A case report of Andersen-Tawil syndrome misdiagnosed with myodystrophy.

Andersen-Tawil syndrome (ATS) is a rare periodic paralysis caused by the KCNJ2 gene mutation. Here, we report on an ATS patient misdiagnosed with myodystrophy. A 66-year-old man presented with a 60-year history of episodic weakness in the proximal muscles of the upper and lower limbs. The man has been diagnosed with muscle pathology and has undergone genetic examinations in many hospitals since childhood. We conducted a correct diagnosis in combination with the patient's history, electrical physiology, and genetic analysis and identified a heterozygous KCNJ2 gene variant (c.220A > G; p.T74A). Patients with ATS can develop permanent myasthenia characterized by chronic progressive myopathy. ATS patients should also pay special attention to the risks of anesthesia in surgery, including malignant hyperthermia (MH), muscle spasms affecting tracheal intubation or ventilation, and ventilator weakness. Early diagnosis and therapy could help delay the onset of myasthenia and prevent risks associated with anesthesia accidents.

DHCR24 reverses Alzheimer's disease-related pathology and cognitive impairment via increasing hippocampal cholesterol levels in 5xFAD mice.

Accumulating evidences reveal that cellular cholesterol deficiency could trigger the onset of Alzheimer's disease (AD). As a key regulator, 24-dehydrocholesterol reductase (DHCR24) controls cellular cholesterol homeostasis, which was found to be downregulated in AD vulnerable regions and involved in AD-related pathological activities. However, DHCR24 as a potential therapeutic target for AD remains to be identified. In present study, we demonstrated the role of DHCR24 in AD by employing delivery of adeno-associated virus carrying DHCR24 gene into the hippocampus of 5xFAD mice. Here, we found that 5xFAD mice had lower levels of cholesterol and DHCR24 expression, and the cholesterol loss was alleviated by DHCR24 overexpression. Surprisingly, the cognitive impairment of 5xFAD mice was significantly reversed after DHCR24-based gene therapy. Moreover, we revealed that DHCR24 knock-in successfully prevented or reversed AD-related pathology in 5xFAD mice, including amyloid-ß deposition, synaptic injuries, autophagy, reactive astrocytosis, microglial phagocytosis and apoptosis. In conclusion, our results firstly demonstrated that the potential value of DHCR24-mediated regulation of cellular cholesterol level as a promising treatment for AD.

DHCR24 Knockdown Lead to Hyperphosphorylation of Tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites by Membrane Lipid-Raft Dependent PP2A Signaling in SH-SY5Y Cells.

Accumulating data suggest that the downregulation of DHCR24 is linked to the pathological risk factors of AD, denoting a potential role of DHCR24 in AD pathogenesis. However, it remains unclear whether the downregulation of DHCR24 affects the abnormal heper-phosphorylation of tau protein, which is involved in tauopathy. In present papers, immunofluorescence and Filipin III fluorescence results showed that DHCR24 knockdown significantly lowered the level of plasma membrane cholesterol and expression level of membrane lipid-raft structural protein caveolin-1; and overexpression of DHCR24 could increase the plasma membrane cholesterol levels and facilitating caveolae structure through increase the expression of caveolin-1. PP2A is the key phosphatase involving in tau phosphorylation, which is localized in cholesterol-dependent caveola/raft lipid domains. Here, the PP2A activity was detected by western blot assay. Interestingly, the level of p-PP2Ac at Y307 (inactive) and p-GSK3ß at Y216 (active) in the downstream of the PP2A signal pathway were both significantly increased in silencing DHCR24 SH-SY5Y cells, which denoted an inhibition of the PP2A and activation of GSK3ß signaling. Conversely, overexpression of DHCR24 blunted the inhibition effect of PP2A and activation of GSK3ß. Besides, in the SH-SY5Y cell lines we demonstrated that DHCR24 knockdown obviously induced hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. In contrast, DHCR24 overexpression protects neuronal SH-SY5Y cells against the hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. Furthermore, PP2A activator D-erythro-Sphingosine (DES) also obviously inhibited the hyperphosphorylation of tau induced by DHCR24 knockdown. Collectively, our findings firstly confirmed that DHCR24 knockdown obviously induced abnormal hyperphosphorylation of tau by a novel lipid raft-dependent PP2A signaling. We propose that DHCR24 downregulation led to altered cholesterol synthesis as a potential mechanism in the progression of tau hyperphosphorylation involving in AD and other tauopathies.

