Maturation Of Cerebellar Granule Cells Research Articles

BmK NT1-induced neurotoxicity is mediated by PKC/CaMKⅡ-dependent ERK1/2 and p38 activation in primary cultured cerebellar granule cells

Voltage-gated sodium channels (VGSCs) represent molecular targets for a number of potent neurotoxins that affect the ion permeation or gating kinetics. BmK NT1, an α-scorpion toxin purified from Buthus martensii Karch (BMK), induces excitatory neurotoxicity by activation of VGSCs with subsequent overloading of intracellular Ca2+ in cerebellar granule cells (CGCs). In the current study, we further investigated signaling pathways responsible for BmK NT1-induced neurotoxicity in CGCs. BmK NT1 exposure induced neuronal death in different development stages of CGCs with similar potencies ranging from 0.21−0.48 μM. The maximal neuronal death induced by BmK NT1 gradually increased from 25.6% at 7 days in vitro (DIVs) to 42.1%, 47.8%, and 67.2% at 10, 13, and 16 DIVs, respectively, suggesting that mature CGCs are more vulnerable to BmK NT1 exposure. Application of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) inhibitors, KN-62 or KN-93, but not Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609, completely abolished BmK NT1-induced neuronal death. Moreover, BmK NT1 exposure stimulated CaMKⅡ phosphorylation. BmK NT1 also stimulated extracellular regulated protein kinases 1/2 (ERK1/2) and p38 phosphorylation which was abolished by tetrodotoxin demonstrating the role of VGSCs on BmK NT1-induced ERK1/2 and p38 phosphorylation. However, BmK NT1 didn’t affect c-Jun N-terminal kinase (JNK) phosphorylation. In addition, both ERK1/2 inhibitor, U0126 and p38 inhibitor, SB203580 attenuated BmK NT1-induced neuronal death. Both PKC inhibitor, Gö 6983 and CaMKⅡ inhibitor, KN-62 abolished BmK NT1-induced ERK1/2 and p38 phosphorylation. Considered together, these data demonstrate that BmK NT1-induced neurotoxicity is through PKC/CaMKⅡ mediated ERK1/2 and p38 activation.

αvβ5 integrin mediates the effect of vitronectin on the initial stage of differentiation in mouse cerebellar granule cell precursors

Vitronectin (VN), one of the extracellular matrix proteins, controls the maturation of cerebellar granule cells (CGCs) through the promotion of the initial differentiation stage progress. However, the receptors of VN in the initial differentiation stage of CGC precursors (CGCPs) have not been clarified. In this study, we characterized the receptor candidates for VN in CGCPs. First, we confirmed that αvβ3 and αvβ5 integrins, which are receptor candidates for VN, were co-localized with VN in the developing cerebellum and primary cultured CGCPs. Next, the knockdown (KD) of αv, β3, and β5 integrins with small interference RNA (siRNA) for each integrin reduced the ratio of Tuj1, a final differentiation marker, -positive CGCPs. We further studied whether αvβ3 and αvβ5 integrins control the initial differentiation stage. The KD of αv and β5, but not β3, integrins significantly increased the ratio of transient axonal glycoprotein 1 (TAG1), an initial differentiation marker, -positive CGCPs, whereas the KD of αv and β3 integrins, not β5 integrin, stimulated the proliferation of CGCPs. Overexpression of β5 integrin stimulated the progress of the initial differentiation stage as well. To confirm the interaction between αvβ5 integrin and VN, VN was added to β5 integrin-KD CGCPs. The promotion of the progress of initial differentiation by VN was abrogated by β5 integrin KD using small hairpin RNA (shRNA). Taken together, our results indicated that αvβ5 integrin, as the very receptor of VN, is responsible for the progress of the initial differentiation stage in mouse CGCPs.

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry.

Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature cerebellar granule cells, including the development of a dense neuritic network, neuronal activity, neurotransmitter release and the expression of neuronal protein markers. Moreover, CGN represent a convenient model for the study of Clostridial Neurotoxins (CNT), most notably known as Tetanus and Botulinum neurotoxins, as they abundantly express both CNT receptors and intraneuronal substrates, i.e., Soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE proteins). Here, we describe a protocol for obtaining a highly pure culture of CGN from postnatal rats/mice and an easy procedure for their intoxication with CNT. We also illustrate handy methods to evaluate CNT activity and their inhibition.

