Ketamine Improves the Glymphatic Pathway by Reducing the Pyroptosis of Hippocampal Astrocytes in the Chronic Unpredictable Mild Stress Model.

Ketamine as a glutamate receptor antagonist has a rapid, potent, and long-lasting antidepressant effect, but its specific mechanism is still not fully understood. Depression is associated with elevated levels of glutamate and astrocyte loss in the brain; the exploration of the relationships between ketamine's antidepressant effect and astrocytes has drawn great attention. Astrocytes and aquaporin 4 (AQP4) are essential components of the glymphatic system, which is a brain-wide perivascular pathway to help transport nutrients to the parenchyma and remove metabolic wastes. In this study, we investigated pyroptosis-associated protein Nlrp3/Caspase-1/Gsdmd-N expression in the hippocampus of mice and the toxic effect of high levels of glutamate on primary astrocytes. On this basis, the protective mechanism of ketamine is explored. A single administration of ketamine (10 mg/kg) remarkably relieved anxious and depressive behaviors in the sucrose preference test, elevated plus maze test, and forced swim test. Meanwhile, ketamine reduced the level of hippocampus Nlrp3 and the expression of its downstream molecules in chronic unpredictable mild stress (CUMS) mice model by western blot and reduced the colocalization of Gfap and Gsdmd by nearly 25% via immunofluorescent staining. Ketamine also increased the Gfap-positive cells and AQP4 expression in the hippocampus of the CUMS mice. More important, ketamine increased the distribution of the fluorescent tracer of CUMS mice. Treatment with 128 mM glutamate in cortical and hippocampus astrocytes increased the level of Nlrp3, and Gsdmd-N, and ketamine alleviated high glutamate-induced pyroptosis-associated proteins. In summary, these results suggest that high glutamate-induced astrocyte pyroptosis through the Nlrp3/Caspase-1/Gsdmd-N pathway which was inhibited by ketamine and ketamine can improve the damaged glymphatic function of the CUMS mice. The present study indicates that inhibiting astrocyte pyroptosis and promoting the glymphatic circulation function are a new mechanism of ketamine's antidepressant effect, and astrocyte pyroptosis may be a new target for other antidepressant medicines.

Ageing related thyroid deficiency increases brain-targeted transport of liver-derived ApoE4-laden exosomes leading to cognitive impairment.

Alzheimer's disease (AD) is the prevalent cause of dementia in the ageing world population. Apolipoprotein E4 (ApoE4) allele is the key genetic risk factor for AD, although the mechanisms linking ApoE4 with neurocognitive impairments and aberrant metabolism remains to be fully characterised. We discovered a significant increase in the ApoE4 content of serum exosomes in old healthy subjects and AD patients carrying ApoE4 allele as compared with healthy adults. Elevated exosomal ApoE4 demonstrated significant inverse correlation with serum level of thyroid hormones and cognitive function. We analysed effects of ApoE4-containing peripheral exosomes on neural cells and neurological outputs in aged or thyroidectomised young mice. Ageing-associated hypothyroidism as well as acute thyroidectomy augmented transport of liver-derived ApoE4 reach exosomes into the brain, where ApoE4 activated nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome by increasing cholesterol level in neural cells. This, in turn, affected cognition, locomotion and mood. Our study reveals pathological potential of exosomes-mediated relocation of ApoE4 from the periphery to the brain, this process can represent potential therapeutic target.

The neuroprotective mechanism of lithium after ischaemic stroke.

Stroke causes degeneration and death of neurones leading to the loss of motor function and frequent occurrence of cognitive impairment and depression. Lithium (Li+), the archetypal mood stabiliser, is neuroprotective in animal models of stroke, albeit underlying mechanisms remain unknown. We discover that Li+ inhibits activation of nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes in the middle cerebral artery occlusion (MCAO) stroke model in mice. This action of Li+ is mediated by two signalling pathways of AKT/GSK3ß/ß-catenin and AKT/FoxO3a/ß-catenin which converge in suppressing the production of reactive oxygen species (ROS). Using immunocytochemstry, MRI imaging, and cell sorting with subsequent mRNA and protein quantification, we demonstrate that Li+ decreases the infarct volume, improves motor function, and alleviates associated cognitive and depressive impairments. In conclusion, this study reveals molecular mechanisms of Li+ neuroprotection during brain ischaemia, thus providing the theoretical background to extend clinical applications of Li+ for treatment of ischemic stroke.

Proteomic characterization of secretory granules in dopaminergic neurons indicates chromogranin/secretogranin-mediated protein processing impairment in Parkinson's disease.

Parkinson's disease (PD) is an aging disorder related to vesicle transport dysfunctions and neurotransmitter secretion. Secretory granules (SGs) are large dense-core vesicles for the biosynthesis of neuropeptides and hormones. At present, the involvement of SGs impairment in PD remains unclear. In the current study, we found that the number of SGs in tyrosine hydroxylase-positive neurons and the marker proteins secretogranin III (Scg3) significantly decreased in the substantia nigra and striatum regions of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) exposed mice. Proteomic study of SGs purified from the dopaminergic SH-sy5Y cells under 1-methyl-4-phenylpyridinium (MPP+) treatments (ProteomeXchange PXD023937) identified 536 significantly differentially expressed proteins. The result indicated that disabled lysosome and peroxisome, lipid and energy metabolism disorders are three characteristic features. Protein-protein interaction analysis of 56 secretory proteins and 140 secreted proteins suggested that the peptide processing mediated by chromogranin/secretogranin in SGs was remarkably compromised, accompanied by decreased candidate proteins and peptides neurosecretory protein (VGF), neuropeptide Y, apolipoprotein E, and an increased level of proenkephalin. The current study provided an extensive proteinogram of SGs in PD. It is helpful to understand the molecular mechanisms in the disease.

Association Analyses of SNAP25, HNMT, FCHSD1, and DBH Single-Nucleotide Polymorphisms with Parkinson's Disease in a Northern Chinese Population.

PURPOSE: Sequencing potentially causal and susceptible genes and genome-wide association studies in samples from Parkinson's disease (PD) patients has revealed several related loci. The genes for synaptosome-associated protein of 25 kDa (SNAP25), histamine-N-methyltransferase (HNMT), FCH and double SH3 domains 1 (FCHSD1) and dopamine ß-hydroxylase (DBH) are candidate loci and have not been studied in a northern Chinese population. We explored the genetic distribution of four single-nucleotide polymorphisms (rs3746544, rs11558538, rs456998, rs129882) located on SNAP25, HNMT, FCHSD1 and DBH, respectively. PATIENTS AND METHODS: A total of 330 patients with sporadic PD and 332 healthy controls (HCs) were recruited from a northern Chinese population. Polymerase chain reaction restriction fragment length polymorphism was used to genotype these four SNPs. RESULTS: After statistical analyses and correction of the genotyping results, the mutant-allele T in rs456998 of FCHSD1 was found to be significantly related to reducing the PD risk (P = 0.029, OR = 0.754, 95% CI = 0.586-0.971, power = 0.591). However, rs3746544, rs11558538, and rs129882 did not show an association with PD. CONCLUSION: FCHSD1 rs456998 may have a protective role in PD in a northern Chinese population, but more studies are needed to support this suggestion.

Iron induces two distinct Ca2+ signalling cascades in astrocytes.

Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe2+), whereas transferrin receptors (TFR) carry transferrin (TF)-bound ferric (Fe3+). In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Administration of Fe2+ or Fe3+ in µM concentrations evoked [Ca2+]i in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca2+]i through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibits astroglial Na+-K+-ATPase, which leads to elevation in cytoplasmic Na+ concentration, thus reversing Na+/Ca2+ exchanger and thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus triggering InsP3 receptor-mediated Ca2+ release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.

Iatrogenic Iron Promotes Neurodegeneration and Activates Self-Protection of Neural Cells against Exogenous Iron Attacks.

