Brain responses to intermittent fasting and the healthy living diet in older adults.

Diet may promote brain health in metabolically impaired older individuals. In an 8-week randomized clinical trial involving 40 cognitively intact older adults with insulin resistance, we examined the effects of 5:2 intermittent fasting and the healthy living diet on brain health. Although intermittent fasting induced greater weight loss, the two diets had comparable effects in improving insulin signaling biomarkers in neuron-derived extracellular vesicles, decreasing the brain-age-gap estimate (reflecting the pace of biological aging of the brain) on magnetic resonance imaging, reducing brain glucose on magnetic resonance spectroscopy, and improving blood biomarkers of carbohydrate and lipid metabolism, with minimal changes in cerebrospinal fluid biomarkers for Alzheimer's disease. Intermittent fasting and healthy living improved executive function and memory, with intermittent fasting benefiting more certain cognitive measures. In exploratory analyses, sex, body mass index, and apolipoprotein E and SLC16A7 genotypes modulated diet effects. The study provides a blueprint for assessing brain effects of dietary interventions and motivates further research on intermittent fasting and continuous diets for brain health optimization. For further information, please see ClinicalTrials.gov registration: NCT02460783.

Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum.

Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age.

Upregulation of Sarcolemmal Hemichannels and Inflammatory Transcripts with Neuromuscular Junction Instability during Lower Limb Unloading in Humans.

Human skeletal muscle atrophy and a disproportionate force loss occur within a few days of unloading in space and on Earth, but the underlying mechanisms are not fully understood. Disruption of neuromuscular junction homeostasis has been proposed as one of the possible causes. Here, we investigated the potential mechanisms involved in this neuromuscular disruption induced by a 10-day unilateral lower limb suspension (ULLS) in humans. Specifically, we investigated hemichannels' upregulation, neuromuscular junction and axonal damage, neurotrophins' receptor downregulation and inflammatory transcriptional signatures. Biomarkers were evaluated at local and systemic levels. At the sarcolemmal level, changes were found to be associated with an increased expression of connexin 43 and pannexin-1. Upregulation of the inflammatory transcripts revealed by deep transcriptomics was found after 10 days of ULLS. The destabilisation of the neuromuscular junction was not accompanied by changes in the secretion of the brain-derived neurotrophic factor and neurotrophin-4, while their receptor, BDNF/NT growth factors receptor (TrkB), decreased. Furthermore, at 5 days of ULLS, there was already a significant upregulation of the serum neurofilament light chain concentration, an established clinical biomarker of axonal injury. At 10 days of ULLS, other biomarkers of early denervation processes appeared. Hence, short periods of muscle unloading induce sarcolemmal hemichannels upregulation, inflammatory transcripts upregulation, neuromuscular junction instability and axonal damage.

Effects of short-term unloading and active recovery on human motor unit properties, neuromuscular junction transmission and transcriptomic profile.

Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these aspects and the underlying molecular mechanisms with short-term unloading and active recovery (AR). Eleven healthy males underwent a 10-day unilateral lower limb suspension (ULLS) period, followed by 21-day AR based on resistance exercise. Quadriceps femoris (QF) cross-sectional area (CSA) and isometric maximum voluntary contraction (MVC) were evaluated. Intramuscular electromyographic recordings were obtained during 10% and 25% MVC isometric contractions from the vastus lateralis (VL). Biomarkers of NMJ molecular instability (serum c-terminal agrin fragment, CAF), axonal damage (neurofilament light chain) and denervation status were assessed from blood samples and VL biopsies. NMJ and ion channel transcriptomic profiles were investigated by RNA-sequencing. QF CSA and MVC decreased with ULLS. Increased CAF and altered NMJ transcriptome with unloading suggested the emergence of NMJ molecular instability, which was not associated with impaired NMJ transmission stability. Instead, increased MUP complexity and decreased motor unit firing rates were found after ULLS. Downregulation of ion channel gene expression was found together with increased neurofilament light chain concentration and partial denervation. The AR period restored most of these neuromuscular alterations. In conclusion, the human NMJ is destabilized at the molecular level but shows functional resilience to a 10-day unloading period at least at relatively low contraction intensities. However, MUP properties are altered by ULLS, possibly due to alterations in ion channel dynamics and initial axonal damage and denervation. These changes are fully reversed by 21 days of AR. KEY POINTS: We used integrative electrophysiological and molecular approaches to comprehensively investigate changes in neuromuscular integrity and function after a 10-day unilateral lower limb suspension (ULLS), followed by 21 days of active recovery in young healthy men, with a particular focus on neuromuscular junction (NMJ) and motor unit potential (MUP) properties alterations. After 10-day ULLS, we found significant NMJ molecular alterations in the absence of NMJ transmission stability impairment. These findings suggest that the human NMJ is functionally resilient against insults and stresses induced by short-term disuse at least at relatively low contraction intensities, at which low-threshold, slow-type motor units are recruited. Intramuscular electromyography analysis revealed that unloading caused increased MUP complexity and decreased motor unit firing rates, and these alterations could be related to the observed changes in skeletal muscle ion channel pool and initial and partial signs of fibre denervation and axonal damage. The active recovery period restored these neuromuscular changes.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Semi-automated 3D segmentation of human skeletal muscle using Focused Ion Beam-Scanning Electron Microscopic images.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1-2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and manual segmentation are two critical problems that limit the more extensive use of FIB-SEM technology. Here, we present an easily accessible robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for semi-automated data segmentation using the open-source machine learning Weka segmentation software, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.