DHCR24 overexpression modulates microglia polarization and inflammatory response via Akt/GSK3ß signaling in Aß25-35 treated BV-2 cells.

Microglial phenotypic polarization, divided into pro-inflammatory "M1" phenotype and anti-inflammatory "M2" phenotype, played a crucial role in the pathogenesis of Alzheimer's disease (AD). Facilitating microglial polarization from M1 to M2 phenotype was shown to alleviate AD-associate pathologic damage, and modulator of the microglial phenotype has become a promising therapeutic approach for the treatment of AD. Previous little evidence showed that DHCR24 (3-ß-hydroxysteroid-Δ-24-reductase), also known as seladin-1 (selective Alzheimer's disease indicator-1), exerted potential anti-inflammatory property, however, the link between DHCR24 and microglial polarization has never been reported. Thus, the role of DHCR24 in microglial polarization in amyloid-beta 25-35 (Aß25-35) treated BV-2 cells was evaluated in this study. Our results demonstrated that Aß25-35 aggravated inflammatory response and facilitated the transition of microglia phenotype from M2 to M1 in BV-2 cells, by upregulating M1 marker (i-NOS, IL-1ß and TNF-α) and downregulating M2 marker (arginase-1, IL-4 and TGF-ß). DHCR24 overexpression by lentivirus transfection could significantly reverse these effects, meanwhile, activated Akt/GSK3ß signaling pathway via increasing the protein expression of P-Akt and P-GSK3ß. Furthermore, when co-treated with Akt inhibitor MK2206, the effect of DHCR24 was obviously reversed. The study exhibited the neuroprotective function of DHCR24 in AD-related inflammatory injury and provided a novel therapeutic target for AD in the future.

Anti-inflammatory effects of doxepin hydrochloride against LPS-induced C6-glioma cell inflammatory reaction by PI3K-mediated Akt signaling.

Recent studies have shown that tricyclic antidepressants (TCAs) may have anti-inflammatory and anticonvulsant effects in addition to its antidepressant effects. So far, the nonantidepressant effects of TCAs and their molecular pharmacological mechanisms remain completely unclear. Chronic inflammation in the brain parenchyma may be related to the pathogenesis and progression of various neurodegenerative diseases. As a common antidepressant and anti-insomnia drug, doxepin also may be a potential anti-inflammatory and anticonvulsant drug, so the study on the anti-inflammatory protective effect of doxepin and its molecular mechanism has become a very important issue in pharmacology and clinical medicine. Further elucidating the anti-inflammatory and neuroprotective effects of doxepin and its molecular mechanism may provide the important theoretical and clinical basis for the prevention and treatment of neurodegenerative disease. This study was designed to understand the glio-protective mechanism of doxepin against the inflammatory damage induced by lipopolysaccharide (LPS) exposure in C6-glioma cells. We found the treatment of C6-glioma cells with LPS results in deleterious effects, including the augmentation of inflammatory cytokine levels (tumor necrosis factor-α, interleukin-1ß), and suppresses the Akt phosphorylation. Furthermore, our outcomes demonstrated that doxepin was able to suppress these effects induced by LPS, through activation of the phosphatidylinositol-3-kinase-mediated protein kinase B (Akt) pathway. To sum up, these results highlight the potential role of doxepin against neuroinflammatory-related disease in the brain.

Melatonin receptor stimulation by agomelatine prevents Aß-induced tau phosphorylation and oxidative damage in PC12 cells.

