Maturing Autophagosomes are Transported Towards the Cell Periphery.

Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson's disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP "tandem-fluorescence" tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

A ß-Wrapin Targeting the N-Terminus of α-Synuclein Monomers Reduces Fibril-Induced Aggregation in Neurons.

Reducing α-synuclein pathology constitutes a plausible strategy against Parkinson's disease. As we recently demonstrated, the ß-wrapin protein AS69 binds an N-terminal region in monomeric α-synuclein, interferes with fibril nucleation, and reduces α-synuclein aggregation in vitro and in a fruit fly model of α-synuclein toxicity. The aim of this study was to investigate whether AS69 also reduces α-synuclein pathology in mammalian neurons. To induce α-synuclein pathology, primary mouse neurons were exposed to pre-formed fibrils (PFF) of human α-synuclein. PFF were also injected into the striatum of A30P-α-synuclein transgenic mice. The extent of α-synuclein pathology was determined by phospho-α-synuclein staining and by Triton X-100 solubility. The degeneration of neuronal somata, dendrites, and axon terminals was determined by immunohistochemistry. AS69 and PFF were taken up by primary neurons. AS69 did not alter PFF uptake, but AS69 did reduce PFF-induced α-synuclein pathology. PFF injection into mouse striatum led to α-synuclein pathology and dystrophic neurites. Co-injection of AS69 abrogated PFF-induced pathology. AS69 also reduced the PFF-induced degeneration of dopaminergic axon terminals in the striatum and the degeneration of dopaminergic dendrites in the substantia nigra pars reticulata. AS69 reduced the activation of astroglia but not microglia in response to PFF injection. Collectively, AS69 reduced PFF-induced α-synuclein pathology and the associated neurodegeneration in primary neurons and in mouse brain. Our data therefore suggest that small proteins binding the N-terminus of α-synuclein monomers are promising strategies to modify disease progression in Parkinson's disease.

Parkinson's disease and translational research.

Parkinson's disease (PD) is diagnosed when patients exhibit bradykinesia with tremor and/or rigidity, and when these symptoms respond to dopaminergic medications. Yet in the last years there was a greater recognition of additional aspects of the disease including non-motor symptoms and prodromal states with associated pathology in various regions of the nervous system. In this review we discuss current concepts of two major alterations found during the course of the disease: cytoplasmic aggregates of the protein α-synuclein and the degeneration of dopaminergic neurons. We provide an overview of new approaches in this field based on current concepts and latest literature. In many areas, translational research on PD has advanced the understanding of the disease but there is still a need for more effective therapeutic options based on the insights into the basic biological phenomena.

An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that over-expression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson's disease, we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP-decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 over-expression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7-induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7-induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson's disease. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Rab7 induces clearance of α-synuclein aggregates.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. Aggregates are degraded by the autophago-lysosomal pathway. Since Rab7 has been shown to regulate trafficking of late endosomes and autophagosomes, we hypothesized that over-expressing Rab7 might be beneficial in Parkinson's disease. To test this hypothesis, we expressed the pathogenic A53T mutant of α-synuclein in HEK293 cells and Drosophila melanogaster. In HEK293 cells, EGFP-Rab7-decorated vesicles contain α-synuclein. Rab7 over-expression reduced the percentage of cells with α-synuclein particles and the amount of α-synuclein protein. Time-lapse microscopy confirmed that particles frequently disappeared with Rab7 over-expression. Clearance of α-synuclein is explained by the increased occurrence of acidified α-synuclein vesicles with Rab7 over-expression, presumably representing autolysosomes. Rab7 over-expression reduced apoptosis and the percentage of dead cells in trypan blue staining. In the fly model, Rab7 rescued the locomotor deficit induced by neuronal expression of A53T-α-synuclein. These beneficial effects were not produced by Rab7 missense mutations causing Charcot Marie Tooth neuropathy, or by the related GTPases Rab5, Rab9, or Rab23. Using mass spectrometry, we identified Rab7 in neuromelanin granules purified from human substantia nigra, indicating that Rab7 might be involved in the biogenesis of these possibly protective, autophagosome-like organelles in dopaminergic neurons. Taken together, Rab7 increased the clearance of α-synuclein aggregates, reduced cell death, and rescued the phenotype in a fly model of Parkinson's disease. These findings indicate that Rab7 is rate-limiting for aggregate clearance, and that Rab7 activation may offer a therapeutic strategy for Parkinson's disease. Cells over-expressing aggregation-prone A53T alpha-synuclein develop cytoplasmic aggregates mimicking changes observed in Parkinson's disease. When following cells in time-lapse microscopy, some few cells are able to remove these aggregates (Opazo et al. 2008). We now show that the percentage of cells clearing all aggregates from their cytosol is greatly increased with Rab7 over-expression, indicating that availability of Rab7 is rate-limiting for autophagic clearance of aggregates. The functional significance of this effect in neurons was confirmed in a Drosophila melanogaster model of Parkinson's disease.

Cellular models for Parkinson's disease.

