Ascorbic acid and its transporter SVCT2, affect radial glia cells differentiation in postnatal stages.

Radial glia (RG) cells generate neurons and glial cells that make up the cerebral cortex. Both in rodents and humans, these stem cells remain for a specific time after birth, named late radial glia (lRG). The knowledge of lRG and molecules that may be involved in their differentiation is based on very limited data. We analyzed whether ascorbic acid (AA) and its transporter SVCT2, are involved in lRG cells differentiation. We demonstrated that lRG cells are highly present between the first and fourth postnatal days. Anatomical characterization of lRG cells, revealed that lRG cells maintained their bipolar morphology and stem-like character. When lRG cells were labeled with adenovirus-eGFP at 1 postnatal day, we detected that some cells display an obvious migratory neuronal phenotype, suggesting that lRG cells continue generating neurons postnatally. Moreover, we demonstrated that SVCT2 was apically polarized in lRG cells. In vitro studies using the transgenic mice SVCT2+/- and SVCT2tg (SVCT2-overexpressing mouse), showed that decreased SVCT2 levels led to accelerated differentiation into astrocytes, whereas both AA treatment and elevated SVCT2 expression maintain the lRG cells in an undifferentiated state. In vivo overexpression of SVCT2 in lRG cells generated cells with a rounded morphology that were migratory and positive for proliferation and neuronal markers. We also examined mediators that can be involved in AA/SVCT2-modulated signaling pathways, determining that GSK3-ß through AKT, mTORC2, and PDK1 is active in brains with high levels of SVCT2/AA. Our data provide new insights into the role of AA and SVCT2 in late RG cells.

Ascorbate insufficiency disrupts glutamatergic signaling and alters electroencephalogram phenotypes in a mouse model of Alzheimer's disease.

Clinical studies have reported that increased epileptiform and subclinical epileptiform activity can be detected in many patients with an Alzheimer's disease (AD) diagnosis using electroencephalogram (EEG) and this may correlate with poorer cognition. Ascorbate may have a specific role as a neuromodulator in AD as it is released concomitantly with glutamate reuptake following excitatory neurotransmission. Insufficiency may therefore result in an exacerbated excitatory/inhibitory imbalance in neuronal signaling. Using a mouse model of AD that requires dietary ascorbate (Gulo-/-APPswe/PSEN1dE9), EEG was recorded at baseline and during 4 weeks of ascorbate depletion in young (5-month-old) and aged (20-month-old) animals. Data were scored for changes in quantity of spike trains, individual spikes, sleep-wake rhythms, sleep fragmentation, and brainwave power bands during light periods each week. We found an early increase in neuronal spike discharges with age and following ascorbate depletion in AD model mice and not controls, which did not correlate with brain amyloid load. Our data also show more sleep fragmentation with age and with ascorbate depletion. Additionally, changes in brain wave activity were observed within different vigilance states in both young and aged mice, where Gulo-/-APPswe/PSEN1dE9 mice had shifts towards higher frequency bands (alpha, beta, and gamma) and ascorbate depletion resulted in shifts towards lower frequency bands (delta and theta). Microarray data supported ascorbate insufficiency altering glutamatergic transmission through the decreased expression of glutamate related genes, however no changes in protein expression of glutamate reuptake transporters were observed. These data suggest that maintaining optimal brain ascorbate levels may support normal brain electrical activity and sleep patterns, particularly in AD patient populations where disruptions are observed.

Knockdown of microglial iron import gene, Slc11a2, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease.

BACKGROUND: Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. METHODS: In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-ß-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1Cre-ERT2;Slc11a2flfl;APP/PS1+or - mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted. RESULTS: DMT1 inhibition in vitro robustly decreased Aß-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2KD APP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2KD cells from APP/PS1 females also exhibited decreased expression of markers associated with subsets of disease-associated microglia (DAMs), such as Apoe, Ctsb, Ly9, Csf1, and Hif1α. CONCLUSIONS: This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

Modulation of microglia activation by the ascorbic acid transporter SVCT2.

