Background and Purpose Though peripheral blood mRNA and microRNA change following ischemic stroke, any role for long noncoding RNA (lncRNA), which comprise most of the genome and have been implicated in various diseases, is unknown. Thus, we hypothesized that lncRNA expression also changes following stroke. Methods lncRNA expression was assessed in 266 whole-blood RNA samples drawn once per individual from ischemic stroke patients and matched vascular risk factor controls. Differential lncRNA expression was assessed by Analysis of Covariance (ANCOVA, p-value < 0.005; fold change > |1.2|), principal components analysis and hierarchical clustering on a derivation set (n=176) and confirmed on a validation set (n=90). Post-stroke temporal lncRNA expression changes were assessed using ANCOVA with confounding factor correction (p<0.005; partial correlation with time since event >|0.4|). Because sexual dimorphism exists in stroke, analyses were performed for each sex separately. Results 299 lncRNAs were differentially expressed between stroke and control males, whereas 97 lncRNAs were differentially expressed between stroke and control females. Significant changes of lncRNA expression with time after stroke were detected for 49 lncRNAs in males and 31 lncRNAs in females. Some differentially expressed lncRNAs mapped close to genomic locations of previously identified putative stroke-risk genes, including Lipoprotein, Lp(A)-Like 2, ABO blood group, Prostaglandin 12 Synthase, and α-Adducins. Conclusions This study provides evidence of altered and sexually dimorphic lncRNA expression in peripheral blood of stroke patients compared to controls and suggests lncRNAs have potential for stroke biomarker development. Some regulated lncRNA could regulate some previously identified putative stroke-risk genes.

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Because our recent studies have demonstrated that miR-122 decreased in whole blood of patients and in whole blood of rats following ischemic stroke, we tested whether elevating blood miR-122 would improve stroke outcomes in rats. Young adult rats were subjected to a temporary middle cerebral artery occlusion (MCAO) or sham operation. A polyethylene glycol-liposome-based transfection system was used to administer a miR-122 mimic after MCAO. Neurological deficits, brain infarction, brain vessel integrity, adhesion molecule expression and expression of miR-122 target and indirect-target genes were examined in blood at 24 h after MCAO with or without miR-122 treatment. miR-122 decreased in blood after MCAO, whereas miR-122 mimic elevated miR-122 in blood 24 h after MCAO. Intravenous but not intracerebroventricular injection of miR-122 mimic decreased neurological deficits and brain infarction, attenuated ICAM-1 expression, and maintained vessel integrity after MCAO. The miR-122 mimic also down-regulated direct target genes (e.g. Vcam1, Nos2, Pla2g2a) and indirect target genes (e.g. Alox5, Itga2b, Timp3, Il1b, Il2, Mmp8) in blood after MCAO which are predicted to affect cell adhesion, diapedesis, leukocyte extravasation, eicosanoid and atherosclerosis signaling. The data show that elevating miR-122 improves stroke outcomes and we postulate this occurs via downregulating miR-122 target genes in blood leukocytes.

Whole transcriptome studies have used 3′-biased expression microarrays to study genes regulated in the blood of stroke patients. However, alternatively spliced messenger RNA isoforms have not been investigated for ischemic stroke or intracerebral hemorrhage (ICH) in animals or humans. Alternative splicing is the mechanism whereby different combinations of exons of a single gene produce distinct mRNA and protein isoforms. Here, we used RNA sequencing (RNA-seq) to determine if alternative splicing differs for ICH and cardioembolic, large vessel and lacunar causes of ischemic stroke compared to controls. RNA libraries from 20 whole blood samples were sequenced to 200 M 2 × 100 bp reads using Illumina sequencing-by-synthesis technology. Differential alternative splicing was assessed using one-way analysis of variance (ANOVA), and differential exon usage was calculated. Four hundred twelve genes displayed differential alternative splicing among the groups (false discovery rate, FDR; p < 0.05). They were involved in cellular immune response, cell death, and cell survival pathways. Distinct expression signatures based on usage of 308 exons (292 genes) differentiated the groups (p < 0.0005; fold change >|1.2|). This pilot study demonstrates that alternatively spliced genes from whole blood differ in ICH compared to ischemic stroke and differ between different ischemic stroke etiologies. These results require validation in a separate cohort.Electronic supplementary materialThe online version of this article (doi:10.1007/s12975-015-0407-9) contains supplementary material, which is available to authorized users.

Understanding cell-specific transcriptome responses following intracerebral hemorrhage (ICH) and ischemic stroke (IS) will improve knowledge of the immune response to brain injury. Transcriptomic profiles of 141 samples from 48 subjects with ICH, different IS etiologies, and vascular risk factor controls were characterized using RNA-seq in isolated neutrophils, monocytes and whole blood. In both IS and ICH, monocyte genes were down-regulated, whereas neutrophil gene expression changes were generally up-regulated. The monocyte down-regulated response to ICH included innate, adaptive immune, dendritic, NK cell and atherosclerosis signaling. Neutrophil responses to ICH included tRNA charging, mitochondrial dysfunction, and ER stress pathways. Common monocyte and neutrophil responses to ICH included interferon signaling, neuroinflammation, death receptor signaling, and NFAT pathways. Suppressed monocyte responses to IS included interferon and dendritic cell maturation signaling, phagosome formation, and IL-15 signaling. Activated neutrophil responses to IS included oxidative phosphorylation, mTOR, BMP, growth factor signaling, and calpain proteases-mediated blood–brain barrier (BBB) dysfunction. Common monocyte and neutrophil responses to IS included JAK1, JAK3, STAT3, and thrombopoietin signaling. Cell-type and cause-specific approaches will assist the search for future IS and ICH biomarkers and treatments.

Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes.Methods: A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 39 untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA.Results: Let7i was decreased in patients with acute ischemic stroke (fold change 21.70, p , 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r 5 20.32, p 5 0.02), infarct volume (r 5 20.21, p 5 0.04), and plasma MMP9 (r 5 20.46, p 5 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling.Conclusions: Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke.Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1. Neurology ® 2016;87:2198-2205 GLOSSARY cDNA 5 complementary DNA; HTA 5 Human Transcriptome Array; IL 5 interleukin; mRNA 5 messenger RNA; NIHSS 5 NIH Stroke Scale; 39UTR 5 39 untranslated region; TNF-a 5 tumor necrosis factor a.Ischemic stroke remains a leading cause of disability. Although early reperfusion therapy can improve outcomes, it is available to a minority of stroke patients. Specific therapies are needed to reduce brain injury and improve outcomes after stroke. The immune system responds rapidly following cerebral ischemia. A range of damage-associated molecular patterns, cytokines, and chemokines are released to activate circulating leukocytes, vasculature, and brain cells.

The histone deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for large vessel atherosclerotic stroke (LVAS). In humans, there remains a need to better understand this HDAC9 polymorphism's contribution to large vessel stroke. In this pilot study, we evaluated whether the HDAC9 polymorphism rs2107595 is associated with differences in leukocyte gene expression in patients with LVAS. HDAC9 SNP rs2107595 was genotyped in 155 patients (43 LVAS and 112 vascular risk factor controls). RNA isolated from blood was processed on whole genome microarrays. Gene expression was compared between HDAC9 risk allele-positive and risk allele-negative LVAS patients and controls. Functional analysis identified canonical pathways and molecular functions associated with rs2107595 in LVAS. In HDAC9 SNP rs2107595 risk allele-positive LVAS patients, there were 155 genes differentially expressed compared to risk allele-negative patients (fold change > |1.2|, p < 0.05). The 155 genes separated the risk allele-positive and risk allele-negative LVAS patients on a principal component analysis. Pathways associated with HDAC9 risk allele-positive status involved IL-6 signaling, cholesterol efflux, and platelet aggregation. These preliminary data suggest an association with the HDAC9 rs2107595 risk allele and peripheral immune, lipid, and clotting systems in LVAS. Further study is required to evaluate whether these differences are related to large vessel atherosclerosis and stroke risk.

Interleukin-1β (IL1) is a sleep regulatory substance. The IL1/IL1 type 1 receptor complex requires a receptor accessory protein (AcP) to signal. There are three isoforms of AcP. In the current experiments, mice lacking a neuron-specific isoform, called AcPb knockout (AcPb KO), or mice lacking AcP + AcPb isoforms (AcP KO) or wild-type (WT) mice were used. Spontaneous sleep and sleep responses to sleep deprivation (SD) between zeitgeber time (ZT) 20–ZT4 and ZT8–ZT16 were characterized. Furthermore, somatosensory cortical protein extracts were examined for phosphorylated (p) proto-oncogene tyrosine-protein kinase sarcoma (Src) and p38MAPK levels at ZT4 and ZT16 and after SD. Spontaneous sleep was similar in the three strains, except rapid eye movement sleep (REMS) duration between ZT12–ZT16 was greater in AcP KO than WT mice. After SD at ZT4, only WT mice had non-REMS (NREMS) rebounds. All mouse strains lacked an NREMS rebound after SD at ZT16. All strains after both SD periods had REMS rebounds. AcPb KO mice, but not AcP KO mice, had greater EEG delta wave (0.5–4 Hz) power during NREMS than WT mice. p-Src was very low at ZT16 but high at ZT4, whereas p-p38MAPK was low at ZT4 and high at ZT16. p-p38MAPK levels were not sensitive to SD. In contrast, p-Src levels were less after SD at the P = 0.08 level of significance in the strains lacking AcPb. We conclude that AcPb is required for NREMS responses to sleep loss, but not for SD-induced EEG delta wave or REMS responses. NEW & NOTEWORTHY Interleukin-1β (IL1), a well-characterized sleep regulatory substance, requires an IL1 receptor accessory protein (AcP); one of its isoforms is neuron-specific (called AcPb). We showed that in mice, AcPb is required for nonrapid eye movement sleep responses following 8 h of sleep loss ending 4 h after daybreak but did not affect rapid eye movement sleep rebound. Sleep loss reduced phosphorylation of proto-oncogene tyrosine-protein kinase sarcoma but not of the less sensitive p38MAPK, downstream IL1 signaling molecules.