Withanone,an Active Constituent from <Emphasis Type="Italic">Withania somnifera</Emphasis>, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells

Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 μM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca²⁺, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.     

Expression of <Emphasis Type="Italic">Withania somnifera</Emphasis> Steroidal Glucosyltransferase gene Enhances Withanolide Content in Hairy Roots

In Withania somnifera, sterol molecules of immense medicinal value are diversified by means of glycosylation. Identifying sterol glycosyltransferases provides an imperative insight of diverse sterol modifications, thereby helping to comprehend the underlying plant mechanisms. In the present study, one of the W. somnifera sterol glycosyltransferase-4 (Ws-Sgtl4) gene was transformed into the W. somnifera leaf explant through Agrobacterium rhizogene. Transformed W. Somnifera Ws-Sgtl4 leaf explants were subjected to hairy root induction and analyzed for biomass accumulation. The analysis of Ws-Sgtl4 gene expression was performed at different time exposures with the application of salicylic acid and methyl jasmonate. The elicitation of W. somnifera hairy root expressing the Ws-Sgtl4 gene was also evaluated for the enhancement if any, in the total withanolide yield as well as the withanolides-A contents. The results suggested that Ws-Sgtl4 gene expression enhanced the production of total withanolide yield and withanolides-A in the hairy root culture of W. somnifera in the response to the elicitors.     

Arbuscular mycorrhizal fungal diversity in high-altitude hypersaline Andean wetlands studied by 454-sequencing and morphological approaches

Withania somnifera, also known as Indian ginseng is known to contain valuable bioactive compounds, called withanolides that structurally resemble ginsenosides of Panax ginseng. These compounds provide the basis of pharmacological relevance in traditional systems of medicine. In the present study, 150 hairy root lines of W. somnifera were induced of which nine fast growing lines were analysed for their growth and withanolide content. Hairy root line W9 was selected due to its high specific growth rate (0.196 ± 0.005 d^?1) and high withanolide content. The response to different concentrations of elicitors (methyl jasmonate and P. indica cell homogenate) and various exposure durations was assessed in the W9 hairy root line. The withanolide content as well as the pattern of gene expression from MVA, MEP and sterol pathway, was evaluated using qPCR. Though gene expression and withanolide content were found to be elevated in almost all MeJ and CHP treatments, the exposure of hairy roots to 15 μM MeJ for 4 h gave the maximum withanolide yield. The results suggest that the elicitation potential of methyl jasmonate was higher than that of P. indica cell homogenate for increasing withanolide levels in hairy roots of W. somnifera.     

An improved method of DNA purification from secondary metabolites rich medicinal plants using certain chaotropic agents

The presented work describes good quality DNA isolation method from mature leaves of some medicinally important plant species, viz. Asparagus racemosus, Withania somnifera, Abrus precatorius, Commiphora wightii and Carissa carandas. These plants hold immense medicinal values due to presence of certain secondary metabolites like polyphenols, terpenes, flavonoids, alkaloids, gums, resins, etc. Although these metabolites are accountable for important medicinal properties and authorize these plants to precedence over others, the same compounds disappoint the researcher while isolating high quality DNA. To overcome this problem, we propose a simple method in which DNA is adroitly bounded to diatomaceous earth in a solution of different chaotropic agent and alienated from intrusive compounds. Presented method affirms that secondary products, along with polysaccharides and proteins, can be perceptibly reduced by using silica matrix along with chaotropic agents. The described method is fast, simple and highly reliable for the isolation of DNA from obstinate plant species.     

Insulin Attenuates Beta-Amyloid-Associated Insulin/Akt/EAAT Signaling Perturbations in Human Astrocytes

The excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), mostly located on astrocytes, are the main mediators for glutamate clearance in humans. Malfunctions of these transporters may lead to excessive glutamate accumulation and subsequent excitotoxicity to neurons, which has been implicated in many kinds of neurodegenerative disorders including Alzheimer’s disease (AD). Yet, the specific mechanism of the glutamate system dysregulation remains vague. To explore whether the insulin/protein kinase B (Akt)/EAAT signaling in human astrocytes could be disturbed by beta-amyloid protein (Aβ) and be protected by insulin, we incubated HA-1800 cells with varying concentrations of Aβ_1–42 oligomers and insulin. Then the alterations of several key substrates in this signal transduction pathway were determined. Our results showed that expressions of insulin receptor, phospho-insulin receptor, phospho-protein kinase B, phospho-mammalian target of rapamycin, and EAAT1 and EAAT2 were decreased by the Aβ_1–42 oligomers in a dose-dependent manner (p < 0.05) and this trend could be recovered by insulin treatment (p < 0.05). However, the expressions of total Akt and mTOR were invariant (p > 0.05), and the mRNA levels of EAAT1 and EAAT2 were also unchanged (p > 0.05). Taken together, this study indicates that Aβ_1–42 oligomers could cause disturbances in insulin/Akt/EAAT signaling in astrocytes, which might be responsible for AD onset and progression. Additionally, insulin can exert protective functions to the brain by modulating protein modifications or expressions.     

