Apoptotic Neurons Research Articles

A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo.

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterized by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live-imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarized vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterized by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.

Anti-apoptotic and neurogenic properties in the hippocampus as possible mechanisms for learning and memory improving impacts of vitamin D in hypothyroid rats during the growth period

The current study was designed to investigate the protective effects of Vitamin D (VD) on hippocampal neurogenesis, apoptosis, and subsequent hippocampal-dependent learning and memory performance in hypothyroid juvenile rats. Twenty eight male Wistar rats were randomly divided into four groups as; control, Hypothyroid (Hypo), Hypo-VD100 and Hypo-VD500. Hypothyroidism was induced by giving 0.05 % propylthiouracil (PTU), and VD (100 or 500 IU/kg) treatment was performed daily by gavage. At the end of treatment, Morris water maze (MWM) was carried out and evaluated hippocampal neurogenesis, apoptosis, and dark neurons (DNs). Our results revealed that the escape latency and the traveled distance to find the platform in the Hypo group were significantly longer but the time spent and distance traveled in the target area in probe trial was lower than the control group. Hypothyroidism was accompanied by a marked decrease in hippocampal neurogenesis, and a significant increase in the number of apoptotic neurons and DNs compared to the control group. VD decreased escape latency and the traveled distance to find the platform but increased the time spent and distance traveled in the target area in probe trial than the Hypo group. VD also increased neurogenesis, reduced apoptosis and DNs production compared to the Hypo group. In conclusion, these results support a role for VD in the restoring hippocampal neurogenesis impairment, reducing neuronal apoptosis, and DNs in hypothyroid rats as well as raise the possibility that VD may contribute as a therapeutic approach to improve the learning and memory deficits associated with hypothyroidism.

Microglia Regulating Neuronal Apoptosis Via Alpha7 Nicotinic Acetylcholine Receptor in HIV-Associated Neurocognitive Disorders

Background: Microglia, the innate immune cells of the central nervous system (CNS), originate from erythromyeloid progenitor cells in the embryonic yolk sac. As the innate immune cells of the CNS, microglia play an important role in innate immunity, neurogenesis, and brain tissue repair by activating the alpha7 nicotinic acetylcholine receptor (α7nAChR). They are the key target cells for HIV-1 infection in the CNS and have been shown to produce a chronic inflammatory response in the brain which underlies HIV-associated neurocognitive disorders (HAND). Apart from the microglial neuroinflammation, recent studies have reported that activated microglia could induce neuronal apoptosis through the release of neurotoxic factors (e.g., ROS and NLRP3 inflammasome) in HAND. In addition, α7nAChR was significantly up-regulated in the striatum which is the most serious part of neuron apoptosis in gp120 transgenic mice brains. However, the underlying mechanism of neuronal cell apoptosis by gp120-activated microglia remains unclear. Here, this study aimed to explore the mechanism by which HIV-1 gp120-activated microglia induce neuronal apoptosis. Materials and methods: The expression of α7nAChR was examined by immunohistochemistry in the brain of HIV-1 LAV gp120 transgenic (Tg) mice (C57BL/6J background, 12-month-old). Methyllycaconitine (MLA, specific α7nAChR agonists) and Nicotine (NT, specific α7nAChR antagonists) were used to evaluate the effect of α7nAChR on apoptosis induced by gp120. Immortalized microglial cells (BV2) were incubated for 24 hours with gp120, NT, and gp120+NT, respectively, with or without preincubation with MLA for 15 minutes to obtain their supernatants. Next, the neuronal cells (SH-SY5Y) were treated with the same as BV2 or with collected BV2 culture supernatants. The apoptosis effect of SH-SY5Y and the level of ROS in the BV2 culture supernatants were assessed via quantitative analysis of flow cytometry. Comparisons between supernatant groups and drug groups were conducted to evaluate the effects of the SH-SY5Y apoptosis induced by microglia. Western blotting of microglia cell lysates was employed for the analysis of cellular signaling pathways. Student's t-tests and ANOVA were applied to determine statistical significance. Results: The expression of α7nAChR in HIV-1 gp120 transgenic mice was significantly upregulated (P < 0.001). The effect of apoptosis was more significant in microglia culture supernatants groups compared to that in drug-treated groups (P < 0.001). Neuronal activity was not changed in the NT group or MLA group than in the control, but HIV-1 gp120 could increase the number of apoptotic neurons (P < 0.01). Significant neuronal apoptosis was observed when treated with BV2 supernatants from the group treated by combining the gp120 with NT (P < 0.001), and this effect was in turn inhibited by MLA (P < 0.001). These results suggested that gp120-activated microglia might induce neuronal apoptosis invitro owing to gp120 upregulating α7nAChR. In addition, the ROS level in microglia was significantly upregulated following gp120 exposure (P < 0.001), and this effect could be enhanced by NT (P < 0.001). Notably, there was a positive correlation between the levels of ROS in microglia culture supernatants and the levels of neuronal apoptosis treated in the same supernatants. This positive relevance indicated that microglia might induce neuronal apoptosis by producing excessive ROS. The expressions of p53, OMI, and caspase-3 significantly upregulated following gp120 exposure with the down-regulate expression of XIAP (P < 0.001). And this effect could be enhanced by NT (P < 0.001). The results of western blotting indicated that gp120 and NT can up-regulate the ROS to activate p53 by α7nAChR, which could lead to an increased OMI and decreased XIAP levels. (P < 0.001). Conclusions: Microglia may play a critical role in regulating neuronal apoptosis in HAND. HIV-1 gp120 and NT could increase the generation of ROS through α7nAChR in microglia, which leads to neuronal apoptosis. Moreover, our results showed that the combined action of NT and gp120 may upregulate p53 and Omi / HtrA2 protein expression, and the downregulation of XIAP protein may significantly activate the level of caspase-3. (Acknowledgements:Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 82172259 to H.C.) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Age-related hearing loss was accelerated by apoptosis of spiral ganglion and stria vascularis cells in ApoE KO mice with hyperglycemia and hyperlipidemia.

