Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease.

The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.

Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Distinct growth regimes of α-synuclein amyloid elongation.

Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in ß-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.

Comparing lipid remodeling of brown adipose tissue, white adipose tissue, and liver after one-week high fat diet intervention with quantitative Raman microscopy.

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.

Chemical catalysis by biological amyloids.

Toxic aggregation of proteins and peptides into amyloid fibers is the basis of several human diseases. In each disease, a particular peptide noncovalently assembles into long thin structures with an overall cross-ß fold. Amyloids are not only related to disease: functional amyloids are found in many biological systems and artificial peptide amyloids are developed into novel nanomaterials. Amyloid fibers can act as template for the generation of more amyloids but are considered nonreactive in chemical catalysis. The perception of amyloids as chemically inert species was recently challenged by in vitro work on three human amyloid systems. With the use of model substrates, amyloid-ß, α-synuclein and glucagon amyloids were found to catalyze biologically relevant chemical reactions. The detected catalytic activity was much less than that of 'real' enzymes, but like that of designed (synthetic) catalytic amyloids. I here describe the current knowledge around this new activity of natural amyloids and the putative connection to metabolic changes in amyloid diseases. These pioneering studies imply that catalytic activity is an unexplored gain-of-function activity of disease amyloids. In fact, all biological amyloids may harbor intrinsic catalytic activity, tuned by each amyloid's particular fold, that await discovery.

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-ß-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aß42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aß42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Single-cell tracking demonstrates copper chaperone Atox1 to be required for breast cancer cell migration.

Copper ions are needed for several hallmarks of cancer. However, the involved pathways, mechanisms, and copper-binding proteins are mostly unknown. We recently found that cytoplasmic Antioxidant 1 copper chaperone (Atox1), which is up-regulated in breast cancer, is localized at the lamellipodia edges of aggressive breast cancer cells. To reveal molecular insights into a putative role in cell migration, we here investigated breast cancer cell (MDA-MB-231) migration by video microscopy as a function of Atox1. Tracking of hundreds of individual cells (per condition) over a 9-h time series revealed that cell migration velocity and directionality are significantly reduced upon Atox1 silencing in the cells. Because silencing of the copper transporter ATP7A also reduced cell migration, these proteins appear to be on the same pathway, suggesting that their well-known copper transport activity is involved. In-cell proximity ligation assays demonstrated that Atox1, ATP7A, and the proenzyme of lysyl oxidase (LOX; copper-loaded via ATP7A) are all in close proximity and that LOX activity is reduced upon Atox1 silencing in the cells. Since LOX is an established player in cancer cell migration, our results imply that Atox1 mediates breast cancer cell migration via coordinated copper transport in the ATP7A-LOX axis. Because individual cell migration is an early step in breast cancer metastasis, Atox1 levels in tumor cells may be a predictive measure of metastasis potential and serve as a biomarker for copper depletion therapy.

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein.

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Amyloid formation of fish ß-parvalbumin involves primary nucleation triggered by disulfide-bridged protein dimers.

Amyloid formation involves the conversion of soluble protein species to an aggregated state. Amyloid fibrils of ß-parvalbumin, a protein abundant in fish, act as an allergen but also inhibit the in vitro assembly of the Parkinson protein α-synuclein. However, the intrinsic aggregation mechanism of ß-parvalbumin has not yet been elucidated. We performed biophysical experiments in combination with mathematical modeling of aggregation kinetics and discovered that the aggregation of ß-parvalbumin is initiated by the formation of dimers stabilized by disulfide bonds and then proceeds via primary nucleation and fibril elongation processes. Dimer formation is accelerated by H2O2 and hindered by reducing agents, resulting in faster and slower aggregation rates, respectively. Purified ß-parvalbumin dimers readily assemble into amyloid fibrils with similar morphology as those formed when starting from monomer solutions. Furthermore, addition of preformed dimers accelerates the aggregation reaction of monomers. Aggregation of purified ß-parvalbumin dimers follows the same kinetic mechanism as that of monomers, implying that the rate-limiting primary nucleus is larger than a dimer and/or involves structural conversion. Our findings demonstrate a folded protein system in which spontaneously formed intermolecular disulfide bonds initiate amyloid fibril formation by recruitment of monomers. This dimer-induced aggregation mechanism may be of relevance for human amyloid diseases in which oxidative stress is often an associated hallmark.

