Therapeutic targeting of STING-TBK1-IRF3 signalling ameliorates chronic stress induced depression-like behaviours by modulating neuroinflammation and microglia phagocytosis.

Stress is well known to contribute to the development of both neurological and psychiatric diseases. In the central nervous system, a role for STING (stimulator of interferon genes) in modulating immunological responses has been widely suggested, and this protein possesses both neurotoxic and neuroprotective properties. However, the potential role of the STING signalling pathway and the underlying regulatory mechanism in chronic stress have not been well established. In this study, C57BL/6 mice were subjected to intermittent restraint stress for 14 days (6 h/day), and sucrose preference, elevated plus maze, and tail suspension tests were performed by mice subjected to chronic restraint stress (RST). Here, we showed that RST mice displayed depression-like behaviours, accompanied by increased levels of proinflammatory cytokines in the brain. We also observed remarkably decreased levels of the pathway components STING, p-TBK1 (phospho-TANK-binding kinase-1), and p-IRF3 (phospho-interferon regulatory factor-3) in the hippocampus and the prefrontal cortex of RST mice. Significant reductions in STING fluorescence intensity were also observed in the hippocampus and the prefrontal cortex of RST mice. Next, fluorescently labelled latex beads, flow cytometry, and CD68-positive cell counts were utilized to evaluate the phagocytic abilities of microglia in vivo and in vitro. Importantly, our results first indicated that activation of the STING pathway by administration of the STING agonist 2'3-cGAMP enhanced microglial phagocytosis and suppressed the release of the proinflammatory cytokines TNF-α, IL-6, and IL-1ß in the brains of RST mice, which further led to antidepressant effects. Based on the results of our study, the amelioration of stress-driven depression-like behaviours by activation of the STING pathway is associated with the suppression of neuroinflammation and enhanced phagocytosis.

Transplantation of platelet-derived mitochondria alleviates cognitive impairment and mitochondrial dysfunction in db/db mice.

Diabetes-associated cognitive impairment (DACI) can increase the risk of major cardiovascular events and death. Neuronal functionality is highly dependent on mitochondria and emerging evidence has shown that mitochondrial transplantation is a potential and effective strategy that can reduce brain injury and associated disorders. Platelets are abundant in blood and can be considered a readily available source of small-size mitochondria. These cells can be easily acquired from the peripheral blood with minimal invasion via simple venipuncture. The present study aimed to investigate whether transplantation of platelet-derived mitochondria (Mito-Plt) could improve DACI. Cognitive behaviors were assessed using the Morris water maze test in db/db mice. The results demonstrated that Mito-Plt was internalized into hippocampal neurons 24 h following intracerebroventricular injection. Importantly, one month following Mito-Plt transplantation, DACI was alleviated in db/db mice and the effect was accompanied with increased mitochondrial number, restored mitochondrial function, attenuated oxidative stress and neuronal apoptosis, as well as decreased accumulation of Aß and Tau in the hippocampus. Taken together, the data demonstrated that transplantation of Mito-Plt attenuated cognitive impairment and mitochondrial dysfunction in db/db mice. This method may be a potential therapeutic application for the treatment of DACI.

Electroacupuncture attenuated cerebral ischemic injury and neuroinflammation through α7nAChR-mediated inhibition of NLRP3 inflammasome in stroke rats.

