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.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Phosphatidylinositol 3'-OH kinase and protein kinase A pathways mediate the anti-apoptotic effect of pituitary adenylyl cyclase-activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol

Cerebellar granule neurons cultured in the presence of 5 mm KCl undergo spontaneous apoptosis, which is reduced by exposure to pituitary adenylyl cyclase-activating polypeptide (PACAP). Previous work has suggested roles for the cyclic AMP/PKA and MAP kinase signaling pathways in the anti-apoptotic effect of PACAP. In the present study, the use of specific inhibitors confirmed the role of the cyclic AMP/PKA pathway, and also demonstrated a role for the phosphatidylinositol 3'-OH kinase (PI 3-kinase) neuroprotective pathway in the action of PACAP. Ethanol exposure accelerates the anti-apoptotic effect of PACAP by a mechanism that involves the PKA and PI-3 kinase pathways. The results demonstrate that ethanol can increase neuroprotection induced by PACAP. As previous work has shown that ethanol can increase apoptosis of cerebellar granule neurons by inhibiting the protective effect of agents such as NMDA or IGF-1, the overall effect of ethanol on cerebellar neuron apoptosis during development may reflect the balance between inhibition and enhancement of the actions of various endogenous neuroprotective agents.     

Neuregulin induces GABAA receptor beta2 subunit expression in cultured rat cerebellar granule neurons by activating multiple signaling pathways

The GABAA receptor beta subunit is required to confer sensitivity to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the CNS. In previous studies we demonstrated that the growth and differentiation factor neuregulin 1 (NRG1) selectively induced expression of the beta2 subunit mRNA and encoded protein in rat cerebellar granule neurons in culture. In the present report we examine the signaling pathways that mediate this effect. These studies demonstrate that the effects of NRG1 on beta2 subunit polypeptide expression require activation of the ErbB4 receptor tyrosine kinase; its effects are inhibited by pharmacological blockade of ErbB4 phosphorylation or reduction of receptor level with an antisense oligodeoxynucleotide. The NRG1-induced activation of ErbB4 stimulates the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and cyclin-dependent kinase-5 (cdk5) pathways. Pharmacological blockade of any of these pathways inhibits increased beta2 subunit expression, demonstrating that all three pathways are required to mediate the effects of NRG1 on GABAA receptor subunit expression in cerebellar granule neurons. These studies provide novel information concerning the actions of NRG1 on GABAA receptor expression in the CNS.     

Laser microirradiation of cerebellar neurons in culture

Electrophysiological and ultrastructural effects of focused laser radiation on neurons from neonatal rat cerebellum in tissue culture are reported. Action potentials were elicited by an extracellular current pulse train. The stimulator voltage required for half-maximum response frequency was measured as a function of the energy delivered by a single laser pulse. Above a “threshold” laser energy, the cell response to stimulation became negligible for all stimulator voltages. Electron micrographs of cells revealed that the mitochondria are preferentially damaged at an energy comparable to the electrophysiological threshold. The damaged mitochondria showed swollen matrix space and disrupted cristae membranes. Higher laser energies resulted in damage to other cytoplasmic structures. The results are consistent with a model that assumes that light interaction with the nerve cells proceeds by local heating of the mitochondria and nearby structures and leads to an increased conductance of the membrane to some ionic species.     

Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons

Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the β6‐β7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG‐loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1‐IIIc‐Ig2‐3 and FGFR2‐IIIb‐Ig2‐3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1‐IIIc‐Ig2‐3 indicating overlapping binding sites. Hexafin‐binding to FGFR1‐IIIc resulted in receptor phosphorylation, but inhibited FGF1‐induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC‐80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

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 asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

Neuroprotection by scatter factor/hepatocyte growth factor and FGF-1 in cerebellar granule neurons is phosphatidylinositol 3-kinase/akt-dependent and MAPK/CREB-independent

Neuroprotective actions of scatter factor/hepatocyte growth factor (SF/HGF) have not been described. We examined the effects of SF/HGF in comparison to acidic fibroblast growth factor-1 (FGF-1) on N-methyl-D-aspartate (NMDA) and quinolinic acid (QUIN)-induced excitotoxicity in primary cerebellar granule neurons. Exposure to NMDA or QUIN for 24 h resulted in concentration-dependent cell death (p < 0.001) that was completely attenuated (p < 0.001) by pre-treatment of cells with SF/HGF (50 ng/mL) or FGF-1 (40 ng/mL). SF/ HGF and FGF-1 activated both Akt and MAP-kinase > threefold (p < 0.001). Neither SF/HGF nor FGF-1 activated cyclic AMP-response element binding protein (CREB), a downstream target of MAP-kinase, whereas brain-derived neurotrophic factor (BDNF) activated both MAP-kinase and CREB in granule neurons. Neuroprotection against NMDA or QUIN by SF/HGF and FGF-1 was negated by the addition of LY294002 (10 microM) or wortmannin (100 microM), two distinct inhibitors of phosphatidylinositol 3-kinase (P13-K), but not by the MAP-kinase kinase (MEK) inhibitor PD98059 (33 microm). Likewise, expression of a dominant-negative mutant of Akt (Akt-kd) completely prevented the neuroprotective actions of SF/HGF and FGF-1. Overexpression of a constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) attenuated excitotoxic cell death. These data show that both SF/HGF and FGF-1 protect cerebellar granule neurons against excitotoxicity with similar potency in a P13-K/Akt-dependent and MAP-kinase/CREB-independent manner.     

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons

In the present study we focused in the PI3K/Akt pathway which plays a key role in neuronal survival. Here we show that inhibition of PI3K/Akt by means of LY294002 induces apoptosis via a caspase-dependent and calpain-independent pathway in cerebellar granule neurons (CGNs). This finding was confirmed using zVAD-fmk, a widely caspase inhibitor that prevents apoptosis. For this purpose, we compared two models of apoptosis in CGNs, namely inhibition of PI3K/Akt, and serum potassium deprivation (S/K deprivation). In contrast to the S/K deprivation model, caspase-3 was not activated when PI3K is inhibited. Likewise, CDK5 activation was not involved in this apoptotic process, because calpain activation is responsible for the formation of CDK5/p25 neurotoxic form. However, S/K deprivation activated calpain, as it is shown by α-spectrin breakdown, and favoured the formation of CDK5/p25. Moreover, although PI3K/Akt inhibition enhanced pRbser780 phosphorylation, no increase in the expression of cell-cycle proteins, namely: cyclin D, cyclin E, CDK2 or CDK4, was detected. Furthermore, BrdU incorporation assay did not shown any increase in DNA synthesis. Likewise, PI3K/Akt inhibition increased GSK3β activity and c-Jun phosphorylation, which implicates these two pathways in this apoptotic route. Although previous reports suggest that apoptosis induced in CGNs by LY294002 and S/K deprivation causes PI3K inhibition and increases GSK3β activity and c-Jun phosphorylation activation, our results demonstrate substantial differences between them and point to a key role of GSK3β in the apoptosis induced in CGNs in the two models tested.     

TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c- jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.     

Chronic ethanol exposure attenuates the anti-apoptotic effect of NMDA in cerebellar granule neurons

Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti-apoptotic effect of NMDA. The in vitro anti-apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti-apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1-3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti-apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca(2+) and induction of brain-derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long-lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.     

Delayed rectifier outward K+ current mediates the migration of rat cerebellar granule cells stimulated by melatonin

Melatonin (MT) may work as a neuromodulator through the associated MT receptors in the central nervous system. Previously, our studies have shown that MT increased the I(K) current via a G protein-related pathway. In the present study, patch-clamp whole-cell recording, transwell migration assays and organotypic cerebellar slice cultures were used to examine the effect of MT on granule cell migration. MT increased the I(K) current amplitude and migration of granule cells. Meanwhile, TEA, the I(K) channel blocker, decreased the I(K) current and slowed the migration of granule cells. Furthermore, the effects of MT on the I(K) current and cell migration were not abolished by pre-incubation with P7791, a specific antagonist of MT(3)R, but were eliminated by the application of the MT(2)R antagonists K185 and 4-P-PDOT. I(K) current and cell migration were decreased by the application of dibutyryl cyclic AMP (dbcAMP), which was in contrast to the MT effect on the I(K) current and cell migration. Incubation with dbcAMP essentially blocked the MT-induced increasing effect. Moreover, incubation of isolated cell cultures in the MT-containing medium also decreased the cAMP immunoreactivity in the granule cells. It is concluded, therefore, that I(K) current, downstream of a cAMP transduction pathway, mediates the migration of rat cerebellar granule cells stimulated by MT.     

Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers

Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 589–603, 2010     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.