Age-related hearing loss (ARHL) is associated with diabetes and/or dyslipidemia in humans. However, the detailed mechanism for the development of ARHL by diabetes and/or dyslipidemia has not been elucidated. In this study, we investigated the etiology of ARHL in apolipoprotein E (ApoE)-deficient mice with diabetes and dyslipidemia. The atherosclerotic CD-STZ (mice fed with a control diet and received an STZ injection), WD-con (mice fed with a western diet), and WD-STZ (mice fed with a western diet and received an STZ injection) mice showed a 2.4-, 4.9-, and 6.8-fold larger area, respectively, occupied by lesions throughout the aorta compared with the CD-con mice. A significantly larger area under the curve (AUC) was observed in the STZ-treated groups than in the non-treated groups based on the oral glucose tolerance test (OGTT). At 20 weeks of age, HbA1c levels were significantly higher in the CD-STZ and WD-STZ mice than in the CD-con and WD-con mice. In all the groups, the auditory brainstem response (ABR) thresholds of the 16-week-old mice were significantly higher compared with those of the 8-week-old mice. In particular, in the WD-STZ mice, the ABR thresholds of the left and right ears reached the maximum decibel peak equivalent sound pressure levels (130 dBpeSPL), which is a sign of deafness. The apoptotic spiral ganglion neurons (SGNs) of the WD-STZ mice were significantly increased compared with those of the other three groups, indicating that SGN apoptosis resulted in hearing loss in STZ-induced diabetic ApoE KO mice fed with a WD. A significant loss of the stria vascularis cells was observed in the WD-STZ group compared with the CD-con mice. In the organ of Corti, few apoptotic hair cells were found in all the groups; however, no significant difference was observed. Therefore, we consider that the reduced hearing ability in the STZ-treated and WD-fed groups was attributed to the damage to the SGN and stria vascularis in the cochlea. Thus, our results indicated that ototoxicity by diabetes and/or dyslipidemia accelerated ARHL in ApoE KO mice, thereby suggesting the importance of appropriate treatment of patients with diabetes and/or dyslipidemia to prevent ARHL.

Gas6 Promotes Microglia Efferocytosis and Suppresses Inflammation Through Activating Axl/Rac1 Signaling in Subarachnoid Hemorrhage Mice.

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is characterized byrapid development of neuron apoptosis and dysregulated inflammatory response. Microglia efferocytosis plays a critical role in the clearance of apoptotic cells, attenuation of inflammation, and minimizing brain injury in various pathological conditions. Here, using a mouse SAH model, we aim to investigate whether microglia efferocytosis is involved in post-SAH inflammation and to determine the underlying signaling pathway. We hypothesized that TAM receptors and their ligands regulate this process. To prove our hypothesis, the expression and cellular location of TAM (Tyro3, Axl, and Mertk) receptors and their ligands growth arrest-specific 6 (Gas6) and Protein S (ProS1) were examined by PCR, western blots, and fluorescence immunostaining. Thirty minutes after SAH, mice received an intraventricular injection of recombinant Gas6 (rGas6) or recombinant ProS1 (rPros1) and underwent evaluations of inflammatory mediator expression, neurological deficits, and blood-brain barrier integrity at 24h. Microglia efferocytosis of apoptotic neurons was analyzed in vivo and in vitro. The potential mechanism was determined by inhibiting or knocking down TAM receptors and Rac1 by specific inhibitors or siRNA. SAH induced upregulation of Axl and its ligand Gas6. The administration of rGas6 but not rPros1 promoted microglia efferocytosis, alleviated inflammation, and ameliorated SAH-induced BBB breakdown and neurological deficits. The beneficial effects of rGas6 were arrogated by inhibiting or knocking down Axl and Rac1. We concluded that rGas6 attenuated the development of early brain injury in mice after SAH by facilitating microglia efferocytosis and preventing inflammatory response, which is partly dependent on activation of Axl and Rac1.