PURPOSE: As a novel antidepressant drug, agomelatine has good therapeutic effect on the mood disorder and insomnia in Alzheimer's disease (AD). Recent studies have shown the neuroprotective function of agomelatine, including anti-oxidative and anti-apoptosis effect. However, it remains unclear whether agomelatine exerts neuroprotection in AD. Thus, the neuroprotective effect of agomelatine against amyloid beta 25-35 (Aß25-35)-induced toxicity in PC12 cells was evaluated in this study. METHODS: The concentration of malondialdehyde (MDA), LDH, and ROS was investigated to evaluate oxidative damage. The expression of P-tau, tau, PTEN, P-Akt, Akt, P-GSK3ß, and GSK3ß proteins was assessed by Western blotting. Our results demonstrated that Aß25-35 significantly increased the content of MDA, LDH, and ROS. Meanwhile, Aß25-35 upregulated the expression of P-tau and PTEN as well as downregulated P-Akt and P-GSK3ß expression. These effects could be blocked by agomelatine pretreatment. Furthermore, luzindole, the melatonin receptor (MT) antagonist, could reverse the neuroprotective effect of agomelatine. CONCLUSION: The results demonstrated that antidepressant agomelatine might prevent the tau protein phosphorylation and oxidative damage induced by Aß25-35 in PC12 cells by activating MT-PTEN/Akt/GSK3ß signaling. This study provided a novel therapeutic target for AD in the future.

Mechanism underlying the effects of doxepin on ß-amyloid -induced memory impairment in rats.

OBJECTIVES: In previous studies, researchers observed that doxepin could improve cognitive processes and has protective effects on the central nervous system. Thus, this study was designed to analyze the effects of doxepin on ß-amyloid (Aß)-induced memory impairment and neuronal toxicity in rat and to explore the underlying mechanism. MATERIALS AND METHODS: Rats were treated with Aß1-42 and doxepin was injected to validate its effects on cognitive function. The Morris water maze test was performed to detect memory function. Aß1-42-treated SH-SY5Y human neuroblastoma cell line was also used to detect the effects of doxepin and to explore the underlying mechanism. Western blotting analysis was used to detect the protein expression levels of PSD-95, synapsin 1, p-AKT and p-mTOR in rats. RESULTS: After treated with 1 mg/kg of doxepin, Aß1-42-treated rats showed markedly lower escape latency and higher platform-finding strategy score. Low doses of doxepin significantly reversed the effects of Aß1-42 on the protein expression levels of PSD-95, synapsin 1, p-AKT and p-mTOR in rats. In vitro experiment showed the consistent results. Besides, PI3K inhibitor (LY294002) treatment could markedly reversed the effects of doxepin on Aß1-42-treated SH-SY5Y cells. CONCLUSION: Our results demonstrated that doxepin could protect against the Aß1-42-induced memory impairment in rats. The protective effect of doxepin was associated with the enhancement of PSD-95 and synapsin 1 expression via PI3K/AKT/mTOR signaling pathway.

Insulin-like growth factor-1 protects SH-SY5Y cells against ß-amyloid-induced apoptosis via the PI3K/Akt-Nrf2 pathway.

Insulin-like growth factor-1 (IGF-1) shows protective effect against Aß-induced cytotoxicity and apoptosis, but the underlying mechanisms are poorly characterized. The present study was conducted to explore the mechanisms involved in the beneficial effect of IGF-1 against Aß-induced apoptosis in SH-SY5Y cells. We found that pretreatment with IGF-1 attenuated Aß25-35-induced loss of cell viability and apoptosis in SH-SY5Y cells in a dose-dependent manner. In addition, IGF-1 inhibited the generation of reactive oxygen species (ROS) and increased the antioxidant activity in Aß25-35-treated cells. Further, IGF-1 significantly promoted the nuclear translocation of Nrf2, and upregulated the expression of its downstream gene heme oxygenase-1 (HO-1). Moreover, LY294002, a specific PI3K inhibitor, was found to completely abolish the protective effect of IGF-1 on Aß25-35-induced apoptosis and ROS generation. Together, our findings suggest that IGF-1 protects SH-SY5Y cells against Aß25-35-induced cell injury by scavenging ROS via the PI3K/Akt-Nrf2 signaling pathway.

