APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR.

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5 minutes of mGluR activation and a robust inhibition of the same within 2 minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15 minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.

Remodeling of ER-plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis.

Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.

Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition.

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min-1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

GPN does not release lysosomal Ca2+ but evokes Ca2+ release from the ER by increasing the cytosolic pH independently of cathepsin C.

The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.

Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons.

Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER-plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER-plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

Similar donors-similar blood?

BACKGROUND: Red blood cell (RBC) storage lesions have been suggested as contributing factors to suboptimal clinical outcomes. While undesirable effects of storage are well documented, their clinical relevance is still debated. Focus on storage time as the sole determinant of RBC quality ignores the variability in cell properties that may depend on factors other than age. Mechanical fragility (MF) aggregately reflects many storage-related functional and structural changes. This study evaluates interdonor versus intradonor variability, throughout storage, of both MF and autohemolysis (AH). STUDY DESIGN AND METHODS: Thirteen uniformly manufactured RBC units were collected initially as whole blood from nonsmoking, group A+, male Caucasian research donors. Mechanical stress was applied using a bead mill with oscillation at 50 Hz over durations varying from 0.5 to 60 minutes. MF profiles were described in terms of percent hemolysis after stresses of specified durations. Two months later, 11 of the 13 donors returned and assays were performed using the same protocol to allow comparison of intradonor versus interdonor variation. RESULTS: At 5 days postcollection, RBC MF profiles exhibited marked interdonor variability (up to twofold) overall. Both autolysis and MF across all units increased during storage-with rates of these increases varying by up to 10-fold for certain MF variables. Especially high AH and MF were observed for an outlier donor (with p < 0.05), for whom follow-up revealed previously undisclosed hereditary hypertriglyceridemia (levels exceeding approx. 1000 mg/dL). CONCLUSIONS: RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.

Protein refolding by pH-triggered chaperone binding and release.

Molecular chaperones are typically either adenosine triphosphate (ATP) dependent or rely heavily on their ATP-dependent chaperone counterparts in order to promote protein folding. This presents a challenge to chaperones that are localized to ATP-deficient cellular compartments. Here we describe a mechanism by which the pH-regulated acid stress chaperone HdeA is capable of independently facilitating the refolding of acid-denatured proteins in the bacterial periplasm, which lacks both ATP and ATP-dependent chaperone machines. Our results are consistent with a model in which HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding. Unlike previously characterized chaperones, HdeA appears to facilitate protein folding by using a single substrate binding-release cycle. This cycle is entirely regulated by the external environment and is therefore energy-neutral for the bacteria.

IP3R, store-operated Ca2+ entry and neuronal Ca2+ homoeostasis in Drosophila.

The IP3R (inositol 1,4,5-trisphosphate receptor) releases Ca2+ from the ER (endoplasmic reticulum) store upon binding to its ligand InsP3, which is thought to be generated by activation of certain membrane-bound G-protein-coupled receptors in Drosophila. Depletion of Ca2+ in the ER store also activates SOCE (store-operated Ca2+ entry) from the extracellular milieu across the plasma membrane, leading to a second rise in cytosolic Ca2+, which is then pumped back into the ER. The role of the IP3R and SOCE in mediating Ca2+ homoeostasis in neurons, their requirement in neuronal function and effect on neuronal physiology and as a consequence behaviour, are reviewed in the present article.

Functional complementation of Drosophila itpr mutants by rat Itpr1.

The Drosophila inositol 1,4,5-trisphosphate receptor (IP(3)R) and mammalian type-1 IP(3)Rs have 57-60% sequence similarity and share major domain homology with each other. Mutants in the single Drosophila IP(3)R gene, itpr, and Itpr1 knockout mice both exhibit lethality and defects in motor coordination. Here the authors show that the rat type-1 IP(3)R, which is the major neuronal isoform, when expressed in the pan-neuronal domain in Drosophila, functionally complements Drosophila IP(3)R function at cellular and systemic levels. It rescues the established neuronal phenotypes of itpr mutants in Drosophila, including wing posture, flight, electrophysiological correlates of flight maintenance, and intracellular calcium dynamics. This is the first in vivo demonstration of functional homology between a mammalian and fly IP(3)R. This study also paves the way for cellular and molecular analyses of the spinocerebellar ataxias in Drosophila, since SCA15/16 is known to be caused by heterozygosity of human ITPR1.

High accuracy alignment facility for the receiver and transmitter of the BepiColombo Laser Altimeter.