Regulation of the apoptosis/autophagy switch by propionic acid in ventromedial hypothalamus of rats with type 2 diabetes mellitus

BackgroundHypothalamic dysregulation may cause abnormal glucose metabolism and type 2 diabetes mellitus (T2DM). The balance between autophagy and apoptosis is important for maintaining cellular/tissue homeostasis and may be disrupted in T2DM. ObjectivesSince propionic acid (PA) exerts neuroprotective effects, the aim was to investigate its effects on apoptosis/autophagy switch in the ventromedial hypothalamus (VMH) of T2DM rats. Materials and methodsMale Wistar rats were divided: 1) control; 2) T2DM; groups that received (14 days, orally): 3) metformin (60 mg/kg); 4) sodium salt of PA (60 mg/kg); 5) PA + metformin. Western blotting (Bax, Bcl-xl, LC3, Beclin-1, caspase-3), RT-PCR (Bax, Bcl-xl, LC3, Beclin-1), transmission electron microscopy and immunohistochemical staining (Bax, Bcl-xl) were performed on the VMH samples. ResultsT2DM-induced apoptosis and mitoptosis, enlarged endoplasmic reticulum (ER) tubules/cisterns were observed in VMH, and accompanied by an imbalance of pro- and anti-apoptotic factors: elevation of pro-apoptotic markers Bax and caspase-3, decrease in autophagy protein LC3 and anti-apoptotic Bcl-xl. Metformin and PA administration partially improved VMH ultrastructural changes by reducing mitochondrial swelling and diminishing the number of apoptotic neurons. Metformin inhibited neuronal apoptosis, however, caused reactive astrogliosis and accumulation of lipofuscin granules. Elevated number of autophagosomes was associated with the LC3, Beclin-1 and Bcl-xl increase and decrease in Bax and caspase-3 vs. T2DM. PA switched cell fate from apoptosis to autophagy by elevating LC3 and Beclin-1 levels, increasing Bcl-xl content that altogether may represent adaptive response to T2DM-induced apoptosis. PA + metformin administration lowered relative area of ER membranes/cisterns vs. control, T2DM and metformin, and was optimal considering ratio between the pro-apoptotic, anti-apoptotic and autophagy markers. ConclusionT2DM was associated with apoptosis activation leading to impairments in VMH. PA in combination with metformin may be effective against diabetes-induced cell death by switching apoptosis to autophagy in VMH.

Gossypetin ameliorates 5xFAD spatial learning and memory through enhanced phagocytosis against Aβ

BackgroundMicroglia are the resident immune cells found in our brain. They have a critical role in brain maintenance. Microglia constantly scavenge various waste materials in the brain including damaged or apoptotic neurons and Aβ. Through phagocytosis of Aβ, microglia prevent the accumulation of Aβ plaque in the brain. However, in Alzheimer’s disease (AD) patients, chronic exposure to Aβ makes microglia to become exhausted, which reduces their phagocytic activity against Aβ. Since microglia play an important role in Aβ clearance, enhancing microglial phagocytic activity against Aβ is a promising target for AD treatment. Therefore, there is a great need for therapeutic candidate that enhances microglial Aβ clearance while inhibiting microglia’s pathogenic properties.MethodsIn vivo studies were conducted with 5xFAD AD model mice by treating gossypetin for 13 weeks through intragastric administration. Their spatial learning and memory were evaluated through behavior tests such as Y-maze and Morris Water Maze test. Hippocampus and cortex were acquired from the sacrificed mice, and they were used for histological and biochemical analysis. Also, mouse tissues were dissociated into single cells for single-cell RNA sequencing (scRNA-seq) analysis. Transcriptome of microglial population was analyzed. Mouse primary microglia and BV2 mouse microglial cell line were cultured and treated with fluorescent recombinant Aβ to evaluate whether their phagocytic activity is affected by gossypetin.ResultsGossypetin treatment improved the spatial learning and memory of 5xFAD by decreasing Aβ deposition in the hippocampus and cortex of 5xFAD. Gossypetin induced transcriptomic modulations in various microglial subpopulations, including disease-associated microglia. Gossypetin enhanced phagocytic activity of microglia while decreasing their gliosis. Gossypetin also increased MHC II+ microglial population.ConclusionsGossypetin showed protective effects against AD by enhancing microglial Aβ phagocytosis. Gossypetin appears to be a novel promising therapeutic candidate against AD.

