Biology subjects

Bezard, E.

Publications and source records attributed to Bezard, E..

13 recordsLinked to original sources

bioRxiv2026

WDR44 drives de novo α-synuclein aggregation at the lysosomal membrane and promotes neuronal dysfunction in Parkinson's Disease

The aggregation of -synuclein (-SYN) into Lewy bodies (LBs) is a central event in the pathogenesis of Parkinsons disease (PD) and related synucleinopathies1,2. Despite significant advances in understanding -SYN self-assembly, the precise sequence of early aggregation steps has not been directly visualized in living neurons. Here, we use an optogenetic-induced protein aggregation system with a high temporal resolution to monitor the onset of -SYN assembly in neurons. We found that the initiation and accumulation of -SYN aggregates occur predominantly at the lysosomal membrane, an event driven by the -SYN N-terminus and modulated by the membrane-associated adaptor protein WD repeat-containing protein 44 (WDR44). Remarkably, we demonstrate that WDR44 knockdown markedly reduced de novo -SYN aggregation in both neuronal cultures and in vivo, whereas WDR44 overexpression enhances -SYN aggregation in PD patient-derived iPSC neurons. Consistent with its potential pathogenic involvement, WDR44 aberrantly accumulates in vivo and in the brains of PD patients, where it colocalizes with LB inclusions. Finally, we show that lysosome-associated -SYN aggregates compromised lysosomal structure and function, leading to neuronal impairment, a phenotype worsened by WDR44 overexpression, linking early aggregation events to downstream toxicity. Together, these findings reveal the earliest dynamic stages of -SYN oligomerization in living neurons and identify the WDR44--SYN interaction as a promising therapeutic target for reducing -SYN pathology and enabling early intervention in PD.

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bioRxiv2025

Decoding Amyloid Plaque Penetrability: Exploring Extracellular Space and Rheology2in Plaque-rich Cortex

A hallmark of Alzheimers disease (AD) is the accumulation of amyloid plaques, primarily composed of misfolded amyloid {beta} (A{beta}) peptides. We employed complementary high-resolution imaging techniques to investigate the plaque penetrability and the extracellular space (ECS) rheology in a mouse model of AD. Two-photon shadow imaging in vivo confirmed that a dense ring of cells surrounds cortical amyloid plaques but highlighted the diffusional penetrability of the amyloid core. Quantum dot tracking unveiled that ECS diffusional parameters are heterogeneous in and around plaques, with an elevated diffusivity within and around plaques compared to WT-tissue. The amyloid core showed low nanoparticle density, varying by plaque phenotype. Carbon nanotube tracking confirmed these altered local rheological properties at the level of the whole cortex of AD mice. Finally, we found the extracellular matrix to be dysregulated within the amyloid plaque, which may account for the observed alterations in diffusivity. Our study provides fresh insights for understanding A{beta} plaque penetration, a prerequisite for therapeutic development.

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bioRxiv2025

Ultra-performance liquid chromatography-mass spectrometry analysis of post-mortem brain tissue reveals specific amino acid profile dysregulation in Parkinson's Disease and Alzheimer's Disease patients

BackgroundCombined metabolomic and HPLC-based analyses have identified significant metabolic alterations in serum and plasma amino acid levels of Parkinsons disease (PD) patients, underscoring their potential as biomarkers. However, it remains unclear whether these biochemical changes also manifest within the central nervous system or are confined to peripheral metabolism, reflecting systemic metabolic disturbances. MethodsTo address this issue, here we measured the levels of 44 different amino acids in post-mortem brain samples from MPTP-intoxicated, L-DOPA-treated monkeys and PD patients at different Braak Lewy body (LB) stages, compared to their respective controls, through targeted Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). ResultsIn MPTP-intoxicated monkeys, UPLC-MS revealed significant elevations in GABA, citrulline, threonine, isoleucine, phenylalanine, valine, glycine, and serine in the putamen, whereas we failed to detect alterations in the superior frontal gyrus (SFG). In PD patients, caudate-putamen (CPu) analysis demonstrated consistent serine upregulation across Braak LB stages 3-4 and 6, with stage 6 specifically showing additional proline increases and phosphoethanolamine decreases. Notably, serine was the sole amino acid significantly altered in both the putamen of MPTP-intoxicated monkeys and the CPu of PD patients. No significant amino acid alterations were observed in the SFG of PD patients, mirroring the findings in monkeys. In contrast, Alzheimers disease (AD) patient SFG samples showed significant increases in tryptophan, phenylalanine, threonine, tyrosine, and methionine relative to controls. ConclusionsThese findings demonstrate that cerebral amino acid alterations in PD are region-specific and primarily localized to brain areas receiving nigrostriatal dopaminergic innervation. Moreover, the cortical amino acid profile in AD differs substantially from that in PD, suggesting disease-specific metabolic signatures in distinct neurodegenerative conditions.

