Biology subjects

Melki, R.

Publications and source records attributed to Melki, R..

30 records · Page 2Linked to original sources

bioRxiv2022

Human pericytes degrade alpha-synuclein aggregates in a strain-dependent manner

Parkinsons disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of -synuclein (-syn) aggregates. Central to disease progression is the gradual spread of pathological -syn. -syn aggregation is closely linked to progressive neuron loss. As such, clearance of -syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy are increasing. Our previous work has shown that pericytes -- vascular mural cells that regulate the blood-brain barrier -- contain -syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar -syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes efficiently break down -syn aggregates in vitro, with clear differences in the number of -syn aggregates/cell and average aggregate size when comparing five pure -syn strains (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110). Furthermore, pericytes derived from PD brains have a less uniform response than those derived from control brains. Our results highlight the vital role brain vasculature may play in reducing -syn burden in PD.

neuroscience↗

bioRxiv2022

Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM

Synucleinopathies are a heterogenous group of neurodegenerative diseases characterized by the progressive accumulation of pathological -synuclein (-Syn). The importance of structural polymorphism of -Syn assemblies for distinct synucleinopathies and their progression is increasingly recognized. However, the underlying mechanisms are poorly understood. Here we use fluorescence lifetime imaging microscopy (FLIM) to investigate seeded aggregation of -Syn in a biosensor cell line. We show that conformationally distinct -Syn polymorphs exhibit characteristic fluorescence lifetimes. FLIM further revealed that -Syn polymorphs were differentially processed by cellular clearance pathways, yielding fibrillar species with increased seeding capacity. Thus, FLIM is not only a powerful tool to distinguish different amyloid structures, but also to monitor the dynamic process of amyloid remodeling by the cellular environment. Our data suggest that the accumulation of highly seeding competent degradation products for particular polymorphs may account for accelerated disease progression in some patients.

molecular biology↗

bioRxiv2021

C9orf72-derived poly-GA DPRs undergo endocytic uptake in iNPC-derived astrocytes and spread to motor neurons

Dipeptide repeat proteins (DPRs) are aggregation-prone polypeptides encoded by the pathogenic G4C2 repeat expansion in the C9orf72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic &[beta]-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and - independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to- motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly- GA cellular uptake and cell-to-cell propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

neuroscience↗

bioRxiv2021

Quantitative study of alpha-synuclein prion-like spreading in fully oriented reconstructed neural networks reveals non-synaptic dissemination of seeding aggregates

The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinsons disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.

neuroscience↗

bioRxiv2021

Cryo-EM structure of alpha-synuclein fibrils amplified by PMCA from PD and MSA patient brains

Synucleinopathies are neurodegenerative diseases related to the aggregation of the protein alpha-synuclein (aSyn). Among these diseases, Parkinsons disease (PD) and multiple system atrophy (MSA) are most prevalent. aSyn can readily form different fibrillar polymorphs, if exposed to an air-water interface or by templating with pre-existing fibrils. We here report the structures of three fibrillar polymorphs that were obtained after seeding monomeric aSyn with PD and MSA patients brain homogenates using protein misfolding cyclic amplification (PMCA). Seeding with a control brain homogenate did not produce fibrils, and seeding with other in vitro generated fibrillar polymorphs as a control faithfully produced polymorphs of a different type. The here determined fibril structures from PD and MSA brain tissue represent new folds, which partly resemble that of previously reported in vitro generated fibrils from Y39 phosphorylated aSyn protein. The relevance of these fibrils for synucleinopathies in humans remains to be further investigated. Impact StatementNeurodegenerative diseases such as Parkinsons disease (PD) and Multiple System Atrophy (MSA) are suspected to be causatively related to the prion-like propagation of aggregates of the protein alpha-synuclein (aSyn). The fibril structures reported here were obtained after seeding from diseased human brain homogenate and differ from all previously published aSyn fibril arrangements. In case these fibrils would turn out to be the long sought causative agents of these diseases, their structures might lead to the development of therapeutic strategies to modify these diseases and to a better understanding of the mechanistic processes that lead to neurodegeneration and spreading of the diseases.

neuroscience↗

bioRxiv2021

Impact of α-synuclein fibrillar strains and β-amyloid assemblies on mouse cortical neurons endo-lysosomal logistics

Endosomal transport and positioning are involved in establishing neuronal compartment architecture, dynamics and function, contributing to neuronal intracellular logistics. Furthermore, endo-lysosomal dysfunction has been identified as a common mechanism in neurodegenerative diseases. Here, we analyzed endolysosomal transport following the external application of -synuclein (-syn) fibrillar polymorphs, {beta}-amyloid (A{beta}) fibrils and oligomers on primary cultures of mouse cortical neurons. We used a simple readout to measure this transport: the spontaneous endocytosis of fluorescent nanodiamonds -- a perfectly stable nano-emitter -- in cultured neurons. We then performed a high-throughput automatic extraction and quantification of the directed motions of these nanodiamonds. -syn fibrillar polymorphs, A{beta} fibrils and oligomers halved the proportion of nanodiamonds transported along microtubules, but only slightly decreased their interactions with cortical neurons. This large decrease in endosomal transport would be expected to have a huge impact on neuronal homeostasis. We then assessed lysosomal dynamics with Lysotracker. The exposure of neurons to A{beta} oligomers led to an increase in the number of lysosomes, a decrease in the fraction of moving lysosomes and an increase in their size, reminiscent of findings for the APP transgenic model of Alzheimers disease. We then analyzed the effect of -syn fibrillar polymorphs, A{beta} fibrils and oligomers on endosomal and lysosomal transport and quantified the directed transport of these assemblies within cortical neurons. We report different impacts on endosomal and lysosomal transport parameters and differences in trajectory length for cargoes loaded with pathogenic protein assemblies. Our results suggest that the internalization and transport of intraneuronal pathogenic protein aggregates are potential targets for novel neuroprotective treatment strategies. Significance StatementNeurodegenerative diseases (NDs) are characterized by the deposition of protein aggregates with broad-range neuronal toxicity. Defects of endolysosomal trafficking are increasingly being seen as key pathological features of NDs, probably contributing to synaptic dysfunction and ultimate neuronal death. We used fast fluorescence videomicroscopy to investigate endosomal and lysosomal dynamics in the branches of mouse cortical neurons in primary cultures following the application of -syn fibrillar polymorphs (fibrils and ribbons) and A{beta} assemblies (oligomers and fibrils). We provide new insight into the differential effects of these pathogenic protein assemblies on endosomal and lysosomal transport, and reveal differences in the transport characteristics of the compartments loaded with these protein assemblies relative to endosomes.

