Intranasal Administration of Apelin-13 Ameliorates Cognitive Deficit in Streptozotocin-Induced Alzheimer's Disease Model via Enhancement of Nrf2-HO1 Pathways.

BACKGROUND: The discovery of novel diagnostic methods and therapies for Alzheimer's disease (AD) faces significant challenges. Previous research has shed light on the neuroprotective properties of Apelin-13 in neurodegenerative disorders. However, elucidating the mechanism underlying its efficacy in combating AD-related nerve injury is imperative. In this study, we aimed to investigate Apelin-13's mechanism of action in an in vivo model of AD induced by streptozocin (STZ). METHODS: We utilized an STZ-induced nerve injury model of AD in mice to investigate the effects of Apelin-13 administration. Apelin-13 was administered intranasally, and cognitive impairment was assessed using standardized behavioral tests, primarily, behavioral assessment, histological analysis, and biochemical assays, in order to evaluate synaptic plasticity and oxidative stress signaling pathways. RESULTS: Our findings indicate that intranasal administration of Apelin-13 ameliorated cognitive impairment in the STZ-induced AD model. Furthermore, we observed that this effect was potentially mediated by the enhancement of synaptic plasticity and the attenuation of oxidative stress signaling pathways. CONCLUSIONS: The results of this study suggest that intranasal administration of Apelin-13 holds promise as a therapeutic strategy for preventing neurodegenerative diseases such as AD. By improving synaptic plasticity and mitigating oxidative stress, Apelin-13 may offer a novel approach to neuroprotection in AD and related conditions.

SRSF10 regulates proliferation of neural progenitor cells and affects neurogenesis in developing mouse neocortex.

Alternative pre-mRNA splicing plays critical roles in brain development. SRSF10 is a splicing factor highly expressed in central nervous system and plays important roles in maintaining normal brain functions. However, its role in neural development is unclear. In this study, by conditional depleting SRSF10 in neural progenitor cells (NPCs) in vivo and in vitro, we found that dysfunction of SRSF10 leads to developmental defects of the brain, which manifest as abnormal ventricle enlargement and cortical thinning anatomically, as well as decreased NPCs proliferation and weakened cortical neurogenesis histologically. Furthermore, we proved that the function of SRSF10 on NPCs proliferation involved the regulation of PI3K-AKT-mTOR-CCND2 pathway and the alternative splicing of Nasp, a gene encoding isoforms of cell cycle regulators. These findings highlight the necessity of SRSF10 in the formation of a structurally and functionally normal brain.

The identities of insulin signaling pathway are affected by overexpression of Tau and its phosphorylation form.

Introduction: Hyperphosphorylated Tau formed neurofibrillary tangles was one of the major neuropathological hallmarks of Alzheimer's disease (AD). Dysfunctional insulin signaling in brain is involved in AD. However, the effect of Tau pathology on brain insulin resistance remains unclear. This study explored the effects of overexpressing wild-type Tau (WTau) or Tau with pseudo-phosphorylation at AT8 residues (PTau) on the insulin signaling pathway (ISP). Methods: 293T cells or SY5Y cells overexpressing WTau or PTau were treated with or without insulin. The elements in ISP or the regulators of IPS were analyzed by immunoblotting, immunofluorescent staining and co-immunoprecipitation. Akt inhibitor MK2206 was used for evaluating the insulin signaling to downstream of mTOR in Tau overexpressing cells. The effects of anti-aging drug lonafarnib on ISP in WTau or PTau cells were also analyzed with immunoblotting. Considering lonafarnib is an inhibitor of FTase, the states of Rhes, one of FTase substrate in WTau or PTau cells were analyzed by drug affinity responsive target stability (DARTS) assay and the cellular thermal shift assay (CETSA). Results: WTau or PTau overexpression in cells upregulated basal activity of elements in ISP in general. However, overexpression of WTau or PTau suppressed the ISP signaling transmission responses induced by insulin simulation, appearing relative higher response of IRS-1 phosphorylation at tyrosine 612 (IRS-1 p612) in upstream IPS, but a lower phosphorylation response of downstream IPS including mTOR, and its targets 4EPB1 and S6. This dysregulation of insulin evoked signaling transmission was more obvious in PTau cells. Suppressing Akt with MK2206 could compromise the levels of p-S6 and p-mTOR in WTau or PTau cells. Moreover, the changes of phosphatases detected in WTau and PTau cells may be related to ISP dysfunction. In addition, the effects of lonafarnib on the ISP in SY5Y cells with WTau and PTau overexpression were tested, which showed that lonafarnib treatment resulted in reducing the active levels of ISP elements in PTau cells but not in WTau cells. The differential effects are probably due to Tau phosphorylation modulating lonafarnib-induced alterations in Rhes, as revealed by DARTS assay. Conclusion and discussion: Overexpression of Tau or Tau with pseudo-phosphorylation at AT8 residues could cause an upregulation of the basal/tonic ISP, but a suppression of insulin induced the phasic activation of ISP. This dysfunction of ISP was more obvious in cells overexpressing pseudo-phosphorylated Tau. These results implied that the dysfunction of ISP caused by Tau overexpression might impair the physiological fluctuation of neuronal functions in AD. The different effects of lonafarnib on ISP between WTau and PTau cells, indicating that Tau phosphorylation mediates an additional effect on ISP. This study provided a potential linkage of abnormal expression and phosphorylation of Tau to the ISP dysfunction in AD.

