A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), US National Institutes of Health (A.M. and A.B.); by the Boehringer Ingelheim Fonds (P.F.); by a European Research Council starting grant (to M.B.D.M. and E.A.A.N.); and by The Cambridge Centre for Misfolding Diseases. N.C. thanks the Spanish Ministry of Economy and Competitiveness (RYC-2012-12068). S.W.C. thanks the Agency for Science, Technology, and Research, Singapore for support

Assessing motor-related phenotypes of Caenorhabditis elegans with the wide field-of-view nematode tracking platform

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously

Co-chaperones are limiting in a depleted chaperone network

To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1

An ALS-Linked Mutant SOD1 Produces a Locomotor Defect Associated with Aggregation and Synaptic Dysfunction When Expressed in Neurons of Caenorhabditis elegans

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carbonyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events