Corrigendum: What can the common fruit fly teach us about stroke?: lessons learned from the hypoxic tolerant Drosophila melanogaster.

[This corrects the article DOI: 10.3389/fncel.2024.1347980.].

What can the common fruit fly teach us about stroke?: lessons learned from the hypoxic tolerant Drosophila melanogaster.

Stroke, resulting in hypoxia and glucose deprivation, is a leading cause of death and disability worldwide. Presently, there are no treatments that reduce neuronal damage and preserve function aside from tissue plasminogen activator administration and rehabilitation therapy. Interestingly, Drosophila melanogaster, the common fruit fly, demonstrates robust hypoxic tolerance, characterized by minimal effects on survival and motor function following systemic hypoxia. Due to its organized brain, conserved neurotransmitter systems, and genetic similarity to humans and other mammals, uncovering the mechanisms of Drosophila's tolerance could be a promising approach for the development of new therapeutics. Interestingly, a key facet of hypoxic tolerance in Drosophila is organism-wide metabolic suppression, a response involving multiple genes and pathways. Specifically, studies have demonstrated that pathways associated with oxidative stress, insulin, hypoxia-inducible factors, NFκB, Wnt, Hippo, and Notch, all potentially contribute to Drosophila hypoxic tolerance. While manipulating the oxidative stress response and insulin signaling pathway has similar outcomes in Drosophila hypoxia and the mammalian middle cerebral artery occlusion (MCAO) model of ischemia, effects of Notch pathway manipulation differ between Drosophila and mammals. Additional research is warranted to further explore how other pathways implicated in hypoxic tolerance in Drosophila, such as NFκB, and Hippo, may be utilized to benefit mammalian response to ischemia. Together, these studies demonstrate that exploration of the hypoxic response in Drosophila may lead to new avenues of research for stroke treatment in humans.

Beclin 1 regulates astrocyte phagocytosis and phagosomal recruitment of retromer.

Phagocytosis plays an important role in maintaining brain homeostasis and when impaired can result in the accumulation of unwanted cellular material. While microglia are traditionally considered the phagocytes of the brain, astrocytes are also capable of phagocytosis and are the most numerous cells in the brain. In Alzheimer's disease (AD), astrocytes can be found surrounding ß-amyloid (Aß) plaques yet they seem unable to eliminate these deposits, suggesting phagocytosis may be impaired in AD. Mechanisms that might diminish astrocyte phagocytosis in AD are currently unclear. Here, we demonstrate that the autophagy protein beclin 1, which is reduced in AD, plays a role in regulating astrocyte phagocytosis. Specifically, we show that reducing beclin 1 in C6 astrocytes impairs the phagocytosis of latex beads, reduces retromer levels, and impairs retromer recruitment to the phagosomal membrane. Furthermore, we show that these beclin 1-mediated changes are accompanied by reduced expression of the phagocytic receptor Scavenger Receptor Class B type I (SR-BI). Collectively, these findings suggest a critical role for the protein beclin 1 in both receptor trafficking and receptor-mediated phagocytosis in astrocytes. Moreover, these findings provide insight into mechanisms by which astrocytes may become impaired in AD.

IL-4 Is Required for Sex Differences in Vulnerability to Focal Ischemia in Mice.

BACKGROUND AND PURPOSE: Interleukin (IL)-4 protects from middle cerebral artery occlusion in male mice. Females generally show less injury in response to the same ischemic challenge, but the underlying mechanisms are not fully understood. We tested the importance of IL-4 in female protection using IL-4 knockout (KO) mice. METHODS: IL-4 KO and wild-type (WT) mice of both sexes were subjected to middle cerebral artery occlusion. Infarct volume was assessed by triphenyltetrazolium chloride staining and neurobehavioral outcome by neuroscore. T cell proliferation was assessed after Concanavalin A exposure. Ischemic brain immune cell populations were analyzed by fluorescence-activated cell sorting and immunostaining. RESULTS: Infarction in WT females during estrus and proestrus phases was significantly smaller than in males; neurological score was better. Infarction volume was larger and neurological score worse in IL-4 KO compared with WT in both sexes, with no sex difference. Proliferation of T cells was inhibited in WT females with higher proliferation and no sex difference in IL-4 KO. Macrophage numbers and total T cells in the ischemic hemisphere were lower in WT females, and monocytes increased markedly in IL-4 KOs with no sex difference. The reduced macrophage infiltration in WT-females was predominantly M2. Loss of IL-4 increased CD68+ and iNOS+ cells and reduced YM1+ and Arg1+ cells in both sexes. CONCLUSIONS: IL-4 is required for female neuroprotection during the estrus phase of the estrus cycle. Protected WT females show a predominance of M2-activated microglia/macrophages and reduced inflammatory infiltration. Increasing macrophage M2 polarization, with or without added inhibition of infiltration, may be a new approach to stroke treatment.