Brain milieu induces early microglial maturation through the BAX-Notch axis

Microglia are derived from primitive myeloid cells and gain their early identity in the embryonic brains. However, the mechanism by which the brain milieu confers microglial maturation signature remains elusive. Here, we demonstrate that the baxcq55 zebrafish and Baxtm1Sjk mouse embryos exhibit similarly defective early microglial maturation. BAX, a typical pro-apoptotic factor, is highly enriched in neuronal cells and regulates microglial maturation through both pro-apoptotic and non-apoptotic mechanisms. BAX regulates dlb via the CaMKII-CREB axis calcium-dependently in living neurons while ensuring the efficient Notch activation in the immigrated pre-microglia by apoptotic neurons. Notch signaling is conserved in supporting embryonic microglia maturation. Compromised microglial development occurred in the Cx3cr1Cre/+Rbpjfl/fl embryonic mice; however, microglia acquire their appropriate signature when incubated with DLL3 in vitro. Thus, our findings elucidate a BAX-CaMKII-CREB-Notch network triggered by the neuronal milieu in microglial development, which may provide innovative insights for targeting microglia in neuronal disorder treatment.

Mechanism of Dexmedetomidine Intervention on Neurogenic Inflammation in Cognitive Impairment Rats after Partial Hepatectomy.

Objective To study the effect of dexmedetomidine on cognitive function in rats with cognitive impairment after partial hepatectomy and its mechanism. Methods 60 SD rats were randomly divided into 4 groups (n = 15): blank control group (CG group), sham operation group (Sham group), cognitive impairment model group (POCD group), and dexmedetomidine + cognitive impairment model group (DEX group). Rats in the POCD group underwent left lobe hepatectomy and intraperitoneal injection of the same amount of normal saline after resuscitation. Rats in the DEX group underwent left lobe hepatectomy and intraperitoneal injection of dexmedetomidine 50 μg/kg. Group CG was not operated on and the same amount of normal saline was injected intraperitoneally. In the Sham group, liver resection was not allowed after the abdominal incision, and normal saline was injected intraperitoneally. Rats were injected every 24 hours for 5 consecutive days. Morris water maze (MWM) were used to evaluate the effects of dexmedetomidine on learning and memory ability of POCD rats. TUNEL method was used to detect apoptotic neurons in the hippocampus. INOS, Arg-1, IL-6, and TNF-αexpression levels were detected. Western blot detects the expression level of TNF-α, Bcl-2, and NF-κB protein. Result Compared with the CG group, the escape latency of the other three groups was prolonged on the 5th day after the operation, and the number of crossing the platform was reduced. Compared with the Sham group, the escape latency of the POCD group and DEX group was significantly prolonged, and the number of crossing the platform was significantly reduced on day 5 (P < 0.05). Compared with the POCD group, the DEX group shortened the escape latency and increased the number of crossing the platform on the 5th day (P < 0.05). It shows that the spatial learning and memory function of rats has been restored to a certain extent.The number of iNOS and Arg-1 positive cells in the POCD group and DEX group was higher than that in the control group, and the number of Arg-1 positive cells in the DEX group was higher than that in the POCD group (P < 0.05). Western blot results the expression of Bcl-2 and NF-κB protein in POCD group, and DEX group was higher than that of the sham group (P < 0.05). The expression of Bcl-2 and NF-κB protein was the most in POCD group. The expression of Bcl-2 and NF-κB protein in DEX group was lower than that in POCD group (P < 0.05). Conclusion Behavioral results showed that the learning and cognitive ability of POCD model rats after hepatectomy was impaired, and inflammatory factors and activated microglia were found in the hippocampus of POCD rats. Dexmedetomidine may improve the brain function of POCD rats by inhibiting neuronal apoptosis,partly through NF-κB apoptosis pathway.

Mafba and Mafbb regulate microglial colonization of zebrafish brain via controlling chemotaxis receptor expression

Microglia are the central nervous system (CNS)-resident macrophages involved in neural inflammation, neurogenesis, and neural activity regulation. Previous studies have shown that naturally occurring neuronal apoptosis plays a critical role in regulating microglial colonization of the brain in zebrafish. However, the molecular signaling cascades underlying neuronal apoptosis-mediated microglial colonization and the regulation of these cascades remain undefined. Here, we show that basic leucine zipper (b-Zip) transcription factors, Mafba and Mafbb, two zebrafish orthologs of mammalian MAFB, are key regulators in neuronal apoptosis-mediated microglial colonization of the brain in zebrafish. We document that the loss of Mafba and Mafbb function perturbs microglial colonization of the brain. We further demonstrate that Mafba and Mafbb act cell-autonomously and cooperatively to orchestrate microglial colonization, at least in part, by regulating the expression of G protein-coupled receptor 34a (Gpr34a), which directs peripheral macrophage recruitment into the brain through sensing the lysophosphatidylserine (lysoPS) released by the apoptotic neurons. Our study reveals that Mafba and Mafbb regulate neuronal apoptosis-mediated microglial colonization of the brain in zebrafish via the lysoPS-Gpr34a pathway.

