ATF5-Mediated Mitochondrial Unfolded Protein Response (UPRmt) Protects Neurons Against Oxygen-Glucose Deprivation and Cerebral Ischemia.

BACKGROUND: The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPRmt may be a potential therapeutic target for ischemic stroke. METHODS: We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPRmt. We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro. RESULTS: Inducing UPRmt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPRmt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPRmt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPRmt in vivo, which reduced infarction and improved neurological outcomes. CONCLUSIONS: These findings suggest that the UPRmt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPRmt mechanism may provide a new therapeutic avenue for ischemic stroke.

Circadian and Diurnal Regulation of Cerebral Blood Flow.

Circadian and diurnal variation in cerebral blood flow directly contributes to the diurnal variation in the risk of stroke, either through factors that trigger stroke or due to impaired compensatory mechanisms. Cerebral blood flow results from the integration of systemic hemodynamics, including heart rate, cardiac output, and blood pressure, with cerebrovascular regulatory mechanisms, including cerebrovascular reactivity, autoregulation, and neurovascular coupling. We review the evidence for the circadian and diurnal variation in each of these mechanisms and their integration, from the detailed evidence for mechanisms underlying the nocturnal nadir and morning surge in blood pressure to identifying limited available evidence for circadian and diurnal variation in cerebrovascular compensatory mechanisms. We, thus, identify key systemic hemodynamic factors related to the diurnal variation in the risk of stroke but particularly identify the need for further research focused on cerebrovascular regulatory mechanisms.

Transcriptomic Profiling Reveals Neuroinflammation in the Corpus Callosum of a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a widespread neurodegenerative disorder characterized by progressive cognitive decline, affecting a significant portion of the aging population. While the cerebral cortex and hippocampus have been the primary focus of AD research, accumulating evidence suggests that white matter lesions in the brain, particularly in the corpus callosum, play an important role in the pathogenesis of the disease. OBJECTIVE: This study aims to investigate the gene expression changes in the corpus callosum of 5xFAD transgenic mice, a widely used AD mouse model. METHODS: We conducted behavioral tests for spatial learning and memory in 5xFAD transgenic mice and performed RNA sequencing analyses on the corpus callosum to examine transcriptomic changes. RESULTS: Our results show cognitive decline and demyelination in the corpus callosum of 5xFAD transgenic mice. Transcriptomic analysis reveals a predominance of upregulated genes in AD mice, particularly those associated with immune cells, including microglia. Conversely, downregulation of genes related to chaperone function and clock genes such as Per1, Per2, and Cry1 is also observed. CONCLUSIONS: This study suggests that activation of neuroinflammation, disruption of chaperone function, and circadian dysfunction are involved in the pathogenesis of white matter lesions in AD. The findings provide insights into potential therapeutic targets and highlight the importance of addressing white matter pathology and circadian dysfunction in AD treatment strategies.

Not open and shut: Complex and prolonged blood-brain barrier responses after stroke.

Blood-brain barrier dysfunction (BBB) occurs rapidly after stroke and contributes to edema, inflammation, and secondary brain injury including haemorrhage. Two recent studies shed light on the temporal extent of post-stroke BBB dysfunction as well as its consequences for drug delivery. Zhang et al. found increases in BBB permeability that persist up to one-year post-ischemia. Despite increased paracellular leakage, Stanton et al. showed that transcellular transporter systems are required to deliver therapeutics into brain parenchyma. Both studies remind us of the complexity of BBB responses after stroke and provide novel entry points for future research into the underlying mechanisms.

O-GlcNAcylation is essential for therapeutic mitochondrial transplantation.

