Differentiation and regulation of CD4+ T cell subsets in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and its hallmark pathological features are the loss of dopaminergic (DA) neurons in the midbrain substantia nigra pars compacta (SNpc) and the accumulation of alpha-synuclein (α-syn). It has been shown that the integrity of the blood-brain barrier (BBB) is damaged in PD patients, and a large number of infiltrating T cells and inflammatory cytokines have been detected in the cerebrospinal fluid (CSF) and brain parenchyma of PD patients and PD animal models, including significant change in the number and proportion of different CD4+ T cell subsets. This suggests that the neuroinflammatory response caused by CD4+ T cells is an important risk factor for the development of PD. Here, we systematically review the differentiation of CD4+ T cell subsets, and focus on describing the functions and mechanisms of different CD4+ T cell subsets and their secreted cytokines in PD. We also summarize the current immunotherapy targeting CD4+ T cells with a view to providing assistance in the diagnosis and treatment of PD.

T Cells Trafficking into the Brain in Aging and Alzheimer's Disease.

The meninges, choroid plexus (CP) and blood-brain barrier (BBB) are recognized as important gateways for peripheral immune cell trafficking into the central nervous system (CNS). Accumulation of peripheral immune cells in brain parenchyma can be observed during aging and Alzheimer's disease (AD). However, the mechanisms by which peripheral immune cells enter the CNS through these three pathways and how they interact with resident cells within the CNS to cause brain injury are not fully understood. In this paper, we review recent research on T cells recruitment in the brain during aging and AD. This review focuses on the possible pathways through which T cells infiltrate the brain, the evidence that T cells are recruited to the brain, and how infiltrating T cells interact with the resident cells in the CNS during aging and AD. Unraveling these issues will contribute to a better understanding of the mechanisms of aging and AD from the perspective of immunity, and hopefully develop new therapeutic strategies for brain aging and AD.

Hepcidin depending on astrocytic NEO1 ameliorates blood-brain barrier dysfunction after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) significantly compromises the blood-brain barrier (BBB) and impairs patient recovery. This study elucidates the critical role of astrocytic Neogenin-1 (NEO1) in BBB integrity post-SAH and examines the regulatory effects of hepcidin on endothelial cell (EC) function amid NEO1-mediated disruptions in iron homeostasis. Proteomic analyses of cerebrospinal fluid (CSF) from SAH patients revealed a substantial decrease in NEO1 expression, identifying it as a key factor in BBB integrity. 111 CSF proteins were significantly reduced in early SAH stages (days 1-3), with NEO1 among the most significantly altered. This dysregulation was linked to poorer patient outcomes, as indicated by a negative correlation between NEO1 levels and Modified Rankin Scale scores six months post-SAH (R = -0.4743, P < 0.0001). Experimental models further highlighted the importance of NEO1: SAH model and NEO1GFAP-Cre mice exhibited exacerbated EC dysfunction and increased BBB permeability, evidenced by significant Evans Blue retention and dextran leakage in the parietal cortex, effects that were mitigated by hepcidin administration. Our findings highlight the complex interplay between astrocytic signaling and endothelial function in SAH pathophysiology. The loss of astrocytic NEO1 led to increased EC proliferation and altered BBB structure, as confirmed by transmission electron microscopy and immunostaining for PECAM-1, indicating heightened blood vessel density in the affected cortex. Hepcidin treatment effectively reversed the EC dysfunction and BBB disruption in both NEO1-cKO mice and the SAH model, highlighting its potential as a therapeutic agent to enhance recovery and improve prognosis following SAH.

Senolytics and Senomorphics Targeting p38MAPK/NF-κB Pathway Protect Endothelial Cells from Oxidative Stress-Mediated Premature Senescence.

Oxidative stress is a prominent causal factor in the premature senescence of microvascular endothelial cells and the ensuing blood-brain barrier (BBB) dysfunction. Through the exposure of an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes, and pericytes to H2O2, this study examined whether a specific targeting of the p38MAPK/NF-κB pathway and/or senescent cells could delay oxidative stress-mediated EC senescence and protect the BBB. Enlarged BMECs, displaying higher ß-galactosidase activity, γH2AX staining, p16 expression, and impaired tubulogenic capacity, were regarded as senescent. The BBB established with senescent BMECs had reduced transendothelial electrical resistance and increased paracellular flux, which are markers of BBB integrity and function, respectively. Premature senescence disrupted plasma-membrane localization of the tight junction protein, zonula occludens-1, and elevated basement membrane-degrading matrix metalloproteinase-2 activity and pro-inflammatory cytokine release. Inhibition of p38MAPK by BIRB796 and NF-κB by QNZ and the elimination of senescent cells by a combination of dasatinib and quercetin attenuated the effects of H2O2 on senescence markers; suppressed release of the pro-inflammatory cytokines interleukin-8, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1; restored tight junctional unity; and improved BBB function. In conclusion, therapeutic approaches that mitigate p38MAPK/NF-κB activity and senescent cell accumulation in the cerebrovasculature may successfully protect BBB from oxidative stress-induced BBB dysfunction.

