Single-cell atlas of ABCA7 loss-of-function reveals impaired neuronal respiration via choline-dependent lipid imbalances.

Loss-of-function (LoF) variants in the lipid transporter ABCA7 significantly increase the risk of Alzheimer's disease (odds ratio ∼2), yet the pathogenic mechanisms and the neural cell types affected by these variants remain largely unknown. Here, we performed single-nuclear RNA sequencing of 36 human post-mortem samples from the prefrontal cortex of 12 ABCA7 LoF carriers and 24 matched non-carrier control individuals. ABCA7 LoF was associated with gene expression changes in all major cell types. Excitatory neurons, which expressed the highest levels of ABCA7, showed transcriptional changes related to lipid metabolism, mitochondrial function, cell cycle-related pathways, and synaptic signaling. ABCA7 LoF-associated transcriptional changes in neurons were similarly perturbed in carriers of the common AD missense variant ABCA7 p.Ala1527Gly (n = 240 controls, 135 carriers), indicating that findings from our study may extend to large portions of the at-risk population. Consistent with ABCA7's function as a lipid exporter, lipidomic analysis of isogenic iPSC-derived neurons (iNs) revealed profound intracellular triglyceride accumulation in ABCA7 LoF, which was accompanied by a relative decrease in phosphatidylcholine abundance. Metabolomic and biochemical analyses of iNs further indicated that ABCA7 LoF was associated with disrupted mitochondrial bioenergetics that suggested impaired lipid breakdown by uncoupled respiration. Treatment of ABCA7 LoF iNs with CDP-choline (a rate-limiting precursor of phosphatidylcholine synthesis) reduced triglyceride accumulation and restored mitochondrial function, indicating that ABCA7 LoF-induced phosphatidylcholine dyshomeostasis may directly disrupt mitochondrial metabolism of lipids. Treatment with CDP-choline also rescued intracellular amyloid ß -42 levels in ABCA7 LoF iNs, further suggesting a link between ABCA7 LoF metabolic disruptions in neurons and AD pathology. This study provides a detailed transcriptomic atlas of ABCA7 LoF in the human brain and mechanistically links ABCA7 LoF-induced lipid perturbations to neuronal energy dyshomeostasis. In line with a growing body of evidence, our study highlights the central role of lipid metabolism in the etiology of Alzheimer's disease.

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aß42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aß uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aß phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on a Lipid Membrane.

The bioinspired design of ligands for nanoparticle coating with remarkable precision in controlling anisotropic connectivity and with universal binding efficiency to the membrane has made a great impact on nanoparticle self-assembly. We utilize the HIV-1-derived trans-activator of transcription peptide (TAT), a member of the cell-penetrating peptides, as a soft shell coating on gold nanoparticles (GNPs) and characterize TAT pepide-mediated binding behaviors of GNPs on the lipid membrane. Whereas the peptides enable GNPs to firmly attach to the membrane, the binding structures are driven by two electrostatic forces: the interparticle peptide repulsion and the peptide-membrane attraction. Although transmission electron microscopy images showed that the densities of membrane-embedded GNPs were almost equal, X-ray reflectivity revealed a significant difference in binding structures of GNPs along the surface normal upon the increase of charge densities (ϕ) of the membrane. In particular, GNPs were densely suspended at ϕ = 70% while they adopted an additional well-defined layer underneath the membrane at ϕ = 100%, in addition to a translocation of the initially bound particles into the membrane. The observed behaviors of GNPs manifest a 3D to 2D transformation of the self-assembled structures from the diffused state to the closely packed state with the increase in the charge density of the membrane. The present study also provides insights on the binding mechanisms of the cell-penetrating peptide-coated nanoparticles to the lipid membranes, which is a common theme of delivery systems in pharmaceutical research.

Demonstration of a time-resolved x-ray scattering instrument utilizing the full-repetition rate of x-ray pulses at the Pohang Light Source.

We report on the development of a new experimental instrument for time-resolved x-ray scattering (TRXS) at the Pohang Light Source (PLS-II). It operates with a photon energy ranging from 5 to 18 keV. It is equipped with an amplified Ti:sappahire femtosecond laser, optical diagnostics, and laser beam delivery for pump-probe experiments. A high-speed single-element detector and high trigger-rate oscilloscope are used for rapid data acquisition. While this instrument is capable of measuring sub-nanosecond dynamics using standard laser pump/x-ray probe techniques, it also takes advantage of the dense 500 MHz standard fill pattern in the PLS-II storage ring to efficiently record nano-to-micro-second dynamics simultaneously. We demonstrate this capability by measuring both the (fast) impulsive strain and (slower) thermal recovery dynamics of a crystalline InSb sample following intense ultrafast laser excitation. Exploiting the full repetition rate of the storage ring results in a significant improvement in data collection rates compared to conventional bunch-tagging methods.

