Biology subjects

Wijenayake, S.

Publications and source records attributed to Wijenayake, S..

8 recordsLinked to original sources

bioRxiv2025

Investigating milk derived extracellular vesicles as mediators of maternal stress and environmental intervention

Parental communication signals are transmitted through nursing and critically shape neurodevelopmental trajectories. Mirroring some well characterized effects of gestational challenges in rodents, maternal immune activation (MIA) during the lactational period disrupts maternal physiology, decreases lipid content, and is associated with adverse neurobehavioral outcomes in offspring. This occurs without MIA significantly affecting maternal care. While gestational MIA models are responsive to environmental interventions, which beneficially alter maternal milk composition and associated offspring outcomes, the bioactive mediators in milk underlying resilience remain poorly understood. Milk-derived extracellular vesicles (MEVs) transport and deposit biologically active cargo, including microRNAs (miRNAs) that induce post-translational regulation of candidate mRNA in the nursing offsprings tissues and cells. Using a rat model, we show that lactational MIA alters MEV-miRNA cargo and the expression of hippocampal miRNAs in offspring. Several miRNAs in MEVs were also found in the hippocampus of matching offspring. Remarkably, the miRNA changes in MEVs and the neonatal hippocampus were rescued when dams were raised in an enriched environment, suggesting environmental enrichment protected from the effects of MIA. This was supported by the behavioral phenotype. RNA-seq of adult offspring hippocampus showed long-term transcriptional changes associated with the gene targets of early-life regulated miRNAs. Our results position MEV-miRNA as dynamic programming signals by which maternal experience is communicated to offspring, encoding both stress-induced and protective cues that influence development. This suggests that breastfeeding interventions can regulate the genetic cargo of the milk, programming the life of developing infants.

neuroscience↗

bioRxiv2025

Milk-derived extracellular vesicles mitigate NF-κB pathway and NLRP3 inflammasome formation in Long Evans neonates.

Exposure to a maternal high fat diet (HFD) during perinatal (prenatal and postnatal combined) life increases offsprings risk of developing metabolic diseases (obesity, type II diabetes and hypertension), impairs immunity, behaviour, and neurodevelopment. Exclusive breast/chest milk feeding is a potential solution to reduce the negative developmental effects of HFD, mainly chronic systemic pro-inflammation. This study focuses on analyzing anti-inflammatory effects of a group of biological nanovesicles found in human milk, entitled milk-derived extracellular vesicles (MEVs). Specifically, we characterized the modulation of the nuclear factor {kappa}B (NF-{kappa}B) signaling pathway and NLR family pyrin domain containing 3 (NLRP3) inflammasome formation by MEVs in male and female neonatal rats with perinatal HFD exposure in the liver and hypothalamus. Female Long Evans dams were placed on a HFD or a control diet (CHD), with matching sucrose levels, 4 weeks before breeding and remained on the diets through gestation and lactation. HFD and CHD offspring received human MEVs through oral gavage twice a day from postnatal day (PND) 4 to 11. Transcript and protein abundance of candidate targets in the NF-{kappa}B signaling pathway and NLRP3 inflammasome were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western immunoblotting, respectively. Our results indicate that MEV treatment attenuates the activation of NF-{kappa}B pathway and NLRP3 inflammasome formation at critical checkpoints, in males and females with perinatal HFD exposure in liver and the hypothalamus. Taken together, our data suggests that MEVs may elicit anti-inflammatory benefits postnatally that mitigates gestational HFD exposure.

developmental biology↗

bioRxiv2025

Human milk-derived extracellular vesicle treatment promotes the heat shock response in neonates with perinatal high fat diet exposure.

