The Special Issue “Glioblastoma: What Do We Know [...]

Glial scar formation is one of the major pathological mechanisms following ischemic stroke. Rapamycin is a potent specific mTOR inhibitor and an autophagy activator. Although it has neuroprotective effects against acute ischemic stroke, it is unknown whether delayed administration of rapamycin can reduce ischemic stroke-induced pathogenesis such as glial scar formation, independent on its effects of acute administration. We recently reported that matrilin-3, an extracellular matrix component, provides neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation and glial scar formation. Here, in rat models of middle cerebral artery occlusion and reperfusion (I/R), rapamycin was administered for consecutive 7 or 14 days starting at day 1 post-reperfusion; and in an oxygen-glucose deprivation and reoxygenation (OGD/Re)-induced primary astrocyte or human astrocyte injury model, rapamycin was given upon reoxygenation. We found that rapamycin improved I/R-mediated rats' neurological dysfunction, accompanied by reduced glial scar formation and neuronal loss. To our surprise, rapamycin increased the levels of matrilin-3 in the peri-infarct region of rats and in OGD/Re-treated astrocytes associating with restoring autophagic flux. In contrast, the autophagy inhibitors wortmannin and bafilomycin A1 blocked autophagic flux, decreased the levels of matrilin-3 and enhanced glial scar formation, respectively. Overexpression of matrilin-3 significantly reduced the glial scar formation. Mechanistically, rapamycin could decrease the ADAMTS-4 and ADAMTS-5 levels, two hydrolases responsible for the breakdown of matrilin-3, thus upregulating the matrilin-3 levels. Our results reveal that delayed administration of rapamycin suppresses the glial scar formation by upregulating the astrocytic matrilin-3 related to restoring autophagic flux in ischemic stroke.

Alzheimer’s disease (AD) and stroke have been identified as risk factors for each other. More than half of AD patients suffer stroke attacks and worse ischemic injuries. There has been a lack of research focus and clinical treatment for the comorbidity of these neurological disorders. AD and ischemic stroke share characteristic pathophysiology, including hyperactivities of excitatory neurons and NMDA receptors (NMDARs), excitotoxicity, and synapse/neurovascular destruction. Our recent investigations identified the deficiency of the NMDAR regulatory GluN3A (NR3A) subunit as a novel pathogenesis of sporadic AD. The present investigation tested a preemptive treatment to prevent AD development in two AD models and, in the meantime, to prime the susceptible brain against upcoming ischemic attacks. In the preclinical stage of 3-month-old GluN3A KO mice, an NMDAR-mediated sporadic AD model, and 5xFAD mice, an amyloid-based familial AD model, treatments with memantine (MEM), an NMDAR antagonist (10 mg/kg/day in drinking water) and a drug-free control were started when cognition of these mice was generally normal. Three months later, the mice were subjected to focal cerebral ischemic surgery, followed by continued 1.5–2.0 months of MEM or vehicle control. Morphological, pathological, and functional assessments were performed and compared at different time points. In both AD models, the early MEM treatment confined AD progression before and after stroke, reduced ischemia-induced brain injury, suppressed neuroinflammation, and improved locomotion, sensorimotor, psychological, and cognitive functions. This is the first report endorsing a shared mechanism of NMDAR hyperactivity in AD and stroke in AD models with distinctive risk factors. The dual therapeutic effects of the preemptive MEM treatment provide a disease-modifying possibility for individuals who are susceptible to sporadic or familial AD as well as ischemic stroke.

