Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA₁ receptor knockout mice

Background

The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory.

Methodology/Principal Findings

Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice.

Conclusions/Significance

These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.     

Effect of Ca2EDTA on Zinc Mediated Inflammation and Neuronal Apoptosis in Hippocampus of an In Vivo Mouse Model of Hypobaric Hypoxia

Background

Calcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage.

Methods

Adult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca₂EDTA (1.25 mM/kg i.p) daily for four days. The effects of Ca₂EDTA on apoptosis (caspases activity and DNA fragmentation), pro-inflammatory markers (iNOS, TNF-α and COX-2), NADPH oxidase activity, poly(ADP ribose) polymerase (PARP) activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain.

Results

Hypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6). Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca₂EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus.

Conclusion

We propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other neurodegenerative conditions can be designed in future.     

Pulmonary Toxicity in Mice Following Exposure to Cerium Chloride

The widespread application of lanthanoids (Lns) in manufacturing industries has raised occupational and environmental health concerns about the possible increased health risks to humans exposed to Lns in their working and living environments. Numerous studies have shown that exposures to Ln cause pulmonary injury in animals, but very little is known about the molecular mechanisms of the pulmonary inflammation caused by cerium chloride (CeCl₃) exposure. In this study, we evaluated the oxidative stress and molecular mechanism underlying with the pulmonary inflammation associated with chronic lung toxicity in mice treated with nasally instilled CeCl₃ for 90 consecutive days. Our findings suggest that significant cerium accumulated in the lung, leading the obvious increase of the lung indices, significant increases in inflammatory cells and levels of lactate dehydrogenase, alkaline phosphate, and total protein, overproduction of reactive oxygen species and peroxidation of lipids, reduced antioxidant capacity, and pulmonary inflammation. CeCl₃ exposure also activated nuclear factor κB, increased the expression of tumor necrosis factor α, cyclooxygenase-2, heme oxygenase 1, interleukin 2, interleukin 4, interleukin 6, interleukin 8, interleukin 10, interleukin 18, interleukin 1β, and CYP1A1. However, CeCl₃ reduced the expression of nuclear factor κB (NF-κB)-inhibiting factor and heat shock protein 70. These findings suggest that the pulmonary inflammation caused by CeCl₃ in mice is closely associated with oxidative stress and inflammatory cytokine expression.     

Fat-1 expression enhance hippocampal memory in scopolamine-induced amnesia

Omega-3 polyunsaturated fatty acids (PUFA) are critical for optimal brain health and are involved in psychiatric and neurological ailments. Here, we report the effects of higher endogenous omega-3 PUFA on memory impairment in the hippocampus by studying mice with transgenic expression of the fat-1 gene that converts omega-6 to omega-3 PUFA. We performed Y-maze and passive avoidance tests to evaluate the memory function of fat-1 mice treated with scopolamine. Fat-1 mice showed induced alternation in the Y-maze test and increased latency in the passive avoidance test. The effects of scopolamine on hippocampal neurogenesis were confirmed by increases in the number of Ki-67- and DCX-positive cells in the fat-1 mice. Western blotting revealed increased brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein levels, and lower scopolamine-induced apoptosis based on the cleaved-caspase 3 protein level in fat-1 mice. These findings suggest that higher endogenous omega-3 PUFA prevented granular cell loss, increased BDNF signaling, and decreased apoptosis signaling in scopolamine-treated fat-1 mice. These processes may underlie granular cell survival and suggest potential therapeutic targets for memory impairment.     

The Impairment of Liver DNA Conformation and Liver Apoptosis of Mice Caused by CeCl3

Cerium (Ce) was shown to cause various toxic effects both in rats and mice; however, the molecular mechanism by which Ce exert theirs toxicity is still understood. In this report, the impairment of liver DNA conformation and liver apoptosis of mice caused by CeCl₃ was studied in vivo using inductively coupled plasma–mass spectrometry, various spectral methods, gel electrophoresis, and transmission electron micrograph. We found that the coefficients of liver to body weight of the mice treated with CeCl₃ were significantly increased. Ce³⁺ could be significantly accumulated in the liver, and it insert itself into DNA base pairs and/or bind to DNA nucleotide, and alter the conformation of DNA. Furthermore, the evaluation by gel electrophoresis and transmission electron micrograph showed that higher dose of Ce³⁺ could cause DNA cleavage and hepatocyte apoptosis in mice. Therefore, our study aroused the attention of Ce application and exposure effects especially on human liver for long-term and low-dose treatment.     

Mapping of adenylyl cyclase genes type I,II, III,IV, V,and VI in mouse

The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I–VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes.     

Involvement of bcl-family expression in the spatial memory impairment induced by repeated ischemia

In the present study, we examined the effects of repeated ischemia (10 min x 2, 1 hr interval) on spatial memory in rats in an 8-arm radial maze test compared with single ischemia (10 min x 1). Repeated ischemia produced more severe impairment of spatial memory and stronger TUNEL-positive immunoreactivity in the hippocampal CA1 region than single ischemia at 7 days after reperfusion. Moreover, repeated ischemia altered bcl-family expression, which is related to apoptosis, while this was not affected by single ischemia. These results suggest that spatial memory impairment at 7 days after repeated ischemia may be related to apoptosis in hippocampal CA1 cells.     

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

Age-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α₁-AR antagonist, is reported to have access to the brain and interact with α₁-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.     

Assembly of different length of polyubiquitins on the catalytic cysteine of E2 enzymes without E3 ligase; a novel application of non-reduced/reduced 2-dimensional electrophoresis

In this study using non-reduced/reduced 2-dimensional electrophoresis (NR/R-2DE), we clearly demonstrated that E3-independent ubiquitination by Ube2K produced not only unanchored but also Ube2K-linked polyubiquitins through thioester and isopeptide bonds. E3-independent assembly of polyubiquitins on the catalytic cysteine of Ube2K strongly supports the possibility of ‘en bloc transfer’ for polyubiquitination. From the same analyses of E3-independent ubiquitination products by other E2s, we also found that different lengths of polyubiquitins were linked to different E2s through thioester bond; longer chains by Cdc34 like Ube2K, short chains by Ube2g2, and mono-ubiquitin by UbcH10. Our results suggest that E2s possess the different intrinsic catalytic activities for polyubiquitination.     

Toward a Broader View of Ube3a in a Mouse Model of Angelman Syndrome: Expression in Brain,Spinal Cord,Sciatic Nerve and Glial Cells

Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted Ube3a gene. Ube3a is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of Ube3a in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.     

Ginsenoside Rg1 Decreases Aβ1–42 Level by Upregulating PPARγ and IDE Expression in the Hippocampus of a Rat Model of Alzheimer's Disease

Background and Purpose

The present study was designed to examine the effects of ginsenoside Rg1 on expression of peroxisome proliferator-activated receptor γ (PPARγ) and insulin-degrading enzyme (IDE) in the hippocampus of rat model of Alzheimer''s disease (AD) to determine how ginsenoside Rg1 (Rg1) decreases Aβ levels in AD.

Experimental Approach

Experimental AD was induced in rats by a bilateral injection of 10 µg soluble beta-amyloid peptide 1–42 (Aβ_1–42) into the CA1 region of the hippocampus, and the rats were treated with Rg1 (10 mg·kg⁻¹, intraperitoneally) for 28 days. The Morris water maze was used to test spatial learning and memory performance. Hematoxylin-eosin staining was performed to analyze the hippocampal histopathological damage. Immunohistochemistry, western blotting, and real-time PCR were used to detect Aβ_1–42, PPARγ, and insulin-degrading enzyme (IDE) expression in the hippocampus.