BACKGROUND: Transplantation of mitochondria is increasingly explored as a novel therapy in central nervous system (CNS) injury and disease. However, there are limitations in safety and efficacy because mitochondria are vulnerable in extracellular environments and damaged mitochondria can induce unfavorable danger signals. METHODS: Mitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase (OGT) and UDP-GlcNAc. O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry and the antiglycation ability of O-GlcNAcylated DJ1 was determined by loss-of-function via mutagenesis. Therapeutic efficacy of O-GlcNAcylated mitochondria was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. To explore translational potential, we evaluated O-GlcNAcylated DJ1 in CSF collected from patients with subarachnoid hemorrhagic stroke (SAH). RESULTS: We show that isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria induce the receptor for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress when transferred into neurons. However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves viability of mitochondria recipient neurons. In a mouse model of stroke, treatment with extracellular mitochondria modified by O-GlcNAcylation reduces neuronal injury and improves neurologic deficits. In cerebrospinal fluid (CSF) samples from SAH patients, levels of O-GlcNAcylation in extracellular mitochondria correlate with better clinical outcomes. CONCLUSIONS: These findings suggest that AGE-modification in extracellular mitochondria may induce danger signals, but O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS.

Mitochondria are the part of a cell that generate most of its energy to perform its functions. In injury or disease, mitochondrial function can become disrupted. Transplantation of healthy mitochondria is being explored as a potential therapy to replace damaged mitochondria and restore normal cellular function. However, this approach is difficult to perform because mitochondria are not able to maintain their healthy state outside of cells. Here, we show that one of the reasons for this is due to a molecular process called advanced glycation end product modification. We show that simple modification of mitochondria with a sugar prevents this process and helps to improve the success of therapeutic mitochondrial transplantation in cells and in a mouse model of stroke. Our findings may help to guide future efforts to develop therapies based on mitochondrial transplantation.

Functional magnetic particle imaging (fMPI) of cerebrovascular changes in the rat brain during hypercapnia.

Objective.Non-invasive functional brain imaging modalities are limited in number, each with its own complex trade-offs between sensitivity, spatial and temporal resolution, and the directness with which the measured signals reflect neuronal activation. Magnetic particle imaging (MPI) directly maps the cerebral blood volume (CBV), and its high sensitivity derives from the nonlinear magnetization of the superparamagnetic iron oxide nanoparticle (SPION) tracer confined to the blood pool. Our work evaluates functional MPI (fMPI) as a new hemodynamic functional imaging modality by mapping the CBV response in a rodent model where CBV is modulated by hypercapnic breathing manipulation.Approach.The rodent fMPI time-series data were acquired with a mechanically rotating field-free line MPI scanner capable of 5 s temporal resolution and 3 mm spatial resolution. The rat's CBV was modulated for 30 min with alternating 5 min hyper-/hypocapnic states, and processed using conventional fMRI tools. We compare our results to fMRI responses undergoing similar hypercapnia protocols found in the literature, and reinforce this comparison in a study of one rat with 9.4T BOLD fMRI using the identical protocol.Main results.The initial image in the time-series showed mean resting brain voxel SNR values, averaged across rats, of 99.9 following the first 10 mg kg-1SPION injection and 134 following the second. The time-series fit a conventional General Linear Model with a 15%-40% CBV change and a peak pixel CNR between 12 and 29, 2-6× higher than found in fMRI.Significance.This work introduces a functional modality with high sensitivity, although currently limited spatial and temporal resolution. With future clinical-scale development, a large increase in sensitivity could supplement other modalities and help transition functional brain imaging from a neuroscience tool focusing on population averages to a clinically relevant modality capable of detecting differences in individual patients.

Ultraflexible endovascular probes for brain recording through micrometer-scale vasculature.

Implantable neuroelectronic interfaces have enabled advances in both fundamental research and treatment of neurological diseases but traditional intracranial depth electrodes require invasive surgery to place and can disrupt neural networks during implantation. We developed an ultrasmall and flexible endovascular neural probe that can be implanted into sub-100-micrometer-scale blood vessels in the brains of rodents without damaging the brain or vasculature. In vivo electrophysiology recording of local field potentials and single-unit spikes have been selectively achieved in the cortex and olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

Aerobic exercise reverses aging-induced depth-dependent decline in cerebral microcirculation.