Developing new therapeutic strategies for Parkinson's disease requires cellular models. Current models reproduce the two most salient changes found in the brains of patients with Parkinson's disease: The degeneration of dopaminergic neurons and the existence of protein aggregates consisting mainly of α-synuclein. Cultured cells offer many advantages over studying Parkinson's disease directly in patients or in animal models. At the same time, the choice of a specific cellular model entails the requirement to focus on one aspect of the disease while ignoring others. This article is intended for researchers planning to use cellular models for their studies. It describes for commonly used cell types the aspects of Parkinson's disease they model along with technical advantages and disadvantages. It might also be helpful for researchers from other fields consulting literature on cellular models of Parkinson's disease. Important models for the study of dopaminergic neuron degeneration include Lund human mesencephalic cells and primary neurons, and a case is made for the use of non-dopaminergic cells to model pathogenesis of non-motor symptoms of Parkinson's disease. With regard to α-synuclein aggregates, this article describes strategies to induce and measure aggregates with a focus on fluorescent techniques. Cellular models reproduce the two most salient changes of Parkinson's disease, the degeneration of dopaminergic neurons and the existence of α-synuclein aggregates. This article is intended for researchers planning to use cellular models for their studies. It describes for commonly used cell types and treatments the aspects of Parkinson's disease they model along with technical advantages and disadvantages. Furthermore, this article describes strategies to induce and measure aggregates with a focus on fluorescent techniques. This article is part of a special issue on Parkinson disease.

Glycosaminoglycans (GAGs) suppress the toxicity of HypF-N prefibrillar aggregates.

A group of diverse human pathologies is associated with proteins unable to retain their native state and convert into prefibrillar and fibrillar amyloid aggregates that are then deposited in the extracellular space. Glycosaminoglycans (GAGs) have been found to physically associate with these deposits and also to promote their formation in vitro. However, the effect of GAGs on the toxicity of these aggregates has been investigated in only one protein system, the amyloid ß peptide associated with Alzheimer's disease. In this study, we investigate whether GAGs affect the toxicity of the N-terminal domain of Escherichia coli HypF (HypF-N) oligomers on Chinese hamster ovarian cells and the mechanism by which such suppression is mediated. The results show that heparin and other GAGs inhibit the toxicity observed by HypF-N oligomers in a dose-dependent manner. GAGs were not found to bind preformed HypF-N oligomers, change their morphological and structural characteristics or disaggregate them. Nevertheless, they were found to bind to the cells' surface and prevent the interaction of the oligomers with the cells. Overall, the results indicate that GAGs have a generic ability to inhibit the toxicity of aberrant protein oligomers and that such toxicity suppression can occur through different mechanisms, such as through binding to the oligomers with consequent loss of interaction of the oligomers to the GAGs present on the cell surface, as proposed previously for amyloid ß aggregates, or through mechanisms independent of direct GAG-oligomer binding, as shown here for HypF-N aggregates.

Trafficking of the phosphoprotein PfCRT to the digestive vacuolar membrane in Plasmodium falciparum.

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site-directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.

Export of PfSBP1 to the Plasmodium falciparum Maurer's clefts.

The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer's clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite's secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite's plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P. falciparum-infected erythrocytes.

A conditional export system provides new insights into protein export in Plasmodium falciparum-infected erythrocytes.

The human malarial parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within its host erythrocyte, including the cytoplasm, the plasma membrane and membrane profiles of parasite origin termed Maurer's clefts. While there is some information regarding the signals that allot proteins for export, the trafficking route itself has remained largely obscure, partly due to technical limitations in following protein trafficking with time. To overcome these shortcomings, we have established a conditional protein export system in P. falciparum, based on the previously described conditional aggregation domain (CAD domain) that self-aggregates in the endoplasmic reticulum in a manner that is reversible by the addition of a small molecule. By fusing the CAD domain to the first 80 amino acids of STEVOR and full-length PfSBP1, we were able to control export of a soluble and a transmembrane protein to the erythrocyte cytosol and the Maurer's clefts respectively. The conditional export system allowed us to study the temporal sequence of events of protein export and identify intermediate steps. We further explored the potential of the conditional export system in identifying factors that interact with exported proteins en route. Our data provide evidence for a physical interaction of exported proteins with the molecular chaperone PfBiP during early export steps.

Diverse adaptations of an ancestral gill: a common evolutionary origin for wings, breathing organs, and spinnerets.

Changing conditions of life impose new requirements on the morphology and physiology of an organism. One of these changes is the evolutionary transition from aquatic to terrestrial life, leading to adaptations in locomotion, breathing, reproduction, and mechanisms for food capture. We have shown previously that insects' wings most likely originated from one of the gills of ancestral aquatic arthropods during their transition to life on land. Here we investigate the fate of these ancestral gills during the evolution of another major arthropod group, the chelicerates. We examine the expression of two developmental genes, pdm/nubbin and apterous, that participate in the specification of insects' wings and are expressed in particular crustacean epipods/gills. In the horseshoe crab, a primitively aquatic chelicerate, pdm/nubbin is specifically expressed in opisthosomal appendages that give rise to respiratory organs called book gills. In spiders (terrestrial chelicerates), pdm/nubbin and apterous are expressed in successive segmental primordia that give rise to book lungs, lateral tubular tracheae, and spinnerets, novel structures that are used by spiders to breathe on land and to spin their webs. Combined with morphological and palaeontological evidence, these observations suggest that fundamentally different new organs (wings, air-breathing organs, and spinnerets) evolved from the same ancestral structure (gills) in parallel instances of terrestrialization.