Meningeal enhancement is observed on cranial and spinal magnetic resonance image in a subset of patients with multiple sclerosis (MS) and is associated with a higher risk of future disease progression, however, the underlying mechanisms by which it promotes such progression remain elusive. By cross-validating clinical MRI datasets with the experimental autoimmune encephalomyelitis model (EAE), we revealed a meningeal lymphatics-innate immune axis that orchestrates a localized signaling cascade driving and promoting the pathogenesis of inflammatory demyelination. We show that the earliest phase of the disease is characterized by a rapid, selective influx of neutrophils recruited directly from the vertebral bone marrow via specialized osteo-channels. Crucially, we identify the glycosphingolipid hexosylceramide (Hex1Cer), which is derived from meningeal lymphatic vessels after drainage of myelin debris and triggers neutrophil extracellular trap (NET) formation. These meningeal NETs utilized a SELPLG-SELL axis to recruit pathogenic lymphocytes, promoting their infiltration into the spinal cord parenchyma. Both pharmacological targeting of the Hex1Cer or NETs formation pathway significantly reduced downstream T cell infiltration and clinical severity. Finally, we demonstrate that low-dose intrathecal repurposing of Sivelestat to target this meningeal niche provides superior protection compared to systemic delivery. Our findings define the meningeal lymphatic endothelial cells (mLECs) as active metabolic gatekeepers of neuroinflammation and establish the mLEC-myelin-Hex1Cer-NETs formation axis as a high-priority therapeutic target for intercepting MS.

BACKGROUND:Brain aging poses a major public health challenge and is the primary risk factor for neurodegenerative diseases. Macrophage lineage cells (MLCs) have emerged as pivotal mediators of brain aging. While fundamental to central nervous system (CNS) homeostasis through their scavenging, detoxification, and neurotrophic functions, their transition to a senescent state is a primary driver of pathology. This shift is marked by a loss of clearance capacity and the adoption of a pro-inflammatory senescence-associated secretory phenotype (SASP). OBJECTIVES:Here, we summarize the distinct and cooperative roles of MLC subsets in brain aging. We examine the key molecular drivers of MLCs senescence and detail how subset-specific dysfunction contributes to the propagation of cellular aging and related neuropathology. Finally, we evaluate current and emerging therapeutic strategies that target MLCs senescence. CONCLUSION:We conclude by proposing a multidimensional management framework for brain aging. This framework positions MLCs as a central therapeutic hub, integrating advanced diagnostics and stratified interventions to preserve brain health and mitigate neurodegenerative pathology.

Communication between the immune system and the brain is critical for neuronal health, yet the molecular signals that mediate this crosstalk are not fully understood. Here, we uncover an unexpected and direct neuroprotective axis linking the thymus to the central nervous system (CNS). We show that the thymus-derived peptide Thymosin alpha-1 (Tα1) functions as a non-canonical ligand for the Hypocretin (Orexin) Receptor 1 (HCRTR1), a classical neuropeptide receptor on neurons. This engagement shields neurons from cell death by suppressing the activity of Receptor-Interacting Protein Kinase 3 (RIPK3), a central executioner of necroptosis. The physiological relevance of this pathway is highlighted in ischemic stroke, where we found that circulating Tα1 levels were significantly reduced in patients and mice, strongly correlating with disease severity. Genetic deletion of the Tα1-encoding gene Ptma exacerbated stroke injury, whereas therapeutic administration of Tα1 conferred robust neuroprotection and improved functional recovery. Our findings identify a novel thymus-brain signaling pathway, revealing a new neuroprotective function for an immune peptide and an unexpected role for a canonical neuronal receptor. This work establishes Tα1 as a promising dual-action therapeutic candidate, capable of both direct neuronal protection and systemic immunomodulation.

