Neuronal Differentiation Of PC12 Cells Research Articles

Metformin inhibits nerve growth factor-induced sympathetic neuron differentiation through p35/CDK5 inhibition.

The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.

Chemoenzymatic Synthesis of GAA-7 Glycan Analogues and Evaluation of Their Neuritogenic Activities.

Ganglioside GAA-7 exhibits higher neurite outgrowth than ganglioside GM1a and most echinodermatous gangliosides (EGs) when tested on neuron-like rat adrenal pheochromocytoma (PC12) cells in the presence of nerve growth factor (NGF). The unique structure of GAA-7 glycan, containing an uncommon sialic acid (8-O-methyl-N-glycolylneuraminic acid) and sialic acid-α-2,3-GalNAc linkage, makes it challenging to synthesize. We recently developed a streamlined method to chemoenzymatically synthesize GAA-7 glycan and employed this modular strategy to efficiently prepare a library of GAA-7 glycan analogues incorporating N-modified or 8-methoxyl sialic acids. Most of these synthetic glycans exhibited moderate efficacy in promoting neuronal differentiation of PC12 cells. Among them, the analogue containing common sialic acid shows greater potential than the GAA-7 glycan itself. This result reveals that methoxy modification is not essential for neurite outgrowth. Consequently, the readily available analogue presents a promising model for further biological investigations.

Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review.

PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-β cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.

A Neurofilament-L reporter cell line for the quantification of early neuronal differentiation: A Bioassay for neurotrophic activities

BackgroundNeurotrophic activity constitutes a crucial factor in the recovery from neurological injuries and is impaired in neurodegenerative disorders. Preclinical studies of neurotrophic factors to improve outcome of neurodegenerative diseases have yielded promising results. However, due to the complexity of these therapies, the clinical translation of this approach was so far not successful and more feasible treatments with neurotrophic activity may be promising alternatives. Therefore, highly sensitive and robust assays for compound screening are required. New methodNerve growth factor is known to induce Neurofilament-L (NF-L) expression in a rat pheochromocytoma cell line (PC12 cells) during early neuronal differentiation. We generated and characterized an enhanced green fluorescent protein (EGFP)-NF-L reporter PC12 cell line for the development of a cell-based assay (designated Neurofilament-L Bioassay) that allows straightforward quantification of early neuronal differentiation based on NF-L expression. ResultsUsing Cerebrolysin® as a role model for a pharmacological compound that stimulates neurotrophic activity in the central nervous system, the Neurofilament-L Bioassay was proved to be a robust, specific, and reproducible method. Comparison with existing method(s)It was already shown that NF-L expression correlates with neurite outgrowth in PC12 cells. Currently, quantification of neurite outgrowth is the most commonly used method to evaluate neuronal differentiation in PC12 cells, an approach that is time-consuming and of high variability. ConclusionsThis work describes the development of an EGFP-NF-L reporter PC12 cell-based assay as a robust and reproducible tool for “high throughput” compound screening for neurotrophic activity.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Overexpression of the dystrophins Dp40 and Dp40L170P modifies neurite outgrowth and the protein expression profile of PC12 cells

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown. Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression. Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated. The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

Engineering 2D approaches fibrous platform incorporating turmeric and polyaniline nanoparticles to predict the expression of βIII-Tubulin and TREK-1 through qRT-PCR to detect neuronal differentiation of PC12 cells

The bioengineering electroactive construct of a nerve-guided conduit for repairing and restoring injured nerves is an exciting biomedical endeavor that has implications for the treatment of peripheral nerve injury. In this study, we report the development the polycaprolactone (PCL) nanofibrous substrate consisting of turmeric (TUR) and polyaniline nanoparticles (PANINPs) exhibits topological and biological features that mimics the natural extracellular matrix (ECM) for nerve cells. We evaluated the morphology of 2-dimensional (2D) fibrous substrates, and their ability of stem cell adhesion, growth and proliferation rate were influenced by use of various concentrations of turmeric in PCL–TUR substrates. The results showed that 0.62 wt% of TUR and 0.28 wt% of PANINPs in PCL nanofibers substrate exhibited the optimal cellular microenvironment to accelerate PC12 cellular activities. The in vitro experiments revealed that PCL–TUR@PANI substrates significantly stimulated the proliferation, differentiation, and spontaneous outgrowth and extension of neurites from the cells. The substrate has the capacity to respond directly to neuronal markers with significant upregulation of βIII-Tubulin and TREK-1 through myelination, and also trigger neurotrophic protein expression, which was confirmed via immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. This study provides a new technique to design substrate of nerve tissue-specific microenvironment for peripheral nerve cell regeneration and could offer promising biomaterials for in vivo peripheral nerve repair.