Amyloids of α-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites.

The assembly of α-synuclein into cross-ß structured amyloid fibers results in Lewy body deposits and neuronal degeneration in Parkinson's disease patients. As the cell environment is highly crowded, interactions between the formed amyloid fibers and a range of biomolecules can occur in cells. Although amyloid fibers are considered chemically inert species, recent in vitro work using model substrates has shown α-synuclein amyloids, but not monomers, to catalyze the hydrolysis of ester and phosphoester bonds. To search for putative catalytic activity of α-synuclein amyloids on biologically relevant metabolites, we here incubated α-synuclein amyloids with neuronal SH-SY5Y cell lysates devoid of proteins. LC-MS-based metabolomic (principal component and univariate) analysis unraveled distinct changes in several metabolite levels upon amyloid (but not monomer) incubation. Of 63 metabolites identified, the amounts of four increased (3-hydroxycapric acid, 2-pyrocatechuic acid, adenosine, and NAD), and the amounts of seventeen decreased (including aromatic and apolar amino acids, metabolites in the TCA cycle, keto acids) in the presence of α-synuclein amyloids. Many of these metabolite changes match what has been reported previously in Parkinson's disease patients and animal-model metabolomics studies. Chemical reactivity of α-synuclein amyloids may be a new gain-of-function that alters the metabolite composition in cells and, thereby, modulates disease progression.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Frustration Dynamics and Electron-Transfer Reorganization Energies in Wild-Type and Mutant Azurins.

Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials, and active-site reorganization energies. Although the minimal frustration of the folding landscape enables this tuning, residual frustration in the vicinity of the metallocofactor can allow conformational fluctuations required for protein function. We show here that the constrained copper site in wild-type azurin is governed by an intricate pattern of minimally frustrated local and distant interactions that together enable rapid electron flow to and from the protein. In contrast, sluggish electron transfer reactions (unfavorable reorganization energies) of active-site azurin variants are attributable to increased frustration near to as well as distant from the copper site, along with an exaggerated oxidation-state dependence of both minimally and highly frustrated interaction patterns.

Macromolecular crowding modulates α-synuclein amyloid fiber growth.

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson's disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.

Orientation of α-Synuclein at Negatively Charged Lipid Vesicles: Linear Dichroism Reveals Time-Dependent Changes in Helix Binding Mode.

The neuronal protein α-synuclein, linked to Parkinson's disease, binds to negatively charged vesicles adopting a partial α-helix structure, but helix arrangement at the vesicle surface is not fully understood. Using linear dichroism spectroscopy (LD), we study the interaction of monomeric α-synuclein with large unilamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) under mild shear flow. The LD data of oriented lipid vesicles show that the long axis of the protein helix is oriented preferentially perpendicular to the membrane normal but deviates from a uniform in-plane distribution. Upon initial binding, a fraction of helices are oriented in the direction of least curvature for all ellipsoid-shaped vesicles at a lipid:protein molar ratio of 100. However, at a lower protein concentration the helices distribute uniformly on DOPS and POPS vesicles. In all cases, the α-synuclein helices rearrange with time (minute time scale) in the shear flow and begin to tilt into the vesicle membrane. Faster reorientation kinetics in the presence of flow suggests that modulation of membrane dynamics, by thermal or shear-dynamic activation, may overcome steric barriers by what may be called "flow catalysis".

C-terminal truncation of α-synuclein alters DNA structure from extension to compaction.

