Cultures Of Cerebellar Granule Neurons Research Articles

Identification of Tetrabromobisphenol A Diallyl Ether as an Emerging Neurotoxicant in Environmental Samples by Bioassay-Directed Fractionation and HPLC-APCI-MS/MS

Brominated flame retardants (BFRs) have been widely used as additives in products to reduce their flammability. Recent findings suggested that some BFRs exhibit neurotoxicity and thus might pose a threat to human health. In this work, a neurotoxicity assay-directed analysis was developed, combining sample cleanup, fractionation, chemical identification, and bioassay. Viability of primary cultured cerebellar granule neurons (CGNs) was used to evaluate the neurotoxicity of extracts or separated fractions from environmental samples. Tetrabromobisphenol A diallyl ether (TBBPA DAE) was identified as the causative toxicant in sediment samples collected from a river near a brominated flame retardant (BFR) manufacturing plant in South China. Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) was optimized to determine TBBPA DAE levels in the potent fractions and to confirm TBBPA DAE as the key neurotoxicant. On the basis of comparison with the structure of other TBBPA derivatives, the 1-propenyl group in TBBPA DAE appears to be the cause for the neurotoxic potency. The levels of TBBPA DAE in samples along the river were found at up to 49 ng/L for river water, 10,183 ng/g dry weight (dw) in surface sediments, and 42 ng/g dw in soils. According to the distribution of TBBPA DAE in the environmental samples, the manufacturing plant was identified as the release source of TBBPA DAE. To our knowledge, this study is the first to demonstrate potential neurotoxicity induced by TBBPA DAE in real environmental samples.

Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5–1 μM/min during 30 min (achieved with 0.1 mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca 2+ channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca 2+ dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca 2+ concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca 2+ influx through L-VOCC. Combined analysis of cytosolic Ca 2+ concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

Altered energy status of primary cerebellar granule neuronal cultures from rats exposed to lead in the pre- and neonatal period

This paper examines the effect of pre- and neonatal exposure of rats to lead (0.1% lead acetate in drinking water, resulting in rat offspring whole blood lead concentration (Pb-B) 4 μg/dL) on the energy status of neuronal mitochondria by measuring changes in ATP, ADP, AMP, adenosine, TAN concentration, adenylate energy charge value (AEC) and mitochondrial membrane potential in primary cerebellar granule neurons (CGC) in dissociated cultures. Fluorescence studies were performed to imaging and evaluate mitochondria mass, mitochondrial membrane potential, intracellular and mitochondrial reactive oxygen species (ROS) production. The Na +/K + ATPase activity in intact CGC was measured spectrophotometrically. Our data shows that pre- and neonatal exposure of rats to Pb, even below the threshold of whole blood Pb value considered safe for people, affects the energy status of cultured primary cerebellar granule neurons through a decrease in ATP and TAN concentrations and AEC value, inhibition of Na +/K + ATPase, and increase in intracellular and mitochondrial ROS concentration. These observations suggest that even these low levels of Pb are likely to induce important alterations in neuronal function that could play a role in neurodegeneration.

Hairless Expression Attenuates Apoptosis in a Mouse Model and the COS Cell Line; Involvement of p53

BackgroundNeurons are more likely to die through apoptosis in the immature brain after injury whereas adult neurons in the mature brain die by necrosis. Several studies have suggested that this maturational change in the mechanism of cell death is regulated, in part, by thyroid hormone. We examined the involvement of the hairless (Hr) gene which has been suspected of having a role in cell cycle regulation and apoptosis in the hair follicle and is strongly regulated by the thyroid hormone in the brain.MethodologyForced expression of Hr by transfection decreased the number of apoptotic nuclei, levels of caspase-3 activity, and cytosolic cytochrome C in COS cells exposed to staurosporine and tunicamycin. Similarly, capsase-3 activity was lower and the decrease in mitochondrial membrane potential was smaller in cultures of adult cerebellar granule neurons from wild type mice compared to Hr knockout mice induced to undergo apoptosis. In vivo, apoptosis as detected by positive TUNEL labeling and caspase 3 activity was lower in wild-type mice compared to Hr knockouts after exposure to trimethyltin. Hr expression lowered levels of p53, p53 mediated reporter gene activity, and lower levels of the pro-apoptotic Bcl2 family member Bax in COS cells. Finally, Hr expression did not attenuate apoptosis in mouse embryonic fibroblasts from p53 knockout mice but was effective in mouse embryonic fibroblasts from wild type mice.Conclusions/SignificanceOverall, our studies demonstrate that Hr evokes an anti-apoptotic response by repressing expression of p53 and pro-apoptotic events regulated by p53.

Glutamine effect on cultured granule neuron death induced by glucose deprivation and chemical hypoxia

Using a specific fluorescent probe of mitochondrial membrane potential (tetramethylrhodamine ethyl ester), we have shown that glucose deprivation (GD) of cultured cerebellar granule neurons (CGN) for 3h lowers mitochondrial membrane potential in these cells. Longer glucose starvation (24h) causes CGN death that is not prevented by blockers of ionotropic glutamate receptors (MK-801 (10µM) and NBQX (10µM)). Glutamine or pyruvate (2mM) maintain membrane potential of mitochondria and decrease CGN death under GD conditions. In the presence of glucose the mitochondrial respiratory chain blocker rotenone induces neuron death potentiated by glutamine. The potentiation effect is completely prevented by blockers of ionotropic glutamate receptors. These results show that glutamine under conditions of GD can be utilized by mitochondria as substrate, but at the same time, in the case of mitochondrial function deterioration, metabolism of this amino acid results in glutamate accumulation to toxic level.

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca 2+] i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca 2+] i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca 2+] i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca 2+] i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca 2+] i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca 2+] i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca 2+] i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.

Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

An imaging assay to analyze primary neurons for cellular neurotoxicity

The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.

Neurotoxic activity of a Topoisomerase-I inhibitor, camptothecin, in cultured cerebellar granule neurons

DNA Topoisomerase-I (Topo-I) is an enzyme involved in DNA rearrangements, transcription and replication and is a potential target for cancer chemotherapy. Camptothecin is one of the chemotherapeutic agents inhibiting the catalytic activity of Topo-I through the formation of single-strand protein–DNA cross-links. Here, we show that camptothecin is toxic to primary cerebellar granule neurons (CGNs), and camptothecin-induced toxicity is not solely due to the inhibition of Topo-I. An analysis of the effect of camptothecin on CGNs in culture showed that camptothecin inhibits the viability of CGNs. The observed inhibition of cell viability is through the induction of a pro-apoptotic pathway that leads to neuronal degeneration. Furthermore, results show that camptothecin inhibits both protein synthesis and the neuritic outgrowth of CGNs. To determine if the observed neurotoxicity was due to inhibition of Topo-I alone, siRNA-mediated Topo-I-downregulated CGNs were analyzed for cell viability, apoptosis, protein synthesis and neurite outgrowth. The results of these experiments demonstrate that Topo-I downregulation affects only neurite outgrowth and has no significant effect on viability, apoptosis and protein synthesis in granule neurons. In conclusion, camptothecin-induced neurotoxicity may be due to the induction of protein–DNA cross-links and other unknown drug-related interactions rather than the inhibition of Topo-I activity alone.

A peptide derived from a trans‐homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis-interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans-interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding.

Acidosis-induced zinc-dependent death of cultured cerebellar granule neurons.

Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase of the intracellular zinc ions ([Zn(2+)](i)) and decrease of [Ca(2+)](i). Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn(2+)](i) and acidosis-induced neuronal death. However, neuronal death was insensitive to blockade of ASIC1 channels with amiloride, as CGNs display considerably lower expression of ASIC1a than other neurons. The antioxidant trolox and menadione significantly protected neurons from acidotic death. Earlier, we demonstrated that menadione rescues neurons from the deleterious effect of inhibition of mitochondrial complex I (Isaev et al. Neuroreport 15:2227-2231, 2004). We speculate that excessive Zn(2+)-dependent production of reactive oxygen species by mitochondrial complex I may be a general motive for the induction of cell death in CGNs under acidotic conditions.

Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress and dysfunction.

Distinct signaling pathways of precursor BDNF and mature BDNF in cultured cerebellar granule neurons

Recent studies have focused on a distinctive contrast between bioactivities of precursor brain-derived neurotrophic factor (proBDNF) and mature BDNF (matBDNF). In this study, using a proteolytic cleavage-resistant proBDNF mutant (CR-proBDNF), signaling mechanisms underlying the proapoptotic effect of proBDNF and antiapoptotic effect of matBDNF on the low potassium (LK)-inducing cell death of cultured cerebellar granule neurons (CGNs) were analyzed. A time course study demonstrated that unlike matBDNF, CR-proBDNF failed to induce TrkB phosphorylation for up to 360 min. CR-proBDNF did not activate ERK-1, ERK-2 and Akt, which are involved in TrkB-induced cell survival signaling, while matBDNF activated these kinases. On the other hand treatment of CGNs with CR-proBDNF led to a rapid activation of Rac-GTPase and phosphorylation of JNK which are involved in p75 NTR-induced apoptosis. In addition, a JNK-specific inhibitor, SP600125, inhibited the CR-proBDNF-induced apoptosis but did not affect the antiapoptotic effect of matBDNF. CR-proBDNF treatment led to an earlier appearance of active caspase-3. In contrast, matBDNF dramatically postponed the appearance of active caspase-3. Not like other signaling molecules, activation of caspase-3 was conversely regulated by both CR-proBDNF and matBDNF. These results thus suggest that in CGNs proBDNF elicits apoptosis via activation of p75 NTR, Rac-GTPase, JNK, and caspase-3, while matBDNF signals cell survival via activation of TrkB, ERKs and Akt, and deactivation of caspase-3.

L-type calcium channels and cytochrome b5 reductase are components of protein complexes tightly associated with lipid rafts microdomains of the neuronal plasma membrane

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14-26], we have shown that cytochrome b(5) reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b(5) reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.

Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events of calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5–30 min of exposure and at doses ≥ 10 µM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations ≥ 3 µg/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 µM) and longer exposures (> 30 min) are required to see cell death. These results show that PBDE mixtures and congeners activate MAPK pathway at concentrations where no significant cytotoxicity was observed, suggesting that perturbed intracellular signaling including MAPK pathway might be involved in the initiation of adverse effects, including learning and memory, related to these persistent chemicals.

Apoptosis-Associated Tyrosine Kinase and Neuronal Cell Death

Apoptosis-associated tyrosine kinase (AATYK) is up-regulated by phosphorylation in cultured cerebellar granule neurons (CGN) undergoing apoptosis upon switch to low KCl-containing medium. However, the underlying signaling pathways remain to be fully characterized. When CGN at culture day 7 were switched from 25 mM KCl (K25) to 5 mM (K5) medium, AATYK band migration on SDS-PAGE shifted to a more slowly migrating position expected for the hyperphosphorylated protein. The apoptosis-inducing agent C(2)-ceramide also caused a mobility shift of the AATYK protein. Exposing CGN (K25) to L-type voltage-dependent Ca(2+) channel antagonists shifted the AATYK band to the K5-induced position, while the Ca(2+) channel activator FPL-64176 had the contrary effect. FK-506, a calcineurin inhibitor caused AATYK hyperphosphorylation under high KCl conditions. CGN death in K5 medium is linked to inhibition of the PI 3-kinase/Akt survival pathway and concomitant activation of the pro-apoptotic downstream target glycogen synthase kinase-3 (GSK-3). GSK-3 inhibitors blocked the K5-induced mobility shift of AATYK. Moreover, CGN cultured from AATYK-deficient mice remained sensitive to death in K5 medium. Thus, AATYK activation may not be a physiologically relevant principal regulatory target of the GSK-3 death pathway in KCl-deprived CGN.

BDNF regulates neuronal sensitivity to endocannabinoids

The diacylglycerol lipases (DAGLα/β) synthesize 2-arachidonylglycerol (2-AG), the major endocannabinoid in the developing and adult brain (eCB). This lipid acts on cannabinoid receptors to regulate axonal growth and guidance, activity-dependent synaptic plasticity and adult neurogenesis, and can also protect neurons from excitotoxicity. 2-AG action is generally terminated by monoacylglycerol lipase (MAGL), however we know very little about the mechanisms that regulate neuronal sensitivity to eCBs. In the present study we show that the brain-derived neurotrophic factor (BDNF) can determine neuronal sensitivity to eCBs. In this context, in cultured cerebellar granule neurons (CGNs) BDNF increases the expression of CB1 receptor transcripts and decreases expression of MAGL transcripts. Using phosphorylation of Akt as the readout, we show that BDNF can promote a stable increase in neuronal sensitivity to eCBs. For example, concentrations of 2-AG and noladin either (NE) that normally do not lead to Akt phosphorylation in control neurons do so in neurons pre-treated with BDNF. In addition, Akt phosphorylation in response to a wide range of concentrations of NE was always greater in neurons pre-treated with BDNF. Our data suggests the existence of a positive feedback loop that might sustain neuronal survival in the normal brain.

Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

BackgroundProneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75NTR) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood.ResultsBased on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons.ConclusionThese results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK‐3beta: connecting neurotransmission with neuroprotection

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.