Slit3 secreted from M2-like macrophages increases sympathetic activity and thermogenesis in adipose tissue.

Beiging of white adipose tissue (WAT) is associated with an increase of anti-inflammatory M2-like macrophages in WAT. However, mechanisms through which M2-like macrophages affect beiging are incompletely understood. Here, we show that the macrophage cytokine Slit3 is secreted by adipose tissue macrophages and promotes cold adaptation by stimulating sympathetic innervation and thermogenesis in mice. Analysing the transcriptome of M2-like macrophages in murine inguinal WAT (iWAT) after cold exposure, we identify Slit3 as a secreted cytokine. Slit3 binds to the ROBO1 receptor on sympathetic neurons to stimulate Ca2+/calmodulin-dependent protein kinase II signalling and norepinephrine release, which enhances adipocyte thermogenesis. Adoptive transfer of Slit3-overexpressing M2 macrophages to iWAT promotes beiging and thermogenesis, whereas mice that lack Slit3 in myeloid cells are cold-intolerant and gain more weight. Our findings shed new light on the integral role of M2-like macrophages for adipose tissue homeostasis and uncover the macrophage-Slit3-sympathetic neuron-adipocyte signalling axis as a regulator of long-term cold adaptation.

Interaction between extracellular ATP5A1 and LPS alleviates LPS-induced neuroinflammation in mice.

Neuroinflammation is one of the main causes of Alzheimer's disease (AD). The presence of Lipopolysaccharide (LPS) in senile plaques (SP) of AD suggests that it plays a role in AD pathogenesis. ATP5A1 (F1F0-ATP synthase F1 α subunit) is abundant in SP. Further, the protein has recently been found to have an anti-infection role in zebrafish embryos. In the present study, we observed that LPS levels were higher in the brains of APP/PS1 mice than in control mice, and LPS co-localised with ATP5A1 in amyloid plaques. The interaction of recombinant ATP5A1(rATP5A1) and LPS was evidenced by cellular thermal shift assay and enzyme-linked immunosorbent assay-based binding assay in vitro. Neuroinflammation in the brain of a mouse model was induced by intracerebroventricular injection of LPS. The addition of rATP5A1 relieved LPS-induced reduction of spontaneous locomotor ability, depressive-like behaviour, and working memory impairment. Furthermore, rATP5A1 suppressed the activation of astrocytes and microglia, IL-1ß accumulation, and tau phosphorylation induced by LPS. Taken together, findings suggest that ATP5A1 is involved in the regulation of LPS-mediated neuroinflammation in AD.

NRSF deficiency leads to abnormal postnatal development of dentate gyrus and impairment of progenitors in subgranular zone of hippocampus.

Neuron-restrictive silencing factor (NRSF) is a zinc-finger transcription factor that regulates expression of a diverse set of genes. However, NRSF function in brain development still remains elusive. In the present study, we generated NRSF-conditional knockout (NRSF-cKO) mice by hGFAP-Cre/loxp system to study the effect of NRSF deficiency on brain development. Results showed that NRSF conditional knockout caused a smaller hippocampus and a thinner granule cell layer (GCL) in mice. Moreover, the reduction and disarrangement of GFAP+ cells in subgranular zone (SGZ) of NRSF-cKO mice was accompanied with the decreased number of premature neurons, neural stem cells (NSCs) and neural progenitor cells (NPCs), as well as compromising the majority of mitotically active cells. The analysis of postnatal development of hippocampus indicated the existence of an abnormality at postnatal day (P) 8, rather than at P1, in NRSF-cKO mice, although the densities of Ki67+ cells with mitotic ability in dentate gyrus were relatively unaffected at P1 and P8. Meanwhile, NRSF deficiency led to abnormal organization of SGZ at P8 during postnatal development. RNA-Seq analysis revealed 79 deregulated genes in hippocampus of NRSF-cKO mice at P8, which were involved in p53 signal transduction, neuron migration and negative regulation of cell proliferation, etc. The deregulation of p53 pathway in NRSF-cKO mice at P1 and P8 was evidenced, of which p21/Cdkn1a was accumulated in a portion of NSCs and NPCs in hippocampus during postnatal development. Together, these results, for the first time, revealed that NRSF could significantly influence the postnatal development of hippocampus, especially the formation of SGZ.

Involvement of RhoA/ROCK Signaling in Aß-Induced Chemotaxis, Cytotoxicity and Inflammatory Response of Microglial BV2 Cells.

Reactive microglia clustering around amyloid plaques in brain is a histopathological feature of Alzheimer's disease (AD) and reflects the contribution of neuroinflammation in AD pathogenesis. ß-Amyloid peptide (Aß) has been shown to induce a range of microglial responses including chemotaxis, cytotoxicity and inflammation, but the underlying mechanism is poorly understood. Considering the fundamental role of RhoA/ROCK signaling in cell migration and its broad implication in AD and neuroinflammation, we hypothesized that RhoA/ROCK signaling might be involved in Aß-induced microglial responses. From in vivo mouse models including APP/PS1 transgene and fibrillar Aß stereotactic injection, we observed the elevated expression level of RhoA in reactive microglia. Through a series in vitro cell migration, cytotoxicity and biochemistry assays, we found that RhoA/ROCK signaling plays an essential role in Aß-induced responses of microglial BV2 cells. Small molecular agents Fasudil and Y27632 showed prominent beneficial effects, which implies the therapeutic potential of RhoA/ROCK signaling inhibitors in AD treatment.

Memantine Differentially Regulates Tau Phosphorylation Induced by Chronic Restraint Stress of Varying Duration in Mice.

Exposure to chronic psychiatric stress has been linked to Alzheimer's disease-related tau hyperphosphorylation and abnormalities in glutamate neurotransmission. However, the pathological relationship between glutamatergic dysfunction and tau phosphorylation in the cerebral cortex under chronic psychiatric stress is not fully understood. The present study investigated the effects of memantine (MEM, 5 and 10 mg/kg), an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, on chronic restraint stress- (CRS-) induced tau phosphorylation in mice. CRS administered for 16 or 28 consecutive days (1 h daily) induced significant tau phosphorylation in the brain. MEM treatment suppressed the elevation of phosphorylated tau (P-tau) levels induced by 16-day CRS in a dose-dependent manner. P-tau reduction was accompanied by the attenuation of the upregulation of GSK3ß and CDK5 expression and the downregulation of PP2A activity induced by CRS. Additionally, MEM reduced CRS-induced upregulation of NMDA receptor subunit levels (GluN2A, GluN2B) in the frontal cortex. However, MEM markedly enhanced tau phosphorylation in the frontal cortex and other cerebral cortical regions following 28 days of CRS. The stimulatory effect of MEM on CRS-induced tau phosphorylation was correlated with increased activities of AKT, JNK, and GSK3ß, inactivation of PP2A, and downregulation of Pin1 and HSP70. Moreover, MEM did not effectively reverse the NMDA receptor upregulation induced by 28-day CRS and even increased GluN2B subunit levels. In contrast to the duration-dependent effects of MEM on P-tau levels, MEM produced an anxiolytic effect in both regimens as revealed by elevated plus maze testing. However, MEM did not affect the body weight reduction induced by CRS. Thus, MEM exerts distinctive effects on CRS-induced tau phosphorylation, which might be related to the expression of GluN2B. The differential effects of MEM on P-tau levels have crucial implications for its clinical application.

Pseudo-phosphorylation at AT8 epitopes regulates the tau truncation at aspartate 421.

