Posttranslational Regulation of BCL2 Levels in Cerebellar Granule Cells: A Mechanism of Neuronal Survival Laura Lossi, 1,2 Graziana Gambino, 1 Francesco Ferrini, 1 Silvia Alasia, 1 Adalberto Merighi 1,2 1 Dipartimento di Morfofisiologia Veterinaria, Universita ` degli Studi di Torino, Italy 2 Istituto Nazionale di Neuroscienze (INN), Torino, Italy Received 22 May 2009; revised 2 July 2009; accepted 6 July 2009 ABSTRACT: Apoptosis can be modulated by K + and Ca 2+ inside the cell and/or in the extracellular mi- lieu. In murine organotypic cultures, membrane potential-regulated Ca 2+ signaling through calcineurin phosphatase has a pivotal role in development and mat- uration of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle- mediated gene transfer in CGCs, to study the posttrans- lational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K + /Ca 2+ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (moni- tored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (in Western blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell mem- brane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K + ] e and [Ca 2+ ] i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K + ] e under conditions of immaturity, is inde- pendent of extracellular Ca 2+ and operates via IP3 channels. The second leads to influx of extracellular Ca 2+ following activation of ryanodine channels in the presence of physiological [K + ] e , when CGCs are main- tained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. ' 2009 Wiley Periodicals, Inc. Develop Neurobiol 69: 855– 870, 2009 Keywords: BCL2; cell death; cerebellum; autophagy; organotypic cultures; gene transfer; endoplasmic reticulum INTRODUCTION In neurons that are excitable cells, regulation of ionic concentrations is crucial to membrane potential and electrical activity. Changes in ionic concentrations inside the cell and/or in the extracellular milieu also modulate apoptosis, a gene-regulated process of cell death. Loss of cytosolic K + and the consequent diminu- tion of the intracellular K + concentration ([K + ] i ) eventually lead to nuclear DNA fragmentation, cell shrinkage, and apoptosis (Dallaporta et al., 1998). Consistently with these findings, several inducers of apoptosis promote death by increasing the efflux of K + from the cell (Bortner et al., 2008). Under certain circumstances, disruption of K + homeostasis is per se sufficient to provoke apoptosis (Yu and Choi, 2000), Correspondence to: L. Lossi (laura.lossi@unito.it). Contract grant sponsor: Italian MiUR; contract grant number: Fondi PRIN 2007 (to L.L.). Contract grant sponsor: University of Turin, and Regione Piemonte; contract grant number: Fondi CIPE 2004 (to L.L.). ' 2009 Wiley Periodicals, Inc. Published online 11 August 2009 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20744 855