Parkinson's disease (PD) is linked to aggregation of the protein α-synuclein (aS) into amyloid fibers. aS is proposed to regulate synaptic activity and may also play a role in gene regulation via interaction with DNA in the cell nucleus. Here, we address the role of the negatively-charged C-terminus in the interaction between aS and DNA using single-molecule techniques. Using nanofluidic channels, we demonstrate that truncation of the C-terminus of aS induces differential effects on DNA depending on the extent of the truncation. The DNA extension increases for full-length aS and the (1-119)aS variant, but decreases about 25% upon binding to the (1-97)aS variant. Atomic force microscopy imaging showed full protein coverage of the DNA at high aS concentration. The characterization of biophysical properties of DNA when in complex with aS variants may provide important insights into the role of such interactions in PD, especially since C-terminal aS truncations have been found in clinical samples from PD patients.

Effects of the Toxic Metals Arsenite and Cadmium on α-Synuclein Aggregation In Vitro and in Cells.

Exposure to heavy metals, including arsenic and cadmium, is associated with neurodegenerative disorders such as Parkinson's disease. However, the mechanistic details of how these metals contribute to pathogenesis are not well understood. To search for underlying mechanisms involving α-synuclein, the protein that forms amyloids in Parkinson's disease, we here assessed the effects of arsenic and cadmium on α-synuclein amyloid formation in vitro and in Saccharomyces cerevisiae (budding yeast) cells. Atomic force microscopy experiments with acetylated human α-synuclein demonstrated that amyloid fibers formed in the presence of the metals have a different fiber pitch compared to those formed without metals. Both metal ions become incorporated into the amyloid fibers, and cadmium also accelerated the nucleation step in the amyloid formation process, likely via binding to intermediate species. Fluorescence microscopy analyses of yeast cells expressing fluorescently tagged α-synuclein demonstrated that arsenic and cadmium affected the distribution of α-synuclein aggregates within the cells, reduced aggregate clearance, and aggravated α-synuclein toxicity. Taken together, our in vitro data demonstrate that interactions between these two metals and α-synuclein modulate the resulting amyloid fiber structures, which, in turn, might relate to the observed effects in the yeast cells. Whilst our study advances our understanding of how these metals affect α-synuclein biophysics, further in vitro characterization as well as human cell studies are desired to fully appreciate their role in the progression of Parkinson's disease.

Folding of copper proteins: role of the metal?

Copper is a redox-active transition metal ion required for the function of many essential human proteins. For biosynthesis of proteins coordinating copper, the metal may bind before, during or after folding of the polypeptide. If the metal binds to unfolded or partially folded structures of the protein, such coordination may modulate the folding reaction. The molecular understanding of how copper is incorporated into proteins requires descriptions of chemical, thermodynamic, kinetic and structural parameters involved in the formation of protein-metal complexes. Because free copper ions are toxic, living systems have elaborate copper-transport systems that include particular proteins that facilitate efficient and specific delivery of copper ions to target proteins. Therefore, these pathways become an integral part of copper protein folding in vivo. This review summarizes biophysical-molecular in vitro work assessing the role of copper in folding and stability of copper-binding proteins as well as protein-protein copper exchange reactions between human copper transport proteins. We also describe some recent findings about the participation of copper ions and copper proteins in protein misfolding and aggregation reactions in vitro.

Copper chaperone blocks amyloid formation via ternary complex.

Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein α-synuclein (αS), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks αS aggregation. Because the same inhibition was found for a truncated version of αS, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in αS. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.

Female Faculty: Why So Few and Why Care?

Despite slow ongoing progress in increasing the representation of women in academia, women remain significantly under-represented at senior levels, in particular in the natural sciences and engineering. Not infrequently, this is downplayed by bringing forth arguments such as inherent biological differences between genders, that current policies are adequate to address the issue, or by deflecting this as being "not my problem" among other examples. In this piece we present scientific evidence that counters these claims, as well as a best-practice example, Genie, from Chalmers University of Technology, where one of the authors is currently employed. We also highlight particular challenges caused by the current COVID-19 pandemic. Finally, we conclude by proposing some possible solutions to the situation and emphasize that we need to all do our part, to ensure that the next generation of academics experience a more diverse, inclusive, and equitable working environment.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.