Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-β (Aβ), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aβ, aggregates rapidly to form amyloids and can expedite the aggregation of Aβ through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aβ to form amyloid aggregates that are less toxic than pure Aβ aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aβ in solution. This is the first evidence that Aα can coassemble with Aβ and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

We used a vesicle scaffold to form a ternary, quasi-panchromatic funnel for electronic excited states oriented from the outer surface towards the membrane. We did so via electronic energy transfer (EET) from two conjugated polyelectrolytes to a phthalocyanine.

Shared research resources, also known as core facilities, serve a crucial role in supporting research, training, and other needs for their respective institutions. In response to the coronavirus disease (COVID-19) pandemic, all but the most critical laboratory research was halted in many institutions around the world. The Association of Biomolecular Resource Facilities conducted 2 surveys to understand and document institutional responses to the COVID-19 pandemic from core facility perspectives. The first survey was focused on initial pandemic response and efforts to sustainably ramp down core facility operations. The second survey, which is the subject of this study, focused on understanding the approaches taken to ramp up core facility operations after these ramp-down procedures. The survey results revealed that many cores remained active during the ramp-down, performing essential COVID-19 research, and had a more coordinated institutional response for ramping up research as a whole. The lessons gained from this survey will be indexed to serve as a resource for the core facility community to understand, plan, and mitigate risk and disruptions in the event of future disasters.

Amyloid-β (Aβ) is an intrinsically disordered peptide thought to play an important role in Alzheimer's disease (AD). It has been the target of most AD therapeutic efforts, which have repeatedly failed in clinical trials. A more predominant peptidic fragment, formed through alternative processing of the amyloid precursor protein, is the p3 peptide. p3 has received little attention, which is possibly due to the prevailing view in the AD field that it is "non-amyloidogenic." By probing the self-assembly of this peptide, we found that p3 aggregates to form oligomers and fibrils and, when compared with Aβ, displays enhanced aggregation rates. Our findings highlight the solubilizing effect of the N-terminus of Aβ and the favorable formation of structures formed through C-terminal hydrophobic peptide interfaces. Based on our findings, we suggest a reevaluation of the current therapeutic approaches targeting only the β-secretase pathway of AD, given that the α- secretase pathway is also amyloidogenic.

Amyloid is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloid and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of approximate to 5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.

Racemic mixtures frequently display properties that are different from those associated with their enantiopure counterparts, and are often characterized by higher propensity to form aggregates. Our previous research established that mixing of the enantiomers of Alzheimer amyloid beta (A beta) 42 peptides is an effective strategy to induce an oligomer-to-fibril conversion, which puts A beta 42 into a substantially less toxic state. Here, new insights into this chiral inactivation effect are presented. In addition to the commonly used Thioflavin T fibril formation assays, the use of the less aggregation-prone A beta 40 system allowed us to monitor peptide aggregation by NMR. Whereas enantiopure peptide was well soluble under the chosen experimental conditions and showed no sign of precipitation, addition of one equivalent of the mirror-image peptide triggered an instant and rapid aggregation, observable through the attenuation of the NMR signal. The racemic A beta 40 fibrils were found by transmission electron microscopy to be distinct in morphology, exhibiting a similar to 2-fold narrowing as compared with their enantiopure counterparts.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aβ) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aβ42 should reduce the concentration of toxic oligomers formed from natural l-Aβ42. We synthesized l- and D-Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aβ42 at concentrations up to 50 μm. The mixing of Aβ42 enantiomers thus accelerates the formation of non-toxic fibrils.

Aggregation of the 140-amino acid protein α-synuclein (α-syn) is linked to the development of Parkinson's disease (PD). α-Syn is a copper binding protein with potential function as a regulator of metal-dependent redox activity. Epidemiological studies suggest that human exposure to excess copper increases the incidence of PD. α-Syn exists in both solution and membrane-bound forms. Previous work evaluated the Cu(2+) uptake for α-syn in solution and identified Met1-Asp2 and His50 as primary contributors to the coordination shell, with a dissociation constant of approximately 0.1 nM. When bound to the membrane bilayer, α-syn takes on a predominantly helical conformation, which spatially separates His50 from the N-terminus of the protein and is therefore incompatible with the copper coordination geometry of the solution state. Here we use circular dichroism and electron paramagnetic resonance (continuous wave and pulsed) to evaluate the coordination of copper to the membrane-bound form of α-syn. In this molecular environment, Cu(2+) binds exclusively to the N-terminus of the protein (Met1-Asp2) with no participation from His50. Copper does not alter the membrane-bound α-syn conformation or enhance the release of the protein from the bilayer. The Cu(2+) affinity is similar to that identified for solution α-syn, suggesting that copper coordination is retained in the membrane. Consideration of these results demonstrates that copper exerts its greatest conformational effect on the solution form of α-syn.