Aging is a major risk factor for cognitive impairment. Aerobic exercise benefits brain function and may promote cognitive health in older adults. However, underlying biological mechanisms across cerebral gray and white matter are poorly understood. Selective vulnerability of the white matter to small vessel disease and a link between white matter health and cognitive function suggests a potential role for responses in deep cerebral microcirculation. Here, we tested whether aerobic exercise modulates cerebral microcirculatory changes induced by aging. To this end, we carried out a comprehensive quantitative examination of changes in cerebral microvascular physiology in cortical gray and subcortical white matter in mice (3-6 vs. 19-21 months old), and asked whether and how exercise may rescue age-induced deficits. In the sedentary group, aging caused a more severe decline in cerebral microvascular perfusion and oxygenation in deep (infragranular) cortical layers and subcortical white matter compared with superficial (supragranular) cortical layers. Five months of voluntary aerobic exercise partly renormalized microvascular perfusion and oxygenation in aged mice in a depth-dependent manner, and brought these spatial distributions closer to those of young adult sedentary mice. These microcirculatory effects were accompanied by an improvement in cognitive function. Our work demonstrates the selective vulnerability of the deep cortex and subcortical white matter to aging-induced decline in microcirculation, as well as the responsiveness of these regions to aerobic exercise.

Ultra-flexible endovascular probes for brain recording through micron-scale vasculature.

Implantable neuroelectronic interfaces have enabled significant advances in both fundamental research and treatment of neurological diseases, yet traditional intracranial depth electrodes require invasive surgery to place and can disrupt the neural networks during implantation. To address these limitations, we have developed an ultra-small and flexible endovascular neural probe that can be implanted into small 100-micron scale blood vessels in the brains of rodents without damaging the brain or vasculature. The structure and mechanical properties of the flexible probes were designed to meet the key constraints for implantation into tortuous blood vessels inaccessible with existing techniques. In vivo electrophysiology recording of local field potentials and single-unit spikes has been selectively achieved in the cortex and the olfactory bulb. Histology analysis of the tissue interface showed minimal immune response and long-term stability. This platform technology can be readily extended as both research tools and medical devices for the detection and intervention of neurological diseases.

Aerobic exercise reverses aging-induced depth-dependent decline in cerebral microcirculation.

Aging is a major risk factor for cognitive impairment. Aerobic exercise benefits brain function and may promote cognitive health in older adults. However, underlying biological mechanisms across cerebral gray and white matter are poorly understood. Selective vulnerability of the white matter to small vessel disease and a link between white matter health and cognitive function suggests a potential role for responses in deep cerebral microcirculation. Here, we tested whether aerobic exercise modulates cerebral microcirculatory changes induced by aging. To this end, we carried out a comprehensive quantitative examination of changes in cerebral microvascular physiology in cortical gray and subcortical white matter in mice (3-6 vs. 19-21 months old), and asked whether and how exercise may rescue age-induced deficits. In the sedentary group, aging caused a more severe decline in cerebral microvascular perfusion and oxygenation in deep (infragranular) cortical layers and subcortical white matter compared with superficial (supragranular) cortical layers. Five months of voluntary aerobic exercise partly renormalized microvascular perfusion and oxygenation in aged mice in a depth-dependent manner, and brought these spatial distributions closer to those of young adult sedentary mice. These microcirculatory effects were accompanied by an improvement in cognitive function. Our work demonstrates the selective vulnerability of the deep cortex and subcortical white matter to aging-induced decline in microcirculation, as well as the responsiveness of these regions to aerobic exercise.

Endothelial cells regulate astrocyte to neural progenitor cell trans-differentiation in a mouse model of stroke.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.

Diurnal Differences in Immune Response in Brain, Blood and Spleen After Focal Cerebral Ischemia in Mice.

