Biology subjects

Outeiro, T. F.

Publications and source records attributed to Outeiro, T. F..

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

Blm10/PA200-activated 20S proteasomes promote α-synuclein degradation and bypass proteasome inhibition in Parkinson disease models

Protein homeostasis is essential for maintaining normal cellular function. However, protein homeostasis efficiency declines with age, leading to the accumulation of aberrant protein structures associated with neurodegenerative diseases such as Parkinsons disease (PD). PD is characterized by the aggregation of alpha-synuclein (Syn) into cytoplasmic inclusions. This process is accompanied by elevated phosphorylation at serine 129 (S129). The accumulation of Syn into aggregates and their propagation disrupts key proteostasis pathways, including the ubiquitin-proteasome system (UPS) or autophagy, contributing to cellular dysfunction and neuronal death. This study identified the proteasome activator Blm10 and its human ortholog PA200 as modulators of Syn degradation and toxicity. The conserved Blm10/PA200 protein plays a key role in regulating proteasome activity and assembly. The Syn expression increases Blm10 protein stability through autophagy inhibition, in a manner dependent on Syn phosphorylation at S129 in yeast. Overexpression of BLM10 or PA200 reduces Syn aggregation and enhances Syn turnover via activation of the 20S proteasome in yeast and mammalian cells. Blm10 and PA200-capped 20S proteasomes efficiently degrade both monomeric as well as oligomeric Syn in vitro. Notably, capped proteasomes retain proteolytic activities in presence of Syn, indicating resistance to Syn-induced inhibition, in contrast to 20S or 26S proteasomes. These results reveal a distinct proteasome subtype that bypasses UPS impairment and restores proteolytic capacity under proteotoxic stress. Our findings establish Blm10/PA200 as critical regulator of Syn proteostasis and highlight its protective role in maintaining protein homeostasis and cell viability under conditions of Syn toxicity.

cell biology↗

bioRxiv2025

Tau oligomer heterogeneity and associated protein profile in slowly versus rapidly progressive Alzheimer's disease

Rapidly progressive Alzheimers disease (rpAD) is a rare but devastating clinical variant characterized by abrupt cognitive decline, yet the molecular features underlying this phenotype remain unknown. Tau oligomers (TauO) are key mediators of tau toxicity, but whether their biochemical properties differ across AD subtypes has not been examined in human brain. We isolated endogenous TauO from frontal cortex of well-characterized control, slowly progressive AD (spAD), and rpAD cases using T22 immunoprecipitation and performed ultrastructural, biochemical, and proteomic characterization. rpAD TauO displayed compact, densely aggregated morphology and exhibited the highest levels of disease-associated phosphorylation (pS396, pS422). Label-free proteomics revealed that control and spAD shared a robust TauO interactome enriched for translation, proteostasis, mitochondrial metabolism, and vesicle trafficking. Strikingly, these modules were absent in rpAD, which instead showed selective enrichment for aldehyde detoxification, amino-acid and carbon metabolism, and actin-regulatory pathways. rpAD TauO demonstrated increased association with SERPINA1, ALDH9A1, MAPRE3, DPYSL2/3, and NFASC, and reduced association with MRPL17 and C9. Functionally, rpAD TauO induced the strongest toxicity in SH-SY5Y cells. Together, these findings indicate that rpAD likely harbors a biochemically distinct TauO species, defining a molecular signature that may underpin its fulminant clinical progression and support the development of subtype-specific therapeutic strategies.

neuroscience↗

bioRxiv2025

A 'brain-first' mouse model of progressive alpha-synuclein pathology via intranasal rotenone administration

The precise aetiology of Parkinsons disease (PD) is still poorly understood, but it is thought to arise due to an intricate relationship between genes and the environment. Our study takes a unique approach to understanding the effect of environmental factors on the onset and progression of -synuclein (aSyn) pathology, a key feature of PD, from the olfactory bulb (OB) to other brain regions. In the present study, we evaluated the time-dependent progression of PD-like pathology by administering rotenone intranasally for 5.5 months in C57BL/6 male mice. We performed olfactory and motor tests and examined the aSyn accumulation, glial cell activation and dopaminergic neurodegeneration after 3, 4 and 5.5 months of rotenone exposure by immunoblotting and immunofluorescence techniques. We observed a time-dependent progression of aSyn accumulation from the OB to other brain regions, including the mid-brain and cortex. Consistently, we observed a time-dependent behavioural impairment, OB atrophy, progression of aSyn pathology, neuroinflammation and neurodegeneration. Our findings also established a link between distinct astrocyte activation and dopaminergic (DAergic) activity. In conclusion, this chronic and progressive mouse model mimics the brain-first type of progression of PD-like pathology in some PD patients, opening the possibility for testing potential disease-modifying interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/677764v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@5a983eorg.highwire.dtl.DTLVardef@1c115ceorg.highwire.dtl.DTLVardef@17eb778org.highwire.dtl.DTLVardef@3f3fa4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

The Impact of Methylglyoxal and SOD1 Mutation on TDP-43 Interaction in ALS Proteinopathy

IntroductionProteinopathy is a key feature of amyotrophic lateral sclerosis (ALS) that causes the loss of motor neurons. Glycated SOD1 increases the levels of phospho-TDP-43, a form that aggregates in the cytosol of neurons experiencing neurodegeneration in most ALS cases. ObjectiveHere, we evaluated whether TDP-43 interacts with SOD1 and the impact of methylglyoxal (MGO) and G93A SOD1, found in patients, on this interaction. MethodologyTDP-43-SOD1 interaction was observed in H4 cells using the bimolecular fluorescence complementation (BiFC) system. ResultsExposure to MGO reduced SOD1 activity and the levels of phospho-TDP-43 only in cells expressing WT SOD1. Our results showed that both WT and G93A SOD1 interact with TDP-43 in the nucleus and cytosol, with a greater proportion of cells showing cytosolic interactions between TDP-43 and the SOD1 mutant. MGO did not affect the interaction between TDP-43 and WT SOD1; however, it did lead to an increase in cytosolic inclusions at 0.4 mM MGO, a stress that resulted in a 50% reduction in cell viability. These inclusions did not colocalize with stress granules. Treatment with Cyclosporin A, an inhibitor of calcineurin (a phosphatase that dephosphorylates TDP-43), reduced the number of cells containing TDP-43 and WT SOD1 inclusions, as well as the cells showing TDP-43 and G93A SOD1 interactions in the cytosol. ConclusionThus, we conclude that damaged SOD1, produced by MGO, or G93A mutation disrupts TDP-43 phosphorylation, altering its location within the cell and inducing its aggregation, which are important markers of ALS. Summary for Social Media If Published@laboratorio_life Amyotrophic lateral sclerosis (ALS) is an incurable, devastating, and progressive neurodegenerative disease. It is characterized by the accumulation of misfolded proteins, such as TDP-43 and SOD1, which causes motor neuron degeneration. The relationship between SOD1 and TDP-43 remains unclear, but glycated SOD1 increases the levels of phospho-TDP-43, a form found in cytosolic inclusions within neurons undergoing neurodegeneration in most ALS cases. Our results indicate that SOD1 interacts with TDP-43 mainly in the nucleus. However, damaged SOD1, produced by methylglyoxal, or G93A SOD1, a mutant found in patients, disrupts TDP-43 phosphorylation, altering its location within the cell and inducing its aggregation, which are important markers of ALS. We therefore conclude that SOD1 plays a crucial role in the development of the disease, making it a potential target for assessing ALS risk and developing treatments.

cell biology↗

bioRxiv2025

Proteo-transcriptomic reprogramming and resource reallocation define the aging mammalian brain

Brain aging is a major risk for neurodegeneration, yet the underlying molecular mechanisms remain poorly understood. Here we performed an integrative proteo-transcriptomic analysis of the aging mouse brain, uncovering molecular signatures of aging through the assessment of protein aggregation, mRNA relocalization, and comparative proteomics across eight models of premature aging and neurodegeneration. We identified dynamic changes in physiological aging highlighting differences in synaptic maintenance and energy-allocation. These were linked to changes associated with fundamental protein biochemical properties such as size and net charge. Network analysis highlighted a decrease in mitochondrial complex I proteins not compensated at the mRNA level. Aggregation of 60S ribosome subunits indicated deteriorating translation efficiency and was accompanied by mitochondrial and proteasomal imbalance. The analysis of the nine models revealed key similarities and differences between physiological aging and pathology. Overall, our study provides an extensive resource on molecular aging, and offers insights into mechanisms predisposing to neurodegeneration, easily accessible at our Brain Aging and Molecular Atlas Project (BrainAging-MAP) website.

