Differential dysregulation of CREB and synaptic genes in transgenic Drosophila melanogaster expressing shaggy (GSK3), TauWT, or Amyloid-beta.

BACKGROUND: Tau, Amyloid-beta (Aß42), and Glycogen synthase kinase 3 (GSK3) contribute to synaptic dysfunction observed in Alzheimer's disease (AD), the most common form of dementia. In the current study, the effect of pan-neuronal expression of TauWT, Aß42, or shaggy (orthologue of GSK3) in Drosophila melanogaster was assessed on the locomotor function, ethanol sensitivity, synaptic genes and CREB expression. The effect of TauWT and Aß42 on the expression of shaggy was also determined. METHODS AND RESULTS: Gene expression analysis was performed using quantitative real-time RT-PCR method. While syt1, SNAP25 and CREB (upstream transcription factor of syt1 and SNAP25) were upregulated in flies expressing TauWT or Aß42, a prominent decline was observed in those genes in shaggy expressing flies. Although all transgenic flies showed climbing disability and higher sensitivity to ethanol, abnormality in these features was significantly more prominent in transgenic flies expressing shaggy compared to TauWT or Aß42. Despite a significant upregulation of shaggy transcription in TauWT expressing flies, Aß42 transgenic flies witnessed no significant changes. CONCLUSIONS: TauWT, Aß42, and shaggy may affect synaptic plasticity through dysregulation of synaptic genes and CREB, independently. However shaggy has more detrimental effect on synaptic genes expression, locomotor ability and sensitivity to ethanol. It is important when it comes to drug discovery. It appears that CREB is a direct effector of changes in synaptic genes expression as they showed similar pattern of alteration and it is likely to be a part of compensatory mechanisms independent of the GSK3/CREB pathway in TauWT or Aß42 expressing flies.

Berberis integerrima hydro-alcoholic root extract and its constituent berberine protect against cisplatin-induced nephro- and hepato-toxicity.

BACKGROUND: This study determined the potential hepato- and renal protective role of berberine and hydroalcohol extract of Berberis integerrima (barberry) against cisplatin-induced acute liver and kidney injury. METHODS: Animals were dedicated into six groups (n = 7 per group): control, control+Ber (berberine, 0.4 mg/kg/day during 10 days, i.p.), control+B.E (barberry extract, 160 mg/kg/day during 10 days, i.p.), Cis (cisplatin, 8 mg/kg on 7th day, i.p.), Cis+Ber (berberine, 100 mg/kg/day during 10 days; cisplatin, 8 mg/kg on 7th day), Cis+B.E (barberry extract, 160 mg/kg/day during 10 days; cisplatin, 8 mg/kg on 7th day). After placing the rats in metabolic cages for 24 h, blood, urine, liver and kidney tissue samples were collected. RESULTS: Compared to control, control+Ber and control+B.E groups, cisplatin administration led to kidney and liver dysfunction. These happened with diminished activities of antioxidant enzymes, increased levels of malondialdehyde, Toll-like receptor 4 gene expression and histological damages in hepatic and renal tissues. Berberine and barberry extract administration decreased all the changes. CONCLUSIONS: An intensification in enzymatic oxidant status, decrease in lipid peroxidation with decrease in TLR4 gene expression level indicate that barberry extract may be a potential candidate in combating cisplatin-induced oxidative stress and inflammation in liver and kidney tissues through its constituent berberine.

Tau and amyloid beta differentially affect the innate immune genes expression in Drosophila models of Alzheimer's disease and ß- D Mannuronic acid (M2000) modulates the dysregulation.