Tau pathology in Alzheimer's disease (AD) includes hyperphosphorylation and truncation of tau. Phosphorylation at S422 is found to suppress truncation of tau at D421 that leading to the generation of ΔTau. However, the interrelation between hyperphosphorylation and generation of ΔTau in AD remains elusive. In current study, staurosporine (Stau) induced ΔTau generation by caspases in SH-SY5Y cells with tau overexpression was found to be accompanied by a dramatic dephosphorylation at S422 and the epitope of the diagnostic antibody AT8 (S199 + S202 + T205), but a moderate dephosphorylation of PHF1 (S396 + S404) epitope. Therefore, to explore the effect of AT8 epitope on tau truncation, the residues in AT8 epitope were mutated to produce "pseudo-phosphorylated" (AT8E) or "pseudo-unphosphorylated" (AT8A) tau constructs. With Stau treatment, the generation of ΔTau from tau-AT8E was significantly attenuated comparing with that from tau-AT8A, which was S422-independent in that addition of S422A mutation still preserved this effect. Interestingly, this modulatory effect was able to be reversed by addition of PHF1E mutation. Moreover, treating the crude tau extracts with recombinant caspase-3 in vitro, also showed that ΔTau level was suppressed by AT8E, and potentiated by AT8E + PHF1E. The results primarily revealed the modulating effects of phosphorylation on ΔTau generation which may have potential implications in tau pathological processes and therapeutic intervention.

Prolonged DADLE exposure epigenetically promotes Bcl-2 expression and elicits neuroprotection in primary rat cortical neurons via the PI3K/Akt/NF-κB pathway.

Both in vivo and in vitro studies have shown the beneficial effects of the delta-opioid receptor (DOR) on neurodegeneration in hypoxia/ischemia. We previously reported that DOR stimulation with [(D-Ala2, D-Leu5) enkephalin] (DADLE), a potent DOR agonist, for both a short (minutes) and long (days) time has notable protective effects against sodium azide (NaN3)-induced cell injury in primary cultured rat cortical neurons. We further demonstrated that short-term DADLE stimulation increased neuronal survival through the PKC-mitochondrial ERK pathway. However, the mechanisms underlying long-term neuroprotection by DADLE remain unclear. Here, we showed that DOR stimulation with DADLE (0.1 µmol/L) for 2 d selectively activates the PI3K/Akt/NF-κB pathway in NaN3-treated neurons; this activation increased Bcl-2 expression, attenuated Cyto c release and promoted neuronal survival. Further investigation revealed that sustained DADLE stimulation increased Bcl-2 expression by enhancing NF-κB binding to the Bcl-2 promoter and upregulating the histone acetylation levels of the Bcl-2 promoter. Our results demonstrate that prolonged DADLE exposure epigenetically promotes Bcl-2 expression and elicits neuroprotective effects in the NaN3 model via the PI3K/Akt/NF-κB pathway.

Aging as a Precipitating Factor in Chronic Restraint Stress-Induced Tau Aggregation Pathology, and the Protective Effects of Rosmarinic Acid.

Stress is an important risk factor of Alzheimer's disease (AD). It has been evidenced that stress could induce tau phosphorylation and increase tau insolubility in brain; however, little is known about the interactional effect of stress with aging on tauopathy. Therefore, we explored the effects of aging on stress-induced tauopathy and the potential mechanism in mouse model of chronic restraint stress (CRS). Here we found that in general, the level of phosphorylated tau (P-tau) was higher in brain of middle-aged mice than that in adult mice under physiological conditions. CRS-induced tau phosphorylation and its insolubility were more prominent in middle-aged mice. The increase of AT8-labeled insoluble P-tau was dramatic in middle-aged mice, which was highly ubiquitinated but did not form PHF structures. The levels of chaperones were relatively lower in middle-aged mice brain; CRS further reduced the expression, especially for HDJ2/HSP40. CRS also suppressed the expression of Pin1, the peptidylprolyl cis/trans isomerase, in middle-aged mice but not in adult mice. Downregulation of HSP40 or Pin1 caused an increase of transfected extraneous tau in 293 cells. Rosmarinic acid (RA) could effectively suppress the elevation of P-tau and insoluble P-tau formation induced by CRS, and reversed the abnormal changes of chaperones and Pin1 particularly in middle-aged mice. Taken together, our findings provided evidence that aging could be a promoting factor in stress-induced tauopathy, which was relevant with malregulation of chaperones and Pin1, and RA might be a promising beneficial agent for stress-induced tauopathy.