Neuronal and NMDA receptor (NMDAR) hyperactivities are common pathophysiology in Alzheimer’s disease (AD). We recently identified that the deficiency of NMDAR regulatory subunit GluN3A (NR3A) cultivated early psychological and olfactory symptoms, followed by cognitive decline and deferred endogenous amyloid/tau pathologies. NMDAR antagonist memantine (MEM) prevented AD-like progression in GluN3A knockout (KO) mice. We tested the hypothesis that AD development of the 5xFAD mouse can be antagonized by the preemptive MEM treatment. MEM (10 or 20 mg/kg per day in drinking water for 3 months) was started in wild-type (WT) and 5xFAD mice at 3 months old. In this preclinical stage, the 5xFAD mouse displayed psychological and olfactory symptoms, yet exhibited no significant cognitive deficits or Aβ42 deposition. The MEM treatment antagonized early symptoms, abated cognitive decline, and amyloid/tau pathology. Early and persistent maintenance of normal neuronal/NMDAR activities in individuals carrying AD risk factors should be considered as a preventive and a possible disease-modifying therapy.

Stroke and Alzheimer's disease are common neurological disorders and often occur in the same individuals. The comorbidity of the two neurological disorders represents a grave health threat to older populations. This review presents a brief background of the development of novel concepts and their clinical potentials. The activity of glutamatergic N-methyl-D-aspartate receptors and N-methyl-D-aspartate receptor-mediated Ca 2+ influx is critical for neuronal function. An ischemic insult induces prompt and excessive glutamate release and drastic increases of intracellular Ca 2+ mainly via N-methyl-D-aspartate receptors, particularly of those at the extrasynaptic site. This Ca 2+ -evoked neuronal cell death in the ischemic core is dominated by necrosis within a few hours and days known as acute excitotoxicity. Furthermore, mild but sustained Ca 2+ increases under neurodegenerative conditions such as in the distant penumbra of the ischemic brain and early stages of Alzheimer's disease are not immediately toxic, but gradually set off deteriorating Ca 2+ -dependent signals and neuronal cell loss mostly because of activation of programmed cell death pathways. Based on the Ca 2+ hypothesis of Alzheimer's disease and recent advances, this Ca 2+ -activated "silent" degenerative excitotoxicity evolves from years to decades and is recognized as a unique slow and chronic neuropathogenesis. The N-methyl-D-aspartate receptor subunit GluN3A, primarily at the extrasynaptic site, serves as a gatekeeper for the N-methyl-D-aspartate receptor activity and is neuroprotective against both acute and chronic excitotoxicity. Ischemic stroke and Alzheimer's disease, therefore, share an N-methyl-D-aspartate receptor- and Ca 2+ -mediated mechanism, although with much different time courses. It is thus proposed that early interventions to control Ca 2+ homeostasis at the preclinical stage are pivotal for individuals who are susceptible to sporadic late-onset Alzheimer's disease and Alzheimer's disease-related dementia. This early treatment simultaneously serves as a preconditioning therapy against ischemic stroke that often attacks the same individuals during abnormal aging.

Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell’s fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.

Our recent investigation revealed that deficiency of N-methyl-D-aspartate (NMDA) receptor subunit GluN3A (NR3A) is a trigger for chronic neuronal hyperactivity and disruptionFfepspof Ca2+ homeostasis, leading to sporadic Alzheimer's disease (AD) phenotypes. The identification of the amyloid-independent pathogenesis was a surprise considering that GluN3A is a much less known NMDA receptor subunit with obscure function in aging adulthood, while the new concept of degenerative excitotoxicity as a decade-long pathogenic mechanism of AD/dementia remains to be further delineated. With negative observations in GRIN3A-/- mouse, Verhaeghe et al. in their letter challenge the "odd" idea that lasting GluN3A deficiency is detrimental and responsible for the spontaneous progression of AD and cognitive decline. We now discuss the potential mouse strain hypothesis and experimental data in these two investigations, and provide additional evidence that further supports the validity and specificity of GluN3A deficiency in the development of AD and associated dementia.