ZIC1 Function in Normal Cerebellar Development and Human Developmental Pathology.

Zic genes are strongly expressed in the cerebellum. This feature leads to their initial identification and their name "zic," as the abbreviation of "zinc finger protein of the cerebellum." Zic gene function in cerebellar development has been investigated mainly in mice. However, association of heterozygous loss of ZIC1 and ZIC4 with Dandy-Walker malformation, a structural birth defect of the human cerebellum, highlights the clinical relevance of these studies. Two proposed mechanisms for Zic-mediated cerebellar developmental control have been documented: regulation of neuronal progenitor proliferation-differentiation and the patterning of the cerebellar primordium. Clinical studies have also revealed that ZIC1 gain of function mutations contribute to coronal craniosynostosis, a rare skull malformation. The molecular pathways contributing to these phenotypes are not fully explored; however, embryonic interactions with sonic hedgehog signaling, retinoic acid signaling, and TGFβ signaling have been described during mouse cerebellar development. Further, Zic1/2 target a multitude of genes associated with cerebellar granule cell maturation during postnatal mouse cerebellar development.

Distinct expression patterns of inwardly rectifying potassium currents in developing cerebellar granule cells of the hemispheres and the vermis.

G-protein-coupled inwardly rectifying potassium (GIRK) channels play a crucial role during the migration and maturation of cerebellar granule cells (GCs) in the vermis. In the cerebellar hemispheres, however, only minor effects on the development of GCs are observed in mice with GIRK channel impairment. This regional difference may reflect distinct ontogenetic expression patterns of GIRK channels. Therefore, inwardly rectifying responses in mice were characterized at different stages of development in the vermis and the hemispheres. In the vermis, GCs in the premigratory zone (PMZ) at P7-P15 exhibit GIRK current but not constitutive inwardly rectifying potassium (CIRK) current, and are relatively depolarized at rest. In contrast, premigratory GCs in the hemispheres express only CIRK channels, which accounts for their more hyperpolarized resting membrane potential. Furthermore, the pattern of voltage-dependent inward currents in the PMZ GCs of cerebellar hemispheres is consistent with a more mature stage of development than the corresponding GCs in the vermis, resulting in robust firing properties mediated by sodium channels. Later in development (P21-P22), CIRK current is then observed in the majority of vermis GCs. This developmental pattern, revealed by electrophysiological recordings, was confirmed by immunohistological experiments that showed greater reactivity for GIRK2 in the PMZ of the vermis than in the hemispheres during development (P7-P15). These findings suggest that regional differences in development are responsible for the differential expression of inwardly rectifying potassium channels in the vermis and in the hemispheres.

Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain

BackgroundProper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.Methods and FindingsIn this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.ConclusionsThe ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.

AMPA receptors serum-dependently mediate GABA A receptor α1 and α6 subunit down-regulation in cultured mouse cerebellar granule cells

Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA A receptor α1, α6 and β2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates α1 and α6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate α1 or α6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA A receptor α1 and α6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA A receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.

Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40-P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na(+) currents in mature CG cells (P66-P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na(+) currents were not modified in the absence of Nav1.6 whereas persistent Na(+) current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na(+) current and ensure a high degree of temporal precision in repetitive firing of CG cells.

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

Suppression of guanylyl cyclase (β1 subunit) expression impairs neurite outgrowth and synapse maturation in cultured cerebellar granule cells

The increased expression of different soluble guanylyl cyclase (sGC) subunits during development is consistent with these proteins participating in the formation and establishment of interneuronal contacts. Functional sGC is generated by the dimerization of an alpha-subunit (sGCalpha1/2) with the beta1-subunit (sGCbeta1), and both depletion of the sGCbeta1 subunit and inhibiting sGC activity impair neurite outgrowth. Similarly, impairing sGC activity diminishes the amount of growth-associated protein (GAP-43) and synapsin I, two proteins that participate in axon elongation and synaptogenesis, suggesting a role for sGC in these processes. Indeed, fewer synapses form when sGC is inhibited, as witnessed by FM1-43 imaging and synapsin I immunostaining, and the majority of synapses that do form remain functionally immature. These findings highlight the importance of sGC in the regulation of neurite outgrowth and synapse formation, and in the functional maturation of cerebellar granule cells in vitro.

Role of Calcineurin Signaling in Membrane Potential-Regulated Maturation of Cerebellar Granule Cells

At the early postnatal period, cerebellar granule cells proliferate, differentiate, migrate, and finally form refined synaptic connections with mossy fibers. During this period, the resting membrane potential of immature granule cells is relatively depolarized, but it becomes hyperpolarized in mature cells. This investigation was conducted to examine the role of this alteration in membrane potential and its downstream signaling mechanism in development and maturation of granule cells. Experiments were designed to precisely characterize the ontogenic processes of developing granule cells by combining organotypic cerebellar cultures with the specific expression of EGFP (enhanced green fluorescent protein) in granule cells by use of DNA transfection. Multiple approaches using morphology, electrophysiology, and immunohistochemistry demonstrated that granule cells developed and matured at the physiological KCl concentration in organotypic cultures in a temporally regulated manner. We addressed how persistent membrane depolarization influences the developmental and maturation processes of granule cells by depolarizing organotypic cultures with high KCl. Depolarization preserved the developmental processes of granule cells up to the stage of formation of immature dendrites but prevented the maturation processes for synaptic formation by granule cells. Importantly, this blockade of the terminal maturation of granule cells was reversed by inactivation of calcineurin with its specific inhibitor. This investigation has demonstrated that alteration of the membrane potential and its downstream calcineurin signaling play a pivotal role in triggering the maturation program for the synaptic organization of postnatally developing granule cells.

Membrane potential-regulated maturation of cerebellar granule cells

Membrane potential-regulated maturation of cerebellar granule cells

Somatostatin and the somatostatin receptor 2 are reciprocally controlled by calcineurin during cerebellar granule cell maturation

Activity-dependent Ca2+ influx into neurones and the subsequent changes in gene expression are thought to be important in shaping neuronal development. In this study, we investigated whether an important mediator of neuronal migration, somatostatin (Srif), alongside its receptors, is controlled in this manner in cerebellar granule cells. We show that Ca2+ influx increases the expression of somatostatin mRNA (srif), while somatostatin receptor 2 (sst2) mRNA expression is decreased. Both genes appear to be regulated independently of each other and in a calcineurin-dependent manner that does not depend on either the ERK1/2 MAP kinase or the cAMP/CREB pathway. Nonetheless, a second pathway is required to induce changes in srif and sst2 expression, since constitutively active calcineurin alone is not sufficient to induce these changes. Furthermore, calcineurin activation reciprocally regulates the expression of brain-derived neurotrophic factor, bdnf, and its receptor trkb, which have also been shown to play a role in neuronal migration. Finally, calcineurin appears to control the expression of the neuronal marker transient axonal glycoprotein 1, tag-1, thereby strongly suggesting that calcineurin activation in vivo occurs during the late stages of neuronal migration, possibly during synaptogenesis with mossy fibres. We therefore propose that calcineurin might play an important role as a switch between transcriptional programs during neuronal development.

Lack of NMDA receptor subunit exchange alters Purkinje cell dendritic morphology in cerebellar slice cultures

Early postnatal developmental changes in N-methyl- d-aspartate (NMDA) receptor (NR) subunits regulate cerebellar granule cell maturation and potentially Purkinje cell development. We therefore investigated Purkinje cell morphology in slice cultures from mice with genetic subunit exchange from NR2C to NR2B (NR2C-2B). NR2C-2B Purkinje cells after 12 days in vitro showed a significantly impaired dendritic arbour complexity with reduced branching density as compared to wild-type cells, a phenotype that was reversed by NMDA treatment. These data support the concept that in cerebellar slice cultures, Purkinje cell dendritic outgrowth is regulated by granule cell inputs.

Tissue specificity and regulation of the N-terminal diversity of reticulon 3.

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

Long-term NR2B expression in the cerebellum alters granule cell development and leads to NR2A down-regulation and motor deficits

N-methyl-D-aspartate receptor (NMDAR) composition in granule cells changes characteristically during cerebellar development. To analyze the importance of NR2B replacement by NR2C and NR2A subunits until the end of the first month of age, we generated mice with lasting NR2B expression but deficiency for NR2C (NR2C-2B mice). Mutant phenotype was different from NR2C knock-out mice as loss of granule cells and morphological changes in NR2C/2B cerebellar architecture were already evident from the second postnatal week. Increased NR2B subunit levels led also to a gradual down-regulation of cerebellar NR2A levels, preceding the development of motor impairment in adult animals. Therefore, cerebellar NR2A is important for proper motor coordination and cannot be replaced by long-term expression of NR2B. Consequently, the physiological exchange of NMDA receptor subunits during cerebellar granule cell maturation is important for accurate postnatal development and function.

Murine numb regulates granule cell maturation in the cerebellum

Notch is a key regulator of vertebrate neurogenesis and the cytoplasmic adaptor protein Numb is a modulator of the Notch signaling pathway. To address the role of murine Numb in development of the central nervous system, we used a conditional gene ablation approach. We show that Numb is involved in the maturation of cerebellar granule cells. Although the specification of neural cell fates in the cerebellum is not affected in the absence of Numb, the transition from a mitotic progenitor to a mature granule cell is aberrant and migration of postmitotic granule cells to the internal granule cell layer is delayed. In some animals, this results in a complete agenesis of granule cells and a strong ataxia. We confirmed these findings in vitro and found that Numb-deficient cerebellar progenitor cells show a marked delay in granule cell maturation. Our results suggest that Numb plays a role in the transition of a mitotic progenitor to a fully differentiated granule cell in the cerebellum. In addition, the maturation of Purkinje cells is also delayed in Numb-deficient mice.

Kainate-induced excitotoxicity is dependent upon extracellular potassium concentrations that regulate the activity of AMPA/KA type glutamate receptors.

In addition to well-known N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, recent studies suggest that non-NMDA type ionotropic glutamate receptors are also important mediators of excitotoxic neuronal death, and that their functional expression can be regulated by the cellular environment. In this study, we used cerebellar granule cells (CGCs) in culture to investigate kainate (KA)-induced excitotoxicity. Although previous reports indicated that KA induces apoptosis of CGCs in culture, no KA-induced excitotoxic cell death was observed in CGCs treated with KA when cells were maintained in high potassium media (24 mm K+). In contrast, when mature CGCs were shifted into low potassium media (3 mm K+), KA produced significant excitotoxicity. In electrophysiological studies, the KA-induced inward current density was significantly elevated in CGCs shifted into low K+ media compared with those maintained in high K+ media. Non-desensitizing aspects of KA currents observed in this study suggest that these responses were mediated by AMPA rather than KA receptors. In immunofluorescence studies, the surface expression of GluR1 subunits increased when mature CGCs were shifted into a low K+ environment. This study suggests that KA-induced excitotoxicity in mature CGCs is dependent upon the extracellular potassium concentration, which modulates functional expression and excitability of AMPA/KA receptors.

Differential mechanisms of glutamate-stimulated perturbations in the kinetics of c-fos mRNA induction are associated with maturation of cerebellar granule cells in primary culture.

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca2+]), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca2+ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca2+], levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca2+ influx. The results are presented in comparison to the actions of KCI and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.

Developmental Expression of Mouse Krüppel-like Transcription Factor KLF7 Suggests a Potential Role in Neurogenesis

To identify potential functions for the Krüppel-like transcription factor KLF7, we have determined the spatiotemporal pattern of gene expression during embryogenesis and in the adult organism. We show that the profile of Klf7 expression predominantly involves the central and peripheral nervous systems and is broadly identified by three separate phases. The first phase occurs early in embryogenesis with increasingly strong expression in the spinal cord, notably in motor neurons of the ventral horn, in dorsal root ganglia, and in sympathetic ganglia. The second robust phase of Klf7 expression is confined to the early postnatal cerebral cortex and is downregulated thereafter. The third phase is characterized by high and sustained expression in the adult cerebellum and dorsal root ganglia. Functionally, these three phases coincide with establishment of neuronal phenotype in embryonic spinal cord, with synaptogenesis and development of mature synaptic circuitry in the postnatal cerebral cortex, and with survival and/or maintenance of function of adult sensory neurons and cerebellar granule cells. Consistent with Klf7 expression in newly formed neuroblasts, overexpression of the gene in cultured fibroblasts and neuroblastoma cells repressed cyclin D1, activated p21, and led to G1 growth arrest. Based on these data, we argue for multiple potential functions for KLF7 in the developing and adult nervous system; they include participating in differentiation and maturation of several neuronal subtypes and in phenotypic maintenance of mature cerebellar granule cells and dorsal root ganglia.