Probing the molecular mechanism of cerium oxide nanoparticles in protecting against the neuronal cytotoxicity of Aß1-42 with copper ions.

The molecular mechanism of CeONP in protecting against neuronal cytotoxicity from amyloid peptides and copper ions was investigated systematically by photoluminescence of [Ru(phen)2dppz](2+), morphology of TEM, mass spectroscopy, cell viability assay, ROS fluorescence assay, and EPR. The results revealed that CeONPs reduced Aß1-42 aggregation, protected from neurotoxicity of ROS induced by Cu(2+) + Aß1-42via blocking the production of free radicals and scavenging the radicals with Ce(3+)/Ce(4+) catalytic cycles, which provides a valuable insight into CeONPs as a therapeutic intervention in oxidative damage in Alzheimer's disease.

Beta amyloid-induced upregulation of death receptor 6 accelerates the toxic effect of N-terminal fragment of amyloid precursor protein.

Amyloid precursor protein (APP) plays essential roles in the development of the Alzheimer's disease. Although full-length APP has been thoroughly studied, the role of the cleavage fragments especially the N-terminal fragments (N-APPs) in Alzheimer's disease pathogenesis was still elusive. In this study, we demonstrated that application of recombinant APP18₋286 could enhance beta amyloid (Aß)-induced neuronal injuries which were related to the activation of apoptosis proteins. Aß treatment could induce a slight increase of N-APPs release. In addition, expression of death receptor 6 (DR6) was increased in Aß-treated neurons and APP transgenic mice. Moreover, the effect of APP18₋286 on Aß-induced injuries could be suppressed by the application of recombinant DR641₋341 and DR6 antibody. Furthermore, pull-down assay revealed that APP18₋286 could bind both exogenous and endogenous DR6. Aß promoted APP18₋286 targeting to neuron which was accompanied with the increase of DR6 expression, whereas downregulation of DR6 by interference RNA could alleviate the binding of N-APPs to neuron and also suppressed Aß-dependent toxic effect with N-APPs. These results suggested that APP N-terminal fragments might play neurotoxic roles in Aß-induced neuronal injuries through cell surface DR6.

[Localization and expression pattern of MDM2 in axon initial segments of neuron in rodent brain].

To investigate the murine double minute 2 (MDM2) localization and expression pattern in brain, immunohistochemistry, immunofluorescent staining and immunoblotting methods were used to analyze it in brains of Kunming mice during postnatal development, in brains of adult SD rats and in primarily cultured neurons. The distribution of MDM2 and markers of axon initial segment (AIS) was analyzed by double immunolabeling. In addition, Nutlin-3, a MDM2 antagonist, was injected into hippocampus to analyze the effect on the distribution of MDM2 and AIS protein Nav1.6 in AIS. The results showed that the dynamic expression patterns of MDM2 protein in cerebral cortex and hippocampus of Kunming mice after birth were different. However, it was similar that MDM2 was gradually enriched to AIS during postnatal development, especially after postnatal day 7. The MDM2 in AIS was also observed in different brain regions of adult SD rat brain and in primarily cultured neurons, where MDM2 was colocalized with AIS markers such as AnkG and Nav1.6. In addition, hippocampal injection of Nutlin-3 could induce the loss of the characteristic distribution of MDM2 in AIS. Moreover, Nutlin-3 not only caused a decrease of Nav1.6 distributing in AIS, but also disrupted the polarized distribution of MAP2 in neurons. These results indicate that MDM2 can be enriched at the AIS of adult rodent brain, which might play a role in regulation of the maintenance of AIS function and neuronal polarity.