Metal implants are used worldwide, with millions of nails, plates, and fixtures grafted during orthopedic surgeries. Iron is the most common element of these metal implants. As time passes, implants can be corroded and iron can be released. Ionized iron permeates the surrounding tissues and enters circulation; importantly, iron ions pass through the blood-brain barrier. Can iron from implants represent a risk factor for neurological diseases? This remains an unanswered question. In this study, we discovered that patients with metal implants delivered through orthopedic surgeries have higher incidence of Parkinson's disease or ischemic stroke compared to patients who underwent similar surgeries but did not have implants. Concentration of serum iron and ferritin was increased in subjects with metal implants. In experiments in vivo, we found that injection of iron dextran selectively decreased the presence of divalent metal transporter 1 (DMT1) in neurons through increasing the expression of Ndfip1, which degrades DMT1 and does not exist in glial cells. At the same time, excess of iron increased expression of DMT1 in astrocytes and microglial cells and triggered reactive astrogliosis and microgliosis. Facing the attack of excess iron, glial cells act as neuroprotectors to accumulate more extracellular iron by upregulating DMT1, whereas neurons limit iron uptake through increasing DMT1 degradation. Cerebral accumulation of iron in animals is associated with impaired cognition, locomotion, and mood. Excess iron from surgical implants thus can affect neural cells and may be regarded as a risk factor for neurodegeneration.

Secretogranin III upregulation is involved in parkinsonian toxin-mediated astroglia activation.

Environmental neurotoxins such as paraquat (PQ), manganese, and 1-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are associated with a higher risk of Parkinson's disease (PD). These parkinsonian toxins exert certain common toxicological effects on astroglia; however, their role in the regulatory functions of astroglial secretory proteins remains unclear. In a previous study, we observed that secretogranin II (SCG2) and secretogranin III (SCG3), which are important components of the regulated secretory pathway, were elevated in PQ-activated U118 astroglia. In the current study, we used the parkinsonian toxins dopamine (DA), active metabolite of MPTP (MPP+), MnCl2, and lipopolysaccharide (LPS) as inducers, and studied the potential regulation of SCG2 and SCG3. Our results showed that all the parkinsonian toxins except LPS affected astroglial viability but did not cause apoptosis. Exposure to DA, MPP+, and MnCl2 upregulated glial fibrillary acidic protein (GFAP), a marker for astrocyte activation, and stimulated the levels of several astrocytic-derived factors. Further, DA, MPP+, and MnCl2 exposure impeded astroglial cell cycle progression. Moreover, the expression of SCG3 was elevated, while its exosecretion was inhibited in astroglia activated by parkinsonian toxins. The level of SCG2 remained unchanged. In combination with our previous findings, the results of this study indicate that SCG3 may act as a cofactor in astrocyte activation stimulated by various toxins, and the regulation of SCG3 could be involved in the toxicological mechanism by which parkinsonian toxins affect astroglia.

Long-term ketamine administration causes Tau protein phosphorylation and Tau protein-dependent AMPA receptor reduction in the hippocampus of mice.

As a recreational drug of abuse and an injectable anesthetic, ketamine has been shown to cause cognitive dysfunction and induce psychotic states. Although the specific mechanism is still unclear, it may be linked to synaptic receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. Recent evidence suggests that Tau protein phosphorylation and targeted delivery to the postsynaptic area is involved in maintaining neuronal plasticity, indicating that the neurotoxicity induced by ketamine may be related to the transfer of Tau protein after phosphorylation. In this study, we established a model of long-term (6 months) ketamine administration in wild-type (C57BL/6) and Tau knockout mice to investigate the effects of different doses of ketamine administration on Tau protein expression and phosphorylation in the mouse hippocampus. We also investigated changes in AMPA receptor expression in the synaptic membrane of wild-type and Tau knockout mice. Our results showed that long-term ketamine administration led to excessive Tau protein phosphorylation at Ser202/Thr205 and Ser396, but not at Ser199, Ser262 and Ser404. Most importantly, long-term ketamine administration decreased AMPA receptor levels in the hippocampal cell membrane in a Tau protein-dependent manner. Our results reveal the role of Tau protein phosphorylation in the mechanism of ketamine neurotoxicity, suggesting that the changes of membrane AMPA receptor and synaptic function induced by ketamine are mediated by abnormal phosphorylation of Tau protein at specific sites.

