Development of polarity in cerebellar granule neurons

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 223–236, 1997.     

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mm potassium (high K(+)), changing to medium containing 5 mm potassium (low K(+); LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells. In vitro kinase assay using glutathione S-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.     

Alpha-synuclein protects cerebellar granule neurons against 6-hydroxydopamine-induced death

The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.     

Neuron-derived FGF9 is essential for scaffold formation of Bergmann radial fibers and migration of granule neurons in the cerebellum

Although fibroblast growth factor 9 (FGF9) is widely expressed in the central nervous system (CNS), the function of FGF9 in neural development remains undefined. To address this question, we deleted the Fgf9 gene specifically in the neural tube and demonstrated that FGF9 plays a key role in the postnatal migration of cerebellar granule neurons. Fgf9-null mice showed severe ataxia associated with disrupted Bergmann fiber scaffold formation, impaired granule neuron migration, and upset Purkinje cell maturation. Ex vivo cultured wildtype or Fgf9-null glia displayed a stellate morphology. Coculture with wildtype neurons, but not Fgf9-deficient neurons, or treating with FGF1 or FGF9 induced the cells to adopt a radial glial morphology. In situ hybridization showed that Fgf9 was expressed in neurons and immunostaining revealed that FGF9 was broadly distributed in both neurons and Bergmann glial radial fibers. Genetic analyses revealed that the FGF9 activities in cerebellar development are primarily transduced by FGF receptors 1 and 2. Furthermore, inhibition of the MAP kinase pathway, but not the PI3K/AKT pathway, abrogated the FGF activity to induce glial morphological changes, suggesting that the activity is mediated by the MAP kinase pathway. This work demonstrates that granule neurons secrete FGF9 to control formation of the Bergmann fiber scaffold, which in turn, guides their own inward migration and maturation of Purkinje cells.     

Roles of Thapsigargin-Sensitive Ca2+ Stores in the Survival of Developing Cultured Neurons

The roles of the intracellular calcium pool involved in regulating the Ca2+ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca2+]i directly in the culture medium, we found a bell-shaped curve for [Ca2+]i versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K+. The progressive increase in [Ca2+]i of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K+] was lowered from 25 to 5 mM, neither the progressively increasing [Ca2+]i nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca2+]i and survival rate of these immature neurons when switched to 5 mM K+ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca2+]i and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum-free medium by decreasing [Ca2+]i to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca2+-ATPase plays a pivotal role in the progressive increase of [Ca2+]i, which is essential for the growth and maturation of cultured granule neurons.     

Ornithine-σ-Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture

To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.     

Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.     

Apoptosis-signal regulating kinase-1 is involved in the low potassium-induced activation of p38 mitogen-activated protein kinase and c-Jun in cultured cerebellar granule neurons

Previously, we reported that p38, which belongs to the mitogen-activated protein kinase (MAPK) superfamily, has an important role in the induction of apoptosis of cultured cerebellar granule neurons. However, the molecular mechanisms upstream of p38 activation remain unclear. Apoptosis signal-regulating kinase-1 (ASK1), a MAPK kinase kinase (MAPKKK) protein, is known to activate both c-Jun N-terminal kinase (JNK) and p38 via MAPK kinase (MKK) 4/7 and MKK3/6, respectively. Here, we examined whether ASK1 is involved in the activation of p38 in the low potassium (LK)-induced apoptosis of cerebellar granule neurons. We found that ASK1 was activated after a change to LK medium. In addition, the expression of ASK1-KM, a dominant-negative form of ASK1, using an adenovirus system was found to inhibit the activation of p38 and c-Jun and to prevent apoptosis. On the other hand, the expression of ASK1-DeltaN, a constitutively active form of ASK1, activated p38 and c-Jun, but not JNK, another possible downstream target of ASK1. Furthermore, we examined the relationship between phosphatidylinositol 3-kinase (PI3-K) and ASK1. The addition of LY294002, a specific inhibitor of PI3-K, enhanced the ASK1 activity. These results indicate that ASK1 works downstream of PI3-K to regulate the p38-c-Jun pathway and apoptosis in cultured cerebellar granule neurons.     

Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellumin vivo

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine--aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.     

Specific Expression of N-Acetylaspartate in Neurons, Oligodendrocyte-Type-2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro

To test the specificity of N-acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type-1 astrocytes, meningeal cells, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type-1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O-2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.     

GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI-anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI-anchored proteins have been found on both the axonal and somatodendritic cell-surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication-incompetent retroviral vector to express a model GPI-anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high-density monolayers, and maintained for 1-9 days under serum-free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP-expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP-expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well-polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI-anchored protein expressed in a well-polarized primary neuronal cell type in vitro; other signals, such as those present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon-specific GPI-anchored proteins.     

Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

Evoked release of [³H]-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and [³H]-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10–100 M glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 M kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.     

Internalization and down-regulation of muscarinic acetylcholine receptors in cerebellar granule cells of tenascin-gene deficient mice

The expression of tenascin-C on oligodendrocytes parallels the migration of granule cells in the developing cerebellum, indicating a role for tenascin-C as a guide for granule neurons to find their proper locations. In this study, cultured cerebellar granule neurons from tenascin-C-knockout mice were used to examine the role of tenascin-C in agonist-induced muscarinic acetylcholine receptor down-regulation. Exposure of granule cells from wild-type or tenascin-C-negative mice to the muscarinic acetylcholine receptor agonist carbachol (1 mM) resulted in normal sequestration of cell-surface muscarinic acetylcholine receptors as assessed by [3H]N-methylscopolamine binding; however, down-regulation of total muscarinic acetylcholine receptors, measured with [3H]quinuclidinyl benzilate, was inhibited in granule cells from tenascin-C-negative mice. Remarkably, incubation of the tenascin-C-negative cells with the microtubule stabilizer taxol (10 microM) restored down-regulation of total muscarinic acetylcholine receptors to normal levels. We speculate that agonist-induced down-regulation of muscarinic acetylcholine receptors is functionally associated with tenascin-C-regulated microtubule structures in the developing cerebellum.     

Evidence for Functional Diversity between the Voltage-Gated Proton Channel Hv1 and Its Closest Related Protein HVRP1

The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs). However, they lack the pore domain typical of these other channels. Na_V, K_V, and Ca_V proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived), which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.     

K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels

Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.     

Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.