Long noncoding RNA H19 knockdown promotes angiogenesis via IMP2 after ischemic stroke.

AIMS: This study aimed to explore the effects of long noncoding RNA (lncRNA) H19 knockdown on angiogenesis and blood-brain barrier (BBB) integrity following cerebral ischemia/reperfusion (I/R) and elucidate their underlying regulatory mechanisms. METHODS: A middle cerebral artery occlusion/reperfusion model was used to induce cerebral I/R injury. The cerebral infarct volume and neurological impairment were assessed using 2,3,5-triphenyl-tetrazolium chloride staining and neurobehavioral tests, respectively. Relevant proteins were evaluated using western blotting and immunofluorescence staining. Additionally, a bioinformatics website was used to predict the potential target genes of lncRNA H19. Finally, a rescue experiment was conducted to confirm the potential mechanism. RESULTS: Silencing of H19 significantly decreased the cerebral infarct volume, enhanced the recovery of neurological function, mitigated BBB damage, and stimulated endothelial cell proliferation following ischemic stroke. Insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) is predicted to be a potential target gene for lncRNA H19. H19 knockdown increased IMP2 protein expression and IMP2 inhibition reversed the protective effects of H19 inhibition. CONCLUSION: Downregulation of H19 enhances angiogenesis and mitigates BBB damage by regulating IMP2, thereby alleviating cerebral I/R injury.

GM130-silencing may aggravate blood-brain barrier damage and affect microglia polarization by down-regulating PD-L1 expression after experimental intracerebral hemorrhage.

BACKGROUND: In addition to primary injury, secondary injuries related to BBB disruption and immune-inflammatory response also play an important role in intracerebral hemorrhage (ICH). And the Golgi apparatus play an important role in the state of ICH. METHODS: ICH model and GM130-silencing ICH model were established in SD rats. The Garcia score was used to score the neurological defects of the rats. Blood-brain barrier (BBB) integrity were assessed by amount of extravasated Evans blue, and tight junction proteins. The expression of PD-L1 and GM130were detected through Western-blot and the subtype of microglia was showing with Immunofluorescence staining. RESULTS: Compared with the ICH group, GM130-silencing ICH rats got a worsened neurological deficit and enlarged volume of the hematoma. Evan's blue extravasation aggravated as well. The expression of GM130 in peri-hematoma tissue was further decreased, and the morphology and structure of the Golgi apparatus were further damaged. Meanwhile, the GM130 deficit resulted in decreased expression of PD-L1 and more polarization of microglia to the M1 subtype. CONCLUSION: We demonstrate that GM130 could influence the integrity of BBB and plays a role in neuroinflammation via regulation of PD-L1 after ICH. The manipulation of GM130 might be a promising therapeutical target in ICH.

circRNAs as Epigenetic Regulators of Integrity in Blood-Brain Barrier Architecture: Mechanisms and Therapeutic Strategies in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease leading to progressive demyelination and neuronal loss, with extensive neurological symptoms. As one of the most widespread neurodegenerative disorders, with an age onset of about 30 years, it turns out to be a socio-health and economic issue, thus necessitating therapeutic interventions currently unavailable. Loss of integrity in the blood-brain barrier (BBB) is one of the distinct MS hallmarks. Brain homeostasis is ensured by an endothelial cell-based monolayer at the interface between the central nervous system (CNS) and systemic bloodstream, acting as a selective barrier. MS results in enhanced barrier permeability, mainly due to the breakdown of tight (TJs) and adherens junctions (AJs) between endothelial cells. Specifically, proinflammatory mediator release causes failure in cytoplasmic exposure of junctions, resulting in compromised BBB integrity that enables blood cells to cross the barrier, establishing iron deposition and neuronal impairment. Cells with a compromised cytoskeletal protein network, fiber reorganization, and discontinuous junction structure can occur, resulting in BBB dysfunction. Recent investigations on spatial transcriptomics have proven circularRNAs (circRNAs) to be powerful multi-functional molecules able to epigenetically regulate transcription and structurally support proteins. In the present review, we provide an overview of the recent role ascribed to circRNAs in maintaining BBB integrity/permeability via cytoskeletal stability. Increased knowledge of the mechanisms responsible for impairment and circRNA's role in driving BBB damage and dysfunction might be helpful for the recognition of novel therapeutic targets to overcome BBB damage and unrestrained neurodegeneration.