Stroke and late-onset Alzheimer’s disease (AD) are risk factors for each other; the comorbidity of these brain disorders in aging individuals represents a significant challenge in basic research and clinical practice. The similarities and differences between stroke and AD in terms of pathogenesis and pathophysiology, however, have rarely been comparably reviewed. Here, we discuss the research background and recent progresses that are important and informative for the comorbidity of stroke and late-onset AD and related dementia (ADRD). Glutamatergic NMDA receptor (NMDAR) activity and NMDAR-mediated Ca 2+ influx are essential for neuronal function and cell survival. An ischemic insult, however, can cause rapid increases in glutamate concentration and excessive activation of NMDARs, leading to swift Ca 2+ overload in neuronal cells and acute excitotoxicity within hours and days. On the other hand, mild upregulation of NMDAR activity, commonly seen in AD animal models and patients, is not immediately cytotoxic. Sustained NMDAR hyperactivity and Ca 2+ dysregulation lasting from months to years, nevertheless, can be pathogenic for slowly evolving events, i.e. degenerative excitotoxicity, in the development of AD/ADRD. Specifically, Ca 2+ influx mediated by extrasynaptic NMDARs (eNMDARs) and a downstream pathway mediated by transient receptor potential cation channel subfamily M member (TRPM) are primarily responsible for excitotoxicity. On the other hand, the NMDAR subunit GluN3A plays a “gatekeeper” role in NMDAR activity and a neuroprotective role against both acute and chronic excitotoxicity. Thus, ischemic stroke and AD share an NMDAR- and Ca 2+ -mediated pathogenic mechanism that provides a common receptor target for preventive and possibly disease-modifying therapies. Memantine (MEM) preferentially blocks eNMDARs and was approved by the Federal Drug Administration (FDA) for symptomatic treatment of moderate-to-severe AD with variable efficacy. According to the pathogenic role of eNMDARs, it is conceivable that MEM and other eNMDAR antagonists should be administered much earlier, preferably during the presymptomatic phases of AD/ADRD. This anti-AD treatment could simultaneously serve as a preconditioning strategy against stroke that attacks ≥ 50% of AD patients. Future research on the regulation of NMDARs, enduring control of eNMDARs, Ca 2+ homeostasis, and downstream events will provide a promising opportunity to understand and treat the comorbidity of AD/ADRD and stroke.

Background: Adequate collateral circulation can remarkably improve patient prognoses for patients experiencing ischemic stroke. Hypoxic preconditioning enhances the regenerative properties of bone marrow mesenchymal stem cells (BMSCs). Rabep2 (RAB GTPase binding effector protein 2) is a key protein in collateral remodeling. We investigated whether BMSCs and hypoxia-preconditioned BMSCs (H-BMSCs) augment collateral circulation poststroke, particularly through Rabep2 regulation. Methods: BMSCs or H-BMSCs (1×10 6 ) were delivered intranasally in ischemic mice with distal middle cerebral artery occlusion at 6 hours poststroke. Two-photon microscopic imaging and vessel painting methods were used to analyze collateral remodeling. Blood flow, vascular density, infarct volume, and gait analysis were assessed to evaluate poststroke outcomes. Expressions of proangiogenic marker VEGF (vascular endothelial growth factor) and Rabep2 were determined by Western blotting. Western blot, EdU (5-ethynyl-2’-deoxyuridine) incorporation, and tube formation assays were conducted on cultured endothelial cells treated with BMSCs. RESULTS: BMSCs were more effectively transplanted in the ischemic brain after hypoxic preconditioning. The ipsilateral collateral diameter was increased by BMSCs and strengthened by H-BMSCs ( P <0.05). BMSCs increased peri-infarct blood flow and vascular density and reduced infarct volume, gait deficits ( P <0.05), and furthermore by H-BMSCs ( P <0.05). VEGF and Rabep2 protein expression was increased by BMSCs ( P <0.05), which was enhanced by preconditioning ( P <0.01). Additionally, BMSCs increased Rabep2 expression, proliferation, and tube formation of endothelial cells in vitro ( P <0.05). H-BMSCs enhanced these effects ( P <0.05), which were annulled by Rabep2 knockdown. CONCLUSIONS: BMSCs increased collateral circulation and improved poststroke outcomes, through the upregulation of Rabep2. These effects were enhanced by hypoxic preconditioning.

