The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure.

Aggregation of α-synuclein is a defining molecular feature of Parkinson's disease, Lewy body dementia, and multiple systems atrophy. Hereditary mutations in α-synuclein are linked to both Parkinson's disease and Lewy body dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy body dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on α-synuclein fibril structure is fundamental to α-synuclein biology. We therefore determined the cryo-electron microscopy (cryo-EM) structure of α-synuclein fibrils containing the hereditary E46K mutation. The 2.5-Å structure reveals a symmetric double protofilament in which the molecules adopt a vastly rearranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which form the E46-K80 salt bridge in the wild-type fibril structure. We hypothesize that, under our conditions, the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts α-synuclein into a kinetic trap-a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure.

Different Amyloid-ß Self-Assemblies Have Distinct Effects on Intracellular Tau Aggregation.

Alzheimer's disease (AD) pathology is characterized by the aggregation of beta-amyloid (Aß) and tau in the form of amyloid plaques and neurofibrillary tangles in the brain. It has been found that a synergistic relationship between these two proteins may contribute to their roles in disease progression. However, how Aß and tau interact has not been fully characterized. Here, we analyze how tau seeding or aggregation is influenced by different Aß self-assemblies (fibrils and oligomers). Our cellular assays utilizing tau biosensor cells show that transduction of Aß oligomers into the cells greatly enhances seeded tau aggregation in a concentration-dependent manner. In contrast, transduced Aß fibrils slightly reduce tau seeding while untransduced Aß fibrils promote it. We also observe that the transduction of α-synuclein fibrils, another amyloid protein, has no effect on tau seeding. The enhancement of tau seeding by Aß oligomers was confirmed using tau fibril seeds derived from both recombinant tau and PS19 mouse brain extracts containing human tau. Our findings highlight the importance of considering the specific form and cellular location of Aß self-assembly when studying the relationship between Aß and tau in future AD therapeutic development.

Structures of fibrils formed by α-synuclein hereditary disease mutant H50Q reveal new polymorphs.

Deposits of amyloid fibrils of α-synuclein are the histological hallmarks of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, with hereditary mutations in α-synuclein linked to the first two of these conditions. Seeing the changes to the structures of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of α-synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two previously unobserved polymorphs of α-synuclein: narrow and wide fibrils, formed from either one or two protofilaments, respectively. These structures recapitulate conserved features of the wild-type fold but reveal new structural elements, including a previously unobserved hydrogen-bond network and surprising new protofilament arrangements. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells and greater cytotoxicity that we observe for H50Q compared to wild-type α-synuclein.

Amyloid ß-protein oligomers promote the uptake of tau fibril seeds potentiating intracellular tau aggregation.

BACKGROUND: Repeated failure of drug candidates targeting Alzheimer's disease (AD) in clinical trials likely stems from a lack of understanding of the molecular mechanisms underlying AD pathogenesis. Recent research has highlighted synergistic interactions between aggregated amyloid-ß (Aß) and tau proteins in AD, but the molecular details of how these interactions drive AD pathology remain elusive and speculative. METHODS: Here, we test the hypothesis that Aß potentiates intracellular tau aggregation, and show that oligomeric Aß specifically exacerbates proteopathic seeding by tau. Using tau-biosensor cells, we show that treatment with sub-toxic concentrations of Aß oligomers, but not monomers or fibrils, "primes" cells, making them more susceptible to tau seeding. The treatment with Aß oligomers enhances intracellular tau aggregation in a dose-dependent manner when the cells are seeded with either recombinant or brain-derived tau fibrils, whereas little or no aggregation is observed in the absence of Aß-oligomer priming. RESULTS: Priming by Aß oligomers appears to be specific to tau, as α-synuclein seeding is unaffected by this treatment. Aß oligomer-enhanced tau seeding also occurs in primary mouse neurons and human neuroblastoma cells. Using fluorescently labeled tau seeds, we find that treatment with Aß oligomers significantly enhances the cellular uptake of tau seeds, whereas a known tau-uptake inhibitor blocks the effect of Aß on tau uptake. CONCLUSION: The ability of Aß to promote tau seeding suggests a specific and plausible mechanism by which extracellular Aß initiates a deleterious cascade that is unique to AD. These data suggest that the Aß-mediated potentiation of tau uptake into cells should also be taken into account when designing Aß-targeted therapeutics.

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice.

Author Correction: Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

In the version of this Article originally published online, the upper right panel of Fig. 5a was mistakenly a repeat of the lower right panel. This has now been corrected in all versions of the Article.

Inhibiting amyloid-ß cytotoxicity through its interaction with the cell surface receptor LilrB2 by structure-based design.

Inhibiting the interaction between amyloid-ß (Aß) and a neuronal cell surface receptor, LilrB2, has been suggested as a potential route for treating Alzheimer's disease. Supporting this approach, Alzheimer's-like symptoms are reduced in mouse models following genetic depletion of the LilrB2 homologue. In its pathogenic, oligomeric state, Aß binds to LilrB2, triggering a pathway to synaptic loss. Here we identify the LilrB2 binding moieties of Aß (16KLVFFA21) and identify its binding site on LilrB2 from a crystal structure of LilrB2 immunoglobulin domains D1D2 complexed to small molecules that mimic phenylalanine residues. In this structure, we observed two pockets that can accommodate the phenylalanine side chains of KLVFFA. These pockets were confirmed to be 16KLVFFA21 binding sites by mutagenesis. Rosetta docking revealed a plausible geometry for the Aß-LilrB2 complex and assisted with the structure-guided selection of small molecule inhibitors. These molecules inhibit Aß-LilrB2 interactions in vitro and on the cell surface and reduce Aß cytotoxicity, which suggests these inhibitors are potential therapeutic leads against Alzheimer's disease.

Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel.

α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent ß-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs.

Structural Consequences of Chromophore Formation and Exploration of Conserved Lid Residues amongst Naturally Occurring Fluorescent Proteins.

Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the ß-sheets.(1) Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

Water Diffusion In And Out Of The ß-Barrel Of GFP and The Fast Maturing Fluorescent Protein, TurboGFP.

The chromophore of fluorescent proteins is formed by an internal cyclization of the tripeptide 65SYG67 fragment and a subsequent oxidation. The oxidation is slow - the kinetics of this step is presumably improved in fast maturing GFPs. Water molecules can aid in the chromophore formation. We have used 50ns molecular dynamics simulations of the mature and immature forms of avGFP and TurboGFP to examine the diffusion of water molecules in-and-out of the protein ß-barrel. Most crystal structures of GFPs have well-structured waters within hydrogen-bonding distance of Glu222 and Arg96. It has been proposed that they have an important role in chromophore formation. Stable waters are found in similar positions in all simulations conducted. The simulations confirm the existence of a pore that leads to the chromophore in the rapidly maturing TurboGFP; decreased water diffusion upon chromophore formation; and increased water diffusion due to the pore formation.