Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

P2X Purinergic Receptors Are Multisensory Detectors for Micro-Environmental Stimuli That Control Migration of Tumoral Endothelium.

The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.

Deletion of calcineurin from astrocytes reproduces proteome signature of Alzheimer's disease and epilepsy and predisposes to seizures.

Calcineurin (CaN), acting downstream of intracellular calcium signals, orchestrates cellular remodeling in many cellular types. In astrocytes, major homeostatic players in the central nervous system (CNS), CaN is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in mice with conditional deletion of CaN in GFAP-expressing astrocytes (astroglial calcineurin KO, ACN-KO), at 1 month of age, transcription was largely unchanged, while the proteome was deranged in the hippocampus and cerebellum. Gene ontology analysis revealed overrepresentation of annotations related to myelin sheath, mitochondria, ribosome and cytoskeleton. Over-represented pathways were related to protein synthesis, oxidative phosphorylation, mTOR and neurological disorders, including Alzheimer's disease (AD) and seizure disorder. Comparison with published proteomic datasets showed significant overlap with the proteome of a familial AD mouse model and of human subjects with drug-resistant seizures. ACN-KO mice showed no alterations of motor activity, equilibrium, anxiety or depressive state. However, in Barnes maze ACN-KO mice learned the task but adopted serial search strategy. Strikingly, beginning from about 5 months of age ACN-KO mice developed spontaneous tonic-clonic seizures with an inflammatory signature of epileptic brains. Altogether, our data suggest that the deletion of astroglial CaN produces features of neurological disorders and predisposes mice to seizures. We suggest that calcineurin in astrocytes may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the central nervous system.

Early Stimulation of TREK Channel Transcription and Activity Induced by Oxaliplatin-Dependent Cytosolic Acidification.

Oxaliplatin-induced peripheral neuropathy is characterized by an acute hyperexcitability syndrome triggered/exacerbated by cold. The mechanisms underlying oxaliplatin-induced peripheral neuropathy are unclear, but the alteration of ion channel expression and activity plays a well-recognized central role. Recently, we found that oxaliplatin leads to cytosolic acidification in dorsal root ganglion (DRG) neurons. Here, we investigated the early impact of oxaliplatin on the proton-sensitive TREK potassium channels. Following a 6-h oxaliplatin treatment, both channels underwent a transcription upregulation that returned to control levels after 42 h. The overexpression of TREK channels was also observed after in vivo treatment in DRG cells from mice exposed to acute treatment with oxaliplatin. Moreover, both intracellular pH and TREK channel transcription were similarly regulated after incubation with amiloride, an inhibitor of the Na+/H+ exchanger. In addition, we studied the role of oxaliplatin-induced acidification on channel behavior, and, as expected, we observed a robust positive modulation of TREK channel activity. Finally, we focused on the impact of this complex modulation on capsaicin-evoked neuronal activity finding a transient decrease in the average firing rate following 6 h of oxaliplatin treatment. In conclusion, the early activation of TREK genes may represent a mechanism of protection against the oxaliplatin-related perturbation of neuronal excitability.

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice.

The pathogenesis of Alzheimer's disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER- and mitochondria-targeted aequorin-based Ca2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca2+ while Ca2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8-10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

Assessment of a Silicon-Photomultiplier-Based Platform for the Measurement of Intracellular Calcium Dynamics with Targeted Aequorin.

Ca2+ is among the most important intracellular second messengers participating in a plethora of biological processes, and the measurement of Ca2+ fluctuations is significant in the phenomenology of the underlying processes. Aequorin-based Ca2+ probes represent an invaluable tool for reliable measurement of Ca2+ concentrations and dynamics in different subcellular compartments. However, their use is limited due to the lack on the market of ready-to-use, cost-effective, and portable devices for the detection and readout of the low-intensity bioluminescence signal produced by these probes. Silicon photomultipliers (SiPMs) are rapidly evolving solid-state sensors for low light detection, with single photon sensitivity and photon number resolving capability, featuring low cost, low voltage, and compact format. Thus, they may represent the sensors of choice for the development of such devices and, more in general, of a new generation of multipurpose bioluminescence detectors suitable for cell biology studies. Ideally, a detector customized for these purposes must combine high dynamic range with high fidelity in reconstructing the light intensity signal temporal profile. In this article, the ability to perform aequorin-based intracellular Ca2+ measurements using a multipurpose, low-cost setup exploiting SiPMs as the sensors is demonstrated. SiPMs turn out to assure performances comparable to those exhibited by a custom-designed photomultiplier tube-based aequorinometer. Moreover, the flexibility of SiPM-based devices might pave the way toward routinely and wide scale application of innovative biophysical protocols.

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+ /K+ ATPase.

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca2+ /calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein-expressing astrocytes (astroglial calcineurin KO [ACN-KO]). Here, we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K+ homeostasis. Indeed, blockade of Na+ /K+ -ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca2+ -sensitive switch.

The interaction of SiO2 nanoparticles with the neuronal cell membrane: activation of ionic channels and calcium influx.

AIM: To clarify the mechanisms of interaction between SiO2 nanoparticles (NPs) and the plasma membrane of GT1-7 neuroendocrine cells, with focus on the activation of calcium-permeable channels, responsible for the long lasting calcium influx and modulation of the electrical activity in these cells. MATERIALS & METHODS: Nontoxic doses of SiO2 NPs were administered to the cells. Calcium imaging and patch clamp techniques were combined with a pharmacological approach. RESULTS: TRPV4, Cx and Panx-like channels are the major components of the NP-induced inward currents. Preincubation with the antioxidant N-acetyl-L-cysteine strongly reduced the [Ca2+]i increase. CONCLUSION: These findings suggest that SiO2 NPs directly activate a complex set of calcium-permeable channels, possibly by catalyzing free radical production.

Oxaliplatin induces pH acidification in dorsal root ganglia neurons.

Oxaliplatin induced peripheral neurotoxicity is characterized by an acute cold-induced syndrome characterized by cramps, paresthesias/dysesthesias in the distal limbs and perioral region, that develops rapidly and lasts up to one week affecting nearly all the patients as well as by long-lasting symptoms. It has been previously shown that pharmacological or genetic ablation of TRPA1 responses reduces oxaliplatin-induced peripheral neurotoxicity in mouse models. In the present report, we show that treatment with concentrations of oxaliplatin similar to those found in plasma of treated patients leads to an acidification of the cytosol of mouse dorsal root ganglia neurons in culture and this in turn is responsible for sensitization of TRPA1 channels, thereby providing a mechanistic explanation to toxicity of oxaliplatin. Reversal of the acidification indeed leads to a significantly reduced activity of TRPA1 channels. Last, acidification occurs also in vivo after a single injection of therapeutically-relevant doses of oxaliplatin.

Susceptibility of different mouse strains to oxaliplatin peripheral neurotoxicity: Phenotypic and genotypic insights.

Peripheral neurotoxicity is one of the most distressing side effects of oxaliplatin therapy for cancer. Indeed, most patients that received oxaliplatin experience acute and/or chronic severe sensory peripheral neuropathy. However, despite similar co-morbidities, cancer stage, demographics and treatment schedule, patients develop oxaliplatin-induced peripheral neurotoxicity with remarkably different severity. This suggests individual genetic variability, which might be used to glean the mechanistic insights into oxaliplatin neurotoxicity. We characterized the susceptibility of different mice strains to oxaliplatin neurotoxicity investigating the phenotypic features of neuropathy and gene expression profiles in dorsal root ganglia of six genetically different mice strains (Balb-c, C57BL6, DBA/2J, AJ, FVB and CD1) exposed to the same oxaliplatin schedule. Differential gene expression in dorsal root ganglia from each mice strain were assayed using a genome-wide expression analysis and selected genes were validated by RT-PCR analysis. The demonstration of consistent differences in the phenotypic response to oxaliplatin across different strains is interesting to allow the selection of the appropriate strain based on the pre-defined read-out parameters. Further investigation of the correlation between gene expression changes and oxaliplatin-induced neurotoxicity phenotype in each strain will be useful to deeper investigate the molecular mechanisms of oxaliplatin neurotoxicity.