New Histophatological Finding About Data Destroying Amyloid Black Holes in Hippocampus Following Olfactory Bulb Lesion Like as the Universe

Background: Many infinite theories have been suggested to explain memory loss in neurodegenerative diseases. However, there is no data that iron-containing neurofibrillary networks can cause neuron death and erase the memory of neurons, just like black holes in space. Objectives: Ths study aimed to investigate the electromagnetic properties of iron-loaded neurofibrillary networks formed in the hippocampus as a result of damage to the olfactory nerves, just like black holes in space, as well as whether they cause neuron death and memory loss. Methods: All rats were tested with star maze performance before, 3 weeks, and 3 months after surgery. The data used in the study were obtained from the subjects in the experimental groups who had been followed up for 3 months with control (GI; n = 5), SHAM (GII; n = 5) with only frontal burr hole, and study (GIII; n = 15) animals with olfactory bulb lesion. All rats were tested with star maze performance before, 3 weeks, and 3 months after surgery. The olfactory bulbs and hippocampus of subjects were examined by stereological methods. Olfactory bulb volumes, degenerated neuron densities of the hippocampus, and numbers of hippocampal black holes were estimated quantitatively, and results were statistically analyzed by a 1-way analysis of variance (ANOVA). The properties of black holes in the brains and the universe were compared theoretically. Results: The mean olfactory bulb volumes, degenerated neuron density, and black holes of the hippocampus were estimated as 4.43 ± 0.22 mm3, 42 ± 9 mm3, and 3 ± 1 mm3 in GI, 4.01 ± 0.19 mm3, 257 ± 78 mm3, and 11 ± 3 mm3 in GII, and 2.4 ± 0.8 mm3, 1675 ± 119 mm3, and 34 ± 7 mm3 in GIII. All animals were tested with star maze performance before, 3 weeks, and 3 months after surgery. Latency, distance, speed, and path efficiency values of all animals were detected. The more diminished olfactory bulb volume (P &lt; 0.00001) causes more apoptotic neurons and black holes in the hippocampus (P &lt; 0.0001) and more memory loss in olfactory bulb lesion (OBL)-applied animals (P &lt; 0.005). Conclusions: Hippocampal black holes, which are similar to black holes in terms of their formation processes, may be responsible for neuronal losses and memory erasures in the brain by acting like black holes in space. These amyloid plaques, which cause neuron death and memory loss, will be called data-deleting amyloid black holes (DADA-Black Holes) in the paper.

IRES-mediated Wnt2 translation in apoptotic neurons triggers astrocyte dedifferentiation

Reactive astrogliosis usually bears some properties of neural progenitors. How injury triggers astrocyte dedifferentiation remains largely unclear. Here, we report that ischemia induces rapid up-regulation of Wnt2 protein in apoptotic neurons and activation of canonical Wnt signaling in reactive astrocytes in mice, primates and human. Local delivery of Wnt2 shRNA abolished the dedifferentiation of astrocytes while over-expressing Wnt2 promoted progenitor marker expression and neurogenesis. Both the activation of Wnt signaling and dedifferentiation of astrocytes was compromised in ischemic caspase-3−/− cortex. Over-expressing stabilized β-catenin not only facilitated neurogenesis but also promoted functional recovery in ischemic caspase-3−/− mice. Further analysis showed that apoptotic neurons up-regulated Wnt2 protein via internal ribosome entry site (IRES)-mediated translation. Knocking down death associated protein 5 (DAP5), a key protein in IRES-mediated protein translation, significantly diminished Wnt activation and astrocyte dedifferentiation. Our data demonstrated an apoptosis-initiated Wnt-activating mechanism which triggers astrocytic dedifferentiation and facilitates neuronal regeneration.

Comprehensive insights into the function and molecular and pharmacological regulation of neuron-derived orphan receptor 1, an orphan receptor.

