Combined Treatment with Three Natural Antioxidants Enhances Neuroprotection in a SH-SY5Y 3D Culture Model

Currently, the majority of cell-based studies on neurodegeneration are carried out on two-dimensional cultured cells that do not represent the cells residing in the complex microenvironment of the brain. Recent evidence has suggested that three-dimensional (3D) in vitro microenvironments may better model key features of brain tissues in order to study molecular mechanisms at the base of neurodegeneration. So far, no drugs have been discovered to prevent or halt the progression of neurodegenerative disorders. New therapeutic interventions can come from phytochemicals that have a broad spectrum of biological activities. On this basis, we evaluated the neuroprotective effect of three phytochemicals (sulforaphane, epigallocatechin gallate, and plumbagin) alone or in combination, focusing on their ability to counteract oxidative stress. The combined treatment was found to be more effective than the single treatments. In particular, the combined treatment increased cell viability and reduced glutathione (GSH) levels, upregulated antioxidant enzymes and insulin-degrading enzymes, and downregulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 in respect to peroxide-treated cells. Our data suggest that a combination of different phytochemicals could be more effective than a single compound in counteracting neurodegeneration, probably thanks to a pleiotropic mechanism of action.

Download Full-text

Hydroxytyrosol Decreases LPS- and α-Synuclein-Induced Microglial Activation In Vitro

Antioxidants ◽

10.3390/antiox9010036 ◽

2019 ◽

Vol 9 (1) ◽

pp. 36 ◽

Cited By ~ 3

Author(s):

Marta Gallardo-Fernández ◽

Ruth Hornedo-Ortega ◽

Isabel M. Alonso-Bellido ◽

José A. Rodríguez-Gómez ◽

Ana M. Troncoso ◽

...

Keyword(s):

Molecular Mechanisms ◽

Biological Activities ◽

Virgin Olive Oil ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Extra Virgin Olive Oil ◽

Mitogen Activated Protein ◽

Anti Inflammatory ◽

The Relationship ◽

Shed Light

Neuroinflammation is a common feature shared by neurodegenerative disorders, such as Parkinson’s disease (PD), and seems to play a key role in their development and progression. Microglia cells, the principal orchestrators of neuroinflammation, can be polarized in different phenotypes, which means they are able to have anti-inflammatory, pro-inflammatory, or neurodegenerative effects. Increasing evidence supports that the traditional Mediterranean dietary pattern is related to the reduction of cognitive decline in neurodegenerative diseases. A considerable intake of plant foods, fish, and extra virgin olive oil (EVOO), as well as a moderate consumption of red wine, all characteristic of the Mediterranean diet (MD), are behind these effects. These foods are especially rich in polyphenols, being the most relevant in the MD hydroxytyrosol (HT) and their derivatives present in EVOO, which have demonstrated a wide array of biological activities. Here, we demonstrate that HT is able to reduce the inflammation induced by two different stimuli: lipopolysaccharide and α-synuclein. We also study the possible molecular mechanisms involved in the anti-inflammatory effect of HT, including the study of nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and inflammasome. Our data support the use of HT to prevent the inflammation associated with PD and shed light into the relationship between MD and this neurological disorder.

Download Full-text

EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction

Nature Communications ◽

10.1038/s41467-021-21258-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Zhao ◽

Alan Blayney ◽

Xiaorong Liu ◽

Lauren Gandy ◽

Weihua Jin ◽

...

Keyword(s):

Large Scale ◽

Molecular Mechanisms ◽

Epigallocatechin Gallate ◽

Cancer Drug ◽

Dynamic Interactions ◽

Cancer Drug Discovery ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Cancerous Cells

AbstractEpigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG’s anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

Download Full-text

Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/163057 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 19

Author(s):

Yan-Fang Xian ◽

Zhi-Xiu Lin ◽

Qing-Qiu Mao ◽

Jian-Nan Chen ◽

Zi-Ren Su ◽

...

Keyword(s):

Signaling Pathway ◽

Pc12 Cells ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

Neuroprotective Effect ◽

Protective Action ◽

Protective Effects ◽

Phosphorylated Akt ◽

Induced Apoptosis

The neurotoxicity of amyloid-β(Aβ) has been implicated as a critical cause of Alzheimer’s disease. Isorhynchophylline (IRN), an oxindole alkaloid isolated fromUncaria rhynchophylla,exerts neuroprotective effect againstAβ25–35-induced neurotoxicityin vitro. However, the exact mechanism for its neuroprotective effect is not well understood. The present study aimed to investigate the molecular mechanisms underlying the protective action of IRN againstAβ25–35-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Pretreatment with IRN significantly increased the cell viability, inhibited the release of lactate dehydrogenase and the extent of DNA fragmentation inAβ25–35-treated cells. IRN treatment was able to enhance the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3β(p-GSK-3β). Lithium chloride blockedAβ25–35-induced cellular apoptosis in a similar manner as IRN, suggesting that GSK-3βinhibition was involved in neuroprotective action of IRN. Pretreatment with LY294002 completely abolished the protective effects of IRN. Furthermore, IRN reversedAβ25–35-induced attenuation in the level of phosphorylated cyclic AMP response element binding protein (p-CREB) and the effect of IRN could be blocked by the PI3K inhibitor. These experimental findings unambiguously suggested that the protective effect of IRN againstAβ25–35-induced apoptosis in PC12 cells was associated with the enhancement of p-CREB expression via PI3K/Akt/GSK-3βsignaling pathway.

Download Full-text

High Matrix Metalloproteinase Production Correlates with Immune Activation and Leukocyte Migration in Leprosy Reactional Lesions

Infection and Immunity ◽

10.1128/iai.00896-09 ◽

2009 ◽

Vol 78 (3) ◽

pp. 1012-1021 ◽

Cited By ~ 17

Author(s):

Rosane M. B. Teles ◽

Rose B. Teles ◽

Thais P. Amadeu ◽

Danielle F. Moura ◽

Leila Mendonça-Lima ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Cultured Cells ◽

Leukocyte Migration ◽

Therapeutic Interventions ◽

Matrix Metalloproteinase 2 ◽

Mrna Levels ◽

Membrane Breakdown ◽

Tnf Α ◽

Ifn Γ

ABSTRACT Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-α. It was observed that IFN-γ, TNF-α, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-α, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

Download Full-text

Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering

Circulation Research ◽

10.1161/res.117.suppl_1.9 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Brisa Pena ◽

Valentina Martinelli ◽

Susanna Bosi ◽

Carmen Sucharov ◽

Mark Jeong ◽

...

Keyword(s):

Tissue Engineering ◽

Body Temperature ◽

Cell Viability ◽

Cultured Cells ◽

Ventricular Myocytes ◽

3D Culture ◽

Polymeric Matrix ◽

Neonatal Rat ◽

Cell Delivery

Background: Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability. Methods and results: We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia. Conclusion: These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.

Download Full-text

Visualization of SARS-CoV-2 infection dynamic

10.1101/2021.06.03.446942 ◽

2021 ◽

Author(s):

Chengjin Ye ◽

Kevin Chiem ◽

Jun-Gyu Park ◽

Jesus Silvas ◽

Desarey Morales Vasquez ◽

...

Keyword(s):

Viral Infection ◽

Cultured Cells ◽

Imaging System ◽

Reporter Genes ◽

Therapeutic Interventions ◽

Wild Type Virus ◽

Nucleocapsid Gene ◽

Recombinant Viruses

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Download Full-text

Alpha-synuclein from patient Lewy bodies exhibits distinct pathological activity that can be propagated in vitro

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01288-2 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Nicholas P. Marotta ◽

Jahan Ara ◽

Norihito Uemura ◽

Marshall G. Lougee ◽

Emily S. Meymand ◽

...

Keyword(s):

Cellular Response ◽

Amyloid Fibrils ◽

Cultured Cells ◽

Biological Activities ◽

Lewy Bodies ◽

Alpha Synuclein ◽

Biological Behavior ◽

Motor Deficits ◽

Original Material

AbstractLewy bodies (LBs) are complex, intracellular inclusions that are common pathological features of many neurodegenerative diseases. They consist largely of aggregated forms of the protein alpha-Synuclein (α-Syn), which misfolds to give rise to beta-sheet rich amyloid fibrils. The aggregation of monomers into fibrils occurs readily in vitro and pre-formed fibrils (PFFs) generated from recombinant α-Syn monomers are the basis of many models of LB diseases. These α-Syn PFFs recapitulate many pathological phenotypes in both cultured cells and animal models including the formation of α-Syn rich, insoluble aggregates, neuron loss, and motor deficits. However, it is not clear how closely α-Syn PFFs recapitulate the biological behavior of LB aggregates isolated directly from patients. Direct interrogation of the cellular response to LB-derived α-Syn has thus far been limited. Here we demonstrate that α-Syn aggregates derived from LB disease patients induce pathology characterized by a prevalence of large somatic inclusions that is distinct from the primarily neuritic pathology induced by α-Syn PFFs in our cultured neuron model. Moreover, these LB-derived aggregates can be amplified in vitro using recombinant α-Syn to generate aggregates that maintain the unique, somatic pathological phenotype of the original material. Amplified LB aggregates also showed greater uptake in cultured neurons and greater pathological burden and more rapid pathological spread in injected mouse brains, compared to α-Syn PFFs. Our work indicates that LB-derived α-Syn from diseased brains represents a distinct conformation species with unique biological activities that has not been previously observed in fully recombinant α-Syn aggregates and demonstrate a new strategy for improving upon α-Syn PFF models of synucleinopathies using amplified LBs.

Download Full-text

Biophysical studies of protein misfolding and aggregation in in vivo models of Alzheimer's and Parkinson's diseases

Quarterly Reviews of Biophysics ◽

10.1017/s0033583520000025 ◽

2020 ◽

Vol 53 ◽

Cited By ~ 2

Author(s):

Tessa Sinnige ◽

Karen Stroobants ◽

Christopher M. Dobson ◽

Michele Vendruscolo

Keyword(s):

Protein Misfolding ◽

Molecular Mechanisms ◽

Amyloid Β ◽

Therapeutic Interventions ◽

In Vivo Models ◽

Disease Modifying Drugs ◽

Parkinson’S Diseases ◽

Protein Misfolding And Aggregation

Abstract Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases. We discuss in particular models based on the transgenic expression of amyloid-β (Aβ) and tau in AD, and α-synuclein in PD. Because biophysical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro, we expect that the further development of these methods to probe directly the corresponding mechanisms in vivo will open effective routes for diagnostic and therapeutic interventions.

Download Full-text

Regulatory Potential of bHLH-Type Transcription Factors on the Road to Rubber Biosynthesis in Hevea brasiliensis

Plants ◽

10.3390/plants9060674 ◽

2020 ◽

Vol 9 (6) ◽

pp. 674

Author(s):

Tomoko Yamaguchi ◽

Yukio Kurihara ◽

Yuko Makita ◽

Emiko Okubo-Kurihara ◽

Ami Kageyama ◽

...

Keyword(s):

Cell Differentiation ◽

Natural Rubber ◽

Hevea Brasiliensis ◽

Time Course ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Early Stage ◽

Rubber Biosynthesis ◽

On The Road

Natural rubber is the main component of latex obtained from laticifer cells of Hevea brasiliensis. For improving rubber yield, it is essential to understand the genetic molecular mechanisms responsible for laticifer differentiation and rubber biosynthesis. Jasmonate enhances both secondary laticifer differentiation and rubber biosynthesis. Here, we carried out time-course RNA-seq analysis in suspension-cultured cells treated with methyljasmonic acid (MeJA) to characterize the gene expression profile. Gene Ontology (GO) analysis showed that the term “cell differentiation” was enriched in upregulated genes at 24 h after treatment, but inversely, the term was enriched in downregulated genes at 5 days, indicating that MeJA could induce cell differentiation at an early stage of the response. Jasmonate signaling is activated by MYC2, a basic helix–loop–helix (bHLH)-type transcription factor (TF). The aim of this work was to find any links between transcriptomic changes after MeJA application and regulation by TFs. Using an in vitro binding assay, we traced candidate genes throughout the whole genome that were targeted by four bHLH TFs: Hb_MYC2-1, Hb_MYC2-2, Hb_bHLH1, and Hb_bHLH2. The latter two are highly expressed in laticifer cells. Their physical binding sites were found in the promoter regions of a variety of other TF genes, which are differentially expressed upon MeJA exposure, and rubber biogenesis-related genes including SRPP1 and REF3. These studies suggest the possibilities that Hb_MYC2-1 and Hb_MYC2-2 regulate cell differentiation and that Hb_bHLH1 and Hb_bHLH2 promote rubber biosynthesis. We expect that our findings will help to increase natural rubber yield through genetic control in the future.

Download Full-text

The Rise of Retinal Organoids for Vision Research

International Journal of Molecular Sciences ◽

10.3390/ijms21228484 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8484 ◽

Cited By ~ 1

Author(s):

Kritika Sharma ◽

Tim U. Krohne ◽

Volker Busskamp

Keyword(s):

Molecular Mechanisms ◽

Cell Types ◽

Therapeutic Interventions ◽

Retinal Cell ◽

Retinal Diseases ◽

Organ Systems ◽

Cell Sources ◽

Retinal Degenerative Diseases

Retinal degenerative diseases lead to irreversible blindness. Decades of research into the cellular and molecular mechanisms of retinal diseases, using either animal models or human cell-derived 2D systems, facilitated the development of several therapeutic interventions. Recently, human stem cell-derived 3D retinal organoids have been developed. These self-organizing 3D organ systems have shown to recapitulate the in vivo human retinogenesis resulting in morphological and functionally similar retinal cell types in vitro. In less than a decade, retinal organoids have assisted in modeling several retinal diseases that were rather difficult to mimic in rodent models. Retinal organoids are also considered as a photoreceptor source for cell transplantation therapies to counteract blindness. Here, we highlight the development and field’s improvements of retinal organoids and discuss their application aspects as human disease models, pharmaceutical testbeds, and cell sources for transplantations.

Download Full-text