Different ataxin-3 amyloid aggregates induce intracellular Ca(2+) deregulation by different mechanisms in cerebellar granule cells.

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Differential expression of two-pore domain potassium channels in rat cerebellar granule neurons.

Two pore domain potassium (K2P) channels are mostly present in the central nervous system (CNS) where they play important roles in modulating neuronal excitability. K2P channels give rise to background K(+) currents (IKSO) a key component in setting and maintaining the resting membrane potential in excitable cells. Here, we studied the expression and relative abundances of K2P channels in cerebellar granule neurons (CGNs), combining molecular biology, electrophysiology and immunologic techniques. The CGN IKSO was very sensitive to external pH, as previously reported. Quantitative determination of mRNA expression level demonstrated the existence of an accumulation pattern of transcripts in CGN that encode K2P9>K2P1>K2P3>K2P18>K2P2=K2P10>K2P4>K2P5 subunits. The presence of the major K2P subunits expressed was then confirmed by Western blot and immunofluorescence analysis, demonstrating robust expression of K2P1 (TWIK-1), K2P3 (TASK-1), K2P9 (TASK-3) and K2P18 (TRESK) channel protein. Based, on these results, it is concluded that K2P1, -3, -9 and -18 subunits represent the majority component of IKSO current in CGN.

C-terminal binding proteins are essential pro-survival factors that undergo caspase-dependent downregulation during neuronal apoptosis.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. Here, we show that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors. Moreover, antisense-mediated downregulation of CtBP1 is sufficient to cause CGN apoptosis. Similarly, the CtBP inhibitor, 4-methylthio-2-oxobutyric acid, induces expression of the CtBP target Noxa and causes actinomycin-sensitive CGN apoptosis. Unexpectedly, we found that the mechanism of CtBP downregulation in CGNs undergoing apoptosis varies in a stimulus-specific manner involving either the proteasome or caspases. In the case of CGNs deprived of depolarizing potassium (5K apoptotic condition), caspases appear to play a dominant role in CtBP downregulation. However, incubation in 5K does not enhance the kinetics of CtBP1 degradation and recombinant CtBP1 is not cleaved in vitro by caspase-3. In addition, 5K has no significant effect on CtBP transcript expression. Finally, mouse embryonic stem cells display caspase-dependent downregulation of CtBP1 following exposure to staurosporine, an effect that is not observed in DGCR8 knockout cells which are deficient in miRNA processing. These data identify caspase-dependent downregulation of CtBPs as an alternative mechanism to the proteasome for regulation of these transcriptional co-repressors in neurons undergoing apoptosis. Moreover, caspases appear to regulate CtBP expression indirectly, at a post-transcriptional level, and via a mechanism that is dependent upon miRNA processing. We conclude that CtBPs are essential pro-survival proteins in neurons and their downregulation contributes significantly to neuronal apoptosis via the de-repression of pro-apoptotic genes.

Neuregulin-1/ErbB4 signaling regulates Kv4.2-mediated transient outward K+ current through the Akt/mTOR pathway.

Neuregulin-1 (NRG-1) is a member of a family of neurotrophic factors that is required for the differentiation, migration, and development of neurons. NRG-1 signaling is thought to contribute to both neuronal development and the neuropathology of schizophrenia, which is believed to be a neurodevelopmental disorder. However, few studies have investigated the role of NRG-1 on voltage-gated ion channels. In this study, we report that NRG-1 specifically increases the density of transient outward K(+) currents (IA) in rat cerebellar granule neurons (CGNs) in a time-dependent manner without modifying the activation or inactivation properties of IA channels. The increase in IA density is mediated by increased protein expression of Kv4.2, the main α-subunit of the IA channel, most likely by upregulation of translation. The effect of NRG-1 on IA density and Kv4.2 expression was only significant in immature neurons. Mechanistically, both Akt and mammalian target of rapamycin (mTOR) signaling pathways are required for the increased NRG-1-induced IA density and expression of Kv4.2. Moreover, pharmacological blockade of the ErbB4 receptor reduced the effect of NRG-1 on IA density and Kv4.2 induction. Our data reveal, for the first time, that stimulation of ErbB4 signaling by NRG-1 upregulates the expression of K(+) channel proteins via activation of the Akt/mTOR signaling pathway and plays an important role in neuronal development and maturation. NRG1 does not acutely change IA and delayed-rectifier outward (IK) of rat CGNs, suggesting that it may not alter excitability of immature neurons by altering potassium channel property.

