Biology subjects

Marchuk, D. A.

Publications and source records attributed to Marchuk, D. A..

7 recordsLinked to original sources

bioRxiv2025

Targeting the ferroptosis pathway: A novel compound, AZD1390, protects the brain after ischemic stroke

BackgroundFerroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. MethodsWe investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo, and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. ResultATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo. ConclusionsThis study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

neuroscience↗

bioRxiv2022

A cross-species approach using an in vivo evaluation platform in mice demonstrates that sequence variation in the human RABEP2 gene modulates ischemic stroke outcomes

Ischemic stroke, caused by vessel blockage, results in cerebral infarction; the death of brain tissue. Previously, quantitative trait locus mapping (QTL) of cerebral infarct volume and collateral vessel number identified a single, strong genetic locus regulating both phenotypes. Additional studies identified the causative gene, encoding RAB GTPase Binding Effector Protein 2 (Rabep2). 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 using adeno-associated virus (AAV) gene replacement and verified that both mouse and human RABEP2 rescue the mouse Rabep2 KO 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 the human RABEP2 gene. 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.

genetics↗

bioRxiv2021

Developmental Venous Anomalies are a Genetic Primer for Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCM) are a neurovascular anomaly that may occur sporadically in otherwise healthy individuals, or be inherited by autosomal dominant mutations in the genes that encode the proteins of the CCM signaling complex (KRIT1, CCM2, or PDCD10)1-4. CCMs have long been known to follow a genetic two-hit model where lesion formation is initiated by somatic mutations resulting in biallelic loss of a CCM complex gene5-8. Recent studies have shown that somatic mutations in MAP3K3 and PIK3CA also contribute to CCM pathogenesis9-11; however, it remains unclear how these mutations contribute to sporadic versus familial cases. Here we show that somatic mutations in MAP3K3 are mutually exclusive with mutations in CCM complex genes and that mutations in MAP3K3 contribute to sporadic, but not familial CCM. Using single-nucleus DNA sequencing, we show that co-occurring MAP3K3 and PIK3CA mutations are present within the same clonal population of cells. Furthermore, we identify PIK3CA mutations in CCM-associated developmental venous anomalies (DVA). It has long been known that sporadic CCM often develop in the vicinity of a DVA. However, the underlying cause of this association is unknown12-14. In this first report of the molecular pathology of CCM-associated DVA, we find that the identical PIKC3A mutation is found in both the DVA and its associated CCM, but that an activating MAP3K3 mutation appears only in the CCM. These results support a mechanism where DVA develop as the result of a PIK3CA mutation, creating a region of the brain vasculature that functions as a genetic primer for CCM development following acquisition of an additional somatic mutation.

genetics↗

bioRxiv2020

A neuroprotective locus modulates ischemic stroke infarction independent of collateral vessel anatomy

Ischemic stroke is caused by a disruption of the blood supply to the brain leading to neuronal cell death. Genetic studies of ischemic stroke have identified numerous gene variants that increase the risk to develop stroke. In stark contrast, genetic studies of stroke outcomes, such as the infarct territory size, are confounded by many uncontrollable variables, leading to a paucity of gene targets for treatment of an incipient stroke. Using genetically diverse inbred strains of mice and a surgically-induced model of ischemic stroke, we used quantitative trait locus mapping to identify novel gene targets modulating infarct size, which varies greatly across inbred strains. Although infarct size is largely determined by the extent of collateral vessel connection between arteries in the brain that enables reperfusion of the ischemic territory, we have identified strain pairs that do not vary in this phenotype, but which nonetheless exhibit large differences in infarct size. In this study we performed QTL mapping in mice from an intercross between two such strains, WSB/EiJ and C57BL/6J. We identified a strong locus on chromosome 8 that overlaps with a locus of similar direction and effect previously mapped in an intercross between C3H/HeJ and C57BL/6J strains. To identify causative genes within the overlapping genetic interval, we surveyed nonsynonymous coding SNPs and performed RNA sequencing data analysis for all three mapping strains. We identified Macrophage Scavenger Receptor 1 (Msr1) as a strong candidate gene that harbors multiple coding SNPs predicted to be damaging. Using Msr1-deficient mice, we demonstrated that cerebral infarct volume after stroke induction is dramatically increased in a strain background where reperfusion effects due to collateral vessels is blunted. Significantly, the identification of neuroprotective genes such as Msr1 provides new genes for future mechanistic studies of infarction following ischemic stroke and provides novel gene/protein targets for therapeutic development. Author summaryThe most common form of stroke arises when a blockage occurs in a blood vessel of the brain, thereby preventing delivery of oxygen and nutrients to areas supplied by the affected vessel, leading to tissue death. The main treatment for this form of stroke is medication to dissolve the blockage; however, more treatment options are required to better reduce the death and disability associated with stroke. In this study, we sought to identify genes that can decrease the amount of damage to brain tissue following a stroke, with a specific focus on examining genes that work to directly protect the neurons, rather than returning blood flow to the affected area. Since it is impossible to precisely control the nature of stroke and the genetic variability in humans, we used mice identify a genetic region that is associated with the amount of tissue damage following stroke. Within this genetic region, we identified a list of candidate genes, including the gene Msr1, which we found is important for controlling tissue damage in one genetic population of mice after stroke. The genes identified here require further follow-up to determine the impact on stroke outcomes and the usefulness of these candidates as therapeutic targets.