Introduction: Stroke-induced immunosuppression (SIIS) can lead to increased susceptibility to infections, which ultimately affects functional outcomes and increases mortality rates. Our previous studies suggest that Bone Marrow Mesenchymal Stem Cell (BM-MSCs) protect against SIIS and prevent post-stroke pneumonia.In this study, we propose that BM-MSCs have the potential to repair thymic atrophy after infarction, thereby offering protection against SIIS. Methods: We conducted a 60-minute transient middle cerebral artery occlusion (tMCAO) on male C57BL/6J mice. Two hours after the tMCAO, separate injections of PBS , BM-MSCs , and GFP-TSPAN4-labeled migrasomes derived from BM-MSCs were administered.The thymuses of the infarcted mice were subsequently collected at 3, 7, and 14 days post-tMCAO for RNA sequencing, and immunofluorescence staining. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to profile the proteome of migrasomes. Result: We found that in tMCAO mice, the mortality rate decreased and thymic weight was restored after injection of BM-MSCs. Cellular mechanism studies using bulk RNA sequencing revealed that BM-MSC treatment downregulates senesence of thymic cells and enhances cell proliferation. Immunofluorescence analysis indicated that after stroke, thymic medullary epithelial cells (TECs) exhibited remarkable senescence, especially those in the medulla. BM-MSC treatment reversed the senesence of thymic cells. Interestingly, we found that BM-MSCs do not directly enter the thymus, while migrasomes derived from BM-MSCs, which are newly discovered organelle that produced during cellular migration, could cross the blood-thymus barrier, reaching and interacting with TECs. Proteomic analysis of BM-MSC-derived migrasomes revealed that Peptidyl-prolyl cis-trans isomerase (Pin1) was packed in the organelle. Pin1 is a vital cell cycle protein that promotes cell proliferation and division. Based on these findings, we hypothesize that migrasomes derived from BM-MSCs may transfer Pin1 protein to TECs, potentially reversing cellular aging. Conclusions: BM-MSCs have the potential to reverse thymic atrophy after infarction and protect against the senscence of TECs. Notably, migrasomes derived from BM-MSCs can cross the blood-thymus barrier and transfer the Pin1 protein to TECs. Based on these findings, we propose that BM-MSC-derived migrasomes can restore thymic atrophy and prevent SIIS.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe neurological disorder characterized by the presence of autoantibodies against the NMDAR and blood-brain barrier (BBB) disruption. This study investigates the involvement of monocytes and interferon regulatory factor 7 (IRF7) in BBB leakage of this disease. In anti-NMDAR encephalitis patients, the circulating monocytes count was positively correlated with BBB disruption, besides, IRF7 was activated in these cells. In peripheral blood and brain of our anti-NMDAR encephalitis mouse model, increased monocytes and elevated IRF7 expression within these cells were found. Additionally, in the blood and brain of this model, the quantity of monocytes and their IRF7 expression level were positively correlated with behavioral deficits. IRF7-KO mice were subjected to anti-NMDAR encephalitis modeling and exhibited milder disease severity and reduced BBB disruption compared to their WT counterparts. Bone marrow derived macrophages (BMDMs) from IRF7-KO mice showed diminished capacity to disrupt BBB compared with BMDMs from WT mice in in vitro study. Our findings suggest that IRF7 plays a critical role in the pathogenesis of anti-NMDAR encephalitis by modulating monocyte's capacity to disrupt BBB. Targeting IRF7 may offer a novel therapeutic strategy for this devastating neurological condition.

AIMS:The aim of this study is to investigate the role of glymphatic function of cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL), the most common monogenic small vessel disease caused by NOTCH3 mutation, and to explore potential therapeutic strategies to improve glymphatic function. METHODS:We assessed glymphatic influx and efflux function in CADASIL mouse models (Notch3R170C) and correlated these findings with brain atrophy in CADASIL patients. We also investigated the underlying mechanisms of glymphatic impairment, focusing the expression of AQP4 in astrocytic endfeet. RESULTS:CADASIL mouse exhibited both impaired glymphatic influx and efflux, which impedes waste clearance and promotes brain senescence. In accordance, brain atrophy in CADASIL patients is associated with perivascular space enlargement, indicating that glymphatic impairment contributes to advanced brain senescence in CADASIL. The glymphatic malfunction in CADASIL is attributed to diminished AQP4 expression in astrocytic endfeet, which is the core mediator of glymphatic activity. Mechanistically, AQP4 expression is regulated by NOTCH3-RUNX1-CMYB signaling. Reinforcing AQP4 expression in astrocytes by AAV-based therapy resumes the glymphatic functions in CADASIL mice, which further prevents brain senescence. CONCLUSION:We propose that to improve glymphatic function by reinforcing AQP4 expression is a promising therapeutic strategy in CADASIL.