The βγ subunit of heterotrimeric G proteins interacts with actin filaments during neuronal differentiation

The βγ subunit of heterotrimeric G proteins, a key molecule in the G protein-coupled receptors (GPCRs) signaling pathway, has been shown to be an important factor in the modulation of the microtubule cytoskeleton. Gβγ has been shown to bind to tubulin, stimulate microtubule assembly, and promote neurite outgrowth of PC12 cells. In this study, we demonstrate that in addition to microtubules, Gβγ also interacts with actin filaments, and this interaction increases during NGF-induced neuronal differentiation of PC12 cells. We further demonstrate that the Gβγ-actin interaction occurs independently of microtubules as nocodazole, a well-known microtubule depolymerizing agent did not inhibit Gβγ-actin complex formation in PC12 cells. A confocal microscopic analysis of NGF-treated PC12 cells revealed that Gβγ co-localizes with both actin and microtubule cytoskeleton along neurites, with specific co-localization of Gβγ with actin at the distal end of these neuronal processes. Furthermore, we show that Gβγ interacts with the actin cytoskeleton in primary hippocampal and cerebellar rat neurons. Our results indicate that Gβγ serves as an important modulator of the neuronal cytoskeleton by interacting with both microtubules and actin filaments, and is likely to participate in various aspects of neuronal differentiation including axon and growth cone formation.

Construction of differential expression plasmids of NGF to detect its influence on PC12 cell neuronal differentiation.

Alongside angiogenesis and lymphangiogenesis, neurogenesis also occurs within the cancer microenvironment. Neurogenesis is a complex process involving multiple factors, among which nerve growth factor (NGF) possesses the dual biological roles of neuron nutrition and axon growth promotion. Thus, NGF might be a key molecule involved in regulating cancer-related neurogenesis, which could play a crucial role in the signal transmission system that controls nerve growth in tumors, and enhances the abilities of migration, invasion and metastasis of tumor cells. The present study aimed to construct differential expression plasmids of NGF, in order to detect whether NGF has a vital role in neurogenesis in breast cancer cells. In the present study, 92 clinical cases of breast cancer were collected and immunohistochemical analysis was performed to verify the existence of neurons in the breast cancer microenvironment. Furthermore, recombinant NGF lentiviral overexpression, knockout and silencing plasmids were constructed, and whether NGF has an effect on neuron growth was preliminarily confirmed, indicating that the successfully constructed plasmids could be used to verify the roles of NGF in cancer-associated neurogenesis.

Accelerated Neurite Outgrowth and Neurogenesis of PC12 Cells on an Fe-doped TiO2 Nanorod Film Triggered by Visible Light.

Acceleration of neurite outgrowth and neuronal differentiation of neural cells are critical for effective neural tissue regeneration. In addition to biochemical cues, biomaterials have proven to be a valuable tool for engineering neural cellular physiological processes. However, strategies with convenient potential spatiotemporal control are still desirable. We here design a novel Fe-doped TiO2 nanorod film using hemoglobin as the Fe source to endow it with visible-light-responsive regulated surface hydroxyl groups (-OH), which was demonstrated as the central role in mediating cell-material interactions in our previous study. The acceleration of neurite outgrowth and neuronal differentiation of PC12 cells might be attributed to the upregulated distinct terminal hydroxyl groups triggered by visible light. We also demonstrate that the actin cytoskeletal system plays a pivotal role during these processes, approved by the inhibition experiment results. This study therefore sheds light on the regulation of neurite outgrowth and neuronal differentiation of neural cells using a convenient spatiotemporal controllable strategy.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells.

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Human placental laminin: Role in neuronal differentiation, cell adhesion and proliferation

Human placental extract has wound healing potential. Immuno-blots revealed presence of laminin in placental extract (70 +/- 0.257 μg/ml; n=3). It was purified using immuno-affinity chromatography. SDS-PAGE and SEHPLC indicated a188 kDa protein with some small peptides. Since placental laminin existed in its truncated form, its roles in cellular migration, differentiation and wound healing were verified. Induction of cellular migration and motility in rat fibroblasts were enhanced by placental laminin as observed from scratch wound assay. Promotion of neuronal differentiation of PC12 cells by placental laminin was observed by phase contrast microscopy. Confocal images showed presence of laminin on the cell surface and along the axonal processes. Significant interaction between integrin receptors and laminin responsible for cellular differentiation was demonstrated from co-localization experiments. Union between integrin receptor and its synthetic antagonist revealed retarded pattern of neurite outgrowth in laminin treated cells. Animal model studies revealed faster wound healing in the presence of placental laminin. Induction of re-epithelialization and angiogenesis in wound area by cellular proliferation and adhesion were observed. The cytokine levels showed an initial rise and gradual fall over the duration of wound healing on application of the fragmented laminin. Thus, roles of placental laminin in neuronal differentiation and wound healing were indicated.

Molecular mechanism of nur77 gene expression and downstream target genes in the early stage of forskolin-induced differentiation in PC12 cells

Forskolin promotes neuronal differentiation of PC12 cells via the PKA-CREB-dependent signaling pathway. Activation of PKA by forskolin phosphorylates CREB, which then binds to CRE sites in numerous gene promoters. However, it is unclear which gene contains the CRE sites responsible for forskolin-induced neuronal differentiation. In this study, we investigated how an immediate early gene, nur77, which has CRE sites in the promoter region, contributes to the early stage of differentiation of forskolin-treated PC12 cells. After treatment with forskolin, expression of Nur77 was upregulated within 1 hr. In addition, knockdown of nur77 inhibited neurite outgrowth induced by forskolin. We also revealed that the specific four CRE sites near the transcriptional start site (TSS) of nur77 were strongly associated with phosphorylated CREB within 1 hr after treatment with forskolin. To analyze the roles of these four sites, reporter assays using the nur77 promoter region were performed. The results showed that nur77 expression was mediated through three of the CRE sites, −242, −222, and −78, and that −78, the nearest of the three to the TSS of nur77, was particularly important. An analysis of neuronal markers controlled by Nur77 after A-CREB-Nur77-Synapsin1 signaling pathway plays a pivotal role in differentiation of forskolin-induced PC12 cells.

ERK‐mediated Phosphorylation of the Egr Transcriptional Coregulator NAB2 at Multiple Sites

The early growth response (Egr) family of transcription factors comprises five members, Egr1‐4 and Wilms Tumor 1 (WT1), which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type‐ and stimulus‐specific manner. Roles for Egr1‐3 are particularly well‐documented in the nervous system. Egr1 and 3 contribute to learning and memory through regulation of genes that mediate synaptic plasticity and long‐term potentiation. Egr2 controls hindbrain development through regulation of Hox gene expression and drives peripheral nerve myelination through, at least in part, activation of the gene encoding myelin protein zero in Schwann cells. Egr1 and 2 also contribute to transcriptional network that drives neuronal differentiation of PC12 cells. However, the mechanisms that regulate Egr1‐3 activities in the nervous system are not fully understood. One mechanism of regulation involves protein‐protein interactions with co‐regulators NAB1 and 2 through a conserved R1 domain in Egr1‐3. Most reports demonstrate that NABs repress Egr1‐3 transactivation activity when bound, however, NABs can also enhance transactivation in a target gene‐specific manner. The mechanisms controlling the effect of NABs on Egr activities are not clear. Here, we provide evidence that NAB2 regulation may involve its phosphorylation at several sites by ERK. Using PC12 cells as a model system, our data demonstrate that nerve growth factor (NGF) induces a NAB2 mobility shift in SDS‐PAGE within 10 minutes of treatment. The mobility shift was reversed when lysates from NGF‐treated cells were treated with lambda phosphatase, suggesting the shift is due to phosphorylation. Inhibition of ERK signaling during NGF treatment blocked the mobility shift, while recombinant ERK phosphorylated recombinant NAB2 in an in vitro kinase assay, together indicating that direct phosphorylation of NAB2 by ERK may be responsible for the NAB2 mobility shift in response to NGF. Nine potential ERK phosphorylation sites were identified in NAB2, which were mutated individually to alanine within a NAB2 expression construct to evaluate phosphorylation at those sites based on their mobility in SDS‐PAGE. Of the nine mutants, three exhibited clear changes in NAB2 mobility, which were additive in double and triple mutants for those sites. Ongoing experiments are evaluating the effect of NAB2 phosphorylation on its localization and regulation of Egr1‐3.Support or Funding InformationBridgewater State University Undergraduate Research Program

Using CRISPR/Cas9 to Investigate the Role of Egr1 and NAB2 in Neuronal Differentiation of PC12 Cells

PC12 cells are a common model system used to study the molecular events underlying neuronal differentiation due to their ability to differentiate into neuronal cells following treatment with nerve growth factor (NGF). NGF induces neuronal differentiation through activation of the Ras/Raf/MEK/ERK signaling pathway, which in turn activates a transcriptional program leading to up‐regulation of neuronal genes, neurite outgrowth, and establish electrical excitability. Interestingly, treatment with epidermal growth factor (EGF) also activates Ras/Raf/MEK/ERK signaling, but induces proliferation rather than differentiation. The differential effects of NGF versus EGF are due to differences in ERK signal duration; EGF induces more transient ERK signaling that lasts 30–60 min, whereas NGF induces more sustained ERK signaling that lasts 4–6 h. Work in our lab and others indicate that sustained ERK signaling in response to NGF drives differentiation in part through sustained activity of the transcription factor Egr1 and, in turn, sustained expression of Egr1 target genes. Egr1 activity in response to EGF and NGF is regulated at two levels. First, Egr1 levels rapidly rise to peak levels by 1 h after treatment and then decline to baseline levels; similar to ERK signaling, Egr1 levels are sustained longer in response to NGF versus EGF. Second, one Egr1 target gene is its corepressor NAB2, which binds and represses Egr1 transactivation once expressed. This project was designed to further test our hypothesis that sustained Egr1 activity is a major driver of PC12 neuronal differentiation by evaluating whether knockout of NAB2 will lead to sustained Egr1 activity in response to EGF and, in turn, neuronal differentiation. To acquire PC12 cells that lack NAB2 protein, we used CRISPR/Cas9 technology to knockout the NAB2 gene. More specifically, a CRISPR NAB2‐KO plasmid was purchased from Santa Cruz Biotechnology, which encodes a green fluorescent protein (GFP) selectable marker, the Cas9 protein, and the guide RNA (gRNA). The plasmid had three variations for the gRNA which target different locations in the NAB2 gene. One day after transfection, GFP‐positive cells were sorted into 96‐well plates to isolate clones. Following clonal expansion, clones were first screened for NAB2 expression by Western blot, which identified eight putative knockout clones. Genomic DNA was then extracted from the eight clones, the NAB2 gene amplified by PCR, and the PCR project then subjected to Sanger sequencing to identify the INDELs responsible for NAB2 knockout. Ongoing work is examining the effects of NAB2 knockout on Egr1 levels and activity in response to EGF and NGF, as well as neuronal differentiation based on neurite outgrowth with the prediction that NAB2 knockout will result in sustained Egr1 activity and neuronal differentiation in response to both growth factors.Support or Funding InformationBridgewater State University Undergraduate Research Program

Effect of bone matrix protein osteocalcin on proliferation and neuronal differentiation of PC12 cells

Effect of bone matrix protein osteocalcin on proliferation and neuronal differentiation of PC12 cells

Guiding neural extensions of PC12 cells on carbon nanotube tracks dielectrophoretically formed in poly(ethylene glycol) dimethacrylate.

The PC12 cell line has been widely used as an in vitro model for studying neuronal differentiation and identifying the factors affecting the process. It has the ability to differentiate in the presence of nerve growth factor (NGF), resulting in neural extensions called dendrites and axons. In this study, first the impact of randomly distributed multi-walled carbon nanotubes (MWCNTs) in poly(ethylene glycol) dimethacrylate (PEGDMA) on PC12 cell differentiation was investigated in terms of neurite length, number of neurite per cell and differentiation marker gene expression profile. Then, dielectrophoretically aligned MWCNTs in PEGDMA was used to guide and support the neuronal differentiation of PC12 cells in the presence of NGF. The method is expected to be useful in revealing the nanotopographical role in fundamental studies and understanding of nanotopographical effects for biomedical applications on nerve regeneration.

Sustained GRK2-dependent CREB activation is essential for α2-adrenergic receptor-induced PC12 neuronal differentiation

Many aspects of neuronal development, such as neuronal survival and differentiation, are regulated by the transcription factor cAMP-response element-binding protein (CREB). We have previously reported that α2-adrenergic receptors (ARs), members of the G protein-coupled receptor (GPCR) superfamily, induce neuronal differentiation of rat pheochromocytoma (PC)-12 cells in a subtype-specific manner, i.e. α2A<α2B<α2C. Herein, we sought to investigate CREB`s involvement in this α2AR-dependent neurogenic process. We used a combination of gene reporter assays and immunoblotting analysis, coupled with co-immunoprecipitation and morphological analysis, in transfected PC12 cell lines. Chronic α2B- or α2C-AR activation results in sustained CREB activation, which is both necessary and sufficient for neuronal differentiation of PC12 cells expressing these two α2ARs. In contrast, chronic α2A activation only leads to transient CREB activation, insufficient for PC12 neuronal differentiation. However, upon CREB overexpression, α2A-AR triggers neuronal differentiation similarly to α2B- or α2C-ARs. Importantly, NGF (Nerve Growth Factor)`s TrkA receptor transactivation is essential for the sustained activation of CREB by all three α2 subtypes in PC12 cells, whereas protein kinase A (PKA), the prototypic kinase that phosphorylates CREB, is not. Instead, TrkA-activated GPCR-kinase (GRK)-2 mediates the sustained CREB activation during α2AR-induced neuronal differentiation of PC12 cells. In conclusion, catecholaminergic-induced neuronal differentiation of PC12 cells through α2ARs uses a non-canonical pathway involving TrkA transactivation and subsequent GRK2-dependent, sustained phosphorylation/activation of CREB. These findings provide novel insights into catecholaminergic neurogenesis and suggest that α2AR agonists, combined with NGF analogs or GRK2 stimulators, may exert neurogenic/neuroprotective effects.

Synthesis of polypyrrole nanorods via sacrificial removal of aluminum oxide nanopore template: A study on cell viability, electrical stimulation and neuronal differentiation of PC12 cells.

Synthesis of nanomaterials having uniform shape and size is a challenging task. Properties exhibited by such substrates would be compatible and homogeneous compared to the average properties displayed by those substrates with heterogeneous size. Herein, we report the synthesis of polypyrrole nanorods (PPy-NRs) of almost uniform size via oxidative chemical polymerization of pyrrole within anodized aluminum oxide nanopores followed by sacrificial removal of the template. Field emission scanning electron microscopy (FE-SEM), fourier transformed infra-red (FT-IR) spectra, X-ray diffraction (XRD), and ultra-violet-visible-near infra-red (UV-Vis-NIR) spectra of the substrate were used to analyze the physicochemical properties of as-synthesized PPy-NRs. PPy-NRs treated MC3T3-E1 and PC12 cells exhibited good biocompatibility in CCK-8 and live/dead assays. The assay showed more cell viability on PC12 cell lines. Electrical stimulation through PPy-NRs treated PC12 cells accelerated neuronal differentiation compared to those without electrical stimulation during in vitro cell culture.

Shexiang Baoxin Pill, a Formulated Chinese Herbal Mixture, Induces Neuronal Differentiation of PC12 Cells: A Signaling Triggered by Activation of Protein Kinase A.

Background: Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), which is commonly used today in treating cardiovascular diseases. SBP consists of seven materials thereof, including Moschus, extract of Ginseng Radix et Rhizoma, Bovis Calculus Artifactus, Cinnamomi Cortex, Styrax, Bufonis Venenum, and Borneolum Syntheticum. Here, we are investigating the potential roles of SBP in inducing neuron differentiation, i.e., seeking possible application in neurodegenerative diseases. Methods: Water and ethanol extracts of SBP, denoted as SBPwater and SBPEtOH, respectively, as well as its individual herbal materials, were standardized and applied onto cultured rat pheochromocytoma PC12 cells. The potential effect of SBP extracts in neuronal differentiation was suggested by following parameters: (i) induction of neurite outgrowth of PC12 cells, (ii) increase of neurofilament expression, and (iii) activation of transcription of neurofilament. Results: The treatments of SBPwater and SBPEtOH, or extracts from individual herbal materials, with or without low concentration of nerve growth factor (NGF), could potentiate the differentiation of cultured PC12 cells. The differentiation was indicated by increase of neurite outgrowth, as well as expression of neurofilaments. In addition, application of H89, a protein kinase A (PKA) inhibitor, suppressed the SBP-induced neurofilament expressions, as well as the phosphorylation of cAMP-responsive element binding protein (CREB) in cultures. Conclusion: SBP is proposed to possess trophic activity in modulating neuronal differentiation of PC12 cells, and this induction is shown to be mediated partly by a cAMP-PKA signaling pathway. These results indicate the neurite-promoting SBP could be useful in developing potential drug in treating or preventing neurodegenerative diseases.