Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Bridging the gap: improving correspondence between low-field and high-field magnetic resonance images in young people.

Background: Portable low-field-strength magnetic resonance imaging (MRI) systems represent a promising alternative to traditional high-field-strength systems with the potential to make MR technology available at scale in low-resource settings. However, lower image quality and resolution may limit the research and clinical potential of these devices. We tested two super-resolution methods to enhance image quality in a low-field MR system and compared their correspondence with images acquired from a high-field system in a sample of young people. Methods: T1- and T2-weighted structural MR images were obtained from a low-field (64mT) Hyperfine and high-field (3T) Siemens system in N = 70 individuals (mean age = 20.39 years, range 9-26 years). We tested two super-resolution approaches to improve image correspondence between images acquired at high- and low-field: (1) processing via a convolutional neural network ('SynthSR'), and (2) multi-orientation image averaging. We extracted brain region volumes, cortical thickness, and cortical surface area estimates. We used Pearson correlations to test the correspondence between these measures, and Steiger Z tests to compare the difference in correspondence between standard imaging and super-resolution approaches. Results: Single pairs of T1- and T2-weighted images acquired at low field showed high correspondence to high-field-strength images for estimates of total intracranial volume, surface area cortical volume, subcortical volume, and total brain volume (r range = 0.60-0.88). Correspondence was lower for cerebral white matter volume (r = 0.32, p = 0.007, q = 0.009) and non-significant for mean cortical thickness (r = -0.05, p = 0.664, q = 0.664). Processing images with SynthSR yielded significant improvements in correspondence for total brain volume, white matter volume, total surface area, subcortical volume, cortical volume, and total intracranial volume (r range = 0.85-0.97), with the exception of global mean cortical thickness (r = 0.14). An alternative multi-orientation image averaging approach improved correspondence for cerebral white matter and total brain volume. Processing with SynthSR also significantly improved correspondence across widespread regions for estimates of cortical volume, surface area and subcortical volume, as well as within isolated prefrontal and temporal regions for estimates of cortical thickness. Conclusion: Applying super-resolution approaches to low-field imaging improves regional brain volume and surface area accuracy in young people. Finer-scale brain measurements, such as cortical thickness, remain challenging with the limited resolution of low-field systems.

Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

Differential Effect of Chronic Morphine on Neuronal Degeneration in Male vs. Female Mice.

Opioid abuse in the United States has been increasing at an alarming rate over the past 20 years. Sex differences are documented for the rates of opioid-related overdoses, abuse patterns, and drug-induced physiological effects. In our previous study, we demonstrated that chronic oxycodone administration in young female rats is associated with neurodegeneration in the brain. Males and females are susceptible to neurodegenerative diseases via differing mechanisms. To investigate whether opioid exposure affects males and females differently, we treated young mice with chronic morphine. We observed that females had stronger antinociceptive responses to acute morphine and showed a delayed development of tolerance. Males had a higher basal Bax level in the brain that correlated with a higher number of apoptotic cells. Morphine increased Bax levels in both males and females without affecting the numbers of apoptotic cells. Morphine increased activated caspase 3 in axons and increased the MBP level in plasma only in females, suggesting a demyelination process. Our data suggest that males are protected from demyelination by having a higher basal BDNF level. Altogether, our results suggest that males and females have different molecular signaling underlying their patterns in the development of morphine tolerance and drug-induced neuronal degeneration.

Associations of social and cognitive-behavioral variables with disinhibited eating and anxiety: An ecological momentary assessment study.

OBJECTIVE: Among adolescents, disinhibited eating and anxiety commonly co-occur. Precision intervention approaches targeting unique mechanistic vulnerabilities that contribute to disinhibited eating and anxiety may therefore be helpful. However, the effectiveness of such interventions hinges on knowledge of between- and within-person associations related to disinhibited eating, anxiety, and related processes. METHOD: A sample of 39 adolescent females (12-17 years) with elevated anxiety and above-average weight (BMI %ile ≥ 75th) completed measures of theoretically driven social and cognitive-behavioral variables, disinhibited eating, and anxiety via ecological momentary assessment over 7 days. Data were analyzed using mixed-effects models. RESULTS: Between-person differences in social stressors were linked to emotional eating, eating in the absence of hunger, and anxiety, whereas between-person differences in negative thoughts were associated with all disinhibited eating variables and anxiety. Between-person differences in avoidance were not related to any outcome. Additionally, between-person differences in social stressors and negative thoughts-as well as within-person deviations (from person-average levels) of social stressors, negative thoughts, and avoidance-were associated with anxiety. In turn, between-person differences in anxiety predicted eating in the absence of hunger and emotional eating, and within-person deviations in anxiety were associated with emotional eating at any given time point. DISCUSSION: Findings support elements of both the interpersonal and cognitive-behavioral models of disinhibited eating. Differential trigger effects on anxiety, both at the between- and within-person levels, and significant associations between anxiety and all eating-related outcomes, highlight the potential utility of interventions targeting individual differences in sensitivity to anxiety triggers. PUBLIC SIGNIFICANCE: Findings provide support for the interpersonal and cognitive-behavioral models of disinhibited eating, highlighting anxiety as a salient vulnerability and potential mechanistic factor underlying disinhibited eating. Social, cognitive, and behavioral variables were differentially related to anxiety across participants, suggesting potential for future intervention tailoring and intervention selection based on adolescents' sensitivity to anxiety as a trigger for disinhibited eating behavior.

PTP1B mediates the inhibitory effect of MFGE8 on insulin signaling through the ß5 integrin.

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.

A comparative evaluation of the strengths and potential caveats of the microglial inducible CreER mouse models.

The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1YFP-CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, and Tmem119CreER), focusing on (1) recombination specificity, (2) leakiness (the degree of tamoxifen-independent recombination in microglia and other cells), (3) the efficiency of tamoxifen-induced recombination, (4) extraneural recombination (the degree of recombination in cells outside of the CNS, particularly myelo/monocyte lineages), and (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines, which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

Epigenetic weapons in plant-herbivore interactions: Sulforaphane disrupts histone deacetylases, gene expression, and larval development in Spodoptera exigua while the specialist feeder Trichoplusia ni is largely resistant to these effects.

Cruciferous plants produce sulforaphane (SFN), an inhibitor of nuclear histone deacetylases (HDACs). In humans and other mammals, the consumption of SFN alters enzyme activities, DNA-histone binding, and gene expression within minutes. However, the ability of SFN to act as an HDAC inhibitor in nature, disrupting the epigenetic machinery of insects feeding on these plants, has not been explored. Here, we demonstrate that SFN consumed in the diet inhibits the activity of HDAC enzymes and slows the development of the generalist grazer Spodoptera exigua, in a dose-dependent fashion. After consuming SFN for seven days, the activities of HDAC enzymes in S. exigua were reduced by 50%. Similarly, larval mass was reduced by 50% and pupation was delayed by 2-5 days, with no additional mortality. Similar results were obtained when SFN was applied topically to eggs. RNA-seq analyses confirm that SFN altered the expression of thousands of genes in S. exigua. Genes associated with energy conversion pathways were significantly downregulated while those encoding for ribosomal proteins were dramatically upregulated in response to the consumption of SFN. In contrast, the co-evolved specialist feeder Trichoplusia ni was not negatively impacted by SFN, whether it was consumed in their diet at natural concentrations or applied topically to eggs. The activities of HDAC enzymes were not inhibited and development was not disrupted. In fact, SFN exposure sometimes accelerated T. ni development. RNA-seq analyses revealed that the consumption of SFN alters gene expression in T. ni in similar ways, but to a lesser degree, compared to S. exigua. This apparent resistance of T. ni can be overwhelmed by unnaturally high levels of SFN or by exposure to more powerful pharmaceutical HDAC inhibitors. These results demonstrate that dietary SFN interferes with the epigenetic machinery of insects, supporting the hypothesis that plant-derived HDAC inhibitors serve as "epigenetic weapons" against herbivores.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

Photoinduced bidirectional switching in lipid membranes containing azobenzene glycolipids.

Following the reaction of biological membranes to external stimuli reveals fundamental insights into cellular function. Here, self-assembled lipid monolayers act as model membranes containing photoswitchable azobenzene glycolipids for investigating structural response during isomerization by combining Langmuir isotherms with X-ray scattering. Controlled in-situ trans/cis photoswitching of the azobenzene N = N double bond alters the DPPC monolayer structure, causing reproducible changes in surface pressure and layer thickness, indicating monolayer reorientation. Interestingly, for monolayers containing azobenzene glycolipids, along with the expected DPPC phase transitions an additional discontinuity is observed. The associated reorintation represents a crossover point, with the surface pressure and layer thickness changing in opposite directions above and below. This is evidence that the azobenzene glycolipids themselves change orientation within the monolayer. Such behaviour suggests that azobenzene glycolipids can act as a bidirectional switch in DPPC monolayers providing a tool to investigate membrane structure-function relationships in depth.

Loss of control eating in relation to blood pressure among adolescent girls with elevated anxiety at-risk for excess weight gain.

Loss of control (LOC)-eating, excess weight, and anxiety are robustly linked, and are independently associated with markers of poorer cardiometabolic health, including hypertension. However, no study has examined whether frequency of LOC-eating episodes among youth with anxiety symptoms and elevated weight status may confer increased risk for hypertension. We examined the relationship between LOC-eating frequency and blood pressure among 39 adolescent girls (14.9 ± 1.8 years; body mass index [BMI] = 29.9 ± 5.6; 61.5 % White; 20.5 % African American/Black; 5 % Multiple Races; 2.5 % Asian; 12.8 % Hispanic/Latino; 30.8 % with reported LOC-eating) with elevated anxiety and above average BMI who enrolled in a clinical trial aimed at preventing excess weight gain. LOC-eating over the past three months was assessed via clinical interview, and blood pressure (systolic and diastolic) was measured with an automatic blood pressure monitor. Adjusting for age, fat mass, and height, LOC-eating episode frequency was significantly, positively associated with diastolic blood pressure (ß = 0.38, p = 0.02), but not with systolic blood pressure (ß = 0.13, p = 0.41). Replication studies, with larger sample sizes, participants of varying weight-strata, and prospective data are required to elucidate the relationship between LOC-eating and cardiovascular functioning in youth with elevated anxiety.

Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models.

The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, Tmem119CreER), focusing on (1) recombination specificity; (2) leakiness - degree of non-tamoxifen recombination in microglia and other cells; (3) efficiency of tamoxifen-induced recombination; (4) extra-neural recombination -the degree of recombination in cells outside the CNS, particularly myelo/monocyte lineages (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

Trends in Drug Tests among Children: A 22-Year Retrospective Analysis.

There are several pathophysiological outcomes associated with substance abuse including metabolic disbalance, neurodegeneration, and disordered redox. Drug use in pregnant women is a topic of great concern due to developmental harm which may occur during gestation and the associated complications in the neonate after delivery. We sought to determine what the trajectory of drug use is like in children aged 0-4 years and mothers of neonates. Urine drug screen (UDS) results were obtained of our target demographic during 1998-2011 and 2012-2019 from LSU Health Sciences Center in Shreveport (LSUHSC-S). Statistical analysis was performed using R software. We observed an increase in cannabinoid-positive UDS results in both Caucasian (CC) and African American (AA) groups between 1998-2011 and 2012-2019 periods. Cocaine-positive UDS results decreased in both cohorts. CC children had higher UDS positive results for opiates, benzodiazepines, and amphetamines, while AA children had a higher percentage for illicit drugs such as cannabinoids and cocaine. Neonate's mothers had similar UDS trends to that in children during 2012-2019. Overall, while percentage of positive UDS results for both AA and CC 0-4 year old children started to decline for opiate, benzodiazepine, and cocaine during 2012-2019, cannabinoid- and amphetamine (CC)-positive UDS steadily increased. These results suggest a shift in the type of drug use by mothers from opiates, benzodiazepines, and cocaine to cannabinoids and/or amphetamines. We also observed that 18-year-old females who tested positive for opiates, benzodiazepine, or cocaine had higher than average chances of testing positive for cannabinoids later in life.

Learning metric volume estimation of fruits and vegetables from short monocular video sequences.

We present a novel approach for extracting metric volume information of fruits and vegetables from short monocular video sequences and associated inertial data recorded with a hand-held smartphone. Estimated segmentation masks from a pre-trained object detector are fused with the predicted change in relative pose obtained from the inertial data to predict the class and volume of the objects of interest. Our approach works with simple RGB video frames and inertial data which are readily available from modern smartphones. It does not require reference objects of known size in the video frames. Using a balanced validation dataset, we achieve a classification accuracy of 95% and a mean absolute percentage error for the volume prediction of 16% on untrained objects, which is comparable to state-of-the-art results requiring more elaborated data recording setups. A very accurate estimation of the model uncertainty is achieved through ensembling and the use of Gaussian negative log-likelihood loss. The dataset used in our experiments including ground-truth volume information is available at https://sst.aau.at/cns/datasets.

SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

Advice on describing Bayesian analysis of neutron and X-ray reflectometry.

As a result of the availability of modern software and hardware, Bayesian analysis is becoming more popular in neutron and X-ray reflectometry analysis. The understandability and replicability of these analyses may be harmed by inconsistencies in how the probability distributions central to Bayesian methods are represented in the literature. Herein advice is provided on how to report the results of Bayesian analysis as applied to neutron and X-ray reflectometry. This includes the clear reporting of initial starting conditions, the prior probabilities, the results of any analysis and the posterior probabilities that are the Bayesian equivalent of the error bar, to enable replicability and improve understanding. It is believed that this advice, grounded in the authors' experience working in the field, will enable greater analytical reproducibility in the work of the reflectometry community, and improve the quality and usability of results.