Pathogenesis of Cerebral Small Vessel Disease: Role of the Glymphatic System Dysfunction.

Cerebral small vessel disease (CSVD) is a group of pathologies that affect the cerebral blood vessels. CSVD accounts for 25% of strokes and contributes to 45% of dementia. However, the pathogenesis of CSVD remains unclear, involving a variety of complex mechanisms. CSVD may result from dysfunction in the glymphatic system (GS). The GS contains aquaporin-4 (AQP-4), which is in the perivascular space, at the endfeet of the astrocyte. The GS contributes to the removal of waste products from the central nervous system, occupying perivascular spaces and regulating the exchange and movement of cerebrospinal fluid and interstitial fluid. The GS involves astrocytes and aquaporin channels, which are components of the blood-brain barrier, and problems with them may constitute the pathogenesis of CSVD. Vascular risk factors, including diabetes, dilate the perivascular space, disrupting the glymphatic system and the active regulation of AQP-4. CSVD exacerbation due to disorders of the GS is associated with multiple vasculopathies. Dysfunction of the glymphatic system and AQP-4 interferes with the functioning of the blood-brain barrier, which exacerbates CSVD. In a long-term follow-up of CSVD patients with microbleeds, lacunar infarcts, and white matter hyperintensity, several vascular risk factors, including hypertension, increased the risk of ischemic stroke. Dysfunction of the GS may be the cause of CSVD; however, the underlying treatment needs to be studied further.

Using Multiphoton Intravital Microscopy to Study Neutrophil Transmigration and Blood-Brain Barrier Permeability in a Mouse Model of Herpes Simplex Virus Encephalitis.

Multiphoton intravital microscopy (MP-IVM) is an imaging technique used for the observation of living organisms at a microscopic resolution. The tissue of interest is exposed through a window allowing imaging of cells in real time. Using MP-IVM, the temporospatial kinetics of leukocyte transendothelial migration can be visualized and quantitated using reporter mice and cell-specific fluorophore-conjugated monoclonal antibodies to track the leukocytes within and outside of vascular beds. Here we describe a method used to study neutrophil transendothelial migration and blood-brain barrier permeability in a mouse model of herpes simplex virus I (HSV) encephalitis.

RAGE mediates hippocampal pericyte responses and neurovascular unit lesions after TBI.

Traumatic brain injury impairs brain function through various mechanisms. Recent studies have shown that alterations in pericytes in various diseases affect neurovascular function, but the effects of TBI on hippocampal pericytes remain unclear. Here, we investigated the effects of RAGE activation on pericytes after TBI using male C57BL/6 J mice. Hippocampal samples were collected at different time points within 7 days after TBI, the expression of PDGFR-ß, NG2 and the HMGB1-S100B/RAGE signaling pathway was assessed by Western blotting, and the integrity of the hippocampal BBB at different time points was measured by immunofluorescence. RAGE-associated BBB damage in hippocampal pericytes occurred early after cortical impact. By culturing primary mouse brain microvascular pericytes, we determined the different effects of HMGB1-S100B on pericyte RAGE. To investigate whether RAGE blockade could protect neurological function after TBI, we reproduced the process of CCI by administering FPS-ZM1 to RAGE-/- mice. TEM images and BBB damage-related assays showed that inhibition of RAGE resulted in a significant improvement in the number of hippocampal vascular basement membranes and tight junctions and a reduction in perivascular oedema compared with those in the untreated group. In contrast, mouse behavioural testing and doublecortin staining indicated that targeting the HMGB1-S100B/RAGE axis after CCI could protect neurological function by reducing pericyte-associated BBB damage. In conclusion, the present study provides experimental evidence for the strong correlation between the pericyte HMGB1-S100B/RAGE axis and NVU damage in the hippocampus at the early stage of TBI and further demonstrates that pericyte RAGE serves as an important target for the protection of neurological function after TBI.