Neuron-derived orphan receptor 1 (NOR1), also called nuclear receptor subfamily 4 group A member 3 (NR4A3), is a nuclear receptor belonging to the NR4A family. Since no endogenous ligand has been identified to date, NOR1 is also referred to as an orphan receptor. NOR1 is expressed in a variety of cells and tissues, including neurons, vascular smooth muscle cells, T lymphocytes, dendritic cells, tumor cells, heart, liver, and pancreas. Because NOR1 was first identified in apoptotic neurons, it is functionally associated with the regulation of cell migration and the growth of neuronal synapses. In-depth studies have shown that NOR1 can be edited by the immediate early gene and functions as a transcription factor. NOR1 has been shown to be rapidly induced by a number of stimulants including growth factors, fatty acids, and neurotransmitters. Elevated NOR1 levels may be involved in a number of pathophysiological processes. These include regulation of cellular apoptosis and regeneration, neuron formation, contextual fearing memory, inflammation, vascular smooth muscle proliferation, insulin secretion, and tumor development, whereby NOR1 mediates the pathogenesis of numerous diseases such as cerebral ischemia, depression, post-traumatic stress disorder, atherosclerosis, abdominal aortic aneurysm, cardiac hypertrophy, diabetes, osteoarthritis, rheumatoid arthritis, and cancer. However, to date, comprehensive insights into the function of NOR1 are not available in sources published online. In this review, we provide a brief overview of the function and molecular and pharmacological regulation of NOR1 in various pathological or physiological conditions to advance the development of NOR1 as a novel target for disease treatment.

Changes in normal and apoptotic pyramidal neuronal cell densities in prefrontal cortex of the male rat following chronic khat use

Long-term Khat consumption is associated with significant neuro-cognitive changes, which have been elucidated in behavioral studies. However, correlation of these neurophysiological changes with experimental structural changes in prefrontal neurons has not been described adequately. Young adult male Winstar rats, aged 2-3 months, weighing 200-300 grams were randomized into four groups of 11 each to correspond with those used as controls, those that received 500mg/kg, 1000mg/kg and 2000mg/kg body weight khat extracts respectively. The control rats were fed on normal diet, while experimental groups were fed on normal diet and khat extracts using oral gavage for 6 weeks. The animals were sacrificed, and their brains removed. Toluidine blue staining was used to elucidate the Nissl substance. Image-Fiji was used to analyse densities of normal and apoptotic pyramidal neuronal densities across the 4 experimental groups. Normal pyramidal neurons were identifiable by their characteristic uniform size and prominent nucleoli, while apoptotic cells displayed prominent eosinophilia and pyknotic nuclei. There was a statistically significant increase in apoptotic pyramidal neurons across the three khat-fed groups compared to controls, and a decrease in normal pyramidal neuronal population from controls with increasing doses of khat. This decrease in normal pyramidal neurons and increase in apoptosis could be a potential surrogate for neurocognitive perturbations associated with chronic khat use.

Ketogenic diet attenuates post-cardiac arrest brain injury by upregulation of pentose phosphate pathway–mediated antioxidant defense in a mouse model of cardiac arrest

ObjectiveThe aim of this study was to investigate the effect of the ketogenic diet (KD) on post-cardiac arrest brain injury in a mouse model of cardiac arrest and cardiopulmonary resuscitation. MethodsMice were fed a KD for 4 wk and then subjected to cardiac arrest and cardiopulmonary resuscitation. The HT22 cells after β-hydroxybutyrate (β-OHB) treatment were exposed to oxygen-glucose deprivation/reoxygenation. Survival and neurologic function were measured after return of spontaneous circulation. Positron emission tomography/computed tomography scanning, 13C-magnetic resonance spectroscopy analysis, and seahorse assay were performed to explore the mechanism underlying the phenotype. ResultsResults of this study demonstrated that KD improved neurologic function and reduced apoptotic neurons in cardiac arrest and cardiopulmonary resuscitation mice. With no alteration of glucose uptake, KD suppressed glucose oxidation in mouse brain. Consistently, the glycolytic capacity of the HT22 cells was also downregulated by β-OHB treatment. Moreover, KD increased nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate and reduced glutathione/oxidized glutathione couples and reduced reactive oxygen species in the brain, probably due to activation of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the pentose phosphate pathway. Pharmacologic inhibition of pentose phosphate pathway totally abolished the influence of β-OHB on glycolysis, and post–oxygen-glucose deprivation/reoxygenation cell viability and reactive oxygen species production in HT22 cells. ConclusionKD improved survival and attenuated post-cardiac arrest brain injury, which was mediated by upregulation of pentose phosphate pathway and related antioxidant defense system.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages

BackgroundApoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model.MethodsMi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage.ResultsASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout.ConclusionConditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

Bergenin has neuroprotective effects in mice with ischemic stroke through antioxidative stress and anti-inflammation via regulating Sirt1/FOXO3a/NF-κB signaling.