Key Results

Injection of soluble Aβ_1–42 into the hippocampus led to significant dysfunction of learning and memory, hippocampal histopathological abnormalities and increased Aβ_1–42 levels in the hippocampus. Rg1 treatment significantly improved learning and memory function, attenuated hippocampal histopathological abnormalities, reduced Aβ_1–42 levels and increased PPARγ and IDE expression in the hippocampus; these effects of Rg1 could be effectively inhibited by GW9662, a PPARγ antagonist.

Conclusions and Implications

Given that PPARγ can upregulate IDE expression and IDE can degrade Aβ_1–42, these results indicate that Rg1 can increase IDE expression in the hippocampus by upregulating PPARγ, leading to decreased Aβ levels, attenuated hippocampal histopathological abnormalities and improved learning and memory in a rat model of AD.     

Sevoflurane Induces Endoplasmic Reticulum Stress Mediated Apoptosis in Hippocampal Neurons of Aging Rats

Elderly patients are more likely to suffer from postoperative memory impairment for volatile anesthetics could induce aging neurons degeneration and apoptosis while the mechanism was still elusive. Therefore we hypothesized that ER stress mediated hippocampal neurons apoptosis might play an important role in the mechanism of sevoflurane-induced cognitive impairment in aged rats. Thirty 18-month-old male Sprague-Dawley rats were divided into two groups: the sham anesthesia group (exposure to simply humidified 30–50% O₂ balanced by N₂ in an acrylic anesthetizing chamber for 5 hours) and the sevoflurane anesthesia group (received 2% sevoflurane in the same humidified mixed air in an identical chamber for the same time). Spatial memory of rats was assayed by the Morris water maze test. The ultrastructure of the hippocampus was observed by transmission electron microscopy (TEM). The expressions of C/EBP homologous protein (CHOP) and caspase-12 in the hippocampus were observed by immunohistochemistry and real-time PCR analysis. The apoptosis neurons were also assessed by TUNEL assay. The Morris water maze test showed that sevoflurane anesthesia induced spatial memory impairment in aging rats (P<0.05). The apoptotic neurons were condensed and had clumped chromatin with fragmentation of the nuclear membrane, verifying apoptotic degeneration in the sevoflurane group rats by TEM observation. The expressions of CHOP and caspase-12 increased, and the number of TUNEL positive cells of the hippocampus also increased in the sevoflurane group rats (P<0.05). The present results suggested that the long time exposure of sevoflurane could induce neuronal degeneration and cognitive impairment in aging rats. The ER stress mediated neurons apoptosis may play a role in the sevoflurane-induced memory impairment in aging rats.     

Impaired Interleukin-1β and c-Fos Expression in the Hippocampus Is Associated with a Spatial Memory Deficit in P2X7 Receptor-Deficient Mice

Recent evidence suggests that interleukin-1β (IL-1β), which was originally identified as a proinflammatory cytokine, is also required in the brain for memory processes. We have previously shown that IL-1β synthesis in the hippocampus is dependent on P2X₇ receptor (P2X₇R), which is an ionotropic receptor of ATP. To substantiate the role of P2X₇R in both brain IL-1β expression and memory processes, we examined the induction of IL-1β mRNA expression in the hippocampus of wild-type (WT) and homozygous P2X₇ receptor knockout mice (P2X₇R^−/−) following a spatial memory task. The spatial recognition task induced both IL-1β mRNA expression and c-Fos protein activation in the hippocampus of WT but not of P2X₇R^−/− mice. Remarkably, P2X₇R^−/− mice displayed spatial memory impairment in a hippocampal-dependant task, while their performances in an object recognition task were unaltered. Taken together, our results show that P2X₇R plays a critical role in spatial memory processes and the associated hippocampal IL-1β mRNA synthesis and c-Fos activation.     

