Understanding the Modulation of α-Synuclein Fibrillation by N-Acetyl Aspartate: A Brain Metabolite.

α-Synuclein (α-Syn) fibrillation is a prominent contributor to neuronal deterioration and plays a significant role in the advancement of Parkinson's Disease (PD). Considering this, the exploration of novel compounds that can inhibit or modulate the aggregation of α-Syn is a topic of significant research. This study, for the first time, elucidated the effect of N-acetyl aspartate (NAA), a brain osmolyte, on α-Syn aggregation using spectroscopic and microscopic approaches. Thioflavin T (ThT) assay revealed that a lower concentration of NAA inhibits α-Syn aggregation, whereas higher concentrations of NAA accelerate the aggregation. Further, this paradoxical effect of NAA was complemented by ANS, RLS, and the turbidity assay. The secondary structure transition was more pronounced at higher concentrations of NAA by circular dichroism, corroborating the fluorescence spectroscopic observations. Confocal microscopy also confirmed the paradoxical effect of NAA on α-Syn aggregation. Interaction studies including fluorescence quenching and molecular docking were employed to determine the binding affinity and critical residues involved in the α-Syn-NAA interaction. The explanation for this paradoxical nature of NAA could be a solvophobic effect. The results offer a profound understanding of the modulatory mechanism of α-Syn aggregation by NAA, thereby suggesting the potential role of NAA at lower concentrations in therapeutics against α-Syn aggregation-related disorders.

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production.

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

Recent advancement in therapeutic strategies for Alzheimer's disease: Insights from clinical trials.

Alzheimer's disease (AD) is the most prevalent form of dementia, characterized by the presence of plaques of amyloid beta and Tau proteins. There is currently no permanent cure for AD; the only medications approved by the FDA for mild to moderate AD are cholinesterase inhibitors, NMDA receptor antagonists, and immunotherapies against core pathophysiology, that provide temporary relief only. Researchers worldwide have made significant attempts to find new targets and develop innovative therapeutic molecules to treat AD. The FDA-approved drugs are palliative and couldn't restore the damaged neuron cells of AD. Stem cells have self-differentiation properties, making them prospective therapeutics to treat AD. The promising results in pre-clinical studies of stem cell therapy for AD seek attention worldwide. Various stem cells, mainly mesenchymal stem cells, are currently in different phases of clinical trials and need more advancements to take this therapy to the translational level. Here, we review research from the past decade that has identified several hypotheses related to AD pathology. Moreover, this article also focuses on the recent advancement in therapeutic strategies for AD treatment including immunotherapy and stem cell therapy detailing the clinical trials that are currently undergoing development.

Inhibitory potential of N-acetylaspartate against protein glycation, AGEs formation and aggregation: Implication of brain osmolyte in glycation-related complications.

Protein glycation and aggregation have a pivotal role in many diseases including diabetes and neurodegenerative disorders. N-acetyl aspartate (NAA), an osmolyte derived from L-aspartic acid, is one of the most abundant metabolites in the mammalian brain. Although NAA is supposed to be a substitute for a neuronal marker, its function is not fully elucidated. Herein, we have investigated the effect of NAA on glycation, AGEs formation and aggregation of irisin. AGE-specific fluorescence showed strong inhibition of AGEs formation in the presence of NAA, demonstrating its anti-glycating property. The aggregates present in MG-modified irisin were also reduced by NAA, which was confirmed by Thioflavin T fluorescence and fluorescence microscopy. Further, for the explanation of the strong anti-glycating potential of NAA, the interaction between irisin and NAA was also examined. Interaction studies involving steady-state fluorescence and molecular docking demonstrated that hydrogen bonding and salt bridges by NAA stabilize the irisin. It was found that glycation-prone residues i.e., lysine and arginine are specifically involved in the interaction which might prevent them from getting modified during the process of glycation. This study for the first time reported the antiglycating potential of NAA which can be implicated in the therapeutic management of various glycation-related complications.

Characterization of advanced glycation end products and aggregates of irisin: Multispectroscopic and microscopic approaches.

Glycation of proteins leading to the formation of advanced glycation end products (AGEs) has been demonstrated to contribute to the pathogenesis of several diseases. Irisin is a clinically significant protein, putatively involved in obesity, diabetes, and neurological disorders. This study aimed to monitor the methyl-glyoxal (MG) induced AGEs and aggregate formation of irisin, as a function of time, employing multispectroscopic and microscopic approaches. ANS fluorescence suggested a molten globule-like state on Day 6, followed by the formation of irisin AGEs adducts, as confirmed by AGE-specific fluorescence. Glycation of irisin led to aggregate formation, which was characterized by Thioflavin T fluorescence, CD spectroscopy, and microscopic studies. These aggregates were confirmed by exploiting fluorescence microscopy, confocal, and transmission electron microscopy. Molecular docking was performed to determine the crucial residues of irisin involved in irisin-MG interaction. Usually, MG is present in trace amounts as a metabolic by-product in the body, which is found to be elevated in diseased conditions viz. diabetes and Alzheimer's disease. This study characterized the AGEs and aggregates of clinically important protein, irisin; and since MG level has been found to be increased in various pathological conditions, this study provides a clinical perspective. There is a possibility that elevated MG concentrations might glycate irisin resulting in reduced irisin levels as reported in pathological conditions. However, further investigations are required to prove it.

Mitochondrial Dysfunction: Pathophysiology and Mitochondria-Targeted Drug Delivery Approaches.

Mitochondria are implicated in a wide range of functions apart from ATP generation, and, therefore, constitute one of the most important organelles of cell. Since healthy mitochondria are essential for proper cellular functioning and survival, mitochondrial dysfunction may lead to various pathologies. Mitochondria are considered a novel and promising therapeutic target for the diagnosis, treatment, and prevention of various human diseases including metabolic disorders, cancer, and neurodegenerative diseases. For mitochondria-targeted therapy, there is a need to develop an effective drug delivery approach, owing to the mitochondrial special bilayer structure through which therapeutic molecules undergo multiple difficulties in reaching the core. In recent years, various nanoformulations have been designed such as polymeric nanoparticles, liposomes, inorganic nanoparticles conjugate with mitochondriotropic moieties such as mitochondria-penetrating peptides (MPPs), triphenylphosphonium (TPP), dequalinium (DQA), and mitochondrial protein import machinery for overcoming barriers involved in targeting mitochondria. The current approaches used for mitochondria-targeted drug delivery have provided promising ways to overcome the challenges associated with targeted-drug delivery. Herein, we review the research from past years to the current scenario that has identified mitochondrial dysfunction as a major contributor to the pathophysiology of various diseases. Furthermore, we discuss the recent advancements in mitochondria-targeted drug delivery strategies for the pathologies associated with mitochondrial dysfunction.

Unraveling the Binding Mechanism of Alzheimer's Drugs with Irisin: Spectroscopic, Calorimetric, and Computational Approaches.

The prevalence of Alzheimer's disease (AD) has been a major health concern for a long time. Despite recent progress, there is still a strong need to develop effective disease-modifying therapies. Several drugs have already been approved to retard the progression of AD-related symptoms; however, there is a need to develop an effective carrier system for the delivery of drugs to combat such diseases. In recent years, various biological macromolecules, including proteins, have been used as carriers for drug delivery. Irisin is a beneficial hormone in such diseases, including AD and related pathologies. Herein, the interaction mechanism of irisin with AD drugs such as memantine, galantamine, and fluoxetine is investigated. Fluorescence studies revealed that the above drugs bind to irisin with significant affinity, with fluoxetine having the highest binding affinity. Isothermal titration calorimetry (ITC) complemented the spontaneous binding of these drugs with irisin, delineating various associated thermodynamic and binding parameters. Molecular docking further validated the fluorescence and ITC results and unfolded the mechanism that hydrogen bonding governs the binding of fluoxetine to irisin with a significant binding score, i.e., -6.3 kcal/mol. We believe that these findings provide a promising solution to fight against AD as well as a platform for further research to utilize irisin in the drug-delivery system for an effective therapeutic strategy.

FNDC5/Irisin: Physiology and Pathophysiology.

A sedentary lifestyle or lack of physical activity increases the risk of different diseases, including obesity, diabetes, heart diseases, certain types of cancers, and some neurological diseases. Physical exercise helps improve quality of life and reduces the risk of many diseases. Irisin, a hormone induced by exercise, is a fragmented product of FNDC5 (a cell membrane protein) and acts as a linkage between muscles and other tissues. Over the past decade, it has become clear that irisin is a molecular mimic of exercise and shows various beneficial effects, such as browning of adipocytes, modulation of metabolic processes, regulation of bone metabolism, and functioning of the nervous system. Irisin has a role in carcinogenesis; numerous studies have shown its impact on migration, invasion, and proliferation of cancer cells. The receptor of irisin is not completely known; however, in some tissues it probably acts via a specific class of integrin receptors. Here, we review research from the past decade that has identified irisin as a potential therapeutic agent in the prevention or treatment of various metabolic-related and other diseases. This article delineates structural and biochemical aspects of irisin and provides an insight into the role of irisin in different pathological conditions.

MAP/Microtubule Affinity Regulating Kinase 4 Inhibitory Potential of Irisin: A New Therapeutic Strategy to Combat Cancer and Alzheimer's Disease.

Irisin is a clinically significant protein playing a valuable role in regulating various diseases. Irisin attenuates synaptic and memory dysfunction, highlighting its importance in Alzheimer's disease. On the other hand, Microtubule Affinity Regulating Kinase 4 (MARK4) is associated with various cancer types, uncontrolled neuronal migrations, and disrupted microtubule dynamics. In addition, MARK4 has been explored as a potential drug target for cancer and Alzheimer's disease therapy. Here, we studied the binding and subsequent inhibition of MARK4 by irisin. Irisin binds to MARK4 with an admirable affinity (K = 0.8 × 107 M-1), subsequently inhibiting its activity (IC50 = 2.71 µm). In vitro studies were further validated by docking and simulations. Molecular docking revealed several hydrogen bonds between irisin and MARK4, including critical residues, Lys38, Val40, and Ser134. Furthermore, the molecular dynamic simulation showed that the binding of irisin resulted in enhanced stability of MARK4. This study provides a rationale to use irisin as a therapeutic agent to treat MARK4-associated diseases.

Multispectroscopic and Molecular Docking Insight into Elucidating the Interaction of Irisin with Rivastigmine Tartrate: A Combinational Therapy Approach to Fight Alzheimer's Disease.

This study was aimed to study the interaction between purified irisin and rivastigmine tartrate (RT), a cholinesterase inhibitor used in Alzheimer's therapy. Irisin mainly promotes brown fat-like features in white adipose tissues; however, it has some important role in the nervous system also, i.e., capable of opposing synapse and memory failure in Alzheimer's disease (AD). The recombinant protein was purified by Ni-NTA chromatography and characterized using spectroscopic and in silico techniques. Further, the mechanism of interaction between irisin and RT was investigated using various biophysical techniques. Fluorescence quenching studies suggested that there exists a moderate binding between irisin and RT with a binding constant (K) of 104 M-1 and the irisin-RT complex is guided by a combination of both static and dynamic modes of quenching. Thermodynamic parameters suggested the reaction to be driven by hydrogen bonding, making it specific. FTIR and CD spectroscopy suggested no secondary structural alterations in irisin in the presence of RT. Molecular docking investigation provided an insight into the important residues that play a key role in irisin-RT interactions. This study delineates an important finding in AD therapy and can provide a platform further to explore the potential of irisin in AD treatment.

An Insight Into Mitochondrial Dysfunction and its Implications in Neurological Diseases.

In the last few years, a massive increase in research has been observed that focuses on investigating the role of mitochondria in the pathogenesis of several neurodegenerative disorders. Mitochondria are vital cell organelles having important roles in different cellular processes, including energy production, calcium signaling, Reactive Oxygen Species (ROS) generation, apoptosis, etc. Therefore, healthy mitochondria are necessary for cell survival and functioning. It would seem feasible that mitochondrial dysfunction will have implications in various pathological conditions. A large body of evidence indicates the role of the mitochondrion as a potential key player in the loss or dysfunction of neurons in various neurodegenerative disorders. In this review, we provide an insight into mitochondrial dysfunction and its involvement in the pathology of several neurological diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, Hypoxic- Ischemic Brain Injury, and many more.