BACKGROUND:Parenchymal border macrophages (PBMs) reside at the interface between the central nervous system and the periphery. They are known to mediate the accessibility of the substances to the brain. However, no one has examined their role in poststroke Aβ (amyloid-β) clearance. METHODS:Permanent focal cerebral ischemia was induced in 8- to 10-week-old C57/Bl6 male mice by distal middle cerebral artery occlusion. The clodronate liposomes were administered into the cerebral spinal fluid at 7 days before stroke to deplete the PBM population. Sensorimotor and cognitive functions, glymphatic system, and Aβ accumulation were assessed for up to 34 days after stroke. RESULTS:The Aβ accumulated along brain blood vessels after stroke in both the ipsilateral and contralateral hemispheres. When PBMs were depleted, glymphatic drainage of Aβ was markedly reduced, and this was accompanied by deterioration of cognitive function, highlighting a critical role for PBMs in poststroke Aβ disposal. A possible mechanism relates to MANF (mesencephalic astrocyte-derived neurotrophic factor). MANF derived from PBMs suppressed astrocytic stress and maintained glymphatic drainage when supplemented into the cerebral spinal fluid. In the chronic phase of stroke, MANF production in PBMs was downregulated, and consequently, glymphatic impairments were exacerbated, which led to ongoing Aβ accumulation and cognitive decline. CONCLUSIONS:In summary, supplementation of MANF not only mitigates the adverse impacts of PBM depletion but also exerts therapeutic effects that improve glymphatic system function. We thus propose that this represents a promising strategy to prevent poststroke cognitive impairment.

Background: Thymosin alpha 1 (Tα1) had been used in kinds of inflammatory and immunodeficiency diseases as an immunomodulator. According to our clinic observation, patients with acute ischemic stroke (AIS) treated with Tα1 to prevent/cure post-stroke infection display favorable stroke outcome. Whether Tα1 exerts direct neuronal protection besides immunomodulation remains elusive. Aim: To explore the effect of Tα1 on neuronal protection after AIS and unveil the underlying mechanisms. Method: In vivo : 8-10 weeks old male Wild type (WT) mice were subjected to 60 minutes middle cerebral artery occlusion (tMCAO) and administrated Tα-1 by intraperitoneal injection. Ex vivo : Primary mouse neuron is subjected to 60 min oxygen and glucose deprivation (OGD), followed by Tα-1 treatment. Results: Compared with untreated group, Tα1 treatment displayed decreased infarct volume, limited neuronal loss, attenuated white matter injury，restricted neuroinflammation and improved neurological functions. Treatment of antibiotics or glucocorticoid failed to substitute the therapeutic effects of Tα1, indicating extra protection of Tα1 treatment besides anti-infection and immunomodulation. As for primary mouse neuron, Tα1 treatment showed fewer neuron death and higher cell viability upon OGD stimulation. Both in vivo and ex vivo experiments revealed that Tα1 directly inhibited ischemia-induced neuronal death. Mechanistically, Tα1 enhanced mitophagy by down-regulating Drp1, thus promoted mitochondrial renewal and supported neuronal viability. Conclusions: Our results identified that Tα1 possessed direct neuronal protection and improved prognosis of AIS. Given the multi-functions of Tα1 including anti-infection, immunomodulation and neuronal protection, we propose that Tα1 is a promising therapy against AIS.

Multiple sclerosis (MS) is characterized by episodes of inflammatory demyelination followed by varying degrees of remyelination. Macrophages play a pivotal role in both processes. Understanding how macrophages modulate their plasticity to facilitate remyelination in MS holds significant therapeutic potential, yet this mechanism remains poorly understood. In this study, we demonstrate that the digestion of myelin debris by macrophages triggers the activation of FOXP3 signaling, which induces a pro-regenerative phenotype. Specifically, we generated macrophage-specific Foxp3 conditional knockout mice and show that FOXP3+ macrophages promote oligodendrocyte progenitor cell differentiation by producing amphiregulin (AREG), a neurotrophic factor. This mechanism enhances recovery in experimental autoimmune encephalomyelitis. These findings suggest that FOXP3+ macrophages could serve as a promising therapeutic target in MS.

Melatonin deficiency in Parkinson's disease (PD) patients correlates with impaired glymphatic function as measured by diffusion tensor imaging along the perivascular space (DTI-ALPS) index, suggesting circadian-regulated waste clearance as a therapeutic target. Unlike previous studies focusing on agomelatine's (AGM) antidepressant and sleep-regulating properties, we demonstrate its novel mechanism in PD treatment through glymphatic enhancement. Retrospective clinical analysis of PD patients revealed that AGM administration restored glymphatic function and improved motor performance. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced (MPTP) PD models, AGM alleviated glymphatic dysfunction and anxiety-like behaviors while reducing PD pathology. Crucially, AGM promoted aquaporin-4 (AQP4) polarization at astrocytic endfeet, as evidenced by RNA sequencing showing enhanced gap junction-related gene expression. The brain and muscle ARNT-like 1 (BMAL1)-mediated transcriptional regulation emerged as the key pathway underlying these effects. Our findings establish AGM as the first melatoninergic agent targeting the glymphatic-AQP4 axis in PD, shifting therapeutic strategies from symptomatic relief to disease modification. This provides clinical rationale for repurposing circadian regulators to decelerate PD progression through enhanced protein clearance.

Aging is a major risk factor for various neurological disorders, including Alzheimer’s disease, and is associated with the accumulation of senescent cells, which can themselves propagate the senescence process through paracrine signaling. Migrasomes are organelles that form during cellular migration, detach from parent cells and mediate intercellular communication. Here we demonstrate that border-associated macrophages (BAMs) acquire senescence-associated properties during early brain aging, possibly due to prolonged exposure to amyloid beta. Senescent-like BAMs show elevated production of migrasomes, which convey senescence-associated signals including the apoptosis inhibitor of macrophage to neighboring cells. We show that microglia are prominent recipients of senescent-like BAM-derived migrasomes, and that through activation of CD16 in recipient cells, the apoptosis inhibitor of macrophage inhibits apoptosis and promotes senescence induction. Blocking migrasome induction in senescent-like BAMs through treatment with Tspan4-targeting siRNA-encapsulated liposomes ameliorates cognitive deficits in aged mice. Our findings suggest that migrasomes are potent vehicles of senescence-regulatory signals and represent a promising target for senomorphic therapy. Hu et al. identify border-associated macrophages as early targets of brain aging. These cells acquire senescence-associated properties, which are transmittable via migrasomes carrying the apoptosis inhibitor of macrophage. Blocking migrasome production attenuates cognitive decline in aged mice.

Current treatments for cerebral amyloid angiopathy are mainly symptomatic and have limited efficacy, and there is a lack of targeted therapies. Mesenchymal stem cell transplantation improves cognitive and motor function in conditions such as Alzheimer’s disease, acute ischemic stroke, and Parkinson’s disease. In addition, mesenchymal stem cell therapy modulates the immune system, reduces neuroinflammation, and improves resolution of brain lesions by cells of the macrophage lineage. Cerebral amyloid angiopathy and Alzheimer’s disease share similar pathologic changes involving amyloid-beta deposition, which contributes to the progression of both diseases and exacerbates cognitive deficits through impaired vascular integrity and neuroinflammation. Therefore, we hypothesized that mesenchymal stem cell therapy could also ameliorate the pathological changes seen in cerebral amyloid angiopathy by modulating the immune response. In this study, we show that bone marrow mesenchymal stem cells have a protective effect in a mouse model of cerebral amyloid angiopathy (Tg-SwDI/B). Bone marrow mesenchymal stem cell treatment improved cognitive function, reduced neuroinflammation, and maintained blood-brain barrier integrity in Tg-SwDI/B mice. Mechanistically, bone marrow mesenchymal stem cell treatment enhanced the expulsion of damaged mitochondria from neutrophils via migrasomes, in a process known as mitocytosis, thereby preserving mitochondrial quality within the neutrophils. Mitochondrial damage in neutrophils leads to cellular injury, including the generation of reactive oxygen species and the formation of neutrophil extracellular traps. Neutrophils activate mitocytosis to promote mitochondrial renewal, which further enhances their own clearance by macrophage lineage cells. Our findings demonstrate that bone marrow mesenchymal stem cells are a promising therapeutic candidate for cerebral amyloid angiopathy, as they play a significant role in migrasome-dependent mitochondrial quality control in neutrophils.

Objective: Incomplete clearance of cell debris in stroke lesion contributes to post-stroke cognitive impairment. Microglia and infiltrated macrophages are the major scavenger of debris in stroke lesion. However, as stroke progresses, their phagocytic capacity is impaired, while secretion of pro-inflammatory factors was enhanced. This study aims to identify the mechanisms driving the phenotypic shift after excessive phagocytosis. Methods: Brain cells from 8-12 week-old male C57BL/6J mice were collected for single-cell RNA sequencing at 1, 5, and 14 days after transient middle cerebral artery occlusion (tMCAO), as well as from sham-operated control group. Key transcription factors mediating the phenotypic changes of microglia and macrophages were identified through bioinformatics analysis, and further validated in vivo and in vitro using immunofluorescence staining, flow cytometry, and Western blot. Additionally, tMCAO models were treated with the key factor inhibitor to evaluate microglia and macrophage phenotypic shifts and the progression of stroke. Results: Through single-cell RNA sequencing analysis, we identified a concurrent activation pattern between the stimulator of interferon genes (STING) mediated Type I interferon signaling and the pro-inflammatory phenotype of microglia/macrophages. In the first 5 days after stroke, microglia and macrophages displayed an inflammatory-resolving phenotype with enhanced phagocytic activity, but from days 5 to 14, they transitioned to a pro-inflammatory phenotype with sustained upregulation of STING-mediated Type I interferon pathway molecules. We validated the phenomenon observed in bioinformatics using tMCAO models. In vitro and in vivo, we found that continuous phagocytosis by macrophages and microglia led to STING activation, triggering Type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. Treatment with STING inhibitor H-151 reversed these detrimental shifts.. Conclusions: These findings suggest that continuous post-stroke phagocytosis by macrophages and microglia led to the activation of STING, thereby triggering type I interferon signaling and driving the phenotypic shift towards a pro-inflammatory state. STING inhibition may serve as a therapeutic strategy for mitigating neuroinflammatory responses following stroke.

Introduction: Clearing necrotic tissue from lesions by microglia/macrophage is crucial for recovery after acute ischemic stroke (AIS). Lysosomal activity is key for efferocytosis. In the chronic phase, reduced lysosomal activity impairs clearance, causing residual debris and worsening cognitive impairment. Migrasomes, formed during cellular migration, help maintain other organelles like mitochondria. Hypothesis: This study explored how migrasomes in microglia/macrophage help load damaged lysosomes for expulsion, preserving lysosomal quality and efferocytosis post-stroke. Methods: Male C57BL/6 mice including wild-type (WT) and Tspan14 conditional knockout ( Tspan14 fl/fl Lyz2 Cre , TSPAN14 CKO) underwent transient middle cerebral artery occlusion (tMCAO). Migrasome production and TSPAN14 expression were evaluated through staining and nano flow cytometry. Neurobehavioral performance was measured by rotarod, foot fault, novel object recognition, and water maze tests up to 14 days post-stroke. In vitro, bone marrow-derived macrophages (BMDM) were cultured with blebbistatin to inhibit migrasome production. BMDM efferocytosis of dead neurons was assessed by staining and flow cytometry. Results: In cultures, 76.9% of migrasomes from BMDM engulfing dead neurons contained damaged lysosomes. Inhibiting migrasome production in BMDM impaired efferocytosis and increased damaged lysosome accumulation. TSPAN14 on the lysosomal membrane mediated lysosome sorting into migrasomes. Migrasomes from TSPAN14 CKO BMDM engulfing dead neurons had 62.3% less lysosomal content than those from WT BMDM, though their numbers were similar ( P <0.05). BMDM from TSPAN14 CKO mice had 212.4% more damaged lysosomes and 54.7% less efferocytosis compared to WT BMDM ( P <0.05). In tMCAO model, migrasome production and TSPAN14 expression increased in Iba1 + microglia/macrophage after stroke, peaking on day 3 and stabilizing by day 7. By day 14, both decreased. TSPAN14 CKO mice had worse sensorimotor and spatial learning deficits than WT mice 14 days after tMCAO ( P <0.05). Microglia/macrophage in TSPAN14 CKO lesions had more damaged lysosomes and reduced efferocytosis ( P <0.01). Conclusions: TSPAN14 regulates lysosomal quality through migrasome-dependent mechanisms. In the chronic stroke phase, reduced TSPAN14 impairs lysosomal control in microglia/macrophage, leading to incomplete clearance and worsened cognitive impairment. Thus, TSPAN14 is a promising target to enhance clearance and improve stroke outcomes.

Confronting post-stroke neural inflammation is regarded as a promising therapeutic strategy for cerebral ischemia. Macrophage inflammasome activation plays a vital role in driving neural inflammation, and thus directly impacts stroke outcomes. Micheliolide (MCL), a newly discovered modifier of macrophage function, mechanistically suppresses NLRP3 macrophage signaling. The purpose of the present study was to evaluate the role of inflammasome suppression in MCL-based treatment for stroke. Decreased lesion volumes and improved neurological function in mice receiving MCL treatment were measured at both 3- and 14- day intervals after stroke. PKM2 (Pyruvate Kinase M2) was identified as the direct target of MCL that was found to efficiently suppress inflammasome activation and downregulate NF-κB (NLR family pyrin domain containing 3) signaling. P65 (subunit of NF-κB) binding of PKM2 increases during inflammasome induction and MCL treatment interfered with this interaction. In conclusion, MCL interferes with the PKM2-NF-κB interaction during inflammasome activation and ameliorates post-stroke inflammation, demonstrating its potential in acute ischemic stroke treatment.

Acute ischemic stroke triggers immunosuppression, yet existing therapies struggle to balance neuroprotection with poststroke immunosuppression. We demonstrated that bone marrow mesenchymal stem cells (BM-MSC) reverse stroke-induced thymic atrophy by promoting T-cell differentiation and restoring peripheral T-cell populations. Bulk RNA sequencing of BM-MSC-treated thymuses revealed enhanced proliferative signatures. Mechanistically, BM-MSC secrete migrasomes (organelles derived from migrating cells) that traverse the blood‒thymus barrier. Single-cell RNA sequencing analysis demonstrated that migrasome-mediated proliferation occurred specifically in medullary thymic epithelial cell I (mTECI) subpopulations. Proteomic profiling via liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) identified Pin1—a cell cycle regulator—as the predominant cargo in BM-MSC-derived migrasomes. In vivo and in vitro studies confirmed migrasome-mediated thymic epithelial proliferation, T-cell niche reconstruction, and immune homeostasis restoration. Migrasome monotherapy improved neurological deficits and survival rates in stroke model mice, demonstrating dual neuroprotective-immunomodulatory efficacy. This work addresses the clinical dilemma between neuroprotection and immunosuppression alleviation, establishing migrasomes as a cell-free therapeutic strategy for poststroke immunotherapy.