Aldose reductase (AR) is involved in the pathogenesis of ischemic stroke; however, the mechanisms are not well understood. This study aimed to evaluate the role and underlying mechanisms of AR inhibition in ischemic stroke. We found that microglial neuroinflammatory responses to oxygen glucose deprivation/reperfusion (OGD/R) were attenuated by either sorbinil-mediated AR inhibition or AR knockdown. Sorbinil (5-20 μM) attenuated OGD/R-induced endoplasmic reticulum (ER) stress and downstream c-Jun N-terminal kinase (JNK) activation in microglia. Further analysis revealed that sorbinil suppressed microglial autophagic hyperactivation, reduced nuclear receptor coactivator 4 expression, and elevated ferritin heavy chain (FTH1) levels. This effect was accompanied by diminished FTH1 colocalization with lysosomes and decreased intracellular iron (Fe2+) concentrations. Critically, FTH1 knockdown attenuated the inhibitory effect of sorbinil on Fe2+ and interlenkin-1β in microglia. Furthermore, sorbinil mitigated OGD/R-triggered oxidative stress in microglia. Moreover, sorbinil had similar effects on microglia exposed to thapsigargin-induced ER stress. Notably, the protective efficacy of sorbinil was attenuated by the pharmacological activation of JNK. In a mouse middle cerebral artery occlusion/reperfusion (MCAO/R) model, sorbinil (4, 8, and 16 mg/kg; i.p.) ameliorated neurological deficits and decreased the volume of cerebral infraction in MCAO/R model mice. This suppression coincided with attenuated neuroinflammation and the activation of ER stress and ferritinophagy in MCAO/R model mice. Overall, this study reveals a novel role of AR inhibition in regulating microglial ER stress-ferritinophagy signaling during cerebral ischemia. AR represents a promising therapeutic target for mitigating cerebral ischemia‒reperfusion injury.

Ischemic stroke (IS) disrupts the blood-brain barrier (BBB), thereby aggravating neurological deterioration. Loss of tight junction (TJ) and adherens junction (AJ) proteins is a key event in BBB breakdown, but the upstream mechanisms remain incompletely understood. Here, we investigated whether LS21013A-06 (A06), a phosphodiesterase-4 inhibitor, protects brain microvascular endothelial cells and preserves BBB integrity after ischemia/reperfusion, with particular focus on whether its protective effects are PKA-dependent and accompanied by changes in leukocyte cell-derived chemotaxin-2 (LECT2). In human brain microvascular endothelial cells subjected to oxygen-glucose deprivation/reperfusion (OGD/R), LECT2 knockdown preserved TJ and AJ proteins, reduced Bax upregulation, restored Bcl-2 expression, and diminished reactive oxygen species accumulation. A06 pretreatment (3 μM) recapitulated these effects and markedly reduced OGD/R-induced LECT2 expression. The PKA inhibitor H89 (5 μM) abolished A06-mediated LECT2 suppression, junctional preservation, and reductions in apoptosis and reactive oxygen species, whereas LECT2 overexpression similarly abrogated the protective effects of A06. In a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model, post-ischemic A06 administration (1 or 3 mg/kg, intraperitoneally) improved neurological scores and motor performance, reduced infarct volume and Evans blue extravasation, and increased ZO-1, VE-cadherin, and Occludin levels in peri-infarct cortex, together with reduced LECT2 expression and attenuated apoptosis-related changes in Bcl-2 and Bax. These findings indicate that A06 preserves BBB integrity after experimental ischemia. Mechanistically, its protective effects were PKA-dependent and accompanied by reduced LECT2 levels, preserved junctional proteins, and attenuated apoptosis-related changes. A06 enhanced PKA signaling, whereas PKA silencing blunted both its protective effects and the associated reduction in LECT2 expression.