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bioRxiv2025

A subset of dorsal raphe dopamine neurons is critical for survival-oriented vigilance.

Defensive behaviors are essential for survival, with risk assessment enabling organisms to evaluate and respond to potential threats. The dorsal raphe nucleus (DRN), a key neuromodulatory center, is crucial for encoding motivational salience and regulating arousal and sleep-wake states through its diverse neuronal populations, including dopaminergic neurons (DRNDA). While the roles of DRNDA neurons have been studied, their specific contributions to threat evaluation are less understood. Recent research identifies a distinct subset of DRNDA neurons that express vasoactive intestinal peptide (VIP) and project to the central amygdala (CeA) and the oval nucleus of the bed nucleus of the stria terminalis (ovBNST). Together, these two regions comprise the central extended amygdala, a key network regulating adaptive responses to threats. We hypothesized that distinct DRNDA subpopulations exert diverging effects on sleep-wake regulation and that DRNVIP neurons play a pivotal role in coordinating activity between the CeA and ovBNST, thereby influencing risk assessment and defensive response. To test this hypothesis, we used a combination of in situ hybridization, immunochemistry, whole-brain mapping, electrophysiology, and cell-specific genetic tools in mice and non-human primates. Our findings reveal that DRNVIP neurons form a key DRNDA neuronal subset, uniquely positioned to regulate the central extended amygdala through a feedback loop. These neurons receive inputs from Protein Kinase C delta (PKC-{delta}) neurons in the ovBNST and CeA and send glutamate-releasing projections back to these regions, modulating PKC-{delta} neuron excitability. Selective ablation of DRNVIP neurons increases activity in both the BNST and CeA, disrupting active-phase sleep architecture and impairing risk assessment and defensive behaviors. Together, these findings suggest DRNVIP neurons control specific phases of sleep and orchestrate the central extended amygdalas role in risk assessment and defensive responses. HIGHLIGHTSO_LIDRNVIP neurons form a subset of DRNDA neurons in mice and non-human primates. C_LIO_LIDRNVIP receive inputs from Protein Kinase C delta (PKC-{delta}) neurons in the ovBNST and CeA and project back to both. C_LIO_LIBy releasing glutamate, DRNVIP neurons regulate PKC-{delta} neuron excitability in the ovBNST and CeA. C_LIO_LIAblating DRNVIP neurons increases BNST and CeA activity, disrupts active-phase sleep architecture, and impairs threat responses. C_LI IN BRIEFDRNVIP neurons, a key subset of DRNDA neurons in mice and primates, are strategically positioned to influence the central extended amygdala via feedback loops. They regulate PKC-{delta} neuron excitability in the ovBNST and CeA through glutamate release, with their ablation heightening activity in these regions and altering active-phase sleep architecture, risk assessment and defensive behaviors.