Alzheimer's disease (AD) is the most common cause of dementia and neuroinflammation is considered as one of the main culprits. The aim of this study was to evaluate the independent role of Aß42 and tau on the inflammatory pathway in the Drosophila models of AD and investigating the potential modulating effect of M2000 as a novel NSAIDs in those flies. The expression levels of relish, orthologs of NF-κB, antimicrobial peptide (AMP) including attacin A, diptericin B and a dual oxidase (Duox) as a ROS mediator, were evaluated in both M2000 treated and untreated groups followed by brain histology analysis to assess the extent of neurodegeneration. The potential inhibitory role of M2000 (ß-D Mannuronic acid) on the aggregation of tau protein was also investigated in vitro. According to the result, there was a significant induction of Duox, AMPs and its transcription factor expression in both aged and Drosophila models of AD which was in accordance with the increase in the number of vacuoles in the brain section of Drosophila models of AD. Interestingly M2000 treatment revealed a significant reduction in all neurodegeneration indexes; in vivo and anti-aggregating property; in vitro. Findings suggest that M2000 has potential to be an AD therapeutic agent.

DNT1 Downregulation and Increased Ethanol Sensitivity in Transgenic Drosophila Models of Alzheimer's Disease.

Two major pathological hallmarks of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau. Aggregation of amyloid-ß (Aß) is considered as the primary insult in AD. However, failure in treatments based on targetingAß without considering the pathologic tau and close correlation between pathological tau and cognitive decline highlighted the crucial role of tau in AD. Loss of synaptic plasticity and cognitive decline, partly due to decrease in Brain Derived Neurotrophic Factor (BDNF), are other hallmarks of AD. Aß and tau downregulate BDNF at both transcriptional and translational levels. The aim of this research was to study the expression levels of Drosophila Neuroteophin 1 (DNT1), as an orthologue of BDNF, in flies expressing Aß42 or tauR406W. Levels of DNT1 were determined using quantitative real time PCR. Behavioral and Biochemical investigations were also performed in parallel. Our results showed that there is a significant decrease in the levels of DNT1 expression in Aß42 or tauR406W expressing flies. Interestingly, a significant increase was observed in sensitivity to ethanol in both transgenic flies. Rise in Reactive Oxygen Species (ROS) levels was also detected. We concluded that both Aß and pathological tau exert their toxic effect on DNT1 expression, ROS production, and response to ethanol, independently. Interestingly, pathological tau showed higher impact on the ROS production compared to Aß. It seems that Aß42 and tauR406W transgenic flies are proper models to investigate the interplay between BDNF and oxidative stress, and also to assess the mechanism underlying behavioral response to ethanol.

Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy.

BACKGROUND: Alzheimer's disease (AD) is one the most common types of dementia. Plaques of amyloid beta and neurofibrillary tangles of tau are two major hallmarks of AD. Metabolism of these two proteins, in part, depends on autophagy pathways. Autophagy dysfunction and protein aggregation in AD may be involved in a vicious circle. The aim of this study was to investigate whether tau or amyloid beta 42 (Aß42) could affect expression of autophagy genes, and whether they exert their effects in the same way or not. METHODS: Expression levels of some autophagy genes, Hook, Atg6, Atg8, and Cathepsin D, were measured using quantitative PCR in transgenic Drosophila melanogaster expressing either Aß42 or Tau R406W. RESULTS: We found that Hook mRNA levels were downregulated in Aß42-expressing flies both 5 and 25 days old, while they were increased in 25-day-old flies expressing Tau R406W. Both Atg6 and Atg8 were upregulated at day 5 and then downregulated in 25-day-old flies expressing either Aß42 or Tau R406W. Cathepsin D expression levels were significantly increased in 5-day-old flies expressing Tau R406W, while there was no significant change in the expression levels of this gene in 5-day-old flies expressing Aß42. Expression levels of Cathepsin D were significantly decreased in 25-day-old transgenic flies expressing Tau R406W or Aß42. CONCLUSION: We conclude that both Aß42 and Tau R406W may affect autophagy through dysregulation of autophagy genes. Interestingly, it seems that these pathological proteins exert their toxic effects on autophagy through different pathways and independently.