Direct-write X-ray lithography using a hard X-ray Fresnel zone plate.

Results are reported of direct-write X-ray lithography using a hard X-ray beam focused by a Fresnel zone plate with an outermost zone width of 40 nm. An X-ray beam at 7.5 keV focused to a nano-spot was employed to write arbitrary patterns on a photoresist thin film with a resolution better than 25 nm. The resulting pattern dimension depended significantly on the kind of underlying substrate, which was attributed to the lateral spread of electrons generated during X-ray irradiation. The proximity effect originated from the diffuse scattering near the focus and electron blur was also observed, which led to an increase in pattern dimension. Since focusing hard X-rays to below a 10 nm spot is currently available, the direct-write hard X-ray lithography developed in this work has the potential to be a promising future lithographic method.

Traumatic brain injury and the neuronal microenvironment: a potential role for neuropathological mechanotransduction.

Traumatic brain injury (TBI) is linked to several pathologies for which there is a lack of understanding of disease mechanisms and therapeutic strategies. To elucidate injury mechanisms, it is important to consider how physical forces are transmitted and transduced across all spatial scales of the brain. Although the mechanical response of the brain is typically characterized by its material properties and biological structure, cellular mechanotransduction mechanisms also exist. Such mechanisms can affect physiological processes by responding to exogenous mechanical forces directed through sub-cellular components, such as extracellular matrix and cell adhesion molecules, to mechanosensitive intracellular structures that regulate mechanochemical signaling pathways. We suggest that cellular mechanotransduction may be an important mechanism underlying the initiation of cell and sub-cellular injuries ultimately responsible for the diffuse pathological damage and clinical symptoms observed in TBI, thereby providing potential therapeutic opportunities not previously explored in TBI.

Demonstration of feasibility of X-ray free electron laser studies of dynamics of nanoparticles in entangled polymer melts.

The recent advent of hard x-ray free electron lasers (XFELs) opens new areas of science due to their exceptional brightness, coherence, and time structure. In principle, such sources enable studies of dynamics of condensed matter systems over times ranging from femtoseconds to seconds. However, the studies of "slow" dynamics in polymeric materials still remain in question due to the characteristics of the XFEL beam and concerns about sample damage. Here we demonstrate the feasibility of measuring the relaxation dynamics of gold nanoparticles suspended in polymer melts using X-ray photon correlation spectroscopy (XPCS), while also monitoring eventual X-ray induced damage. In spite of inherently large pulse-to-pulse intensity and position variations of the XFEL beam, measurements can be realized at slow time scales. The X-ray induced damage and heating are less than initially expected for soft matter materials.

Coherent X-ray scattering beamline at port 9C of Pohang Light Source II.

The coherent X-ray scattering beamline at the 9C port of the upgraded Pohang Light Source (PLS-II) at Pohang Accelerator Laboratory in Korea is introduced. This beamline provides X-rays of 5-20 keV, and targets coherent X-ray experiments such as coherent diffraction imaging and X-ray photon correlation spectroscopy. The main parameters of the beamline are summarized, and some preliminary experimental results are described.

Nanostructures of small-molecule organic crystals on capillary wave surfaces with controllable capillary lengths.

We report on the nanostructures of organic small-molecule pentacene crystals that have been vapor-deposited onto the capillary wave surfaces of thin liquid films. The characteristic lateral length of the capillary wave surface or the capillary length can be controlled by changing the thickness of the liquid films and, thus, the van der Waals interaction with the substrate. We find that the morphology of the organic crystals gradually varies from fractals to compact islands as the liquid film thickness increases. The square of average distance between organic crystal grains was also found to be proportional to the liquid film thickness. We discuss the possibility that these effects are driven by capillary fluctuations at the air-liquid interface.

Phase separation of phospholipid multilayers incorporated with cell penetrating peptides.

We used X-ray reflectivity to investigate the structures of phospholipid multilayers with transcription-activating-factor-derived peptide (TDP) as a function of the membrane charge density. Mixed phospholipid multilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl -sn-glycero-3-phosphoserine (DPPS) with different mixing ratios (C:S) were used to elucidate the various charge densities in a plasma membrane. We fixed the peptide/lipid molar ratio (P/L) and varied the DPPC/DPPS molar ratio in the mixed multilayer. In the pure DPPC multilayer, the incorporation of TDP had nearly no effect on the bilayer thickness of the mixed lipid multilayer. However, in the mixed DPPC/DPPS multilayer, the incorporation of TDP decreased the bilayer thickness, suggesting that the TDP peptide had a stronger interaction with DPPS than with DPPC and caused disorder in the lamellar structure. Combining this with the refined X-ray reflectivity (XR) data, we concluded that the TDP existed more in the headgroup region of the TDP-induced segregated DPPS in the mixed multilayer and caused significant membrane thinning.

