Golam Sadik

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14‐3‐3 proteins were reported to bind with tau. 14‐3‐3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14‐3‐3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14‐3‐3 in vitro. The affinity between tau and 14‐3‐3 is increased 12‐ to 14‐fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation‐mediated interaction of tau with 14‐3‐3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14‐3‐3. As the phosphorylation at Ser214 is up‐regulated in fetal brain, tau’s interaction with 14‐3‐3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up‐regulated in Alzheimer’s disease brain, tau’s interaction with 14‐3‐3 might be involved in the pathology of this disease.

A comparative study on the antioxidant activity of methanolic extracts from different parts of Morus alba L. (Moraceae)

BackgroundAntioxidants play an important role to protect damage caused by oxidative stress (OS). Plants having phenolic contents are reported to possess antioxidant properties. The present study was designed to investigate the antioxidant properties and phenolic contents (total phenols, flavonoids, flavonols and proanthrocyanidins) of methanolic extracts from Morus alba (locally named as Tut and commonly known as white mulberry) stem barks (TSB), root bark (TRB), leaves (TL) and fruits (TF) to make a statistical correlation between phenolic contents and antioxidant potential.MethodsThe antioxidant activities and phenolic contents of methanolic extractives were evaluated by in vitro standard method using spectrophotometer. The antioxidant activities were determined by total antioxidant capacity, DPPH (1,1-diphenyl-2-picrylhydrazine) radical scavenging assay, hydroxyl radical scavenging assay, ferrous reducing antioxidant capacity and lipid peroxidation inhibition assay methods.ResultsAmong the extracts, TSB showed the highest antioxidant activity followed by TRB, TF and TL. Based on DPPH and hydroxyl radical scavenging activity, the TSB extract was the most effective one with IC50 37.75 and 58.90 μg/mL, followed by TRB, TF and TL with IC50 40.20 and 102.03; 175.01 and 114.63 and 220.23 and 234.63 μg/mL, respectively. The TSB extract had the most potent inhibitory activity against lipid peroxidation with IC50 145.31 μg/mL. In addition, the reducing capacity on ferrous ion was in the following order: TSB > TRB > TL > TF. The content of phenolics, flavonoids, flavonols and proanthocyanidins of TSB was found to be higher than other extractives.ConclusionThe results indicate high correlation and regression (p-value <0 .001) between phenolic contents and antioxidant potentials of the extracts, hence the Tut plant could serve as effective free radical inhibitor or scavenger which may be a good candidate for pharmaceutical plant-based products. However, further exploration is necessary for effective use in both modern and traditional system of medicines.

Evaluation of antioxidant and anticancer properties of the seed extracts of Syzygium fruticosum Roxb. growing in Rajshahi, Bangladesh

BackgroundThe use of plants and their derived substances increases day by day for the discovery of therapeutic agents owing to their versatile applications. Current research is directed towards finding naturally-occurring antioxidants having anticancer properties from plant origin since oxidants play a crucial role in developing various human diseases. The present study was designed to investigate the antioxidant and anticancer properties of Sygygium fruticosum (Roxb.) (abbreviated as SF).MethodsThe dried coarse powder of seeds of SF was exhaustively extracted with methanol and the resulting crude methanolic extract (CME) was successively fractionated with petroleum ether, chloroform and ethyl acetate to get petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and lastly aqueous (AQF) fraction. The antioxidant activities were determined by several assays: total antioxidant capacity assay, DPPH free radical scavenging assay, hydroxyl radical scavenging assay, ferrous reducing antioxidant capacity and lipid peroxidation inhibition assay. The in vivo anticancer activity of SF was determined on Ehrlich’s Ascite cell (EAC) induced Swiss albino mice.ResultsAll the extractives showed strong antioxidant activities related to the standard. The total antioxidant capacity (TAC) of the fractions was in the following order: EAF>AQF>CME>PEF>CHF. The TAC of EAF at 320 μg/mL was 2.60±0.005 which was significantly higher (p < 0.01) than that of standard catechin (1.37 ± 0.005). The ferrous reducing antioxidant capacity of the extracts was in the following order: EAF>AQF>CME>AA>CHF>PEF. In DPPH free radical scavenging assay, the IC50 value of EAF was 4.85 μg/mL, whereas that of BHT was 9.85 μg/mL. In hydroxyl radical scavenging assay and lipid peroxidation inhibition assay, the EAF showed the most potent inhibitory activity with IC50 of 43.3 and 68.11 μg/mL, respectively. The lipid peroxidation inhibition assay was positively correlated (p < 0 .001) with both DPPH free radical scavenging and hydroxyl radical scavenging assay. The total phenolic contents of SF were also positively correlated (p < 0 .001) with DPPH free radical scavenging, hydroxyl radical scavenging and lipid peroxidation inhibition assay. Based on antioxidant activity, EAF was selected for cytotoxic assay and it was found that EAF inhibited 67.36% (p < 0.01) cell growth at a dose of 50 mg/kg (ip) on day six of EAC cell incubation.ConclusionsOur results suggest that EAF of seeds of SF possess significant antioxidant and moderate anticancer properties. Seeds of SF may therefore be a good source for natural antioxidants and a possible pharmaceutical supplement.

Post‐marketing survey of donepezil hydrochloride in Japanese patients with Alzheimer's disease with behavioral and psychological symptoms of dementia (BPSD)

Background:  To investigate the efficacy and safety of donepezil hydrochloride (Aricept®; Eisai Co., Ltd, Tokyo, Japan), we conducted a post‐marketing survey in Japanese patients with Alzheimers disease (AD) who also had behavioral and psychological symptoms of dementia (BPSD), such as hallucinations/delusions, wandering, and aggression, which cause the greatest burden on caregivers.