genetics↗

bioRxiv2020

Novel hemorrhage models of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are ectatic capillary-venous malformations that develop in approximately 0.5% of the population. Patients with CCMs may develop headaches, focal neurologic deficits, seizures, and hemorrhages. While symptomatic CCMs, depending upon the anatomic location, can be surgically removed, there is currently no pharmaceutical therapy to treat CCMs. Several mouse models have been developed to better understand CCM pathogenesis and test therapeutics. The most common mouse models induce a large CCM burden that is anatomically restricted to the cerebellum and contributes to lethality in the early days of life. These inducible models thus have a relatively short period for drug administration. We developed an inducible CCM3 mouse model that develops CCMs after weaning and provides a longer period for potential therapeutic intervention. Using this new model, three recently proposed CCM therapies - fasudil, tempol, vitamin D3, and a combination of the three drugs failed to substantially reduce CCM formation when treatment was administered for five weeks, from postnatal day 21 (P21) to P56. We next restricted Ccm3 deletion to the brain vasculature and provided greater time (121 days) for CCMs to develop chronic hemorrhage, recapitulating the human lesions. We also developed the first model of acute CCM hemorrhage by injecting mice harboring CCMs with lipopolysaccharide. These efficient models will enable future drug studies to more precisely target clinically relevant features of CCM disease: CCM formation, chronic hemorrhage, and acute hemorrhage.

genetics↗

bioRxiv2019

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Biallelic Loss of ENG or ACVRL1

Hereditary Hemorrhagic Telangiectasia (HHT) is a Mendelian disease characterized by vascular malformations including visceral arteriovenous malformations and mucosal telangiectasia. HHT is caused by loss-of-function mutations in one of 3 genes; ENG, ACVRL1 or SMAD4 and is inherited as an autosomal dominant condition. Intriguingly, the constitutional mutation causing HHT is present throughout the body, yet the multiple vascular malformations in HHT patients occur focally, rather than manifesting as a systemic vascular defect. This disconnect between genotype and phenotype suggests that a local event is necessary for the development of vascular malformations. We investigated the hypothesis that local somatic mutations seed the formation HHT-related telangiectasia in a genetic two-hit mechanism. We identified low-frequency somatic mutations in 9/19 telangiectasia using high depth next-generation sequencing. We established phase for 7 of 9 samples using long-read sequencing, which confirm that the germline and somatic mutations in all 7 samples exist in trans configuration; consistent with a genetic two-hit mechanism. These combined data suggest that biallelic loss of ENG or ACVRL1 may be a required event in the development of telangiectasia, and that rather than haploinsufficiency, vascular malformations in HHT are caused by a Knudsonian two-hit mechanism.

genetics↗

bioRxiv2018

Cerebral Cavernous Malformations Develop through Clonal Expansion of Mutant Endothelial Cells

RationaleVascular malformations arise in vessels throughout the entire body. Causative genetic mutations have been identified for many of these diseases; however, little is known about the mutant cell lineage within these malformations.\n\nObjectiveWe utilize an inducible mouse model of cerebral cavernous malformations (CCMs) coupled with a multi-color fluorescent reporter to visualize the contribution of mutant endothelial cells (ECs) to the malformation.\n\nMethods and ResultsWe combined a Ccm3 mouse model with the confetti fluorescent reporter to simultaneously delete Ccm3 and label the mutant EC with one of four possible colors. We acquired Z-series confocal images from serial brain sections and created 3D reconstructions of entire CCMs to visualize mutant ECs during CCM development. We observed a pronounced pattern of CCMs lined with mutant ECs labeled with a single confetti color (n=42). The close 3D distribution, as determined by the nearest neighbor analysis, of the clonally dominant ECs within the CCM was statistically different than the background confetti labeling of ECs in non-CCM control brain slices as well as a computer simulation (p<0.001). Many of the small (<100m diameter) CCMs consisted, almost exclusively, of the clonally dominant mutant ECs labeled with the same confetti color whereas the large (>100m diameter) CCMs contained both the clonally dominant mutant cells and wildtype ECs. We propose of model of CCM development in which an EC acquires a second somatic mutation, undergoes clonal expansion to initiate CCM formation, and then incorporates neighboring wildtype ECs to increase the size of the malformation.\n\nConclusionsThis is the first study to visualize, with single-cell resolution, the clonal expansion of mutant ECs within CCMs. The incorporation of wildtype ECs into the growing malformation presents another series of cellular events whose elucidation would enhance our understanding of CCMs and may provide novel therapeutic opportunities.

genetics↗