The distinctive role of tau and amyloid beta in mitochondrial dysfunction through alteration in Mfn2 and Drp1 mRNA Levels: A comparative study in Drosophila melanogaster.

Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Aggregation of Aß42 and hyperphosphorylated tau are two major hallmarks of AD. Whether different forms of tau (soluble or hyperphosphorylated) or Aß are the main culprit in the events observed in AD is still under investigation. Here, we examined the effect of wild-type, prone to hyperphosphorylation and hyperphosphorylated tau, and also Aß42 peptide on the brain antioxidant defense system and two mitochondrial genes, Marf (homologous to human MFN2) and Drp1 involved in mitochondrial dynamics in transgenic Drosophila melanogaster. AD is an age associated disease. Therefore, the activity of antioxidant agents, CAT, SOD, and GSH levels and the mRNA levels of Marf and Drp1 were assessed in different time points of the flies lifespan. Reduction in cognitive function and antioxidant activity was observed in all transgenic flies at any time point. The most and the least effect on the eye phenotype was exerted by hyperphosphorylated tau and Aß42, respectively. In addition, the most remarkable alteration in Marf and Drp1 mRNA levels was observed in transgenic flies expressing hyperphosphorylated tau when pan neuronal expression of transgenes was applied. However, when the disease causing gene expression was confined to the mushroom body, Marf and Drp1 mRNA levels alteration was more prominent in tauWT and tauE14 transgenic flies, respectively. In conclusion, in spite of antioxidant deficiency caused by different types of tau and Aß42, it seems that tau exerts more toxic effect on the eye phenotype and mitochondrial genes regulation (Marf and Drp1). Moreover, different mechanisms seem to be involved in mitochondrial genes dysregulation when Aß or various forms of tau are expressed.

Effect of Magnesium Sulfate Added to Tincture of Opium and Buprenorphine on Pain and Quality of Life in Women with Dysmenorrhea: A Prospective, Randomized, Double-blind, Placebo-controlled Trial.

BACKGROUND: Adding magnesium sulfate (MgSO4) to opioid receptor agonists increases the opioid analgesic effects via blocking this receptor. The current study aimed to evaluate the effectiveness of adding MgSO4 to tincture of opium (TOP) and buprenorphine (BUP) on pain and quality of life (QOL). METHODS: In prospective, randomized, double-blind, placebo-controlled clinical trial, one hundred and sixty-three women with secondary dysmenorrhea caused by endometriosis were selected using a respondent-driven sampling (RDS) and assigned into six groups using block randomization. Patients received 50 mg/kg MgSO4 in 100 ml saline by micro set in six monthly menstrual periods and completed the visual analogue scale (VAS) and QOL Questionnaire (QOLQ). Data were analyzed by repeated measures analysis of variance (ANOVA) and hierarchical regression. FINDINGS: The primary outcomes showed that pain scores in magnesium (MAG) + opium tincture (OT) [F = 5.7(1,162), P = 0.004] and MAG+ BUP [F = 4.5(1,162), P = 0.006] groups showed a significant decrease compared with control group. Also, QOL scores in MAG + OT [F = 4.8(1,162), P = 0.005] and MAG + BUP [F = 5.9(1,162), P = 0.003] showed a significant increase. However, there was no significant difference between the two groups (P = 0.140) and the changes did not persist until follow-up (P = 0.810). Secondary outcomes indicated that the low scores of the two components of QOL including physical and psychological components were predictors of pain (P = 0.011, Beta > 3.09). CONCLUSION: Simultaneous use of MAG with opioids is associated with pain reduction and the improvement of QOL. However, this hypothesis requires careful handling in a randomized controlled trial.

Remote Ischemic Perconditioning Modulates Apelin Expression After Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia/reperfusion injury (IRI) can result in impaired ability of urine concentration and increased sodium fractional excretion. Apelin, a (neuro) vasoactive peptide, enhances diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of arginine vasopressin on the tubules. However, changes in renal apelin expression in renal IRI rat model have not been elucidated. Remote ischemic perconditioning (RIPerC) improves renal sodium and water handling after IRI. Here, we investigated whether RIPerC prevents dysregulation of renal sodium and water handling in response to IRI by apelin signaling pathway in rats. MATERIALS AND METHODS: Renal IRI was induced by 45-min clamping of renal arteries followed by 24 h reperfusion. RIPerC was created by applying four cycles of 2-min ischemia of the left femoral artery followed by 3-min reperfusion at the start of renal ischemia. Rats were randomly divided into sham, ischemia/reperfusion, and RIPerC + ischemia/reperfusion groups. Urine and blood were sampled after reperfusion period. The kidney was harvested for mRNA isolation and histopathological study. RESULTS: IRI resulted in decreased clearance of creatinine, increased sodium fractional excretion, and reduced urine osmolality compared with sham animals. This occurred with an increase in mRNA expression levels of apelin and histological damages in both cortical and medullary regions of kidney tissues. RIPerC treatment ameliorated all these changes. CONCLUSIONS: This study showed that RIPerC has protective effects against dysregulation of renal sodium and water handling after renal IRI, which might be related with inhibition of apelin signaling pathway.

Kinetics and mechanism of antibacterial activity and cytotoxicity of Ag-RGO nanocomposite.

Nowadays, nanomaterials with remarkable antibacterial activity and low cytotoxicity attract much interest in research. By considering the antibacterial activity of Ag and graphene oxide (GO), the Ag-RGO nanocomposite was prepared by a one-pot and facile technique and it was used to evaluate its antibacterial activity and cytotoxicity against Escherichia coli and glioblastoma cancer cells (U87MG), respectively. The antibacterial activity was studied by micro-dilution and colony counting methods to investigate cell viability. The viability of glioblastoma cells was determined using MTT assay. Since MIC and MBC values of the nanocomposite are 20 and 40µg/mL, respectively, it acts as a bactericidal agent. The antibacterial properties of nanocomposite are time and concentration dependent. The kinetics and mechanism of the antibacterial activity of the nanocomposite were investigated. The antibacterial activity for Ag-RGO nanocomposite is induced by capturing-killing process. From the results, we concluded that Ag-RGO nanocomposite can simultaneously induce apoptosis. Our results bring up a new plan for the use of silver nanoparticles in the form of nanocomposite with reduced graphene oxide in antibacterial applications. Also, Ag-RGO nanocomposite can reduce the viability of U87MG in a dose dependent manner which may show its anticancer potential.

The structural alteration and aggregation propensity of glycated lens crystallins in the presence of calcium: Importance of lens calcium homeostasis in development of diabetic cataracts.

The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an important risk factor for development of cataract diseases. In the current study, the impact of elevated levels of calcium ions were investigated on structure and aggregation propensity of glycated lens crystallins using gel electrophoresis and spectroscopic assessments. The glycated proteins indicated significant resistance against calcium-induced structural insults and aggregation. While, glycated crystallins revealed an increased conformational stability; a slight instability was observed for these proteins upon interaction with calcium ions. Also, in the presence of calcium, the proteolytic pattern of native crystallins was altered and that of glycated protein counterparts remained almost unchanged. According to results of this study it is suggested that the structural alteration of lens crystallins upon glycation may significantly reduce their calcium buffering capacity in eye lenses. Therefore, under chronic hyperglycemia accumulation of this cataractogenic metal ion in the lenticular tissues may subsequently culminate in activation of different pathogenic pathways, leading to development of lens opacity and cataract diseases.

The Structural Alteration and Aggregation of Bovine Lens Gamma-Crystallin by Homocysteinylation; The Pathomechanism Underlying Cataract Development During Hyperhomocysteinimia.

A significant association between increased level of blood homocysteine (hyperhomocysteinimia) and various eye pathological disorders including cataract has been reported. This metabolic byproduct is converted into a highly reactive cyclic thioester compound, homocysteine thiolactone (HCTL), which can potentially react with free amino groups in protein. In the current study, as bovine lens γ-Crystallin (γ-Cry) was incubated with HCTL, various spectroscopic techniques, gel mobility shift assay, and microscopic analysis were applied to characterize structural variation and aggregation of this protein. According to the fluorescence results, HCTL-induced structural alteration was accompanied with the significant enhancement in surface hydrophobicity of γ-Cry. Also, this cyclic thioester was indicated to alter γ-Cry secondary structures and to induce aggregation of this protein. The results of gel mobility shift assay suggest the involvement of disulfide bond cross-linking in formation of the protein aggregates. In conjunction with Thioflavin T and Congo red assays, the microscopic analysis also suggests that HCTL can induce formation of ordered aggregate entities in bovine lens γ-Cry. The relationship between γ-Cry insolubilization/aggregation and growth of cataract disorders has been already reported. Therefore, the induction of structural alteration and aggregation of γ-Cry by HCTL can elucidate the pathomechanism underlying cataract disorders particularly in hyperhomocysteinimia.

Partial and complete microdeletions of Y chromosome in infertile males from South of Iran.

Y chromosome microdeletions are the second genetic cause of male infertility. The incidence of Y chromosome microdeletions can vary considerably depending on several factors, including patient selection criteria, population composition, and diagnostic protocols. They are associated with spermatogenic failure and lead to azoospermia or oligozoospermia. The advance in assisted reproductive technology and intracytoplasmic sperm injection, and the possibility of genetic defect transmission to the next generation make it necessary to improve our knowledge about the various factors leading to spermatogenic impairment. This study was designed to determine the frequency of microdeletions of Y chromosome in a population from South of Iran. 81 infertile males with non-obstructive azoospermia or oligozoospermia were selected. Multiplex PCR using several STS markers was carried out to detect the complete or partial microdeletions. The frequency of both complete and partial microdeletions in men with azoospermia or severe oligozoospermia was 7.4%. All microdeletions were observed in AZFc region. There was 1.25% complete microdeletions and after excluding complete microdeletions, we detected 5% gr/gr and 1.25% b2/b3 microdeletions. In our control group of fertile males, 4% gr/gr microdeletions was detected while there was no b2/b3 microdeletions. We concluded that there is a low frequency of Y chromosome microdeletions in a population of infertile males from South of Iran. b2/b3 microdeletions was detected only in infertile males and not in the control group. Screening a population with larger sample size is necessary to determine the involvement of this partial microdeletion in infertility of this population.

Increased pro-nerve growth factor and decreased brain-derived neurotrophic factor in non-Alzheimer's disease tauopathies.

Alterations in the expression and signaling of brain-derived neurotrophic factor (BDNF) and the precursor to nerve growth factor (NGF), proNGF, play a role in the neuronal and cognitive dysfunction of Alzheimer's disease. Aggregated amyloid-ß has been shown to down-regulate specific BDNF transcripts in Alzheimer's disease, but the role of tau pathology in neurotrophin dysregulation has not been investigated. We measured levels of BDNF mRNA and protein using real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay and proNGF protein using Western blotting in parietal cortex of subjects with tauopathies, neurodegenerative diseases exhibiting tau pathology without amyloid-ß accumulation. We observed a significant increase in the level of proNGF protein in Pick's disease and a significant decrease in BDNF mRNA and protein levels in Pick's disease and corticobasal degeneration, but no neurotrophin alterations in progressive supranuclear palsy. The decrease in total BDNF mRNA levels in these tauopathies was predominantly due to down-regulation of transcript IV. These findings implicate tau pathology in neurotrophin dysregulation, which may represent a mechanism through which tau confers toxicity in Alzheimer's disease and related non-Alzheimer's dementias.

Biological activity of nerve growth factor precursor is dependent upon relative levels of its receptors.

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.