BACKGROUND: The immune response to acute cerebral ischemia is a major factor in stroke pathobiology. Circadian biology modulates some aspects of immune response. The goal of this study is to compare key parameters of immune response during the active/awake phase versus inactive/sleep phase in a mouse model of transient focal cerebral ischemia. METHODS: Mice were housed in normal or reversed light cycle rooms for 3 weeks, and then they were blindly subjected to transient focal cerebral ischemia. Flow cytometry was used to examine immune responses in blood, spleen, and brain at 3 days after ischemic onset. RESULTS: In blood, there were higher levels of circulating T cells in mice subjected to focal ischemia during zeitgeber time (ZT)1-3 (inactive or sleep phase) versus ZT13-15 mice (active or awake phase). In the spleen, organ weight and immune cell numbers were lower in ZT1-3 versus ZT13-15 mice. Consistent with these results, there was an increased infiltration of activated T cells into brain at ZT1-3 compared with ZT13-15. CONCLUSIONS: This proof-of-concept study indicates that there are significant diurnal effects on the immune response after focal cerebral ischemia in mice. Hence, therapeutic strategies focused on immune targets should be reassessed to account for the effects of diurnal rhythms and circadian biology in nocturnal rodent models of stroke.

Fingolimod Does Not Reduce Infarction After Focal Cerebral Ischemia in Mice During Active or Inactive Circadian Phases.

BACKGROUND: It has been reported that the S1P (sphingosine 1-phosphate) receptor modulator fingolimod reduces infarction in rodent models of stroke. Recent studies have suggested that circadian rhythms affect stroke and neuroprotection. Therefore, this study revisited the use of fingolimod in mouse focal cerebral ischemia to test the hypothesis that efficacy might depend on whether experiments were performed during the inactive sleep or active wake phases of the circadian cycle. METHODS: Two different stroke models were implemented in male C57Bl/6 mice-transient middle cerebral artery occlusion and permanent distal middle cerebral artery occlusion. Occlusion occurred either during inactive or active circadian phases. Mice were treated with 1 mg/kg fingolimod at 30- or 60-minute postocclusion and 1 day later for permanent and transient middle cerebral artery occlusion, respectively. Infarct volume, brain swelling, hemorrhagic transformation, and behavioral outcome were assessed at 2 or 3 days poststroke. Three independent experiments were performed in 2 different laboratories. RESULTS: Fingolimod decreased peripheral lymphocyte number in naive mice, as expected. However, it did not significantly affect infarct volume, brain swelling, hemorrhagic transformation, or behavioral outcome at 2 or 3 days after transient or permanent focal cerebral ischemia during inactive or active circadian phases of stroke onset. CONCLUSIONS: Outcomes were not improved by fingolimod in either transient or permanent focal cerebral ischemia during both active and inactive circadian phases. These negative findings suggest that further testing of fingolimod in clinical trials may not be warranted unless translational studies can identify factors associated with fingolimod's efficacy or lack thereof.

Transcriptome Profiling of Mouse Corpus Callosum After Cerebral Hypoperfusion.

White matter damage caused by cerebral hypoperfusion is a major hallmark of subcortical ischemic vascular dementia (SIVD), which is the most common subtype of vascular cognitive impairment and dementia (VCID) syndrome. In an aging society, the number of SIVD patients is expected to increase; however, effective therapies have yet to be developed. To understand the pathological mechanisms, we analyzed the profiles of the cells of the corpus callosum after cerebral hypoperfusion in a preclinical SIVD model. We prepared cerebral hypoperfused mice by subjecting 2-month old male C57BL/6J mice to bilateral carotid artery stenosis (BCAS) operation. BCAS-hypoperfusion mice exhibited cognitive deficits at 4 weeks after cerebral hypoperfusion, assessed by novel object recognition test. RNA samples from the corpus callosum region of sham- or BCAS-operated mice were then processed using RNA sequencing. A gene set enrichment analysis using differentially expressed genes between sham and BCAS-operated mice showed activation of oligodendrogenesis pathways along with angiogenic responses. This database of transcriptomic profiles of BCAS-hypoperfusion mice will be useful for future studies to find a therapeutic target for SIVD.