Parratt-based and model-independent X-ray reflectivity fitting procedure for nanoscale thin film characterization.

A general-purpose fitting procedure is presented for X-ray reflectivity data. The Parratt formula was used to fit the low-angle region of the reflectivity data and the resulting electron density profile (continuous base EDP or cbEDP) was then divided into a series of electron density slabs of width 1 angstroms (discrete base EDP or dbEDP), which is then easily incorporated into the Distorted Wave Born Approximation (DWBA). An additional series of density slabs of resolution-limited width are overlapped to the dbEDP, and the density value of the each additional slab is allowed to vary to further fit the data model-independently using DWBA. Because this procedure combines the Parratt formula and the model-independent DWBA fitting, each fitting method can always be employed depending on the type of thin film. Moreover, it provides a way to overcome the difficulties when both fitting methods do not work well for certain types of thin films. Simulations show that this procedure is suitable for nanoscale thin film characterization.

Characterization of crystalline structure and morphology of NiO thin films.

We investigated the relation of sputtering powers with structural and morphological properties of nickel oxide (NiO) thin films. NiO thin films were fabricated by using an rf-reactive sputtering method on Si(100) substrates with a Ni target in a partial pressure of oxygen and argon. The films were deposited by various rf-sputtering powers from 100 to 200 W at room temperature. The phases and crystalline structures of the deposited films were investigated by using grazing incident X-ray diffraction (XRD). The thickness and surface morphology of the films were investigated by using a field emission-scanning electron microscopy (FE-SEM). The different sputtering conditions drastically affected the crystallinity and the surface morphology of NiO thin films. A combined analysis of the data obtained from X-ray diffraction and SEM images demonstrates that the preferred orientation of NiO films tends to grow from (111) to (200) direction as increasing the sputtering power, which can be explained by in terms of the surface energy along the indexing plane in an fcc structure. As increasing the rf power, lattice constants decreased from 4.26 to 4.20 angstroms and samples became high-quality crystals. Under our experimental condition, NiO films prepared at 150 W with 20% partial pressure of oxygen and 7 cm distance from the sample to the target show the best quality of the crystal.

Structural and magnetic properties of TiZrNi thin films prepared by magnetron sputtering and thermal annealing.

Distinctive thin layers of TiZr and Ni were deposited by using a magnetron sputtering method and a thermal annealing was applied to discover metallic films of quasicrystals. After a heat treatment in vacuum, 70 nm thick deposited layers were well mixed with nominal compositions of 49.7, 29.3 and 21.0 for Ti, Zr and Ni, respectively, which is very close with the one forming a quasicrystalline phase. The magnetization values were significantly decreased from 0.286 to 0.142 emu/mm3 at 2000 Oe, after annealing, while a shape of magnetic hysteresis was maintained. It is believed that a different magnetic behavior after thermal annealing is due to the homogeneous mixing of atomic elements and possible existence of a metastable phase.

Note: Construction of x-ray scattering and x-ray absorption fine structure beamline at the Pohang Light Source.

A new hard x-ray beamline, 10B KIST-PAL beamline (BL10B), has been designed and constructed at the Pohang Light Source (PLS) in Korea. The beamline, operated by Pohang Accelerator Laboratory-Korean Institute of Science and Technology consortium, is dedicated to x-ray scattering (XRS) and x-ray absorption fine structure (XAFS) experiments. X rays with photon energies from 4.0 to 16.0 keV are delivered to the experimental station passing a collimating mirror, a fixed-exit double-crystal Si(111) monochromator, and a toroidal mirror. Basic experimental equipment for XAFS measurement, a high resolution diffractometry, an image plate detector system, and a hot stage have been prepared for the station. From our initial commissioning and performance testing of the beamline, it is observed that BL10B beamline can perform XRS and XAFS measurements successfully.

Observation of a liquid-to-layered transition in thin liquid films when surface and interface regions overlap.

We have used x-ray reflectivity to study the coupling of surface and interface layering in a molecularly thin normal liquid [tetrakis(2-ethylhexoxy)silane (TEHOS)], as a function of temperature and film thickness. The best fits to the data were obtained with an electron density model that consists of a uniform density component superimposed upon molecular-scale density oscillations (layers). The two types of layer profiles were observed to vary with temperature from 187-286 K . The amount of material in the molecular layers increases as that in the uniform density layer decreases, with the onset of liquid-to-layered transition occurring at a total film thickness of approximately 40 A (about twice the bulk correlation length of TEHOS).