Maternal consumption of a high-fat diet (mHFD) during perinatal life (the collective prenatal and postnatal periods) influences neonatal development, initiates hypothalamic-pituitary-adrenal (HPA) axis activation, and impacts the long-term physiological and metabolic health of offspring. Milk-derived extracellular vesicles (MEVs) are lipid-coated nanovesicles found in mammalian milk that survive intestinal degradation and cross complex biological barriers, including the blood-brain barrier. MEVs have known cytoprotective activity in peripheral organs; however, their pro-survival functions in response to chronic pro-inflammation stemming from early life nutrient stress remain unknown in the neonatal brain. Further, sex differences resulting from MEV treatment require investigation, as male and female neonates illicit variable responses to early life nutrient stress. We investigated whether MEVs promote the heat shock response (HSR), a principal pro-survival mechanism responsible for refolding or degrading misfolded protein aggregates through the action of heat shock protein (HSP) chaperones. We investigated the interaction between MEVs and the HSR in the liver, hypothalamus, and prefrontal cortex in male and female neonatal rats exposed to perinatal mHFD within the stress hyporesponsive period at postnatal day 11. MEV treatment robustly modulated the HSR in female neonates with the largest response recorded in the prefrontal cortex. Specifically, in the prefrontal cortex, MEV treatment led to an upregulation of the main transcription factor (HSF1), while downregulating the negative regulators of HSF1 (Hsp70 and Hsp90). These results suggest that MEVs may influence pro-survival outcomes in the prefrontal cortex by activating HSF1-mediated pro-survival in a sex specific manner in response to mHFD.

molecular biology↗

bioRxiv2025

Human milk-derived extracellular vesicles promote the heat shock response in polarized microglia.

Proteotoxic stress induces microglia polarization and attenuates cytoprotective, pro-survival cellular cascades. Milk-derived extracellular vesicles (MEVs) are lipid-coated nanovesicles that combat pro-inflammation in peripheral cells and tissues; however, the cytoprotective potential of MEVs remains unknown in brain macrophages. We investigated whether MEVs reduce neuroinflammation in human microglia by activating the heat shock response (HSR). The HSR triggers the upregulation of molecular chaperones (heat shock proteins; HSPs) to restore proteostasis by refolding or degrading misfolded aggregates. MEVs were isolated from unpasteurized human donor milk. Human microglia clone 3 (HMC3) cells were primed with 10 ng/mL IFN-{gamma} to induce polarization, and a subset of cells were supplemented with 200 {micro}g of MEVs. The abundance of HSF1 and candidate HSPs (Hsp70, Hsp90, Hsp40, Hsp27) were analyzed via RT-qPCR and western immunoblotting at 6h, 12h, and 24h post-MEV supplementation. We found that MEV supplementation promoted the HSR in polarized microglia, compared to homeostatic cells. Furthermore, MEVs increased the duration of the HSR in response to pro-inflammatory stress, exerting robust and continued pro-survival benefits.

molecular biology↗

bioRxiv2024

The glia club: Validation of polarization biomarkers for human microglia (HMC3) using quantitative real time RT-qPCR.

Microglia are the primary immune cells of the brain and play critical roles in neurodevelopment, neuroprotection, the maintenance of homeostasis, and neurotoxicity. Classification of microglia polarization, however, remains contentious. Identifying suitable biomarkers for gene expression analysis of microglia polarization is crucial for characterizing the biological significance of microglia in health and disease. In this study, we use human microglia clone 3 (HMC3) cells to validate and test suitable internal controls (GAPDH, PKM, 18S, ACTB, PGK1, TKT1, TPI1), homeostatic (CD68, TGF-{beta}, IBA1, BIN1, RGS10), proinflammatory (IL-6, CXCL10, CCL5, SAA, IL-1{beta}) and anti-inflammatory (CCL2, SOCS3, IL-10, CD200R1, ARG1) markers along with their transcriptional profiles upon interferon-gamma (IFN-{gamma}) stimulation to characterize the microglia polarization spectrum. Our study is the first to present a comprehensive list of biomarkers with detailed methodology on gene selection, primer design, RT-qPCR parameters, and transcript abundance in baseline and polarized HMC3 cells. Our study will increase the rigor of gene expression analysis and target selection in a widely used brain macrophage.

cell biology↗

bioRxiv2020

Maternal high fat diet alters lactation-specific miRNA expression and programs the DNA methylome in the amygdala of female offspring

Adverse maternal diets high in saturated fats are associated with impaired neurodevelopment and epigenetic modifications in offspring. Maternal milk, the primary source of early life nutrition in mammals, contains lactation-specific microRNAs (miRNAs). Lactation-specific miRNAs have been found in various offspring tissues in early life, including the brain. We examined the effects of maternal high saturated fat diet (mHFD) on lactation-specific miRNAs that inhibit DNA methyltransferases (DNMTs), enzymes that catalyze DNA methylation modifications, in the amygdala of female offspring during early life and adulthood. Offspring exposed to mHFD showed reduced miR-148/152 and miR-21 transcripts in stomach milk and amygdala in the first week of life. This was associated with increased DNMT1 expression, DNMT activity, and global DNA methylation in the amygdala. In addition, persistent DNA methylation modifications from early life to adulthood were observed in pathways involved in neurodevelopment as well as genes regulating the DNMT machinery and protein function in mHFD offspring. The findings indicate a novel link between exogenous, lactation-specific miRNAs and developmental programming of the neural DNA methylome in offspring.

molecular biology↗

bioRxiv2020

Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

Milk is a highly complex, heterogeneous biological fluid that contains bioactive, membrane-bound extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, exosome isolation, and RNA extraction. In this study, we tested: 1) three pre-processing methods to remove cream, fat, and casein proteins from bovine milk to determine whether pre-processing of whole milk, prior to long-term storage, improves MDE isolations, 2) two commonly-used exosome isolation methods, and 3) four extraction protocols for obtaining high quality MDE RNA from bovine and human milk. MDEs were characterized via Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). We also present an optimized method of TEM sample preparation and isolation of total soluble protein from MDEs. Our results indicated that: 1) pre-processing of bovine milk prior to storage does not affect the final exosome yield or the purity, 2) ExoQuick precipitation is better suited for MDE isolation than ultracentrifugation for bovine and human milk, and 3) TRIzol LS produced the highest RNA yield in bovine milk, whereas TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for human milk.

molecular biology↗

bioRxiv2019

Maternal high-fat diet induces sex-specific effects on inflammatory responses to corticosterone and lipopolysaccharide challenge in adult rat offspring

BackgroundAcute elevations in endogenous corticosterone (CORT) with psychosocial stress or exogenous administration potentiate inflammatory gene expression. Maternal obesity as a result of high-fat diet (HFD) consumption has been linked to higher basal levels of neuroinflammation, including increased expression of pro-inflammatory genes in the amygdala. These findings suggest that exposure to maternal HFD may elicit pro-inflammatory responses in the presence of an immune stressor such as lipopolysaccharide (LPS), a component of gram-negative bacteria, as well as acute elevated CORT. MethodsRat offspring were exposed to maternal HFD or control diet (CHD) throughout pre and postnatal development until weaning, when all offspring were provided CHD until adulthood. In adulthood, offspring were challenged with administration of exogenous CORT, to simulate an acute physiological stress, LPS, to induce an immune stress, or both. qPCR was used to measure transcript abundance of CORT receptors and downstream inflammatory genes in the amygdala, hippocampus and prefrontal cortex, brain regions that mediate neuroendocrine and behavioral responses to stress. ResultsHFD female offspring exhibited elevations in anti-inflammatory transcripts, whereas HFD male offspring responded with greater pro-inflammatory gene expression to simultaneous CORT and LPS administration. ConclusionsThese findings suggest that exposure to maternal HFD leads to sex-specific alterations that may alter inflammatory responses in the brain, possibly as an adaptive response to basal inflammation.

neuroscience↗