Regulation of Tau Protein on the Antidepressant Effects of Ketamine in the Chronic Unpredictable Mild Stress Model.

Tau protein is known to play an important role in maintaining microtubule assembly and stabilization, and maintaining the normal morphology of neurons, but several studies have found that chronic stress leads to Tau hyperphosphorylation. A large number of clinical trials have found that ketamine, which is an N-methyl-D-aspartate receptor antagonist, produces a rapid, long-lasting, and potent antidepressant effect in patients suffering from major depression. This rapid antidepressant effect of ketamine, which involves many mechanisms, has attracted wide attention. However, the relationship between ketamine's antidepressant effects and Tau protein has rarely been examined. We used C57BL/6 and Tau KO mice exposed to 42 days of chronic unpredictable mild stress (the CUMS model) to investigate the effect of ketamine on behavioral changes and synaptic functioning of the hippocampus. The results showed that a single treatment of ketamine rapidly relieved the CUMS-induced anhedonia, depression-like, and anxious behaviors of the C57BL/6 mice. The abnormal behaviors were accompanied by increased levels of specific alterations of hyperphosphorylated Tau protein in cytoplasm and synapse in the hippocampus of the C57BL/6 mice, but ketamine reduced the aggregation of hyperphosphorylated Tau protein only in the synapse. We also found that CUMS exposure reduced the levels of GluA1 and PSD95 in the hippocampus of the C57BL/6 mice and that these deficits were reversed by ketamine. However, the Tau KO mice did not develop any stress-induced depressive behaviors or deficits of hippocampal function. The antidepressant effect of ketamine may decrease the levels of hyperphosphorylated Tau protein in synapse of C57BL/6 mice.

Secretogranin III may be an indicator of paraquat-induced astrocyte activation and affects the recruitment of BDNF during this process.

Astrocyte activation has been described as a multi­stage defensive response, which is characterized by the morphological alteration of astrocytes and the overexpression of intermediate filament proteins. However, the functional mechanism of the secretion system in activated astroglia remains unclear. It has previously been demonstrated that secretogranin II, a member of the granin family, may be involved in the sorting and expression of inflammatory factors and excitatory neurotransmitters in paraquat (PQ)­induced astroglial activation. Secretogranin III (SCG3) has been reported to represent an important component of the regulated secretory pathway in neuroendocrine cells; however, its role as an anchor protein of dense­core vesicles in astrocytes remains to be elucidated. In the present study, a PQ­activated U118MG astrocytoma cell model established in our previous study was used to investigate the effects of SCG3. The results revealed that SCG3 was highly expressed and subsequently released from cells in response to PQ. Inhibition of SCG3 expression via transfection with small interfering RNA partially restored astrocyte morphology, but did not affect the expression of astrocytic factors. Further studies investigating the association between SCG3 and other cellular factors were conducted, in order to determine the expression levels and subcellular localization of these proteins. Neurotrophins and inflammatory factors exhibited an increase in characteristic expression patterns, paralleling the alterations in SCG3 expression. The results further demonstrated that brain­derived neurotrophic factor partially colocalized with SCG3­positive vesicles; however, the localization of interleukin­6 was not affected. In conclusion, SCG3 may be involved in PQ­induced astrocyte activation via regulation of the expression and selective recruitment of cellular factors, thus suggesting that SCG3 may represent an indicator of astrocyte activation.

Forensic characterization of 15 autosomal STRs in four populations from Xinjiang, China, and genetic relationships with neighboring populations.

The Xinjiang Uyghur Autonomous Region of China (XUARC) harbors 47 ethnic groups including the Manchu (MCH: 0.11%), Mongols (MGL: 0.81%), Kyrgyz (KGZ: 0.86%) and Uzbek (UZK: 0.066%). To establish DNA databases for these populations, allele frequency distributions for 15 autosomal short tandem repeat (STR) loci were determined using the AmpFlSTR Identifiler PCR amplification kit. There was no evidence of departures from Hardy-Weinberg equilibrium (HWE) in any of the four populations and minimal departure from linkage equilibrium (LE) for a very small number of pairwise combinations of loci. The probabilities of identity for the different populations ranged from 1 in 1.51 × 1017 (MCH) to 1 in 9.94 × 1018 (MGL), the combined powers of discrimination ranged from 0.99999999999999999824 (UZK) to 0.9999999999999999848 (MCH) and the combined probabilities of paternal exclusion ranged from 0.9999979323 (UZK) to 0.9999994839 (MCH). Genetic distances, a phylogenetic tree and principal component analysis (PCA) revealed that the MCH, KGZ and UZK are genetically closer to the Han population of Liaoning and the Mongol population of Mongolia while the MGL are closer to Han, Japanese, Korean, Malaysian, Hong Kong Han and Russians living in China.

Population data and phylogenetic structure of Han population from Jiangsu province of China on GlobalFiler STR loci.

Forensic statistical parameters based on allelic frequencies of commonly used short tandem repeats were estimated for the Han population of Jiangsu province from P.R. China. The 6-dye GlobalFiler™ PCR amplification kit incorporates 21 autosomal STRs, providing reliable DNA typing results with enhanced the power of discrimination. Here, we analyzed the GlobalFiler™ STR loci in 516 unrelated individuals from Jiangsu Han population. A total of 256 alleles were observed ranging between 5 and 35.2 repeat units, and SE33 showed the greatest power of discrimination (34 alleles) in Jiangsu Han population. Most of the loci were found to be in the Hardy-Weinberg equilibrium after the Bonferroni correction with the exception of D3S1358. The combined power of exclusion (CPE) was 0.999999996353609, and the combined match probability (CMP) was 3.64 × 10-25. Phylogenetic parameters including pairwise genetic distances showed that Han population living in Jiangsu had closest genetic relationship with other East Asian populations. The present study provides precise reference database for forensic applications and population genetic studies.

Effects of PQ's cytotoxicity on secretory vesicles in astroglia: Expression alternation of secretogranin II and its potential interaction with intracellular factors.

It has been extensively characterized that paraquat (PQ) selectively targets to the substantia nigra and exerts neurotoxic actions on dopaminergic neurons. However, a little knowledge is available about astroglia in PQ exposure, especially its complex secretory machinery. To explore this point, we built up a PQ-induced model in cultural U118 astrocyte. Since the granin family is considered as a master regulator of cargo sorting and large dense core vesicles (LDCVs) biogenesis in the regulated secretory pathway of nervous and neuroendocrine cells, the current study focused on one member, secretogranin II (SCG2) and investigated its alternation and potential relationship with other astrocyte-derived factors under PQ insult. We found that PQ upregulated SCG2 expression on both RNA and protein levels and stimulated the mRNA expression of neurotrophic factors, cytokines and glutamine synthetase (GS) simultaneously. RNAi knockdown of SCG2 did not rescue the cell cycle arrest induced by PQ but affected expressions of IL-6 and GS on mRNA and protein levels. Further studies on subcellular location showed that SCG2-positive secretory granules were partially colocalized with IL-6 but not GS in PQ exposure astrocyte. Taken together, our findings indicate that the expression alternation of SCG2 under astroglial activation by PQ may be necessary compensation for cargo sorting and LDCV biogenesis. The involvement of the IL-6 and GS suggests that the SCG2 may potentially regulate inflammatory factors and excitatory neurotransmitter to the cytotoxicity of PQ on astroglia.

Genetic analysis of indel markers in three loci associated with Parkinson's disease.

The causal mutations and genetic polymorphisms associated with susceptibility to Parkinson's disease (PD) have been extensively described. To explore the potential contribution of insertion (I)/deletion (D) polymorphisms (indels) to the risk of PD in a Chinese population, we performed genetic analyses of indel loci in ACE, DJ-1, and GIGYF2 genes. Genomic DNA was extracted from venous blood of 348 PD patients and 325 age- and sex-matched controls without neurodegenerative disease. Genotyping of the indel loci was performed by fragment length analysis after PCR and DNA sequencing. Our results showed a statistically significant association for both allele X (alleles without 5) vs. 5 (odds ratio = 1.378, 95% confidence interval = 1.112-1.708, P = 0.003) and genotype 5/X+X/X vs. 5/5 (odds ratio = 1.681, 95% confidence interval = 1.174-2.407, P = 0.004) in the GIGYF2 locus; however, no significant differences were detected for the ACE and DJ-1 indels. After stratification by gender, no significant differences were observed in any indels. These results indicate that the GIGYF2 indel may be associated with increased risk of PD in northern China.

Clathrin-Dependent Uptake of Paraquat into SH-SY5Y Cells and Its Internalization into Different Subcellular Compartments.

The herbicide paraquat (PQ) is an exogenous toxin that allows the selective activation of dopaminergic neurons in the mesencephalon to induce injury and also causes its apoptosis in vitro. However, uptake mechanisms between PQ and neurons remain elusive. To address this issue, we undertook a study of PQ endocytosis in a dopaminergic SH-SY5Y cell line as well as explored the subsequent subcellular location and potential functional analysis of PQ. The PQ was found to bind the SH-SY5Y cell membrane and then became internalized via a clathrin-dependent pathway. PQ was internalized by many subcellular organelles in a time- and dose-dependent manner. Interestingly, the taken up PQ and secretogranin III (SCG3), which became dysregulated with PQ treatment that induced SH-SY5Y apoptosis in our previous study, colocalized in cytoplasmic vesicles. Taken together, our findings indicate that PQ is endocytosed by SH-SY5Y cells and that its multiple, subcellular localizations indicate PQ may potentially be involved in subcellular-level functions. More importantly, PQ distributing preferentially into SCG3-positive vesicles demonstrates its selective targeting which may affect SCG3 and cargoes carried by SCG3-positive vesicles. Therefore, it is reasonable to infer that PQ toxic insults may potentially interfere with neurotransmitter storage and transport associated with secretory granules.

Female genetic distribution bias in mitochondrial genome observed in Parkinson's Disease patients in northern China.

Genetic polymorphisms associated with susceptibility to Parkinson's disease (PD) have been described in mitochondrial DNA (mtDNA). To explore the potential contribution of mtDNA mutations to the risk of PD in a Chinese population, we examined the linkage relationship between several single nucleotide polymorphisms (SNPs) and haplotypes in mtDNA and PD. We genotyped 5 SNPs located on coding genes using PCR-RFLP analysis. A specific allele 10398G demonstrated an increased risk of PD (OR 1.30; 95% CI 0.95-1.76; P = 0.013). After stratification by gender, the increased risk appeared to be more significant in females (OR 1.91; 95% CI 1.16-3.16; P = 0.001). But the significance only appeared in females under Bonferroni correction. No significant differences were detected for other SNPs (T4336C, G5460A, G9055A, and G13708A). Individual haplotype composed of 4336T-5460G-9055G-10398A-13708G was found to be associated with protective effect regarding PD (P = 0.0025). The haplotypes 4336T-5460G-9055G-10398G-13708G and 4336T-5460G-9055G-10398A-13708G were more significantly associated in females (P = 0.0036 for risk and P = 0.0006 for protective effects). These data suggest that the A10398G and two haplotypes coupled with 10398A or 10398G are closely associated with susceptibility to PD in a northern Chinese population. This association demonstrated a female genetic distribution bias.