Biology subjects

Kandimalla, K. K.

Publications and source records attributed to Kandimalla, K. K..

13 recordsLinked to original sources

bioRxiv2026

Disruptions in glucose and amyloid-beta transport in mouse models manifesting metabolic syndrome

Studies in humans and murine models have pointed towards a possible link between metabolic syndrome, which shows insulin resistance and metabolic dysregulation, and Alzheimer's disease (AD) pathology marked by amyloid-beta (A{beta}) accumulation and hypometabolism in the brain. Yet, the underlying biological mechanisms by which metabolic syndrome affects these pathological changes in AD brain remain unknown. We hypothesized that insulin resistance is responsible for alterations in blood-brain barrier (BBB) transport of A{beta} peptides and glucose. This hypothesis was tested by employing radiolabeled ligands (125I-A{beta}40, 125I-A{beta}42, and 18F-FDG) in high-fat diet (HFD)-fed mouse models that manifest metabolic syndrome. Further, we assessed alterations in the expression of various molecular mediators within the brain microcapillaries harvested from both low-fat diet (LFD)-fed and HFD-fed mice. Our findings show that HFD-fed mice developed peripheral insulin resistance and obesity. In addition, HFD-fed mice demonstrated an increase in the influx rate of A{beta} peptides and a reduction in 18F-FDG (a glucose surrogate) influx rate compared to LFD-fed mice. These transport changes are associated with the increase in the BBB endothelial expression of RAGE (receptor to traffic A{beta} from plasma-to-brain) and reduction of GLUT1 (glucose transporter) expression in HFD-fed mice compared to LFD-fed mice. Moreover, disruption in insulin signaling, as indicated by reduced pAKT and pERK expression, was observed in HFD-fed mice. Inhibiting AKT or ERK phosphorylation resulted in similar changes in A{beta} and glucose uptake in polarized BBB endothelial cell monolayers in vitro. These results indicate that high-fat diet induced metabolic syndrome may lead to BBB dysfunction, characterized by increased plasma-to-brain A{beta} trafficking and diminished glucose transport at the BBB, thereby aggravating the expression of AD pathological hallmarks.

neuroscience↗

bioRxiv2025

Molecular Mechanisms Underlying the Regulation of VCAM-1 Expression by the Short-Chain Fatty Acid Butyrate

Over the past decade, cerebrovascular inflammation has been increasingly recognized as a contributor to the progression of neurodegenerative diseases, particularly Alzheimers disease (AD). One of the molecular hallmarks of cerebrovascular inflammation is the increased expression of vascular cell adhesion molecule (VCAM)-1 on blood-brain barrier (BBB) endothelial cells. Exposure to amyloid beta (A{beta}) peptides, one of the primary hallmarks of AD, and pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-) induces VCAM-1 expression on the BBB endothelium, which facilitates extravasation of leukocytes into the brain thereby promoting an inflammatory response. Therefore, it is crucial to explore therapeutic agents that can inhibit VCAM-1 expression induced by A{beta} and TNF-. Short-chain fatty acids, such as butyrate, produced by the gut microbiota as byproducts of dietary fiber metabolism, are recognized for their anti-inflammatory properties. In this study, we successfully tested the hypothesis that butyrate mitigates A{beta} and TNF--induced VCAM-1 expression in polarized human cerebral microvascular endothelial cell monolayers, a widely used BBB in vitro model. Our findings indicated that pre-treatment with butyrate significantly reduced A{beta}42 and TNF- mediated upregulation of VCAM-1. Furthermore, we have shown STAT3/GATA6 axis as a key mediator of anti-inflammatory effects of butyrate. These findings provide mechanistic insight into butyrates protective role and highlight its potential to mitigate A{beta} and TNF--induced cerebrovascular inflammation in AD.

molecular biology↗

bioRxiv2025

NVUAtlas: A Comprehensive Single-Nucleus RNA-Seq Resource for the Human Neurovascular Unit in Alzheimer's Disease

BackgroundNeurovascular unit (NVU) dysfunction is being recognized as one of the earliest contributors to Alzheimers disease (AD) pathogenesis. However, systematic investigation of NVU dysfunction is currently limited by lack of access to molecular level information due to underrepresentation of vascular and mural cells in standard single-nucleus RNA sequencing (snRNA-seq) datasets. Consequently, existing transcriptomic atlases lack the resolution necessary to capture the coordinated intercellular signaling and dysfunction across vascular components of NVU, including endothelial cells and pericytes. MethodsWe constructed the Human NVU Atlas by integrating 11 publicly available snRNA-seq datasets, including vascular-enriched samples, from the human prefrontal cortex. This comprehensive dataset aggregates over 4.2 million nuclei from 748 donors, including AD patients and age-matched controls. We utilized a unified probabilistic pipeline based on deep generative models (SCVI) to perform batch-aware integration and employed an ensemble of supervised and deep-learning classifiers to rigorously re-annotate cell types. Differential expression and ligand-receptor interaction analyses were subsequently performed to identify cell-type-specific disruptions in males versus females. ResultsThe atlas successfully curated vascular populations from 11 studies to assemble the largest publically available NVU cohort of endothelial cells (2.8%) and pericytes (1.9%) alongside astrocytes and neurons. Differential expression analysis revealed that while neurons predominantly exhibited gene downregulation in AD, vascular cells displayed a pattern of transcriptional hyperactivity with significant gene upregulation. We also identified pronounced sex-specific vulnerabilities; females exhibited distinct inflammatory signatures and downregulation of basement membrane collagen genes (e.g., COL4A1, COL4A2) in pericytes, whereas these changes were not observed in males. Moreover, cell-cell interaction analysis revealed a widespread loss of collagen-integrin signaling between pericytes and neurons, suggesting the involvement of extracellular matrix disruptions in NVU dysfunction observed in AD. ConclusionThe Human NVU Atlas provides a high-resolution, integrated transcriptomic framework for dissecting the cellular heterogeneity of the neurovascular unit. By uncovering sex-specific vascular mechanisms and disrupted intercellular communication, this resource highlights the critical role of vascular cells in AD progression and serves as a foundational reference for investigating cerebrovascular contribution to AD.

neuroscience↗

bioRxiv2025

Endothelial-pericyte interactions activate insulin signaling and its implications for blood-brain barrier dysfunction in Alzheimers disease

PurposeThis study aimed to investigate how pericyte degeneration contributes to BBB disruption in Alzheimers disease, focusing on the roles of insulin signaling and the imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMPs (TIMPs). MethodsWe employed an in vitro BBB model by co-culturing brain-specific microvascular endothelial-like cells (iBMECs) differentiated from human induced pluripotent stem cells (hiPSCs) and primary human brain vasculature pericytes (hBVPs). Protein expression under solo- versus co-culture conditions was assessed by western blot. MMP enzymatic activity in the culture media was measured by fluorometric assay. Exosomes were isolated from conditioned media and brain derived neurotrophic growth factor (BDNF) concentrations were determined using ELISA assays. ResultsTIMP1 and collagen-IV expression was significantly increased in co-cultured BBB endothelial cells and pericytes compared to solo-cultures. However, a greater effect was observed in cells co-cultured for 2 days than 7 days. Elevated TIMP1 in co-culture media significantly inhibited MMP activity. The AKT and ERK pathways were activated in both cell types after 7 days of co-culture, and the ERK signaling mediated TIMP1 upregulation in endothelial cells. BDNF was significantly enriched in exosomes isolated from co-culture media on the abluminal side compared to the solo-cultures. Endothelial cells also protected pericytes from accumulation of toxic amyloid-beta 42 by downregulating low density lipoprotein receptor-related protein 1 (LRP1) expression. ConclusionsThese findings provide mechanistic insights into BBB disruption due to pericyte degeneration and highlight the important role of BBB insulin resistance in causing cerebrovascular dysfunction in AD.

molecular biology↗

bioRxiv2025

ACUTE AMYLOID-B 40 EXPOSURE DISRUPTS METABOLIC INSULIN SIGNALING IN BLOOD-BRAIN BARRIER ENDOTHELIAL CELLS

BackgroundBrain insulin resistance and cerebrovascular dysfunction emerge early in late-onset Alzheimers disease, but how amyloid-{beta} (A{beta}) disrupts insulin signaling at the cerebrovascular blood-brain barrier--the principal site of insulin receptor signaling and transport into the brain--remains unclear. MethodsWe exposed two distinct human blood-brain-barrier endothelial cell models to soluble A{beta}40 or A{beta}42 for 1 h, followed by 100 nM insulin for 10 min. Protein and phosphoprotein responses were quantified by reverse-phase protein array, and differential expression was evaluated using empirical-Bayes-stabilized linear models with FDR correction. ResultsA{beta}40 reduced insulin-stimulated Akt phosphorylation and converted insulins normal inhibition of AMPK into modest stimulation, increasing AMPK Thr172 and ACC Ser79 phosphorylation. A{beta}42 did not alter insulin-stimulated Akt signaling, indicating isoform-specific disruption of endothelial insulin responses. Without insulin, both A{beta}40 and A{beta}42 showed modest stimulation of ribosomal protein s6. ConclusionsThese findings show that A{beta}40 acutely impairs insulin signal transduction in BBB endothelial cells, supporting a model in which vascular amyloid exposure contributes directly to the early development of brain insulin resistance in AD.

molecular biology↗

bioRxiv2025

Butyrate regulates the blood-brain barrier transport and intra-endothelial accumulation of Alzheimers disease Amyloid-beta peptides

Alzheimers disease (AD) is characterized by the pathological deposition of amyloid beta (A) proteins as amyloid plaques, tau aggregates, and cerebrovascular dysfunction that drive disease progression. Butyrate, a gut microbial metabolite, has been found to be reduced in AD patients; butyrate supplementation improved cognition and decreased amyloid burden in animal models. However, the precise underlying mechanisms are unclear. Our previous studies have demonstrated that insulin signaling impacts A transport kinetics at the blood-brain barrier (BBB). In this study, we investigated the effect of butyrate treatment on intra-endothelial A accumulation and BBB integrity by modulating the insulin signaling pathway. The effect of butyrate on A accumulation was assessed by flow cytometry in BBB cell culture models. Insulin signaling activation and the expression of various receptors and transporters at the BBB were evaluated by Western blots and confocal microscopy. The roles of various molecular mediators were confirmed using specific inhibitors (MK2206, Trametinib, Rapamycin, VX-745). The effect of butyrate on the expression of BBB receptors and transporters that play a critical role in A trafficking was examined in mouse brains colonized with butyrate-producing bacteria via immunohistochemistry. Butyrate significantly decreased A{beta}42 accumulation in endothelial cells. This effect was associated with insulin signaling pathway activation, particularly AKT and ERK phosphorylation. Inhibitor studies established the critical role of these specific arms, as co-incubation with MK2206 (AKT inhibitor) or Trametinib (ERK inhibitor) reversed the protective effect of butyrate and increased A{beta}42 accumulation. However, mTOR and p38 inhibitors did not show a similar effect. In addition, butyrate restored P-glycoprotein efflux transporter expression and claudin-5 tight junction protein levels that were reduced with A{beta} treatment. These effects were supported by in vivo work, which demonstrated the upregulation of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2). This protein is associated with AKT activation and extracellular matrix stabilization in mice colonized with butyrate-producing bacteria. In conclusion, we have demonstrated that butyrate decreases A{beta}42 uptake at the BBB endothelium by activating the AKT and ERK arms of the insulin signaling pathway. These changes may also improve the integrity of BBB tight junctions by increasing claudin-5 expression and extracellular matrix, and by upregulating TIMP-2 expression. This study highlights butyrates potential as a therapeutic modulator of AD-related BBB dysfunction.

neuroscience↗

bioRxiv2025

Characterization of transcriptomic changes in the neurovascular unit of Alzheimiers transgenic mouse models using digital spatial profiling

Alzheimers disease (AD) affects 40 million individuals globally and is characterized by the accumulation of amyloid-beta (A{beta}) proteins, which aggregate and form plaques. BBB dysfunction drives AD cerebrovascular pathology and BBB integrity is maintained by neurovascular unit (NVU). Specifically, within the NVU, the cerebral endothelial cells maintain vascular homeostasis. In this study, we isolated endothelial-enriched regions of interest (ROIs) using the Nanostring GeoMx digital spatial profiler and employed a deconvolution model to evaluate transcriptomic changes. We observed dysregulation of cellular signaling potentially disrupting the APP+ BBB integrity. Analysis of ligand-receptor pairings that are the foundation of the NVU intercellular signaling indicated that the endothelial vasculature completes a feedback loop with the NVU to the regulating astroctyes. Further, we identified potentially antagonistic signaling roles for opioid receptor species that should be further investigated for potential therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/640886v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@d89b56org.highwire.dtl.DTLVardef@be2d96org.highwire.dtl.DTLVardef@1a2e7bforg.highwire.dtl.DTLVardef@1cdc9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Influence of Gut Microbiota on Oral Drug Absorption and Metabolism

Gut bacteria influence host intestinal phenotype in ways that impact drug absorption and metabolism. By comparing colonic tissue from germ-free mice to that of mice colonized with human microbiota, this study evaluates microbiota-driven differences in gene expression, mucosal permeability, and P-gp efflux capacity. Transcriptomic analysis revealed upregulation of genes coding ATP-binding cassette drug transporters P-gp (Abcb1a, Abcb1b), BCRP (Abcg2), and MRP3 (Abcc3), along with increased expression of solute carriers MCT1 (Slc16a1) and OCTN2 (Slc22a5). Immunohistochemistry indicated greater P-gp expression and apical localization in humanized mice. No relevant gene expression changes were observed in human homologs of drug-metabolizing cytochrome P450 enzymes, though cytochrome P450 oxidoreductase (Por) upregulation suggests increased cytochrome activity. Other phase I drug-metabolizing enzymes, including multiple homologs of human carboxylesterase 2 (CES2) and several reductases, were upregulated with high significance. Regarding phase II metabolism, genes encoding most UDP-glucuronosyltransferases and glutathione S-transferases were upregulated, along with the enzymes responsible for synthesizing their corresponding co-substrates, UDP-glucuronic acid and glutathione. Small intestinal mucosal explants demonstrated higher permeability to 14C-labeled polyethylene glycol 4000 (14C-PEG4000) in germ-free mice than in humanized mice. The 14C-PEG4000 permeability differences are in agreement with changes in paracellular tight junctional proteins such as claudins, zonula occludens, and myosin II-related genes. These results demonstrate the broader impact of gut microbiome on oral drug absorption and metabolism and implicate the contributions of gut microbiome to individual variations in drug efficacy and toxicity.

pharmacology and toxicology↗

bioRxiv2025

Elucidating Molecular Mechanisms Governing TNF-Alpha-Mediated Regulation of Amyloid Beta 42 Uptake in Blood-Brain Barrier Endothelial Cells

Cerebrovascular inflammation is prevalent in a majority of Alzheimers patients. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), circulating in the plasma have been shown to cause the inflammation of blood-brain barrier (BBB) endothelium lining the cerebral microvasculature. The BBB inflammation has been implicated in the increase of toxic A{beta} accumulation within Alzheimers disease (AD) brain. TNF-alpha in the peripheral circulation can aggravate the accumulation of amyloid-beta (A{beta}) peptides in Alzheimers disease brain. In the current study, we have shown that the exposure to TNF-alpha leads to an increase in A{beta}42 accumulation in mice and BBB endothelial cells in vitro. Moreover, dynamic SPECT/CT imaging in wild-type (WT) mice infused with TNF-alpha increased the permeability and influx of A{beta}42 into the mice brain. In addition, our results show that TNF-alpha modifies the expression of cofilin, actin, and dynamin, which are critical components for A{beta} endocytosis by BBB endothelial cells. These results offer a mechanistic understanding of how TNF-alpha may promote A{beta} accumulation at the BBB and the underlying interactions between inflammation and A{beta} exposure that drives BBB dysfunction. Hence, a therapeutic intervention aimed at addressing cerebrovascular inflammation in Alzheimers disease may potentially reduce A{beta} induced cerebrovascular toxicity in Alzheimers disease brain. Significance statementIncreased levels of TNF-alpha circulating in the plasma are considered significant factors in the consequences of A{beta} pathology in Alzheimers disease, where it can promote cerebrovascular inflammation and BBB dysfunction. However, the role of TNF-alpha, in exacerbating A{beta} pathology by increasing A{beta} accumulation at the BBB endothelial cells remains only partially understood. In this study, we demonstrated that TNF-alpha enhances A{beta}42 accumulation in the BBB endothelium by altering the expression of the BBB endocytosis machinery, specifically cofilin, actin, and dynamin. These findings are anticipated to contribute to the development of therapeutic approaches aimed at addressing elevated cytokine levels in Alzheimers disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/635286v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@6b27a4org.highwire.dtl.DTLVardef@f7b37dorg.highwire.dtl.DTLVardef@28efbcorg.highwire.dtl.DTLVardef@193408c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2022

Amyloid Beta Peptides Inhibit Glucose Transport at the Blood-brain Barrier by Disrupting Insulin-Akt Pathway in Alzheimer's Disease

Disruptions in glucose uptake and metabolism in the brain are implicated in metabolic disorders and Alzheimers disease (AD). Toxic soluble amyloid-beta (sA{beta}) peptides accumulating in the brain and plasma of AD patients were suggested to promote blood-brain barrier (BBB) dysfunction, brain hypometabolism, and cognitive decline. Exposure to sA{beta} peptides is reported to interfere with glucose metabolism in the brain parenchyma, although their effects on the BBB have not been fully characterized. Our data showed that the brain uptake of glucose surrogate, [18F]-fluorodeoxyglucose (18FDG), was reduced significantly in APP/PS1 transgenic mice (overproduce A{beta}) compared to wild-type (WT) mice. In addition, the influx rate of 18FDG was also decreased in both A{beta}40 and A{beta}42 pre-infused mice compared to control mice. Glucose is primarily delivered from blood into the brain via glucose transporter 1 (GLUT1). The confocal microscopy experiment showed that A{beta}40 and A{beta}42 peptides significantly decreased GLUT1 expression in polarized human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. Insulin-AKT pathway has been observed to induce glucose uptake via regulating the expression of TXNIP, the only -arrestin protein known to bind to thioredoxin. We found that A{beta}40 and A{beta}42 peptides decreased p-AKT and increased TXNIP expression in the hCMEC/D3 cell monolayers. MK2206, a kinase inhibitor of AKT, was used to confirm that inhibition of insulin/AKT pathway reduced GLUT1 expression in an insulin-independent manner in the hCMEC/D3 cell monolayers. These results suggest that inhibitory effects of sA{beta} on GLUT1 expression are mediated by inhibition of the insulin/AKT pathway. The role of TXNIP on endothelial GLUT1 expression was investigated using resveratrol, which has been reported to downregulate TXNIP overexpression. Consistently, resveratrol treatment led to a significant increase in GLUT1 expression in the hCMEC/D3 cell monolayers. Furthermore, by co-incubation of resveratrol and sA{beta} peptides in hCMEC/D3 cell monolayers, we found that resveratrol rectified the aberrant TXNIP expression caused by sA{beta} peptides. Together, these findings provide novel evidence that toxic sA{beta} peptide exposure inhibits glucose transport at the BBB by decreasing GLUT1 expression via the insulin/Akt/TXNIP axis.

pharmacology and toxicology↗

bioRxiv2022

Amyloid-beta peptides 40 and 42 employ distinct molecular pathways for cell entry and intracellular transit at the BBB endothelium

Blood-brain barrier (BBB) is a critical portal regulating the bidirectional transport of amyloid beta (A{beta}) proteins between blood and brain. Disrupted trafficking at the BBB may not only promote the build-up of A{beta} plaques in the brain parenchyma, but also facilitate A{beta} accumulation within the BBB endothelium, which aggravates BBB dysfunction. Soluble A{beta}42:A{beta}40 ratios in plasma and cerebrospinal fluid have been reported to decrease during Alzheimers disease (AD) progression. Our previous publications demonstrated that trafficking of A{beta}42 and A{beta}40 at the BBB is distinct and is disrupted under various pathophysiological conditions. However, the intracellular mechanisms that allow BBB endothelium to differentially handle A{beta}40 and A{beta}42 have not been clearly elucidated. In this study, we identified mechanisms of fluorescently labeled A{beta} (F-A{beta}) endocytosis in polarized human cerebral microvascular endothelial (hCMEC/D3) cell monolayers using pharmacological inhibition and siRNA knock-down approaches. Further, intracellular transit of F-A{beta} following endocytosis was tracked using live cell imaging. Our studies demonstrated that both F-A{beta} peptides were internalized by BBB endothelial cells via energy, dynamin and actin dependent endocytosis. Interestingly, endocytosis of F-A{beta}40 is found to be clathrin-mediated, whereas F-A{beta}42 endocytosis is caveolae-mediated. Following endocytosis, both isoforms were sorted by the endo-lysosomal system. While A{beta}42 was shown to accumulate more in the lysosome which could lead to its higher degradation and/or aggregation at lower lysosomal pH, A{beta}40 demonstrated robust accumulation in recycling endosomes which may facilitate its transcytosis across the BBB. These results provide a mechanistic insight into the selective ability of BBB endothelium to transport A{beta}40 versus A{beta}42. This knowledge contributes to the understanding of molecular pathways underlying A{beta} accumulation in the BBB endothelium and associated cerebrovascular dysfunction as well as amyloid deposition in the brain parenchyma which are implicated in AD pathogenesis.

neuroscience↗

bioRxiv2022

Deconvolution of plasma pharmacokinetics from dynamic heart imaging data obtained by SPECT/CT imaging

Plasma pharmacokinetic (PK) data is required as an input function for graphical analysis (e.g., Patlak plot) of single positron emission computed tomography/computed tomography (SPECT/CT) and positron emission tomography/CT (PET/CT) data to evaluate tissue influx rate of radiotracers. Dynamic heart imaging data is often used as a surrogate of plasma PK. However, accumulation of radiolabel (representing both intact and degraded tracer) in the heart tissue may interfere with accurate prediction of plasma PK from the heart data. Therefore, we developed a compartmental model, which involves forcing functions to describe intact and degraded radiolabeled proteins in plasma and their accumulation in heart tissue, to deconvolve plasma PK of 125I-amyloid beta 40 (125I-A{beta}40) and 125I-insulin from their dynamic heart imaging data. The three-compartment model was shown to adequately describe the plasma concentration-time profile of intact/degraded proteins and the heart radioactivity time data obtained from SPECT/CT imaging for both tracers. The model was successfully applied to deconvolve the plasma PK of both tracers from their naive datasets of dynamic heart imaging. In agreement with our previous observations made by conventional serial plasma sampling, the deconvolved plasma PK of 125I-A{beta}40 and 125I-insulin in young mice exhibited lower area under the curve (AUC) than the aged mice. Further, Patlak plot parameters (Ki) extracted using deconvolved plasma PK as input function successfully recapitulated age-dependent blood-to-brain influx kinetics changes for both 125I-A{beta}40 and 125I-insulin. Therefore, the compartment model developed in this study provides a novel approach to deconvolve plasma PK of radiotracers from their noninvasive dynamic heart imaging. This method facilitates the application of preclinical SPECT or PET imaging data to characterize distribution kinetics of tracers where simultaneous plasma sampling is not feasible.

pharmacology and toxicology↗

bioRxiv2021

Clustering-based time series analysis on insulin response in the blood-brain barrier

BackgroundCritical functions of the blood-brain barrier (BBB), including cerebral blood flow and vascular response, are regulated by insulin signaling pathways. Therefore, endothelial insulin resistance could lead to vascular dysfunction, which is associated with neurodegenerative diseases such as Alzheimers disease (AD). ObjectiveThe objective of the current study is to map the dynamics of insulin-responsive pathways in polarized human cerebral microvascular endothelial cells (hCMEC/D3) cell monolayers, a widely used BBB cell culture model, to identify molecular mechanisms underlying BBB dysfunction in AD. MethodsRNA-Sequencing (RNA-Seq) was performed on hCMEC/D3 cell monolayers with and without insulin treatment at various time points. The Short Time-series Expression Miner (STEM) method was used to identify clusters of genes with distinct and representative patterns. Functional annotation and pathway analysis of the genes from top clusters were conducted using the Webgestalt and Ingenuity Pathway Analysis (IPA) software, respectively. ResultsQuantitative expression differences of 19,971 genes between the insulin-treated and control monolayers at five-time points were determined. STEM software identified 11 clusters with 3061 genes across that displayed various temporal patterns. Gene ontology enrichment analysis performed using the top 5 clusters demonstrated that these genes were enriched in various biological processes associated with AD pathophysiology. The IPA analyses revealed that signaling pathways exacerbating AD pathology such as inflammation were downregulated after insulin treatment (clusters 1 to 3). In contrast, pathways attenuating AD pathology were upregulated, including synaptogenesis and BBB repairment (clusters 4 and 5). ConclusionsThese findings unravel the dynamics of insulin action on the BBB endothelium and inform about downstream signaling cascades that potentially regulate neurovascular unit (NVU) functions that are disrupted in AD.

bioinformatics↗