PD-1 mediates microglia polarization via the MAPK signaling pathway to protect blood-brain barrier function during cerebral ischemia/reperfusion.

BACKGROUND: Cerebral ischemia is characterized by its rapid onset and high rates of recurrence, morbidity, and mortality, with blood-brain barrier (BBB) permeability playing a vital role in brain injury. Therefore, it is important to understand the molecular mechanism which regulates the BBB during cerebral ischemia. MATERIALS AND METHODS: An in vitro model of oxygen-glucose deprivation (OGD) and an in vivo model of cerebral ischemia/reperfusion (I/R) were constructed. PD-1 overexpression vectors and vectors containing si-RNA were transfected and injected into in vitro and in vivo models. Western blotting, real-time quantitative PCR (qPCR), immunofluorescence (IF) analysis, and immunohistochemical staining were employed to evaluate the expression levels of programmed cell death-1 (PD-1), microglia M1 and M2 biomarkers, and tight junction proteins. Flow cytometry and ELISA were used to measure the levels of pro-inflammatory cytokines. The BBB permeability of brain tissues was evaluated by Evans blue dye (EBD) extravasation and transendothelial electrical resistance (TEER). Brain water content was measured to assess the extent of inflammatory exudation. The infarct volume and neurological severity score (NSS) were used to assess the severity of brain injury. Brain cell apoptosis was assessed by the TUNEL assay and hematoxylin-eosin (H&E) staining. RESULTS: PD-1 helped to convert the microglia M1 phenotype to the M2 phenotype and to reduce BBB permeability both in vitro and in vivo. Overexpression of PD-1 promoted a shift of the M1 phenotype to the M2 phenotype and reduced BBB permeability via the ERK and p38 MAPK signaling pathways. PD-1 reduced inflammatory exudation, BBB permeability, cell apoptosis, and brain injury in vivo. CONCLUSION: Our present study verified that PD-1 exerts an anti-inflammatory effect by converting the microglia M1 phenotype to the M2 phenotype, reducing BBB permeability, and thereby relieves brain injury caused by cerebral ischemia. PD-1 is potential therapeutic target for brain injury caused by cerebral ischemia.

AD16 attenuates neuroinflammation induced by cerebral ischemia through down-regulating astrocytes A1 polarization.

A1 polarization of astrocytes mediated prolonged inflammation contributing to brain injury in ischemic stroke. We have previously shown that AD16 protects against neonatal hypoxic-ischemic brain damage in vivo and oxygen-glucose deprivation in vitro. More recently, AD16 has demonstrated safety, tolerability, and favorable pharmacokinetics in a randomized controlled phase I trial. In this study, we utilized a rat model of transient middle cerebral artery occlusion (tMCAO) to explore whether the anti-inflammatory compound AD16 protects against ischemic brain injury by regulating A1 polarization and its underlying mechanisms. Our results showed that AD16 treatment significantly reduced the brain infarcted volume and improved neurological function in tMCAO rats. GO analysis results show that differential genes among the Sham, tMCAO and AD16 treatment groups are involved in the regulation of cytokine and inflammatory response. KEGG enrichment pathways analysis mainly enriched in cytokine-cytokine receptor interaction, viral protein interaction with cytokine-cytokine receptor, TNF, chemokine, NF-κB and IL-17 signaling pathway. Furthermore, AD16 treatment decreased the permeability of the blood-brain barrier and suppressed neuroinflammation. AD16 treatment also significantly reduced the polarization of A1 and inhibited NF-κB and JAK2/STAT3 signaling pathways. This study demonstrates that AD16 protects against brain injury in ischemic stroke by reducing A1 polarization to suppress neuroinflammation through downregulating NF-κB and JAK2/STAT3 signaling. Our findings uncover a potential molecular mechanism for AD16 and suggest that AD16 holds promising therapeutic potential against cerebral ischemia.

Immune-mediated disruption of the blood-brain barrier after intracerebral hemorrhage: Insights and potential therapeutic targets.