The molecular mechanisms regulating fate divergence of closely related, but distinct, layer 6 corticothalamic and layer 5 subcerebral projection neurons are largely unknown. We present evidence for central transcriptional mechanisms that regulate fate specification of corticothalamic (layer 6) and subcerebral (layer 5) projection neurons. We found that TBR1 promotes the identity of corticothalamic neurons and represses subcerebral fates through reducing expression ofFezf2and CTIP2. These conclusions are based on the following: (1) InTbr1−/−mice, the number of cells expressing layer 6 markers was reduced, and the number of cells expressing layer 5 markers was increased. Early-born (birthdated on E11.5) neurons ectopically expressed subcerebral neuronal markers, and extended their axons into subcerebral targets. (2) EctopicTbr1expression in layer 5 neurons prevented them from extending axons into the brainstem and the spinal cord. (3) Chromatin immunoprecipitation analysis using TBR1 antibodies showed that TBR1 bound to a conserved region in theFezf2gene. (4) Analysis ofFezf2mutants andTbr1−/−;Fezf2−/−compound mutants provided evidence thatFezf2blocks corticothalamic fate in layer 5 by reducingTbr1expression in subcerebral neurons. All neocortical regions appear to use this core transcriptional program to specify corticothalamic (layer 6) and subcerebral (layer 5) projection neurons.

PHR [PAM (protein associated with Myc)–HIW (Highwire)–RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel β sandwich fold composed of 11 antiparallel β-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092→Glu, observed in the Caenorhabditis elegans ortholog RPM-1.

This is a continuation of a study begun in 2005 when the authors first reported on the theoretical possibility of a non-trivial error in calibration of radon chambers caused by the current use of scintillation cells. The ramifications of this error are just now being felt by the radon industry as experts around the world attempt to better define radon concentration standards and insist upon measurement results more accurate than the traditional 25 %. In this report, the authors conclude their study by comparing the theoretical error and the actual experimental error of using a certain popular scintillation cell made by Pylon™ (1) with a volume of 271 ml. A graph is presented which predicts the cell calibration error, as a function of the difference in elevation of any secondary chamber and the primary calibration facility, which would be introduced using this Pylon™ cell. The authors summarize their study by presenting a second graph that predicts the cell calibration error introduced by using any right cylinder scintillation cell of any dimensions that can be modeled as a scaled-up or scaled-down version of the popular Rocky Mountain Glassworks cell.

Caenorhabditis elegans RPM-1 is a member of a conserved protein family, the PHR proteins, that includes human Pam, mouse Phr1, zebrafish Esrom, and Drosophila Highwire. PHR proteins play important roles in the development of the nervous system. In particular, mutations in rpm-1 cause a disruption of synaptic architecture, affecting the distribution of synaptic vesicles and the number of presynaptic densities. Using antibodies against RPM-1, we determined the localization of the endogenous RPM-1 protein in wild-type and in several mutants that affect synaptic development. Our analyses show that, in mature neurons, RPM-1 resides in a distinct region that is close to, but does not overlap with, the synaptic exo- and endocytosis domains. The localization of RPM-1 occurs independently of several proteins that function in the transport or assembly of synapse components, and its abundance is partially dependent on its binding partner the F-box protein FSN-1. RPM-1 has been shown to target the MAPKKK DLK-1 for degradation. We show that activated DLK-1 may be preferentially targeted for degradation. Furthermore, using transgene analysis, we identified a critical role of the conserved PHR domain of RPM-1 in its subcellular localization.

Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.

During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-β family members between presynaptic and postsynaptic termini are proposed to govern synapse formation1,2,3. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. Here we report the identification of FSN-1, a novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes4,5,6,7. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains8,9. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. We provide evidence that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase10), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation.