scholarly journals Free circular introns with an unusual branchpoint in neuronal projections

2019 ◽  
Author(s):  
Harleen Saini ◽  
Alicia A. Bicknell ◽  
Sean R. Eddy ◽  
Melissa J. Moore
Keyword(s):  
Hippocampal Neurons ◽  
Neuronal Cells ◽  
Rna Localization ◽  
Sequence Conservation ◽  
Total Rna ◽  
Polyadenylated Rnas

AbstractThe polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

scholarly journals Free circular introns with an unusual branchpoint in neuronal projections

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Harleen Saini ◽  
Alicia A Bicknell ◽  
Sean R Eddy ◽  
Melissa J Moore
Keyword(s):  
Hippocampal Neurons ◽  
Neuronal Cells ◽  
Rna Localization ◽  
Sequence Conservation ◽  
Total Rna ◽  
Polyadenylated Rnas

The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

scholarly journals Comprehensive catalog of dendritically localized mRNA isoforms from sub-cellular sequencing of single mouse neurons

2018 ◽  
Author(s):  
Sarah A. Middleton ◽  
James Eberwine ◽  
Junhyong Kim
Keyword(s):  
Hippocampal Neurons ◽  
Rna Localization ◽  
Synaptic Potentiation ◽  
Protein Structure Analysis ◽  
Mrna Isoforms ◽  
Neuronal Dendrites ◽  
Specific Localization ◽  
Dendritic Rna ◽  
Alternative Isoforms

AbstractRNA localization to neuronal dendrites is critical step for long-lasting synaptic potentiation, but there is little consensus regarding which RNAs are localized and the role of alternative isoforms in localization. Using independent RNA-sequencing from soma and dendrites of the same neuron, we deeply profiled the sub-cellular transcriptomes to assess the extent and variability of dendritic RNA localization in individual hippocampal neurons, including an assessment of differential localization of alternative 3’UTR isoforms. We identified 2,225 dendritic RNAs, including 298 cases of 3’UTR isoform-specific localization. We extensively analyzed the localized RNAs for potential localization motifs, finding that B1 and B2 SINE elements are up to 5.7 times more abundant in localized RNA 3’UTRs than non-localized, and also functionally characterized the localized RNAs using protein structure analysis. Finally, we integrate our list of localized RNAs with the literature to provide a comprehensive list of known dendritically localized RNAs as a resource.

Embryonic Rat Hippocampal Neurons and GABA A Receptor Subunit-Transfected Non-neuronal Cells Release GABA Tonically

1998 ◽  
Vol 164 (3) ◽  
pp. 239-251 ◽  
Author(s):  
A.Y. Valeyev ◽  
A.E. Schaffner ◽  
P. Skolnick ◽  
V.S. Dunlap ◽  
G. Wong ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Neuronal Cells ◽  
Receptor Subunit ◽  
Gaba A

scholarly journals Object-based analyses in FIJI/ImageJ to measure local RNA translation sites in neurites in response to Aβ1-42 oligomers

2020 ◽  
Author(s):  
María Gamarra ◽  
Maite Blanco-Urrejola ◽  
Andreia F.R. Batista ◽  
Josune Imaz ◽  
Jimena Baleriola
Keyword(s):  
Protein Synthesis ◽  
Protein Delivery ◽  
Hippocampal Neurons ◽  
Rna Localization ◽  
Laplacian Operator ◽  
Local Translation ◽  
Local Protein Synthesis ◽  
Rna Translation ◽  
Object Based ◽  
Local Protein

AbstractSubcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. However, protein delivery can also rely on localization of mRNAs that are translated at target sites. Although once considered heretical, RNA localization has proven to be highly conserved in eukaryotes.RNA localization and localized translation are especially relevant in polarized cells like neurons where neurites extend dozens to hundreds of centimeters away from the soma. Local translation confers dendrites and axons the capacity to respond to their environment in an acute manner without fully relying on somatic signals. The relevance of local protein synthesis in neuron development, maintenance and disease has not been fully acknowledged until recent years, partly due to the limited amount of locally produced proteins. For instance, in hippocampal neurons levels of newly-synthesized somatic proteins can be more than 20-30 times greater than translation levels of neuritic proteins. Thus local translation events can be easily overlooked under the microscope.Here we describe an object-based analysis used to visualize and quantify local RNA translation sites in neurites. Newly-synthesized proteins are tagged with puromycin and endogenous RNAs labelled with SYTO. After imaging, signals corresponding to neuritic RNAs and proteins are filtered with a Laplacian operator to enhance the edges. Resulting pixels are converted into objects and selected by automatic masking followed by signal smoothing. Objects corresponding to RNA or protein and colocalized objects (RNA and protein) are quantified along individual neurites. Colocalization between RNA and protein in neurites correspond to newly-synthesized proteins arising from localized RNAs and represent localized translation sites. To test the validity of our analyses we have compared control neurons to Aβ1-42-treated neurons. Aβ is involved in the pathology of Alzheimer’s disease and was previously reported to induce local translation in axons and dendrites which in turn contributes to the disease. We have observed that Aβ increases the synthesis of neuritic proteins as well as the fraction of translating RNAs in distal sites of the neurite, suggesting an induction of local protein synthesis. Our results thus confirm previous reports and validate our quantification method.

scholarly journals Neuronal Cells Confinement by Micropatterned Cluster-Assembled Dots with Mechanotransductive Nanotopography

2018 ◽  
Author(s):  
Carsten Schulte ◽  
Jacopo Lamanna ◽  
Andrea Stefano Moro ◽  
Claudio Piazzoni ◽  
Francesca Borghi ◽  
...  
Keyword(s):  
Neural Networks ◽  
Hippocampal Neurons ◽  
Neural Circuit ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Deep Understanding ◽  
Glass Substrates ◽  
Wide Range ◽  
Controlled Structure

ABSTRACTThe in vitro fabrication of neural networks able to simulate brain circuits and to maintain their native connectivity is of strategic importance to gain a deep understanding of neural circuit physiology and brain natural computational algorithm(s). This would also enable a wide-range of applications including the development of efficient brain-on-chip devices or brain-computer interfaces. Chemical and mechanotransductive cues cooperate to promote proper development and functioning of neural networks. Since the 80’s, controlled growth of mammalian neuronal cells on micrometric patterned chemical cues with the development of synaptic connections and electrical activity has been reported, however the role of mechanotransductive signaling on the growth/organization of neural networks has not been investigated so far. Here we report the fabrication and characterization of patterned substrates for neuronal culture with a controlled structure both at the nano- and microscale suitable for the selective adhesion of neuronal cells. Nanostructured micrometric dots were patterned on passivated cell-repellent glass substrates by supersonic cluster beam deposition of zirconia nanoparticles through stencil masks. Cluster-assembled nanostructured zirconia surfaces are characterized by nanotopographical features that can direct the maturation of neural networks by mechanotransductive signaling. Our approach produces a controlled microscale pattern of adhesive areas with predetermined nanoscale morphology. We have validated these micropatterned substrates using a neuronal cell line (PC12 cells) and cultured hippocampal neurons. While cells have been uniformly plated on the substrates, they adhered only on the nanostructured zirconia regions, remaining effectively confined inside the nanostructured dots on which they were found to grow, move and differentiate.

scholarly journals Early exercise induces long-lasting morphological changes in cortical and hippocampal neurons throughout of a sedentary period of rats

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fernando Tadeu Serra ◽  
Andrea Dominguez Carvalho ◽  
Bruno Henrique Silva Araujo ◽  
Laila Brito Torres ◽  
Fabrizio dos Santos Cardoso ◽  
...  
Keyword(s):  
Physical Exercise ◽  
Hippocampal Neurons ◽  
Hippocampal Formation ◽  
Morphological Changes ◽  
Neuronal Cells ◽  
Exercise Session ◽  
Childhood And Adolescence ◽  
Early Exercise ◽  
Brain Functions ◽  
Survival Proteins

Abstract Life experiences at early ages, such as physical activity in childhood and adolescence, can result in long-lasting brain effects able to reduce future risk of brain disorders and to enhance lifelong brain functions. However, how early physical exercise promotes these effects remains unclear. A possible hypothesis is that physical exercise increases the expression of neurotrophic factors and stimulates neuronal growth, resulting in a neural reserve to be used at later ages. Basing our study on this hypothesis, we evaluated the absolute number and morphology of neuronal cells, as well as the expression of growth, proliferation and survival proteins (BDNF, Akt, mTOR, p70S6K, ERK and CREB) in the cerebral cortex and hippocampal formation throughout of a sedentary period of rats who were physically active during youth. To do this, male Wistar rats were submitted to an aerobic exercise protocol from the 21st to the 60th postnatal days (P21–P60), and evaluated at 0 (P60), 30 (P90) and 60 (P120) days after the last exercise session. Results showed that juvenile exercise increased, and maintained elevated, the number of cortical and hippocampal neuronal cells and dendritic arborization, when evaluated at the above post-exercise ages. Hippocampal BDNF levels and cortical mTOR expression were found to be increased at P60, but were restored to control levels at P90 and P120. Overall, these findings indicate that, despite the short-term effects on growth and survival proteins, early exercise induces long-lasting morphological changes in cortical and hippocampal neurons even during a sedentary period of rats.

scholarly journals Focus on the Role of Klotho Protein in Neuro-Immune Interactions in HT-22 Cells Upon LPS Stimulation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1231 ◽  
Author(s):  
Kinga Rusinek ◽  
Przemysław Sołek ◽  
Anna Tabęcka-Łonczyńska ◽  
Marek Koziorowski ◽  
Jennifer Mytych
Keyword(s):  
Er Stress ◽  
Neurodegenerative Disorders ◽  
Hippocampal Neurons ◽  
Nitrosative Stress ◽  
Defense Mechanism ◽  
Mapk Pathway ◽  
Apoptotic Cell ◽  
Neuronal Cells ◽  
Lps Challenge

Neuroinflammation is defined as the activation of the brain’s innate immune system in response to an inflammatory challenge and is considered to be a prominent feature of neurodegenerative diseases. The contribution of overactivated neuroglial cells to neuroinflammation and neurodegenerative disorders is well documented, however, the role of hippocampal neurons in the neuroinflammatory process remains fragmentary. In this study, we show for the first time, that klotho acts as a signal transducer between pro-survival and pro-apoptotic crosstalk mediated by ER stress in HT-22 hippocampal neuronal cells during LPS challenge. In control HT-22 cells, LPS treatment results in activation of the IRE1α-p38 MAPK pathway leading to increased secretion of anti-inflammatory IL-10, and thus, providing adaptation mechanism. On the other hand, in klotho-deficient HT-22 cells, LPS induces oxi-nitrosative stress and genomic instability associated with telomere dysfunctions leading to p53/p21-mediated cell cycle arrest and, in consequence, to ER stress, inflammation as well as of apoptotic cell death. Therefore, these results indicate that klotho serves as a part of the cellular defense mechanism engaged in the protection of neuronal cells against LPS-mediated neuroinflammation, emerging issues linked with neurodegenerative disorders.

scholarly journals K6PC-5 Activates SphK1-Nrf2 Signaling to Protect Neuronal Cells from Oxygen Glucose Deprivation/Re-Oxygenation

2018 ◽  
Vol 51 (4) ◽  
pp. 1908-1920 ◽  
Author(s):  
Hua Liu ◽  
Zhiqing Zhang ◽  
Min Xu ◽  
Rong Xu ◽  
Zhichun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Ischemia Reperfusion ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Glucose Deprivation ◽  
Cell Counting ◽  
Oxygen Glucose Deprivation ◽  
Related Factor ◽  
Cell Protection ◽  
Neuroprotective Strategy

Background/Aims: New strategies are required to combat neuronal ischemia-reperfusion injuries. K6PC-5 is a novel sphingosine kinase 1 (SphK1) activator whose potential activity in neuronal cells has not yet been tested. Methods: Cell survival and necrosis were assessed with a Cell Counting Kit-8 assay and lactate dehydrogenase release assay, respectively. Mitochondrial depolarization was tested by a JC-1 dye assay. Expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling components were examined by quantitative real-timePCR and western blotting. Results: K6PC-5 protected SH-SY5Y neuronal cells and primary murine hippocampal neurons from oxygen glucose deprivation/re-oxygenation (OGDR). K6PC-5 activated SphK1, and SphK1 knockdown by targeted short hairpin RNA (shRNA) almost completely abolished K6PC-5-induced neuronal cell protection. Further work showed that K6PC-5 inhibited OGDR-induced programmed necrosis in neuronal cells. Importantly, K6PC-5 activated Nrf2 signaling, which is downstream of SphK1. Silencing of Nrf2 by targeted shRNA almost completely nullified K6PC-5-mediated neuronal cell protection against OGDR. Conclusion: K6PC-5 activates SphK1-Nrf2 signaling to protect neuronal cells from OGDR. K6PC-5 might be a promising neuroprotective strategy for ischemia-reperfusion injuries.

scholarly journals Artemisinin Attenuated Hydrogen Peroxide (H2O2)-Induced Oxidative Injury in SH-SY5Y and Hippocampal Neurons via the Activation of AMPK Pathway

2019 ◽  
Vol 20 (11) ◽  
pp. 2680 ◽  
Author(s):  
Xia Zhao ◽  
Jiankang Fang ◽  
Shuai Li ◽  
Uma Gaur ◽  
Xingan Xing ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Neurodegenerative Diseases ◽  
Hippocampal Neurons ◽  
Neuronal Cells ◽  
Adenosine Monophosphate ◽  
Dependent Manner ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Ampk Pathway

Oxidative stress is believed to be one of the main causes of neurodegenerative diseases such as Alzheimer’s disease (AD). The pathogenesis of AD is still not elucidated clearly but oxidative stress is one of the key hypotheses. Here, we found that artemisinin, an anti-malarial Chinese medicine, possesses neuroprotective effects. However, the antioxidative effects of artemisinin remain to be explored. In this study, we found that artemisinin rescued SH-SY5Y and hippocampal neuronal cells from hydrogen peroxide (H2O2)-induced cell death at clinically relevant doses in a concentration-dependent manner. Further studies showed that artemisinin significantly restored the nuclear morphology, improved the abnormal changes in intracellular reactive oxygen species (ROS), reduced the mitochondrial membrane potential, and caspase-3 activation, thereby attenuating apoptosis. Artemisinin also stimulated the phosphorylation of the adenosine monophosphate -activated protein kinase (AMPK) pathway in SH-SY5Y cells in a time- and concentration-dependent manner. Inhibition of the AMPK pathway attenuated the protective effect of artemisinin. These data put together suggested that artemisinin has the potential to protect neuronal cells. Similar results were obtained in primary cultured hippocampal neurons. Cumulatively, these results indicated that artemisinin protected neuronal cells from oxidative damage, at least in part through the activation of AMPK. Our findings support the role of artemisinin as a potential therapeutic agent for neurodegenerative diseases.

scholarly journals Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

1997 ◽  
Vol 17 (8) ◽  
pp. 4633-4643 ◽  
Author(s):  
W L Kuo ◽  
K C Chung ◽  
M R Rosner
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Hippocampal Neurons ◽  
Alternative Pathway ◽  
Neuronal Cells ◽  
Neuronal Cell ◽  
Mek Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.

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