neuroscience↗

bioRxiv2021

The JAK2-STAT3 pathway controls a beneficial proteostasis response of reactive astrocytes in Huntington disease

Huntingtons disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntingtons disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntingtons disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models. Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntingtons disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntingtons disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons. Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntingtons disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.

neuroscience↗

bioRxiv2021

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies

While the initial pathology of Parkinsons disease and other -synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of -synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analyzed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with -synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of -synuclein pathology ex vivo. The overall survival of A53T -synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of -synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of -synucleinopathies is not universally valid.

neuroscience↗

bioRxiv2020

a-Synuclein strains influence multiple system atrophy via central and peripheral mechanisms

Multiple system atrophy (MSA) is a progressive neurodegenerative disease with prominent autonomic and motor features. Different disease subtypes are distinguished by their predominant parkinsonian or cerebellar signs. The pathognomonic feature of MSA is the presence of -synuclein (Syn) protein deposits in glial cells of the central and peripheral nervous system. It is unclear why MSA, that invariably presents with Syn pathology, is clinically so heterogeneous, why it progresses at varying rates and how neuroinflammation affects disease progression. Recently, it was shown that different strains of Syn can assemble in unique disease environments but also that a variety of strains might exist in the brain of MSA patients. We therefore investigated if different Syn strains might influence MSA disease progression. To this aim, we injected two recombinant strains of Syn in MSA transgenic mice and found that they significantly impact MSA disease progression in a strain-dependent way via oligodendroglial, neurotoxic and immune-related mechanisms. Neurodegeneration and brain atrophy were accompanied by unique microglial and astroglial responses and the recruitment of central and peripheral immune cells. The differential activation of microglial cells correlated with the structural features of Syn strains both in vitro and in vivo. By injecting Syn strains in MSA mice we could more closely mimic a comprehensive MSA phenotype in an experimental setting. This study therefore shows that i) MSA phenotype is governed by both the Syn strain nature and the host environment and ii) Syn strains can directly trigger a detrimental immune response related to disease progression in MSA.

neuroscience↗

bioRxiv2019

Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species

The accumulation of amyloid Tau aggregates is implicated in Alzheimers disease and other Tauopathies. Molecular chaperones are known for their function in maintaining protein homeostasis by preventing the formation or promoting the disaggregation of amorphous and amyloid protein aggregates. Here we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro. This function is mediated by the core chaperone Hsc70, assisted by specific co-chaperones, in particular class B J-domain proteins and an Hsp110-type NEF. Recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell culture were processed by the Hsp70 disaggregation machinery, demonstrating the ability of this machinery to recognize a broad range of Tau aggregates. Chaperone treatment released monomeric, and small oligomeric Tau species, which induced the aggregation of self-propagating Tau species in a Tau cell culture model. We infer from these results that the activity of the Hsp70 disaggregation machinery is a double-sided sword as it attempts to eliminate Tau amyloids but with the price of generating new seeds. The Hsp70 disaggregase therefore has a crucial function in the Tau propagation cycle, rendering it a potential drug target in Tauopathies.

molecular biology↗

bioRxiv2019

Two new polymorphic structures of alpha-synuclein solved by cryo-electron microscopy

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinsons disease (PD). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 [A] and 3.4 [A] resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability. Impact statementTwo new polymorphic structures of recombinant human alpha-synuclein fibrils show striking differences to previous structures, while familial PD mutation sites remain crucial for protofilament interaction and fibril stability.

neuroscience↗

bioRxiv2019

LRRK2 modifies α-syn pathology and spread in mouse models and human neurons

Progressive aggregation of the protein alpha-synuclein (-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinsons disease (PD). Accruing evidence suggests that -syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of -syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors have been identified in studies of both familial and sporadic forms of PD. However, how these genes affect -syn aggregation and pathological spread, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of -syn aggregation, using an -syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated -syn aggregation in mouse primary neurons. Conversely, -syn aggregation increased in primary neurons from transgenic mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, we investigated -syn spreading in a LRRK2 G2019S transgenic mouse model and observed acceleration of -syn aggregation and degeneration of dopaminergic neurons in the SNpc. Furthermore, LRRK2 G2019S transgenic mice displayed more pronounced degeneration-associated neuroinflammation and exacerbated behavioral deficits. To validate our findings in a human context, we established a human -syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs). Recombinant human -syn PFFs triggered aggregation of endogenous -syn in a time-dependent manner in human iNs. In isogenic lines of human iNs, the G2019S mutation enhanced -syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and -syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, not just in the cases caused by LRRK2 mutations.

neuroscience↗