MicroRNA-200c contributes to injury from transient focal cerebral ischemia by targeting Reelin.

BACKGROUND AND PURPOSE: MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin. METHODS: Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot. RESULTS: Pretreatment with miR-200c antagomir decreased post-middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3' untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection. CONCLUSIONS: These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.

The use of microRNAs to modulate redox and immune response to stroke.

SIGNIFICANCE: Cerebral ischemia is a major cause of death and disability throughout the world, yet therapeutic options remain limited. The interplay between the cellular redox state and the immune response plays a critical role in determining the extent of neural cell injury after ischemia and reperfusion. Excessive amounts of reactive oxygen species (ROS) generated by mitochondria and other sources act both as triggers and effectors of inflammation. This review will focus on the interplay between these two mechanisms. RECENT ADVANCES: MicroRNAs (miRNAs) are important post-transcriptional regulators that interact with multiple target messenger RNAs coordinately regulating target genes, including those involved in controlling mitochondrial function, redox state, and inflammatory pathways. This review will focus on the regulation of mitochondria, ROS, and inflammation by miRNAs in the chain of deleterious intra- and intercellular events that lead to brain cell death after cerebral ischemia. CRITICAL ISSUES: Although pretreatment using miRNAs was effective in cerebral ischemia in rodents, testing treatment after the onset of ischemia is an essential next step in the development of acute stroke treatment. In addition, miRNA formulation and delivery into the CNS remain a challenge in the clinical translation of miRNA therapy. FUTURE DIRECTIONS: Future research should focus on post-treatment and potential clinical use of miRNAs.

Mitigation of murine focal cerebral ischemia by the hypocretin/orexin system is associated with reduced inflammation.

BACKGROUND AND PURPOSE: Brain ischemia causes immediate and delayed cell death that is exacerbated by inflammation. Recent studies show that hypocretin-1/orexin-A (Hcrt-1) reduces ischemic brain injury, and Hcrt-positive neurons modulate infection-induced inflammation. Here, we tested the hypothesis that Hcrt plays a protective role against ischemia by modulating inflammation. METHODS: Orexin/ataxin-3 (AT) mice, a transgenic strain in which Hcrt-producing neurons degenerate in early adulthood, and wild-type mice were subjected to transient middle cerebral artery occlusion (MCAO). Infarct volume, neurological score, and spontaneous home cage activity were assessed. Inflammation was measured using immunohistochemistry, ELISA, and assessment of cytokine mRNA levels. RESULTS: Infarct volumes 24 and 48 hours after MCAO were significantly larger, neurological score was worse, and spontaneous activity decreased in AT compared with wild-type mice. Macrophage/microglial infiltration and myeloperoxidase-positive cells were higher in AT compared with wild-type mice. Pre-MCAO intracerebroventricular injection of Hcrt-1 significantly reduced infarct volume and macrophage/microglial infiltration in both genotypes and improved neurological score in AT mice. Post-MCAO treatment decreased infarct size in both wild-type and AT mice, but had no effect on neurological score in either genotype. Microglia express the Hcrt-1 receptor after MCAO. Tumor necrosis factor-α production by lipopolysaccharide-stimulated microglial BV2 cells was significantly reduced by Hcrt-1 pretreatment. Sham AT mice exhibit increased brain tumor necrosis factor-α and interleukin-6 mRNA, suggesting chronic inflammation. CONCLUSIONS: Loss of Hcrt neurons in AT mice resulted in worsened stroke outcomes, which were reversed by administration of exogenous Hcrt-1. The mechanism underlying Hcrt-mediated neuroprotection includes attenuation of inflammatory responses after ischemic insult.

Effects of heat shock protein 72 (Hsp72) on evolution of astrocyte activation following stroke in the mouse.

