Differential effects of AMPA receptor activation on survival and neurite integrity during neuronal development.
ABSTRACT: While neuronal cultures are an established model for analyzing excitotoxic brain injury in the adult, in vitro systems have not been extensively employed to study how developing neurons respond to levels of excitatory compounds that are lethal to mature neurons. Recently, we reported that the in vivo differentiation programs of cerebellar granule cells (CGNs) are recapitulated in purified CGN cultures [Manzini M.C., Ward M.S., Zhang Q., Lieberman M.D., Mason C.A. (2006) The stop-signal revised: immature cerebellar granule neurons in the external germinal layer arrest pontine mossy fiber growth. J. Neurosci. 26:6040-6051]. Here, we have used this model system to compare the response of immature and mature neurons to excitotoxic compounds. We found that immature CGNs are less sensitive to AMPA receptor (AMPA-R) activation than mature cells and that levels of AMPA-R expression on the plasma membrane are critical in regulating the balance between death and survival during maturation of these neurons. However, the majority of immature cells that survive excitotoxic treatment bear a degenerating neurite, suggesting that AMPA-R activation can still cause damage in the absence of cell death.
Project description:Developing neurons undergo a series of maturational stages, and the timing of these events is critical for formation of synaptic circuitry. Here we addressed temporal regulation of the Gabra6 gene, which is expressed in a delayed manner during dendritogenesis in maturing cerebellar granule neurons (CGNs). Developmental up-regulation of Gabra6 transcription required a binding site for nuclear factor I (NFI) proteins. The amounts and DNA binding activities of NFI proteins were similar in immature and mature CGNs; however, NFI occupancy of the Gabra6 promoter in native chromatin was temporally delayed in parallel with Gabra6 gene expression, both in vivo and in culture. The trans-repressor RE1 silencing transcription factor (REST) occupied the Gabra6 proximal promoter in CGN progenitors and early postmitotic CGNs, and its departure mirrored the initial onset of NFI binding as CGNs differentiated. Furthermore constitutive REST expression blocked both Gabra6 expression and NFI occupancy in mature CGNs, whereas REST knockdown in immature CGNs accelerated the initiation of both events. These studies identify a novel mechanism for controlling the timing of dendritogenesis-associated gene expression in maturing neurons through delayed binding of NFI proteins to chromatin. They also establish a temporal function for REST in preventing premature promoter occupancy by NFI proteins in early-stage postmitotic neurons.
Project description:Expression analysis was performed with in vitro cultured cerebellar granule neurons (CGNs) isolated from rat brain. The CGNs were culture for four weeks. Each sample was collected after interval of seven days. No treatment was given to any cultured neurons at any time point. The purpose of the experiment was to identify the genes differentially expressed during the senescence of CGNs. The experiment is useful in revealing the senescence associated genetic markers in neurons.
Project description:Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.
Project description:In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.
Project description:The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.
Project description:MAG (Myelin-associated glycoprotein) is a type I transmembrane glycoprotein expressed by Schwann cells and oligodendrocytes, that has been implicated in the control of axonal growth in many neuronal populations including cerebellar granule neurons (CGNs). However, it is unclear whether MAG has other functions in central nervous system, in particular, in cerebellar development and patterning. We find that MAG expression in the cerebellum is compartmentalised resulting in increased MAG protein levels in the cerebellar white matter. MAG induces apoptosis in developing CGNs through p75NTR signalling. Deletion of p75NTR in vivo reduced the number of apoptotic neurons in cerebellar white matter during development leading to reduction in the size of white matter in the adulthood. Furthermore, we show that MAG impairs CGNs neurite outgrowth as consequence of MAG-induced apoptosis in CGNs. Mechanistically, we find that MAG/NgR1-induced cell death is dependent of p75NTR-mediated activation of JNK/cell death signalling pathway. Together, these findings identify the mechanisms by which MAG induces CGNs apoptotic activity, a crucial event that facilitates cerebellar layer refinement during development.
Project description:Reactive oxygen species are implicated in age-associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2-like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age-associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria-targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age-matched normal control mice or SkQ1-treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4-hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age-associated neurodegeneration.
Project description:Two-pore domain potassium (K2P) channel expression is believed to underlie the developmental emergence of a potassium leak conductance [IK(SO)] in cerebellar granule neurons (CGNs), suggesting that K2P function is an important determinant of the input conductance and resting membrane potential. To investigate the role that different K2P channels may play in the regulation of CGN excitability, we generated a mouse lacking TASK-1, a K2P channel known to have high expression levels in CGNs. In situ hybridization and real-time PCR studies in wild-type and TASK-1 knock-outs (KOs) demonstrated that the expression of other K2P channels was unaltered in CGNs. TASK-1 knock-out mice were healthy and bred normally but exhibited compromised motor performance consistent with altered cerebellar function. Whole-cell recordings from adult cerebellar slice preparations revealed that the resting excitability of mature CGNs was no different in TASK-1 KO and littermate controls. However, the modulation of IK(SO) by extracellular Zn2+, ruthenium red, and H+ was altered. The IK(SO) recorded from TASK-1 knock-out CGNs was no longer sensitive to alkalization and was blocked by Zn2+ and ruthenium red. These results suggest that a TASK-1-containing channel population has been replaced by a homodimeric TASK-3 population in the TASK-1 knock-out. These data directly demonstrate that TASK-1 channels contribute to the properties of IK(SO) in adult CGNs. However, TASK channel subunit composition does not alter the resting excitability of CGNs but does influence sensitivity to endogenous modulators such as Zn2+ and H+.
Project description:PACT and its murine ortholog RAX were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase PKR. Recent studies indicated that RAX played a role in embryogenesis and neuronal development. In this study, we investigated the expression of RAX during the postnatal development of the mouse cerebellum and its role in the migration of cerebellar granule neurons (CGNs). High expression of RAX was observed in the cerebellum from postnatal day (PD) 4 to PD9, a period when the CGNs migrate from the external granule layer (EGL) to the internal granule layer (IGL). The migration of the EGL progenitor cells in vivo was inhibited by RAX knockdown on PD4. This finding was confirmed by in vitro studies showing that RAX knockdown impaired the migration of CGNs in cerebellar microexplants. PACT/RAX-regulated migration required its third motif and was independent of PKR. PACT/RAX interacted with focal adhesion kinase (FAK) and PACT/RAX knockdown disturbed the FAK phosphorylation in CGNs. These findings demonstrated a novel function of PACT/RAX in the regulation of neuronal migration.
Project description:Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.