Power Grid Protection of the Muscle Mitochondrial Reticulum.

Mitochondrial network connectivity enables rapid communication and distribution of potential energy throughout the cell. However, this connectivity puts the energy conversion system at risk, because damaged elements could jeopardize the entire network. Here, we demonstrate the mechanisms for mitochondrial network protection in heart and skeletal muscle (SKM). We find that the cardiac mitochondrial reticulum is segmented into subnetworks comprising many mitochondria linked through abundant contact sites at highly specific intermitochondrial junctions (IMJs). In both cardiac and SKM subnetworks, a rapid electrical and physical separation of malfunctioning mitochondria occurs, consistent with detachment of IMJs and retraction of elongated mitochondria into condensed structures. Regional mitochondrial subnetworks limit the cellular impact of local dysfunction while the dynamic disconnection of damaged mitochondria allows the remaining mitochondria to resume normal function within seconds. Thus, mitochondrial network security is comprised of both proactive and reactive mechanisms in striated muscle cells.

Mitochondrial reticulum for cellular energy distribution in muscle.

Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion; however, genetic evidence suggests that facilitated diffusion is not critical for normal function. We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.

Maturation of the HIV-1 core by a non-diffusional phase transition.

The formation of the HIV-1 core is the final step in the viral maturation pathway, resulting in the formation of infectious virus. Most current models for HIV-1 core formation suggest that, upon proteolytic cleavage from the immature Gag, capsid (CA) dissociates into the viral interior before reforming into the core. Here we present evidence for an alternate view of core formation by taking advantage of our serendipitous observation of large membrane-enclosed structures in HIV-1 supernatants from infected cells. Cryo-electron tomographic studies show that these structures, which contain ordered arrays of what is likely the membrane-associated matrix protein, contain multiple cores that can be captured at different stages of maturation. Our studies suggest that HIV maturation involves a non-diffusional phase transition in which the detaching layer of the cleaved CA lattice is gradually converted into a roll that ultimately forms the surface of the mature conical core.

Visualizing cells and humans in 3D: biomedical image analysis at nanometer and meter scales.

Researchers analyzed and presented volume data from the Visible Human Project (VHP) and data from high-resolution 3D ion-abrasion scanning electron microscopy (IA-SEM). They acquired the VHP data using cryosectioning, a destructive approach to 3D human anatomical imaging resulting in whole-body images with a field of view approaching 2 meters and a minimum resolvable feature size of 300 microns. IA-SEM is a type of block-face imaging microscopy, a destructive approach to microscopic 3D imaging of cells. The field of view of IA-SEM data is on the order of 10 microns (whole cell) with a minimum resolvable feature size of 15 nanometers (single-slice thickness). Despite the difference in subject and scale, the analysis and modeling methods were remarkably similar. They are derived from image processing, computer vision, and computer graphics techniques. Moreover, together we are employing medical illustration, visualization, and rapid prototyping to inform and inspire biomedical science. By combining graphics and biology, we are imaging across nine orders of magnitude of space to better promote public health through research.

Determination of molecular structures of HIV envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) (2). Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor (5,9). The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine. The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Šresolution (9,14). This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

Correlative 3D imaging of whole mammalian cells with light and electron microscopy.

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus.

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells.

The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T cells and virus-bearing antigen-presenting cells such as macrophages or dendritic cells via specialized structures known as virological synapses. Using ion abrasion SEM, electron tomography, and superresolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T cells. We demonstrate the striking envelopment of T cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T cells, but virions are not released passively at the synapse; instead, virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T cells from the extracellular milieu, the burial of the site of HIV transfer, and the receptor-dependent initiation of virion transfer by T cells highlight unique aspects of cell-cell HIV transmission.

Ion-abrasion scanning electron microscopy reveals surface-connected tubular conduits in HIV-infected macrophages.

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of approximately 150-200 nm, and lengths of up to approximately 5 microm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.

Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy.

Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW.

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes.

We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor.

The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular-vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN.