Maternal diabetes is associated with increased systemic inflammation and has been linked to adverse neonatal outcomes, including developmental delays that persist into early childhood. In this study we sought to characterize and compare the maternal levels and fetal cord-blood levels of the inflammatory markers C-reactive protein (CRP) and IL-6, as well as the neurotrophin brain-derived neurotrophic factor (BDNF) between mothers with pre-gestational Type-1 diabetes (T1DM) or Type-2 diabetes (T2DM), and non-diabetic controls (nonDM).A prospective cohort design was employed, analyzing biomarker concentrations during the third trimester in 98 pregnant women ages 18-40 years of age including 16 participants with T1DM, 49 participants with T2DM and 33 control participants matched for gestational age and body mass index (BMI) to control for confounding factors such as obesity. Plasma samples were collected at 28-30 weeks, 34-36 weeks, delivery, and from cord blood. The biomarkers CRP, IL-6, and BDNF were measured using standardized assays, and concentrations were compared among groups using ANOVA.In T2DM mothers, CRP levels were 2x higher in the third trimester as compared to nonDM controls. In T1DM mothers, IL6 levels were 3x lower than nonDM controls and 3.4x lower than T2DM. While not reaching statistical significance, cord-blood levels of IL6 were higher in T2DMs than other groups (p = 0.052). When examining BDNF levels, no differences were observed between groups.This study emphasizes the importance of addressing inflammation-related risks in pregnancies affected by diabetes. Targeted interventions may mitigate adverse neonatal outcomes and improve health trajectories. Future research should explore direct pathways linking maternal inflammation to fetal neural function to inform clinical strategies.

BACKGROUND:Studies implicating dysfunctional mitochondrial respiration in metabolic tissues in the development of insulin resistance in obesity have only included adults. Peripheral blood mononuclear cells (PBMCs) and platelets have been found to reflect systemic mitochondrial fitness and bioenergetic health. We sought to identify bioenergetic differences in PBMCs and platelets from children with obesity and insulin resistance and determine associations with whole-body metabolism and/or biomarkers of metabolic health and inflammation. METHODS:We stratified prepubertal children (ages 5-10 years) into three groups: normal weight insulin sensitive (N-IS; n = 20), overweight/obese insulin sensitive (O-IS; n = 28) and overweight/obese insulin resistant (O-IR; n = 17). We measured oxygen consumption rate and proton efflux rate in PBMCs and platelets. We estimated whole-body resting metabolic rate by bioimpedance and dietary fatty acid oxidation by oral deuterated palmitate and quantifying recovery of D2O in urine. We used ANOVA for comparisons among groups and Spearman correlations for associations between circulating cell bioenergetics and whole-body metabolism and biomarkers. RESULTS:O-IS and O-IR PBMCs exhibited increased maximal mitochondrial respiration and spare respiratory capacity compared to N-IS. Bioenergetics shifted towards glycolysis in O-IS PBMCs as compared to both N-IS and O-IR PBMCs. In platelets, glycolysis and ATP production rates were decreased in O-IR compared to O-IS children. PBMC respiration positively correlated with BMIz, HOMA-IR and fasting glucose and insulin, but negatively correlated with inflammatory cytokines. Dietary fatty acid oxidation was higher in O-IS compared to N-IS children and positively correlated with PBMC spare respiratory capacity. Resting metabolic rate correlated positively with several parameters of PBMC mitochondrial respiration. CONCLUSIONS:PBMCs from young children with overweight/obesity exhibit adaptations to the metabolic stressors associated with insulin resistance, and PBMC metabolism correlates well with whole-body metabolism.

Late onset Alzheimer disease is a complex syndrome, genetically, clinically and pathogenetically heterogeneous. Genome Wide association studies have identified risk alleles for AD harboring genes in the endolysosomal network (ELN). We hypothesize that aggregate burden of these endolysosomal risk alleles impacts cell type specific ELN function, thus contributing to LOAD pathogenesis. To test this hypothesis, we first developed an endolysosomal polygenic risk score (ePRS) using a curated list of established GWAS AD risk loci. We identified a cohort in the top quartile (high) and bottom quartile (low) of ePRS scores within our LOAD biobank with rich clinical and pathological phenotype data, enabling brain tissue omics and cell based functional studies from the same donor. To explore cell type specific implications of ePRS burden, we studied frozen cortical brain tissue and primary cells derived from the same set of LOAD cases. We identified cell type-specific gene expression and regulatory network shifts associated with ePRS burden. Immunohistochemistry revealed that high ePRS was associated with enlarged neuronal endosomes and microglial lysosomes. We explored whether ePRS itself predicted ELN changes in cells that are not exposed to pathologic protein deposition. Leptomeningeal cells isolated from the ePRS cohort also manifested changes to ELN morphology and bulk RNA sequencing revealed ePRS-dependent gene expression changes, suggesting that ePRS directly influences ELN related transcriptomic phenotype. Taken together, our data suggests that AD risk loci implicating genes within the endolysosomal system do in fact impact the cellular ELN, giving more confidence to the pathogenic relevance of “endolysosomal” risk loci. Dysfunction of endolysosomal pathways likely contributes to AD pathogenesis in LOAD and may highlight more specific points of intervention. Pathway-specific polygenic risk scores can enhance our understanding of the complex mechanisms contributing to AD and pave the way for more specific targeted therapies in the clinic.

Circulating blood cells such as platelets represent a readily available sample type to determine mitochondrial function in humans. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform on the respiratory function of platelets isolated from human blood. Approximately 50 mL of whole blood was collected from healthy adults (n = 16) following an overnight (>12 h) fast. Platelets were immediately isolated from whole blood by centrifugation for respirometry. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer in either RPMI or MIR05 (containing 5 mM glucose, 1 mM pyruvate, and 2 mM glutamine), or the participant's own plasma. In addition, respiratory function was determined in intact platelets using a Seahorse Extracellular Flux analyzer (XFe96) in RPMI buffer containing 1 mM pyruvate, 2 mM glutamine, and variable glucose concentrations (5, 10, and 10 mM). In assays performed in an O2K, routine and ATP-linked respiration were greater in cells assayed in RPMI compared to MIR05 (p < 0.001). However, compared to cells assayed in RPMI or MIR05, routine and ATP-linked respiration were higher in intact platelets assayed in their own plasma (p < 0.001). In digitonin-permeabilized platelets, state 3 respiration was greater when assayed in MIR05 compared to RPMI (p < 0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K versus an XFe96 (p < 0.05), whereas respiration available for ADP phosphorylation was greater in cells assayed on an O2K versus an XFe96 (p < 0.001), due to a diminished coupling response to oligomycin in cells assayed on the XFe96 (p < 0.001). Platelet respiratory function is influenced by assay buffer composition and instrumental platform. Consideration of these factors should be made by investigators planning to use platelet respiratory function as a readout of cellular energetics.

Background: Prior work established that about a third of ASD-derived LCLs show excessive mitochondrial respiration and stress vulnerability—features divergent from both controls and classical mitochondrial disease. This study explores how mRNA and microRNA (miRNA) expression profiles distinguish subtypes of autism spectrum disorder (ASD) defined by mitochondrial function. Methods: Lymphoblastoid cell lines (LCLs) from boys with ASD were classified into two groups: those with abnormal (AD-A) and normal (AD-N) mitochondrial function. RNA-seq compared mRNA and miRNA expression differences. Results: 24 mRNA differentially expressed genes (DEGs) (14 downregulated, 10 upregulated in AD-N vs. AD-A) were identified, implicating processes such as mRNA processing, immune response, cancer biology, and crucially, mitochondrial and nuclear activities. Notably, genes such as DEPTOR (an mTOR modulator) were upregulated in AD-A, highlighting dysregulation in the mTOR pathway—a central regulator of cellular metabolism, protein synthesis, autophagy, and mitochondrial function. miRNA analysis revealed 18 differentially expressed miRNAs (DEMs) upregulated and one downregulated in AD-N compared to AD-A. Several miRNAs (including hsa-miR-1273h-3p, hsa-miR-197-3p, and hsa-miR-199a-5p) targeted both the differentially expressed genes and pathways previously linked to ASD, such as mTOR, Calmodulin Kinase II, and mitochondrial regulation. Enrichment analyses indicated involvement regulation of cell growth and division, gene expression, immune regulation and cellular stress as well as mTOR signaling. Conclusions: These molecular signatures support the idea that mitochondrial dysfunction in ASD is tied to specific disruptions in the mTOR and PI3K/AKT signaling axes, influencing cell growth, autophagy, oxidative stress handling, and neuronal metabolism. The findings highlight a miRNA-mRNA regulatory network that may underpin mitochondrial dysfunction and ASD heterogeneity, suggesting avenues for subtype-specific biomarkers and targeted therapies that address energy metabolism and cellular stress in ASD.

Late-onset Alzheimer's disease (LOAD) has a complex genomic architecture with risk variants in multiple pathways, including the endolysosomal network (ELN). Whether genetic risk in specific pathways correlates with corresponding biological dysfunction remains largely unknown. We developed an endolysosomal pathway-specific polygenic risk score (ePRS) using 13 established AD GWAS loci containing ELN genes. We investigated the association between ePRS and AD neuropathology, then examined cell-specific endolysosomal morphology and transcriptomic profiles in post-mortem dorsolateral prefrontal cortex samples from donors stratified by ePRS burden. We found that the ePRS was significantly associated with AD diagnosis and neuropathological measures, comparable to a pathway-agnostic PRS despite representing far fewer loci. High ePRS correlated with increased neuronal endosome volume, number and perinuclear aggregation, as well as enlarged microglial lysosomes, independent of AD pathology. Single-nucleus RNA sequencing revealed cell-type transcriptomic changes associated with ePRS status, including glutamatergic signaling, protein homeostasis, responses to DNA damage and immune function. Neurons, astrocytes, oligodendrocytes, and microglia showed varied gene expression patterns associated with ePRS burden. Conclusions: This study provides evidence that AD genetic risk variants harboring ELN genes correlate with endolysosomal dysfunction in human brain tissue. These findings suggest that pathway-specific genetic risk contributes to corresponding cellular pathology in AD and nominates candidate mechanisms by which ELN AD variants contribute to pathogenesis.

Background: During gestation, the brain development of the fetus is affected by many biological markers, where inflammatory processes and neurotrophic factors have been of particular interest in the past decade. Aim: This exploratory study is the first attempt to explore the relationships between biomarker levels in maternal and cord-blood samples and human fetal brain activity measured with non-invasive fetal magnetoencephalography (fMEG). Method: Twenty-three women were enrolled in this study for collection of maternal serum and fMEG tracings immediately prior to their scheduled cesarean delivery. Twelve of these women had a preexisting diabetic condition. At the time of delivery, umbilical cord blood was also collected. Biomarker levels from both maternal and cord blood were measured and subsequently analyzed for correlations with fetal brain activity in four frequency bands extracted from fMEG power spectral densities. Results: Relative power in the delta, alpha, and beta frequency bands exhibited moderate-sized correlations with maternal BDNF and cord-blood CRP levels before and after adjusting for confounding diabetic status. These correlations were negative for the delta band, and positive for the alpha and beta bands. Maternal CRP and cord-blood BDNF and IL-6 exhibited negligible correlations with relative power in all four bands. Diabetes did not appear to be a strong confounding factor affecting the studied biomarkers. Conclusions: Maternal BDNF levels and cord-blood CRP levels appear to have a direct correlation to fetal brain activity. Our findings indicate the potential use of these biomarkers in conjunction with fetal brain electrophysiology to track fetal neurodevelopment.

Background: Obesity-associated inflammation drives the development of insulin resistance and type 2 diabetes. We sought to identify associations of circulating regulatory T cells (Treg) with the degree of obesity (eg, body mass index Z-score [BMIz]), insulin resistance (homeostatic model of insulin resistance [HOMA-IR]), and glycemic control (HbA1c) in children and adolescents. We further sought to examine associations among bioenergetics of peripheral blood mononuclear cells (PBMCs) and CD4 T cells and BMIz, HOMA-IR, and HbA1c. Methods: A total of 65 children and adolescents between the ages 5 and 17 years were studied. HbA1c and fasting levels of plasma glucose and insulin were measured. We quantified circulating Tregs (CD3+CD4+CD25+CD127-FoxP3+) by flow cytometry, and measured mitochondrial respiration (oxygen consumption rate [OCR]) and glycolysis (extracellular acidification rate [ECAR]) in PBMCs and isolated CD4 T cells by Seahorse extracellular flux analysis. Results: Tregs (% CD4) are negatively associated with BMIz but positively associated with HOMA-IR. In PBMCs, OCR/ECAR (a ratio of mitochondrial respiration to glycolysis) is positively associated with BMIz but negatively associated with HbA1c. Conclusions: In children, Tregs decrease as body mass index increases; however, the metabolic stress and inflammation associated with insulin resistance may induce a compensatory increase in Tregs. The degree of obesity is also associated with a shift away from glycolysis in PBMCs but as HbA1c declines, metabolism shifts back toward glycolysis. Comprehensive metabolic assessment of the immune system is needed to better understand the implications immune cell metabolic alterations in the progression from a healthy insulin-sensitive state toward glucose intolerance in children. Trial registration: This observational study was registered at the ClinicalTrials.gov (NCT03960333, https://clinicaltrials.gov/study/NCT03960333?term=NCT03960333&rank=1).

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

The SORL1 gene has recently emerged as a strong Alzheimer’s Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Background: Studies have linked autism spectrum disorder (ASD) to physiological abnormalities including mitochondrial dysfunction. Mitochondrial dysfunction may be linked to a subset of children with ASD who have neurodevelopmental regression (NDR). We have developed a cell model of ASD which demonstrates a unique mitochondrial profile with mitochondrial respiration higher than normal and sensitive to physiological stress. We have previously shown similar mitochondrial profiles in individuals with ASD and NDR.Methods: Twenty-six ASD individuals without a history of NDR (ASD-NoNDR) and 15 ASD individuals with a history of NDR (ASD-NDR) were recruited from 34 families. From these families, 30 mothers, 17 fathers and 5 typically developing (TD) siblings participated. Mitochondrial respiration was measured in peripheral blood mononuclear cells (PBMCs) with the Seahorse 96 XF Analyzer. PBMCs were exposed to various levels of physiological stress for 1 h prior to the assay using 2,3-dimethoxy-1,4-napthoquinone.Results: ASD-NDR children were found to have higher respiratory rates with mitochondria that were more sensitive to physiological stress as compared to ASD-NoNDR children, similar to our cellular model of NDR. Differences in mitochondrial respiration between ASD-NDR and TD siblings were similar to the differences between ASD-NDR and ASD-NoNDR children. Interesting, parents of children with ASD and NDR demonstrated patterns of mitochondrial respiration similar to their children such that parents of children with ASD and NDR demonstrated elevated respiratory rates with mitochondria that were more sensitive to physiological stress. In addition, sex differences were seen in ASD children and parents. Age effects in parents suggested that mitochondria of older parents were more sensitive to physiological stress.Conclusion: This study provides further evidence that children with ASD and NDR may have a unique type of mitochondrial physiology that may make them susceptible to physiological stressors. Identifying these children early in life before NDR occurs and providing treatment to protect mitochondrial physiology may protect children from experiencing NDR. The fact that parents also demonstrate mitochondrial respiration patterns similar to their children implies that this unique change in mitochondrial physiology may be a heritable factor (genetic or epigenetic), a result of shared environment, or both.

A recent study by the Amal team published in this journal in May 2023 proved for the first time the link of nitric oxide (NO) with autism spectrum disorder (ASD), thereby opening new venues for the potential use of neuronal nitric oxide synthase (nNOS) inhibitors as therapeutics for improving the neurological and behavioral symptoms of ASD. The authors conclude that their findings demonstrate that NO plays a significant role in ASD. Indeed, earlier studies support elevated NO and its metabolites, nitrite, and peroxynitrite, in individuals diagnosed with ASD. Dysregulated NOS activity may underlie the well-documented mitochondrial dysfunction in a subset of individuals with ASD. Strategies for treating ASD shall also consider NO effects on mitochondrial respiration in modulating NOS activity. Further experimental evidence and controlled clinical trials with NOS modifiers are required for assessing their therapeutic potential for individuals with ASD.

Objectives: This study aims to show the relation between biomarkers in maternal and cord-blood samples and fetal heart rate variability (fHRV) metrics through a non-invasive fetal magnetocardiography (fMCG) technique. Methods: Twenty-three women were enrolled for collection of maternal serum and fMCG tracings immediately prior to their scheduled cesarean delivery. The umbilical cord blood was collected for measurement of biomarker levels. The fMCG metrics were then correlated to the biomarker levels from the maternal serum and cord blood. Results: Brain-derived neurotrophic factor (BDNF) had a moderate correlation with fetal parasympathetic activity (0.416) and fetal sympathovagal ratios (-0.309; -0.356). Interleukin (IL)-6 also had moderate-sized correlations but with an inverse relationship as compared to BDNF. These correlations were primarily in cord-blood samples and not in the maternal blood. Conclusions: In this small sample-sized exploratory study, we observed a moderate correlation between fHRV and cord-blood BDNF and IL-6 immediately preceding scheduled cesarean delivery at term. These findings need to be validated in a larger population.

Platelets are anucleated cells that circulate in the bloodstream. Historically, platelets were thought to perform a singular function—stop bleeding via clotting. Although platelets do play a key role in hemostasis and thrombosis, recent studies indicate that platelets also modulate inflammation, and this platelet-induced inflammation contributes to the pathophysiology of various diseases such as atherosclerosis and diabetes mellitus. Thus, in recent years, our understanding of platelet function has broadened. In this review, we revisit the classic role of platelets in hemostasis and thrombosis and describe the newly recognized function of platelets in modulating inflammation. We cover the potential use of purinergic receptor antagonists to prevent platelet-modulated inflammation, particularly in patients with chronic kidney disease, and finally, we define key questions that must be addressed to understand how platelet-modulated inflammation contributes to the pathophysiology of chronic kidney disease.

Microglia, the innate immune cells of the brain, influence Alzheimer’s disease (AD) progression and are potential therapeutic targets. However, microglia exhibit diverse functions, the regulation of which is not fully understood, complicating therapeutics development. To better define the transcriptomic phenotypes and gene regulatory networks associated with AD, we enriched for microglia nuclei from 12 AD and 10 control human dorsolateral prefrontal cortices (7 males and 15 females, all aged >60 years) before single-nucleus RNA sequencing. Here we describe both established and previously unrecognized microglial molecular phenotypes, the inferred gene networks driving observed transcriptomic change, and apply trajectory analysis to reveal the putative relationships between microglial phenotypes. We identify microglial phenotypes more prevalent in AD cases compared with controls. Further, we describe the heterogeneity in microglia subclusters expressing homeostatic markers. Our study demonstrates that deep profiling of microglia in human AD brain can provide insight into microglial transcriptional changes associated with AD.

In humans, circulating blood cells such as platelets are being increasingly used as a readily available and minimally invasive biospecimen to determine mitochondrial respiratory function. Here, we set out to determine the influence of sample preparation, assay buffer composition, and instrumental platform, on the respiratory function of platelets isolated from human blood.Approximately 50 mL of whole blood was collected from healthy adults (n=16) following an overnight (>12 hr.) fast. Platelets were immediately isolated from whole blood by centrifugation. Respiratory function was assayed in intact and permeabilized platelets using an Oxygraph-2K (O2K) high-resolution respirometer (Oroboros Instruments). Respiratory function was assayed in intact platelets suspended in either culture (RPMI) or respiration (MIR05) media (both supplemented with 5 mM glucose, 1 mM pyruvate and 2 mM glutamine), or the participants own plasma. Respiratory function was also assayed in digitonin-permeabilized platelets suspended in both RPMI and MIR05. In addition, respiratory function was determined in intact platelets using a Seahorse Extra-Cellular Flux analyzer (XFe) in RPMI buffer containing 5 mM glucose, 1 mM pyruvate and 2 mM glutamine.In intact platelets assayed in suspension using an O2K, routine and ATP-linked respiration were 35 and 34% greater in cells assayed in RPMI compared to MIR05, respectively (P<0.001). However, coupling control in response to oligomycin was comparable in cells assayed in RPMI and MIR05 (92±4 vs. 94±4%). Compared to cells assayed in RPMI (0.19±0.01 pmol/s/100 million cells) or MIR05 (0.14±0.02 pmol/s/100 million cells), ATP-linked respiration was greater in intact cells assayed in their own plasma (0.24±0.02 pmol/s/100 million cells, both P<0.001). In digitonin-permeabilized platelets, ATP-linked respiration was greater when assayed in MIR05 compared to RPMI (0.36±0.08 vs. 0.30±0.07 pmol/s/100 million cells, P<0.05). Across instrumental platforms, routine and leak respiration were lower in intact platelets assayed on an O2K vs. XFe (P<0.05), whereas ATP-linked respiration was greater in cells assayed on an O2K. (P<0.001), due to a diminished coupling response to oligomycin in cells assayed in the XFe (P<0.001). Platelet respiratory function is influenced by sample preparation, assay buffer and protocol, and the instrumental platform used. Consideration of these factors is necessary when using platelet respiratory function as a readout of cellular energetics. Supported by USDA-ARS Project 6026-51000-012-06S, NIH 5P20GM109096-07 and 5R35GM142744-02. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.