Late-onset Alzheimer’s disease (AD) and related dementia (ADRD) are serious neurodegenerative disorders among aging populations. The progress of AD/ADRD cultivates over years to decades in humans and several months in animal models. Ca2+ homeostasis is a core function of neurons where the N-methyl-D-aspartate (NMDA) receptor plays a major role in excitatory neuronal activities. The Ca2+ hypothesis of AD proposes that even slight but sustained Ca2+ dyshomeostasis in the brain is a critical pathophysiology or pathogenesis of AD. However, instigating factors like the trigger and time/duration of the Ca2+ dysregulation in AD progression have been largely obscure, while β-amyloid (Aβ) peptides are often indicated as the trigger. Hyperactivities of excitatory neurons and NMDARs have been implicated in AD as a main mediating mechanism caused by AD pathologies such as Aβ deposition. NMDAR overactivation is restrained by the unique regulatory GluN3 subunits (GluN3A and GluN3B; previously known as NR3A and NR3B). Expression of GluN3A in the receptor complex reduces NMDAR currents and Ca2+ influx, while deletion of GluN3A causes larger NMDA currents and elevated intracellular Ca2+. Significant GluN3A levels are detected in both rodent and human adult/aging brains. We hypothesized that the “gatekeeper” role of GluN3A is constantly required for Ca2+ homeostasis and normal aging; its deficiency can lead to slowly evolved “degenerative excitotoxicity”. Our in vitro, ex vivo, and in vivo studies using GluN3A knockout (KO) mice of young and older ages revealed neuronal hyperactivity, moderate but sustained elevation of cytosolic Ca2+, chronic inflammation, neuronal loss/apoptosis, synaptic impairments and progressive cognitive deficits. The AD hallmarks of Aβ and tau pathology were identified after, but not before, cognition decline. In the “gain of function” experiment, expression of GluN3A in the GluN3A KO brain prevented AD progression. Specific regional knockdown of GluN3A in the cortex and hippocampus of wild-type mice at the adult age (3 months old) resulted in similar AD/ADRD phenotypic alterations in the following 3-6 months. The NMDAR antagonist memantine (MEM) is approved by FDA as a symptomatic treatment for moderate-severe AD patients. According to the chronic neurohyperactivity in AD progression and the modified Ca2+ hypothesis that Ca2+ dysregulation is an early and “life-long” pathogenesis, the maintenance of NMDAR normal activity by MEM or other safe NMDAR antagonists could be a disease-modifying early treatment in preclinical/prodromal stages. This prediction is endorsed by clinical trials using MEM and other NMDAR antagonists in mild cognitive impairment (MCI) and early AD patients, showing beneficial effects of maintaining cognitive functions. Consistently, we showed that, in GluN3A KO mice and 5xFAD mice, early and chronic MEM treatment (started at 3-month of age and lasted for 3-6 months) prevented or attenuated AD phenotypes. Meanwhile, the chronic MEM therapy in mice showed preconditioning effect of neuroprotection against ischemic stroke that strikes over 50% AD patients. Large clinical trials of long-term and more systematic examinations on AD/ADRD progression and the comorbidity of stroke are warranted for this innovative therapy. Our results support the modification of Ca2+ hypothesis of AD with the novel amyloid-independent mechanism. Specifically, the deficiency of GluN3A alone causes lifelong Ca2+-related progression of AD pathophysiology and amyloid pathology. The long-term GluN3A modulation of NMDARs signifies new therapeutic targets and possible preventive interventions for late-onset AD/ ADRD.

