Neurite Outgrowth In SH-SY5Y Cells Research Articles

Different concentrations of adapalene induce differentiation and apoptosis of SH-SY5Y cells

To investigate the effects of different concentrations of adapalene on the morphology and functions of neuroblastoma cell line SH-SY5Y, as well as its role in inducing cell differentiation and apoptosis. SH-SY5Y cells were divided into control group, low concentration (0.1 μM and 1 μM) adapalene groups, and high concentration (10 μM) adapalene group. Time-lapse microscopy was used to observe the morphological changes of SH-SY5Y cells. Immunofluorescence staining was performed to detect the expression of neuronal specific marker βIII-tubulin and mature neuronal marker neurofilament heavy polypeptide (NFH). Multi-electrode array was used to record the electrophysiological features of SH-SY5Y cells. Cell apoptosis was evaluated using a cell apoptosis detection kit. Low concentrations of adapalene promoted the formation of neurite outgrowth in SH-SY5Y cells, with the neurites interconnected to form a network. Spontaneous discharge activity was observed in SH-SY5Y cells treated with low concentrations of adapalene. Compared to the control group, the expression of βIII-tubulin and NFH increased in the 1 μM adapalene group, while the level of cell apoptosis increased in the high concentration adapalene group (P<0.05). Low concentrations of adapalene can induce differentiation of SH-SY5Y cells into mature functional neurons, while high concentrations of adapalene can induce apoptosis in SH-SY5Y cells.

Neurite outgrowth induced by stimulation of angiotensin II AT2 receptors in SH-SY5Y neuroblastoma cells involves c-Src activation

Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and βIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.

Bile Acids Induce Neurite Outgrowth in Nsc-34 Cells via TGR5 and a Distinct Transcriptional Profile.

Increasing evidence supports a neuroprotective role for bile acids in major neurodegenerative disorders. We studied major human bile acids as signaling molecules for their two cellular receptors, farnesoid X receptor (FXR or NR1H4) and G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5), as potential neurotrophic agents. Using quantitative image analysis, we found that 20 μM deoxycholic acid (DCA) could induce neurite outgrowth in NSC-34 cells that was comparable to the neurotrophic effects of the culture control 1 μM retinoic acid (RA), with lesser effects observed for chenodexoycholic acid (CDCA) at 20 μM, and similar though less robust neurite outgrowth in SH-SY5Y cells. Using chemical agonists and antagonists of FXR, LXR, and TGR5, we found that TGR5 agonism was comparable to DCA stimulation and stronger than RA, and that neither FXR nor liver X receptor (LXR) inhibition could block bile acid-induced neurite growth. RNA sequencing identified a core set of genes whose expression was regulated by DCA, CDCA, and RA. Our data suggest that bile acid signaling through TGR5 may be a targetable pathway to stimulate neurite outgrowth.

Flavones 7,8-DHF, Quercetin, and Apigenin Against Tau Toxicity via Activation of TRKB Signaling in ΔK280 TauRD-DsRed SH-SY5Y Cells

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with memory loss and cognitive decline. Neurofibrillary tangles (NFTs) formed by hyperphosphorylated Tau protein are one of the pathological hallmarks of several neurodegenerative diseases including AD. Heat shock protein family B (small) member 1 (HSPB1) is a molecular chaperone that promotes the correct folding of other proteins in response to environmental stress. Nuclear factor erythroid 2-like 2 (NRF2), a redox-regulated transcription factor, is the master regulator of the cellular response to excess reactive oxygen species. Tropomyosin-related kinase B (TRKB) is a membrane-bound receptor that, upon binding brain-derived neurotrophic factor (BDNF), phosphorylates itself to initiate downstream signaling for neuronal survival and axonal growth. In this study, four natural flavones such as 7,8-dihydroxyflavone (7,8-DHF), wogonin, quercetin, and apigenin were evaluated for Tau aggregation inhibitory activity and neuroprotection in SH-SY5Y neuroblastoma. Among the tested flavones, 7,8-DHF, quercetin, and apigenin reduced Tau aggregation, oxidative stress, and caspase-1 activity as well as improved neurite outgrowth in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. Treatments with 7,8-DHF, quercetin, and apigenin rescued the reduced HSPB1 and NRF2 and activated TRKB-mediated extracellular signal-regulated kinase (ERK) signaling to upregulate cAMP-response element binding protein (CREB) and its downstream antiapoptotic BCL2 apoptosis regulator (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of these three flavones. Our results suggest 7,8-DHF, quercetin, and apigenin targeting HSPB1, NRF2, and TRKB to reduce Tau aggregation and protect cells against Tau neurotoxicity and may provide new treatment strategies for AD.

Effects of Oxytocin Analogs on Neurite Outgrowth and Cellular Signaling in Human SH‐SY5Y Neuroblastoma Cell Line

Introduction In the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G-protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression. Methods The human neuroblastoma derived SH-SY5Y cell line demonstrates morphological characteristics of neurons including neurites, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8-AM to measure ligand-induced Ca2+mobilization. Sigmoidal dose-response curves for OT analogs were generated using three-parameter nonlinear regression analysis in GraphPad Prism 8.3 software. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of the consensus mammalian OT sequence (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly; Leu8-OT) and a new world monkey analog (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Pro-Gly; Pro8-OT) for 24 hours, fixed and stained with phalloidin and DAPI. The Nikon A1R confocal microscope was used to collect 60X images of GFP-expressing neurons assessed for effects on neurite outgrowth using the Image J package FIJI with the NeuronJ plugin. Results In SH-SY5Y cells, both OT analogs were more potent than AVP at inducing Ca2+ mobilization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L-371,257. Additionally, both OT analogs show a dose-dependent increase in neurite outgrowth. Conclusions Together, these data suggest that OTR mediated signaling pathways lead to neurite outgrowth in SH-SY5Y cells. Integrative studies of behavior, genetics and ligand-receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.

An appropriate ratio of unsaturated fatty acids is the constituent of hickory nut extract for neurite outgrowth in human SH-SY5Y cells.

Hickory nuts (Carya cathayensis Sarg, CCS), a well‐known Chinese medicinal nut, is thought to improve memory in Chinese folks. However, functional constituents have not been scientifically identified. In this study, human SH‐SY5Y cells, combined with Q‐TOF mass spectrometry (Q‐TOF‐MS) and standard substances, were used to evaluate the function in neuronal development and to identify constituents of CCS hydrophobic extracts (CCS‐HE). Data showed that CCS‐HE but not the control induced neurite outgrowth of SH‐SY5Y cells in a dose‐dependent manner, supported by which CCS‐HE induced the expression of nerve growth factor (NGF), neurofilament 160 (NF160), and neuronal peptide Y (NPY) mRNA. Q‐TOF‐MS analysis with standard substances indicated that linolenic acid (LNA), linoleic acid (LA), and oleic acid (OA) were the main constituents in CCS‐HE. Furthermore, mixtures of these unsaturated fatty acids (UFAs) at the natural ratio (1:8:16) significantly induced neurite outgrowth and gene expression of NGF, NF160, and NPY in a dose‐dependent manner. However, the individual and alternative ratios were not effective to induce the neurite outgrowth and gene expression of NGF, NF160, and NPY. These data implicate that an appropriate ratio of UFAs is the main constituent for the neurite outgrowth.

The Effect of Glyceraldehyde-Derived Advanced Glycation End Products on β-Tubulin-Inhibited Neurite Outgrowth in SH-SY5Y Human Neuroblastoma Cells.