Antimicrobial and cytotoxic constituents of Loranthus globosus.

(+)-Catechin, 3,4-dimethoxycinnamyl alcohol and 3,4,5-trimethoxycinnamyl alcohol were isolated from the barks of Loranthus globosus. All compounds showed significant antimicrobial and cytotoxic activities.

In vitro acetylcholinesterase inhibitory activity and the antioxidant properties of Aegle marmelos leaf extract: implications for the treatment of Alzheimer's disease.

Alzheimers disease (AD) is a progressive neurodegenerative disorder clinically characterized by loss of memory and cognition. The effective therapeutic options for AD are limited and thus there is a demand for new drugs. Aegle marmelos (Linn.) (A. marmelos) leaves have been used in traditional medicine to promote intellect and enhance memory. In this study, we evaluated A. marmelos for its acetylcholinesterase (AChE) inhibitory activity and antioxidant property in vitro in the treatment of AD.

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3zeta. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3zeta is approximately 3-folds higher than that between unphosphorylated 4R-tau and 14-3-3zeta. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3zeta to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3zeta. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3zeta exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3zeta suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells, and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau

In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH‐SY5Y human neuroblastoma cells and COS‐7 cells transfected with the tau gene. When cells were treated with 0.1–10 µmol/L of lactacystin and 1.0–20 µmol/L of MG‐132 (inhibitors of proteasome), 0.1–10 µmol/L of CA‐074Me (a cathepsin inhibitor), and 0.1–2 µmol/L of puromycin (a puromycin‐sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse‐chase experiments demonstrated that the proteolysis of tau protein in SH‐SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH‐SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH‐SY5Y cells. The protein metabolism of tau‐containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) was also investigated using pulse‐chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation‐dependent antibodies revealed that phosphorylation levels of tau at Thr231, Ser396, and Ser409 were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP‐17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.

Mild cognitive impairment and subjective cognitive impairment

Until a decade ago, the common understanding was that forgetfulness in the elderly is a physiological phenomenon and that all elderly people show memory loss to some extent, with considerable variations in the severity of memory loss between individuals. Commonly observed memory loss in the elderly was termed ‘benign senescent forgetfulness’, which was regarded as different from the pathological state of dementia. Kral described benign senescent forgetfulness as follows: (i) impossible to recall unimportant things and part of the experience; (ii) able to recall these forgotten things at other times and on other occasions; (iii) more memory loss with older things compared with recent things; and (iv) subjects recognize this forgetfulness and try to compensate for it by several means. In the 1980s, there was an interest in ‘age-associated memory impairment’ (AAMI), which was also regarded as different from Alzheimer’s disease, in which only memory function is disturbed without an affect on other cognitive functions. In those days, memory loss in the elderly was regarded as common and different from the pathological conditions leading to dementia, including Alzheimer’s disease. Recent findings in neuroscience do not necessarily support the notions described above, and it is conceived that memory loss in the elderly is not inevitable, because newer findings show fully functioning brain activity in the elderly. For example, there is no significant decrease in the number of neurons in the elderly brain, even though the size of the neurons is smaller, and there is no reduction in cerebral blood flow and cerebral glucose metabolism in the brain of healthy elderly people compared with the younger brain. Neurogenesis is observed in the dentate gyrus of the hippocampus of brains over 50 years of age. We have now abundant data that support the hypothesis that the human brain can function properly over 50 years of age if the elderly person is not affected by dementia. Furthermore, this new understanding has been accelerated by research into the pathogenesis of Alzheimer’s disease that explores possible means of intervention and prevention. Since 1996, drugs for the symptomatic treatment of Alzheimer’s disease have been used widely, including acetylcholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and an N-methyl-D-aspartate (NMDA) antagonist (memantine), and we are now at the stage of developing disease-modifying drugs, which will give more benefit to the patients if administrated in earlier stages of the disease. Considering these situations, the initial stage, or even prestage, of the disease attracts more attention in research because it would be the period when the disease-modifying drugs would exert their maximum effect.

Toll-like receptor 3 mediated hyperphosphorylation of tau in human SH-SY5Y neuroblastoma cells

Abstract  Neurofibrillary tangles of abnormally phosphorylated tau are one of the characteristic pathological hallmarks of Alzheimers disease (AD). In addition, immunological and inflammatory changes including complements and activated microglia are also common phenomena in AD. However, these pathological changes are yet to be interlinked in a common explainable background. In this study, the relevant mechanism of phosphorylation of tau protein and an innate immune signal transduction system were investigated. Toll‐like receptor 3 (TLR3) is a receptor working in the innate immune system and its expression in the brain has already been reported. Total RNA was isolated from SH‐SY5Y cells and reverse transcriptase polymerase chain reaction was done to see endogenous expression of TLR3 in SH‐SY5Y cells that was further confirmed at protein level by Western blot analysis. Cells were treated with 50 µg/mL of polyinosinic–polycytidylic acid (pIpC), a synthetic analog of dsRNA and the changes of phosphorylation of tau protein were investigated. Further the level of phosphorylation of tau protein was investigated after the cells had been previously treated with 10 ng/mL of lipopolysaccharide (LPS) for 6 h to induce over‐expression of TLR3. Increased phosphorylation of tau protein at PHF‐1 site (Ser396/404), activation of Jun N‐terminal kinase and p38 MAPK were observed in cells treated with pIpC. These effects were enhanced when cells were pretreated with LPS, a known transducer of TLR3. These data suggest that toll‐like receptor 3, an innate immune molecule, might be a potential link to mediate hyperphosphorylation of tau in neurodegenerative processes of AD.