Background Alzheimer’s disease (AD) is a complex systemic disorder that extends beyond the central nervous system, exerting pathological effects on the heart. Epidemiological studies have consistently shown that individuals with AD often exhibit impaired cardiac function. While amyloid-beta (Aβ) is a key pathological hallmark of AD, primarily known for forming oligomers and fibrils in the brain, emerging evidence suggests that Aβ also exerts detrimental effects on the myocardium. Despite these observations, the precise mechanisms through which AD contributes to the onset or progression of heart failure (HF) remain poorly understood. This study aims to elucidate the underlying links between AD and HF, with a specific focus on the pathogenic role of Aβ in promoting cardiac dysfunction within experimental models of AD. Methods Cardiomyocytes and 3 × Tg-AD mouse models were used to investigate Aβ-induced cardiotoxicity and to determine the mode of myocardial cell death. We assessed cell viability, intracellular copper levels, and markers of cuproptosis. Mitochondrial oxidative respiration, ATP production, and reactive oxygen species (ROS) levels were also evaluated. Myocardial pathology and cuproptosis-related proteins were detected by histochemistry and immunoblotting. Results In 3 × Tg-AD mice, elevated cardiac Aβ paralleled cardiac dysfunction, promoted cuproptosis in cardiomyocytes, and this effect was counteracted by the copper chelator TTM which inhibited myocardial copper uptake and protected cardiac function. Building on this in vivo observation, we further investigated the mechanism in vitro and found that Aβ upregulated the copper importer SLC31A1 in vitro. Furthermore, Aβ1-42 acted synergistically with CuCl₂ or elesclomol-CuCl₂ to exacerbate cardiomyocyte death. This synergy increased intracellular copper accumulation, triggered Fe-S cluster protein loss, and promoted DLAT oligomerization-hallmarks of cuproptosis. These cuproptosis-associated changes suppressed mitochondrial oxidative respiration, decreased ATP synthesis, and elevated ROS levels. Importantly, interference with SLC31A1 expression in vivo and in vitro partially inhibited cuproptosis and protected mitochondrial or cardiac function. Conclusion Aβ1-42 disrupts copper homeostasis by upregulating SLC31A1, thereby exacerbating myocardial cuproptosis and impairing cardiac function in AD. This novel mechanism highlights SLC31A1-mediated cuproptosis as a potential therapeutic target for preserving cardiac health in AD.

Background Alzheimer's disease (AD) is characterized by cortical atrophy, glutamatergic neuron loss, and cognitive decline. However, large-scale quantitative assessments of cellular changes during AD pathology remain scarce. Objective This study aims to integrate single-nuclei sequencing data from the Seattle Alzheimer's Disease Cortical Atlas (SEA-AD) with spatial transcriptomics to quantify cellular changes in the prefrontal cortex and temporal gyrus, regions vulnerable to AD neuropathological changes (ADNC). Methods We mapped differentially expressed genes (DEGs) and analyzed their interactions with pathological factors such as APOE expression and Lewy bodies. Cellular proportions were assessed, focusing on neurons, glial cells, and immune cells. Results RORB-expressing L4-like neurons, though vulnerable to ADNC, exhibited stable cell numbers throughout disease progression. In contrast, astrocytes displayed increased reactivity, with upregulated cytokine signaling and oxidative stress responses, suggesting a role in neuroinflammation. A reduction in synaptic maintenance pathways indicated a decline in astrocytic support functions. Microglia showed heightened immune surveillance and phagocytic activity, indicating their role in maintaining cortical homeostasis. Conclusions The study underscores the critical roles of glial cells, particularly astrocytes and microglia, in AD progression. These findings contribute to a better understanding of cellular dynamics and may inform therapeutic strategies targeting glial cell function in AD.

A high-resolution spatial physiological atlas of cortical neurons serves as an essential reference for studying neurological diseases and is crucial for a comprehensive understanding of the cortical functions and physiological mechanisms. Although previous studies have elucidated the cellular basis of individual cortex, the molecular characteristics and precise spatial organization of neural cells within multiple human cortex remain incompletely understood. Subcellular-level spatial transcriptomic sequencing and snRNA-seq enabled mapping of 14 human cortical regions, creating a single-cell atlas with transcriptomic data from 1,121,772 nuclei and spatial profiles from 1,888,306 cells to characterize neural cell organization. The atlas reveals distinct expression patterns and spatial arrangements of neural cell types. Glutamatergic neurons show precise laminar patterns, with similar expression in adjacent cortex. SST neurons fall into two transcriptional categories, corresponding to superficial and deep layer distributions. The atlas, integrated with functional networks, highlights correlations between neural cell types and cortical functions, uncovering cell-cell interactions and ligand-receptor patterns with regional differences in neuron-glia communication. It also deciphers transcriptomic differences and cellular composition in layer 4 and the stable subplate (layer 6b) across regions. Our findings offer insights into the cellular foundations of complex and intelligent regions within the human cortex.

