scholarly journals Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons

2018 ◽  
Vol 19 (8) ◽  
pp. 2185 ◽  
Author(s):  
Michael Paolillo ◽  
Stefanie Peters ◽  
Andrea Schramm ◽  
Jens Schlossmann ◽  
Robert Feil
Keyword(s):  
Real Time ◽  
Primary Cultures ◽  
Granule Cell Layer ◽  
Cerebellar Granule Neurons ◽  
Type I ◽  
Granule Neurons ◽  
No Donor ◽  
Cerebellar Granule ◽  
Real Time Imaging ◽  
Cgmp Signaling

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP–Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

scholarly journals Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Susana González-Reyes ◽  
Silvia Guzmán-Beltrán ◽  
Omar Noel Medina-Campos ◽  
José Pedraza-Chaverri
Keyword(s):  
Protective Effect ◽  
Heme Oxygenase ◽  
Curcuma Longa ◽  
Primary Cultures ◽  
Buthionine Sulfoximine ◽  
Antioxidant Response ◽  
Cerebellar Granule Neurons ◽  
Heme Oxygenase 1 ◽  
Granule Neurons ◽  
Cerebellar Granule

Curcumin is a bifunctional antioxidant derived fromCurcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs) of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1) expression and by 5.6–14.3-fold glutathione (GSH) levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS) production, by 94% the reduction of GSH/glutathione disulfide (GSSG) ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death.

scholarly journals Neuroglobin Plays a Protective Role in Arsenite-Induced Cytotoxicity by Inhibition of Cdc42 and Rac1GTPases in Rat Cerebellar Granule Neurons

2015 ◽  
Vol 36 (4) ◽  
pp. 1613-1627 ◽  
Author(s):  
Xiaona Liu ◽  
Yanhui Gao ◽  
Yuan An ◽  
Xiaoyan Fu ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Rho Gtpases ◽  
Primary Cultures ◽  
Protective Role ◽  
Cell Counting ◽  
Cerebellar Granule Neurons ◽  
Tunel Staining ◽  
Granule Neurons ◽  
Cytotoxic Effects ◽  
Cerebellar Granule

Background and Aims: We have previously shown that neuroglobin (Ngb) expression can be regulated by sodium arsenite (NaAsO2) exposure in rat cerebellar granule neurons (CGNs). However, the precise molecular mechanisms of Ngb action are largely unknown. Ras homolog (Rho) guanosine triphosphatases (Rho GTPases) are involved in the regulation of a number of cellular processes, including cell cytotoxicity. It has been reported that Ngb can act as a guanine nucleotide dissociation inhibitior (GDI) role to inactivate Rho GTPases. Therefore, we investigated Rho GTPases activation induced by NaAsO2 exposure in rat CGNs and effects of Rho GTPases activation on the cells. We also investigated the role of Ngb in this process. Methods: Primary cultures of CGNs were prepared from 7-day-old Wistar rat pups. The cytotoxic effects of NaAsO2 on CGNs were evaluated using the Cell Counting Kit-8 assay and TUNEL staining. RNA interference technology was used to silence Ngb, and the subsequent effects were evaluated by quantitative RT-PCR and Western blot. Cdc42 and Rac1 activation were measured by pull-down assay and Western blot. Results: NaAsO2 induced cytotoxicity in rat CGNs, increased GTP-bound form of Cdc42 and Rac1 GTPases in the cells. Furthermore, inhibition of Cdc42 or Rac1 activity using the inhibitor ZCL278 or NSC23766 decreased apoptosis and increased cell viability in the cells exposed to NaAsO2. Using siRNA-mediated knockdown, we show that NaAsO2-induced cytotoxicity was exacerbated, activation of Cdc42 (GTP-Cdc42) and Rac1 (GTP-Rac1) was increased in Ngb RNA silencing cells. Conclusions: cytotoxic effects of NaAsO2 on rat CGNs is induced at least partly by Cdc42 and Rac1 activation, and Ngb can inhibit Cdc42 and Rac1 activation to play protective role in rat CGNs exposed to NaAsO2.

scholarly journals Binding of Amyloid β(1–42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

2021 ◽  
Vol 22 (4) ◽  
pp. 1984
Author(s):  
Joana Poejo ◽  
Jairo Salazar ◽  
Ana M. Mata ◽  
Carlos Gutierrez-Merino
Keyword(s):  
Lipid Rafts ◽  
Calcium Homeostasis ◽  
Binding Capacity ◽  
Primary Cultures ◽  
Amyloid Β ◽  
Cytosolic Calcium ◽  
Cerebellar Granule Neurons ◽  
Granule Neurons ◽  
Microscopy Imaging ◽  
Cerebellar Granule

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer’s disease (AD). In a previous work, we showed that Aβ(1–42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aβ(1–42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ(1–42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aβ(1–42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ(1–42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ(1–42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ(1–42) concentration in this range was achieved by 2 h incubation of CGN with 2 μM Aβ(1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ(1–42) dimers, whose activity is inhibited by CaM:Aβ(1–42) complexes bound to lipid rafts.

scholarly journals AraC binds the p75NTR transmembrane domain to induce neurodegeneration in mature neurons

2021 ◽  
Author(s):  
Vanessa Lopes-Rodrigues ◽  
Pia Boxy ◽  
Eunice Sim ◽  
Dong Ik Park ◽  
Josep Carbonell ◽  
...  
Keyword(s):  
Cell Death ◽  
Transmembrane Domain ◽  
Primary Cultures ◽  
Cerebellar Granule Neurons ◽  
High Dose ◽  
Dependent Manner ◽  
Granule Neurons ◽  
Cerebellar Granule ◽  
Mature Neurons ◽  
Neurite Degeneration

AbstractBackgroundCytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer including acute myeloid leukaemia. However, after high dose AraC chemotherapy regime, patients develop severe neurotoxicity and neurodegeneration in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells, however, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesized that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukemia patients undergoing AraC treatment.MethodsTo determine the role of AraC-p75NTR signalling in degeneration of mature cerebellar granule neurons, we used primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurodegeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the direct interaction between AraC and p75NTR, we performed isothermal dose response-cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging.ResultsWe show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, AraC binds to Proline 252 and Cysteine 256 of the p75NTR transmembrane domain and selectively uncouples p75NTR from the NFκB survival pathway. This in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR.ConclusionOur findings identify p75NTR as a novel molecular target to develop treatments to counteract AraC-mediated neurodegeneration.

scholarly journals Neuroprotection Comparison of Rosmarinic Acid and Carnosic Acid in Primary Cultures of Cerebellar Granule Neurons

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2956 ◽  
Author(s):  
Faten Taram ◽  
Elizabeth Ignowski ◽  
Nathan Duval ◽  
Daniel Linseman
Keyword(s):  
Rosmarinic Acid ◽  
Nitrosative Stress ◽  
Primary Cultures ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Carnosic Acid ◽  
Cerebellar Granule Neurons ◽  
Granule Neurons ◽  
Neuroprotective Effects ◽  
Cerebellar Granule

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease, are characterized by the progressive loss of neurons in specific regions of the brain and/or spinal cord. Neuronal cell loss typically occurs by either apoptotic or necrotic mechanisms. Oxidative stress and nitrosative stress, along with excitotoxicity and caspase activation, have all been implicated as major underlying causes of neuronal cell death. Diverse nutraceuticals (bioactive compounds found in common foods) have been shown to have neuroprotective effects in a variety of in vitro and in vivo disease models. In the current study, we compared the neuroprotective effects of two polyphenolic compounds, rosmarinic acid and carnosic acid, which are both found at substantial concentrations in the herb rosemary. The capacity of these compounds to rescue primary cultures of rat cerebellar granule neurons (CGNs) from a variety of stressors was investigated. Both polyphenols significantly reduced CGN death induced by the nitric oxide donor, sodium nitroprusside (nitrosative stress). Rosmarinic acid uniquely protected CGNs from glutamate-induced excitotoxicity, while only carnosic acid rescued CGNs from caspase-dependent apoptosis induced by removal of depolarizing extracellular potassium (5K apoptotic condition). Finally, we found that carnosic acid protects CGNs from 5K-induced apoptosis by activating a phosphatidylinositol 3-kinase (PI3K) pro-survival pathway. The shared and unique neuroprotective effects of these two compounds against diverse modes of neuronal cell death suggest that future preclinical studies should explore the potential complementary effects of these rosemary polyphenols on neurodegenerative disease progression.

Sign in / Sign up

Export Citation Format

Share Document