Nanosized TiO2 is internalized by dorsal root ganglion cells and causes damage via apoptosis.

Titanium dioxide (TiO2) is widely used as ingredient in several products in the nanoform. TiO2-nanoparticles (NPs) are also currently studied for different medical applications. A large debate exists on possible adverse health effects related to their exposure. While there is some evidence of TiO2-NP central nervous system toxicity, their effects on peripheral neurons have been poorly explored. In this study we investigated the effects of TiO2-NPs on dorsal root ganglion (DRG) sensory neurons and satellite glial cells that may be reached by nanoparticles from the bloodstream. We found that TiO2-NPs are internalized in DRG cells and induce apoptosis in a dose dependent manner in both types of cells, ROS production and changes in expression of proinflammatory cytokine IL-1ß. Furthermore, we found that the axonal retrograde transport is altered in neurons upon exposure to TiO2-NPs. Overall, the results indicate a potential neurotoxic effect of TiO2-NPs on DRG cells. FROM THE CLINICAL EDITOR: Exposure to titanium dioxide nanoparticles is increasing in medical practice. Little is known about their potential toxic effects on the peripheral nervous system. The authors studied this aspect and showed that titanium nanoparticles might potentially cause toxicity over long term.

Activation of TRPV4 channels reduces migration of immortalized neuroendocrine cells.

Calcium is a universal signal, and its capacity to encode intracellular messages via spatial, temporal and amplitude characteristics allows it to participate in most cellular events. In a specific context, calcium plays a pivotal role in migration, although its role has not been elucidated fully. By using immortalized gonadotropin-releasing hormone-secreting neurons (GN11), we have now investigated the role of TRPV4, a member of the vanilloid family of Ca(2+) channels, in neuronal migration. Our results show that TRPV4 channels are present and functional in GN11 cells and their localization is polarized and enriched in lamellipodial structures. TRPV4 activation leads to a retraction of the lamellipodia and to a decrease in migratory behaviour; moreover cells migrate slower and in a more random manner. We therefore provide evidence for a new regulation of gonadotropin-releasing hormone neurons and a new role for calcium at the leading edge of migratory cells.

Novel adenosine and cAMP signalling pathways in migrating glial cells.

This study was aimed at characterizing the effect of purinergic transmission on migration of embryonic ciliary ganglion satellite glial cells. Application of adenosine significantly decreased the rate of migration of glial cells whereas no differences were observed in the presence of ATP. The A(2B) receptor antagonist reverted this action, but application of an A(2A) receptor antagonist or a cAMP-protein kinase inhibitor had no effect on the agonist's stimulation. Forskolin, which stimulates adenylate cyclase activity, and the cAMP analogue 8-CPT-2'-O-Me-cAMP, which selectively activates the guanine exchange factor Epac1, mimicked the effect of adenosine. In addition, intracellular calcium measurements studies revealed that application of either adenosine or ATP induced an increase in [Ca(2+)]i and that the adenosine-induced [Ca(2+)]i response was due to Ca(2+) entry and was blocked by an A(2A) receptor antagonist, SCH 58261, or by high Gd(3+) concentrations. Furthermore, forskolin, but not 8-CPT-2'-O-Me-cAMP, activated the Ca(2+) entry which was blocked by Gd(3+) and was independent of cAMP-protein kinase activity. These results demonstrate the involvement of purinergic P1 signalling in the regulation of cellular migration, and point to the importance of adenosine as a negative modulator of migration of peripheral developing glial cells and as an activator of Ca(2+) entry.

Calcium signals activated by ghrelin and D-Lys(3)-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells.

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys(3)-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca(2+)](i) followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca(2+) entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys(3)-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca(2+) activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys(3)-GHRP-6 ghrelin antagonist features ghrelin independent activities.

Calcium signals and the in vitro migration of chick ciliary ganglion cells.

We have studied calcium signals and their role in the migration of neuronal and nonneuronal cells of embryonic chick ciliary ganglion (CG). In vitro, neurons migrate in association with nonneuronal cells to form cellular aggregates. Changes in the modulus of the velocity of the neuron-nonneuronal cell complex were observed in response to treatments that increased or decreased intracellular calcium concentration. In addition, both cell types generated spontaneous calcium activity that was abolished by removal of extracellular calcium. Calcium signals in neurons could be characterized as either spikes or waves. Neuronal spikes were found to be related to action potential generation whereas neuronal waves were due to voltage-independent calcium influx. Nonneuronal cells generated calcium oscillations that were dependent on calcium release from intracellular stores and on voltage-independent calcium influx. Application of thimerosal, a compound that stimulates calcium mobilization from internal stores, increased: (1) the amplitude of spontaneous nonneuronal oscillations; (2) the area of migrating nonneuronal cells; and (3) the velocity of the neuronal-nonneuronal cell complex. We conclude that CG cell migration is a calcium dependent process and that nonneuronal cell calcium oscillations play a key role in the modulation of velocity.

Calcium signals activated by arachidonic acid in embryonic chick ciliary ganglion neurons.

Arachidonic acid (AA, 20:4) has been reported to modulate a variety of calcium-permeable ionic channels, both in the plasma membrane and in the endoplasmic reticulum. We have studied the effects of AA on calcium signaling in a well-characterized model of developing peripheral neurons, embryonic chick ciliary ganglion neurons in culture. When given at low non-micellar concentrations (5 microM), in the majority of cells AA directly activated a delayed and long-lasting increase in [Ca2+]i, involving both the cytoplasm and the nucleoplasm, that was completely reversed by abolition of extracellular calcium. Other fatty acids (FAs), either saturated like arachidic acid (20:0), or unsaturated like linoleic (18:2) and docosahexaenoic acid (22:6), shared its ability to activate calcium influx. This entry was not suppressed by voltage-dependent calcium channel inhibitors omega-conotoxin and nifedipine, by the voltage-independent calcium channel antagonist LOE-908, by pre-treatment with blockers of AA metabolic pathways or with pertussis toxin. The arachidonate-activated calcium pathway was permeable to Mn2+ and blocked by La3+, Gd3+ and Ni2+. In a neuronal subpopulation, AA at the same concentration was also able to elicit calcium release from thapsigargin-sensitive intracellular stores; we provide evidence that cytochrome P450 epoxygenase is involved in this process.

GDNF and bFGF are differentially involved in glial cell differentiation and neurite bundle formation in cultures from chick embryonic ciliary ganglia.

We have shown that the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) exert different effects on glial cells in cultures from chick embryo ciliary ganglia. bFGF acts as a mitogen on glial cells, and induces their aggregation to neuronal bodies; after 48 h of culture no glial cells could be observed along neurites. GDNF has no proliferative role; in contrast, it promotes the expression of the differentiative marker O4 and the association of glial cell bodies to neurites to form robust bundles.

A K(+) channel activated by cholinergic muscarinic receptors in chick ciliary ganglion neurons at early developmental stage.

Embryonic chick ciliary ganglion (CG) neurons obtained from E7-E8 ganglia maintained in serum-free medium were stimulated with 50 microM muscarine. A fast hyperpolarization of the membrane potential was observed in 25% of the cells tested, that in some cases was associated with a slower depolarization. Accordingly, in voltage clamp experiments, either an outward current or a biphasic current response could be observed. Single-channel experiments provide evidence that these signals can be associated to the activation of a K(+) channel whose conductance is 20 pS.

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons.

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.

In vitro analysis of neuron-glial cell interactions during cellular migration.

We used time-lapse microscopy to study the in vitro migration of neuronal cells from developing chick ciliary ganglion. These cells, when dissociated and cultured in a chemically defined medium, are able to migrate and to associate into clusters. We focused our attention on the study of the distribution of neuronal velocity components. Quantitative analysis of cell trajectories allowed us to demonstrate that, in many cells, velocities are well described by the Langevin equation, when deterministic components of the forces acting on the cells are taken into account. We also have shown that the majority of neurons whose movement is not purely random migrate in association with glial cells. We conclude that glial cells, by guiding neurons during migration, play an important role in the cell organization in vitro.