Dynamin-related protein 1 (Drp1) regulates mitochondrial fission and participates in neuronal apoptosis during the pathology of cerebral ischemia. We have previously shown that inhibition of phosphodiesterase-4 (PDE4) protects against neuronal apoptosis in models of ischemic stroke. However, it remains unclear whether PDE4 inhibition affects Drp1-mediated mitochondrial dysfunction and apoptosis under cerebral ischemia conditions. This study aimed to determine whether ZX21011, a novel PDE4 inhibitor synthesized in our laboratory, can act on Drp1 to counteract ischemic brain injury and to elucidate its mechanism of action. We demonstrated that ZX21011 effectively reduced neuronal apoptosis caused by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells and ameliorated neurological deficits caused by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats. ZX21011 enhanced glycogen synthase kinase-3(3 (GSK3(3) phosphorylation (Ser9), GSK3(3 (S9A) mutation blocked the protective effects of ZX21011. Simultaneously, ZX21011 reduced the levels of reactive oxygen species (ROS), restored the morphology of mitochondria, and inhibited the phosphorylation of Drp1(Ser616). The Drp1(S616E) mutation blocked the protective effects of ZX21011 on ROS production and mitochondrial morphology function after cerebral ischemia. What's more, co-immunoprecipitation analysis revealed that ZX21011 decreased the binding of GSK3(3 to Drp1, and GSK3(3(S9A) mutation reversed the effects of ZX21011 on Drp1 phosphorylation and cell viability. Moreover, ZX21011 decreased Drp1(Ser616) phosphorylation within the ischemic penumbra of rats following cerebral ischemia/reperfusion. In summary, ZX21011 counteracts ischemic stroke-induced oxidative stress and neuronal death, and its action is related to decreased Drp1 phosphorylation at Ser616. Thus, ZX21011 is a potential compound for the intervention of stroke.

The dynamic balance of mitochondrial fusion and fission directly contributes to mitochondrial homeostasis, which influences numerous cellular functions in addition to adenosine triphosphate (ATP) homeostasis. Therefore, assessing mitochondrial morphology under stress conditions is essential for mechanistic research. This study describes a detailed protocol for analyzing mitochondrial morphology, encompassing the preparation of a MitoTracker solution, staining of mitochondria, optimization of imaging parameters, and detection of morphological features. MitoTrackers are commonly used, cost-effective mitochondrion-specific dyes. However, some changes in mitochondrial morphology may occur owing to inappropriate handling, which can be unperceivable and fail to reflect the true state of mitochondria. Therefore, it is necessary to understand how to analyze changes in mitochondrial morphology using MitoTrackers. The protocol utilized SH-SY5Y cells stimulated with 1-methyl-4-phenylpyridinium iodide (MPP+) to illustrate the protocol of mitochondrial morphological analysis. Compared with control cells, MPP+-stimulated cells exhibited smaller and more fragmented mitochondria, with morphological parameters indicating decreased mitochondrial footprint. These results suggest that MitoTracker staining is an effective and feasible method for mitochondrial morphological analysis that (with minor modifications) can be applied to study various conditions.

Discovery of new natural products with both anti-inflammatory effects on activated microglia and protective activity on dopaminergic neurons is a new strategy to find new drug leads against neuroinflammation in Parkinson's disease. In this work, nine new limonoids, named thaigranatumins A-I (1-9), and two new protolimonoids, named thaigranatumins J (10) and K (11), were obtained from seeds of the Thai mangrove, Xylocarpus granatum. The structures of these compounds were established by analysis of spectroscopic data, single-crystal X-ray diffraction (Cu Kα), and comparison of experimental and calculated ECD spectra. Thaigranatumin A (1), containing a C-16/C-30 δ-lactone ring-D and a tetra-substituted C8-O-C17-bridged tetrahydrofuran ring-F, is the first limonoid featuring a unique 6/6/6/6/6/5/5-fused heptacyclic framework. Thaigranatumin G (7) exhibited both inhibitory effects on the protein expression of iNOS, COX2, and IL-1β in lipopolysaccharide-stimulated mouse microglia BV2 cells and neuroprotective activity against rotenone-induced injury in mouse midbrain dopaminergic neuron MN9D cells in a dose-dependent manner. Preliminary bioassays indicated that thaigranatumin G might be a valuable lead against neuroinflammation, thus warranting further studies.

