ISDN2014_0394: DENND5A regulates NGF‐induced neurite outgrowth in PC12 cells and dendrite patterning of primary hippocampal neurons

Abstract Dp40 is ubiquitously expressed, including in the central nervous system. Dp40 mRNA and protein are detected in the early stages and postnatal stages of the mouse brain, respectively. 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 (a mutant of Dp40) during the neuronal differentiation process 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 carried out in nerve growth factor-differentiated PC12-Dp40L170P cells showed that the protein expression profile was modified compared to that of the control cells (PC12 Tet-On). The proteins with the highest upregulated expression were α-internexin and S100a6, which are involved in cytoskeletal structure. 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. HspB1 was absent in undifferentiated cells and weakly detected in all differentiated cells. These results suggest that the subcellular distribution and expression of Dp40 has an important role in the neurite outgrowth of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

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1,800 MHz Radiofrequency Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells

Frontiers in Public Health ◽

10.3389/fpubh.2021.771508 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yanqi Li ◽

Ping Deng ◽

Chunhai Chen ◽

Qinlong Ma ◽

Huifeng Pi ◽

...

Keyword(s):

Protein Expression ◽

Cell Viability ◽

Brain Development ◽

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neurite Length ◽

Primary Mouse ◽

Primary Hippocampal Neurons ◽

Neuro2a Cells

Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development.Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway.Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells.Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF-EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h.Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus.

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S100A12 protein is a strong inducer of neurite outgrowth from primary hippocampal neurons

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2001.00605.x ◽

2008 ◽

Vol 79 (4) ◽

pp. 767-776 ◽

Cited By ~ 25

Author(s):

Sanne E. Mikkelsen ◽

Vera Novitskaya ◽

Marina Kriajevska ◽

Vladimir Berezin ◽

Elisabeth Bock ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

S100a12 Protein ◽

Primary Hippocampal Neurons

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KLHL1/MRP2 Mediates Neurite Outgrowth in a Glycogen Synthase Kinase 3β-Dependent Manner

Molecular and Cellular Biology ◽

10.1128/mcb.02167-05 ◽

2006 ◽

Vol 26 (22) ◽

pp. 8371-8384 ◽

Cited By ~ 20

Author(s):

Seyha Seng ◽

Hava Karsenty Avraham ◽

Shuxian Jiang ◽

Saritha Venkatesh ◽

Shalom Avraham

Keyword(s):

Neurite Outgrowth ◽

Pc12 Cells ◽

Hippocampal Neurons ◽

Glycogen Synthase ◽

Growth Cones ◽

Glycogen Synthase Kinase ◽

Actin Binding ◽

Dependent Manner ◽

Glycogen Synthase Kinase 3Β ◽

Expression Levels

ABSTRACT The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3β (GSK3β) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3β inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3β overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3β through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3β and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3β-dependent manner.

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Roles of Vitamin E in Energy Metabolism During Neurite Outgrowth

Current Developments in Nutrition ◽

10.1093/cdn/nzaa057_051 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1235-1235

Author(s):

Zachary Stratton ◽

Abigail Davis ◽

Elizabeth Jonas ◽

Kristi Crowe-White ◽

Han-A Park

Keyword(s):

Vitamin E ◽

Energy Metabolism ◽

Neurite Outgrowth ◽

Group Treatment ◽

Hippocampal Neurons ◽

Brain Injuries ◽

Control Group ◽

Morphological Development ◽

Central Target ◽

Primary Hippocampal Neurons

Abstract Objectives Neurite outgrowth is a pivotal process of brain development and recovery after brain injury. This metabolically demanding process requires assembly of the cytoskeleton and formation and maintenance of synapses. We have recently found that treatment with alpha-tocotrienol, an antioxidant and a member of the vitamin E family, prevents loss of mitochondrial inner membrane potential during oxidative stress. In this study, we hypothesize that the mitochondrion is the central target of alpha-tocotrienol-mediated neuroprotection, and treatment with alpha-tocotrienol may improve neuronal energy metabolism and promote neurite outgrowth. Methods Primary hippocampal neurons were grown in neurobasal media with or without alpha-tocotrienol for 3 weeks. Then, the morphological development of neurites, including polarity and arborization, was analyzed. We also assayed the ATP: ADP ratio in neurites using PercevalHR fluorescence biosensor after treatment with alpha-tocotrienol. Results Neurons grown with alpha-tocotrienol achieved neuronal polarity prior to the control group, and alpha-tocotrienol treated neurons showed longer and more branched neurites compared to the control group. Treatment with alpha-tocotrienol enhanced ATP levels in primary hippocampal neurons. Conclusions Our data show that alpha-tocotrienol improves mitochondria-mediated ATP production and supports the metabolically demanding process of neurite growth. This study also suggests that alpha-tocotrienol may be beneficial for recovery after brain injuries associated with neurite loss. Funding Sources RG14811 (University of Alabama).

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Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons

Toxicology and Applied Pharmacology ◽

10.1016/j.taap.2013.11.023 ◽

2014 ◽

Vol 274 (3) ◽

pp. 372-382 ◽

Cited By ~ 25

Author(s):

Daniella M. Pizzurro ◽

Khoi Dao ◽

Lucio G. Costa

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Primary Hippocampal Neurons

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Vitamin E Improves Neurite Complexity by Enhancing Mitochondrial Function

Current Developments in Nutrition ◽

10.1093/cdn/nzab049_028 ◽

2021 ◽

Vol 5 (Supplement_2) ◽

pp. 915-915

Author(s):

Han-A Park ◽

Kristi Crowe-White ◽

Abigail Davis ◽

Sydni Bannerman ◽

Garret Burnett ◽

...

Keyword(s):

Oxidative Stress ◽

Vitamin E ◽

Neurite Outgrowth ◽

Mitochondrial Function ◽

Hippocampal Neurons ◽

Brain Diseases ◽

Control Group ◽

University Of Alabama ◽

Protein Levels ◽

Primary Hippocampal Neurons

Abstract Objectives Neurite outgrowth is a foundational process in brain development and recovery from brain injury. Assembly of the cytoskeleton and formation of new synapses during neurite outgrowth requires an abundance of energy. We have reported that the mitochondrial protein Bcl-xL is necessary for neurite outgrowth and arborization. However, Bcl-xL undergoes post-translational cleavage during oxidative stress resulting in a product that impairs mitochondrial function. Our recent publication demonstrated that treatment with alpha-tocotrienol, an antioxidant member of the vitamin E family, prevents cleavage of Bcl-xL and protects neurons from oxidative stress. In this study, we hypothesize that treatment with alpha-tocotrienol improves mitochondrial function to support the energy demanding processes of growth and development in the neurons. Methods Primary hippocampal neurons were grown in neurobasal media with or without alpha-tocotrienol for 3 weeks. Then, the number of neurite branches was quantified applying Sholl analysis. We also assayed the ATP/ADP ratio at neurites using the PercevalHR fluorescence biosensor. mRNA and protein levels of total Bcl-xL and cleaved Bcl-xL were measured using real time PCR and immunoblotting. Results Neurons grown with alpha-tocotrienol achieved more advanced neurite complexity than the control group. Treatment with alpha-tocotrienol enhanced both total ATP and local neurite ATP levels in primary hippocampal neurons. Furthermore, we found that alpha-tocotrienol Increased mRNA and protein levels of Bcl-xL without enhancing post-translational cleavage of Bcl-xL, consistent with our previous study. Conclusions Our data show that alpha-tocotrienol improves mitochondria-mediated ATP production by enhancing Bcl-xL to support metabolically demanding processes in neurons. We suggest a novel function of alpha-tocotrienol in normal physiological development of the brain. This study also suggests a potential therapeutic role of alpha-tocotrienol in brain diseases associated with neurite injury. Funding Sources RGC Program (University of Alabama) Crenshaw Research Fund (University of Alabama).

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ATRA-induced increase in intracellular Ca2+concentration in primary hippocampal neurons

Cell Biology International ◽

10.1042/cbi034s074b ◽

2010 ◽

Vol 34 (8) ◽

pp. S74-S74

Author(s):

Tingyu Li ◽

Xiaojuan Zhang ◽

Xuan Zhang ◽

Jian Hea ◽

Yang Bi Youxue Liu ◽

...

Keyword(s):

Hippocampal Neurons ◽

Primary Hippocampal Neurons

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