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bioRxiv2024

Lipidomic Profiling Reveals Shared and Distinct Pathological Signatures in Sporadic Parkinson's Disease and GBA Mutation Carriers: Implications for Disease Mechanisms

Parkinsons Disease (PD) is a neurodegenerative disorder characterised by the deposition of protein-lipid inclusions, containing alpha-synuclein, neuronal cell loss and disruptions in lipid metabolism such as those associated with GBA mutations. GBA mutations are together an important genetic risk factor for PD and are associated with a decrease in glucocerebrosidase, a lysosomal glycoprotein encoded by GBA, increase in alpha-synuclein and changes in sphingolipids levels and composition. However, the extent of lipid metabolism disruptions associated to PD and their contributions to disease progression remain unclear. In this study, we used a combination of biochemical and lipidomic analyses of amygdala from healthy controls (HC) and people with sporadic (sPD) or GBA-associated PD (PD-GBA) to investigate the correlation between alpha-synuclein, glucocerebrosidase and lipids. We found extensive metabolic remodelling of brain lipids, including increased free cholesterol, diacylglycerides, sphingolipids and specific glycerophospholipids in amygdala from people with sPD and disease duration above 30 years (sPD>30y) and from people with PD carriers of a GBA risk mutation (PD-GBArisk) relative to HC. The levels of free cholesterol, diacylglycerides, sphingolipids and specific glycerophospholipids all correlated positively with pathological S and negatively with GCase activity. In contrast, the levels of phosphatidylethanolamine and cardiolipin only correlated positively with GCase activity. Moreover, we observed changes in the distribution of species for sphingolipids and glycerophospholipids in opposite directions for two categories of PD cases. We found a shift from short to long sphingomyelin and ceramide and from long to short phosphatidylserine and phosphatidylethanolamine in sPD>30y and PD-GBArisk cases and the opposite in sPD<10y and PD-GBAsevere cases. The relative proportion of lipid species affected in these samples all correlated with glucocerebrosidase activity and pathological alpha-synuclein levels. Together, these findings highlight the correlation between glucocerebrosidase, pathological alpha-synuclein and lipid levels in PD. Moreover, the identified opposite changes in lipid distribution for two categories of people with sPD and PD-GBA underscore the importance of patient stratification in clinical trials aiming at reverting PD-related lipid changes.

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bioRxiv2024

1.94 Angstrom structure of synthetic alpha-synuclein fibrils seeding MSA neuropathology

Summary ParagraphMultiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease of unknown cause, typically affecting individuals aged 50-60 and leading to death within a decade1-3. It is characterized by glial cytoplasmic inclusions (GCIs) composed of fibrillar alpha-synuclein (aSyn)4-8, whose formation shows parallels with prion propagation9,10. While fibrils extracted from MSA brains have been structurally characterized11, their ability to replicate in a "protein-only" manner has been questioned12, and their capacity to induce GCIs in vivo remains unexplored. By contrast, the synthetic fibril strain 1B13,14, assembled from recombinant human aSyn, self-replicates in vitro and induces GCIs in mice15 - suggesting direct relevance to MSA - but awaited scrutiny at an atomic scale. Here, we report high-resolution structural analyses of 1B fibrils and of fibrils extracted from diseased mice injected with 1B that developed GCIs (1BP). We show in vivo that conformational templating enables fibril strain replication, resulting in MSA-like inclusion pathology. Remarkably, the structures of 1B and 1BP are highly similar and mimic the fold of aSyn observed in one protofilament of fibrils isolated from MSA patients11. Moreover, reinjection of crude mouse brain homogenates containing 1BP into new mice reproduces the same MSA-like pathology induced by the parent synthetic seed 1B. Our findings identify 1B as a synthetic pathogen capable of self-replication in vivo and reveal structural features of 1B/1BP that may underlie MSA pathology, offering insights for therapeutic strategies.

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bioRxiv2022

Encoding social preference by interhemispheric neurons in the Insula

The Insula is a multisensory relay that participates in socio-emotional processing through multiple projections to sensory, cognitive, emotional, and motivational regions. Interestingly, the Insula interhemispheric projection to the contralateral Insula is a strong but understudied projection. Using cutting-edge neuroanatomy, ex vivo and in vivo electrophysiology associated with specific circuit manipulation, we unraveled the nature and role of Insula interhemispheric communication in social and anxiety processing in mice. In this study, we 1) characterized the anatomical and molecular profile of the interhemispheric neurons of the Insula, 2) highlighted that stimulation of this neuronal subpopulation triggers excitation in the Insula interhemispheric circuit 3) uncovered their engagement in social processing. In conclusion, this study demonstrates that interhemispheric neurons of the Insula constitute a unique class of Insula neurons and proposes new meaningful insights into the neuronal mechanisms underlying social behavior.

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bioRxiv2021

Similar neuronal imprint and absence of cross-seeded partner fibrils in α-synuclein aggregates from MSA and Parkinson's disease brains

Aggregated alpha-synuclein (-syn) is a principal constituent of Lewy bodies (LBs) and glial cytoplasmic inclusions (GCIs) observed respectively inside neurons in Parkinsons disease (PD) and oligodendrocytes in multiple system atrophy (MSA). Yet, the cellular origin, the pathophysiological role, and the mechanism of formation of these inclusions bodies (IBs) remain to be elucidated. It has recently been proposed that -syn IBs eventually cause the demise of the host cell by virtue of the cumulative sequestration of partner proteins and organelles. In particular, the hypothesis of a local cross-seeding of other fibrillization-prone proteins like tau or TDP-43 has also been put forward. We submitted sarkosyl-insoluble extracts of post-mortem brain tissue from PD, MSA and control subjects to a comparative proteomic analysis to address these points. Our studies indicate that: i) -syn is by far the most enriched protein in PD and MSA extracts compared to controls; ii) PD and MSA extracts share a striking overlap of their sarkosyl-insoluble proteomes, consisting of a vast majority of mitochondrial and neuronal synaptic proteins, and (iii) other fibrillization-prone protein candidates possibly cross-seeded by -syn are neither found in PD nor MSA extracts. Thus, our results (i) support the idea that pre-assembled building blocks originating in neurons serve to the formation of GCIs in MSA, (ii) show no sign of amyloid cross-seeding in either synucleinopathy, and (iii) point to the sequestration of mitochondria and of neuronal synaptic components in both LBs and GCIs.

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bioRxiv2021

Amyloid beta oligomers modulate neuronal autophagy through the primary cilium

The major neurodegenerative diseases, like Alzheimers disease (AD), accumulate neuropathogenic proteins that compromise autophagic function. In AD, autophagy contributes to intracellular APP processing and amyloid beta (A{beta}) generation by mutant presenilin-1 (PS1). However, how extracellular soluble A{beta} oligomers (A{beta}o) impact intracellular autophagy is not well understood. The primary cilium (PC), a signaling organelle on the surface of mature neurons and glia, is able to bind A{beta}. Since PC signaling pathways knowingly modify autophagy in non-brain cells, we here investigated the role of neuronal PC in the modulation of autophagy during acute extracellular A{beta}o overload. Our results show that, in vivo, recombinant A{beta}o require the presence of neuronal PC to modulate early autophagy and to induce the accumulation of autophagic vacuoles in an age-dependent manner. We show that activated Akt mediates these effects in an age-dependent manner, and that ciliary p75NTR receptor is required to block autophagy by A{beta}o. These findings demonstrate that neuronal PC in the adult brain participates in the deleterious effects mediated by soluble A{beta}o. The PC should therefore be considered as a target organelle to modulate autophagy for the treatment of neurodegenerative diseases. HighlightsO_LIA{beta}o requires the neuronal PC to impair learning in young and old mice. C_LIO_LIAutophagy in whole hippocampus differs from autophagy response in hippocampal neurons. C_LIO_LIA{beta}o induce autophagolysosome accumulation through primary cilia- and age-dependent Akt phosphorylation. C_LI

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bioRxiv2020

Dopaminergic Co-transmission with Sonic Hedgehog Inhibits Expression of Abnormal Involuntary Movements

L-Dopa induced dyskinesia (LID) is a debilitating side effect of dopamine replacement therapy for Parkinsons Disease. The mechanistic underpinnings of LID remain obscure. Here we report that diminshed sonic hedgehog (Shh) signaling in the basal ganglia caused by the degeneration of midbrain dopamine neurons (DANs) facilitates the formation and expression of LID. We demonstrate that augmenting Shh signaling with agonists of the Shh effector Smoothened attenuates LID in mouse and macaque models of PD. Employing conditional genetic loss-of-function approaches, we show that reducing Shh secretion from DANs or Smo activity in cholinergic interneurons (CINs) promotes LID. Conversely, the selective expression of constitutively active Smo (SmoM2) in CINs is sufficient to render the sensitized aphakia model of PD resistant to LID. Furthermore, acute depletion of Shh from DANs through prolonged optogenetic stimulation in otherwise intact mice and in the absence of L-Dopa produces LID-like involuntary movements. These findings indicate that augmenting Shh signaling in the L-Dopa treated brain may be a promising and unexpected novel therapeutic approach for mitigating the dyskinetic side effects of long-term treatment with L-Dopa

