Differentiated SH-SY5Y Cells Research Articles

Electrophysiological Profile Remodeling via Selective Suppression of Voltage-Gated Currents by CLN1/PPT1 Overexpression in Human Neuronal-Like Cells.

CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.

Toward Spontaneous Neuronal Differentiation of SH-SY5Y Cells Using Novel Three-Dimensional Electropolymerized Conductive Scaffolds

Neuroblastoma-derived SH-SY5Y cells have become an excellent model for nervous system regeneration to treat neurodegenerative disorders. Many approaches achieved a mature population of derived neurons in in vitro plates. However, the importance of the third dimension in tissue regeneration has become indispensable to achieve a potential implant to replace the damaged tissue. Therefore, we have prepared porous 3D structures composed uniquely of carbon nanotubes (CNT) and poly(3,4-ethylenedioxythiophene) (PEDOT) that show great potential in the tridimensional differentiation of SH-SY5Y cells into mature neurons. The scaffolds have been manufactured through electropolymerization by applying 1.2 V in a three-electrode cell using a template of sucrose/CNT as a working electrode. By this method, PEDOT/CNT 3D scaffolds were obtained with homogeneous porosities and high conductivity. In vitro analyses showed that an excellent biocompatibility of the scaffold and the presence of high amount of β-tubulin class III and MAP-II target proteins that mainly expresses in neurons, suggesting the differentiation into neuronal cells already after a week of incubation.

Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.

Trichostatin A increases BDNF protein expression by improving XBP-1s/ATF6/GRP78 axis in Schwann cells of diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is the common complication of diabetes mellitus. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) is reported to ameliorate the peripheral nerves degeneration of DPN. However, the exact mechanism is still not well elucidated. Here, we first revealed that TSA promoted nerve conduction and brain derived neurotrophic factor (BDNF) expression in the sciatic nerves of diabetic mice. In line, TSA also reversed high glucose-reduced mature BDNF expression in vitro cultured rat Schwann cells (RSC96). Then unexpectedly, the downstream targets of TSA HDAC1 and HDAC5 were not involved in TSA-improved BDNF expression. Furthermore, unfolded protein response (UPR) chaperone GRP78 was revealed to be downregulated with high glucose stimulation in RSC96 cells, which was avoided with TSA treatment. Also, GRP78 upregulation mediated TSA-improved mature BDNF expression in high glucose-cultured RSC96 cells by binding with BDNF. As well, TSA treatment enhanced the binding of GRP78 with BDNF in RSC96 cells. Again, UPR-associated transcription factors XBP-1s and ATF6 were involved in TSA-increased GRP78 expression in high glucose-stimulated RSC96 cells. Finally, conditioned medium from high glucose-cultured RSC96 cells delayed neuron SH-SY5Y differentiation and that from TSA-treated high glucose-cultured RSC96 cells promoted SH-SY5Y cell differentiation. Taken together, our findings suggested that TSA increased BDNF expression to ameliorate DPN by improving XBP-1s/ATF6/GRP78 axis in Schwann cells.

HuD regulates SOD1 expression during oxidative stress in differentiated neuroblastoma cells and sporadic ALS motor cortex

The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3′ untranslated region (3′UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.

Contemporary circulating enterovirus D68 strains have acquired the capacity for viral entry and replication in human neuronal cells

Background: Enterovirus D68 (EV-D68) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. EV-D68 infection has historically been associated with respiratory illnesses in humans. However, in the summers of 2014, 2016, and 2018 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis (AFM) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. Methods and materials: To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a cell culture model to determine if differential infection is observed for different EV-D68 isolates. Viral replication and infectivity in SH-SY5Y was assessed using four different assays-infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Results: In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to virus binding and entry were at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. Conclusion: Taken together, these results suggest that EV-D68 has evolved to acquire novel neurotropic characteristics that may contribute to the development of neurological disease in humans. In addition to supporting virus replication and other functional studies, the cell culture model we’ve developed may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Dynamics of Internalization and Intracellular Interaction of Tau Antibodies and Human Pathological Tau Protein in a Human Neuron-Like Model.

We and others have shown in various in vivo, ex vivo and cell culture models that several tau antibodies interact with pathological tau within neurons. To further clarify this interaction in a dynamic human model, we differentiated SH-SY5Y cells with retinoic acid and BDNF to create a neuron-like model. Therein, tau antibodies were primarily taken up by receptor-mediated endocytosis, and prevented toxicity of human brain-derived paired helical filament-enriched tau (PHF). Subsequently, we monitored in real-time the interaction of antibodies and PHF within endocytic cellular compartments. Cells were pre-treated with fluorescently-tagged PHF and then incubated with tau antibodies, 4E6, 6B2, or non-specific isotype control IgG1 labeled with a pH sensitive dye. The uptake and binding of the efficacious antibody, 4E6, to PHF occurred mainly within the soma, whereas the ineffective antibody, 6B2, and ineffective control IgG1, were visualized via the processes and showed limited colocalization with PHF within this period. In summary, we have developed a neuron-like model that clarifies the early intracellular dynamics of the interaction of tau antibodies with pathological tau, and identifies features associated with efficacy. Since the model is entirely human, it is suitable to verify the therapeutic potential of humanized antibodies prior to extensive clinical trials.

Safflower Yellow Compounds Alleviate Okadaic Acid-Induced Impairment of Neurite Outgrowth in Differentiated SH-SY5Y Cells

Safflower Yellow Compounds Alleviate Okadaic Acid-Induced Impairment of Neurite Outgrowth in Differentiated SH-SY5Y Cells

Loss of DEK Expression Induces Alzheimer's Disease Phenotypes in Differentiated SH-SY5Y Cells.

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by the buildup of β-amyloid plaques and neurofibrillary Tau tangles. This leads to decreased synaptic efficacy, cell death, and, consequently, brain atrophy in patients. Behaviorally, this manifests as memory loss and confusion. Using a gene ontology analysis, we recently identified AD and other age-related dementias as candidate diseases associated with the loss of DEK expression. DEK is a nuclear phosphoprotein with roles in DNA repair, cellular proliferation, and inhibiting apoptosis. Work from our laboratory determined that DEK is highly expressed in the brain, particularly in regions relevant to learning and memory, including the hippocampus. Moreover, we have also determined that DEK is highly expressed in neurons. Consistent with our gene ontology analysis, we recently reported that cortical DEK protein levels are inversely proportional to dementia severity scores in elderly female patients. However, the functional role of DEK in neurons is unknown. Thus, we knocked down DEK in an in vitro neuronal model, differentiated SH-SY5Y cells, hypothesizing that DEK loss would result in cellular and molecular phenotypes consistent with AD. We found that DEK loss resulted in increased neuronal death by apoptosis (i.e., cleaved caspases 3 and 8), decreased β-catenin levels, disrupted neurite development, higher levels of total and phosphorylated Tau at Ser262, and protein aggregates. We have demonstrated that DEK loss in vitro recapitulates cellular and molecular phenotypes of AD pathology.

Neuroprotective Effects of Methyl Caffeate against Hydrogen Peroxide-Induced Cell Damage: Involvement of Caspase 3 and Cathepsin D Inhibition.

Finding effective neuroprotective strategies to combat various neurodegenerative disorders still remain a clinically unmet need. Methyl caffeate (MC), a naturally occurring ester of caffeic acid, possesses antioxidant and anti-inflammatory activities; however, its role in neuroprotection is less investigated. In order to better characterize neuroprotective properties of MC, we tested its effectiveness in various models of neuronal cell injury in human neuroblastoma SH-SY5Y cells and in mouse primary neuronal cell cultures. MC at micromolar concentrations attenuated neuronal cell damage induced by hydrogen peroxide (H2O2) in undifferentiated and neuronal differentiated SH-SY5Y cells as well as in primary cortical neurons. This effect was associated with inhibition of both caspase-3 and cathepsin D but without involvement of the PI3-K/Akt pathway. MC was neuroprotective when given before and during but not after the induction of cell damage by H2O2. Moreover, MC was protective against 6-OHDA-evoked neurotoxicity in neuronal differentiated SH-SY5Y cells via inhibition of necrotic and apoptotic processes. On the other hand, MC was ineffective in models of excitotoxicity (induced by glutamate or oxygen–glucose deprivation) and even moderately augmented cytotoxic effects of the classical apoptotic inducer, staurosporine. Finally, in undifferentiated neuroblastoma cells MC at higher concentrations (above 50 microM) induced cell death and when combined with the chemotherapeutic agent, doxorubicin, it increased the cell damaging effects of the latter compound. Thus, neuroprotective properties of MC appear to be limited to certain models of neurotoxicity and depend on its concentrations and time of administration.

Effect of Chemical Vapor Deposition WS2 on Viability and Differentiation of SH-SY5Y Cells.

In recent years, transition metal dichalcogenides have been attracting an increasing interest in the biomedical field, thus implying the need of a deeper understanding of their impact on cell behavior. In this study we investigate tungsten disulfide (WS2) grown via chemical vapor deposition (CVD) on a transparent substrate (sapphire) as a platform for neural-like cell culture. We culture SH-SY5Y human neuroblastoma cells on WS2, using graphene, sapphire and standard culture well as controls. The quality, thickness and homogeneity of the materials is analyzed using atomic force microscopy and Raman spectroscopy. The cytocompatibility of CVD WS2 is investigated for the first time by cell viability and differentiation assessment on SH-SY5Y cells. We find that cells differentiated on WS2, displaying a viability and neurite length comparable with the controls. These findings shine light on the possibility of using WS2 as a cytocompatible material for interfacing neural cells.

The synthesis and cholinesterase inhibitory activities of solasodine analogues with seven-membered F ring

Solasodine analogues containing a seven-membered F ring with a nitrogen atom placed at position 22a were prepared from diosgenin or tigogenin in a four-step synthesis comprising of the simultaneous opening of the F-ring and introduction of cyanide in position 22α, activation of the 26-hydroxyl group as mesylate, nitrile reduction, and N-cyclization. Solasodine, six obtained 22a(N)-homo analogues, as well as four 26a-homosolasodine derivatives and their open-chain precursors (13 in total) were tested as potential inhibitors of acetyl- and butyryl-cholinesterases and showed activity at micromolar concentrations. The structure-activity relationship study revealed that activities against studied esterases are affected by the structure of E/F rings and the substitution pattern of ring A. The most potent compound 8 acted as non-competitive inhibitors and exerted IC50 = 8.51 μM and 7.05 μM for eeAChE and eqBChE, respectively. Molecular docking studies revealed the hydrogen bond interaction of 8 with S293 of AChE; further rings are stabilized via hydrophobic interaction (ring A) or interaction with Y341 and W286 (rings B and C). Biological experiments showed no neurotoxicity of differentiated SH-SY5Y cells. More importantly, results from neuroprotective assay based on glutamate-induced cytotoxicity revealed that most derivatives had the ability to increase the viability of differentiated SH-SY5Y cells in comparison to galantamine and lipoic acid assayed as standards. The newly synthesized solasodine analogues are able to inhibit and to bind cholinesterases in noncompetitive mode of inhibition and exhibited neuroprotection potential of differentiated neuroblastoma cells after Glu-induced toxicity.

Influence of serum concentration in retinoic acid and phorbol ester induced differentiation of SH-SY5Y human neuroblastoma cell line.

Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum used. In this paper, we compared the effects of high (10%), low (3%) and descending (2.5%/1%) serum concentration in differentiation medium containing different proportion of retinoic acid (RA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) or RA-only on the undifferentiated SH-SY5Y cells with regards to cell morphology, biochemical and gene expression alterations. Cells differentiated in culture medium containing low and descending serum concentrations showed increased number of neurite projections and reduced proliferation rates when compared to undifferentiated cells. The SH-SY5Y cells differentiated in culture medium containing 3% RA and low serum or descending (2.5%/1% RA/TPA) were found to be more susceptible to 6-hydroxydopamine (6-OHDA) induced cytotoxicity. Cells differentiated with RA/TPA or RA differentiated showed increased production of the α-synuclein (SNCA) neuroprotein and dopamine neurotransmitter compared to undifferentiated cells, regardless serum concentrations used. There was no significant difference in the expression of tyrosine hydroxylase (TH) gene between undifferentiated and differentiated SH-SY5Y cells. However, the expression of dopamine receptor D2 (DRD2) gene was markedly increased (p<0.05) in differentiated cells with 3% serum and RA only when compared to undifferentiated cells. In conclusion, to terminally differentiate SH-SY5Y cells to be used as a cell-based model to study Parkinson's disease (PD) to investigate molecular mechanisms and drug discovery, the optimal differentiation medium should contain 3% serum in RA-only.

The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis.

Simple SummaryThe study demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our results also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

Low dose lead exposure induces alterations on heterochromatin hallmarks persisting through SH-SY5Y cell differentiation

Lead (Pb) is a commonly found heavy metal due to its historical applications. Recent studies have associated early-life Pb exposure with the onset of various neurodegenerative disease. The molecular mechanisms of Pb conferring long-term neurotoxicity, however, is yet to be elucidated. In this study, we explored the persistency of alteration in epigenetic marks that arise from exposure to low dose of Pb using a combination of image-based and gene expression analysis. Using SH-SY5Y as a model cell line, we observed significant alterations in global 5-methycytosine (5 mC) and histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 9 tri-methylation (H3K9me3) levels in a dose-dependent manner immediately after Pb exposure. The changes are partially associated with alterations in epigenetic enzyme expression levels. Long term culturing (14 days) after cease of exposure revealed persistent changes in 5 mC, partial recovery in H3K9me3 and overcompensation in H3K27me3 levels. The observed alterations in H3K9me3 and H3K27me3 are reversed after neuronal differentiation, while reduction in 5 mC levels are amplified with significant changes in patterns as identified via texture clustering analysis. Moreover, correlation analysis demonstrates a strong positive correlation between trends of 5 mC alteration after differentiation and neuronal morphology. Collectively, our results suggest that exposure to low dose of Pb prior to differentiation can result in persistent epigenome alterations that can potentially be responsible for the observed phenotypic changes. Our work reveals that Pb induced changes in epigenetic repressive marks can persist through neuron differentiation, which provides a plausible mechanism underlying long-term neurotoxicity associated with developmental Pb-exposure.

Distinct alpha-Synuclein species induced by seeding are selectively cleared by the Lysosome or the Proteasome in neuronally differentiated SH-SY5Y cells.

A major pathological feature of Parkinson's disease (PD) is the aberrant accumulation of misfolded assemblies of alpha-synuclein (α-Syn). Protein clearance appears as a regulator of the 'α-Syn burden' underlying PD pathogenesis. The picture emerging is that a combination of pathways with complementary roles, including the Proteasome System and the Autophagy-Lysosome Pathway, contributes to the intracellular degradation of α-Syn. This study addresses the mechanisms governing the degradation of α-Syn species seeded by exogenous fibrils in neuronally differentiated SH-SY5Y neuroblastoma cells with inducible expression of α-Syn. Using human α-Syn recombinant fibrils (pre-formed fibrils, PFFs), seeding and aggregation of endogenous Proteinase K (PK)-resistant α-Syn species occurs within a time frame of 6days, and is still prominent after 12days of PFF addition. Clearance of α-Syn assemblies in this inducible model was enhanced after switching off α-Syn expression with doxycycline. Lysosomal inhibition led to accumulation of SDS-soluble α-Syn aggregates 6days after PFF-addition or when switching off α-Syn expression. Additionally, the autophagic enhancer, rapamycin, induced the clearance of α-Syn aggregates 13days post-PFF addition, indicating that autophagy is the major pathway for aggregated α-Syn clearance. SDS-soluble phosphorylated α-Syn at S129 was only apparent at 7days of incubation with a higher amount of PFFs.Proteasomal inhibition resulted in further accumulation of SDS-soluble phosphorylated α-Syn at S129, with limited PK resistance. Our data suggest that in this inducible model autophagy is mainly responsible for the degradation of fibrillar α-Syn, whereas the proteasome system is responsible, at least in part, for the selective clearance of phosphorylated α-Syn oligomers.

Anticancer Properties of Platinum Nanoparticles and Retinoic Acid: Combination Therapy for the Treatment of Human Neuroblastoma Cancer.

Neuroblastoma is the most common extracranial solid tumor in childhood. The different treatments available for neuroblastoma are challenged by high rates of resistance, recurrence, and progression, most notably in advanced cases and highly malignant tumors. Therefore, the development of more targeted therapies, which are biocompatible and without undesired side effects, is highly desirable. The mechanisms of actions of platinum nanoparticles (PtNPs) and retinoic acid (RA) in neuroblastoma have remained unclear. In this study, the anticancer effects of PtNPs and RA on neuroblastoma were assessed. We demonstrated that treatment of SH-SY5Y cells with the combination of PtNPs and RA resulted in improved anticancer effects. The anticancer effects of the two compounds were mediated by cytotoxicity, oxidative stress (OS), mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis-associated networks. Cytotoxicity was confirmed by leakage of lactate dehydrogenase (LDH) and intracellular protease, and oxidative stress increased the level of reactive oxygen species (ROS), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and nitric oxide (NO), and protein carbonyl content (PCC). The combination of PtNPs and RA caused mitochondrial dysfunction by decreasing the mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, number of mitochondria, and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Endoplasmic reticulum-mediated stress and apoptosis were confirmed by upregulation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), p53, Bax, and caspase-3 and down regulation of B-cell lymphoma 2 (BCl-2). PtNPs and RA induced apoptosis, and oxidative DNA damage was evident by the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG). Finally, PtNPs and RA increased the differentiation and expression of differentiation markers. Differentiated SH-SY5Y cells pre-treated with PtNPs or RA or the combination of both were more sensitive to the cytotoxic effect of cisplatin than undifferentiated cells. To our knowledge, this is the first study to demonstrate the effect of the combination of PtNPs and RA in neuroblastoma cells. PtNPs may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. The results of this study provide a rationale for clinical evaluation of the combination of PtNPs and RA for the treatment of children suffering from high-risk neuroblastoma.

Gateways for Glutamate Neuroprotection in Parkinson's Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD.

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

Neuroprotective Effects of 7-Geranyloxycinnamic Acid from Melicope lunu ankenda Leaves.

Neurodegenerative diseases (NDDs) are chronic conditions that have drawn robust interest from the scientific community. Phytotherapeutic agents are becoming an important source of chemicals for the treatment and management of NDDs. Various secondary metabolites have been isolated from Melicope lunu-ankenda plant leaves, including phenolic acid derivatives. However, their neuroprotective activity remains unclear. Thus, the aim of this study is to elucidate the in vitro neuroprotective activity of 7-geranyloxycinnamic acid isolated from Melicope lunu-ankenda leaves. The neuroprotective activity was evaluated in differentiated human neuroblastoma (SH-SY5Y) cells by monitoring cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Moreover, the potential to impair apoptosis in differentiated cells was investigated employing the Annexin V-FITC assay, acridine orange and propidium iodide (AO/PI) staining, and fluorescence microscopy. Morphological assessment and ultrastructural analysis were performed using scanning and transmission electron microscopy to evaluate the effect of 7-geranyloxycinnamic acid on surface morphology and internal features of the differentiated cells. Pre-treatment of neuronal cells with 7-geranyloxycinnamic acid significantly protected the differentiated SH-SY5Y cells against H2O2-induced apoptosis. Cytoskeleton and cytoplasmic inclusion were similarly protected by the 7-geranyloxycinnamic acid treatment. The present findings demonstrate the neuroprotective potential of 7-geranyloxycinnamic acid against H2O2-induced neurotoxicity in neuronal cells, which is an established hallmark of neuronal disorders.

NCX1 and EAAC1 transporters are involved in the protective action of glutamate in an in vitro Alzheimer's disease-like model

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer’s disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities.We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid β protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection.Remarkably, our results indicate, as proof‐of‐principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.