Neuroinflammation is a major characteristic of pathology in several neurodegenerative diseases. Microglia, the brain's resident myeloid cells, shift between activation states under neuroinflammatory conditions, both responding to, but also driving damage in the brain. Vitamin C (ascorbate) is an essential antioxidant for central nervous system function that may have a specific role in the neuroinflammatory response. Uptake of ascorbate throughout the central nervous system is facilitated by the sodium-dependent vitamin C transporter 2 (SVCT2). SVCT2 transports the reduced form of ascorbate into neurons and microglia, however the contribution of altered SVCT2 expression to the neuroinflammatory response in microglia is not well understood. In this study we demonstrate that SVCT2 expression modifies microglial response, as shown through changes in cell morphology and mRNA expression, following a mild traumatic brain injury (mTBI) in mice with decreased or increased expression of SVCT2. Results were supported by in vitro studies in an immortalized microglial cell line and in primary microglial cultures derived from SVCT2-heterozygous and transgenic animals. Overall, this work demonstrates the importance of SVCT2 and ascorbate in modulating the microglial response to mTBI and suggests a potential role for both in response to neuroinflammatory challenges.

Microglial-specific knockdown of iron import gene, Slc11a2, blunts LPS-induced neuroinflammatory responses in a sex-specific manner.

Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1Cre-ERT2 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1ß, and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease.

The Pathophysiology and Biomarkers of Delirium.

Delirium is a major disturbance in the mental state characterized by fluctuations in arousal, deficits in attention, distorted perception, and disruptions in memory and cognitive processing. Delirium affects approximately 18% to 25% of hospital inpatients, with even higher rates observed during critical illness. To develop therapies to shorten the duration and limit the adverse effects of delirium, it is important to understand the mechanisms underlying its presentation. Neuroimaging modalities such as magnetic resonance imaging (MRI), positron emission tomography, functional MRI, and near-infrared spectroscopy point to global atrophy, white matter changes, and disruptions in cerebral blood flow, oxygenation, metabolism, and connectivity as key correlates of delirium pathogenesis. Electroencephalography demonstrates generalized slowing of normal background activity, with pathologic decreases in variability of oscillatory patterns and disruptions in functional connectivity among specific brain regions. Elevated serum biomarkers of inflammation, including interleukin-6, C-reactive protein, and S100B, suggest a role of dysregulated inflammatory processes and cellular metabolism, particularly in perioperative and sepsis-related delirium. Emerging animal models that can mimic delirium-like clinical states will reveal further insights into delirium pathophysiology. The combination of clinical and basic science methods of exploring delirium shows great promise in elucidating its underlying mechanisms and revealing potential therapeutic targets.

Altered EEG, disrupted hippocampal long-term potentiation and neurobehavioral deficits implicate a delirium-like state in a mouse model of sepsis.

Sepsis and systemic inflammation are often accompanied by severe encephalopathy, sleep disruption and delirium that strongly correlate with poor clinical outcomes including long-term cognitive deficits. The cardinal manifestations of delirium are fluctuating altered mental status and inattention, identified in critically ill patients by interactive bedside assessment. The lack of analogous assessments in mouse models or clear biomarkers is a challenge to preclinical studies of delirium. In this study, we utilized concurrent measures of telemetric EEG recordings and neurobehavioral tasks in mice to characterize inattention and persistent cognitive deficits following polymicrobial sepsis. During the 24-hour critical illness period for the mice, slow-wave EEG dominance, sleep disruption, and hypersensitivity to auditory stimuli in neurobehavioral tasks resembled clinical observations in delirious patients in which alterations in similar outcome measurements, although measured differently in mice and humans, are reported. Mice were tested for nest building ability 7 days after sepsis induction, when sickness behaviors and spontaneous activity had returned to baseline. Animals that showed persistent deficits determined by poor nest building at 7 days also exhibited molecular changes in hippocampal long-term potentiation compared to mice that returned to baseline cognitive performance. Together, these behavioral and electrophysiological biomarkers offer a robust mouse model with which to further probe molecular pathways underlying brain and behavioral changes during and after acute illness such as sepsis.

Serotonin Transporter Ala276 Mouse: Novel Model to Assess the Neurochemical and Behavioral Impact of Thr276 Phosphorylation In Vivo.

The serotonin (5-HT) transporter (SERT) is a key regulator of 5-HT signaling and is a major target for antidepressants and psychostimulants. Human SERT coding variants have been identified in subjects with obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD) that impact transporter phosphorylation, cell surface trafficking and/or conformational dynamics. Prior to an initial description of a novel mouse line expressing the non-phosphorylatable SERT substitution Thr276Ala, we review efforts made to elucidate the structure and conformational dynamics of SERT with a focus on research implicating phosphorylation at Thr276 as a determinant of SERT conformational dynamics. Using the high-resolution structure of human SERT in inward- and outward-open conformations, we explore the conformation dependence of SERT Thr276 exposure, with results suggesting that phosphorylation is likely restricted to an inward-open conformation, consistent with prior biochemical studies. Assessment of genotypes from SERT/Ala276 heterozygous matings revealed a deviation from Mendelian expectations, with reduced numbers of Ala276 offspring, though no genotype differences were seen in growth or physical appearance. Similarly, no genotype differences were evident in midbrain or hippocampal 5-HT levels, midbrain and hippocampal SERT mRNA or midbrain protein levels, nor in midbrain synaptosomal 5-HT uptake kinetics. Behaviorally, SERT Ala276 homozygotes appeared normal in measures of anxiety and antidepressant-sensitive stress coping behavior. However, these mice displayed sex-dependent alterations in repetitive and social interactions, consistent with circuit-dependent requirements for Thr276 phosphorylation underlying these behaviors. Our findings indicate the utility of SERT Ala276 mice in evaluation of developmental, functional and behavioral consequences of regulatory SERT phosphorylation in vivo.

Targeted and Untargeted Mass Spectrometry Reveals the Impact of High-Fat Diet on Peripheral Amino Acid Regulation in a Mouse Model of Alzheimer's Disease.

Recent research regarding amino acid metabolism has shown that there may be a link between obesity and Alzheimer's disease (AD). This work reports a metabolomics study using targeted and untargeted mass spectrometry-based metabolomic strategies to investigate this link. Targeted hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry and untargeted reversed-phase liquid chromatography-high resolution tandem mass spectrometry assays were developed to analyze the metabolic changes that occur in AD and obesity. APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice (to represent familial or early-onset AD) and wild-type littermate controls were fed either a high-fat diet (HFD, 60% kcal from lard) or a low-fat diet (LFD, 10% kcal from lard) from 2 months of age or a reversal diet (HFD, followed by LFD from 9.5 months). For targeted analyses, we applied the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) LC-MS C62-A document and the U.S. Food and Drug Administration (FDA) bioanalytical method validation guidance for industry to evaluate the figures of merit of the assays. Our targeted and untargeted metabolomics results suggest that numerous peripheral pathways, specifically amino acid metabolism and fatty acid metabolism, were significantly affected by AD and diet. Multiple amino acids (including alanine, glutamic acid, leucine, isoleucine, and phenylalanine), carnitines, and members of the fatty acid oxidation pathway were significantly increased in APP/PSEN1 mice on HFD compared to those on LFD. More substantial effects and changes were observed in the APP/PSEN1 mice than in the WT mice, suggesting that they were more sensitive to an HFD. These dysregulated peripheral pathways include numerous amino acid pathways and fatty acid beta oxidation and suggest that obesity combined with AD further enhances cognitive impairment, possibly through aggravated mitochondrial dysfunction. Furthermore, partial reversibility of many altered pathways was observed, which highlights that diet change can mitigate the metabolic effects of AD. The same trends in individual amino acids were observed in both strategies, highlighting the biological validity of the results.

Brain manganese and the balance between essential roles and neurotoxicity.

Manganese (Mn) is an essential micronutrient required for the normal development of many organs, including the brain. Although its roles as a cofactor in several enzymes and in maintaining optimal physiology are well-known, the overall biological functions of Mn are rather poorly understood. Alterations in body Mn status are associated with altered neuronal physiology and cognition in humans, and either overexposure or (more rarely) insufficiency can cause neurological dysfunction. The resultant balancing act can be viewed as a hormetic U-shaped relationship for biological Mn status and optimal brain health, with changes in the brain leading to physiological effects throughout the body and vice versa. This review discusses Mn homeostasis, biomarkers, molecular mechanisms of cellular transport, and neuropathological changes associated with disruptions of Mn homeostasis, especially in its excess, and identifies gaps in our understanding of the molecular and biochemical mechanisms underlying Mn homeostasis and neurotoxicity.

Ascorbate deficiency decreases dopamine release in gulo-/- and APP/PSEN1 mice.

Dopamine (DA) has important roles in learning, memory, and motivational processes and is highly susceptible to oxidation. In addition to dementia, Alzheimer's disease (AD) patients frequently exhibit decreased motivation, anhedonia, and sleep disorders, suggesting deficits in dopaminergic neurotransmission. Vitamin C (ascorbate, ASC) is a critical antioxidant in the brain and is often depleted in AD patients as a result of disease-related oxidative stress and dietary deficiencies. To probe the effects of ASC deficiency and AD pathology on the DAergic system, gulo-/- mice, which like humans depend on dietary ASC to maintain adequate tissue levels, were crossed with APP/PSEN1 mice and provided sufficient or depleted ASC supplementation from weaning until 12 months of age. Ex vivo fast-scan cyclic voltammetry showed that chronic ASC depletion and APP/PSEN1 genotype both independently decreased dopamine release in the nucleus accumbens, a hub for motivational behavior and reward, while DA clearance was similar across all groups. In striatal tissue containing nucleus accumbens, low ASC treatment led to decreased levels of DA and its metabolites 3,4-dihydroxyohenyl-acetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA). Decreased enzyme activity observed through lower pTH/TH ratio was driven by a cumulative effect of ASC depletion and APP/PSEN1 genotype. Together the data show that deficits in dopaminergic neurotransmission resulting from age and disease status are magnified in conditions of low ASC which decrease DA availability during synaptic transmission. Such deficits may contribute to the non-cognitive behavioral changes observed in AD including decreased motivation, anhedonia, and sleep disorders.

Cell-free hemoglobin augments acute kidney injury during experimental sepsis.

Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.

Behavioral analyses of animal models of intellectual and developmental disabilities.

Intellectual and developmental disabilities (IDDs) are a common group of disorders that frequently share overlapping symptoms, including cognitive deficits, altered attention, seizures, impaired social interactions, and anxiety. The causes of these disorders are varied ranging from early prenatal/postnatal insults to genetic variants that either cause or are associated with an increased likelihood of an IDD. As many of the symptoms observed in individuals with IDDs are a manifestation of altered nervous system function resulting in altered behaviors, it should not be surprising that the field is very dependent upon in vivo model systems. This special issue of Neurobiology of Learning and Memory is focused on the methods and approaches that are being used to model and understand these disorders in mammals. While surveys by the Pew Foundation continue to find a high degree of confidence/trust in scientists by the public, several recent studies have documented issues with reproducibility in scientific publications. This special issue includes both primary research articles and review articles in which careful attention has been made to transparently report methods and use rigorous approaches to ensure reproducibility. Although there have been and will continue to be remarkable advances for treatment of subset of IDDs, it is clear that this field is still in its early stages. There is no doubt that the strategies being used to model IDDs will continue to evolve. We hope this special issue will support this evolution so that we can maintain the trust of the public and elected officials, and continue developing evidence-based approaches to new therapeutics.

Reversal of high fat diet-induced obesity improves glucose tolerance, inflammatory response, ß-amyloid accumulation and cognitive decline in the APP/PSEN1 mouse model of Alzheimer's disease.

This study assessed the extent to which high fat diet (HFD)-induced ß-amyloid accumulation and cognitive decline in APP/PSEN1 mice are reversible through control of fat intake. Ten months of HFD (60% calories from fat) led to significant deficits in a 2-trial Y maze task, and nest building assay, and decreased voluntary locomotor activity. The HFD induced an inflammatory response, indicated by increased expression of several inflammatory markers. Substituting a low fat diet led to pronounced weight loss and correction of glucose intolerance, decreases in the inflammatory response, and improved performance on behavioral tasks in both wild-type and APP/PSEN1 transgenic mice. Insoluble ß-amyloid levels, and extent of tau phosphorylation were also lower following dietary reversal in APP/PSEN1 mice compared to high fat-fed animals, indicating that the inflammatory response may have contributed to key pathogenic pathways in the Alzheimer's disease model. The data suggest that weight loss can be a vital strategy for cognitive protection, but also highlight potential mechanisms for intervention when sustained weight loss is not possible.

Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Differential proteomic and behavioral effects of long-term voluntary exercise in wild-type and APP-overexpressing transgenics.

Physical exercise may provide protection against the cognitive decline and neuropathology associated with Alzheimer's disease, although the mechanisms are not clear. In the present study, APP/PSEN1 double-transgenic and wild-type mice were allowed unlimited voluntary exercise for 7months. Consistent with previous reports, wheel-running improved cognition in the double-transgenic mice. Interestingly, the average daily distance run was strongly correlated with spatial memory in the water maze in wild-type mice (r(2)=.959), but uncorrelated in transgenics (r(2)=.013). Proteomics analysis showed that sedentary transgenic mice differed significantly from sedentary wild-types with respect to proteins involved in synaptic transmission, cytoskeletal regulation, and neurogenesis. When given an opportunity to exercise, the transgenics' deficiencies in cytoskeletal regulation and neurogenesis largely normalized, but abnormal synaptic proteins did not change. In contrast, exercise enhanced proteins associated with cytoskeletal regulation, oxidative phosphorylation, and synaptic transmission in wild-type mice. Soluble and insoluble Aß40 and Aß42 levels were significantly decreased in both cortex and hippocampus of active transgenics, suggesting that this may have played a role in the cognitive improvement in APP/PSEN1 mice. ß-secretase was significantly reduced in active APP/PSEN1 mice compared to sedentary controls, suggesting a mechanism for reduced Aß. Taken together, these data illustrate that exercise improves memory in wild-type and APP-overexpressing mice in fundamentally different ways.

Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease.

Background: Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. Methods: In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-ß-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1 Cre - ERT2 ;Slc11a2 flfl;APP/PS1 + or - mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b + microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted. Results: DMT1 inhibition in vitro robustly decreased Aß-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2 KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2 KD APP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2 KD cells from APP/PS1 females also exhibited decreased expression of markers associated with disease-associated microglia (DAMs), such as Apoe, Ctsb, Csf1, and Hif1α. Conclusions: This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2 KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

Systemic inflammation and delirium during critical illness.

PURPOSE: The purpose of this study was to determine associations between markers of inflammation and endogenous anticoagulant activity with delirium and coma during critical illness. METHODS: In this prospective cohort study, we enrolled adults with respiratory failure and/or shock treated in medical or surgical intensive care units (ICUs) at 5 centers. Twice per day in the ICU, and daily thereafter, we assessed mental status using the Richmond Agitation Sedation Scale (RASS) and the Confusion Assessment Method-Intensive Care Unit (CAM-ICU). We collected blood samples on study days 1, 3, and 5, measuring levels of C-reactive protein (CRP), interferon gamma (IFN-γ), interleukin (IL)-1 beta (IL-1ß), IL-6, IL-8, IL-10, IL-12, matrix metalloproteinase-9 (MMP-9), tumor necrosis factor-alpha (TNF-α), tumor necrosis factor receptor 1 (TNFR1), and protein C using validated protocols. We used multinomial logistic regression to analyze associations between biomarkers and the odds of delirium or coma versus normal mental status the following day, adjusting for age, sepsis, Sequential Organ Failure Assessment (SOFA), study day, corticosteroids, and sedatives. RESULTS: Among 991 participants with a median age (interquartile range, IQR) of 62 [53-72] years and enrollment SOFA of 9 [7-11], higher concentrations of IL-6 (odds ratio [OR] [95% CI]: 1.8 [1.4-2.3]), IL-8 (1.3 [1.1-1.5]), IL-10 (1.5 [1.2-1.8]), TNF-α (1.2 [1.0-1.4]), and TNFR1 (1.3 [1.1-1.6]) and lower concentrations of protein C (0.7 [0.6-0.8])) were associated with delirium the following day. Higher concentrations of CRP (1.4 [1.1-1.7]), IFN-γ (1.3 [1.1-1.5]), IL-6 (2.3 [1.8-3.0]), IL-8 (1.8 [1.4-2.3]), and IL-10 (1.5 [1.2-2.0]) and lower concentrations of protein C (0.6 [0.5-0.8]) were associated with coma the following day. IL-1ß, IL-12, and MMP-9 were not associated with mental status. CONCLUSION: Markers of inflammation and possibly endogenous anticoagulant activity are associated with delirium and coma during critical illness.

Behavioral and monoamine changes following severe vitamin C deficiency.

Severe vitamin C deficiency (ascorbic acid; AA) was induced in gulo-/- mice incapable of synthesizing their own AA. A number of behavioral measures were studied before and during the deprivation period, including a scorbutic period, during which weight loss was observed in the mice. Mice were then resuscitated with AA supplements. During the scorbutic period, gulo-/- mice showed decreased voluntary locomotor activity, diminished physical strength, and increased preference for a highly palatable sucrose reward. These behaviors all returned to control levels following resuscitation. Altered trial times in subordinate mice in the tube test for social dominance in the AA-deprived mice persisted following resuscitation and may signify a depressive-like behavior in these mice. Biochemical analyses were undertaken following a second deprivation period. AA deficiency was accompanied by decreased blood glucose levels, oxidative damage to lipids and proteins in the cortex, and decreases in dopamine and serotonin metabolites in both the cortex and striatum. Given the reasonably high proportions of the population that do not consume sufficient AA in the diet, these data have important implications for physical and psychological function in the general population.

Combined vitamin C and E deficiency induces motor defects in gulo(-/-)/SVCT2(+/-) mice.

OBJECTIVES: Key antioxidants, vitamins C and E, are necessary for normal brain development and neuronal function. In this study, we depleted both of these vitamins in two mouse models to determine if oxidative stress due to combined vitamin C and E dietary deficiency altered their neurological phenotype. The first model lacked both alleles for the Gulonolactone oxidase gene (Gulo(-/-)) and therefore was unable synthesize vitamin C. To obtain an additional cellular deficiency of vitamin C, the second model also lacked one allele for the cellular vitamin C transporter gene (Gulo(-/-)/SVCT2(+/-)). METHODS: The experimental treatment was 16 weeks of vitamin E deprivation followed by 3 weeks of vitamin C deprivation. Mice were assessed for motor coordination deficits, vitamin levels, and oxidative stress biomarkers. RESULTS: In the first model, defects in motor performance were more apparent in both vitamin C-deficient groups (VE+VC-, VE-VC-) compared to vitamin C-supplemented groups (VE+VC+, VE-VC+) regardless of vitamin E level. Analysis of brain cortex and liver confirmed decreases of at least 80% for each vitamin in mice on deficient diets. Vitamin E deficiency doubled oxidative stress biomarkers (F2-isoprostanes and malondialdehyde). In the second model, Gulo(-/-)/SVCT2(+/-) mice on the doubly deficient diets showed deficits in locomotor activity, Rota-rod performance, and other motor tasks, with no concomitant change in anxiety or spatial memory. DISCUSSION: Vitamin E deficiency alone caused a modest oxidative stress in brain that did not affect motor performance. Adding a cellular deficit in vitamin C to dietary deprivation of both vitamins significantly impaired motor performance.