Methamphetamine‐induced changes in the mice hippocampal neuropeptide Y system: implications for memory impairment

Methamphetamine (METH) is a psychostimulant drug that causes irreversible brain damage leading to several neurological and psychiatric abnormalities, including cognitive deficits. Neuropeptide Y (NPY) is abundant in the mammalian central nervous system (CNS) and has several important functions, being involved in learning and memory processing. It has been demonstrated that METH induces significant alteration in mice striatal NPY, Y₁ and Y₂ receptor mRNA levels. However, the impact of this drug on the hippocampal NPY system and its consequences remain unknown. Thus, in this study, we investigated the effect of METH intoxication on mouse hippocampal NPY levels, NPY receptors function, and memory performance. Results show that METH increased NPY, Y₂ and Y₅ receptor mRNA levels, as well as total NPY binding accounted by opposite up‐ and down‐regulation of Y₂ and Y₁ functional binding, respectively. Moreover, METH‐induced impairment in memory performance and AKT/mammalian target of rapamycin pathway were both prevented by the Y₂ receptor antagonist, BIIE0246. These findings demonstrate that METH interferes with the hippocampal NPY system, which seems to be associated with memory failure. Overall, we concluded that Y₂ receptors are involved in memory deficits induced by METH intoxication.     

Cdc42 signaling regulated by dopamine D2 receptor correlatively links specific brain regions of hippocampus to cocaine addiction

BackgroundHippocampus plays critical roles in drug addiction. Cocaine-induced modifications in dopamine receptor function and the downstream signaling are important regulation mechanisms in cocaine addiction. Rac regulates actin filament accumulation while Cdc42 stimulates the formation of filopodia and neurite outgrowth. Based on the region specific roles of small GTPases in brain, we focused on the hippocampal subregions to detect the regulation of Cdc42 signaling in long-term morphological and behavioral adaptations to cocaine.MethodsGenetically modified mouse models of Cdc42, dopamine receptor D1 (D1R) and D2 (D2R) and expressed Cdc42 point mutants that are defective in binding to and activation of its downstream effector molecules PAK and N-WASP were generated, respectively, in CA1 or dentate gyrus (DG) subregion.ResultsCocaine induced upregulation of Cdc42 signaling activity. Cdc42 knockout or mutants blocked cocaine-induced increase in spine plasticity in hippocampal CA1 pyramidal neurons, leading to a decreased conditional place preference (CPP)-associated memories and spatial learning and memory in water maze. Cdc42 knockout or mutants promoted cocaine-induced loss of neurogenesis in DG, leading to a decreased CPP-associated memories and spatial learning and memory in water maze. Furthermore, by using D1R knockout, D2R knockout, and D2R/Cdc42 double knockout mice, we found that D2R, but not D1R, regulated Cdc42 signaling in cocaine-induced neural plasticity and behavioral changes.ConclusionsCdc42 acts downstream of D2R in the hippocampus and plays an important role in cocaine-induced neural plasticity through N-WASP and PAK-LIMK-Cofilin, and Cdc42 signaling pathway correlatively links specific brain regions (CA1, dentate gyrus) to cocaine-induced CPP behavior.     

The role of ubiquitin-conjugating enzyme Ube2j1 phosphorylation and its degradation by proteasome during endoplasmic stress recovery

The human Ube2j1 and Ube2j2 are the only ubiquitin-conjugating enzymes (E2s) that are localized to endoplasmic reticulum (ER) through its C-terminal transmembrane domains. Ube2j1 is a known substrate of MAPK signalling pathway and it is phosphorylated at serine-184 during ER stress. Here, we demonstrate that Ube2j1, not Ube2j2 is essential for the recovery of cells from transient ER stress. The ectopic expression of wild-type Ube2j1 and phospho-mimic mutant, Ube2j1^S184D but not phospho-mutant Ube2j1^S184A can recover cells from ER stress. We also found that ubiquitin-ligase (E3), c-IAP1 preferentially interacts with phosphorylated Ube2j1. Moreover, we noticed that phosphorylated Ube2j1 is rapidly degraded by the proteasome during ER stress cell recovery. Taken together, these data suggest that Ube2j1 and its phosphorylation is important for transient ER stress cell recovery and the phosphorylated Ube2j1 is degraded by the proteasome.