Oxytosis/Ferroptosis in Neurodegeneration: the Underlying Role of Master Regulator Glutathione Peroxidase 4 (GPX4).

Oxytosis/ferroptosis is an iron-dependent oxidative form of cell death triggered by lethal accumulation of phospholipid hydroperoxides (PLOOHs) in membranes. Failure of the intricate PLOOH repair system is a principle cause of ferroptotic cell death. Glutathione peroxidase 4 (GPX4) is distinctly vital for converting PLOOHs in membranes to non-toxic alcohols. As such, GPX4 is known as the master regulator of oxytosis/ferroptosis. Ferroptosis has been implicated in a number of disorders such as neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), etc.), ischemia/reperfusion injury, and kidney degeneration. Reduced function of GPX4 is frequently observed in degenerative disorders. In this study, we examine how diminished GPX4 function may be a critical event in triggering oxytosis/ferroptosis to perpetuate or initiate the neurodegenerative diseases and assess the possible therapeutic importance of oxytosis/ferroptosis in neurodegenerative disorders. These discoveries are important for advancing our understanding of neurodegenerative diseases because oxytosis/ferroptosis may provide a new target to slow the course of the disease.

Network Pharmacology Integrated Molecular Docking and Dynamics to Elucidate Saffron Compounds Targeting Human COX-2 Protein.

Background and Objectives: Cyclooxygenase-2 (COX-2) is mostly linked to inflammation and has been validated as a molecular target for treating inflammatory diseases. The present study aimed to identify novel compounds that could inhibit COX-2, which is associated with various diseases including inflammation, and in such a scenario, plant-derived biomolecules have been considered as attractive candidates. Materials and Methods: In the present study, physiochemical properties and toxicity of natural compounds/drugs were determined by SWISSADME and ProTox-II. In the present study, the molecular docking binding features of saffron derivatives (crocetin, picrocrocin, quercetin, safranal, crocin, rutin, and dimethylcrocetin) against human COX-2 protein were assessed. Moreover, protein-protein interactions, topographic properties, gene enrichment analysis and molecular dynamics simulation were also determined. Results: The present study revealed that picrocrocin showed the highest binding affinity of -8.1 kcal/mol when docked against the COX-2 protein. PROCHECK analysis revealed that 90.3% of the protein residues were found in the most favored region. Compartmentalized Protein-Protein Interaction identified 90 interactions with an average interaction score of 0.62, and the highest localization score of 0.99 found in secretory pathways. The Computed Atlas of Surface Topography of Proteins was used to identify binding pockets and important residues that could serve as drug targets. Use of WEBnmα revealed protein dynamics by using normal mode analysis. Ligand and Receptor Dynamics used the Molecular Generalized Born Surface Area approach to determine the binding free energy of the protein. Gene enrichment analysis revealed that ovarian steroidogenesis, was the most significant enrichment pathway. Molecular dynamic simulations were executed for the best docked (COX-2-picrocrocin) complex, and the results displayed conformational alterations with more pronounced surface residue fluctuations in COX-2 with loss of the intra-protein hydrogen bonding network. The direct interaction of picrocrocin with various crucial amino-acid residues like GLN203, TYR385, HIS386 and 388, ASN382, and TRP387 causes modifications in these residues, which ultimately attenuates the activity of COX-2 protein. Conclusions: The present study revealed that picrocrocin was the most effective biomolecule and could be repurposed via computational approaches. However, various in vivo and in vitro observations are still needed.

The Emerging Landscape of Natural Small-molecule Therapeutics for Huntington's Disease.

Huntington's disease (HD) is a rare and fatal neurodegenerative disorder with no diseasemodifying therapeutics. HD is characterized by extensive neuronal loss and is caused by the inherited expansion of the huntingtin (HTT) gene that encodes a toxic mutant HTT (mHTT) protein having expanded polyglutamine (polyQ) residues. Current HD therapeutics only offer symptomatic relief. In fact, Food and Drug Administration (FDA) approved two synthetic small-molecule VMAT2 inhibitors, tetrabenazine (1) and deutetrabenazine (2), for managing HD chorea and various other diseases in clinical trials. Therefore, the landscape of drug discovery programs for HD is evolving to discover disease- modifying HD therapeutics. Likewise, numerous natural products are being evaluated at different stages of clinical development and have shown the potential to ameliorate HD pathology. The inherent anti-inflammatory and antioxidant properties of natural products mitigate the mHTT-induced oxidative stress and neuroinflammation, improve mitochondrial functions, and augment the anti-apoptotic and pro-autophagic mechanisms for increased survival of neurons in HD. In this review, we have discussed HD pathogenesis and summarized the anti-HD clinical and pre-clinical natural products, focusing on their therapeutic effects and neuroprotective mechanism/s.