Anti-N-methyl-d-aspartate receptor encephalitis (NMDARE) is the most prevalent autoimmune encephalitis caused by antibodies against the NMDAR subunit GluN1. Clinical evidence suggests that NMDARE is characterized by microglial activation, but the role of this activation remains unclear. In this study, single-nucleus RNA sequencing of the hippocampus from NMDARE mice revealed an upregulation of microglial complement 1q (C1q) levels. Clinically, we observed elevated C1q in the cerebrospinal fluid of NMDARE patients. Our studies showed that microglial cells express NMDAR, and antibodies from NMDARE patients act on microglia, inducing NMDAR internalization at the microglial membrane and triggering microglial C1q expression. Notably, silencing C1q attenuated the microglial activation induced by NMDAR antibodies, whereas C1q overexpression exacerbated this process. In vivo, C1q microglial knockout and knockdown in mice both showed reduced damage following NMDARE, while C1q overexpression in the hippocampus intensified pathological effects. Most promising is that neutralizing antibodies against C1q significantly mitigated injury in NMDARE mice. In summary, our findings highlight the therapeutic potential of inhibiting C1q to counteract NMDARE-induced injury.

Abnormality in transactivating response region DNA binding protein 43 (TDP43) is well-recognized as the pathological hallmark of neurodegenerative diseases. However, the role of TDP43 in neuromyelitis optica spectrum disorder (NMOSD) remains unknown. Here, our observations demonstrate an upregulation of TDP43 in both in vitro and in vivo models of NMOSD, as well as in biological samples from NMOSD patients. Single-nucleus RNA sequencing revealed that NMOSD induced A1-like reactive astrocytes and astrocyte mitochondrial dysfunction in mice. We further found that NMOSD provoked the translocation of TDP43 to mitochondria and the release of mitochondrial DNA (mtDNA) into the cytoplasm. NMOSD caused activation of mtDNA/cyclic GMP-AMP synthase (cGAS) / stimulator of interferon genes (STING) pathway and A1-type inflammatory activation in astrocytes. Crucially, the knockdown of TDP43 markedly ameliorated NMOSD-induced mitochondrial dysfunction and the activation of the cGAS/STING pathway in astrocytes. Conversely, overexpression of TDP43 exacerbated these pathological changes. Specific silencing astrocytic TDP43 ameliorated NMOSD-induced injury in mice, and conversely, TDP43 overexpression intensified the injury. Meanwhile, both cGAS and STING inhibitors attenuated NMOSD-induced injury in mice. In summary, our data suggest that TDP43 exacerbates inflammatory activation of astrocytes in NMOSD through upregulating the mtDNA/cGAS/STING signaling pathway. Therefore, targeting TDP43 represents a compelling therapeutic strategy for NMOSD.

Microglial activation and complement-mediated synaptic pruning are involved in depression development. We previously found that the inhibition of phosphodiesterase 4 (PDE4) inhibits microglial activation and increases synaptic plasticity. However, the role of PDE4 in microglia phagocytosis and complement-mediated synaptic pruning during depression remains unclear. Here, we investigated the effect of PDE4 on the expression of complement component 1q (C1q) and C3. We also designed and synthesized a novel PDE4 inhibitor LS21013A-06 (A06), and examined whether A06 exerts antidepressant-like effects by regulating microglia phagocytosis and complement-mediated synaptic pruning. We found that treatment with high-mobility group box-1 (HMGB1) triggered an inflammatory response, enhanced levels of complement component 1q (C1q) and C3, and promoted microglial phagocytosis both in vitro and in vivo. Notably, PDE4B knockdown reduced the levels of HMGB1, C1q, and C3 in lipopolysaccharide (LPS)-treated BV2 cells. Inhibition of PDE4 by A06 reduced the levels of HMGB1, suppressed neuroinflammation and microglial phagocytosis. In addition, A06 alleviated LPS-induced depressive-like behaviors in mice, reduced the levels of HMGB1, C1q, and C3 in the hippocampus, elevated the level of postsynaptic density protein-95, and reduced excessive microglial phagocytosis and engulfment of synapses. Moreover, C1q overexpression inhibited the effects of A06 on microglial activation and synaptic pruning. In conclusion, we demonstrated for the first time that PDE4 regulates the expression of C1q/C3, and A06 reduces microglial activation and ameliorates depressive-like behavior in mice. This mechanism involves complement C1q/C3-mediated excessive microglia phagocytosis and synaptic pruning.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play pivotal roles in the pathology of cerebral ischemia. In this study, we investigated whether phelligridimer A (PA), an active compound isolated from the medicinal and edible fungus Phellinus igniarius, ameliorates ischemic cerebral injury by restoring mitochondrial function and restricting ER stress. An in vitro cellular model of ischemic stroke-induced neuronal damage was established by exposing HT-22 neuronal cells to oxygen-glucose deprivation/reoxygenation (OGD/R). An in vivo animal model was established in rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). The results showed that PA (1–10 μM) dose-dependently increased HT-22 cell viability, reduced OGD/R-induced lactate dehydrogenase release, and reversed OGD/R-induced apoptosis. PA reduced OGD/R-induced accumulation of reactive oxygen species, restored mitochondrial membrane potential, and increased ATP levels. Additionally, PA reduced the expression of the 78-kDa glucose-regulated protein (GRP78) and the phosphorylation of inositol-requiring enzyme-1α (p-IRE1α) and eukaryotic translation-initiation factor 2α (p-eIF2α). PA also inhibited the activation of the mitogen-activated protein kinase (MAPK) pathway in the OGD/R model. Moreover, treatment with PA restored the expression of mitofusin 2 (Mfn-2), a protein linking mitochondria and ER. The silencing of Mfn-2 abolished the protective effects of PA. The results from the animal study showed that PA (3–10 mg/kg) significantly reduced the volume of cerebral infarction and neurological deficits, which were accompanied by an increased level of Mfn-2, and decreased activation of the ER stress and MAPK pathways in the penumbra of the ipsilateral side after MCAO/R in rats. Taken together, these results indicate that PA counteracts cerebral ischemia-induced injury by restoring mitochondrial function and reducing ER stress. Therefore, PA might be a novel protective agent to prevent ischemia stroke-induced neuronal injury.

Given the established role of nuclear receptor corepressor 1 (NCoR1) in sensing environmental cues and the importance of inflammation in neurodegenerative diseases, elucidation of NCoR1 involvement in neuroinflammation has notable implications. Yet, its regulatory mechanism remains largely unclear. Under in vitro conditions, NCoR1 expression peaked and then decreased at 12 h after lipopolysaccharides (LPS) stimulation in BV2 cells, However, NCoR1 knockdown using si-RNA attenuated microglial inflammation, evident by reduced the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), phosphorylated-JNK and high mobility group box-1 (HMGB1). Furthermore, NCoR1 suppression could counteract the decline in mitochondrial membrane potential while simultaneously enhancing the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). Under in vivo conditions, microglia-specific NCoR1 knockout (MNKO) mice after LPS injections alleviated the symptoms of anhedonia, diminished autonomic activity and cognitive impairment. Additionally, MNKO mice showed attenuation of microglial activation, downregulated HMGB1 and COX2, and upregulated PGC-1 alpha expression in the cortex. In conclusion, these findings suggest that NCoR1 deficiency leads to a modest reduction in neuroinflammation, possibly attributed to the increased expression of PGC-1 alpha.

We have previously shown that inhibition of phosphodiesterase 4 (PDE4) protects against cerebral ischemia/reperfusion injury. However, it remains unclear whether and how PDE4 affects ferroptosis under cerebral ischemia/reperfusion conditions. In this study, we found that overexpression of PDE4B in HT-22 cells exacerbated the detrimental effects of oxygen-glucose deprivation/reoxygenation (OGD/R), including a decrease in cell viability and glutathione (GSH) levels and an increase in Fe2+ content. PDE4B knockdown mitigated the effects of OGD/R, as evidenced by decreased oxidative stress, lactate dehydrogenase (LDH) release, Fe2+ content, and nuclear receptor coactivator 4 (NCOA4) expression. PDE4B knockdown also enhanced the levels of GSH, ferroportin (FPN), and ferritin heavy chain 1 (FTH1). Consistently, inhibition of PDE4 by roflumilast (Roflu) produced similar effects as PDE4B knockdown. Roflu also ameliorated the morphology and membrane potential of the mitochondria. Glutathione peroxidase 4 (GPX4) knockdown blocked the effects of Roflu on cell viability and lipid peroxidation. Moreover, we found that nuclear factor erythroid 2-related factor 2 (Nrf-2) knockdown decreased GPX4 expression. In addition, Nrf-2 knockdown led to enhanced lipid peroxidation, LDH release, and iron levels, while the GSH and FPN levels decreased. More crucially, PDE4 inhibition decreased infarct volume, alleviated oxidative stress, and restored the expression levels of ferroptosis-associated proteins in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Interestingly, the GPX4 inhibitor RSL3 blocked the neuroprotective effects of Roflu in rats subjected to MCAO/R. Thus, PDE4 inhibition significantly inhibits neuronal ferroptosis by activating the Nrf-2/GPX4 pathway. These data indicate the existence of a novel mechanism underlying the neuroprotective effects of PDE4 inhibition.

We have previously shown that phosphodiesterase 4 (PDE4) inhibition protects against neuronal injury in rats following middle cerebral artery occlusion/reperfusion (MCAO/R). However, the effects of PDE4 on brain edema and astrocyte swelling are unknown. In this study, we showed that inhibition of PDE4 by Roflumilast (Roflu) reduced brain edema and brain water content in rats subjected to MCAO/R. Roflu decreased the expression of aquaporin 4 (AQP4), while the levels of phosphorylated protein kinase B (Akt) and forkhead box O3a (FoxO3a) were increased. In addition, Roflu reduced cell volume and the expression of AQP4 in primary astrocytes undergoing oxygen and glucose deprivation/reoxygenation (OGD/R). Consistently, PDE4B knockdown showed similar effects as PDE4 inhibition; and PDE4B overexpression rescued the inhibitory role of PDE4B knockdown on AQP4 expression. We then found that the effects of Roflu on the expression of AQP4 and cell volume were blocked by the Akt inhibitor MK2206. Since neuroinflammation and astrocyte activation are the common events that are observed in stroke, we treated primary astrocytes with interleukin-1β (IL-1β). Astrocytes treated with IL-1β showed decreased AQP4 and phosphorylated Akt and FoxO3a. Roflu significantly reduced AQP4 expression, which was accompanied by increased phosphorylation of Akt and FoxO3a. Furthermore, overexpression of FoxO3a partly reversed the effect of Roflu on AQP4 expression. Our findings suggest that PDE4 inhibition limits ischemia-induced brain edema and astrocyte swelling via the Akt/FoxO3a/AQP4 pathway. PDE4 is a promising target for the intervention of brain edema after cerebral ischemia.

Sepsis can quickly result in fatality for critically ill individuals, while liver damage can expedite the progression of sepsis, necessitating the exploration of new strategies for treating hepatic sepsis. PDE4 has been identified as a potential target for the treatment of liver damage. The scaffold hopping of lead compounds FCPR16 and Z19153 led to the discovery of a novel 7-methoxybenzofuran PDE4 inhibitor 4e, demonstrating better PDE4B (IC50 = 10.0 nM) and PDE4D (IC50 = 15.2 nM) inhibitor activity as a potential anti-hepatic sepsis drug in this study. Compared with FCPR16 and Z19153, 4e displayed improved oral bioavailability (F = 66%) and longer half-life (t1/2 = 2.0 h) in SD rats, which means it can be more easily administered and has a longer-lasting effect. In the D-GalN/LPS-induced liver injury model, 4e exhibited excellent hepatoprotective activity against hepatic sepsis by decreasing ALT and AST levels and inflammatory infiltrating areas.

An abnormal increase in the expression of nuclear receptor subfamily 6 group A member 1 (NR6A1) in the hippocampus has been reported to result in depressive-like behavior in mice. However, the role of NR6A1 in the progression of neuronal death induced by ischemic stroke remains unknown. In this study, we observed an increase in NR6A1 in neurons in both in vivo and in vitro cerebral ischemic models. We found that knocking down NR6A1 in HT-22 neuronal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) attenuated mitochondrial dysfunction and endoplasmic reticulum (ER) stress. Conversely, NR6A1 overexpression exacerbated neuronal damage following OGD/R. NR6A1 hindered the transcription of mitonfusin 2 (MFN2), leading to a decrease in its expression. In contrast, MFN2 conferred the protective effect of NR6A1 silencing against both mitochondrial dysfunction and ER stress. In addition, NR6A1 silencing also attenuated brain infarction, ER stress, neuronal apoptosis, and loss of MFN2 in mice subjected to middle cerebral artery occlusion/reperfusion. These findings indicate that NR6A1 is a promising target for the treatment of neuronal death following cerebral ischemia. Furthermore, these results confirm the involvement of MFN2 in the effects of NR6A1 silencing. Therefore, targeting NR6A1 has potential as a viable strategy for the treatment of ischemic stroke.

In our pursuit of creating a comprehensive human cortical atlas to understand human intelligence, we examined the single-nuclei transcriptomes of 307,738 cells alongside spatial transcriptomics data from 46,948 VISIUM spots and 1,355,582 Stereo cells. Atlases reveal distinct expression patterns and spatial arrangements of cortical neural cell types. Glutamatergic neurons exhibit precise laminar patterns, often mirroring expression patterns in adjacent cortical regions. Overlaying our atlas with functional networks delineated substantial correlations between neural cell types and cortical region function. Notably, regions involved in processing sensory information (pain) display a pronounced accumulation of extratelencephalic neurons. Additionally, our atlas enabled precise localization of the thicker layer 4 of the visual cortex and an in-depth study of the stabilize the subplate structure, known as layer 6b, revealed specific marker genes and cellular compositions. Collectively, our research sheds light on the cellular foundations of the intricate and intelligent regions within the human cortex.### Competing Interest StatementThe authors have declared no competing interest.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characteristized by the presence of dyskinesia and the progressive loss of dopaminergic neurons. Although certain drugs can mitigate the symptoms of PD, they are unable to delay the disease progression, and their prolonged use may result in complications. Therefore, there exists an urgent necessity to identify potential agents that can effectively delay PD progression with fewer side effects. Recent research has unveiled that several traditional Chinese medicines (TCM) exhibit neuroprotective properties in various models pertinent to PD. Forsythoside A (FSA), the primary bioactive compound derived from TCM Lianqiao, has undergone extensive research in animal models of Alzheimer's disease and cerebral ischemia. However, the investigation into the impact of FSA on PD is limited in existing research. In this study, we aimed to evaluate the neuroprotective effects of FSA on MPTP-induced PD mouse model. FSA demonstrated significant improvements in the behavioral and neuropathological changes triggered by MPTP in mice. Furthermore, it exerted a suppressive effect on the activations of astrocyte and microglia. Meanwhile, Tandem mass tag (TMT)-based quantitative proteomics of striatal tissue and bioinformatics analysis were performed to elucidate the underlying mechanisms of FSA on PD mouse model. Proteomics demonstrated a total of 68 differentially expressed proteins (DEPs) were identified between HFSA and MPTP groups including 26 upregulated and 42 downregulated. Systematic bioinformatics analysis of the 68 DEPs illustrated that they were predominantly related to estrogen signaling pathway and calcium signaling pathway. The related DEPs (PLCβ4, Grm2, HPAC and Cox4i1) expression levels were verified by Western blot. FSA effectively restored the altered expression of the four DEPs induced by MPTP. Summarily, FSA exerted remarkable neuroprotective effects in MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of FSA as a potential treatment for PD.