Nutritional factors can affect the risk of developing neurological disorders and their rate of progression. In particular, abnormalities of carbohydrate metabolism in diabetes mellitus patients lead to an increased risk of neurological disorders such as Alzheimer’s disease (AD). In this study, we investigated the relationship between nervous system disorder and the pathogenesis of AD by exposing SH-SY5Y neuroblastoma cells to glyceraldehyde (GA). We previously reported that GA-derived toxic advanced glycation end products (toxic AGEs, TAGE) induce AD-like alterations including intracellular tau phosphorylation. However, the role of TAGE and their target molecules in the pathogenesis of AD remains unclear. In this study, we investigated the target protein for TAGE by performing two-dimensional immunoblot analysis with anti-TAGE antibody and mass spectrometry and identified β-tubulin as one of the targets. GA treatment induced TAGE-β-tubulin formation and abnormal aggregation of β-tubulin, and inhibited neurite outgrowth in SH-SY5Y cells. On the other hand, glucose-derived AGEs were also involved in developing AD. However, glucose did not make abnormal aggregation of β-tubulin and did not inhibit neurite outgrowth. Understanding the underlying mechanism of TAGE-β-tubulin formation by GA and its role in neurodegeneration may aid in the development of novel therapeutics and neuroprotection strategies.

Effect of (m)RVD-hemopressin against Aβ1-42-induced apoptosis and inhibition of neurite outgrowth in SH-SY5Y cells

Alzheimer's disease (AD) is a serious neurodegenerative disease. Senile plaques (SPs) in the extracellular space and neurofibrillary tangles (NFTs) in the intracellular areas of the brain are two typical features of AD. SPs and NFTs are composed of amyloid-β (Aβ) aggregates and hyperphosphorylated Tau, respectively. (m)RVD-hemopressin (RVD), which is derived from mouse brain peptide, binds to the cannabinoid 1 receptor (CB1R) as an agonist. Our previous study indicated that RVD reversed Aβ1–42-induced memory impairment in mice. Here, we investigated the underlying molecular mechanism of RVD on Aβ1–42-induced neurotoxicity in retinoic acid-differentiated human neuroblastoma SH-SY5Y cells. Cell viability and neurite outgrowth were investigated by live cell imaging and analysis instrument. We found that RVD reversed Aβ1–42-induced Tau phosphorylation, apoptosis and suppression of neurite outgrowth and the synapse-associated protein postsynaptic density protein 95 (PSD-95) by inhibiting the activity of protein kinase A (PKA) and glycogen synthase kinase 3β (GSK-3β). Combined treatment with AM251 (a CB1R antagonist) blocked the effects of RVD. In conclusion, RVD may be a potential therapeutic agent for the treatment of cognitive dysfunctions, such as Alzheimer's disease.

The oligomerization mediated by the alanine 397 residue in the transmembrane domain is crucial to sydecan-3 functions

Syndecans are single-pass transmembrane proteins on the cell surface that are involved in various cellular functions. Previously, we reported that both homo- and hetero-form of syndecan dimers affected their functionality. However, little is known about the structural role of the transmembrane domain of syndecan-3. A series of glutathione-S-transferase syndecan-3 proteins showed that syndecan-3 formed SDS-resistant dimers and oligomers. SDS-resistant oligomer formation was barely observed in the syndecan deletion mutants lacking the transmembrane domain. Interestingly, the presence of an alanine 397 residue in the transmembrane domain correlated with SDS-resistant oligomer, and its replacement by phenylalanine (AF mutant) significantly reduced SDS-resistant oligomer formation. Beside the AF mutant significantly reduced syndecan-3 mediated cellular processes such as cell adhesion, migration and neurite outgrowth of SH-SY5Y neuroblastoma. Furthermore, the alanine residue regulated hetero-oligomer formation of syndecan-3, and hetero-oligomer formation significantly reduced syndecan-3-mediated neurite outgrowth of SH-SY5Y cells. Taken together, all these data suggest that syndecan-3 has a specific feature of oligomerization by the transmembrane domain and this oligomerization tendency is crucial for the function of syndecan-3.

NOX2-Dependent Reactive Oxygen Species Regulate Formyl-Peptide Receptor 1-Mediated TrkA Transactivation in SH-SY5Y Cells.

Several enzymes are capable of producing reactive oxygen species (ROS), but only NADPH oxidases (NOX) generate ROS as their primary and sole function. In the central nervous system, NOX2 is the major source of ROS, which play important roles in signalling and functions. NOX2 activation requires p47phox phosphorylation and membrane translocation of cytosolic subunits. We demonstrate that SH-SY5Y cells express p47phox and that the stimulation of Formyl-Peptide Receptor 1 (FPR1) by N-fMLP induces p47phox phosphorylation and NOX-dependent superoxide generation. FPR1 is a member of the G protein-coupled receptor (GPCR) family and is able to transphosphorylate several tyrosine kinase receptors (RTKs). This mechanism requires ROS as signalling intermediates and is necessary to share information within the cell. We show that N-fMLP stimulation induces the phosphorylation of cytosolic Y490, Y751, and Y785 residues of the neurotrophin receptor TrkA. These phosphotyrosines provide docking sites for signalling molecules which, in turn, activate Ras/MAPK, PI3K/Akt, and PLC-γ1/PKC intracellular cascades. N-fMLP-induced ROS generation plays a critical role in FPR1-mediated TrkA transactivation. In fact, the blockade of NOX2 functions prevents Y490, Y751, and Y785 phosphorylation, as well as the triggering of downstream signalling cascades. Moreover, we observed that FPR1 stimulation by N-fMLP also improves proliferation, cellular migration, and neurite outgrowth of SH-SY5Y cells.

Somatostatin-Mediated Changes in Microtubule-Associated Proteins and Retinoic Acid-Induced Neurite Outgrowth in SH-SY5Y Cells.

Somatostatin (SST) is a growth hormone inhibitory peptide involved in regulation of several physiological responses of cells including neurotransmission, cell migration, maturation, and neurite formation. In the present study, we examined the role of SST in all-trans retinoic acid (RA)-induced progression of neurite outgrowth in SH-SY5Y cells. We also determined the morphological and developmental changes in prominent intracellular markers of neurite growth including microtubule-associated protein 2 (MAP2), neuron-specific III β-tubulin (TUJ1), and Tau. Here, we present evidence that SST is a molecular determinant in regulating the transition of SH-SY5Y cells from non-neuronal entity to neuronal phenotype in response to RA. The results from present study reveal that SST changes the distributional pattern of MAP2/Tau and TUJ1, and activates extracellular signal-regulated kinase (ERK1/2) signaling pathway through SST receptors (SSTRs). The expression of MAP2 and Tau remains elevated upon treatment with RA and SST alone or in combination. Importantly, we identified that the cells displaying strong co-expression of SST and TUJ1 are more likely to bear elongated neurite formation than cells devoid of such expression. These findings show that the site-specific expression of MAP2 and TUJ1 is an essential determinant of neurite outgrowth in SH-SY5Y cells in RA-mediated differentiation. Taken together, results presented here further substantiates the role of SST in the promotion of neurite formation and elongation in SH-SY5Y cells in combination with RA. Investigating how SST can improve neurite formation in neurodegenerative disease may help to develop new therapeutic approach in improving cognitive function and memory loss.

Effect of Graphene Nanoribbons (TexasPEG) on locomotor function recovery in a rat model of lumbar spinal cord transection.

A sharply transected spinal cord has been shown to be fused under the accelerating influence of membrane fusogens such as polyethylene glycol (PEG) (GEMINI protocol). Previous work provided evidence that this is in fact possible. Other fusogens might improve current results. In this study, we aimed to assess the effects of PEGylated graphene nanoribons (PEG-GNR, and called “TexasPEG” when prepared as 1wt% dispersion in PEG600) versus placebo (saline) on locomotor function recovery and cellular level in a rat model of spinal cord transection at lumbar segment 1 (L1) level. In vivo and in vitro experiments (n = 10 per experiment) were designed. In the in vivo experiment, all rats were submitted to full spinal cord transection at L1 level. Five weeks later, behavioral assessment was performed using the Basso Beattie Bresnahan (BBB) locomotor rating scale. Immunohistochemical staining with neuron marker neurofilament 200 (NF200) antibody and astrocytic scar marker glial fibrillary acidic protein (GFAP) was also performed in the injured spinal cord. In the in vitro experiment, the effects of TexasPEG application for 72 hours on the neurite outgrowth of SH-SY5Y cells were observed under the inverted microscope. Results of both in vivo and in vitro experiments suggest that TexasPEG reduces the formation of glial scars, promotes the regeneration of neurites, and thereby contributes to the recovery of locomotor function of a rat model of spinal cord transfection.

Identification of function-regulating antibodies targeting the receptor protein tyrosine phosphatase sigma ectodomain.

Receptor tyrosine phosphatase sigma (RPTPσ) plays an important role in the regulation of axonal outgrowth and neural regeneration. Recent studies have identified two RPTPσ ligands, chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPG), which can modulate RPTPσ activity by affecting its dimerization status. Here, we developed a split luciferase assay to monitor RPTPσ dimerization in living cells. Using this system, we demonstrate that heparin, an analog of heparan sulfate, induced the dimerization of RPTPσ, whereas chondroitin sulfate increased RPTPσ activity by inhibiting RPTPσ dimerization. Also, we generated several novel RPTPσ IgG monoclonal antibodies, to identify one that modulates its activity by inducing/stabilizing dimerization in living cells. Lastly, we demonstrate that this antibody promotes neurite outgrowth in SH-SY5Y cells. In summary, we demonstrated that the split luciferase RPTPσ activity assay is a novel high-throughput approach for discovering novel RPTPσ modulators that can promote axonal outgrowth and neural regeneration.

Alpha-Synuclein Suppresses Retinoic Acid-Induced Neuronal Differentiation by Targeting the Glycogen Synthase Kinase-3β/β-Catenin Signaling Pathway

Alpha-synuclein (α-SYN) is expressed during neuronal development and is mainly involved in the modulation of synaptic transmission. Missense mutations and amplifications of this gene have been associated with the pathogenesis of Parkinson's disease. Here, we evaluate whether α-SYN plays a detrimental role in the phenotypic and morphological regulation of neurons. We also identify the underlying mechanisms of this process in all-trans-retinoic acid (RA)-induced differentiated SH-SY5Y cells, which represents dopaminergic (DAergic) phenotype. Our results indicate that overexpression of wild-type or mutant A53T α-SYN attenuated the RA-induced upregulation of tyrosine hydroxylase and dopamine transporter as well as neurite outgrowth in SH-SY5Y cells. In addition, GSK-3β inactivation and downstream β-catenin stabilization were associated with RA-induced differentiation, which was attenuated by α-SYN. Moreover, protein phosphatase 2A was positively regulated by α-SYN and was implicated in the α-SYN-mediated interference with RA signaling. The results obtained from SH-SY5Y cells were verified in primary cultures of mesencephalic DAergic neurons from A53T α-SYN transgenic mice, which represent high levels of α-SYN and protein phosphatase 2A in the midbrain. The number and length of neurites in tyrosine hydroxylase-positive as well as Tau-positive cells from A53T α-SYN transgenic mice were significantly lower than those in littermate controls. The current results provide novel insight into the role of α-SYN in the regulation of neuronal differentiation, including DAergic neurons. Identifying the signaling pathway involved in the α-SYN-mediated dysregulation of neuronal differentiation could lead to a better understanding of the developmental processes underlying α-SYN-related pathologies and facilitate the discovery of specifically targeted therapeutics.

Design, Synthesis, and Biological Evaluation of Ganglioside Hp-s1 Analogues Varying at Glucosyl Moiety.

Ganglioside Hp-s1 is isolated from the ovary of sea urchin Diadema setosum. It exhibited better neuritogenic activity than GM1 in pheochromocytoma 12 cells. To explore the roles of glucosyl moiety of Hp-s1 in contributing to the neurogenic activity, we developed feasible procedures for synthesis of Hp-s1 analogues (2a-2f). The glucosyl moiety of Hp-s1 was replaced with α-glucose, α-galactose, β-galactose, α-mannose, and β-mannose, and their biological activities on SH-SY5Y cells and natural killer T (NKT) cells were evaluated. We found that the orientation of C-2 hydroxyl group at glucosyl moiety of Hp-s1 plays an important role to induce neurite outgrowth of SH-SY5Y cells. Surprisingly, compound 2d could activate NKT cells to produce interleukin 2, although it did not show great activity on neurite outgrowth of SH-SY5Y cells. In general, the Hp-s1 might be considered as a lead compound for the development of novel drugs aimed at modulating the activity of neuronal cells.

Hypoxia-inducible factor-1α upregulates tyrosine hydroxylase and dopamine transporter by nuclear receptor ERRγ in SH-SY5Y cells.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor relevant to the development of many mammalian organs including the brain. However, the molecular mechanisms by which signaling events mediate neuronal differentiation have not been fully elucidated. In the present study, we show for the first time that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) is upregulated by HIF-1α and plays essential roles in HIF-1α-induced upregulation of dopaminergic marker molecules such as tyrosine hydroxylase and dopamine transporter. We found that deferoxamine upregulated HIF-1α and enhanced the dopaminergic phenotype and neurite outgrowth of SH-SY5Y cells. Deferoxamine activated transcription and protein expression of ERRγ, and deferoxamine-induced upregulation of tyrosine hydroxylase and dopamine transporter was attenuated by using the ERRγ inverse agonist or silencing ERRγ. Altogether, these results suggest that HIF-1α can positively regulate the dopaminergic phenotype through ERRγ. This study could provide new perspectives for understanding the mechanisms underlying the promotion of dopaminergic neuronal differentiation by hypoxia.

Estrogen-related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH-SY5Y cells.

The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding dopaminergic differentiation.

The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells.

The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.

Beyond the mitochondrion: cytosolic PINK1 remodels dendrites through Protein Kinase A

The subcellular compartmentalization of kinase activity allows for regulation of distinct cellular processes involved in cell differentiation or survival. The PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, is a neuroprotective kinase localized to cytosolic and mitochondrial compartments. While mitochondrial targeting of PINK1 is important for its activities regulating mitochondrial homeostasis, the physiological role of the cytosolic pool of PINK1 remains unknown. Here, we demonstrate a novel role for cytosolic PINK1 in neuronal differentiation/neurite maintenance. Over-expression of wild-type PINK1, but not a catalytically inactive form of PINK1(K219M), promoted neurite outgrowth in SH-SY5Y cells and increased dendritic lengths in primary cortical and midbrain dopaminergic neurons. To identify the subcellular pools of PINK1 involved in promoting neurite outgrowth, we transiently transfected cells with PINK1 constructs designed to target PINK1 to the outer mitochondrial membrane (OMM-PINK1) or restrict PINK1 to the cytosol (ΔN111-PINK1). Both constructs blocked cell death associated with loss of endogenous PINK1. However, transient expression of ΔN111-PINK1, but not of OMM-PINK1 or ΔN111-PINK1(K219M), promoted dendrite outgrowth in primary neurons, and rescued the decreased dendritic arborization of PINK1-deficient neurons. Mechanistically, the cytosolic pool of PINK1 regulated neurite morphology through enhanced anterograde transport of dendritic mitochondria and amplification of protein kinase A-related signaling pathways. Our data support a novel role for PINK1 in regulating dendritic morphogenesis.

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.