Genotype independent and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the medicinal plant <Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD₆₀₀ 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36?±?13.2 to 277.6?±?22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T₀ transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.     

Growth kinetics and withanolide production in novel transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis> and measurement of their antioxidant potential using chemiluminescence

Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75?±?0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.     

Ashwagandha—An ancient Indian drug

Ashwagandha, the root ofWithania somnifera Dunal, is an ancient drug used in India by practitioners of Ayurvedic mediicne and as a household remedy. The drug is now official in the Indian Pharmacopoeia. Results of a survey regarding the cultivation, collection and marketing of this drug are described.     

<Emphasis Type="Italic">Withania somnifera</Emphasis> (Linn.) Dunal: a review of chemical and pharmacological diversity

Withania somnifera Dunal, is a commonly used herb in Indian Ayurvedic medicine system. Due to its pharmacological value and an inexhaustible source of novel biologically active compounds, it has been a great interest for researchers. The plant is known to possess anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory and hemopoetic properties. Various withanolides, steroidal lactones, have been isolated from W. somnifera and were known to have high therapeutic value. Based on the differences in the substitution patterns of withanolides the species has been classified into various chemotypes. So far, three different chemotypes have been identified, which have been further classified into ecotypes based on the contents of withanolides. Present review summarizes the phytochemical variability and pharmacological advances reported in literature.     

Dopamine protects neurons against glutamate-induced excitotoxicity

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.     

<Emphasis Type="Italic">S</Emphasis>-Nitrosylation Regulates Cell Survival and Death in the Central Nervous System

Nitric oxide (NO), which is produced from nitric oxide synthase, is an important cell signaling molecule that is crucial for many physiological functions such as neuronal death, neuronal survival, synaptic plasticity, and vascular homeostasis. This diffusible gaseous compound functions as an effector or second messenger in many intercellular communications and/or cell signaling pathways. Protein S-nitrosylation is a posttranslational modification that involves the covalent attachment of an NO group to the thiol side chain of select cysteine residues on target proteins. This process is thought to be very important for the regulation of cell death, cell survival, and gene expression in the central nervous system (CNS). However, there have been few reports on the role of protein S-nitrosylation in CNS disorders. Here, we briefly review specific examples of S-nitrosylation, with particular emphasis on its functions in neuronal cell death and survival. An understanding of the role and mechanisms underlying the effects of protein S-nitrosylation on neurodegenerative/neuroprotective events may reveal a novel therapeutic strategy for rescuing neurons in neurodegenerative diseases.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress,Dysfunction of Calcium Signalling,and Inhibition of Autophagy–Lysosomal Pathway

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-d-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic–lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.     

Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain

The glutamate-induced excitotoxicity pathway has been reported in several neurodegenerative diseases. Molecules that inhibit the release of glutamate or cause the overactivation of glutamate receptors can minimize neuronal cell death in these diseases. Osmotin, a homolog of mammalian adiponectin, is a plant protein from Nicotiana tabacum that was examined for the first time in the present study to determine its protective effects against glutamate-induced synaptic dysfunction and neurodegeneration in the rat brain at postnatal day 7. The results indicated that glutamate treatment induced excitotoxicity by overactivating glutamate receptors, causing synaptic dysfunction and neuronal apoptosis after 4 h in the cortex and hippocampus of the postnatal brain. In contrast, post-treatment with osmotin significantly reversed glutamate receptor activation, synaptic deficit and neuronal apoptosis by stimulating the JNK/PI3K/Akt intracellular signaling pathway. Moreover, osmotin treatment abrogated glutamate-induced DNA damage and apoptotic cell death and restored the localization and distribution of p53, p-Akt and caspase-3 in the hippocampus of the postnatal brain. Finally, osmotin inhibited glutamate-induced PI3K-dependent ROS production in vitro and reversed the cell viability decrease, cytotoxicity and caspase-3/7 activation induced by glutamate. Taken together, these results suggest that osmotin might be a novel neuroprotective agent in excitotoxic diseases.     

Propensity of <Emphasis Type="Italic">Withania somnifera</Emphasis> to Attenuate Behavioural,Biochemical, and Histological Alterations in Experimental Model of Stroke

The present study was designed to evaluate the beneficial effects of Withania somnifera (WS) pre-supplementation on middle cerebral artery occlusion (MCAO) model of ischemic stroke. Ischemic stroke was induced in the rats by inserting intraluminal suture for 90 min, followed by reperfusion injury for 24 h. The animals were assessed for locomotor functions (by neurological deficit scores, narrow beam walk and rotarod test), cognitive and anxiety-like behavioural functions (by morris water maze and elevated plus maze test). MCAO animals showed significant impairment in locomotor and cognitive functions. Neurobehavioural changes were accompanied by decreased acetylcholinesterase activity, increased oxidative stress in terms of enhanced lipid peroxidation and lowered thiol levels in the MCAO animals. In addition, MCAO animals had cerebral infarcts and the presence of pycnotic nuclei. Single-photon emission computerized tomography (SPECT) of MCAO animals revealed a cerebral infarct as a hypoactive area. On the other hand, pre-supplementation with WS (300 mg/kg body weight) for 30 days to MCAO animals was effective in restoring the acetylcholinesterase activity, lipid peroxidation, thiols and attenuated MCAO induced behavioural deficits. WS significantly reduced the cerebral infarct volume and ameliorated histopathological alterations. Improved blood flow was observed in the SPECT images from the brain regions of ischemic rats pre-treated with WS. The results of the study showed a protective effect of WS supplementation in ischemic stroke and are suggestive of its potential application in stroke management.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

<Emphasis Type="Italic">Sip1</Emphasis> mutation suppresses the resistance of cerebral cortex neurons to hypoxia through the disturbance of mechanisms of hypoxic preconditioning

Mutation of Sip1 plays a key role in pathogenesis of the Mowat–Wilson syndrome characterized by the presence of pronounced epileptic signs in patients. As a rule, neurodegenerative processes are accompanied by hypoxic phenomena, glutamate toxicity, and death of nerve cells. The molecular mechanisms underlying these phenomena are multifaceted and complex. Hypoxia causes the leakage of glutamate and other neurotransmitters and thus activates glutamate receptors and channels of plasma membrane. Hypoxia is accompanied by increased synthesis and secretion of proteins-regulators of oxidative stress, inflammation, apoptosis, and synaptic transmission. In this work, we investigated the effect of Sip1 mutations on the neuronal sensitivity of the brain to hypoxia and the formation of the hypoxic preconditioning phenomenon. The preconditioning effect was evaluated by the degree of suppressing activity of NMDA receptors by hypoxic episodes. Using fluorescent microscopy, we showed that cortical neurons from the brain of heterozygous (Sip1 ^wt/fI) and homozygous (Sip1 ^fI/fI) mice are characterized by the absence of the hypoxic preconditioning effect, whereas in Sip1 ^wt/wt neurons the amplitudes of Ca²⁺ responses to the application of NMDA are suppressed after transient episodes of hypoxia. In addition, hypoxia exerted a significant toxic effect on Sip1fI/fI neurons, which was manifested not only by an increased sensitivity to a decrease of the oxygen partial pressure (pO₂) and increased amplitudes of Ca2+ responses to application of NMDA after each hypoxic episode, but also by death of a considerable number of cells.     

Kainic acid-mediated excitotoxicity as a model for neurodegeneration

Neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mechanism in neurodegenerative disorders. Excitation resulting from stimulation of the ionotropic glutamate receptors is known to cause the increase in intracellular calcium and trigger calcium-dependent pathways that lead to neuronal apoptosis. Kainic acid (KA) is an agonist for a subtype of ionotropic glutamate receptor, and administration of KA has been shown to increase production of reactive oxygen species, mitochondrial dysfunction, and apoptosis in neurons in many regions of the brain, particularly in the hippocampal subregions of CA1 and CA3, and in the hilus of dentate gyrus (DG). Systemic injection of KA to rats also results in activation of glial cells and inflammatory responses typically found in neurodegenerative diseases. KA-induced selective vulnerability in the hippocampal neurons is related to the distribution and selective susceptibility of the AMPA/kainate receptors in the brain. Recent studies have demonstrated ability of KA to alter a number of intracellular activities, including accumulation of lipofuscin-like substances, induction of complement proteins, processing of amyloid precursor protein, and alteration of tau protein expression. These studies suggest that KA-induced excitotoxicity can be used as a model for elucidating mechanisms underlying oxidative stress and inflammation in neurodegenerative diseases. The focus of this review is to summarize studies demonstrating KA-induced excitotoxicity in the central nervous system and possible intervention by anti-oxidants.     

Mitochondrial proteomic analysis and characterization of the intracellular mechanisms of bis(7)-tacrine in protecting against glutamate-induced excitotoxicity in primary cultured neurons

Increasing evidence supports that the mitochondrial dysfunction, mainly caused by abnormal changes in mitochondrial proteins, plays a pivotal role in glutamate-induced excitotoxicity, which is closely associated with the pathogenesis of acute and chronic neurodegenerative disorders, such as stroke and Alzheimer's disease. In this study, post-treatment of cerebellar granule neurons with bis(7)-tacrine significantly reversed declines in mitochondrial membrane potential, ATP production, and neuronal cell death induced by glutamate. Moreover, this reversal was independent of NMDA antagonism, acetylcholinesterase inhibition, and cholinergic pathways. Using two-dimensional differential in-gel electrophoresis, we conducted a comparative analysis of mitochondrial protein patterns. In all, 29 proteins exhibiting significant differences in their abundances were identified in the glutamate-treated group when compared with the control. The expression patterns in 22 out of these proteins could be reversed by post-treatment with bis(7)-tacrine. Most of the differentially expressed proteins are involved in energy metabolism, oxidative stress, and apoptosis. In particular, the altered patterns of four of these proteins were further validated by Western blot analysis. Our findings suggest that multiple signaling pathways initiated by the altered mitochondrial proteins may mediate glutamate-induced excitotoxicity and also offer potentially useful intracellular targets for the neuroprotection provided by bis(7)-tacrine.