Bergenin (BGN) is a C-glycoside of 4-O-methylgallic acid with anti-inflammatory, antioxidant, and tissue-repairing abilities. Here, we probed the roles and mechanisms of BGN in ischemic stroke-mediated cerebral injury. The middle cerebral artery occlusion (MCAO) model was established in mice, which were injected intraperitoneally with varying concentrations of BGN (10, 20, and 40 mg/kg). The modified neurological severity score (mNSS) and the water maze experiment were adopted to evaluate mice's neural functions (movement and memory). The brain edema was assessed by the dry and wet method. TdT-mediated dUTP nick end labeling (TUNEL)-labeled apoptotic neurons and Iba1-labeled microglia in the cortex were measured by immunohistochemistry (IHC). Quantitative reverse transcription-PCR and ELISA were implemented to determine the expression of inflammatory cytokines (TNFα, IL-1β, and IL-6), neurotrophic factors (BDNF and VEGF), and oxidative stress factors (SOD and MDA) in brain tissues. The profiles of Sirt1, FOXO3a, Nrf2, NF-κB, and STAT6 in brain tissues were checked by western blot. BGN significantly improved MCAO mice's cognitive, learning, and motor functions, reduced brain edema, hampered the production of inflammatory factors and oxidative stress mediators, and suppressed neuronal apoptosis. Additionally, BGN dampened the expression of proinflammatory cytokines and upregulated neurotrophic factors and oxidative stress factors in ischemic brain tissues of MCAO mice. Meanwhile, BGN reduced the expression of inflammatory cytokines and oxidative stressors in oxygen-glucose deprivation/reoxygenation-induced BV2 microglia. Further mechanistic studies revealed that BGN concentration dependently elevated the profiles of Sirt1, FOXO3a, STAT6, and Nrf2, and abated the NF-κB phosphorylation. BGN protects against ischemic stroke in mice by boosting the Sirt1/FOXO3a pathway, suggesting its potential as a therapeutic agent for ischemic stroke.

Saphenous vein phlebotomy alleviates neuroinflammatory response and oxidative stress following traumatic brain injury

ObjectiveIn spite of the great advances in the field of traumatic brain injury (TBI), it still remains as a global health problem. Phlebotomy has been mentioned as an effective treatment for TBI in Persian medicine. This study aimed to evaluate the possible effect of saphenous vein phlebotomy (SVP) on the alleviation of neuroinflammation and oxidative stress after TBI. MethodsSeventy-two adult male Wistar rats were randomly divided into three main groups: sham, TBI and TBI + SVP group. Animal model of TBI was made through a controlled cortical impactor (CCI) device and SVP treatment was applied 10 min after TBI. Then, neurological function, body temperature, cerebral edema, blood–brain barrier integrity, brain apoptosis, oxidative stress, and inflammation were assessed. ResultsOur results indicated that SVP was associated with improvement of neurological scores 24 h post-TBI. In addition, SVP application significantly alleviated proinflammatory cytokines (IL- 1β, IL-17, and TNF-α) and oxidative stress factors as shown by reduction of malondialdehyde (MDA) and elevation of superoxide dismutase (SOD) with no significant effect on glutathione peroxidase (GPx) and nitric oxide (NO) in the brain 6 h after TBI. Besides, it reduced body temperature, attenuated severity of brain edema, and also reduced caspase-3 and number of apoptotic neurons. ConclusionThis study showed that SVP could ameliorate the severity of acute TBI, partly through alleviation of neuroinflammation, oxidative stress, and apoptosis. Further studies are necessary for more understanding the involved mechanism(s).

Exosomes derived from bone marrow mesenchymal stem cells attenuate neurological damage in traumatic brain injury by alleviating glutamate-mediated excitotoxicity

