Browsing by Author "Lee, Han Kyu"
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Item Open Access A cross-species approach using an in vivo evaluation platform in mice demonstrates that sequence variation in human RABEP2 modulates ischemic stroke outcomes.(American journal of human genetics, 2022-10) Lee, Han Kyu; Kwon, Do Hoon; Aylor, David L; Marchuk, Douglas AIschemic stroke, caused by vessel blockage, results in cerebral infarction, the death of brain tissue. Previously, quantitative trait locus (QTL) mapping of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified RAB GTPase-binding effector protein 2 (Rabep2) as the casual gene. However, there is yet no evidence that variation in the human ortholog of this gene plays any role in ischemic stroke outcomes. We established an in vivo evaluation platform in mice by using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 knockout ischemic stroke volume and collateral vessel phenotypes. Importantly, this cross-species complementation enabled us to experimentally investigate the functional effects of coding sequence variation in human RABEP2. We chose four coding variants from the human population that are predicted by multiple in silico algorithms to be damaging to RABEP2 function. In vitro and in vivo analyses verify that all four led to decreased collateral vessel connections and increased infarct volume. Thus, there are naturally occurring loss-of-function alleles. This cross-species approach will expand the number of targets for therapeutics development for ischemic stroke.Item Open Access A Neuroprotective Locus Modulates Ischemic Stroke Infarction Independent of Collateral Vessel Anatomy.(Frontiers in neuroscience, 2021-01) Lee, Han Kyu; Wetzel-Strong, Sarah E; Aylor, David L; Marchuk, Douglas AAlthough studies with inbred strains of mice have shown that infarct size is largely determined by the extent of collateral vessel connections between arteries in the brain that enable reperfusion of the ischemic territory, we have identified strain pairs that do not vary in this vascular phenotype, but which nonetheless exhibit large differences in infarct size. In this study we performed quantitative trait locus (QTL) mapping in mice from an intercross between two such strains, WSB/EiJ (WSB) and C57BL/6J (B6). This QTL mapping revealed only one neuroprotective locus on Chromosome 8 (Chr 8) that co-localizes with a neuroprotective locus we mapped previously from F2 progeny between C3H/HeJ (C3H) and B6. The allele-specific phenotypic effect on infarct volume at the genetic region identified by these two independent mappings was in the opposite direction of the parental strain phenotype; namely, the B6 allele conferred increased susceptibility to ischemic infarction. Through two reciprocal congenic mouse lines with either the C3H or B6 background at the Chr 8 locus, we verified the neuroprotective effects of this genetic region that modulates infarct volume without any effect on the collateral vasculature. Additionally, we surveyed non-synonymous coding SNPs and performed RNA-sequencing analysis to identify potential candidate genes within the genetic interval. Through these approaches, we suggest new genes for future mechanistic studies of infarction following ischemic stroke, which may represent novel gene/protein targets for therapeutic development.Item Open Access Natural allelic variation of the IL-21 receptor modulates ischemic stroke infarct volume.(The Journal of clinical investigation, 2016-08) Lee, Han Kyu; Keum, Sehoon; Sheng, Huaxin; Warner, David S; Lo, Donald C; Marchuk, Douglas ARisk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.Item Open Access Natural genetic variation of integrin alpha L (Itgal) modulates ischemic brain injury in stroke.(PLoS genetics, 2013-01) Keum, Sehoon; Lee, Han Kyu; Chu, Pei-Lun; Kan, Matthew J; Huang, Min-Nung; Gallione, Carol J; Gunn, Michael D; Lo, Donald C; Marchuk, Douglas ADuring ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms.Item Open Access Neuronal IL-4Rα modulates neuronal apoptosis and cell viability during the acute phases of cerebral ischemia.(The FEBS journal, 2018-08) Lee, Han Kyu; Koh, Sehwon; Lo, Donald C; Marchuk, Douglas AIschemic stroke caused by an embolus or local thrombosis results in neural tissue damage (an infarct) in the territory of the occluded cerebral artery. Decades of studies have increased our understanding of the molecular events during cerebral infarction; however, translation of these discoveries to druggable targets for ischemic stroke treatment has been largely disappointing. Interleukin-4 (IL-4) is a multifunctional cytokine that exerts its cellular activities via the interleukin-4 receptor α (IL-4Rα). This cytokine receptor complex is associated with diverse immune and inflammatory responses. Recent studies have suggested a role of the cytokine IL-4 in long-term ischemic stroke recovery, involving immune cell activity. In contrast, the role of the receptor, IL-4Rα especially in the acute phase of infarction is unclear. In this study, we determined that IL-4Rα is expressed on neurons and that during the early phases of cerebral infarction (24 h) levels of this receptor are increased to regulate cellular apoptosis factors through activation of STAT6. In this context, we show a neuroprotective role for IL-4Rα in an in vivo surgical model of cerebral ischemia and in ex vivo brain slice explants, using both genetic knockout of this receptor and RNAi-mediated gene knockdown. IL-4Rα may therefore represent a novel target and pathway for therapeutic development in ischemic stroke.Item Open Access Novel Neuroprotective Loci Modulating Ischemic Stroke Volume in Wild-Derived Inbred Mouse Strains.(Genetics, 2019-11) Lee, Han Kyu; Widmayer, Samuel J; Huang, Min-Nung; Aylor, David L; Marchuk, Douglas ATo identify genes involved in cerebral infarction, we have employed a forward genetic approach in inbred mouse strains, using quantitative trait loci (QTL) mapping for cerebral infarct volume after middle cerebral artery occlusion. We had previously observed that infarct volume is inversely correlated with cerebral collateral vessel density in most strains. In this study, we expanded the pool of allelic variation among classical inbred mouse strains by utilizing the eight founder strains of the Collaborative Cross and found a wild-derived strain, WSB/EiJ, that breaks this general rule that collateral vessel density inversely correlates with infarct volume. WSB/EiJ and another wild-derived strain, CAST/EiJ, show the highest collateral vessel densities of any inbred strain, but infarct volume of WSB/EiJ mice is 8.7-fold larger than that of CAST/EiJ mice. QTL mapping between these strains identified four new neuroprotective loci modulating cerebral infarct volume while not affecting collateral vessel phenotypes. To identify causative variants in genes, we surveyed nonsynonymous coding SNPs between CAST/EiJ and WSB/EiJ and found 96 genes harboring coding SNPs predicted to be damaging and mapping within one of the four intervals. In addition, we performed RNA-sequencing for brain tissue of CAST/EiJ and WSB/EiJ mice and identified 79 candidate genes mapping in one of the four intervals showing strain-specific differences in expression. The identification of the genes underlying these neuroprotective loci will provide new understanding of genetic risk factors of ischemic stroke, which may provide novel targets for future therapeutic intervention of human ischemic stroke.