Ronald Melki

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein-containing (α-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology.

α-Synuclein strains cause distinct synucleinopathies after local and systemic administration

Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions. The molecular hallmark of synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson’s disease and dementia with Lewy bodies. The discovery of α-SYN assemblies with different structural characteristics or ‘strains’ has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson’s disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood–brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.

Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates.

Sequence-specific nucleated protein aggregation is closely linked to the pathogenesis of most neurodegenerative diseases and constitutes the molecular basis of prion formation. Here we report that fibrillar polyglutamine peptide aggregates can be internalized by mammalian cells in culture where they gain access to the cytosolic compartment and become co-sequestered in aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. Remarkably, these internalized fibrillar aggregates are able to selectively recruit soluble cytoplasmic proteins with which they share homologous but not heterologous amyloidogenic sequences, and to confer a heritable phenotype on cells expressing the homologous amyloidogenic protein from a chromosomal locus.

Structural and functional characterization of two alpha-synuclein strains

α-Synuclein aggregation is implicated in a variety of diseases including Parkinsons disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

G51D α‐synuclein mutation causes a novel Parkinsonian–pyramidal syndrome

To date, 3 rare missense mutations in the SNCA (α‐synuclein) gene and the more frequent duplications or triplications of the wild‐type gene are known to cause a broad array of clinical and pathological symptoms in familial Parkinson disease (PD). Here, we describe a French family with a parkinsonian–pyramidal syndrome harboring a novel heterozygous SNCA mutation.

Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

The small heat shock protein (smHSP) and α-crystallin genes encode a family of 12–43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the α-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegansHSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the N-terminal domain but not most of the the C-terminal extension which follows the α-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.

Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport

The lesions of Parkinson disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α‐synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α‐synuclein to misfold. Here, we characterized and quantified the axonal transport of α‐synuclein fibrils and showed that fibrils could be transferred from axons to second‐order neurons following anterograde transport.

Structural Characterization of Saccharomyces cerevisiae Prion-like Protein Ure2

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

Structure of tubulin C‐terminal domain obtained by subtilisin treatment The major α and β tubulin isotypes from pig brain are glutamylated

Limited subtilisin digestion of the tubulin α, β heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin‐cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C‐terminus of the molecule. The C‐terminal peptides of both α and β subunits of tubulin were purified by anion‐exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp‐438 and His‐406 of α and Gln‐433 and His‐396 of β tubulin chains. Quantitative analysis of our data show that cleavage at positions His‐406 (α) and His‐396 (β) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu‐445 or −435 of α and β tubulin, respectively.

The yeast prion Ure2p retains its native α‐helical conformation upon assembly into protein fibrils in vitro

The yeast inheritable phenotype [URE3] is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p. In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH. Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross β‐structure of amyloid, but instead are formed by the polymerization of native‐like helical subunits that retain the ability to bind substrate analogues. We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils. By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native‐like monomers, driven by interactions between the N‐terminal glutamine and asparagine‐rich region and the C‐terminal functional domain. The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.