BACKGROUND: Our previous research confirmed that electroacupuncture (EA) stimulus elicits neuroprotective effects against cerebral ischemic injury through α7 nicotinic acetylcholine receptor (α7nAChR)-mediated inhibition of high-mobility group box 1 release mechanism. This study investigated whether the signal transducer of α7nAChR and inhibition of NLRP3 inflammasome are involved in the neuroprotective effects of EA stimulus. METHODS: In adult male Sprague-Dawley rats, the focal cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) models for 1.5 h. The expression of NLRP3 inflammasome in the penumbral tissue following reperfusion was assessed by western blotting and immunoflourescent staining. The infarct size, neurological deficit score, TUNEL staining and the expression of proinflammatory factors or anti-inflammatory cytokines were evaluated at 72 h after reperfusion in the presence or absence of either α7nAChR antagonist (α-BGT) or agonist (PHA-543,613). RESULTS: The contents of inflammasome proteins were gradually increased after cerebral ischemia/reperfusion (I/R). EA stimulus attenuated NLRP3 inflammasome mediated inflammatory reaction and regulated the balance between proinflammatory factors and anti-inflammatory cytokines. The agonist of α7nAChR induced similar neuroprotective effects as EA stimulus. In contrast, α7nAChR antagonist reversed not only the neuroprotective effects, but also the inhibitory effects of NLRP3 inflammasome and the regulatory effects on the balance between proinflammatory factors and anti-inflammatory cytokines. CONCLUSIONS: These results provided compelling evidence that α7nAChR played a pivotal role in regulating the activation and expression of NLRP3 inflammasome in neurons after cerebral I/R. These findings highlighted a novel anti-inflammatory mechanism of EA stimulus by α7nAChR modulating the inhibition of NLRP3 inflammasome, suggesting that α7nAChR-dependent cholinergic anti-inflammatory system and NLRP3 inflammasome in neurons might act as potential therapeutic targets in EA induced neuroprotection against cerebral ischemic injury.

Reprogramming astrocytic NDRG2/NF-κB/C3 signaling restores the diabetes-associated cognitive dysfunction.

BACKGROUND: Dementia is a serious complication in patients with diabetes-associated cognitive dysfunction (DACD). In this study, we aim to explore the protective effect of exercise on DACD in diabetic mice, and the role of NDRG2 as a potential guarder for reversing the pathological structure of neuronal synapses. METHODS: Seven weeks of standardized exercise at moderate intensity was carried out using an animal treadmill in the vehicle + Run and STZ + Run groups. Based on quantitative transcriptome and tandem mass tag (TMT) proteome sequencing, weighted gene co-expression analysis (WGCNA) and gene set enrichment analysis (GSEA) were used to investigate the activation of complement cascades to injury neuronal synaptic plasticity. Golgi staining, Western blotting, immunofluorescence staining, and electrophysiology were used to verify the reliability of sequencing data. The role of NDRG2 was assessed by overexpressing or inhibiting the NDRG2 gene in vivo. Moreover, we estimated the cognitive function in diabetic or normal patients using DSST scores. FINDINGS: Exercise reversed the injury of neuronal synaptic plasticity and the downregulation of astrocytic NDRG2 in diabetic mice, which succeeded in attenuating DACD. The deficiency of NDRG2 aggravated the activation of complement C3 by accelerating the phosphorylation of NF-κB, ultimately leading to synaptic injury and cognitive dysfunction. Conversely, the overexpression of NDRG2 promoted astrocytic remodeling by inhibiting complement C3, thus attenuating synaptic injury and cognitive dysfunction. Meanwhile, C3aR blockade rescued dendritic spines loss and cognitive deficits in diabetic mice. Moreover, the average DSST score of diabetic patients was significantly lower than that of non-diabetic peers. Levels of complement C3 in human serum were elevated in diabetic patients compared to those in non-diabetic patients. INTERPRETATION: Our findings illustrate the effectiveness and integrative mechanism of NDRG2-induced improvement of cognition from a multi-omics perspective. Additionally, they confirm that the expression of NDRG2 is closely related to cognitive function in diabetic mice and the activation of complement cascades accelerated impairment of neuronal synaptic plasticity. NDRG2 acts as a regulator of astrocytic-neuronal interaction via NF-κB/C3/C3aR signaling to restore synaptic function in diabetic mice. FUNDING: This study was supported by the National Natural Science Foundation of China (No. 81974540, 81801899, 81971290), the Key Research and Development Program of Shaanxi (Program No. 2022ZDLSF02-09) and Fundamental Research Funds for the Central Universities (Grant No. xzy022019020).

Label-free in vivo assessment of brain mitochondrial redox states during the development of diabetic cognitive impairment using Raman spectroscopy.

Mitochondrial redox imbalance has been recognized as a unifying cause for diabetic cognitive impairment. Currently, a robust method for the in vivo assessment of brain mitochondrial redox imbalance is still lacking. Here, we conducted a spectral study to assess brain mitochondrial redox imbalance in the process of diabetic cognitive impairment by using label-free resonance Raman spectroscopy (RRS). Our findings showed that mitochondrial redox imbalance in cultured neurons and organotypic cortical slices exposed to high glucose were quantified by the reduction of Raman peak area at 750 cm-1 and 1128 cm-1, which were also associated with synaptic injury and neuron apoptosis. Raman peak area at 750 cm-1 and 1128 cm-1 were also decreased in db/db mice at the age of 8, 16 and 24 weeks, and had a high correlation with the mitochondrial NAD+/NADH redox couple. Of note, this mitochondrial redox imbalance occurred before measurable cognitive decline in 8-week-old diabetic mice, and might signal impending diabetic cognitive impairment. In summary, RRS-based mitochondrial redox states assay enabled the in vivo assessment of brain mitochondrial redox imbalance, and might provide an early indicator to enhance the prediction of diabetic cognitive impairment and inform on the response to therapies targeting mitochondrial redox imbalance.

Involvement of Paired Immunoglobulin-like Receptor B in Diabetes-Associated Cognitive Dysfunction Through Modulation of Axon Outgrowth and Dendritic Remodeling.

Type 2 diabetic patients have high risk of developing cognitive dysfunction, in which neural structural plasticity has played a pivotal role. Paired immunoglobulin-like receptor B (PirB), a receptor mainly expressed in neurons, acts as a critical inhibitor of neurite outgrowth and neural plasticity. However, the role of PirB in type 2 diabetes-associated cognitive dysfunction remains unknown. In this study, learning and memory impairment was observed in 24-week-old db/db mice by performing Morris water maze task, and the number of synapses along with the length of postsynaptic density by transmission electron microscopy were reduced in the hippocampus of db/db mice. Furthermore, PirB expression in the hippocampus of db/db mice was significantly upregulated using western blotting and immunofluorescence analysis. In cultured hippocampal neurons, high glucose treatment reduced the length of the longest neurite as well as axon initial segment (AIS), whereas silencing PirB expression rescued high glucose-induced neurite outgrowth inhibition, but not AIS. Additionally, cognitive deficits, dendrite morphology defects, and synapse-related proteins loss in db/db mice were alleviated when PirB knockdown was performed by adeno-associated virus injection. In conclusion, PirB is involved in diabetes-associated cognitive dysfunction through modulation of axon outgrowth and dendritic remodeling, providing a potential therapeutic target for diabetes-associated cognitive dysfunction.

Muscle-Derived Mitochondrial Transplantation Reduces Inflammation, Enhances Bacterial Clearance, and Improves Survival in Sepsis.

BACKGROUND: Mitochondrial transplantation is a promising strategy for the treatment of several diseases. However, the effects of mitochondrial transplantation on the outcome of polymicrobial sepsis remain unclear. METHODS: The distribution of transplanted mitochondria in cecal ligation and puncture (CLP)-operated mice was detected at 2 and 12 h after intravenous injection in the tail (n = 3). Then, the effects of mitochondrial transplantation on bacterial clearance (n = 7), systemic inflammation (n = 10), organ injury (n = 8), and mortality (n = 19) during CLP-induced sepsis were explored. Microarray analysis (n = 3) was used to testify the molecular changes associated with decreased systemic inflammation and multiorgan dysfunction in sepsis. RESULTS: The extraneous mitochondria were distributed in the lung, liver, kidney, and brain of CLP-operated mice at 2 and 12 h after intravenous injection in the tail. Mitochondrial transplantation increased the survival rate of septic mice, which was associated with decreased bacterial burden, systemic inflammation, and organ injury. Spleen samples were utilized for microarray analysis. Pathway analysis revealed that in polymicrobial sepsis, gene expression was significantly changed in processes related to inflammatory response, complement and coagulation cascades, and rejection reaction. CONCLUSIONS: These data displayed that mitochondrial replenishment reduces systemic inflammation and organ injury, enhances bacterial clearance, and improves the survival rate in sepsis. Thus, extraneous mitochondrial replenishment may be an effective adjunctive treatment to reduce sepsis-related mortality.

Early label-free analysis of mitochondrial redox states by Raman spectroscopy predicts septic outcomes.

BACKGROUND: Sepsis remains an unacceptably high mortality due to the lack of biomarkers for predicting septic outcomes in the early period. Mitochondrial redox states play a pivotal role in this condition and are disturbed early in the development of sepsis. Here, we hypothesized that visualizing mitochondrial redox states via resonance Raman spectroscopy (RRS) could identify septic outcomes at an early time point. Sepsis was induced by cecal ligation and puncture (CLP). We applied RRS analysis at baseline and 30 min, 1 h, 2 h, 4 h, and 6 h after CLP, and the mitochondrial redox states were identified. The levels of blood lactate as a predictor in sepsis were assessed. Our study is the first to reveal the possibility of in vivo detection of the mitochondrial redox state by using RRS in septic mice. The peak area for the Raman reduced mitochondrial fraction, the indicator of mitochondrial redox states, fluctuated significantly at 2 h after CLP. This fluctuation occurred earlier than the change in lactate level. Moreover, this fluctuation had higher prognostic accuracy for mortality than the lactate level during sepsis and could be a novel diagnostic marker for predicting septic outcomes according to the cutoff value of 1.059, which had a sensitivity of 80% and a specificity of 90%. OBJECTIVES: To explore an effective indicator concerning mitochondrial redox states in the early stage of sepsis and to predict septic outcomes accurately in vivo using non-invasive and label-free Resonance Raman spectroscopy (RRS) analysis. METHODS: Mitochondria, primary skeletal muscle cells andex-vivo muscles harvested from gastrocnemius were detected mitochondrial redox states respectively by using RRS. Sepsis was induced by cecal ligation and puncture (CLP). We applied RRS analysis at baseline and 30 min, 1 h, 2 h, 4 h, and 6 h after CLP, and the mitochondrial redox states were identified. The levels of blood lactate as a predictor in sepsis were assessed. The predictive correlation of mitochondrial redox states on mortality, inflammation and organ dysfunction was further assessed. RESULTS: Mitochondrial redox states were clearly recognized in ex-vivo gastrocnemius muscles as well as purified mitochondria and primary skeletal muscle cells by using RRS. The peak area for the Raman reduced mitochondrial fraction, the indicator of mitochondrial redox states, fluctuated significantly at 2 h after CLP. This fluctuation occurred earlier than the change in lactate level. Moreover, this fluctuation had higher prognostic accuracy for mortality than the lactate level during sepsis and could be a novel diagnostic marker for predicting septic outcomes according to the cutoff value of 1.059, which had a sensitivity of 80% and a specificity of 90%. CONCLUSIONS: This study demonstrated that monitoring mitochondrial redox states using RRS as early as 2 h could indicate outcomes in septic mice. These data may contribute to developing a non-invasive clinical device concerning mitochondrial redox states by using bedside-RRS.

Muscle-derived autologous mitochondrial transplantation: A novel strategy for treating cerebral ischemic injury.

The available evidence showed that mitochondrial transfer by releasing the extracellular vesicles containing mitochondria from astrocytes to neurons exerted a neuroprotective effect after stroke. Whether extracellular mitochondrial replenishment could rescue the tissues from cerebral ischemic injury still needs to be explored completely. It was hypothesized that the augmentation of mitochondrial damage after cerebral ischemia could be resolved by timely replenishment of exogenous mitochondria. A stroke model of middle cerebral artery occlusion (MCAO) was used in this study to verify this hypothesis. This study found that the number of extracellular mitochondria increased in rat cerebrospinal fluid after MCAO, and a higher proportion of mitochondria were associated with good neurological outcomes. Following 90-min ischemia, autologously derived mitochondria (isolated from autologous pectoralis major) or vehicle alone was infused directly into the lateral ventricles, and the rats were allowed to recover for 4 weeks. A plenty of infused mitochondria were found to be distributed in the boundary and ischemic penumbra areas. Furthermore, the transplantation of mitochondria reduced cellular oxidative stress and apoptosis, attenuated reactive astrogliosis, and promoted neurogenesis after stroke. Moreover, the transplantation of mitochondria decreased brain infarct volume and reversed neurological deficits. The findings suggested that the delivery of mitochondria through the lateral ventricles resulted in their widespread distribution throughout the brain and exerted a neuroprotective effect after ischemia-reperfusion injury.