Astrocyte activation is a hallmark of the response to brain ischemia consisting of changes in gene expression and morphology. Heat shock protein 72 (Hsp72) protects from cerebral ischemia, and although several protective mechanisms have been investigated, effects on astrocyte activation have not been studied. To identify potential mechanisms of protection, microarray analysis was used to assess gene expression in the ischemic hemispheres of wild-type (WT) and Hsp72-overexpressing (Hsp72Tg) mice 24 h after middle cerebral artery occlusion or sham surgery. After stroke both genotypes exhibited changes in genes related to apoptosis, inflammation, and stress, with more downregulated genes in Hsp72Tg and more inflammation-related genes increased in WT mice. Genes indicative of astrocyte activation were also upregulated in both genotypes. To measure the extent and time course of astrocyte activation after stroke, detailed histological and morphological analyses were performed in the cortical penumbra. We observed a marked and persistent increase in glial fibrillary acidic protein (GFAP) and a transient increase in vimentin. No change in overall astrocyte number was observed based on glutamine synthetase immunoreactivity. Hsp72Tg and WT mice were compared for density of astrocytes expressing activation markers and astrocytic morphology. In animals with comparable infarct size, overexpression of Hsp72 reduced the density of GFAP- and vimentin-expressing cells, and decreased astrocyte morphological complexity 72 h following stroke. However, by 30 days astrocyte activation was similar between genotypes. These data indicate that early modulation of astrocyte activation provides an additional novel mechanism associated with Hsp72 overexpression in the setting of ischemia.

Mice lacking the ß2 adrenergic receptor have a unique genetic profile before and after focal brain ischaemia.

The role of the ß2AR (ß2 adrenergic receptor) after stroke is unclear as pharmacological manipulations of the ß2AR have produced contradictory results. We previously showed that mice deficient in the ß2AR (ß2KO) had smaller infarcts compared with WT (wild-type) mice (FVB) after MCAO (middle cerebral artery occlusion), a model of stroke. To elucidate mechanisms of this neuroprotection, we evaluated changes in gene expression using microarrays comparing differences before and after MCAO, and differences between genotypes. Genes associated with inflammation and cell deaths were enriched after MCAO in both genotypes, and we identified several genes not previously shown to increase following ischaemia (Ccl9, Gem and Prg4). In addition to networks that were similar between genotypes, one network with a central core of GPCR (G-protein-coupled receptor) and including biological functions such as carbohydrate metabolism, small molecule biochemistry and inflammation was identified in FVB mice but not in ß2KO mice. Analysis of differences between genotypes revealed 11 genes differentially expressed by genotype both before and after ischaemia. We demonstrate greater Glo1 protein levels and lower Pmaip/Noxa mRNA levels in ß2KO mice in both sham and MCAO conditions. As both genes are implicated in NF-κB (nuclear factor κB) signalling, we measured p65 activity and TNFα (tumour necrosis factor α) levels 24 h after MCAO. MCAO-induced p65 activation and post-ischaemic TNFα production were both greater in FVB compared with ß2KO mice. These results suggest that loss of ß2AR signalling results in a neuroprotective phenotype in part due to decreased NF-κB signalling, decreased inflammation and decreased apoptotic signalling in the brain.

MicroRNA-320 induces neurite outgrowth by targeting ARPP-19.

MicroRNAs are important in the development, functioning, and pathophysiology of the central nervous system. Here, we show that increasing the levels of microRNA-320 (miR-320) for 3 days markedly increases neurite length, and at 4 days, reduces the total cell number in Neuro-2A cells. In-silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a as potential targets that could be involved. ARPP-19 was validated by showing reduced mRNA and protein levels when miR-320 was overexpressed, whereas miR-320 had no effect on semaphorin 3a expression. ARPP-19 is known to inhibit protein phosphatase-2A activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus, increased levels of miR-320 lead to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury.

miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo.

MicroRNAs (miRNA) are short (~22nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

Transforming growth factor α transforms astrocytes to a growth-supportive phenotype after spinal cord injury.

Astrocytes are both detrimental and beneficial for repair and recovery after spinal cord injury (SCI). These dynamic cells are primary contributors to the growth-inhibitory glial scar, yet they are also neuroprotective and can form growth-supportive bridges on which axons traverse. We have shown that intrathecal administration of transforming growth factor α (TGFα) to the contused mouse spinal cord can enhance astrocyte infiltration and axonal growth within the injury site, but the mechanisms of these effects are not well understood. The present studies demonstrate that the epidermal growth factor receptor (EGFR) is upregulated primarily by astrocytes and glial progenitors early after SCI. TGFα directly activates the EGFR on these cells in vitro, inducing their proliferation, migration, and transformation to a phenotype that supports robust neurite outgrowth. Overexpression of TGFα in vivo by intraparenchymal adeno-associated virus injection adjacent to the injury site enhances cell proliferation, alters astrocyte distribution, and facilitates increased axonal penetration at the rostral lesion border. To determine whether endogenous EGFR activation is required after injury, SCI was also performed on Velvet (C57BL/6J-Egfr(Vel)/J) mice, a mutant strain with defective EGFR activity. The affected mice exhibited malformed glial borders, larger lesions, and impaired recovery of function, indicating that intrinsic EGFR activation is necessary for neuroprotection and normal glial scar formation after SCI. By further stimulating precursor proliferation and modifying glial activation to promote a growth-permissive environment, controlled stimulation of EGFR at the lesion border may be considered in the context of future strategies to enhance endogenous cellular repair after injury.