Circadian Biology and Stroke.

Circadian biology modulates almost all aspects of mammalian physiology, disease, and response to therapies. Emerging data suggest that circadian biology may significantly affect the mechanisms of susceptibility, injury, recovery, and the response to therapy in stroke. In this review/perspective, we survey the accumulating literature and attempt to connect molecular, cellular, and physiological pathways in circadian biology to clinical consequences in stroke. Accounting for the complex and multifactorial effects of circadian rhythm may improve translational opportunities for stroke diagnostics and therapeutics.

Biphasic roles of pentraxin 3 in cerebrovascular function after white matter stroke.

Recent clinical studies suggest that pentraxin 3 (PTX3), which is known as an acute-phase protein that is produced rapidly at local sites of inflammation, may be a new biomarker of disease risk for central nervous system disorders, including stroke. However, the effects of PTX3 on cerebrovascular function in the neurovascular unit (NVU) after stroke are mostly unknown, and the basic research regarding the roles of PTX3 in NVU function is still limited. In this reverse translational study, we prepared mouse models of white matter stroke by vasoconstrictor (ET-1 or L-Nio) injection into the corpus callosum region to examine the roles of PTX3 in the pathology of cerebral white matter stroke. PTX3 expression was upregulated in GFAP-positive astrocytes around the affected region in white matter for at least 21 days after vasoconstrictor injection. When PTX3 expression was reduced by PTX3 siRNA, blood-brain barrier (BBB) damage at day 3 after white matter stroke was exacerbated. In contrast, when PTX3 siRNA was administered at day 7 after white matter stroke, compensatory angiogenesis at day 21 was promoted. In vitro cell culture experiments confirmed the inhibitory effect of PTX3 in angiogenesis, that is, recombinant PTX3 suppressed the tube formation of cultured endothelial cells in a Matrigel-based in vitro angiogenesis assay. Taken together, our findings may support a novel concept that astrocyte-derived PTX3 plays biphasic roles in cerebrovascular function after white matter stroke; additionally, it may also provide a proof-of-concept that PTX3 could be a therapeutic target for white matter-related diseases, including stroke.

AKAP12 Supports Blood-Brain Barrier Integrity against Ischemic Stroke.

A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.

Transcriptomic characterization of microglia activation in a rat model of ischemic stroke.

Microglia are key regulators of inflammatory response after stroke and brain injury. To better understand activation of microglia as well as their phenotypic diversity after ischemic stroke, we profiled the transcriptome of microglia after 75 min transient focal cerebral ischemia in 3-month- and 12-month-old male spontaneously hypertensive rats. Microglia were isolated from the brains by FACS sorting on days 3 and 14 after cerebral ischemia. GeneChip Rat 1.0ST microarray was used to profile the whole transcriptome of sorted microglia. We identified an evolving and complex pattern of activation from 3 to 14 days after stroke onset. M2-like patterns were extensively and persistently upregulated over time. M1-like patterns were only mildly upregulated, mostly at day 14. Younger 3-month-old brains showed a larger microglial response in both pro- and anti-inflammatory pathways, compared to older 12-month-old brains. Importantly, our data revealed that after stroke, most microglia are activated towards a wide spectrum of novel polarization states beyond the standard M1/M2 dichotomy, especially in pathways related to TLR2 and dietary fatty acid signaling. Finally, classes of transcription factors that might potentially regulate microglial activation were identified. These findings should provide a comprehensive database for dissecting microglial mechanisms and pursuing neuroinflammation targets for acute ischemic stroke.

Author Correction: Potential circadian effects on translational failure for neuroprotection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Potential circadian effects on translational failure for neuroprotection.

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches-normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-D-aspartic acid (NMDA) antagonist MK801-in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in 'active-phase' than in 'inactive-phase' rodent neurons. αPBN and MK801 reduced neuronal death only in 'inactive-phase' neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.