The accurate co-alignment of the transmitter to the receiver of the BepiColombo Laser Altimeter is a challenging task for which an original alignment concept had to be developed. We present here the design, construction and testing of a large collimator facility built to fulfill the tight alignment requirements. We describe in detail the solution found to attenuate the high energy of the instrument laser transmitter by an original beam splitting pentaprism group. We list the different steps of the calibration of the alignment facility and estimate the errors made at each of these steps. We finally prove that the current facility is ready for the alignment of the flight instrument. Its angular accuracy is 23 µrad.

Can red blood cell function assays assess response to red cell-modifying therapies?

BACKGROUND: Red blood cell (RBC)-modifying therapies have provided new opportunities for patients with sickle cell disease, although the absence of validated biomarkers of RBC function is a barrier to FDA approval and clinical adoption. Flow Adhesion (FA) and Mechanical Fragility (MF) biomarkers objectively stratify individuals with SCD into pro-adhesive vs pro-hemolytic phenotypes respectively, which may potentially help predict therapeutic responses. OBJECTIVE: A Phase 3 clinical trial to determine the effectiveness of vepoloxamer, an RBC-modifying therapy in sickle cell disease (SCD), failed to meet its primary clinical outcome. The aim of this study was to determine whether standardized flow adhesion and mechanical fragility bioassays could differentiate cellular level "responders" from "non-responders" to vepoloxamer treatment. METHODS: Standardized biomarkers of RBC function (adhesion and mechanical fragility) were utilized in this study to assess the effect of veploxamer on blood samples collected from SCD subjects and to determine whether our assays could differentiate cellular-level "responders" from "non-responders" to vepoloxamer treatment. A Wilcoxon signed-rank test was used to test for differences in adhesion in response to varying vepoloxamer treatments and a Wilcoxon Mann-Whitney test was used to assess differences in mechanical fragility, pre- and post-vepoloxamer treatment. A p-value<0.05 was considered significant. RESULTS: In this study, we report that in vitro treatment with vepoloxamer reduced adhesion by >75%in 54%of patient samples and induced changes in the membranes of sickle erythrocytes (SSRBCs) making sickle cells behave more like normal erythrocytes (AARBCs) in terms of their resistance to hemolysis. CONCLUSION: This study demonstrates that the standardized flow adhesion and mechanical fragility biomarkers described here may be useful tools to predict clinical responders to RBC-modifying therapies.

Baseline cell proliferation rates and response to UV differ in lymphoblastoid cell lines derived from healthy individuals of extreme constitution types.

Differences in human phenotypes and susceptibility to complex diseases are an outcome of genetic and environmental interactions. This is evident in diseases that progress through a common set of intermediate patho-endophenotypes. Precision medicine aims to delineate molecular players for individualized and early interventions. Functional studies of lymphoblastoid cell line (LCL) model of phenotypically well-characterized healthy individuals can help deconvolute and validate these molecular mechanisms. In this study, LCLs are developed from eight healthy individuals belonging to three extreme constitution types, deep phenotyped on the basis of Ayurveda. LCLs were characterized by karyotyping and immunophenotyping. Growth characteristics and response to UV were studied in these LCLs. Significant differences in cell proliferation rates were observed between the contrasting groups such that one type (Kapha) proliferates significantly slower than the other two (Vata, Pitta). In response to UV, one of the fast growing groups (Vata) shows higher cell death but recovers its numbers due to an inherent higher rates of proliferation. This study reveals that baseline differences in cell proliferation could be a key to understanding the survivability of cells under UV stress. Variability in baseline cellular phenotypes not only explains the cellular basis of different constitution types but can also help set priors during the design of an individualized therapy with DNA damaging agents. This is the first study of its kind that shows variability of intermediate patho-phenotypes among healthy individuals with potential implications in precision medicine.

Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase.

There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudomonas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumannii enzyme, an example of the alternative group. The reductase and oxygenase from P. aeruginosa were expressed in Escherichia coli. The reductase was purified as a stable C-terminally His-tagged yellow protein containing weakly bound FAD, and the oxygenase was purified as a stable colorless N-terminally His-tagged protein. The reductase catalyzes the reduction of FAD by NADH and releases the FADH(-) product into solution, but unlike the reductase from A. baumannii, this catalysis is not influenced by HPA. The oxygenase binds the released FADH(-) and catalyzes the oxygenation of HPA to form 3,4-dihydroxyphenylacetate, after which the FAD dissociates to be re-reduced by the reductase, a common overall pattern for two-component flavin-dependent oxygenases. With this system, it appears that interactions between the reductase and the oxygenase can facillitate the transfer of FADH(-) to the oxygenase, although they are not required. We show that the P. aeruginosa oxygenase system in complex with FADH(-) reacts with O(2) to form a quasi-stable, unusually high-extinction flavin hydroperoxide species that binds HPA and reacts to form the product. The resultant flavin hydroxide decomposes to FAD and water while still bound to the oxygenase and then releases product and FAD from the protein. Unlike the enzyme from A. baumannii, during normal catalysis involving both the reductase and oxygenase, the rate-determining step in catalysis is the dissociation of FAD from the oxygenase in a process that is independent of the concentration of HPA. Structures for the reductases and oxygenases from A. baumannii and from Thermus thermophilus (similar to the P. aeruginosa system) form a basis for interpreting the molecular origins of the differences between the two groups of flavin-dependent two-component oxygenases.