Effect of ginsenoside Rg3 on tyrosine hydroxylase and related mechanisms in the forced swimming-induced fatigue rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg3 has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, little is known about the cellular and molecular mechanisms of Rg3 on anti-fatigue and the effect of Rg3 on dopaminergic system has not been reported yet. The major aim of this study is to investigate the effect of Rg3 on TH expression and the related biochemical parameters, such as PKAα, ERK1/2, Akt and α-synuclein in brain of fatigue rats. MATERIALS AND METHODS: Weight-loaded forced swimming was performed to establish an animal model of fatigue. Rg3 (10mg/kg, 50mg/kg and 100mg/kg) was intragastrically administrated before swimming. The effect of Rg3 on the expression and phosphorylation of TH and TH-related proteins in fatigue rats or in SH-SY5Y cells was assessed with western blotting. HPLC was used to examine the level of DA and DOPAC in the fatigue rats tissues. RESULTS: TH and phosphorylated TH were decreased in different brain regions of which ventral midbrain were less affected in weight-loaded forced swimming rats. Pretreatment with Rg3 significantly suppressed fatigue-induced decrease expression of TH and TH phosphorylation. Also treatment with Rg3 reversed the decrease expression of PKAα as well as the phosphorylation of ERK1/2 and Akt which were induced by weight-loaded forced swimming. Moreover, weight-loaded swimming could induce the increase expression of α-synuclein in hippocampus and midbrain, while suppressed α-synuclein expression in striatum and prefrontal cortex. Furthermore, Rg3 could induce the increase of TH expression and phosphorylation which was accompanied with elevated expression and phosphorylation of related kinase proteins in vitro, while the inhibitors of kinase proteins could suppress these effects of Rg3. In addition, HPLC results showed that Rg3 could reverse the weight-loaded swimming-induced increase of DOPAC/DA ratio. CONCLUSION: Our data suggest that fatigue can induce the decrease of DA which might partially result from the change of TH expression and phosphorylation, and Rg3 can reverse these fatigue-induced changes. The underling mechanisms may include the activity changes of PKAα, ERK1/2, Akt and α-synuclein.

[Potential involvement of abnormal increased SUMO-1 in modulation of the formation of Alzheimer's disease senile plaques and neuritic dystrophy in APP/PS1 transgenic mice].

Small ubiquitin-related modifiers (SUMOs) belong to an important class of ubiquitin like proteins. SUMOylation is a post-translational modification process that regulates the functional properties of many proteins, among which are several proteins implicated in neurodegenerative diseases. This study was aimed to investigate the changes of SUMO-1 expression and modification, and the relationship between SUMO-1 and Alzheimer's disease (AD) pathology in APP/PS1 transgenic AD mice. Using Western blot, co-immunoprecipitation and immunofluorescent staining methods, the SUMO-1 expression and modification and its relation to tau, amyloid precursor protein (APP) and ß-amyloid protein (Aß) in the 12-month-old APP/PS1 transgenic AD mice were analyzed. The results showed that: (1) Compared with the normal wild-type mice, the expression and modification of SUMO-1 increased in brain of AD mice, which was accompanied by an increase of ubiquitination; (2) In RIPA soluble protein fraction of cerebral cortex, co-immunoprecipitation analysis showed tau SUMOylated by SUMO-1 increased in AD mice, however, AT8 antibody labeled phosphorylated tau was less SUMOylated whereas PS422 antibody labeled phosphorylated tau was similar to control mice; (3) Double immunofluorescent staining showed that SUMO-1 could distributed in amyloid plaques, appearing that some of SUMO-1 diffused in centre of some plaques and some of SUMO-1 co-localized with AT8 labeled phosphorylated tau forming punctate aggregates around amyloid plaques which was concerned as dystrophic neurites, however, less Aß, APP and PS422 labeled phosphorylated tau were found co-localized with SUMO-1. These results suggest that SUMO-1 expression and modification increase abnormally in transgenic AD mice, which may participate in modulation of the formation of senile plaques and dystrophic neurites.

Effects of puerarin on cholinergic enzymes in the brain of ovariectomized guinea pigs.

Estrogen has beneficial effects on neurodegenerative disorders and cognitive function of postmenopausal women. Puerarin, isolated from Pueraria lobota, has been classified as a phytoestrogen, which can be highly effective against cerebrovascular diseases. In this study, the effects of puerarin on neural cholinergic system in the brain of ovariectomized guinea pigs were studied. The puerarin at the doses used (15 mg/kg body weight (bw)/day and 30 mg/kg bw/day) for 10 days had the estrogenic activity indicated by the attenuation of the reduction of uterine weight induced by ovariectomy. In brain, puerarin treatment increased choline acetyltransferase (ChAT) activity and expression in hippocampus, and increased ChAT immnuopositive signals in septal diagonal region. Puerarin treatment could suppress the increase of acetylcholinesterase expression and activity to the levels of the intact group, although they were not significantly different from those of the ovariectomized animals. Moreover, puerarin decreased the ß-amyloid immunopositive staining in hippocampus. In brief, the present study suggests that puerarin prevents the dysfunction of the neuronal cholinergic system and ameliorates the increase of ß-amyloid caused by estrogen deficiency.

Exploring the differences between mouse mAß(1-42) and human hAß(1-42) for Alzheimer's disease related properties and neuronal cytotoxicity.

The differences between mouse mAß(1-42) and human hAß(1-42), explored using CD and fluorescence spectroscopy, transmission electron microscopy, ROS fluorescent assay, and neuronal cell viability, revealed that mAß(1-42) as a three-site mutant (R5G, Y10F and H13R) of hAß(1-42) altered the metal (copper and zinc) binding sites, reduced the proneness to form ß-sheet structures and aggregated fibrils, alleviated the generation of ROS, and decreased the cytotoxicity, in contrast to hAß(1-42).

The molecular mechanism for human metallothionein-3 to protect against the neuronal cytotoxicity of Aß(1-42) with Cu ions.

Aggregation and cytotoxicity of Aß with redox-active metals in neuronal cells have been implicated in the progression of Alzheimer disease. Human metallothionein (MT) 3 is highly expressed in the normal human brain and is downregulated in Alzheimer disease. Zn(7)MT3 can protect against the neuronal toxicity of Aß by preventing copper-mediated Aß aggregation, abolishing the production of reactive oxygen species (ROS) and the related cellular toxicity. In this study, we intended to decipher the roles of single-domain proteins (α/ß) and the α-ß domain-domain interaction of Zn(7)MT3 to determine the molecular mechanism for protection against the neuronal cytotoxicity of Aß(1-42) with copper ions. With this in mind, the α and ß single-domain proteins, heterozygous ß(MT3)-α(MT1), and a linker-truncated mutant ∆31-34 were prepared and characterized. In the presence/absence of various Zn(7)MT3 proteins, the Aß(1-42)-Cu(2+)-mediated aggregation, the production of ROS, and the cellular toxicity were investigated by transmission electron microscopy, ROS assay by means of a fluorescent probe, and SH-SY5Y cell viability, respectively. The ß domain cannot abolish Aß(1-42)-Cu(2+)-induced aggregation, and neither the ß domain nor the α domain can quench the production of ROS because of the redox cycling of Aß-Cu(2+). Similarly to wild-type Zn(7)MT3, the heterozygous ß(MT3)-α(MT1) possesses the characteristic of alleviating Aß(1-42) aggregation and oxidative stress to neuronal cells. Therefore, the two domains through the linker Lys-Lys-Ser form a cooperative unit, and each of them is indispensable in conducting its bioactivity. The α domain plays an important role in modulating the stability of the metal-thiolate cluster, and the α-ß domain-domain interaction through the linker is critical for its protective role in the brain.

NRSF/REST neuronal deficient mice are more vulnerable to the neurotoxin MPTP.

Parkinson's disease (PD) is characterized by progressing loss of dopaminergic neurons in the midbrain. Abnormal gene expression plays a critical role in its pathogenesis. Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST), a member of the zinc finger transcription factors, inhibits the expression of neuron-specific genes in nonneuronal cells, and regulates neurogenesis. Our previous work showed that 1-methyl-4-phenyl-pyridinium ion triggers dynamic changes of messenger RNA and protein expression of NRSF in human dopaminergic SH-SY5Y cells, and alteration of NRSF expression exacerbates 1-methyl-4-phenyl-pyridinium ion-induced cell death. The purpose of this study was to explore the in vivo role of NRSF in the progress of PD by using NRSF/REST neuron-specific conditional knockout mice (cKO). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in the cKO mice and wild type littermates. At 1, 3, 7, 14, 21, and 28 days after MPTP injection, behavioral tests were performed, and cKO mice displayed some impairments in locomotor activities. Also, the reduction of tyrosine hydroxylase protein in the striatum and the loss of dopaminergic neurons in the substantia nigra were more severe in the cKO mice. Meanwhile, the cKO mice exhibited a more dramatic depletion of striatal dopamine, accompanied by an increase in glial fibrillary acidic protein (GFAP) expression and sustained interleukin-1ß transcription. These results suggested that NRSF/REST neuronal cKO mice are more vulnerable to the dopaminergic neurotoxin MPTP. Disturbance of the homeostasis of NRSF and its target genes, gliogenesis, and inflammation may contribute to the higher MPTP sensitivity in NRSF/REST neuronal cKO mice.