Alzheimer’s disease (AD) is the most common dementia. It is known that women with one ApoE4 allele display greater risk and earlier onset of AD compared with men. In mice, we previously showed that follicle–stimulating hormone (FSH), a gonadotropin that rises in post–menopausal females, activates its receptor FSHR in the hippocampus, to drive AD–like pathology and cognitive impairment. Here we show in mice that ApoE4 and FSH jointly trigger AD-like pathogenesis by activating C/EBPβ/δ-secretase signaling. ApoE4 and FSH additively activate C/EBPβ/δ-secretase pathway that mediates APP and Tau proteolytic fragmentation, stimulating Aβ and neurofibrillary tangles. Ovariectomy-provoked AD-like pathologies and cognitive defects in female ApoE4-TR mice are ameliorated by anti-FSH antibody treatment. FSH administration facilitates AD-like pathologies in both young male and female ApoE4-TR mice. Furthermore, FSH stimulates AD-like pathologies and cognitive defects in ApoE4-TR mice, but not ApoE3-TR mice. Our findings suggest that in mice, augmented FSH in females with ApoE4 but not ApoE3 genotype increases vulnerability to AD-like process by activating C/EBPβ/δ-secretase signalling.

Background: Although Alzheimer's disease (AD) is the most common form of dementia, the effective treatment of AD is not available currently. Multiple trials of drugs, which were developed based on the amyloid hypothesis of AD, have not been highly successful to improve cognitive and other symptoms in AD patients, suggesting that it is necessary to explore additional and alternative approaches for the disease-modifying treatment of AD. The diverse lines of evidence have revealed that lithium reduces amyloid and tau pathology, attenuates neuronal loss, enhances synaptic plasticity and improves cognitive function. Clinical studies have shown that lithium reduces the risk of AD and deters the progress of mild cognitive impairment and early AD. Summary: Our recent study has revealed that lithium stabilizes disruptive calcium homeostasis, and subsequently, attenuates the down-stream neuropathogenic processes of AD. Through these therapeutic actions, lithium produces therapeutic effects on AD with potential to modify the disease process. This review critically analyzed the preclinical and clinical studies for the therapeutic effects of lithium on AD. We suggest that disruptive calcium homeostasis is likely to be the early neuropathological mechanism of AD, and the stabilization of disruptive calcium homeostasis by lithium would be associated with its therapeutic effects on neuropathology and cognitive deficits in AD. Key Messages: Lithium is likely to be efficacious for AD as a disease modifying drug by acting on multiple neuropathological targets including disruptive calcium homeostasis.

Abstract Cerebral amyloid angiopathy (CAA), amyloid-β (Aβ) deposits in cerebral vessel walls, is commonly associated with Alzheimer’s disease (AD) and contributes to cerebrovascular dysfunctions. APOE4 is a strong genetic risk factor for both AD and CAA. However, the mechanisms underlying this genetic susceptibility remain incompletely understood. Here we show that ApoE4 and 27-hydroxycholesterol (27-OHC) additively drive C/EBPβ/AEP signaling activation in macrophages, which impairs Aβ uptake and clearance and stimulates CAA formation, leading to cerebrovascular dysfunctions and microbleeds. To explore the pathological roles of ApoE4/27-OHC in CAA formation during AD pathogenesis, we crossed smooth muscle cells specific SMA-GFP mice with 5xFAD mice and found reduced blood flow, decreased vessel length and elevated oxidative stress and hemorrhagic lesions, which were exacerbated by ApoE4/27-OHC. Notably, ApoE4/27-OHC promoted Aβ deposits in leptomeningeal, cortical and hippocampal arteries and increased parenchymal Aβ load as compared to ApoE3/27-OHC, associated with strong C/EBPβ/AEP activation in macrophages that surrounded and infiltrated the microvessels. Depletion of C/EBPβ or AEP from 5xFAD/SMA-GFP mice ameliorated ApoE4/27-OHC-triggered CAA-associated vasculopathies and cerebrovascular disorders and senile plaques, alleviating cognitive disorders. Therefore, ApoE4 and 27-OHC jointly facilitate both CAA and AD pathologies via activating C/EBPβ/AEP pathway.