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bioRxiv2020

Emergence of stealth polymorphs that escape α-synuclein amyloid monitoring, take over and acutely spread in neurons

The conformational strain diversity characterizing -synuclein (-syn) amyloid fibrils is possibly at the origin of the different clinical presentations of synucleinopathies. Experimentally, various -syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe Thioflavin T (ThT). We report here that besides classical ThT positive products, fibrillization in saline simultaneously gives rise to competing fibril polymorphs that are invisible to ThT (stealth polymorphs), and that can take over. Due to competition, spontaneous generation of such stealth polymorphs bears on the apparent fibrillization kinetics and on the final plateau values. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. Compared to their ThT-positive counterparts, and as judged from their chemical shift resonances fingerprint, these new stealth polymorphs present a yet undescribed atomic organization and show an exacerbated propensity (approx. 20-fold) towards self-replication in cortical neurons. They also trigger a long distance synucleinopathic spread along nigro-striatal projections in vivo. In order to rapidly screen fibrillization products for the presence of such stealth polymorphs, we designed a simple multiplexed assay that can be easily and rapidly operated. This assay allows us to demonstrate the sustainability of the conformational replication of these novel and particularly invasive strains. It should also be of help to avoid erroneous upstream interpretations of fibrillization rates based on sole ThT, and to expedite further structural and functional characterization of stealth amyloid assemblies. One Sentence Summarystealth -synuclein fibrils take over

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bioRxiv2020

In vivo electrophysiological validation of DREADD-based inhibition of pallidal neurons in the non-human primate.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are widely used in rodents to manipulate neuronal activity and establish causal links between structure and function. Their utilization in non-human primates (NHPs) is however limited and their efficacy still debated. Here, we tested DREADD expression in the NHP external globus pallidus (GPe) and electrophysiologically validated DREADD-based inhibition of GPe neurons in the anesthetized monkey.To do so, we performed intracerebral injections of viral construct expressing hM4Di receptor under a neuron-specific promoter into the GPe. Then, we recorded the neuronal activity in the DREADD-transduced (test condition) and DREADD-free (control condition) GPe of two anesthetized animals following local intra-GPe microinjection of clozapine-N-oxide (CNO). In total, 19 and 8 well-isolated and stable units were recorded in the DREADD-transduced and DREADD-free GPe, respectively. Overall, we found that almost half (9/19) of the units modulated their activity following CNO injection in DREADD-transduced GPe. Surprisingly, neuronal activity of the GPe units exhibited diverse patterns in timing and polarity (increase/decrease) of firing rate modulations during and after CNO injection. Nevertheless, decreases were exclusive and stronger after CNO injection. In contrast, only one unit modulated its activity after CNO injection in DREADD-free GPe. Moreover, post-mortem histochemical analysis revealed that hM4Di DREADDs were expressed at high level in the GPe neurons located in the vicinity of the viral construct injection sites. Our results therefore show in vivo DREADD-based inhibition of pallidal neurons in the NHP model and reinforce the view that DREADD technology can be effective in NHPs.

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bioRxiv2019

Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in non-human primates

AO_SCPLOWBSTRACTC_SCPLOWDopaminergic neuronal cell death, associated with intracellular -synuclein (-syn)-rich protein aggregates (termed Lewy bodies), is a well-established characteristic of Parkinsons disease. Much evidence, accumulated from multiple experimental models has suggested that -syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct -syn pathogenic structures derived from PD patients. Unexpectedly, our results show that, in non-human primates, a small amount of singular -syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD as multiple causes can induce similar outcome regarding dopaminergic neurodegeneration.

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