Role of Withaferin A as a Neuroprotectant against Beta Amyloid Induced Toxicity and associated mechanism

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers

Role of Extracellular Vesicles in Substance Abuse and HIV-Related Neurological Pathologies

Extracellular vesicles (EVs) are a broad, heterogeneous class of membranous lipid-bilayer vesicles that facilitate intercellular communication throughout the body. As important carriers of various types of cargo, including proteins, lipids, DNA fragments, and a variety of small noncoding RNAs, including miRNAs, mRNAs, and siRNAs, EVs may play an important role in the development of addiction and other neurological pathologies, particularly those related to HIV. In this review, we summarize the findings of EV studies in the context of methamphetamine (METH), cocaine, nicotine, opioid, and alcohol use disorders, highlighting important EV cargoes that may contribute to addiction. Additionally, as HIV and substance abuse are often comorbid, we discuss the potential role of EVs in the intersection of substance abuse and HIV. Taken together, the studies presented in this comprehensive review shed light on the potential role of EVs in the exacerbation of substance use and HIV. As a subject of growing interest, EVs may continue to provide information about mechanisms and pathogenesis in substance use disorders and CNS pathologies, perhaps allowing for exploration into potential therapeutic options

Withaferin A Suppresses Beta Amyloid in APP Expressing Cells: Studies for Tat and Cocaine Associated Neurological Dysfunctions

Neurological disorders are the biggest concern globally. Out of ~36 million human immunodeficiency virus (HIV) positive people, about 30%–60% exhibit neurological disorders, including dementia and Alzheimer’s disease (AD) like pathology. In AD or AD like neurological disorders, the pathogenesis is mainly due to the abnormal accumulation of extracellular amyloid beta (Aβ). In this era of antiretroviral therapy, the life span of the HIV-infected individuals has increased leading towards increased neurocognitive dysfunction in nearly 30% of HIV-infected individuals, specifically older people. Deposition of the Aβ plaques in the CNS is one the major phenomenon happening in aging HIV patients. ART suppresses the viral replication, but the neurotoxic protein (Tat) is still produced and results in increased levels of Aβ. Furthermore, drugs of abuse like cocaine (coc) is known to induce the HIV associated neurocognitive disorders as well as the Aβ secretion. To target the Tat and coc induced Aβ secretion, we propose a potent bifunctional molecule Withaferin A (WA) which may act as a neuro-protectant against Aβ neurotoxicity. In this study, we show that WA reduces secreted Aβ and induced neurotoxicity in amyloid precursor protein (APP)-plasmid transfected SH-SY5Y cells (SH-APP). In this study, we show that in SH-APP cells, Aβ secretion is induced in the presence of HIV-1 Tat (neurotoxic) and drug of abuse coc. Our fluorescent microscopy studies show the increased concentration of Aβ40 in Tat (50 ng/ml) and coc (0.1 μM) treated SH-APP cells as compared to control. Our dose optimization study show, lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40 levels, without inducing cytotoxicity in the SH-APP cells. Additionally, WA reduces the Tat and cocaine induced Aβ levels. Therefore, we propose that Aβ aggregation is induced by the presence of Tat and coc and WA is potent in reducing the secreted Aβ and induced neurotoxicity. Our study provides new opportunities for exploring the pathophysiology and targeting the neurological disorders

Japanese encephalitis virus: an emerging pathogen

Japanese Encephalitis Virus (JEV) is a flavivirus maintained in a zoonotic cycle which involves pigs, birds and Culex species of mosquitoes causing fatal encephalitis endemic most of Asia and as far as Australia from its putative origin in Indonesia and Malaysia. The principle vector is Culex mosquito, most important being Culex tritaenorhynchus, present in greatest density in rainy season (June to November) Humans are accidental dead-end-hosts as they do not develop a level of viraemia sufficient to infect mosquitoes. The natural cycle of JEV consists of pig-mosquito-pig or bird-mosquito-bird and pigs serve as a biological amplifiers and reservoirs. The risk for Japanese encephalitis varies by appropriate ecological conditions and season to cause epidemics and epizootics. Disease control by vaccination is considered to be most effective. The Envelope (E) protein is dominant antigen including immunologic responses in infected host and eliciting virus neutralizing antibodies. Large scale immunization of susceptible human population is highly important to prevent this deadly infection. Attempts are being made to develop enhanced vaccines using the recombinant DNA technology. Since the existing inactivated, live attenuated or killed vaccines have side effects such as neurological disorders and systemic hypersensitivity, DNA based vaccines might aid the purpose of combating against JEV which are presently under clinical trials. Protection at personal level would help to reduce the incidence of the disease. In India vaccination against Japanese encephalitis are administered in areas where the disease is hyper-endemic.