Cerebellar Granule Neuron

In this study we report the coupling of nucleotide receptors to GSK-3 signalling, a relevant survival pathway in cerebellar granule neurons. P2X7 agonist BzATP induced a 3–4-fold increase in GSK-3 phosphorylation, which is reported to be associated with the catalytic activity inhibition. This effect was dependent on extracellular calcium and PKC, and independent of PI3-K (phosphatidyl-inositol-3-kinase)/Akt, the main survival route of neurotrophins. BzATP also prevented the apoptosis of granule neurons induced by the pharmacological inhibition of the PI3-K signalling. Both effects, BzATP-mediated GSK-3 phosphorylation and neuroprotection, were abolished by P2X7 receptor antagonists, BBG, PPADS and A-438079. We found that BzATP prevented the progressive GSK-3 dephosphorylation and caspase-3 activation occurring under conditions of sustained PI3-K inhibition. These results reveal that P2X7 receptor activation could provide a relevant survival route alternative to classical neurotrophic factors.

The beta-amyloid precursor protein APP and the microtubule-associated protein Tau play a crucial role in the pathogenesis of Alzheimer's disease (AD). However, the possible molecular events linking these two proteins are still unknown. Here, we show that Fe65, one of the ligands of the APP cytodomain, is associated with Tau in vivo and in vitro, as demonstrated by co-immunoprecipitation, co-localization, and FRET experiments. Deletion studies indicated that the N-terminal domain of Tau and the PTB1 domain of Fe65 are required for this association. This interaction is regulated by the phosphorylation of Tau at selected sites, by glycogen synthase kinase-3beta (GSK3beta) and cyclin-dependent kinase 5 (Cdk5), and requires an intact microtubule network. Furthermore, laser scanner microscopy and co-immunoprecipitation experiments provide preliminary evidence of possible complex(es) involving Tau, Fe65, APP. These findings open new perspectives for the study of the possible crosstalk between these proteins in the pathogenesis of AD.

We have previously shown that herpes simplex virus type 1 (HSV-1) infection is associated with early destabilization/degradation of infected cell mRNAs and consequent shutoff of host protein synthesis by the activity of the virion-associated host shutoff (vhs) UL41 protein. Wild-type (wt) virus destabilized/degraded the housekeeping β-actin and α-tubulin mRNAs as well host stress functions, like the heat shock 70 protein induced postinfection. vhs mutants did not degrade the mRNAs. Elaborate studies by others have been concerned with the mode of mRNA degradation and the mRNAs affected. We now describe vhs activity in primary cultures of mouse cerebellar granule neurons (CGNs). Specifically, (i) upon infection in the presence of actinomycin D to test activity of input viral particles, there was a generalized inhibition of protein synthesis, which depended on the input multiplicity of infection (MOI). (ii) Low-MOI infection with vhs-1 mutant virus was associated with increased synthes...

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Cytidine 5'-diphosphocholine (CDP-choline) has been shown to reduce neuronal degeneration induced in central nervous system (CNS) injury. However, the precise mechanism underlying the neuroprotective properties of this molecule is still unknown. Excitotoxicity causes cell death in CNS injury (trauma or ischemia) and has also been involved in neurodegenerative diseases. We have examined whether CDP-choline prevents glutamate-mediated cell death, determined by trypan blue exclusion and lactate dehydrogenase activity assays. Pretreatment of rat cerebellar granule cells (CGCs) with CDP-choline causes a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Cell death is prevented >50% when 100 microM CDP-choline is added 6 d before the glutamate excitotoxic insult but less than 20% when added concomitantly with glutamate. Pretreatment of CGCs with CDP-choline reduces almost completely (>80%) the number of apoptotic cells analyzed by flow cytometry, suggesting ...

The type I metabotropic glutamate receptor (mGlu2) messenger RNA and protein are known to be widely expressed in rat brain, but knowledge of the regional expression of splice variants other than mGlu1a is limited. Probes were designed for in situ hybridization that specifically recognize each of the carboxy-terminal splice variants mGlu1a, -1b, -1c and -1d. The novel rat mGlu1d sequence

Rett syndrome (RTT) is associated with mutations in the transcriptional repressor gene MeCP2. Although the clinical and neuropathological signs of RTT suggest disrupted synaptic function, the specific role of methyl-CpG binding protein 2 (MeCP2) in postmitotic ...

Although extracellular purines may have both trophic and apoptotic functions in the brain depending on the targeted purine receptor and cell type, little is known about the role of specific purine receptors on neurons. In this study, we demonstrate that both ADP and its stable analogue 2-methyl-thio-ADP (2MeSADP) induce up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1). Selective inhibition of 2MeSADP-responsive receptors P2Y1 and P2Y13 with their respective antagonists MRS2179 and MRS2211 and the use of pertussis toxin demonstrated a role of the purinergic P2Y13 receptor in this response. Moreover, luciferase assays demonstrated that ectopic expression of the P2Y13 receptor in neuroblastoma N2A cells resulted in 2MeSADP-dependent induction of antioxidant response elements from the HO-1 promoter. The transcription factor Nrf2 was critical for HO-1 activation and translocated from the cytosol to the nucleus in response to 2MeSADP. In cerebellar granule neurons (CGNs) derived from Nrf2-knockout mice this purine did not activate the Nrf2/HO-1 axis and did not protect against H2O2-induced cell death. The relevance of HO-1 in 2MeSADP-induced neuroprotection was further demonstrated by the evidence that HO-1 inhibition with tin protoporphyrin (SnPP) prevented protection against H2O2-induced oxidative stress and cell death. These observations reveal a previously unrecognized role in protection against oxidative stress by extracellular purines acting on the metabotropic P2Y13 receptor and provide new perspectives for neuroprotective therapies.

Abstract : Caspase-3 enzyme activity is induced, and cell death follows, when cerebellar granule neurons (CGNs) from 8-day-old rats are transferred from an extracellular concentration of 25 mM K+ (25 mM [K+]e) to 5 mM [K+]e. Death of these neurons is diminished by an inhibitor of caspase-3 but not by an inhibitor of caspase-1. Actinomycin D and cycloheximide inhibit induction of caspase-3 and prevent death. Experiments in which CGN intracellular Ca2+ concentration ([Ca2+]i) was manipulated by either changing [K+]e or adding a voltage-gated Ca2+ channel antagonist or a Ca2+ ionophore to the medium showed that caspase-3 mRNA rises 2.5-fold when [Ca2+]i is diminished from 300 to 150 nM, with a corresponding rise in peak caspase enzyme activity. Whereas the caspase-3 mRNA level does not rise further with a still greater diminution in [Ca2+]i, peak caspase enzyme activity continues to increase, reaching sevenfold induction when [Ca2+]i is reduced to 55 nM. In CGNs in which [Ca2+]i is set at 55 nM by incubation in 5 mM [K+]e, treatment with forskolin or dibutyryl 3′,5′-cyclic adenosine-5′-monophosphate delays caspase-3 induction and diminishes death but does not alter [Ca2+]i. We conclude that, in immature CGNs, both caspase-3 transcription and the subsequent processing of caspase-3 are induced by a fall in [Ca2+]i. Elevating cyclic AMP content delays caspase-3 induction by a mechanism that does not require an increase in [Ca2+]i.

Effects of prolonged (48 h) inhibition of glutamate reuptake on the relative abundance of mRNAs coding for N-methyl-d-aspartate (NMDA) receptor subunits, and the expression of corresponding proteins were investigated in primary cultures of rat cerebellar granule neurons. In cells exposed to the glutamate transport blocker, l-trans-pyrrolidine-2,4-dicarboxylate (PDC), the expression of the C1 exon-positive NR1 mRNA variant was reduced by about 40% whereas, the expression of C1-negative mRNA was increased leading to significant reduction of the +C1/−C1 ratio. The expression of the N1-negative NR1 variants was slightly reduced following exposure to PDC, indicating that increased medium levels of glutamate changed the relative abundance of NR1 splice-variant expression but did not reduce the overall NR1 transcription. Expression of NR2A and NR2B mRNAs was 40–50% lower in PDC-treated cells as compared to control. Immunoblot experiments revealed that PDC exposure reduced the expression of NR1 and all NR2 proteins with NR2A and NR2B proteins being reduced to a greater extent than NR1. The overall decrease in NMDA receptor subunit protein expression was considerably more pronounced than the reduction of their corresponding mRNAs, suggesting involvement of a post-transcriptional regulation. Our data support the hypothesis that functional activity and number of NMDA receptors are regulated by strength of the glutamatergic input. Thus, reduced glutamate uptake resulting in increased concentration of ambient glutamate initiate a series of adaptive responses manifested as a gradual down-regulation of the functional activity and expression of NMDA receptors.