AIMS: Intracerebral hemorrhage (ICH) is a condition that arises due to the rupture of cerebral blood vessels, leading to the flow of blood into the brain tissue. One of the pathological alterations that occurs during an acute ICH is an impairment of the blood-brain barrier (BBB), which leads to severe perihematomal edema and an immune response. DISCUSSION: A complex interplay between the cells of the BBB, for example, pericytes, astrocytes, and brain endothelial cells, with resident and infiltrating immune cells, such as microglia, monocytes, neutrophils, T lymphocytes, and others accounts for both damaging and protective mechanisms at the BBB following ICH. However, the precise immunological influence of BBB disruption has yet to be richly ascertained, especially at various stages of ICH. CONCLUSION: This review summarizes the changes in different cell types and molecular components of the BBB associated with immune-inflammatory responses during ICH. Furthermore, it highlights promising immunoregulatory therapies to protect the integrity of the BBB after ICH. By offering a comprehensive understanding of the mechanisms behind BBB damage linked to cellular and molecular immunoinflammatory responses after ICH, this article aimed to accelerate the identification of potential therapeutic targets and expedite further translational research.

Assessing blood-brain barrier dysfunction and its association with Alzheimer's pathology, cognitive impairment and neuroinflammation.

BACKGROUND: Blood-brain barrier (BBB) alterations may contribute to AD pathology through various mechanisms, including impaired amyloid-ß (Aß) clearance and neuroinflammation. Soluble platelet-derived growth factor receptor beta (sPDGFRß) has emerged as a potential biomarker for BBB integrity. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) offers a direct assessment of BBB permeability. However, the relationship between BBB dysfunction, cognitive impairment, and AD pathology remains unclear, with inconsistent findings in the literature. METHODS: We conducted a cross-sectional study using data from the DELCODE and DESCRIBE cohorts to investigate BBB dysfunction in participants with normal cognition (NC), mild cognitive impairment (MCI), and AD dementia. BBB function was assessed using DCE-MRI and sPDGFRß levels in cerebrospinal fluid and AD biomarkers Aß and tau were measured. In a subset of patients, the CSF/plasma-ratio of albumin (QAlb) as a standard marker of BBB integrity and markers of neuroinflammation were analyzed. RESULTS: 91 participants (NC: 44, MCI: 21, AD: 26) were included in the analysis. The average age was 74.4 years, 42% were female. Increased hippocampal BBB disruption was observed in the AD-group (Ktrans: 0.55 × 10- 3 min- 1 ± 0.74 × 10- 3 min- 1) but not the MCI-group (Ktrans: 0.177 × 10- 3 min- 1 ± 0.22 × 10- 3 min- 1), compared to the NC group (Ktrans: 0.19 × 10- 3 min- 1 ± 0.37 × 10- 3 min- 1, p < .01). sPDGFRß was not significantly different between the cognitive groups. However, sPDGFRß levels were significantly associated with age (r = .33, p < .01), independent of vascular risk factors. Further, sPDGFRß showed significant positive associations with soluble Aß levels (Aß40: r = .57, p < .01; Aß42: r = .39, p < .01) and YKL-40 (r = .53, p < .01), a marker of neuroinflammation. sPDGFRß/DCE-MRI was not associated with overall AD biomarker positivity or APOE-status. CONCLUSION: In dementia, but not MCI, hippocampal BBB disruption was observed. sPDGFRß increased with age and was associated with neuroinflammation independent of cognitive impairment. The association between Aß and sPDGFRß may indicate a bidirectional relationship reflecting pericytes' clearance of soluble Aß and/or vasculotoxic properties of Aß.

Blood-brain barrier alterations and their impact on Parkinson's disease pathogenesis and therapy.

There is increasing evidence for blood-brain barrier (BBB) alterations in Parkinson's disease (PD), the second most common neurodegenerative disorder with rapidly rising prevalence. Altered tight junction and transporter protein levels, accumulation of α-synuclein and increase in inflammatory processes lead to extravasation of blood molecules and vessel degeneration. This could result in a self-perpetuating pathophysiology of inflammation and BBB alteration, which contribute to neurodegeneration. Toxin exposure or α-synuclein over-expression in animal models has been shown to initiate similar pathologies, providing a platform to study underlying mechanisms and therapeutic interventions. Here we provide a comprehensive review of the current knowledge on BBB alterations in PD patients and how rodent models that replicate some of these changes can be used to study disease mechanisms. Specific challenges in assessing the BBB in patients and in healthy controls are discussed. Finally, a potential role of BBB alterations in disease pathogenesis and possible implications for therapy are explored. The interference of BBB alterations with current and novel therapeutic strategies requires more attention. Brain region-specific BBB alterations could also open up novel opportunities to target specifically vulnerable neuronal subpopulations.

The Role of Glutamate and Blood-Brain Barrier Disruption as a Mechanistic Link between Epilepsy and Depression.