neuroscience↗

bioRxiv2025

Dual effect of alpha-synuclein disease variants on condensate formation

-synuclein is a pre-synaptic protein1,2 implicated in synucleinopathies like Parkinsons disease and Dementia with Lewy Bodies, where it accumulates in intracellular aggregates termed Lewy bodies and Lewy neurites3. Recent studies have reported that -synuclein undergoes phase separation to form biomolecular condensates both in vitro and in mammalian cells4-7. -synuclein condensates are thought to contribute to disease through progressive aggregation5,8,9. Here we show that specific PD-associated -synuclein variants fail to form biomolecular condensates. We demonstrate that only two -synuclein variants associated with familial disease, E46K and E83Q, enhance condensate formation in vitro and in cells. However, variants including A30G, G51D, and A53E fail to form or have reduced levels of condensate formation in cells. The same phenotypes are reflected in the budding yeast model showing differential inclusion formation. In iPSC-derived neurons, the propensity of -synuclein variants to undergo VAMP2-mediated phase separation reflects the level of synaptic enrichment. We show that the intrinsic propensity of -synuclein to form condensates and the ability to bind lipid membranes are important to mediate condensate formation in cells. Our results emphasize that -synuclein pathology follows divergent pathways, with both increased and decreased condensate formation contributing to disease. This study establishes biomolecular condensates as a key intermediate in -synuclein dysfunction, providing a novel foundation for translational research.

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

Lead as a toxic environmental toxicant in models of synucleinopathies

Lead, a toxic heavy metal, is prevalent in various industrial applications, contributing to environmental contamination and significant health concerns. Lead affects various body systems, especially the brain, causing long-lasting cognitive and behavioral changes. While most studies have focused on continuous lead exposure, intermittent exposure, such as that caused by migration or relocations, has received less attention. Importantly, lead exposure intensifies the severity of Parkinsons disease (PD) and dementia with Lewy bodies, diseases involving the accumulation of alpha-synuclein (aSyn) in the brain and in the gut. Although, the precise mechanisms underlying these observations remain unclear, oxidative stress and mitochondrial dysfunction likely play a role. Here, we investigated how two different profiles of lead exposure - continuous and intermittent - affect models of synucleinopathies. We found that lead exposure enhances the formation of aSyn inclusions, resulting in an increase in both their number and size in cell models. In addition, we found that animals injected with aSyn pre-formed fibrils display serine 129-phosphorylated aSyn inclusions and a reduction in astrocytes in the substantia nigra. These animals also display neuronal damage and alterations in locomotor activity, exploratory behavior, anxiety, memory impairments and hypertension. Our results suggest a mechanistic link between environmental lead exposure and the onset and progression of diseases associated with aSyn pathology. Understanding the molecular and cellular interactions between lead and aSyn is crucial for shaping public health policies and may provide novel insight into strategies for mitigating the impact of environmental toxins on neurodegenerative processes involved in Parkinsons disease and related synucleinopathies. Key points summaryO_LILead exposure increases the number and size of alpha-synuclein (aSyn) inclusions in cell models.[~] C_LIO_LIAnimals exposed to aSyn fibrils and lead show increased phosphorylation of aSyn, neuronal damage and reduced astrogliosis in the substantia nigra. C_LIO_LILead-exposed animals exhibit impaired locomotion, anxiety, memory deficits, hypertension and chemoreceptor reflex hypersensitivity. C_LIO_LIThe study suggests a mechanistic link between environmental lead exposure and the progression of diseases involving aSyn pathology, such as Parkinsons disease. C_LI

physiology↗

bioRxiv2024

The epitranscriptomic m6A RNA modification modulates synaptic function in ageing and in a mouse model of synucleinopathy

N6-methyladenosine (m6A) is the most abundant and conserved transcriptional modification in eukaryotic RNA, regulating RNA fate. While the functions of m6A in the development of the mammalian brain have been extensively studied, its roles in synaptic plasticity, cognitive decline, motor function, or other brain circuits remain underexplored. To date, the role of this modification in Parkinsons disease (PD) and other synucleinopathies has been largely unknown. Here, we investigated the m6A epitranscriptome in a mouse model of synucleinopathy. We performed m6A RNA immunoprecipitation sequencing (meRIP-seq) to obtain the m6A epitranscriptome of the midbrain in young (3 mo) and aged (15 mo) A30P-aSyn transgenic mice (aSyn Tg) and C57BL6 control wild type (Wt) mice. We observed hypermethylation of synaptic genes in 3 mo aSyn Tg mice compared to age-matched Wt mice. This methylation was reduced during ageing, with synaptic genes becoming increasingly hypomethylated. Using immunofluorescence imaging alongside biochemical analysis, we further investigated the expression of m6A regulatory enzymes -- writer, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3); reader, YTH N6-methyladenosine RNA-binding protein (YTHDF1); and eraser, fat mass and obesity-associated protein (FTO) -- in the cortex, striatum, hippocampus, and cerebellum of Wt and aSyn Tg mice, as well as in primary cortical neuronal cultures. We observed that the levels of METTL3, YTHDF1 and FTO were similar between Wt and aSyn Tg mice. Interestingly, the writer protein METTL3 was found in both the nucleus and in the post-synaptic compartment in neuronal cultures. Our findings suggest that alterations in the regulation of m6A RNA methylation may be associated with neurodegeneration and ageing and that this level of epitranscriptomic regulation plays a significant role at the synapse.