Alzheimer’s disease (AD) is a neurodegenerative disease, evolving amyloidosis and tau protein hyperphosphorylation, synapse dysfunction, neuron degeneration and cognitive deficits. Investigations based on the Aβ/tau hypothesis have been performed in transgenic animal models expression familial/mutated AD genes. However, treatments based on this hypothesis have not provided effective clinical therapies. Hyperactivity of the glutamatergic NMDA receptor (NMDAR) is a common pathophysiology of AD and has been viewed as a consequence of Aβ pathology. Among NMDAR subunits, GluN3A is a unique inhibitory subunit that suppresses NMDA currents and Ca 2+ influx. Recent evidence from rodent and human studies shows that GluN3A is widely expressed and plays important functional roles in neonatal and adult brains. Ca 2+ overload is a trigger of excitotoxicity, while GluN3A plays a regulatory role of Ca 2+ homeostasis. The role of GluN3A regulation in AD is unknown. GluN3A knockout mice, gene modifications, immunohistochemistry, Western blotting, MEA recordings, Ca2+ imaging, pathological examinations, functional and behavioral tests. Chronic neuronal hyperactivity and Ca 2+ dysregulation caused by GluN3A deficiency lead to slowly advanced “neurodegenerative excitotoxicity”, dementia and AD pathology in an age-dependent manner. Ca 2+ imaging and MultiElectrode Array recordings in GluN3A KO brain slices revealed moderate but sustained elevation of cytosolic Ca 2+ levels, neuronal hyperactivity, and impaired synaptic plasticity compared to age matched controls. Aging GluN3A KO mice showed increased Ca 2+ -dependent signals, chronic inflammation, neuronal loss and disease symptoms including early onset of olfactory dysfunction followed by cognitive deficits. β-Amyloid deposition and tau protein hyperphosphorylation were identified as a late pathology in the cortex and hippocampus. Expression of GluN3A in the GluN3A KO brain prevented AD progression. Early treatment at the pre-clinical stage using the NMDAR antagonist memantine prevented symptoms in GluN3A KO mice. Western blot analysis of AD patient’s brains showed lower levels of GluN3A in the cortex compared to normal aging subjects. Our results identified a novel amyloid-independent mechanism that the deficiency of GluN3A alone causes a slight lifelong Ca 2+ dyshomeostasis, leading to AD pathophysiology and amyloid pathology. The long-term GluN3A modulation of NMDARs signifies new therapeutic targets and possible preventive interventions for a sporadic type of AD.

ApoE4 is a major genetic risk determinant for Alzheimer's disease (AD) and drives its pathogenesis via Aβ-dependent and -independent pathways. C/EBPβ, a proinflammatory cytokine-activated transcription factor, is upregulated in AD patients and increases cytokines and δ-secretase expression. Under physiological conditions, ApoE is mainly expressed in glial cells, but its neuronal expression is highly elevated under pathological stresses. However, how neuronal ApoE4 mediates AD pathologies remains incompletely understood. Here we show that ApoE4 activates C/EBPβ that subsequently regulates APP, Tau and BACE1 mRNA expression in mouse neurons, driving AD-like pathogenesis. To interrogate the pathological roles of both human ApoE4 and C/EBPβ elevation in neurons in the aged brain, we develop neuronal specific Thy1-ApoE4/C/EBPβ double transgenic mice. Neuronal ApoE4 strongly activates C/EBPβ and augmented δ-secretase subsequently cleaves increased mouse APP and Tau, promoting AD-like pathologies. Notably, Thy1-ApoE4/C/EBPβ mice develop amyloid deposits, Tau aggregates and neurodegeneration in an age-dependent manner, leading to synaptic dysfunction and cognitive disorders. Thus, our findings demonstrate that neuronal ApoE4 triggers AD pathogenesis via activating the crucial regulator C/EBPβ.