BackgroundTraumatic brain injury (TBI) is one of the major contributors to disability and death worldwide. Glutamate-mediated excitotoxicity, one of the secondary injuries occurring after TBI, leads to extreme neuronal apoptosis, and can be a potential target for intervention. Bone marrow mesenchymal stem cells-derived exosomes (BMSCs-Exos) have demonstrated neuroprotective effects on TBI. However, their precise role and the underlying mechanism by which they regulate glutamate-mediated excitotoxicity have not yet been determined. Therefore, this study aimed to determine whether BMSCs-Exos alleviate glutamate excitotoxicity post-TBI and their associated mechanism. MethodsBMSCs-Exos were extracted from the BMSCs incubation medium and identified by transmission electron microscopy, nanoparticle trafficking analysis, and western blotting. The neuroprotective effects of BMSCs-Exos on glutamate excitotoxicity were investigated in the glutamate-mediated excitotoxicity neuronal cell model and the TBI rat model (TBI induced by controlled cortical impact) using western blotting and TUNEL assay. Cortical lesion samples were collected post-TBI on day-1 and day-14 to study histology. In addition, cortical lesion volume on days 1, 3 and 7 following TBI was determined using T2-weighted magnetic resonance imaging (MRI), and cognitive function was assessed at 4 weeks following TBI using the Morris water maze (MWM) test. ResultsBMSCs-Exos were observed to be spherical with a mean diameter of 109.9 nm, and expressed exosomal markers CD9, CD81 and TSG101. BMSCs-Exos were efficiently endocytosed by astrocytes after co-incubation for 24 h. In vitro studies revealed that 125 μM of glutamate significantly induced neuronal apoptosis, which was attenuated by BMSCs-Exos in astrocyte–neuron co-cultures. This attenuation was mediated by the upregulation of glutamate transporter-1 (GLT-1) level and the downregulation of p-p38 MAPK level in astrocytes. Similar results were obtained in vivo, wherein we verified that PKH67-labeled BMSCs-Exos administered intravenously could reach the perilesional cortex crossing the blood-brain barrier and significantly reduce glutamate levels in the perilesional cortex of the TBI rat, accompanied by increased GLT-1 level and downregulation in p-p38 MAPK level. Additionally, western blotting and TUNEL staining also revealed that BMSCs-Exos significantly downregulated the expression of pro-apoptosis markers, including cleaved caspase-3 and cleaved caspase-9, and attenuated neuronal apoptosis following TBI. Immunohistochemical analysis and Nissl staining showed that BMSCs-Exos significantly increased GLT-1-positive cells, and the number of apoptotic neurons decreased in the perilesional cortex. Moreover, MRI and MWM results revealed that BMSCs-Exos significantly minimized cortical lesion volume and ameliorated cognitive function after TBI. The underlying neuroprotective mechanism of BMSCs-Exos may be due to an increase in GLT-1 level in astrocytes by blocking the p38 MAPK signaling pathway. ConclusionTaken together, our findings demonstrate that the implementation of BMSCs-Exos may be an effective prospective therapy for attenuating post-TBI neurological damage.

The role of microglial/macrophagic salt-inducible kinase 3 on normal and excessive phagocytosis after transient focal cerebral ischemia

Previous studies suggested that anti-inflammatory microglia/macrophages (Mi/MΦ) play a role in “normal phagocytosis,” which promoted the rapid clearance of necrotic substances and apoptotic cells. More recently, a few studies have found that Mi/MΦ also play a role in “pathological phagocytosis” in the form of excessive or reduced phagocytosis, thereby worsening damage induced by CNS diseases. However, the underlying mechanisms and the Mi/MΦ subtypes related to this pathological phagocytosis are still unknown. Salt-inducible kinase 3 (SIK3), a member of the 5’ adenosine monophosphate-activated protein kinase (AMPK) family, has been shown to regulate inflammation in several peripheral diseases. Whether SIK3 also regulates the inflammatory response in CNS diseases is currently unknown. Therefore, in this study, we created a transgenic tamoxifen-induced Mi/MΦ-specific SIK3 conditional knockout (SIK3-cKO) mouse to examine SIK3’s role in phagocytotic function induced by transient focal cerebral ischemia (tFCI). By single-cell RNA-seq, we found the pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function, but the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. We found that SIK3-cKO caused Mi/MΦ heterogenization from the transitional phenotype to the anti-inflammatory phenotype after tFCI. This phenotypic shift corresponded with enhanced phagocytosis of both apoptotic and live neurons. Interestingly, SIK3-cKO enhanced normal phagocytosis of myelin debris but attenuating excessive phagocytosis of non-damaged myelin sheath, thereby protecting white matter integrity after tFCI. CD16, a pro-inflammation marker, was decreased significantly by SIK3-cKO and correlated with “excessive phagocytosis.” SIK3-cKO promoted long-term recovery of white matter function and neurological function as assessed with electrophysiological compound action potential (CAPs) and behavioral analysis. This study is the first to show a role of SIK3 in Mi/MΦ phagocytosis in CNS diseases, and reveals that promoting Mi/MΦ anti-inflammatory heterogenization inhibits “excessive phagocytosis” of live cells and facilitates “normal phagocytosis” of apoptotic cells. Therefore, inhibition of SIK3 in Mi/MΦ may be a potential therapeutic target in stroke and other CNS diseases with accompanying white matter destruction.Graphical abstractIn the acute stage of tFCI, Mi/MΦ polarized into different phenotypes. The pro-inflammatory Mi/MΦ phenotype performed an excessive phagocytotic function. In contrast, the anti-inflammatory Mi/MΦ phenotype performed a normal phagocytotic function. After tFCI, SIK3-cKO promoted anti-inflammatory phenotypic heterogenization of Mi/MΦ. SIK3-cKO promoted Mi/MΦ phagocytosis of apoptotic (normal phagocytosis) and living neuronal cell bodies (excessive phagocytosis) in gray matter. Interestingly, SIK3-cKO specifically increased normal phagocytosis of myelin debris concurrent with an attenuation of excessive phagocytosis of myelin sheath in white matter. These changes induced by SIK3-cKO were associated with protection of white matter integrity and long-term neurofunctional recovery after tFCI.