neuroscience↗

bioRxiv2024

Seed-competent alpha-synuclein pathology in metachromatic leukodystrophy: the expanding spectrum of alpha-synucleinopathy in sphingolipidoses

Metachromatic leukodystrophy (MLD) is a rare - typically paediatric - sphingolipid storage disorder resulting from bi-allelic pathogenic variants in the ARSA gene, encoding the lysosomal arylsulphatase A (ASA). Heterozygous variants in ARSA are associated with risk of Lewy body diseases (LBD), a group of age-associated neurodegenerative disorders characterised by the accumulation of the protein -synuclein; however, no study has yet determined whether -synuclein with putative pathological features is observed in MLD brain tissue. We examined post-mortem brain tissue from MLD cases (N=5, age 2-33) compared to matched control cases using histological approaches and -synuclein seeding amplification assay (SAA). Juvenile-onset MLD cases exhibited granular -synuclein deposits in neurons of regions prone to neuronal pathology in MLD, and seed-competent conformers that generated atypical short, twisted fibrils on SAA. In contrast, infantile-onset MLD cases gave only variably positive reactions on SAA. In summary, this study suggests MLD cases manifest -synuclein pathology reminiscent of that observed in LBD, even in juvenile populations, further expanding the spectrum of sphingolipid storage disorders associated with the aggregation of -synuclein. These findings have important implications for understanding the disease process of both LBD and MLD, potentially highlighting novel pathways for therapeutic interventions in both conditions.

neuroscience↗

bioRxiv2024

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3ex{Delta}7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

cell biology↗

bioRxiv2024

Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies.

DNA damage and DNA damage repair (DDR) dysfunction are insults with broad implications on cellular physiology, including in proteostasis, and have been recently implicated in many neurodegenerative diseases. Alpha-synuclein (aSyn), a pre-synaptic and nuclear protein associated with neurodegenerative disorders known as synucleinopathies, has been implicated in DNA double strand break (DSB) repair function. Consistently, DSB induction has been demonstrated in cell and animal models of synucleinopathy. Nevertheless, the types of DNA damage and the contribution of DNA damage towards Lewy body (LB) formation in synucleinopathies are unknown. Here, we demonstrate the increase of DSB in neuronal and non-neuronal cellular populations of post-mortem temporal cortex tissue from dementia with Lewy body (DLB) patients and demonstrate increases in DSBs early at a presymptomatic age of aSyn transgenic mice. Strikingly, in postmortem DLB tissue, DNA damage-derived ectopic cytoplasmic genomic material (eCGM) was evident within the majority of LBs examined. The observed cellular pathology was consistent with nucleoproteasomal upregulation of associated DNA damage repair proteins, particularly in base excision repair and DSB repair pathways. Collectively our study demonstrates the early occurrence of DNA damage and associated nucleoproteasomal changes in response to nuclear aSyn pathology. Furthermore, the data suggests a potential involvement for DNA damage derived eCGM for the facilitation of cytoplasmic aSyn aggregates. Ultimately, uncovering pathological mechanisms underlying DNA damage in DLB sheds light into novel disease mechanisms and opens novel possibilities for diagnosing and treating synucleinopathies.

neuroscience↗

bioRxiv2023

Autophagic cargo in Lewy bodies: are Lewy bodies a compartment for spatial protein quality control?

Lewy bodies are neuropathologically associated with Lewy body dementia (LBD), but little is known about why they form or their role in the disease process. We previously reported Lewy bodies are a common feature of older individuals with primary mitochondrial diseases. However, as they are not an invariant finding, understanding differences between those with and without Lewy bodies may provide insights into factors that govern the formation of Lewy bodies in Lewy body disease (LBD). The present study sought to investigate whether deficient mitophagy in the context of mitochondrial dysfunction may underlie Lewy body formation. Post-mortem tissue was obtained from the cingulate gyrus and dorsal motor nucleus of the vagal nerve (DMV) of mitochondrial disease cases with Lewy bodies, primary mitochondrial disease cases without Lewy bodies, and control cases, in addition to LBD cases as comparison. An array of mitophagy and autophagy markers were quantified in 50 individual neurons per cingulate gyrus and all neurons per DMV using immunofluorescent analysis. No significant differences were found between groups, although there was a striking enrichment of markers of autophagic mitochondria and autophagic vesicles within Lewy bodies. Evaluation of diffuse -synuclein aggregates, thought to precede Lewy body formation, suggested only autophagic mitochondria were present in early aggregates, perhaps suggesting sequestration of dysfunctional mitochondria is an early step in Lewy body formation. To characterise the composition of Lewy bodies, discovery proteomics was performed on isolated insoluble proteins from frozen cingulate gyrus, which identified up-regulation of markers of aggresomes, a regulated cellular response that occurs when protein degradative pathways become overwhelmed, a mechanism of spatial protein quality control (sPQC). Taken together, these findings are consistent with impairment of cellular waste handling pathways in Lewy body-bearing neurons, and that the formation of a Lewy body could be a deliberate cellular response to compartmentalise such waste.

neuroscience↗

bioRxiv2023

Impact of Phosphorylation on alpha-Synuclein Structural Determinants

Serine 129 can be phosphorylated in pathological inclusions formed by the intrinsically disordered protein human -synuclein (AS), a key player in Parkinsons disease and other synucleinopathies. Here, molecular simulations provide insight into the structural ensemble of phosphorylated AS. The simulations suggest that phosphorylation does not impact the structural content of the physiological AS conformational ensemble in aqueous solution, as the phosphate group is mostly solvated. The hydrophobic region of AS contains {beta}-hairpin structures, which may increase the propensity of the protein to undergo amyloid formation, as seen in the non-physiological (non-acetylated) form of the protein in a recent molecular simulation study. Our findings are consistent with existing experimental data, with the caveat of the observed limitations of the force field for the phosphorylated moiety. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/531864v4_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@108c32dorg.highwire.dtl.DTLVardef@45e5d7org.highwire.dtl.DTLVardef@f49cf6org.highwire.dtl.DTLVardef@16ceb61_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Alpha-synuclein oligomers activate nuclear factor of activated T-cells (NFAT) modulating apoptosis and synaptic homeostasis

Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinsons disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth and survival. Here, we investigate NFATs role in neuronal degeneration induced by aSyn-O. aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies.

neuroscience↗

bioRxiv2022

Expansion microscopy at one nanometer resolution

Fluorescence imaging is one of the most versatile and widely-used tools in biology1. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving2, 3, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context4, 5. Here we report a solution to this problem, in the form of one-step nanoscale expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis6, 7 to enable the description of cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the [~]17 kDa protein calmodulin upon Ca2+ binding were readily observable. We also imaged and classified molecular aggregates in cerebrospinal fluid samples from Parkinsons Disease (PD) patients, which represents a promising new development towards improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

biophysics↗

bioRxiv2022

Brain-wide sensory aberrations in a Parkinson's Disease mouse model revealed by functional MRI

Parkinsons disease (PD) is associated with irreparable damage to dopaminergic neurons in brain areas involved in movement. Frequently, a very early symptom of PD involves an impaired sense of smell, while other studies suggest a broader impairment of other sensory modalities. Still, the associated brain-wide mechanisms underlying this phenomenon remain unclear. Here, we harness in vivo functional Magnetic Resonance Imaging (fMRI) at 9.4T to investigate how the olfactory and visual systems are affected in an -Synuclein (Syn) mouse model of PD. We find significant aberrations in fMRI responses along both sensory pathways including decreases in activation extent and in activation amplitude. Deficits in the olfactory system were larger than in the visual pathway, but nevertheless both sensory systems were clearly impacted, suggesting a more overarching mechanism may be involved. To further ensure that the aberrations observed in the fMRI signals were not driven solely by putative vascular deficiency, we quantified c-FOS expression in the olfactory system and found statistically significant decreases in the PD group when compared to age-matched controls. Hence, our findings indicate a neural origin for the observed fMRI aberrations. Our findings thus demonstrate a more global deficiency in sensory systems in the (Syn) mouse model. One Sentence SummaryParkinsons disease (PD) is associated with an impaired sense of smell and more generally sensory deficits, but the associated brain-wide mechanisms remain unclear; here, we investigate aberrations in BOLD-fMRI responses along sensory pathways in an SYN PD mouse model.

neuroscience↗