Atherosclerosis (ATH) and Alzheimer’s disease (AD) are both age-dependent inflammatory diseases, associated with infiltrated macrophages and vascular pathology and overlapping molecules. C/EBPβ, an Aβ or inflammatory cytokine-activated transcription factor, and AEP (asparagine endopeptidase) are intimately implicated in both ATH and AD; however, whether C/EBPβ/AEP signaling couples ATH to AD pathogenesis remains incompletely understood. Here we show that C/EBPβ/AEP pathway mediates ATH pathology and couples ATH to AD. Deletion of C/EBPβ or AEP from primary macrophages diminishes cholesterol load, and inactivation of this pathway reduces foam cell formation and lesions in aorta in ApoE−/− mice, fed with HFD (high-fat-diet). Knockout of ApoE from 3xTg AD mouse model augments serum LDL and increases lesion areas in the aorta. Depletion of C/EBPβ or AEP from 3xTg/ApoE−/− mice substantially attenuates these effects and elevates cerebral blood flow and vessel length, improving cognitive functions. Strikingly, knockdown of ApoE from the hippocampus of 3xTg mice decreases the cerebral blood flow and vessel length and aggravates AD pathologies, leading to cognitive deficits. Inactivation of C/EBPβ/AEP pathway alleviates these events and restores cognitive functions. Hence, our findings demonstrate that C/EBPβ/AEP signaling couples ATH to AD via mediating vascular pathology.

OBJECTIVE:Stroke is a leading cause of human death and disability. Effective early treatments with reasonable therapeutic windows remain critically important to improve the outcomes of stroke. Transcranial magnetic stimulation (TMS) is an established noninvasive technique that has been applied clinically and in animal research for multiple brain disorders, but few studies have examined acute neuroprotection against ischemic stroke. The present investigation tested the novel approach of low-frequency repetitive TMS (rTMS) as an acute treatment after ischemic stroke.METHODS:Adult male rats received focal ischemic surgery through occlusion of the right middle cerebral artery for 60 minutes. The rats received either rTMS or sham treatment with 1.5-, 3-, 4-, or 7-hour delay after the onset of stroke. Low-frequency and low-intensity rTMS was applied to the rat brain for two 30-minute episodes separated by a 1-hour interval.RESULTS:Three days after stroke, compared to stroke controls, rats receiving rTMS treatment with a 1.5-hour delay showed a 35% reduction of infarct volume. Protective effects were also seen with 3- or 4-hour-delayed treatments by rTMS, shown as reduced infarct volume and cell death. rTMS treatment upregulated the antiapoptotic factor Bcl-2 and downregulated the proapoptotic caspase-3 cleavage, expressions of Bax and matrix metallopeptidase-9. In sensorimotor functional assessments 3 to 21 days after stroke, rats receiving rTMS treatment with a 1.5- or 3-hour delay showed significantly better performance compared to stroke controls.INTERPRETATION:These results support the inference that low-frequency rTMS may be feasible as a neuroprotective acute treatment after ischemic stroke. ANN NEUROL 2023;93:336-347.

Ischemic stroke is a leading cause of morbidity and mortality, with limited treatments that can facilitate brain regeneration. Neural progenitor cells (NPCs) hold promise for replacing tissue lost to stroke, and biomaterial approaches may improve their efficacy to overcome hurdles in clinical translation. The immune response and its role in stroke pathogenesis and regeneration may interplay with critical mechanisms of stem cell and biomaterial therapies. Cellular therapy can modulate the immune response to reduce toxic neuroinflammation early after ischemia. However, few studies have attempted to harness the regenerative effects of neuroinflammation to augment recovery. Our previous studies demonstrated that intracerebrally transplanted NPCs encapsulated in a chondroitin sulfate-A hydrogel (CS-A + NPCs) can improve vascular regeneration after stroke. In this paper, we found that CS-A + NPCs affect the microglia/macrophage response to promote a regenerative phenotype following stroke in mice. Following transplantation, PPARγ-expressing microglia/macrophages, and MCP-1 and IL-10 protein levels are enhanced. Secreted immunomodulatory factor expression of other factors was altered compared to NPC transplantation alone. Post-stroke depression-like behavior was reduced following cellular and material transplantation. Furthermore, we showed in cultures that microglia/macrophages encapsulated in CS-A had increased expression of angiogenic and arteriogenic mediators. Neutralization with anti-IL-10 antibody negated these effects in vitro. Cumulatively, this work provides a framework for understanding the mechanisms by which immunomodulatory biomaterials can enhance the regenerative effects of cellular therapy for ischemic stroke and other brain injuries.