Molecular mechanism of Oxr1p mediated disassembly of yeast V-ATPase.

The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p induces V-ATPase disassembly in vitro. Whether and how Oxr1p is involved in enzyme disassembly in vivo, however, is not known. Here, using yeast genetics and fluorescence microscopy, we show that Oxr1p is essential for efficient V-ATPase disassembly in the cell. Supporting biochemical and biophysical in vitro experiments show that whereas Oxr1p-driven holoenzyme disassembly can occur in the absence of nucleotides, the presence of ATP greatly accelerates the process. ATP hydrolysis is needed, however, for subsequent release of Oxr1p so that the free V1 can adopt the autoinhibited conformation. Overall, our study unravels the molecular mechanism of Oxr1p-induced disassembly that occurs in vivo as part of the canonical V-ATPase regulation by reversible disassembly.

Human V-ATPase function is positively and negatively regulated by TLDc proteins.

Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.

Corrigendum: 6-Shogaol inhibits oxidative stress-induced rat vascular smooth muscle cell apoptosis by regulating OXR1-p53 axis.

[This corrects the article DOI: 10.3389/fmolb.2022.808162.].

TLDc Domain-Containing Genes in Autism Spectrum Disorder: New Players in the Oxidative Stress Response.

Oxidative stress (OS) plays a key role in autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by deficits in social communication, restricted interests, and repetitive behaviors. Recent evidence suggests that the TLDc [Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic] domain is a highly conserved motif present in proteins that are important players in the OS response and in neuroprotection. Human proteins sharing the TLDc domain include OXR1, TLDC1, NCOA7, TBC1D24, and C20ORF118. This study was aimed at understanding whether TLDc domain-containing mRNAs together with specific microRNAs (200b-3p and 32-5p) and long noncoding RNAs (TUG1), known to target TLDc proteins, contributed to regulate the OS response in ASD. Data showed a significant increase in the levels of OXR1 and TLDC1 mRNAs in peripheral blood mononuclear cells (PBMCs) of ASD children compared to their neurotypically developing (NTD) counterparts, along with an increase in TUG1 mRNA expression levels, suggesting its possible role in the regulation of TLDc proteins. A positive correlation between the expression of some TLDc mRNAs and the Childhood Autism Rating Scale (CARS) global score as well as inflammatory gene expression was found. In conclusion, our data suggest a novel biological pathway in the OS response of ASD subjects that deserves further exploration.

Omaveloxolone ameliorates cognitive dysfunction in APP/PS1 mice by stabilizing the STAT3 pathway.

AIMS: To determine the availability and the potential molecular mechanisms underlying the therapeutic effect of omaveloxolone (RTA408) on Alzheimer's Disease (AD). MATERIALS AND METHODS: This study employed network pharmacology to assess the feasibility of drug treatment of AD. To determine the cognitive status and emotional state of APPswe/PS1dE9 (APP/PS1) mice after the RTA408 treatment, three classical behavioral experiments (water maze, Y-maze, and open field test) were conducted. Immunofluorescence and immunohistochemical staining were utilized to evaluate hippocampal neuronal status and amyloid (Aß) deposition in mice. RNA-seq and transcription factor prediction analyses were performed to explore the potential molecular mechanisms regulating the therapeutic effects of RTA408. Molecular docking was employed to predict the direct drug targets. To validate these molecular mechanisms, quantitative reverse transcription PCR (qRT-PCR), Western blotting, and immunofluorescence analyses were performed in two instrumental cell lines, i.e., mouse hippocampal neuronal cells (HT22) and microglia (BV2). RESULTS: RTA408 was revealed with the capability to reduce Aß plaque deposition and to restore damaged neurons in the hippocampal region of APP/PS1 mice, ultimately leading to an improvement in cognitive function. This beneficial effect was achieved by balancing the STAT3 pathway. Specifically, RTA408 facilitated the activations of both STAT3/OXR1 and NRF2/ARE axes, thereby enhancing the compromised resistance in neurons to oxidative stress. RTA408 inhibited the NFκB/IL6/STAT3 pathway, effectively countering the neuroinflammation triggered by microglial activation. CONCLUSION: RTA408 is revealed with promising potential in the treatment of AD based on preclinical data.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Tender love and disassembly: How a TLDc domain protein breaks the V-ATPase.

Vacuolar ATPases (V-ATPases, V1 Vo -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V1 -ATPase disengages from Vo proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V1 and Vo subcomplexes.

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock.

Real-time three-dimensional (3-D) imaging is crucial for quantifying correlations among various molecules under acute ischemic stroke. Insights into such correlations may be decisive in selecting molecules capable of providing a protective effect within a shorter period. The major bottleneck is maintaining the cultures under severely hypoxic conditions while simultaneously 3-D imaging intracellular organelles with a microscope. Moreover, comparing the protective effect of drugs and reoxygenation remains challenging. To address this, we propose a novel workflow for the induction of gas-environment-based hypoxia in the HMC-3 cells along with 3-D imaging using laser-scanning-confocal microscopy. The imaging framework is complemented with a pipeline for quantifying time-lapse videos and cell-state classification. First, we show an imaging-based assessment of the in vitro model for hypoxia using a steep gradient in O2 with time. Second, we demonstrate the correlation between mitochondrial superoxide production and cytosolic calcium under acute hypoxia. We then test the efficacy of an L-type calcium channel blocker, compare the results with reoxygenation, and show that the blocker alleviates hypoxic conditions in terms of cytosolic calcium and viability within an acute window of one hour. Furthermore, we show that the drug reduces the expression of oxidative stress markers (HIF1A and OXR1) within the same time window. In the future, this model can also be used to investigate drug toxicity and efficacy under ischemic conditions.

Oxr1a prevents the premature ovarian failure by regulating oxidative stress and mitochondrial function in zebrafish.

Premature ovarian failure (POF) is characterized as the ovarian dysfunction and defective oocyte development. In POF patients, ROS level is reported to be significantly higher than normal individuals. However, the involvement of oxidative stress in POF and the regulatory mechanisms underlying the antioxidative process in oocyte development remain largely unknown. Here, we discover that oxidation resistance 1a (Oxr1a), the ortholog of mammalian Oxr1, protects the oocytes of female zebrafish against oxidative stress and thus represses the POF phenotype. Oxr1a was widely expressed in oocytes at different developmental stages, of which the mRNA expression levels were significantly upregulated upon follicle activation and oocyte maturation. Oxr1a knockout exacerbated the POF phenotype, as evidenced by the decreased number and quality of oocytes. Moreover, the oocytes of oxr1a knockout zebrafish exhibited excessive ROS, increased mitochondrial DNA damage, reduced mitochondria, and abnormal morphology. Mechanistically, instead of decomposing ROS directly, Oxr1a participated in the process of oxidative stress through regulating the mRNA expression levels of the key antioxidant enzymes Cat and Sod1. Moreover, treatment with antioxidant N-Acetyl-l-cysteine attenuated the mitochondrial oxidative damage and improved the fertility of mutant females, indicating that Oxr1a may mediates the Sod1/Cat pathway to metabolize the intracellular ROS and avoid the mitochondrial oxidative damage, thus ensuring the normal development and maturation of oocytes. Taken together, these findings are useful for the elucidation of molecular mechanisms underlying the oxidative damage in oocytes and beneficial to the clinical therapeutics of POF.

LncRNA SNHG4 sponges miR-200b to inhibit cell apoptosis in diabetic retinopathy.

This study aimed to investigate the role of long non-coding RNA (lncRNA) small nucleolar RNA host gene 4 (SNHG4) in diabetic retinopathy (DR). We found that SNHG4 was downregulated in DR. SNHG4 could directly interact with miR-200b, while overexpression of miR-200b did not affect the expression of SNHG4 in human retinal pigment epithelial cells ARPE-19. In contrast, overexpression of SNHG4 led to the upregulation of oxidation resistance 1 (Oxr1), a target of miR-200b. Cell apoptosis analysis showed that overexpression of miR-200b increased the apoptotic rate of ARPE-19 cells under high glucose treatment. Oxr1 and SNHG4 played opposite roles and reduced the effects of overexpression of miR-200b. In conclusion, SNHG4 may sponge miR-200b to inhibit cell apoptosis in DR by upregulating Oxr1.

MicroRNA-668-3p regulates oxidative stress and cell damage induced by Aß1-42 by targeting the OXR1/p53-p21 axis.

Background: Alzheimer's disease (AD) is the most common type of dementia in old age and has become a serious social and medical problem threatening human health. We aimed to explore the mechanisms underlying AD development by screening for microRNAs (miRNAs) that affect AD progression and examining their role in AD development. Methods: Hematoxylin-eosin (HE) staining, immunohistochemistry, and immunofluorescence (IF) were used to analyze the characteristics of the hippocampus, neuron cell separation, and related protein expression in mice. We used Gene Expression Omnibus (GEO) data analysis to screen miRNAs and mRNAs that affect AD progression, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot analysis to determine changes in miRNA and mRNA levels before and after amyloid ß (Aß)1-42 induction. In addition, we used luciferase analysis to examine miRNA and mRNA binding and the effect of miRNA/mRNA interaction on neuronal cell proliferation. Apoptosis and reactive oxygen species (ROS) levels were examined using Cell Counting Kit-8 analysis and flow cytometry (FCM), respectively. The enzyme-linked immunosorbent assay was used to analyze changes in neuronal cell-secreted oxidative stress-related protein levels through miRNA/mRNA interaction. Results: Oxidative stress levels were significantly increased in the AD mouse model. GEO data analysis revealed 67 dysregulated miRNAs, and miR-668-3p was identified as a potential therapeutic target for AD. We found that the AD and Aß1-42-induced models showed an increase in miR-668-3p and a decrease in oxidation resistance 1 (OXR1) expression. The luciferase analysis results revealed that miR-668-3p may play a role in AD development by targeting OXR1 and promoting intracellular oxidative stress by activating p53-p21 signaling. The final rescue experiment also confirmed that Aß1-42-induction decreased cell proliferation, increased apoptosis, increased cell cycle arrest, and promoted oxidative stress. Tenovin-1 (TEN) enhanced the effect of Aß1-42, and the miR-668-3p inhibitor partially alleviated it, although the effect of the miR-668-3p inhibitor was weakened by TEN. Conclusions: MiR-668-3p negatively regulated OXR1 expression by targeting OXR1, affecting p53-p21 protein signaling, and regulating cell damage and oxidative stress induced by Aß1-42. Therefore, miR-668-3p may be a potential therapeutic target for AD.

Gene therapy targeting inflammatory pericytes corrects angiopathy during diabetic wound healing.

Wound healing is impaired in the diabetic status, largely attributable to diabetes-associated angiopathy. Pericytes play critical roles in the stabilization of the formed vessels. The loss and dysfunction of pericytes have been reported in inflammation during diabetes and associated with the pathology of diabetic angiopathy. However, a practical approach that targets inflammatory pericytes to improve diabetic wound healing is lacking. In the current study, we showed that the inflammatory pericytes from wound skin of diabetic patients were impaired in growth potential and underwent oxidative stress and apoptosis. Expression of antioxidant gene oxidation resistance protein 1 (OXR1) specifically in pericytes through an adenovirus carrying OXR1 under a pericyte-specific neuron glia antigen-2 (NG2) promoter (AV-NG2p-OXR1) relieved the oxidative stress, reduced the apoptosis, and recovered the growth potential in diabetic pericytes. Moreover, expression of OXR1 in diabetic pericytes retrieved their potential of both suppressing the migration of co-cultured HUVECs and inducing cell aggregates at the branching points, indicating a functional recovery. In vivo gene therapy using this AV-NG2p-OXR1 to DB/DB mice, the mouse model for type 2 diabetes, significantly improved wound healing, likely through enhancing blood flow at the wound rather than increasing vessel density. Together, our data suggest that gene therapy targeting inflammatory pericytes may improve diabetes-associated impaired wound healing.

OXR1 signaling pathway as a possible mechanism of elastase-induced oxidative damage in pulmonary cells: the protective role of ellagic acid.

BACKGROUND AND OBJECTIVE: Oxidative stress is a process that occurs through free radicals on the cell membranes which causes damage to the cell and intracellular organelles, especially mitochondria membranes. H2O2 induced oxidative stress in human cells is of interest in toxicological research since oxidative stress plays a main role in the etiology of several pathological conditions. Neutrophil Elastase (Serine proteinase) is involved in the pathology process of emphysema as a respiratory disease through lung inflammation, and destruction of alveolar walls. The present study investigated the direct oxidative stress effects of Elastase in comparison with H2O2 on human lung epithelial cells (A549 cells) concerning the generation of reactive oxygen species (ROS) and modulation of oxidation resistance 1 (OXR1) and its downstream pathway using the well-known antioxidant Ellagic acid as an activator of antioxidant genes. MATERIALS AND METHODS: The human pulmonary epithelial cells (A549) were divided into the nine groups including Negative control, Positive control (H2O2), Elastase (15, 30, and 60 mU/mL), Ellagic acid (10 µmol/L), and Elastase + Ellagic acid. Cytotoxicity, ROS generation, oxidative stress profile, level of reactive metabolites, and gene expression of OXR1 and its downstream genes were measured in all groups. RESULTS: The obtained data demonstrated that Elastase exposure caused oxidative stress damage in a dose-depended manner which was associated with decreases in antioxidant defense system genes. Conversely, treatment with Ellagic acid as a potent antioxidant showed improved antioxidant enzyme activity and content which was in line with the upregulation of OXR1 signaling pathway genes. CONCLUSIONS: The present findings can highlight the novel mechanism underlying the oxidative stress induced by Neutrophil Elastase through OXR1 and related genes. Moreover, the benefit of Ellagic acid on cytoprotection, resulting from its antioxidant properties was documented.

6-Shogaol Inhibits Oxidative Stress-Induced Rat Vascular Smooth Muscle Cell Apoptosis by Regulating OXR1-p53 Axis.

Apoptosis of vascular smooth muscle cells (VSMCs) is closely related to the pathogenesis of cardiovascular diseases, and oxidative stress is an important cause of VSMCs' death. Inhibiting VSMCs apoptosis is an effective preventive strategy in slowing down the development of cardiovascular disease, especially for atherosclerosis. In this study, we found that oxidation resistance protein 1 (OXR1), a crucial participator for responding to oxidative stress, could modulate the expression of p53, the key regulator of cell apoptosis. Our results revealed that oxidative stress promoted VSMCs apoptosis by overexpression of the OXR1-p53 axis, and 6-shogaol (6S), a major biologically active compound in ginger, could effectively attenuate cell death by preventing the upregulated expression of the OXR1-p53 axis. Quantitative proteomics analysis revealed that the degradation of p53 mediated by OXR1 might be related to the enhanced assembly of SCF ubiquitin ligase complexes, which is reported to closely relate to the modification of ubiquitination or neddylation and subsequent degradation of p53.

Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.

The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.

Prognostic significance of OXR1 in urothelial carcinoma: low OXR1 expression is associated with worse survival.

Background: Bioinformatic analysis has revealed that OXR1 is significantly downregulated in muscle-invasive bladder cancer. Patients & methods: The expression of OXR1 in patients with urothelial carcinoma was evaluated by immunohistochemistry, including 340 cases with urothelial carcinoma in the upper urinary tract and 295 in the urinary bladder. Results: Low expression of OXR1 was significantly correlated with adverse pathological parameters including high primary tumor (pT) stage, high node stage, high histological grade, high mitotic activity and increased vascular or perineural invasion (all p < 0.05). Low expression of OXR1 independently predicted worse metastasis-free survival (p = 0.033) in urothelial carcinoma of the upper urinary tract and worse disease-specific survival (p = 0.022) and metastasis-free survival (p < 0.001) in urothelial carcinoma of the urinary bladder. Conclusion: Low expression of OXR1 is an adverse prognostic factor in urothelial carcinoma.

MicroRNA-29a Mitigates Osteoblast Senescence and Counteracts Bone Loss through Oxidation Resistance-1 Control of FoxO3 Methylation.

Senescent osteoblast overburden accelerates bone mass loss. Little is understood about microRNA control of oxidative stress and osteoblast senescence in osteoporosis. We revealed an association between microRNA-29a (miR-29a) loss, oxidative stress marker 8-hydroxydeoxyguanosine (8-OHdG), DNA hypermethylation marker 5-methylcystosine (5mC), and osteoblast senescence in human osteoporosis. miR-29a knockout mice showed low bone mass, sparse trabecular microstructure, and osteoblast senescence. miR-29a deletion exacerbated bone loss in old mice. Old miR-29a transgenic mice showed fewer osteoporosis signs, less 5mC, and less 8-OHdG formation than age-matched wild-type mice. miR-29a overexpression reversed age-induced senescence and osteogenesis loss in bone-marrow stromal cells. miR-29a promoted transcriptomic landscapes of redox reaction and forkhead box O (FoxO) pathways, preserving oxidation resistance protein-1 (Oxr1) and FoxO3 in old mice. In vitro, miR-29a interrupted DNA methyltransferase 3b (Dnmt3b)-mediated FoxO3 promoter methylation and senescence-associated ß-galactosidase activity in aged osteoblasts. Dnmt3b inhibitor 5'-azacytosine, antioxidant N-acetylcysteine, or Oxr1 recombinant protein attenuated loss in miR-29a and FoxO3 to mitigate oxidative stress, senescence, and mineralization matrix underproduction. Taken together, miR-29a promotes Oxr1, compromising oxidative stress and FoxO3 loss to delay osteoblast aging and bone loss. This study sheds light on a new antioxidation mechanism by which miR-29a protects against osteoblast aging and highlights the remedial effects of miR-29a on osteoporosis.

Expression profiling and functional characterization of the duplicated Oxr1b gene in zebrafish.

Oxidation Resistance Gene 1 (OXR1) is a conserved gene family involved in protecting various species against oxidative stress. The zebrafish expresses a pair of OXR1 paralogs (i.e., oxr1a and oxr1b). Our previous work has revealed the importance of oxr1a in regulating antioxidant defenses during oxidative stress, but the role of oxr1b is remains unknown. Herein we reported the spatial-temporal expression of oxr1b and revealed its function through reverse genetics. The results showed that, as with oxr1a, oxr1b is a typical maternal-zygotic gene. Its mRNA is mainly distributed in the eye, brain and nervous system (e.g., anterior/posterior lateral line ganglion, neuromasts and spinal cord neuron). Although oxr1a and oxr1b genes have similar expression patterns during embryonic development, the latter have higher levels at the corresponding stages. Subsequently, a viable oxr1b-/- mutant was generated by the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system. Oxr1b knockout caused multiple antioxidant genes (i.e., gpx4a, gpx4b, sod1 and sod3b) to be downregulated, resulting in hypersensitive to oxidative stress. Furthermore, by comparative transcriptome analysis, we found that oxr1b knockout inhibits multiple signal transduction pathways (e.g., MAPK signaling pathway, calcium signaling pathway, cAMP signaling pathway and ErbB signaling pathway) during oxidative stress, thereby suppressing early stress response and ultimately impairing the anti-apoptosis pathway. In conclusion, our findings demonstrate that the duplicated oxr1b gene has an important role in regulating the antioxidant defenses by modulating signaling transduction and early stress response during oxidative stress.

Delivery of pOXR1 through an injectable liposomal nanoparticle enhances spinal cord injury regeneration by alleviating oxidative stress.

Oxidation resistance 1 (OXR1) is regarded as a critical regulator of cellular homeostasis in response to oxidative stress. However, the role of OXR1 in the neuronal response to spinal cord injury (SCI) remains undefined. On the other hand, gene therapy for SCI has shown limited success to date due in part to the poor utility of conventional gene vectors. In this study, we evaluated the function of OXR1 in SCI and developed an available carrier for delivering the OXR1 plasmid (pOXR1). We found that OXR1 expression is remarkably increased after SCI and that this regulation is protective after SCI. Meanwhile, we assembled cationic nanoparticles with vitamin E succinate-grafted ε-polylysine (VES-g-PLL) (Nps). The pOXR1 was precompressed with Nps and then encapsulated into cationic liposomes. The particle size of pOXR1 was compressed to 58 nm, which suggests that pOXR1 can be encapsulated inside liposomes with high encapsulation efficiency and stability to enhance the transfection efficiency. The agarose gel results indicated that Nps-pOXR1-Lip eliminated the degradation of DNA by DNase I and maintained its activity, and the cytotoxicity results indicated that pOXR1 was successfully transported into cells and exhibited lower cytotoxicity. Finally, Nps-pOXR1-Lip promoted functional recovery by alleviating neuronal apoptosis, attenuating oxidative stress and inhibiting inflammation. Therefore, our study provides considerable evidence that OXR1 is a beneficial factor in resistance to SCI and that Nps-Lip-pOXR1 exerts therapeutic effects in acute traumatic SCI.

Layer 3 Pyramidal Cells in the Medial Entorhinal Cortex Orchestrate Up-Down States and Entrain the Deep Layers Differentially.

Up-down states (UDS) are synchronous cortical events of neuronal activity during non-REM sleep. The medial entorhinal cortex (MEC) exhibits robust UDS during natural sleep and under anesthesia. However, little is known about the generation and propagation of UDS-related activity in the MEC. Here, we dissect the circuitry underlying UDS generation and propagation across layers in the MEC using both in vivo and in vitro approaches. We provide evidence that layer 3 (L3) MEC is crucial in the generation and maintenance of UDS in the MEC. Furthermore, we find that the two sublayers of the L5 MEC participate differentially during UDS. Our findings show that L5b, which receives hippocampal output, is strongly innervated by UDS activity originating in L3 MEC. Our data suggest that L5b acts as a coincidence detector during information transfer between the hippocampus and the cortex and thereby plays an important role in memory encoding and consolidation.