MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Design and development of benzyl piperazine linked 5-phenyl-1,2,4-triazole-3-thione conjugates as potential agents to combat Alzheimer's disease.

Our present work demonstrates the molecular hybridization-assisted design, synthesis, and biological evaluation of 22 benzylpiperazine-linked 1,2,4-triazole compounds (PD1-22) as AD modifying agents. All the compounds were tested for their in vitro hChEs, hBACE-1, and Aß-aggregation inhibition properties. Among them, compound PD-08 and PD-22 demonstrated good hChE and hBACE-1 inhibition as compared to standards donepezil and rivastigmine. Both compounds displaced PI from PAS at 50 µM concentration which was comparable to donepezil and also demonstrated anti-Aß aggregation properties in self- and AChE-induced thioflavin T assay. Both compounds have shown excellent BBB permeation via PAMPA-BBB assay and were found to be non-neurotoxic at 80 µM concentration against differentiated SH-SY5Y cell lines. Compound PD-22 demonstrated an increase in rescued eye phenotype in Aß-phenotypic drosophila AD model and amelioration of behavioral deficits in the Aß-induced rat model of AD. The in-silico docking studies of compound PD-22 revealed a good binding profile towards CAS and PAS residues of AChE and the catalytic dyad of the BACE-1. The 100 ns molecular dynamics simulation studies of compound PD-22 complexed with AChE and BACE-1 enzymes suggested stable ligand-protein complex throughout the simulation run. Based on our findings compound PD-22 could further be utilized as a lead to design a promising candidate for AD therapy.

Extraction, isolation, synthesis, and biological evaluation of novel piperic acid derivatives for the treatment of Alzheimer's disease.

In this paper, we developed a series of piperic acid (PA) analogs with the aim of overcoming the limitations associated with the natural products for the management of Alzheimer's disease (AD). A comprehensive SAR study was performed to enhance cholinesterase inhibition of PA. The acetylcholinesterase inhibition and its kinetic data suggested 6j as the lead molecule (AChE IC50 = 2.13 ± 0.015 µM, BChE = 28.19 ± 0.20%), in comparison to PA (AChE = 7.14 ± 0.98%) which was further selected for various biological studies in AD models. 6j, exhibited interaction with the peripheral anionic site of AChE, BBB permeability (Pe = 7.98), and antioxidant property (% radical scavenging activity = 35.41 ± 1.09, 2.43 ± 1.65, for 6j and PA at 20 M[Formula: see text], respectively). The result from the metal chelation study suggests that 6j did not effectively chelate iron. The molecular modeling studies suggested that 6j could effectively interact with Ser293, Phe295, Arg296, and Tyr34 of AChE. In the cell-based cytotoxicity studies, 6j exhibited cytocompatibility at the different tested concentrations. The acute toxicity data on mice suggested that compound 6j had no renal and hepatotoxicity at 500 mg/kg. Moreover, 6j could effectively reverse scopolamine-induced amnesia by improving spatial and cognitive memory in mice. The above results strongly suggest that compound 6j may act as a novel multi-targeted lead for AD therapy.

Development of luminescent atacamite nanoclusters for bioimaging and photothermal applications.

Fluorescent atacamite nanoclusters (FANCs) have been developed and modified with silica for Drosophila salivary gland tissue imaging and photothermally induced cell death of osteosarcoma MG-63 cells. FANCs were synthesized with Moringa oleifera leaf extract without using any hazardous reducing and external capping agents. FANC was further used to evaluate light absorption, fluorescence emission, band gap, and magnetic properties as the first report on such nanoclusters. Upon excitation with a 350 nm light source, FANCs exhibited fluorescence at 460 nm, with a relative quantum yield of 0.3%. Besides, silica-encapsulated fluorescent atacamite nanoclusters (SEFANC) manifested remarkable improvement in emission, quantum yield (1.7%), shelf-life (15 d), biocompatibility, and photostability. Concomitantly, it has also increased the absorption in the near-infrared region and demonstrated high heat generation potential (42 °C → 50 °C). The above results suggest that FANC can be a potential candidate in the area of nanomedicine for a number of applications such as bioimaging, photothermal therapy, etc.

GABA mediated reduction of arsenite toxicity in rice seedling through modulation of fatty acids, stress responsive amino acids and polyamines biosynthesis.