Astrocytes: targets for neuroprotection in stroke.

In the past two decades, over 1000 clinical trials have failed to demonstrate a benefit in treating stroke, with the exception of thrombolytics. Although many targets have been pursued, including antioxidants, calcium channel blockers, glutamate receptor blockers, and neurotrophic factors, often the focus has been on neuronal mechanisms of injury. Broader attention to loss and dysfunction of non-neuronal cell types is now required to increase the chance of success. Of the several glial cell types, this review will focus on astrocytes. Astrocytes are the most abundant cell type in the higher mammalian nervous system, and they play key roles in normal CNS physiology and in central nervous system injury and pathology. In the setting of ischemia astrocytes perform multiple functions, some beneficial and some potentially detrimental, making them excellent candidates as therapeutic targets to improve outcome following stroke and in other central nervous system injuries. The older neurocentric view of the central nervous system has changed radically with the growing understanding of the many essential functions of astrocytes. These include K+ buffering, glutamate clearance, brain antioxidant defense, close metabolic coupling with neurons, and modulation of neuronal excitability. In this review, we will focus on those functions of astrocytes that can both protect and endanger neurons, and discuss how manipulating these functions provides a novel and important strategy to enhance neuronal survival and improve outcome following cerebral ischemia.

Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice.

Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice 3 days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post-injury (DPI). BrdU-labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all timepoints. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations that could be targeted by interventions.

Don't fence me in: harnessing the beneficial roles of astrocytes for spinal cord repair.

Astrocytes comprise a heterogeneous cell population that plays a complex role in repair after spinal cord injury. Reactive astrocytes are major contributors to the glial scar that is a physical and chemical barrier to axonal regeneration. Yet, consistent with a supportive role in development, astrocytes secrete neurotrophic factors and protect neurons and glia spared by the injury. In development and after injury, local cues are modulators of astrocyte phenotype and function. When multipotent cells are transplanted into the injured spinal cord, they differentiate into astrocytes and other glial cells as opposed to neurons, which is commonly viewed as a challenge to be overcome in developing stem cell technology. However, several examples show that astrocytes provide support and guidance for axonal growth and aid in improving functional recovery after spinal cord injury. Notably, transplantation of astrocytes of a developmentally immature phenotype promotes tissue sparing and axonal regeneration. Furthermore, interventions that enhance endogenous astrocyte migration or reinvasion of the injury site result in greater axonal growth. These studies demonstrate that astrocytes are dynamic, diverse cells that have the capacity to promote axon growth after injury. The ability of astrocytes to be supportive of recovery should be exploited in devising regenerative strategies.

TGF-alpha increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice.

Astrocytes respond to environmental cues and play a multifaceted role in the response to trauma in the central nervous system. As the most prevalent contributors to the glial scar, astrocytes are targeted as barriers to regeneration. However, there is also strong evidence that astrocytes are vital for neuroprotection and metabolic support after injury. In addition, consistent with their role during development, astrocytes may be capable of supporting the growth of injured axons. Therefore, we hypothesized that with appropriate stimulation, the reparative functions of endogenous astrocytes could be harnessed to promote axon growth and recovery after spinal cord injury. Transforming growth factor-alpha (TGF-alpha) is a mitogenic growth factor that is active on astrocytes and is poised to contribute to such a strategy. Recombinant TGF-alpha was administered intrathecally to adult C57BL/6 mice for two weeks following a moderate mid-thoracic spinal cord contusion. By three weeks post-injury, TGF-alpha infusion had not affected locomotor recovery, but promoted extensive axon growth and altered the composition of the lesion site. The center of the lesion in the treated mice contained greater numbers of new cells and increased astrocyte invasion. Despite the expression of inhibitory proteoglycans, there was a marked increase in axons expressing neurofilament and GAP-43 immunoreactivity, and the new axons were closely associated with increased laminin expression within and beyond the astrocyte matrix. The results demonstrate that astrocytes are dynamic players in the response to spinal cord injury, and the growth-supportive role of these cells can be enhanced by TGF-alpha infusion.