Cerebellar Granular Neurons Research Articles

Analysis of antibodies to neuronal surface antigens in adult opsoclonus–myoclonus

Antibodies against neuronal surface antigens (NSA-ab) have been described in pediatric opsoclonus–myoclonus syndrome (OMS). We analyzed the presence of NSA-ab by flow cytometry and immunocytochemistry of live cerebellar granular neurons (CGN) in the serum of 25 adult patients with idiopathic (14) and paraneoplastic (11) OMS. Paraneoplastic, but not idiopathic, OMS sera showed a CGN surface binding by flow cytometry higher than that of controls (mean MFI (median fluorescence intensity): 29 ± 6.9 vs. 20 ± 5.8; p = 0.001) but only one serum had a binding greater than three standard deviations of controls. OMS sera did not label live CGN by immunocytochemistry. Unlike pediatric OMS, NSA-ab were not detected in adult cases suggesting that the immunity to NSA in OMS is heterogeneous.

Low Endogenous G-Protein-Coupled Receptor Kinase 2 Sensitizes the Immature Brain to Hypoxia–Ischemia-Induced Gray and White Matter Damage

Hypoxic-ischemic brain injury is regulated in part by neurotransmitter and chemokine signaling via G-protein-coupled receptors (GPCRs). GPCR-kinase 2 (GRK2) protects these receptors against overstimulation by inducing desensitization. Neonatal hypoxic-ischemic brain damage is preceded by a reduction in cerebral GRK2 expression. We determined the functional importance of GRK2 in hypoxic-ischemic brain damage. Nine-day-old wild-type and GRK2(+/-) mice with a approximately 50% reduction in GRK2 protein were exposed to unilateral carotid artery occlusion and hypoxia. In GRK2(+/-) animals, gray and white matter damage was aggravated at 3 weeks after hypoxia-ischemia. In addition, cerebral neutrophil infiltration was increased in GRK2(+/-) animals. Neutrophil depletion reduced brain damage, but neuronal loss was still more pronounced in GRK2(+/-) animals. Onset of neuronal loss was advanced in GRK2(+/-) animals regardless of neutrophil depletion. White matter injury was advanced in GRK2(+/-) animals and was not affected by neutrophil depletion. Activation/infiltration of microglia/macrophages was stronger in GRK2(+/-) brains but only occurred 24 h after hypoxia-ischemia and is therefore not the primary cause of increased damage. During hypoxia, cerebral blood flow was reduced to the same extent in both genotypes. In vitro, GRK2(+/-) hippocampal slices and cerebellar granular neurons were more sensitive to glutamate-induced death. We propose the novel concept that the kinase GRK2 regulates onset and magnitude of hypoxic-ischemic brain damage. Increased gray and white matter damage in GRK2(+/-) animals was not dependent on infiltrating neutrophils and occurred before microglia/macrophage activation was detected. Collectively, our data suggest that cerebral GRK2 has an important endogenous neuroprotective role in ischemic cerebral damage.

Inactivation of glycogen synthase kinase-3beta and up-regulation of LINGO-1 are involved in LINGO-1 antagonist regulated survival of cerebellar granular neurons.

LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO-1-Fc) inhibited low potassium-induced cerebellar granular neurons (CGNs) apoptosis. In the present study, we examined the neuroprotective mechanism of LINGO-1-Fc by Western blot and in situ GST pull-down assay. CGN cultures were preincubated in medium with LINGO-1-Fc or control protein at the concentration of 10 mug/ml for 2 h and then switched to low potassium medium in the presence of corresponding proteins. Cultures were harvested at indicated time intervals for successive analysis. Several apoptosis-associated signaling factors, GSK-3beta, ERK1/2, and Rho GTPases, were observed to be activated in response to potassium deprivation and the activation/dephosphorylation of GSK-3beta was suppressed by LINGO-1-Fc pretreatment compared with control group. Besides, the endogenous LINGO-1 expression level of CGN cultures was augmented by low potassium stimuli and restrained by LINGO-1 antagonist treatment. Although the protein level of p75(NTR) and Nogo-A were down-regulated in different patterns during apoptosis, neither of them was affected by LINGO-1-Fc application. Taken together, these results suggest a new mechanism of LINGO-1 antagonist regulated neuronal survival involving protein synthesis of LINGO-1 and inactivation of GSK-3 pathway.

DNA low-density array analysis of colchicine neurotoxicity in rat cerebellar granular neurons

Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimer's or Parkinson's disease. Colchicine is a microtubule-disrupting agent that binds to tubuline, inhibiting microtubule assembly, and which triggers apoptosis. The present research describes the transcriptional activation of molecules related to alternative forms of apoptosis, in an acute colchicine model of apoptosis in rat cerebellar granule neurons (CGNs). Treatment with colchicine up-regulated significantly the activity of genes related to oxidative stress: glutathione peroxidase 1 and catalase; altered significantly genes related to cell cycle control (cyclin D1 and cyclin-dependent kinase 2), genes related to classical apoptosis pathway (caspase 3) and a neuronal cell-related gene (pentraxin 1). Colchicine treatment also down-regulated the gene expression of calpain 1. In conclusion, our experiments demonstrate that the cell damage caused by exposure to colchicine activates the classical apoptosis pathway, but also promotes the up-regulation of several genes related to oxidative stress and cell cycle control. Present data may help to a better understanding of the molecular mechanisms involved in cytoskeletal degradation-induced apoptosis in neurons.

Bone Morphogenetic Protein 2 Opposes Shh-mediated Proliferation in Cerebellar Granule Cells through a TIEG-1-based Regulation of Nmyc

Nmyc is a potent regulator of cell cycle in cerebellar granular neuron precursors (CGNPs) and has been proposed to be the main effector of Shh (Sonic hedgehog) proliferative activity. Nmyc ectopic expression is sufficient to promote cell autonomous proliferation and can lead to tumorigenesis. Bone morphogenetic protein 2 (BMP2) antagonizes Shh proliferative effect by promoting cell cycle exit and differentiation in CGNPs. Here we report that BMP2 opposes Shh mitogenic activity by blocking Nmyc expression. We have identified TIEG-1 (KLF10) as the intermediary factor that blocks Nmyc expression through the occupancy of the Sp1 sites present in its promoter. We also demonstrate that TIEG-1 ectopic expression in CGNPs induces cell cycle arrest that can lead to apoptosis but fails to promote differentiation. Moreover, TIEG-1 synergizes with BMP2 activity to terminally differentiate CGNPs and independent differentiator signals such as dibutyryl cAMP and prevents apoptosis in TIEG-1 arrested cells. All together, these data strongly suggest that the BMP2 pathway triggers cell cycle exit and differentiation as two separated but coordinated processes, where TIEG-1 acts as a mediator of the cell cycle arrest.

Neural cell adhesion molecule‐180‐mediated homophilic binding induces epidermal growth factor receptor (EGFR) down‐regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28, 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.

New Autoantibodies in Pediatric Opsoclonus–Myoclonus Syndrome

Opsoclonus-myoclonus syndrome (OMS) is a rare neurologic disorder comprising the main symptoms of eye-movement disturbances, muscle jerks, and severe ataxia. In children and adults, some cases are associated with a tumor as a paraneoplastic syndrome, whereas in children the paraneoplastic form is almost exclusively associated with neuroblastoma. The detection of autoantibodies in some OMS sera led to the hypothesis that the syndrome is of autoimmune origin. Beside autoantibodies against intracellular proteins, such as anti-Hu, alpha-enolase, and KHSRP, specific binding of autoantibodies to the surface of neuroblastoma cells and cerebellar granular neurons have been found. Antiproliferative and proapoptotic effects of these autoantibodies on neuroblastoma cell lines were noted as well. These results support the concept of a humoral autoimmune process in the pathogenesis of OMS.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Lifeguard/neuronal membrane protein 35 regulates Fas ligand‐mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)-receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up-regulated during postnatal development and in the adult. We show that over-expression of LFG protected cortical neurons from FasL-induced apoptosis and decreased caspase-activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL-induced cell death and caspase-8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL-induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL-mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL-activated signaling pathways.

Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with α5β1 Integrin Initiates Neurite Outgrowth

Previous studies demonstrated that cross-linking of GM1 ganglioside with multivalent ligands, such as B subunit of cholera toxin (CtxB), induced Ca2+ influx through an unidentified, voltage-independent channel in several cell types. Application of CtxB to undifferentiated NG108-15 cells resulted in outgrowth of axon-like neurites in a Ca2+ influx-dependent manner. In this study, we demonstrate that CtxB-induced Ca2+ influx is mediated by TRPC5 channels, naturally expressed in these cells and primary neurons. Both Ca2+ influx and neurite induction were blocked by TRPC5 small interfering RNA (siRNA). Pretreatment of NG108-15 cells with neuraminidase increased cell-surface GM1 and greatly enhanced the signal. GM1 was not directly associated with TRPC5 but rather with alpha5beta1 integrin, which opened the channel through a signaling sequence after cross-linking of the GM1/integrin complex. This cascade included autophosphorylation of focal adhesion kinase and subsequent activation of phospholipase Cgamma (PLCgamma) and phosphoinositide-3 kinase [PI(3)K]. Pharmacological blockers that inhibited tyrosine kinase, PLC, and PI(3)K suppressed both CtxB-induced Ca2+ influx and neurite outgrowth. These were also suppressed by SK&F96365, a nonspecific transient receptor potential channel blocker. Confocal immunocytochemistry revealed that GM1 cross-linking induced colocalization of GM1 with these signaling elements in sprouting regions of plasma membrane. In primary cerebellar granular neurons (CGNs), TRPC5 was detected at 2 d in vitro (2 DIV), a stage corresponding to CtxB-stimulated Ca2+ influx. Neurite outgrowth in CGNs, determined at 3 DIV, was accelerated by CtxB and suppressed by TRPC5 siRNA and the above blockers. The crucial role of GM1 was indicated with CGNs from ganglio-series null mice, in which growth of axons was significantly retarded.

Calcium Influx and Mitochondrial Alterations at Synapses Exposed to Snake Neurotoxins or Their Phospholipid Hydrolysis Products

Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.

Protease nexin 1 and its receptor LRP modulate SHH signalling during cerebellar development

Development of the postnatal cerebellum relies on the tight regulation of cell number by morphogens that control the balance between cell proliferation, survival and differentiation. Here, we analyze the role of the serine-protease inhibitor protease nexin 1 (PN-1; SERPINE2) in the proliferation and differentiation of cerebellar granular neuron precursors (CGNPs) via the modulation of their main mitogenic factor, sonic hedgehog (SHH). Our studies show that PN-1 interacts with low-density lipoprotein receptor-related proteins (LRPs) to antagonize SHH-induced CGNP proliferation and that it inhibits the activity of the SHH transcriptional target GLI1. The binding of PN-1 to LRPs interferes with SHH-induced cyclin D1 expression. CGNPs isolated from Pn-1-deficient mice exhibit enhanced basal proliferation rates due to overactivation of the SHH pathway and show higher sensitivity to exogenous SHH. In vivo, the Pn-1 deficiency alters the expression of SHH target genes. In addition, the onset of CGNP differentiation is delayed, which results in an enlarged outer external granular layer. Furthermore, the Pn-1 deficiency leads to an overproduction of CGNPs and to enlargement of the internal granular layer in a subset of cerebellar lobes during late development and adulthood. We propose that PN-1 contributes to shaping the cerebellum by promoting cell cycle exit.

CAMP/protein kinase A signalling pathway protects against neuronal apoptosis and is associated with modulation of Kv2.1 in cerebellar granule cells

Previously, we have reported that apoptosis of cerebellar granular neurons induced by incubation in 5 mm K(+) and serum-free medium (LK-S) was associated with an increase in the delayed rectifier K(+) current (I(K)). Here, we show that I(K) associated with apoptotic neurons is mainly encoded by a Kv2.1 subunit. Silencing Kv2.1 expression by small interfering RNA reduces I(K) and increases neuron viability. Forskolin is able to decrease the I(K) amplitude recording from neurons of both the LK-S and control group, and prevents apoptosis of granule cells that are induced by LK-S. Dibutyryl cAMP mimicks the effect of forskolin on the modulation of I(K) and, accordingly, the inhibitor of protein kinase A, H-89, aborts the neuron-protective effect induced by forskolin. Whereas the expression of Kv2.1 was silenced by Kv2.1 small interfering RNA, the inhibition of forskolin on the current amplitude was significantly reduced. Quantitative RT-PCR and whole-cell recording revealed that the expression of Kv2.1 was elevated in the apoptotic neurons, and forskolin significantly depressed the expression of Kv2.1. We conclude that the protection against apoptosis via the protein kinase A pathway is associated with a double modulation on I(K) channel properties and its expression of alpha-subunit that is mainly encoded by the Kv2.1 gene.

The Interaction between Cytoplasmic Prion Protein and the Hydrophobic Lipid Core of Membrane Correlates with Neurotoxicity

Prion protein (PrP), normally a cell surface protein, has been detected in the cytosol of a subset of neurons. The appearance of PrP in the cytosol could result from either retro-translocation of misfolded PrP from the endoplasmic reticulum (ER) or impaired import of PrP into the ER. Transgenic mice expressing cytoplasmic PrP (cyPrP) developed neurodegeneration in cerebellar granular neurons, although no detectable pathology was observed in other brain regions. In order to understand why granular neurons in the cerebellum were most susceptible to cyPrP-induced degeneration, we investigated the subcellular localization of cyPrP. Interestingly, we found that cyPrP is membrane-bound. In transfected cells, it binds to the ER and plasma/endocytic vesicular membranes. In transgenic mice, it is associated with synaptic and microsomal membranes. Furthermore, the cerebellar neurodegeneration in transgenic mice correlates with the interaction between cyPrP and the hydrophobic lipid core of the membrane but not with either the aggregation status or the dosage of cyPrP. These results suggest that lipid membrane perturbation could be a cellular mechanism for cyPrP-induced neurotoxicity and explain the seemingly conflicting results concerning cyPrP.

Cell Survival through Trk Neurotrophin Receptors Is Differentially Regulated by Ubiquitination

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.

NO Deadly Signal

Reminiscent of mild-mannered Clark Kent, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an alter ego with potent and deadly effects. When translocated to the nucleus, GAPDH has been associated with transcriptional regulation, and Hara et al. present results implicating it in control of apoptosis. A yeast two-hybrid screen for proteins interacting with GAPDH turned up Siah1, an E3 ubiquitin ligase. In transfected cells, the authors detected interaction of the proteins and increased localization of GAPDH to the nucleus, an effect that required the nuclear localization signal of Siah1. In human embryonic kidney cells undergoing apoptosis in response to staurosporine, the authors detected modification of GAPDH by mass spectrometry and went on to show in vitro that modification of GAPDH by S -nitrosylation (often a consequence of increased generation of nitric oxide within cells) enhanced association with Siah1. These events appear to be important in regulation of apoptosis. In a macrophage cell line undergoing apoptosis in response to lipopolysaccharide, GAPDH became S -nitrosylated and associated with endogenous GAPDH, and the complex moved to the nucleus. All of these effects were blocked by an inhibitor of the nitric oxide-generating enzyme iNOS (inducible nitric oxide synthase). Depletion of GAPDH with small interfering RNA (siRNA) blocked the appearance of phosphorylated histone H2B, a marker associated with apoptosis. In cerebellar granular neurons undergoing apoptosis in response to activation of glutamate receptors, similar effects were observed. Increased binding of Siah1 to GAPDH was blocked in cells from neuronal NOS (nNOS) knockout mice, and depletion of either GAPDH or Siah1 from wild-type cells with siRNA inhibited apoptosis. Exactly how nuclear Siah1 promotes apoptosis remains to be explored, but its action appears to require its RING finger domain, indicating that Siah1-mediated ubiquitination and consequent degradation of nuclear proteins is one likely mechanism. Nitric oxide-mediated modification of metabolic enzymes has been shown to contribute to cell death through energy depletion. Hara et al. propose that this may be a slower process and one associated more closely with necrosis, whereas the faster nuclear actions of GAPDH, which reflect translocation of only a small portion of cytoplasmic enzyme, appear to be independent of effects on glycolysis. M. R. Hara, N. Agrawal, S. F. Kim, M. B. Cascio, M. Fujimuro, Y. Ozeki, M. Takahashi, J. H. Cheah, S. K. Tankou, L. D. Hester, C. D. Ferris, S. D. Hayward, S. H. Snyder, A. Sawa, S -nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7 , 665-674 (2005). [PubMed]

Surface‐binding autoantibodies to cerebellar neurons in opsoclonus syndrome

Childhood opsoclonus-myoclonus syndrome can occur with or without associated neuroblastoma. An autoimmune pathogenesis has been discussed for both forms. We show here that the majority of children with opsoclonus-myoclonus syndrome (10/14) have autoantibodies binding to the surface of isolated rat cerebellar granular neurons. In some patients, these antibodies are masked by IgG binding to ubiquitous surface antigens, which could be removed by preincubation with the nonneuronal control cell line HEK 293. A newly introduced competitive binding assay showed that the surface binding is directed against the same autoantigen in different patients. Therefore, we hypothesize that opsoclonus-myoclonus syndrome may be the result of an autoimmune process against a neuronal surface protein.

2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty

To evaluate the ability of a tiered quantitative morphological approach to reveal developmental neurotoxicity, morphometric parameters were measured in the offspring of rats treated with methylazoxymethanol (MAM) during days 13–15 of pregnancy. Treatment was aimed at inhibiting the proliferation phase of hippocampal neurons while leaving cerebellar neurons unaffected. 2D and 3D assessment of brain morphology combined with straightforward measurement of brain size, weight and volume, and the usefulness of estimation of total neuron numbers were studied. Each tier indicated major effects of MAM, from macroscopic effects in the cerebrum (first tier) to a considerable loss of neurons in the hippocampal CA1 pyramidal layer (third tier). The cerebellum and the number of cerebellar granular neurons were not changed. Along with each step of the proposed tiered approach (brain size → linear morphometry → stereology), the discriminative strength of the endpoints, and thus the probability to pinpoint the extent and location of developmental brain lesions increased.

Developmental changes in the neuronal protein composition: A study by high resolution 2D-gel electrophoresis

Cerebellar granular neurons were grown in culture up to 21 days and the protein compositions of undifferentiated (day 1), partially differentiated (day 7) and fully differentiated (day 21) neurons were analyzed by high-resolution 2D-gel electrophoresis. During neuronal differentiation there were not only increase in the amount of several known proteins, viz. actin, tubulin (both alpha and beta subunits), myosin (heavy and light chains), but very interesting changes were also observed in the expressions of different subunits and isoforms of those proteins. Furthermore, both in the acidic (pI 4.0-4.5) and alkaline (pI 7.0-8.5) regions interesting up and down regulations of several unidentified proteins were observed during the neuronal differentiation. These results indicated that there were several unidentified proteins that might be very valuable targets for studying regulation of neuronal differentiation. Research is going on for further characterization of those proteins using recently developed proteomics technology.

2‐Iodomelatonin prevents apoptosis of cerebellar granule neurons via inhibition of A‐type transient outward K+ currents

Compelling evidence indicates that excessive K+ efflux and intracellular K+ depletion are key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granular neurons induced by incubation under low K+ (5 mM) conditions was associated with an increase in delayed rectifier outward K+ current (IK) amplitude and caspase-3 activity. Moreover, the melatonin receptor antagonist 4P-PDOT abrogated the effects of 2-iodomelatonin on IK augmentation, caspase-3 activity and apoptosis. Here, we show that incubation under low K+/serum-free conditions for 6 hr led to a dramatic increase in the A-type transient outward K+ current (IA) (a 27% increase; n=31); in addition, fluorescence staining showed that under these conditions, cell viability decreased by 30% compared with the control. Treatment with 2-iodomelatonin inhibited the IA amplitude recorded from control and apoptotic cells in a concentration-dependent manner and modified the IA channel activation kinetics of cells under control conditions. Moreover, 2-iodomelatonin increased the viability of cell undergoing apoptosis. Interestingly, 4P-PDOT did not abrogate the effect of 2-iodomelatonin on IA augmentation under these conditions; in the presence of 4P-PDOT (100 microm), 2-iodomelatonin reduced the average IA by 41+/-4%, which was similar to the effect of 2-iodomelatonin alone. These results suggest that the neuroprotective effects of 2-idomelatonin are not only because of its antioxidant or receptor-activating properties, but rather that 2-iodomelatonin may inhibit IA channels by acting as a channel blocker.