γ-aminobutyric acid (GABA) is a free amino acid, which helps to counteract biotic and abiotic stresses in plants. In the present study, two concentrations of GABA, i.e., 0.5 mM and 1 mM were applied to examine the tolerance of rice seedlings against As(III) (25 µM) toxicity, through the modulations of fatty acids (FAs), stress responsive amino acids (AAs) and polyamines (PAs) biosynthesis. Exogenous GABA (0.5 mM) application significantly reduced the H2O2 and TBARS levels and recovered the growth parameters against As(III) stressed rice seedlings. Simultaneously, co-application of GABA (0.5 and 1 mM) and As(III), consistently enhanced the level of unsaturated fatty acids (USFA) (cis-10-pentadecanoic acid, oleic acid, α-linolenic acid and γ-linolenic acid), which was higher than saturated fatty acid (SFA). Among the USFAs, level of linolenic acid was found to be always higher with GABA application. Similarly, elevated level of AAs (proline, methionine, glutamic acid and cysteine) was also observed with the application of GABA (0.5 and 1 mM) in As(III) stressed seedlings. GABA also enhanced the expression of genes involved in the polyamine synthesis pathway namely arginine decarboxylase (AD), spermine (SPM) and spermidine (SPD) synthase against As(III) treatments, which was higher in roots than in shoots, resulting in enhanced root PAs level. Contrarily, the expression of S-adenosylmethionine decarboxylase (S-AMD) was significantly higher in shoots. Among all the PAs, level of putrescine (PUT) was found to be highest with GABA application. Overall, the study demonstrates that GABA (0.5 mM) at lower concentration plays a vital role in As(III) tolerance by enhancing the biosynthesis of USFA, AA and PA, reducing the level of TBARS and H2O2 in rice seedlings.

Folic acid supplementation rescues anomalies associated with knockdown of parkin in dopaminergic and serotonergic neurons in Drosophila model of Parkinson's disease.

parkin loss associated early-onset of Parkinson's disease, involves mitochondrial dysfunction and oxidative stress as the plausible decisive molecular mechanisms in disease pathogenesis. Mitochondrial dysfunction involves several up/down regulation of gene products, one of which being p53 is found to be elevated. Elevated p53 is involved in mitochondrial mediated apoptosis of neuronal cells in Parkinson's patients who are folate deficient as well. The present study therefore attempts to examine the effect of Folic acid (FA) supplementation in alleviation of anomalies associated with parkin knockdown using RNAi approach, specific to Dopaminergic (DA) neurons in Drosophila model system. Here we show that FA supplementation provide protection against parkin RNAi associated discrepancies, thereby improves locomotor ability, reduces mortality and oxidative stress, and partially improves Zn levels. Further, metabolic active cell status and ATP levels were also found to be improved thereby indicating improved mitochondrial function. To corroborate FA supplementation in mitochondrial functioning further, status of p53 and spargel was checked by qRT-PCR. Here we show that folic acid supplementation enrich mitochondrial functioning as depicted from improved spargel level and lowered p53 level, which was originally vice versa in parkin knockdown flies cultured in standard media. Our data thus support the potential of folic acid in alleviating the behavioural defects, oxidative stress, augmentation of zinc and ATP levels in parkin knock down flies. Further, folic acid role in repressing mitochondrial dysfunction is encouraging to further explore its possible mechanistic role to be utilized as potential therapeutics for Parkinson's disease.

Folic Acid Supplementation Ameliorates Oxidative Stress, Metabolic Functions and Developmental Anomalies in a Novel Fly Model of Parkinson's Disease.

Mutations in parkin cause early-onset Parkinson's disease. Studies involving Drosophila model have emphasised mitochondrial dysfunction as a critical event in disease pathogenesis. In this context, we employed a novel recessive allele of parkin, park (c00062) , for the current study. The piggyBac insertion at 3rd intron of parkin in park (c00062) was confirmed by PCR. Homozygous park (c00062) has diminished levels of truncated parkin transcript with no detectable protein as confirmed by qRT-PCR and western blot analysis, respectively. The homozygous park (c00062) displayed severe developmental anomalies involving reduced body size, ~45 % pupal lethality, high mortality with locomotory defect, elevated oxidative stress, low metabolic active cell status with low mitochondrial respiration as reflected from reduced ATP levels. Further, folic acid therapeutic potential was analysed in park (c00062) . Here we show that dietary folic acid provided protection against disparities involving pupal lethality, high mortality, locomotory defect, elevated oxidative stress and low metabolic active cell status associated with park (c00062) . Further mitochondrial respiration was enhanced as reflected from improved ATP levels in folate supplemented park (c00062) . To corroborate mitochondrial functioning further our analysis regarding transcript status of p53 and spargel by qRT-PCR, revealed down regulation of p53 and up regulation of spargel in folate supplemented park (c00062) , which was originally vice a versa. Our data thus support the potential of FA in alleviating the disparities associated with parkin loss of function in fly model. Further, FA role in alleviating mitochondrial dysfunction is encouraging to further explore FA mechanistic role to be utilized as potential therapeutics for parkin mediated neurodegenerative diseases.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy.

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), ß secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aß aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 µM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 µM) along with good anti-Aß aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 µM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aß-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand-protein confirmation throughout the simulation run.

Lead optimization based design, synthesis, and pharmacological evaluation of quinazoline derivatives as multi-targeting agents for Alzheimer's disease treatment.

The complexity and multifaceted nature of Alzheimer's disease (AD) have driven us to further explore quinazoline scaffolds as multi-targeting agents for AD treatment. The lead optimization strategy was utilized in designing of new series of derivatives (AK-1 to AK-14) followed by synthesis, characterization, and pharmacological evaluation against human cholinesterase's (hChE) and ß-secretase (hBACE-1) enzymes. Amongst them, compounds AK-1, AK-2, and AK-3 showed good and significant inhibitory activity against both hAChE and hBACE-1 enzymes with favorable permeation across the blood-brain barrier. The most active compound AK-2 revealed significant propidium iodide (PI) displacement from the AChE-PAS region and was non-neurotoxic against SH-SY5Y cell lines. The lead molecule (AK-2) also showed Aß aggregation inhibition in a self- and AChE-induced Aß aggregation, Thioflavin-T assay. Further, compound AK-2 significantly ameliorated Aß-induced cognitive deficits in the Aß-induced Morris water maze rat model and demonstrated a significant rescue in eye phenotype in the Aꞵ-phenotypic drosophila model of AD. Ex-vivo immunohistochemistry (IHC) analysis on hippocampal rat brains showed reduced Aß and BACE-1 protein levels. Compound AK-2 suggested good oral absorption via pharmacokinetic studies and displayed a good and stable ligand-protein interaction in in-silico molecular modeling analysis. Thus, the compound AK-2 can be regarded as a lead molecule and should be investigated further for the treatment of AD.

Design, Synthesis, and Biological Evaluation of Ferulic Acid Template-Based Novel Multifunctional Ligands Targeting NLRP3 Inflammasome for the Management of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia, which arises due to low levels of acetyl and butyrylcholines, an increase in oxidative stress, inflammation, metal dyshomeostasis, Aß and tau aggregations. The currently available drugs for AD treatment can provide only symptomatic relief without interfering with pathological hallmarks of the disease. In our ongoing efforts to develop naturally inspired novel multifunctional molecules for AD, systematic SAR studies on EJMC-4e were caried out to improve its multifunctional properties. The rigorous medicinal efforts led to the development of 12o, which displayed a 15-fold enhancement in antioxidant properties and a 2-fold increase in the activity against AChE and BChE over EJMC-4e. Molecular docking and dynamics studies revealed the binding sites and stability of the complex of 12o with AChE and BChE. The PAMPA-BBB assay clearly demonstrated that 12o can easily cross the blood-brain barrier. Interestingly, 12o also expresses promising metal chelation activity, while EJMC-4e was found to be devoid of this property. Further, 12o inhibited metal-induced or self Aß1-42 aggregation. Observing the neuroprotection ability of 12o against H2O2-induced oxidative stress in the PC-12 cell line is noteworthy. Furthermore, 12o also inhibited NLRP3 inflammasome activation and attenuated mitochondrial-induced ROS and MMP damage caused by LPS and ATP in HMC-3 cells. In addition, 12o is able to effectively reduce mitochondrial and cellular oxidative stress in the AD Drosophila model. Finally, 12o could reverse memory impairment in the scopolamine-induced AD mice model, as evident through in vivo and ex vivo studies. These findings suggest that this compound may act as a promising candidate for further improvement in the management of AD.

Design, Synthesis, and Biological Evaluation of Ferulic Acid-Piperazine Derivatives Targeting Pathological Hallmarks of Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent cause of dementia and is characterized by low levels of acetyl and butyrylcholine, increased oxidative stress, inflammation, accumulation of metals, and aggregations of Aß and tau proteins. Current treatments for AD provide only symptomatic relief without impacting the pathological hallmarks of the disease. In our ongoing efforts to develop naturally inspired novel multitarget molecules for AD, through extensive medicinal chemistry efforts, we have developed 13a, harboring the key functional groups to provide not only symptomatic relief but also targeting oxidative stress, able to chelate iron, inhibiting NLRP3, and Aß1-42 aggregation in various AD models. 13a exhibited promising anticholinesterase activity against AChE (IC50 = 0.59 ± 0.19 µM) and BChE (IC50 = 5.02 ± 0.14 µM) with excellent antioxidant properties in DPPH assay (IC50 = 5.88 ± 0.21 µM) over ferulic acid (56.49 ± 0.62 µM). The molecular docking and dynamic simulations further corroborated the enzyme inhibition studies and confirmed the stability of these complexes. Importantly, in the PAMPA-BBB assay, 13a turned out to be a promising molecule that can efficiently cross the blood-brain barrier. Notably, 13a also exhibited iron-chelating properties. Furthermore, 13a effectively inhibited self- and metal-induced Aß1-42 aggregation. It is worth mentioning that 13a demonstrated no symptom of cytotoxicity up to 30 µM concentration in PC-12 cells. Additionally, 13a inhibited the NLRP3 inflammasome and mitigated mitochondrial-induced reactive oxygen species and mitochondrial membrane potential damage triggered by LPS and ATP in HMC-3 cells. 13a could effectively reduce mitochondrial and cellular reactive oxygen species (ROS) in the Drosophila model of AD. Finally, 13a was found to be efficacious in reversing memory impairment in a scopolamine-induced AD mouse model in the in vivo studies. In ex vivo assessments, 13a notably modulates the levels of superoxide, catalase, and malondialdehyde along with AChE and BChE. These findings revealed that 13a holds promise as a potential candidate for further development in AD management.

DNA binding and anti-cancer activity of redox-active heteroleptic piano-stool Ru(II), Rh(III), and Ir(III) complexes containing 4-(2-methoxypyridyl)phenyldipyrromethene.

The synthesis of four novel heteroleptic dipyrrinato complexes [(η(6)-arene)RuCl(2-pcdpm)] (η(6)-arene = C6H6, 1; C10H14, 2) and [(η(5)-C5Me5)MCl(2-pcdpm)] (M = Rh, 3; Ir, 4) containing a new chelating ligand 4-(2-methoxypyridyl)-phenyldipyrromethene (2-pcdpm) have been described. The complexes 1-4 have been fully characterized by various physicochemical techniques, namely, elemental analyses, spectral (ESI-MS, IR, (1)H, (13)C NMR, UV/vis) and electrochemical studies (cyclic voltammetry (CV) and differential pulse voltammetry (DPV)). Structures of 3 and 4 have been determined crystallographically. In vitro antiproliferative and cytotoxic activity of these complexes has been evaluated by trypan blue exclusion assay, cell morphology, apoptosis, acridine orange/ethidium bromide (AO/EtBr) fluorescence staining, and DNA fragmentation assay in Dalton lymphoma (DL) cell lines. Interaction of 1-4 with calf thymus DNA (CT DNA) has also been supported by absorption titration and electrochemical studies. Our results suggest that in vitro antitumor activity of 1-4 lies in the order 2 > 1 > 4 > 3.

Deferoxamine Ameliorates Cypermethrin-Induced Iron Accumulation and Associated Alterations.

Iron is widely linked with the onset and development of Parkinson's disease (PD). Accumulation of iron induces free radical generation and promotes α-synuclein aggregation, oxidative stress, and autophagy impairment. Deferoxamine, an iron chelator, is shown to ameliorate iron dyshomeostasis in rodents and humans. However, the role of deferoxamine in cypermethrin-induced iron accumulation is not yet known. Although an iron accumulation and impaired chaperone-mediated autophagy (CMA) contribute to PD, a link between the two is not yet widely understood. Current study is undertaken to explore the possible association between an iron accumulation and CMA in cypermethrin model of PD in the presence of deferoxamine. Level of iron, iron transporter proteins, oxidative stress, and CMA proteins along with indicators of Parkinsonism were measured. Deferoxamine attenuated cypermethrin-induced iron accumulation and number of iron-positive cells and ameliorated the demise of dopaminergic cells and dopamine content. Deferoxamine significantly normalizes cypermethrin-induced changes in iron transporter proteins, α-synuclein, lysosome-associated membrane protein-2A, and oxidative stress. The results demonstrate that deferoxamine ameliorates cypermethrin-induced iron dyshomeostasis and impairment in CMA.

Dyshomeostasis of Iron and Its Transporter Proteins in Cypermethrin-Induced Parkinson's Disease.

The etiology of Parkinson's disease (PD) is highly complex and is still indefinable. However, a number of studies have indicated the involvement of pesticides and transition metals. Copper, magnesium, iron, and zinc have emerged as important metal contributors. Exposure to pesticides causes an accumulation of transition metals in the substantia nigra (SN) region of the brain. The cypermethrin model of PD is characterized by mitochondrial dysfunction, autophagy impairment, oxidative stress, etc. However, the effect of cypermethrin on metal homeostasis is not yet explored. The study was designed to delineate the role of metals and their transporter proteins in cypermethrin-induced animal and cellular models of PD. The level of copper, magnesium, iron, and zinc was checked in the nigrostriatal tissue and serum by atomic absorption spectroscopy. Since cypermethrin consistently increased iron content in the nigrostriatal tissue and serum after 12 weeks of exposure, the level of iron transporter proteins, such as divalent metal transporter-1 (DMT-1), ceruloplasmin, transferrin, ferroportin, and hepcidin, and their in silico interaction with cypermethrin were checked. 3,3'-Diaminobenzidine-enhanced Perl's staining showed an elevated number of iron-positive cells in the SN of cypermethrin-treated rats. Molecular docking studies revealed a strong binding affinity between cypermethrin and iron transporter protein receptors of humans and rats. Furthermore, cypermethrin increased the expression of DMT-1 and hepcidin while reducing the expression of transferrin, ceruloplasmin, and ferroportin in the nigrostriatal tissue and human neuroblastoma cells. These observations suggest that cypermethrin alters the expression of iron transporter proteins leading to iron dyshomeostasis, which could contribute to dopaminergic neurotoxicity.

Neuroprotective Activity of a Novel Synthetic Rhodamine-Based Hydrazone against Cu2+-Induced Alzheimer's Disease in Drosophila.

A new rhodamine-based probe 3,5-di-tert-butylsalicylaldehyde rhodamine hydrazone (RHTB) has been synthesized and well characterized using spectroscopic techniques and single-crystal X-ray crystallography. Among several metal ions, it selectively detects Cu2+ ions as monitored by UV-Vis and emission spectral titrations. It displays "turn on" behavior owing to the opening of a spirolactum ring and the presence of 3,5-di-tert-butyl as an electron releasing group. Further, Cu2+ ions play a pivotal role in extracellular aggregation of Aß42 peptides. So far, we know probably that there are no promising drugs available in this regard. Hence, countering the Cu2+ ions by RHTB chelation against orally administered Cu2+ ion-induced neurotoxicity in the eye tissue of Drosophila expressing human Aß42 (amyloid-ß42) has been tested. The present study involves in vivo and in silico approaches. They reveal the therapeutic potential of RHTB against Cu2+ ion-induced Aß42 toxicity in Alzheimer's disease (AD) model of Drosophila.

Studies on Regulation of Global Protein Profile and Cellular Bioenergetics of Differentiating SH-SY5Y Cells.

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

Neuroprotective potential of flavonoid rich Ascophyllum nodosum (FRAN) fraction from the brown seaweed on an Aß42 induced Alzheimer's model of Drosophila.

BACKGROUND: In Alzheimer Disease (AD) pathogenesis, aggregation of Aß42 fibrils strongly correlates with memory dysfunction and neurotoxicity. Till date, no promising cures for AD. Report shows that flavonoids contributed anti-oxidant, anti-cancer and neuroprotection activity by regulating the mitochondrial machinery. Here, we first report the identification of flavonoids from Ascophyllum nodosum as having the ability to dissolve Aß42 fibrils in an AD model of Drosophila. FRAN could be superior anti-AD agents for neuroprotection, their underlying mechanism and how they collectively halted amyloidogenesis is currently being investigated. PURPOSE: This study aimed to investigate the neuroprotective role of FRAN in the Aß42 expressing AD model of Drosophila. METHODS: Drosophila stocks: OregonR+, ey-GAL4/CyO, elavc155-GAL4, UAS-mitoGFP, UAS-mcherry.mito.OMM, UAS-Aß42/CyO were used, cultured at 28±1 °C in a BOD incubator. Ascophyllum extract rich in flavonoids as revealed by LC-MS study and employed against the AD flies. The validation of Aß42 expression was done by immunostaining and q-RT PCR. The eye roughness of AD flies was scored in a dose-dependent manner. Further, In vivo and in silico studies of FRAN extract was executed against Aß42 induced neurotoxicity. RESULTS: In order to determine the most effective lethal dose of FRAN extract concentration 1, 2, 5, 10 mg/ml were screened using OregonR+flies. Extract 1 and 2 mg/ml did not show any lethality. Hence, extract 2 mg/ml was employed on AD flies and a ≥ 50% rescue in the eye phenotype was observed using SEM images. This dose had a strong effect on cell apoptosis, viability, longevity, mitochondrial dysfunction and oxidative stress by regulating mitochondrial dynamic markers in comparable to control. Extract also scavenging free radicals in order to maintain in situ cellular ROS and prevent Aß42-induced neurotoxicity in vivo and in silico. Hence, we suggest its great potential as a future therapeutic agent for AD treatment. CONCLUSION: In conclusion, FRAN extract rich in flavonoids as having largest neuroprotective activity against Aß42 aggregation in eye tissue of Drosophila. Extract shows strong effect against Aß42-induced neurotoxicity by altering the various cellular and molecular events. So, it could be considered as strong anti-AD agents for neuroprotection.

Exploration of Neuroprotective Properties of a Naturally Inspired Multifunctional Molecule (F24) against Oxidative Stress and Amyloid ß Induced Neurotoxicity in Alzheimer's Disease Models.

The pathological hallmarks of Alzheimer's disease (AD) are manifested as an increase in the level of oxidative stress and aggregation of the amyloid-ß protein. In vitro, in vivo, and in silico experiments were designed and carried out with multifunctional cholinergic inhibitor, F24 (EJMC-7a) to explore its neuroprotective effects in AD models. The neuroprotection ability of F24 was tested in SH-SY5Y cells, a widely used neuronal cell line. The pretreatment and subsequent co-treatment of SH-SY5Y cells with different doses of F24 was effective in rescuing the cells from H2O2 induced neurotoxicity. F24 treated cells were found to be effective in the reduction of cellular reactive oxygen species, DNA damage, and Aß1-42 induced neurotoxicity, which validated its neuroprotective effectiveness. F24 exhibited efficacy in an in vivoDrosophila model by rescuing eye phenotypes from degeneration caused by Aß toxicity. Further, computational studies were carried out to monitor the interaction between F24 and Aß1-42 aggregates. The computational studies corroborated our in vitro and in vivo studies suggesting Aß1-42 aggregation modulation ability of F24. The brain entry ability of F24 was studied in the parallel artificial membrane permeability assay. Finally, F24 was tested at doses of 1 and 2.5 mg/kg in the Morris water maze AD model. The neuroprotective properties shown by F24 strongly suggest that multifunctional features of this molecule provide symptomatic relief and act as a disease-modifying agent in the treatment of AD. The results from our experiments strongly indicated that natural template-based F24 could serve as a lead molecule for further investigation to explore multifunctional therapeutic agents for AD management.

Retraction of "Fabrication of Graphene Nanoplatelet-Incorporated Porous Hydroxyapatite Composites: Improved Mechanical and in Vivo Imaging Performances for Emerging Biomedical Applications".

[This retracts the article DOI: 10.1021/acsomega.8b03473.].

scribble (scrib) knockdown induces tumorigenesis by modulating Drp1-Parkin mediated mitochondrial dynamics in the wing imaginal tissues of Drosophila.

scrib loss of function is associated with various human-cancers. Most of the human-cancers have been characterized by mitochondrial dysfunction with elevated oxidative stress. However, the role of scrib to mitochondrial dysfunction in cancer has not been investigated earlier. Here, we have shown that scrib knockdown leads to mitochondrial depolarization, fragmentation and perinuclear-clustering along with disruption of the redox homeostasis. Moreover, the scrib abrogated tumor showed the elevation of Drp-1 and reduced expression of Marf, which suggests enhanced mitochondrial-fission. Further, the reduced expression of Parkin and HtrA2 interpret defective mitophagy leading to clustering of fragmented mitochondria and apoptotic inhibition in scrib knockdown tumors. Also, Parkin immunostaining depicted its reduced expression and mislocalization in the tumor cells in comparison to wild type. Moreover, the genetic study revealed the epistatic interactions of parkin and scrib. Thus, for the first time our results suggested that scrib loss induced mitochondrial-dysfunction modulates cancer progression by altering the mitochondrial dynamics regulators.