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Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM [K.sup.+]. We have found that the growth-related gene product [Beta] (GRO[Beta]) partially prevents the [K.sup.+]-depletion-induced cell death, and that the neuroprotective action of GRO[Beta] on granule cells is mediated through the [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GRO[Beta]-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 [micro]M) treatment. Moreover, GRO[Beta]-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GRO[Beta] is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. chemokines | apoptosis | cerebellar granule neurons | glutamate receptors

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis. [ABSTRACT FROM AUTHOR]

Apoptosis triggered by exogenous or endogenous stimuli is a crucial phenomenon to determine the fate of neurons, both in physiological and in pathological conditions. Our previous study established that gastric inhibitory polypeptide (Gip) is a neurotrophic factor capable of preventing apoptosis of cerebellar granule neurons (CGNs), during its pre-commitment phase. In the present study, we conducted whole-genome expression profiling to obtain a comprehensive view of the transcriptional program underlying the rescue effect of Gip in CGNs. By using DNA microarray technology, we identified 65 genes, we named survival related genes, whose expression is significantly de-regulated following Gip treatment. The expression levels of six transcripts were confirmed by real-time quantitative polymerase chain reaction. The proteins encoded by the survival related genes are functionally grouped in the following categories: signal transduction, transcription, cell cycle, chromatin remodeling, cell death, antioxidant activity, ubiquitination, metabolism and cytoskeletal organization. Our data outline that Gip supports CGNs rescue via a molecular framework, orchestrated by a wide spectrum of gene actors, which propagate survival signals and support neuronal viability. [ABSTRACT FROM AUTHOR]

A failure in the control of proliferation of cerebellar granule neuron precursor cells (GCPs), located in the external granular layer (EGL) of the cerebellum, gives rise to medulloblastoma. To investigate the process of neoplastic transformation of GCPs, we generated a new medulloblastoma model by crossing Patchedl heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Overexpression of Tis21 is known to inhibit proliferation and trigger differentiation of GCPs; its expression decreases in human medulloblastomas. Double-knock-out mice show a striking increase in the frequency of medulloblastomas and hyperplastic EGL lesions, formed by preneoplastic GCPs. Tis21 deletion does not affect the proliferation of GCPs but inhibits their differentiation and, chiefly, their intrinsic ability to migrate outside the EGL. This defect of migration may represent an important step in medulloblastoma formation, as GCPs, remaining longer in the EGL proliferative niche, may become more prone to transformation. By genome-wide analysis, we identified the chemokine Cxcl3 as a target of Tis21. Cxcl3 is downregulated in Ti's2i-null GCPs of EGL and lesions; addition of Cxcl3 to cerebellar slices rescues the defective migration of T/s2/-null GCPs and, remarkably, reduces the area of hyperplastic lesions. As Tis21 activates Cxcl3 transcription, our results suggest that Tis21 induces migration of GCPs through Cxcl3, which may represent a novel target for medulloblastoma therapy. [ABSTRACT FROM AUTHOR]

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including b-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the bactin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs. [ABSTRACT FROM AUTHOR]

Dopamine D2 receptor (D2DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D2 receptors in this brain area are essentially obscure. We have studied the physiological responses of the D2DR stimulations in mice lacking the brain cytoplasmicRNABC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D2-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence onD2 receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB1 receptors was intact in the striatum of BC1 knock-out (KO) mice despite D2 and CB1 receptors mediated similar electrophysiological actions. Interestingly, the fragileXmental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D2-mediated transmission. Because D2DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D2DR indirectly, probably regulating translation of molecules involved in D2DR turnover and/or stability. [ABSTRACT FROM AUTHOR]

Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Mattel. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively. [ABSTRACT FROM AUTHOR]

Features a study which found that the chemokine growth-related gene product beta protects rat cerebellar granule cells from apoptotic cell death through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors. Methodology of the study; Results and discussion; Conclusion.

A failure in the control of proliferation of cerebellar granule neuron precursor cells (GCPs), located in the external granular layer (EGL) of the cerebellum, gives rise to medulloblastoma. To investigate the process of neoplastic transformation of GCPs, we generated a new medulloblastoma model by crossing Patched1 heterozygous mice, which develop medulloblastomas with low frequency, with mice lacking the Tis21 gene. Overexpression of Tis21 is known to inhibit proliferation and trigger differentiation of GCPs; its expression decreases in human medulloblastomas. Double-knock-out mice show a striking increase in the frequency of medulloblastomas and hyperplastic EGL lesions, formed by preneoplastic GCPs. Tis21 deletion does not affect the proliferation of GCPs but inhibits their differentiation and, chiefly, their intrinsic ability to migrate outside the EGL. This defect of migration may represent an important step in medulloblastoma formation, as GCPs, remaining longer in the EGL proliferative niche, may become more prone to transformation. By genome-wide analysis, we identified the chemokine Cxcl3 as a target of Tis21. Cxcl3 is downregulated in Tis21-null GCPs of EGL and lesions; addition of Cxcl3 to cerebellar slices rescues the defective migration of Tis21-null GCPs and, remarkably, reduces the area of hyperplastic lesions. As Tis21 activates Cxcl3 transcription, our results suggest that Tis21 induces migration of GCPs through Cxcl3, which may represent a novel target for medulloblastoma therapy.

Dopamine D(2) receptor (D(2)DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D(2) receptors in this brain area are essentially obscure. We have studied the physiological responses of the D(2)DR stimulations in mice lacking the brain cytoplasmic RNA BC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D(2)-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence on D(2) receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB(1) receptors was intact in the striatum of BC1 knock-out (KO) mice despite D(2) and CB(1) receptors mediated similar electrophysiological actions. Interestingly, the fragile X mental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D(2)-mediated transmission. Because D(2)DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D(2)DR indirectly, probably regulating translation of molecules involved in D(2)DR turnover and/or stability.

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.