Epilepsy is associated with substantial neuropsychiatric impairments that persist long after the onset of the condition, significantly impacting quality of life. The goal of this review was to uncover how the pathological consequences of epilepsy, such as excessive glutamate release and a disrupted blood-brain barrier (BBB), contribute to the emergence of neuropsychiatric disorders. We hypothesize that epilepsy induces a dysfunctional BBB through hyperexcitation, which then further amplifies post-ictal glutamate levels and, thus, triggers neurodegenerative and neuropsychiatric processes. This review identifies the determinants of glutamate concentration levels in the brain and explores potential therapeutic interventions that restore BBB integrity. Our focus on therapeutic BBB restoration is guided by the premise that it may improve glutamate regulation, consequently mitigating the neurotoxicity that contributes to the onset of neuropsychiatric symptoms.

Increased Permeability of the Blood-Brain Barrier in a Diabetic Mouse Model (Leprdb/db Mice).

Type 2 Diabetes Mellitus (T2DM) is linked to multiple complications, including cognitive impairment, and the prevalence of memory-related neurodegenerative diseases is higher in T2DM patients. One possible theory is the alteration of the microvascular and macrovascular environment of the blood-brain barrier (BBB). In this study, we employed different approaches, including RT-PCR, functional pharmacokinetic studies using sodium fluorescein (NaFL), and confocal microscopy, to characterize the functional and molecular integrity of the BBB in a T2DM animal model, leptin receptor-deficient mutant mice (Leprdb/db mice). As a result, VCAM-1, ICAM-1, MMP-9, and S100b (BBB-related markers) dysregulation was observed in the Leprdb/db animal model compared to littermate wild-type mice. The brain concentration of sodium fluorescein (NaFL) increased significantly in Leprdb/db untreated mice compared to insulin-treated mice. Therefore, the permeability of NaFL was higher in Leprdb/db control mice than in all remaining groups. Identifying the factors that increase the BBB in Leprdb/db mice will provide a better understanding of the BBB microvasculature and present previously undescribed findings of T2DM-related brain illnesses, filling knowledge gaps in this emerging field of research.

mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

A dispensable role of mural cell-derived laminin-α5 in intracerebral hemorrhage.

Although most laminin isoforms are neuroprotective in stroke, mural cell-derived laminin-α5 plays a detrimental role in an ischemia-reperfusion model. To determine whether this deleterious effect is an intrinsic feature of mural cell-derived laminin-α5 or unique to ischemic stroke, we performed loss-of-function studies using middle-aged mice with laminin-α5 deficiency in mural cells (α5-PKO) in an intracerebral hemorrhage (ICH) model. Control and α5-PKO mice exhibited comparable changes in all parameters examined, including hematoma size, neuronal death, neurological function, blood-brain barrier integrity, and reactive gliosis. These findings highlight a minimal role of mural cell-derived laminin-α5 in ICH. Together with the detrimental role of mural cell-derived laminin-α5 in ischemic stroke, these negative results in ICH model suggest that mural cell-derived laminin-α5 may exert distinct functions in different diseases.

Chronic consequences of ischemic stroke: Profiling brain injury and inflammation in a mouse model with reperfusion.

Stroke is a pervasive and debilitating global health concern, necessitating innovative therapeutic strategies, especially during recovery. While existing literature often focuses on acute interventions, our study addresses the uniqueness of brain tissue during wound healing, emphasizing the chronic phase following the commonly used middle cerebral artery (MCA) occlusion model. Using clinically relevant endpoints in male and female mice such as magnetic resonance imaging (MRI) and plasma neurofilament light (NFL) measurement, along with immunohistochemistry, we describe injury evolution. Our findings document significant alterations in edema, tissue remodeling, and gadolinium leakage through MRI. Plasma NFL concentration remained elevated at 30 days poststroke. Microglia responses are confined to the region adjacent to the injury, rather than continued widespread activation, and boron-dipyrromethene (BODIPY) staining demonstrated the persistent presence of foam cells within the infarct. Additional immunohistochemistry highlighted sustained B and T lymphocyte presence in the poststroke brain. These observations underscore potentially pivotal roles played by chronic inflammation brought on by the lipid-rich brain environment, and chronic blood-brain barrier dysfunction, in the development of secondary neurodegeneration. This study sheds light on the enduring consequences of ischemic stroke in the most used rodent stroke model and provides valuable insights for future research, clinical strategies, and therapeutic development.