Function of Thymosin Beta-4 in Ethanol-Induced Microglial Activation.

BACKGROUND/AIMS: Neuroinflammation mediated by activated microglia may play a pivotal role in a variety of central nervous system (CNS) pathologic conditions, including ethanol-induced neurotoxicity. The purpose of this study was to investigate the function of Tß4 in ethanol-induced microglia activation. METHODS: Quantitative real-time PCR was conducted to assess the expression of Tß4 and miR-339-5p. Western blot analysis was used to measure the expression of Tß4, phosphorylated p38, ERK, JNK, Akt, and NF-x03BA;B p65. The concentration of TNF-α and IL-1ß was determined using ELISA. NO concentration was measured using a nitric oxide colorimetric BioAssay Kit. Double immunofluorescence was performed to determine Tß4 expression, in order to assess microglial activation in neonatal mouse FASD model. RESULTS: Increased Tß4 expression was observed in ethanol treated microglia. Knockdown of Tß4 enhanced ethanol-induced inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) and nitric oxide (NO) in BV-2 cells was performed. Exogenous Tß4 treatment significantly inhibited expression and secretion of these inflammatory mediators. Tß4 treatment attenuated p38, ERK MAPKs, and nuclear factor-kappa B (NF-x03BA;B) pathway activation, and enhanced miR-339-5p expression induced by ethanol exposure in microglia. A neonatal mouse fetal alcohol spectrum disorders (FASD) model showed that Tß4 expression in the microglia of the hippocampus was markedly enhanced, while Tß4 treatment effectively blocked the ethanol-induced increase in inflammatory mediators, to the level expressed in vehicle-treated control animals. CONCLUSION: This study is the first to demonstrate the function of Tß4 in ethanol-induced microglia activation, thus contributing to a more robust understanding of the role of Tß4 treatment in CNS disease.

Protective Effect of DHT on Apoptosis Induced by U18666A via PI3K/Akt Signaling Pathway in C6 Glial Cell Lines.

Various useful animal models, such as Alzheimer's disease and Niemann-Pick disease, were provided by U18666A. However, the pathogenesis of U18666A-induced diseases, including U18666A-mediated apoptosis, remains incompletely elucidated, and therapeutic strategies are still limited. Dihydrotestosterone (DHT) has been reported to contribute to the prevention and treatment of neurodegenerative disorders. Our study investigated the neuroprotective activity of DHT in U18666A-related diseases. Apoptosis of C6 cells was detected by Hoechst 33258 fluorescent staining and flow cytometry with annexin V-FITC/PI dual staining. Cell viability was assessed using Cell Counting Kit-8. Expression of apoptosis-related proteins, such as Akt, seladin-1, Bcl-2 family proteins, and caspase-3, was determined using Western blot. Our results demonstrated that the apoptotic rate of C6 cells significantly increased after U18666A addition, but was remarkably reduced after DHT treatment. Pretreatment with DHT attenuated U18666A-induced cell viability loss. PI3K inhibitor LY294002 could suppress DHT anti-apoptotic effect. Furthermore, we discovered that U18666A could significantly downregulate seladin-1 expression in a dose-dependent manner, but no significant change was observed in Bcl-xL, Bax, and P-Akt protein expressions. Compared with U18666A-treated group, the expression of P-Akt, seladin-1, and Bcl-xL significantly increased, and the expression of Bax and caspase-3 remarkably reduced after DHT treatment. However, in the presence of LY294002, the effect of DHT was reversed. In conclusion, we found that seladin-1 may take part in U18666A-induced apoptosis. DHT may inhibit U18666A-induced apoptosis by regulating downstream apoptosis-related proteins including seladin-1, caspase-3, Bcl-xL, and Bax through activation of the PI3K/Akt signal pathway.

Exendin-4, a glucagon-like peptide-1 receptor agonist, inhibits Aß25-35-induced apoptosis in PC12 cells by suppressing the expression of endoplasmic reticulum stress-related proteins.

Neurodegenerative disorders are chronic and progressive disease. Exendin-4 (Ex-4) can function as a neuroprotective agent and has novel therapeutic ability for the treatment of neurodegenerative disorders. In this study, we aimed to explore the neuroprotective effect of Ex-4 on PC12 cell apoptosis induced by Aß25-35 in molecular level. The apoptosis of PC12 cells was detected by MTT assay, TUNEL staining and flow cytometry. The expression of ERS (endoplasmic reticulum stress, ERS) related proteins such as CHOP, GRP78 and Caspase-12 were determined by Western blot and cell immunocytochemistry. Results showed the apoptotic rate of PC12 cells significantly increased after Aß25-35 addition, which was remarkably reduced after Ex-4 treatment. The expression of CHOP, GRP78 and Caspase-12 were significantly upregulated, and then remarkably reduced after Ex-4 treatment, while in the presence of Exendin9-39, the effect of Ex-4 was reversed. In conclusion, endoplasmic reticulum stress might be involved in the apoptosis process of PC12 cell induced by Aß25-35 and Ex-4 might provide a potential strategy for the treatment and prevention of cell apoptosis-associated disorders.

DHT inhibits the Aß25-35-induced apoptosis by regulation of seladin-1, survivin, XIAP, bax, and bcl-xl expression through a rapid PI3-K/Akt signaling in C6 glial cell lines.

Previous evidences indicate that androgen is neuroprotective in the brain. However, the underling mechanisms remain to be fully elucidated. Moreover, it is controversial whether dihydrotestosterone (DHT) modulates the expression of apoptosis-related effectors, such as survivin, XIAP, bax, and bcl-xl proteins mediated by the PI3-K/Akt pathway, which contributes to androgen neuroprotection. In this study using a C6 glial cell model, apoptotic cells were detected by flow cytometry. Akt, seladin-1, survivin, XIAP, bcl-xl, and bax protein expression is investigated by Western blot. After amyloid ß-protein fragment (Aß25-35) treatment, apoptotic cells at early (annexin V+, PI-) and late (annexin V+, PI+) stages were significantly increased. Apoptosis at early and late was obviously inhibited in the presence of DHT. The effect of DHT was markedly blocked by PI3-K inhibitor LY294002.To elicit the mechanism of DHT protection, the expression of seladin-1, survivin, XIAP, bax, and bcl-xl protein was determined in C6 cells treated with Aß25-35, DHT, or LY294002. Aß25-35 significantly downregulated the expression of seladin-1, survivin, XIAP, bcl-xl protein and upregulated the expression of bax protein. DHT significantly inhibited the expression of bax, seladin-1, survivin, XIAP, and bcl-xl protein induced by Aß25-35. Further, we found the effect of DHT was significantly inhibited by LY294002. Collectively, in a C6 glial cell model, we firstly found that DHT inhibits Aß25-35-induced apoptosis by a rapid nongenic PI-3K/Akt activation as well as regulation of seladin-1, survivin, XIAP, bcl-xl, and bax proteins.

Testosterone up-regulates seladin-1 expression by iAR and PI3-K/Akt signaling pathway in C6 cells.

The previous study indicated that DHCR24/seladin-1 was an important neuroprotective effector. However, the molecular mechanisms that androgen modulates the expression of seladin-1 remain incompletely defined. In this paper, we showed that the expression of seladin-1 was significantly increased by testosterone at all concentrations tested at the protein and mRNA levels in C6 cells, the selective AR antagonist flutamide obviously inhibited the effect in a concentration-dependent manner. Furthermore, we found that testosterone significantly increased the phosphorylation level of V-akt murine thymoma viral oncogene (Akt), a key effector of the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway, while a specific PI3-K inhibitor LY294002 obviously prevented the activation of Akt phosphorylation. In addition, the PI3-K inhibitor LY294002 also markedly blocked the up-regulation expression of seladin-1 gene induced by testosterone at the protein and mRNA levels. Collectively, the above results suggested that testosterone regulated the expression of seladin-1 by the intracellular androgen receptor (iAR)-mediated genomic signaling pathway and the non-genomic PI3-K/Akt signaling pathway in C6 glial cells.