Mitochondrial dysfunctions, oxidative stress and neuroinflammation as therapeutic targets for neurodegenerative diseases: An update on current advances and impediments.

Neurodegenerative diseases (NDs) such as Alzheimer disease (AD), Parkinson disease (PD), and Huntington disease (HD) represent a major socio-economic challenge in view of their high prevalence yet poor treatment outcomes affecting quality of life. The major challenge in drug development for these NDs is insufficient clarity about the mechanisms involved in pathogenesis and pathophysiology. Mitochondrial dysfunction, oxidative stress and inflammation are common pathways that are linked to neuronal abnormalities and initiation of these diseases. Thus, elucidating the shared initial molecular and cellular mechanisms is crucial for recognizing novel remedial targets, and developing therapeutics to impede or stop disease progression. In this context, use of multifunctional compounds at early stages of disease development unclogs new avenues as it acts on act on multiple targets in comparison to single target concept. In this review, we summarize overview of the major findings and advancements in recent years focusing on shared mechanisms for better understanding might become beneficial in searching more potent pharmacological interventions thereby reducing the onset or severity of various NDs.

Functional Deficits of 5×FAD Neural Stem Cells Are Ameliorated by Glutathione Peroxidase 4.

Alzheimer's disease (AD) is the most common cause of dementia affecting millions of people around the globe. Impaired neurogenesis is reported in AD as well as in AD animal models, although the underlying mechanism remains unclear. Elevated lipid peroxidation products are well-documented in AD. In current study, the role of lipid peroxidation on neural stem cell (NSCs) function is tested. Neural stem cells (NSCs) from 5×FAD mice, a widely used AD model with impaired neurogenesis, were observed to have increased levels of lipid reactive oxygen species compared to NSCs from control WT mice. 5×FAD NSCs exhibited altered differentiation potential as revealed by their propensity to differentiate into astrocytic lineage instead of neuronal lineage compared to WT NSCs. In addition, 5×FAD NSCs showed a reduced level of Gpx4, a key enzyme in reducing hydroperoxides in membrane lipids, and this reduction appeared to be caused by enhanced autophagy-lysosomal degradation of Gpx4 protein. To test if increasing Gpx4 could restore differentiation potential, NSCs from 5×FAD and Gpx4 double transgenic mice, i.e., 5×FAD/GPX4 mice were studied. Remarkably, upon differentiation, neuronal linage cells increased significantly in 5×FAD/GPX4 cultures compared to 5×FAD cultures. Taken together, the findings suggest that deficiency of lipid peroxidation defense contributes to functional decline of NSCs in AD.

Enhanced defense against ferroptosis ameliorates cognitive impairment and reduces neurodegeneration in 5xFAD mice.

Oxidative damage including lipid peroxidation is widely reported in Alzheimer's disease (AD) with the peroxidation of phospholipids in membranes being the driver of ferroptosis, an iron-dependent oxidative form of cell death. However, the importance of ferroptosis in AD remains unclear. This study tested whether ferroptosis inhibition ameliorates AD. 5xFAD mice, a widely used AD mouse model with cognitive impairment and robust neurodegeneration, exhibit markers of ferroptosis including increased lipid peroxidation, elevated lyso-phospholipids, and reduced level of Gpx4, the master defender against ferroptosis. To determine if enhanced defense against ferroptosis retards disease development, we generated 5xFAD mice that overexpress Gpx4, i.e., 5xFAD/GPX4 mice. Consistent with enhanced defense against ferroptosis, neurons from 5xFAD/GPX4 mice showed an augmented capacity to reduce lipid reactive oxygen species. In addition, compared with control 5xFAD mice, 5xFAD/GPX4 mice showed significantly improved learning and memory abilities and had reduced neurodegeneration. Moreover, 5xFAD/GPX4 mice exhibited attenuated markers of ferroptosis. Our results indicate that enhanced defense against ferroptosis is effective in ameliorating cognitive impairment and decreasing neurodegeneration of 5xFAD mice. The findings support the notion that ferroptosis is a key contributor to AD pathogenesis.

Neurodegenerative diseases and Withania somnifera (L.): An update.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal also known as 'Ashwaghanda' in Sanskrit and as 'Indian Winter Cherry' in english. is an important medicinal herb in India. It is widely used in Indian systems of medicine as an adaptogen, nerve tonic, anti-stress, memory enhancer and against cognitive deficits, insomnia, anxiety, infectious diseases, infertility, rheumatoid arthritis and gout over thousands of years. Its formulations are mainly used in Unani and Ayurvedic system of medicine. It is a remarkable centuries old herbal Rasayana used to treat neuronal ailments and is known as ''Sattvic Kapha Rasayana. AIM OF THE STUDY: To review neuroprotective properties of Withania somnifera (L.)extract as well as its active constituents in neurodegenerative diseases and other neurological ailments. MATERIALS AND METHODS: The sources of information used in present article include Indian system of Medicine reports on the use of natural products, Medicinal books, research articles and scientific databases like PubMed, Google Scholar, Web of Science, Science-Direct, SciFinder, ACS Publications and Wiley Online Library. RESULTS: Research reports based largely on preclinical studies as well as few clinical trials have highlighted the neuroprotective role of Ashwagandha against many neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease. The protective effects of Ashwagandha were accomplished by restoring mitochondrial and endothelial function, mitigation of apoptosis, inflammation and oxidative stress mechanisms. CONCLUSION: In this review, we recapitulated neuroprotective properties of Ashwagandha extracts and/or its major constituents and discussed their mechanisms of action and potential therapeutic applications. The pre-clinical as well as clinical studies suggest the use of Withania somnifera (L.) against neurodegenerative disease. However, extensive studies are warranted to validate the use of extract or its single constituents for its clinical use.

Deciphering the neuroprotective and neurogenic potential of soluble amyloid precursor protein alpha (sAPPα).

Amyloid precursor protein (APP) is a transmembrane protein expressed largely within the central nervous system. Upon cleavage, it does not produce the toxic amyloid peptide (Aß) only, which is involved in neurodegenerative progressions but via a non-amyloidogenic pathway it is metabolized to produce a soluble fragment (sAPPα) through α-secretase. While a lot of studies are focusing on the role played by APP in the pathogenesis of Alzheimer's disease, sAPPα is reported to have numerous neuroprotective effects and it is being suggested as a candidate with possible therapeutic potential against Alzheimer's disease. However, the mechanisms through which sAPPα precisely works remain elusive. We have presented a comprehensive review of how sAPPα is regulating the neuroprotective effects in different biological models. Moreover, we have focused on the role of sAPPα during different developmental stages of the brain, neurogenic microenvironment in the brain and how this metabolite of APP is regulating the neurogenesis which is regarded as a compelling approach to ameliorate the impaired learning and memory deficits in dementia and diseases like Alzheimer's disease. sAPPα exerts beneficial physiological, biochemical and behavioral effects mitigating the detrimental effects of neurotoxic compounds. It has shown to increase the proliferation rate of numerous cell types and promised the synaptogenesis, neurite outgrowth, cell survival and cell adhesion. Taken together, we believe that further studies are warranted to investigate the exact mechanism of action so that sAPPα could be developed as a novel therapeutic target against neuronal deficits.

Attenuation of Glutamate-Induced Excitotoxicity by Withanolide-A in Neuron-Like Cells: Role for PI3K/Akt/MAPK Signaling Pathway.

Glutamate-induced excitotoxicity is one of the major underlying mechanisms for neurodegenerative diseases. Efforts are being made to treat such conditions with an array of natural compounds that can modulate the release of glutamate or the underlying mechanisms associated with it. Withania somnifera extract has potent pharmacologic activity similar to that of Korean Ginseng tea and is used to treat several neuronal disorders. However, to date, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to investigate withanolide-A, one of the active constituents of Withania somnifera against glutamate-induced excitotoxicity in retinoic acid differentiated Neuro2a neuroblastoma cells. The results indicated that glutamate treatment for 2 h induced death in cells that was significantly attenuated by pre-treatment with MK-801 (specific NMDA receptor antagonist) and different concentrations of withanolide-A. Withanolide-A abated the glutamate-induced influx of intracellular calcium and excessive ROS production significantly. Further on, glutamate treatment resulted in increased levels of pro-apoptotic and decreased levels of anti-apoptotic proteins, and these protein levels were normalized by various doses of withanolide-A. All of these protective effects were partly due to inhibition of MAPK family proteins and activation of PI3K/Akt signaling. Thus, our results suggest that withanolide-A may serve as potential neuroprotective agent.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Withanone, an Active Constituent from Withania somnifera, 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.

Toxicogenetic evaluation of dichlorophene in peripheral blood and in the cells of the immune system using molecular and flow cytometric approaches.

Dichlorophene; a halogenated phenolic compound with wide applications as a fungicide, bactericide and antiprotozoan. Dichlorophene spray also has therapeutic use in the disease digital dermatitis. In guinea pigs, a few studies obtained mixed results in dicholorophene sensitization tests. In consideration of the fact, that the mechanism of its genotoxicity has not been adequately elucidated lead to present study assessing the acute in vivo toxicological impact in Rattus norvegicus. A systematic research has been made encompassing the use of molecular and flow cytometric approaches. The study was designed on blood cells for comet assay which revealed dichlorophene induced DNA damage in all exposures understandable in time dependent manner. The feasibility of this assay was also established as an effective, fast and accurate method with a great potential in biomonitoring. Contemporary molecular techniques were further engaged using leukocytes for the cell apoptosis/cycle and mitochondrial membrane potential employing propidium iodide staining and rhodamine 123 respectively. The effect on cell cycle phases and mitochondrial membrane permeability was analyzed through flow cytometry. These indicators exposed that dichlorophene decreased the mitochondrial membrane potential, altered the cell cycle and confirmed the DNA damage leading to apoptosis of the cells of the immune system accountable for immunotoxic effects of dichlorophene on rat leukocytes.

Endophytes and Neurodegenerative Diseases: A Hope in Desperation.

Neurodegeneration is progressive loss of functional and structural integrity of the central nervous system. Neurodegenerative disorders are yet without any reliable therapy because the neurons of the central nervous system have limited ability to regenerate. Current therapeutic approaches rely mainly on abrogation of symptoms and leave the dying neurons to their fate. Protective and/or rescuing treatments need to be explored fully to suppress neuronal death that will automatically alleviate the symptoms. Adequate precedent exists in literature regarding the neuroprotective activity of endophytes. Endophytes are a class of microorganisms which colonize healthy plant tissues without causing any apparent harm to the host. Chemical moieties from known endophytes have been used against many disease models including neurodegenerative diseases. There is great hope that novel bioactive molecules from newer endophytes can impede pathogenic mechanisms and progression of many diseases. In this review, we will discuss promising pharmacological or clinical relevance of endophytes against various neurodegenerative diseases.

Promise of Retinoic Acid-Triazolyl Derivatives in Promoting Differentiation of Neuroblastoma Cells.

Retinoic acid induces differentiation in various types of cells including skeletal myoblasts and neuroblasts and maintains differentiation of epithelial cells. The present study demonstrates synthesis and screening of a library of retinoic acid-triazolyl derivatives for their differentiation potential on neuroblastoma cells. Click chemistry approach using copper(I)-catalyzed azide-alkyne cycloaddition was adopted for the preparation of these derivatives. The neurite outgrowth promoting potential of retinoic acid-triazolyl derivatives was studied on neuroblastoma cells. Morphological examination revealed that compounds 8a, 8e, 8f, and 8k, among the various derivatives screened, exhibited promising neurite-outgrowth inducing activity at a concentration of 10 µM compared to undifferentiated and retinoic acid treated cells. Further on, to confirm this differentiation potential of these compounds, neuroblastoma cells were probed for expression of neuronal markers such as NF-H and NeuN. The results revealed a marked increase in the NF-H and NeuN protein expression when treated with 8a, 8e, 8f, and 8k compared to undifferentiated and retinoic acid treated cells. Thus, these compounds could act as potential leads in inducing neuronal differentiation for future studies.

Pharmacologic overview of Withania somnifera, the Indian Ginseng.

Withania somnifera, also called 'Indian ginseng', is an important medicinal plant of the Indian subcontinent. It is widely used, singly or in combination, with other herbs against many ailments in Indian Systems of Medicine since time immemorial. Withania somnifera contains a spectrum of diverse phytochemicals enabling it to have a broad range of biological implications. In preclinical studies, it has shown anti-microbial, anti-inflammatory, anti-tumor, anti-stress, neuroprotective, cardioprotective, and anti-diabetic properties. Additionally, it has demonstrated the ability to reduce reactive oxygen species, modulate mitochondrial function, regulate apoptosis, and reduce inflammation and enhance endothelial function. In view of these pharmacologic properties, W. somnifera is a potential drug candidate to treat various clinical conditions, particularly related to the nervous system. In this review, we summarize the pharmacologic characteristics and discuss the mechanisms of action and potential therapeutic applications of the plant and its active constituents.