific (amygdala), early imaging biomarker in ALS. This means that people at risk could be identified early and stratified for clinical trials prior to substantial neuronal cell loss and symptom onset.

neuroscience↗

bioRxiv2023

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, analysis of deeply-phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism, but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 aggregation and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply-phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic aggregation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic aggregation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS. Short AbstractRecent identification of cryptic-splicing events such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism in amyotrophic lateral sclerosis (ALS). However, the temporal nature of TDP-43 loss and its relation to clinical phenotype is not known. Here, we used a novel RNA aptamer to detect TDP-43 aggregation and used single molecule ISH to sensitively reveal TDP-43 loss-of-function, applying these methods in a deeply-phenotyped human post-mortem tissue cohort. We show that nuclear TDP-43 pathology is an early event, that coincides with STMN-2 cryptic splicing. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics and intervention prior to symptom onset in ALS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/563701v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1e49c3forg.highwire.dtl.DTLVardef@1ce05b0org.highwire.dtl.DTLVardef@d77205org.highwire.dtl.DTLVardef@7ed2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimens fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies - compatible with imaging - to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation - inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes - not limited to liquid-liquid phase transition - and we anticipate their widespread application in future life-science research.

biophysics↗

bioRxiv2022

A Theoretical Model reveals RNA sequestration in Alpha Synuclein Aggregates

Nucleic acids can act as potent modulators of protein aggregation, and RNA is able to either hinder or facilitate protein assembly depending on the molecular context. Here we used a computational approach to characterize the physico-chemical properties of regions involved in amyloid aggregation. In different experimental datasets we observed that, while the core is hydrophobic and highly ordered, external regions, more disordered, display a distinct tendency to interact with nucleic acids. To validate our predictions, we performed aggregation assays with -synuclein (aS140), a non-nucleic acid binding amyloidogenic protein, and a mutant truncated at the acidic C-terminus (aS103) that is predicted to sequester RNA. For both aS140 and aS103 we observed acceleration of the aggregation upon RNA addition with a significantly stronger effect for aS103. Due to the favorable electrostatics, we observed enhanced nucleic-acid sequestration ability for aS103 that entrapped a larger amount of RNA. Overall, our research suggests that RNA sequestration is a rather common phenomenon linked to protein aggregation and constitutes a gain-of-function mechanism to be further investigated. STATEMENT OF SIGNIFICANCEOur study indicates that aggregation confers RNA-binding ability to non-RNA-binding proteins such as alpha synuclein. The sequestration of RNA upon protein aggregation might alter RNA homeostasis and impact multiple biochemical cascades.

biochemistry↗

bioRxiv2022

Discovering host protein interactions specific for SARS-CoV-2 RNA genome

SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5 and 3 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.

molecular biology↗