Calcium Deregulation: Novel Insights to Understand Friedreich's Ataxia Pathophysiology.

Friedreich's Ataxia (FRDA) is a neurodegenerative disorder, characterized by degeneration of dorsal root ganglia, cerebellum and cardiomyopathy. Heart failure is one of the most common causes of death for FRDA patients. Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA. The focus of our study was to investigate the unexplored Ca2+ homeostasis in cerebellar granule neurons (CGNs) and in cardiomyocytes of FRDA cellular models to understand the pathogenesis of degeneration. Ca2+ homeostasis in neurons and cardiomyocytes is not only crucial for the cellular wellbeing but more importantly to generate action potential in both neurons and cardiomyocytes. By challenging Ca2+ homeostasis in CGNs, and in adult and neonatal cardiomyocytes of FRDA models, we have assessed the impact of frataxin decrease on both neuronal and cardiac physiopathology. Interestingly, we have found that Ca2+ homeostasis is altered both cell types. CGNs showed a Ca2+ mishandling under depolarizing conditions and this was also reflected in the endoplasmic reticulum (ER) content. In cardiomyocytes we found that the sarcoplasmic reticulum (SR) Ca2+ content was pathologically reduced, and that mitochondrial Ca2+ uptake was impaired. This phenomenon is due to the excess of oxidative stress under FRDA like conditions and the consequent aberrant modulation of key players at the SR/ER and mitochondrial level that usually restore the Ca2+ homeostasis. Our findings demonstrate that in both neurons and cardiomyocytes the decreased Ca2+ level within the stores has a comparable detrimental impact in their physiology. In cardiomyocytes, we found that ryanodine receptors (RyRs) may be leaking and expel more Ca2+ out from the SR. At the same time mitochondrial uptake was altered and we found that Vitamin E can restore this defect. Moreover, Vitamin E protects from cell death induced by hypoxia-reperfusion injury, revealing novel properties of Vitamin E as potential therapeutic tool for FRDA cardiomyopathy.

Chlorpyrifos Toxicity in Mouse Cultured Cerebellar Granule Neurons at Different Stages of Development: Additive Effect on Glutamate-Induced Excitotoxicity.

OBJECTIVE: Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Its mechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicity in mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity. MATERIALS AND METHODS: This study was an in vitro experimental study performed on mice cultured CGNs. Immature and mature neurons were exposed to different concentrations of CPF (1-1000 µM) and glutamate (10-600 µM) for 48 hours after which we used the MTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 days in order to evaluate the cytotoxic effect on developing neurons. Mature neurons received sub-lethal concentrations of CPF (10, 100 µM) combined with different concentrations of glutamate. AChE activity and reactive oxygen species (ROS) generation were assessed after treatments. RESULTS: Immature CGNs had increased sensitivity to CPF toxicity compared to mature neurons. We observed significantly greater ROS production in immature compared to mature neurons, however AChE activity was more inhibited in mature neurons. Although CPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, however glutamate induced ROS production was not affected. The results suggested that CPF potentiated glutamate toxicity by mechanisms other than oxidative stress. CONCLUSION: CPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease.

L1 modulates PKD1 phosphorylation in cerebellar granule neurons.

The neural cell adhesion molecule L1 (L1CAM) is crucial for the development of the nervous system, with an essential role in regulating multiple cellular activities. Protein kinase D1 (PKD1) serves as a key kinase given its diverse array of functions within the cell. Here, we investigated various aspects of the functional relationship between L1 and phosphorylated PKD1 (pPKD1) in cerebellar granule neurons. To study the relationship between L1 and PKD1 phosphorylation, human cerebellar tissue microarrays were subject to immunofluorescence staining. We observed a positive correlation between L1 protein levels and PKD1 phosphorylation. In addition, L1 also co-localized with pPKD1. To analyze the regulatory role of L1 on PKD1 phosphorylation, primary mouse cerebellar granule neurons were treated with various concentrations of rL1 for 48 h. Using Western blot, we revealed that L1 significantly increased PKD1 phosphorylation compared with vehicle control, with the maximal effect observed at 5 nM. ERK1/2 phosphorylation was significantly increased by 2.5 nM and 10nM L1, with no apparent change in SRC phosphorylation. However, SRC expression was markedly reduced by 10nM rL1. AKT1 expression and phosphorylation levels were significantly increased by rL1, with the maximal effect observed at 2.5 and 5 nM, respectively. Our combined data revealed a positive relationship between L1 and pPKD1 in both cultured cerebellar neurons and human cerebellar tissue, suggesting that L1 functions in the modulation of PKD1 phosphorylation.

Neuroprotective Effect of Total and Sequential Extract of Scrophularia striata Boiss. in Rat Cerebellar Granule Neurons Following Glutamate- Induced Neurotoxicity: An In-vitro Study.

Neuroprotective effect of the extract from aerial parts of Scrophularia striata Boiss (Scrophulariaceae) was investigated against glutamate-induced neurotoxicity on cultured rat pups Cerebellar Granule Neurons (CGNs). CGNs from 8 days old Sprague-Dawley rat were prepared and cultured. The experiments were performed after 8 days in culture. The plant was collected from the northeastern part (Ruin region) of Iran and air-dried at room temperature. The total extract was prepared with maceration of prepared powder in ethanol 80% for three times. SEQUENTIAL EXTRACTS WERE OBTAINED USING DRIED AND POWDERED AERIAL PARTS WITH INCREASINGLY POLAR SOLVENTS: petroleum ether, chloroform, ethyl acetate and methanol 80% solution. Cultured cells were exposed to 125 µM of glutamate for 12 h following a 24 h of incubation with test fractions at concentration of 10 mcg/mL. Morphological assay was performed using invert light microscope after fixation and staining with haematoxylin. Neuronal viability was measured using MTT assay. Statistical analysis was done using SPSS software. One way analysis of variance (ANOVA) was performed by Tukey post-hoc test. Values were considered statistically significant when p-value ≤ 0.05. Results of this study showed a significant neuroprotective activity of high polarity methanolic fraction of aerial parts of Scrophularia striata against glutamate-induced neurotoxicity in a dosedependent manner. Treatment with 10 mcg/mL of the fractions showed the best result.

Lysophosphatidylcholine enhances NGF-induced MAPK and Akt signals through the extracellular domain of TrkA in PC12 cells.

Lysophosphatidylcholine (LPC) is one of the major lysophospholipids mainly generated by phospholipase A2 (PLA2)-mediated hydrolysis of phosphatidylcholine (PC). We previously found that LPC displays neurotrophin-like activity in the rat pheochromocytoma PC12 cells and in cerebellar granule neurons, but the molecular mechanism remains unclear. We report here that LPC specifically enhances nerve growth factor (NGF)-induced signals in PC12 cells. When PC12 cells were treated with NGF, MAPK was phosphorylated, but this phosphorylation was significantly elevated when LPC was added together. In accordance, NGF-induced expression of immediate early genes, c-fos and NGF-IA, was upregulated by LPC. Phosphorylation of the upstream components, MEK and NGF receptor TrkA, was also promoted by LPC, which was in line with increased phosphorylation of Akt. In contrast, LPC did not enhance epidermal growth factor (EGF)-, basic fibroblast growth factor-, or insulin-like growth factor-1-induced signals. Studies using TrkA/EGF receptor chimeras demonstrated that the extracellular domain, but not the transmembrane or intracellular domains, of TrkA is responsible for the effect of LPC. Exogenously-added secretory PLA2 (sPLA2) enhanced NGF-induced MAPK phosphorylation at a comparable level to LPC, suggesting that LPC generated in situ by sPLA2-mediated hydrolysis of membrane PC stimulated NGF-TrkA signal. Taken together, these results indicate a specific role and function of LPC on NGF-TrkA signaling pathway.

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain.

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion.