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Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

ABSTRACT: Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p < 0.05), of which 119 (40%) and 179 (60%) genes were abundantly expressed in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three DE genes, which were more highly expressed in male than female MVs. Synthesis of glycosylphosphatidylinositol (GPI), biosynthesis of GPI-anchored proteins, steroid and cholesterol synthesis, were the top three significantly enriched canonical pathways in male MVs. In contrast, respiratory chain, ribosome, and 3 ́-UTR-mediated translational regulation were the top three enriched canonical pathways in female MVs. Different gene functions of MVs were validated by proteomic analysis and western blotting. Our novel findings reveal major sex disparities in gene expression and canonical pathways of MVs and these differences provide a foundation to study the underlying mechanisms and consequences of sex-dependent differences in cerebrovascular and other neurological diseases.

SUBMITTER: Chandra PK

PROVIDER: S-EPMC8392780 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

Chandra Partha K PK Cikic Sinisa S Baddoo Melody C MC Rutkai Ibolya I Guidry Jessie J JJ Flemington Erik K EK Katakam Prasad Vg PV Busija David W DW

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20210309 9

Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p < 0.05), of which 119 (40%) and 179 (60%) gen ...[more]

PMID: 33715494

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Project description:The noninfectious HIV-1 transgenic (HIV-1Tg) rat was developed as a model of AIDs-related pathology and immune dysfunction by manipulation of a noninfectious HIV-1(gag-pol) virus with a deleted 3-kb SphI-MscI fragment containing the 3' -region of gag and the 5' region of pol into F344 rats. Our previous studies revealed significant behavioral differences between HIV-1Tg and F344 control rats in their performance in the Morris water maze and responses to psychostimulants. However, the molecular mechanisms underlying these behavioral differences remain largely unknown. The primary goal of this study was to identify differentially expressed genes and enriched pathways affected by the gag-pol-deleted HIV-1 genome. Using RNA deep sequencing, we sequenced RNA transcripts in the prefrontal cortex, hippocampus, and striatum of HIV-1Tg and F344 rats. A total of 72 RNA samples were analyzed (i.e., 12 animals per group × 2 strains × 3 brain regions). Following deep-sequencing analysis of 50-bp paired-end reads of RNA-Seq, we used Bowtie/Tophat/Cufflinks suites to align these reads into transcripts based on the Rn4 rat reference genome and to measure the relative abundance of each transcript. Statistical analyses on each brain region in the two strains revealed that immune response- and neurotransmission-related pathways were altered in the HIV-1Tg rats, with brain region differences. Other neuronal survival-related pathways, including those encoding myelin proteins, growth factors, and translation regulators, were altered in the HIV-1Tg rats in a brain region-dependent manner. This study is the first deep-sequencing analysis of RNA transcripts associated the HIV-1Tg rat. Considering the functions of the pathways and brain regions examined in this study, our findings of abnormal gene expression patterns in HIV-1Tg rats suggest mechanisms underlying the deficits in learning and memory and vulnerability to drug addiction and other psychiatric disorders observed in HIV-positive patients.

Project description:BackgroundProtein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, isolation of brain microvessels from human brain tissue, particularly in small quantities, poses significant challenges. This study presents a method for isolating brain microvessels from a small amount of frozen human brain tissue, adapting techniques from an established mouse brain capillary isolation method.MethodsBrain microvessel fractions were obtained from approximately 0.3 g of frozen human brain tissue (frontal cortex) using a bead homogenizer for homogenization, followed by purification with a combination of cell strainers and glass beads. Protein expression in the isolated human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.ResultsMicroscopic imaging confirmed the successful isolation of brain microvessels from frozen human brain tissue. Protein quantification assays demonstrated that the microvessel fraction yielded sufficient protein for detailed expression analysis. Western blot analysis revealed an enrichment of BBB-selective proteins including multidrug resistance 1 (MDR1)/ATP-binding cassette sub-family B member 1 (ABCB1), glucose transporter protein type 1 (GLUT1)/solute carrier family 2 member 1 (SLC2A1), and claudin 5 (CLDN5), in the brain microvessel fraction compared to whole-brain lysates. Multiple reaction monitoring quantification of six BBB-selective proteins-MDR1, breast cancer resistance protein (BCRP)/ATP binding cassette subfamily G member 2 (ABCG2), GLUT1, monocarboxylate transporter 1 (MCT1)/solute carrier family 16 member 1 (SLC16A1), transferrin receptor, and CLDN5-revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further demonstrated significant enrichment of human microvascular endothelial cells in the isolated fraction, corroborating the findings from mouse models.ConclusionsWe successfully developed a method for isolation of brain microvessels from a small amount of frozen human brain tissue, facilitating detailed study of BBB proteome in aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and holds potential for improving brain-targeted drug delivery strategies.

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Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

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Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.

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