Telomere length is associated with types of chromosome 21 nondisjunction: a new insight into the maternal age effect on Down syndrome birth.

Advanced maternal age is a well-documented risk factor of chromosome 21 nondisjunction in humans, but understanding of this association at the genetic level is still limited. In particular, the state of maternal genetic age is unclear. In the present study, we estimated maternal genetic age by measuring telomere length of peripheral blood lymphocytes among age-matched mothers of children with Down syndrome (cases: N = 75) and mothers of euploid children (controls: N = 75) in an age range of 18-42 years. All blood samples were taken within 1 week of the birth of the child in both cases and controls. The telomere length estimation was performed by restriction digestion--Southern blot hybridization method. We stratified the cases on the basis of centromeric STR genotyping into maternal meiosis I (N = 48) and maternal meiosis II (N = 27) nondisjunction groups and used linear regression to compare telomere length as a function of age in the euploid, meiosis I and meiosis II groups. Our results show that all three groups have similar telomere length on average for younger mothers. As age increases, all groups show telomere loss, but that loss is largest in the meiosis II mother group and smallest in the euploid mother group with the meiosis I mother group in the middle. The regression lines for all three were statistically significantly different from each other (p < 0.001). Our results do not support the theory that younger women who have babies with Down syndrome do so because are 'genetically older' than their chronological age, but we provide the first evidence that older mothers who have babies with Down syndrome are 'genetically older' than controls, who have euploid babies at the same age. We also show for the first time that telomere length attrition may be associated in some way with meiosis I and meiosis II nondisjunction of chromosome 21 and subsequent Down syndrome births at advanced maternal age.

Choline Is an Intracellular Messenger Linking Extracellular Stimuli to IP3-Evoked Ca2+ Signals through Sigma-1 Receptors.

Sigma-1 receptors (Sig-1Rs) are integral ER membrane proteins. They bind diverse ligands, including psychoactive drugs, and regulate many signaling proteins, including the inositol 1,4,5-trisphosphate receptors (IP3Rs) that release Ca2+ from the ER. The endogenous ligands of Sig-1Rs are unknown. Phospholipase D (PLD) cleaves phosphatidylcholine to choline and phosphatidic acid (PA), with PA assumed to mediate all downstream signaling. We show that choline is also an intracellular messenger. Choline binds to Sig-1Rs, it mimics other Sig-1R agonists by potentiating Ca2+ signals evoked by IP3Rs, and it is deactivated by metabolism. Receptors, by stimulating PLC and PLD, deliver two signals to IP3Rs: IP3 activates IP3Rs, and choline potentiates their activity through Sig-1Rs. Choline is also produced at synapses by degradation of acetylcholine. Choline uptake by transporters activates Sig-1Rs and potentiates Ca2+ signals. We conclude that choline is an endogenous agonist of Sig-1Rs linking extracellular stimuli, and perhaps synaptic activity, to Ca2+ signals.

The FAD cofactor of RebC shifts to an IN conformation upon flavin reduction.

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 A movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction.

Store-Operated Ca2+ Entry in Drosophila Primary Neuronal Cultures.

Intracellular calcium signals in neurons frequently derive from the stimulation of G protein-coupled receptors (GPCR) by neurotransmitters, neuropeptides, and neurohormones. GPCR stimulation in neurons leads to generation of inositol 1,4,5-trisphosphate (IP3), which in turn activates endoplasmic reticulum (ER)-localized IP3 receptors. The IP3 receptor (IP3R) is a ligand-gated Ca2+ channel, which releases Ca2+ from ER stores. In Drosophila neurons it has been shown that depletion of ER Ca2+ store is followed by store-operated Ca2+ entry (SOCE) through STIM and Orai, the ER Ca2+ sensor and the plasma membrane Ca2+ channel respectively. The elucidation of this Ca2+ signaling pathway in neurons has in part been possible due to the ease of genetic manipulation in Drosophila, which has allowed neuron-specific knockdown of various proteins of interest. This has been followed by standardization of conditions for culturing neurons from dissected brains of the relevant genotypes, such that they could be used for robust Ca2+ measurements by imaging with standard Ca2+ indicator dyes. Protocols for measurement of IP3-mediated Ca2+ release, passive depletion of ER Ca2+ store, and SOCE in primary cultures of Drosophila neurons are described here.