Death Of Cerebellar Granule Neurons Research Articles

Copper Ions Reduced Toxicity of Sodium Azide and Lipopolysaccharide on Cultured Cerebellar Granule Neurons

Introduction. Copper ions (Cu2+) are structural elements of proteins such as cytochrome с oxidase (Complex IV), an enzyme that catalyzes the final step of electron transfer to oxygen during oxidative phosphorylation in the mitochondria. With Cu2+ homeostasis being of utmost importance, its disturbances in the central nervous system are involved in the mechanisms of many neurodegenerative and other brain disorders.
 This study aimed to assess the effects of non-toxic copper ion levels on death of cerebellar granule neurons associated with lipopolysaccharide (LPS; in vitro inflammation model) or azide sodium (NaN3; cytochrome с oxidase inhibitor).
 Materials and methods. LPS (10 μg/mL) or NaN3 (250 μM) was added on day 7 to 8 to the culture medium with rat cerebellar cells for 24 hours in vitro. Nitrite concentrations were measured in the culture medium by Griess assay; absorbance was recorded with a spectrophotometer at 540 nm, and morphologically intact cells were counted as survived neurons.
 Results. Added to the culture medium, LPS or NaN3 reduced neuron survival to 15 ± 2% or 20 ± 3% vs. control, respectively. Cu2+ (0.5 to 5.0 μM) increased neuron survival in a dose-dependent manner to 78 ± 4% with toxic levels of LPS and to 86 ± 6% with NaN3 with 5 μM Cu2+. The concentration of nitrites in the control culture medium was 2.0 ± 0.2 μM. Added to the cell cultures, LPS increased the concentration of nitrites to 8.5 ± 0.5 μM. Cu2+ 5 μM did not show any significant effects on nitrite accumulation in the culture medium.
 Conclusions. We showed that copper ions can exert protective effects on neurons against LPS-induced or NaN3-induced toxicity. This protection is likely to be associated rather with Cu2+ interaction with Complex IV of the electron transfer chain in the mitochondria than with inhibition of NO production. Effects of Cu2+ on apoptosis pathway proteins also cannot be ruled out.

Toxic Effect of Zinc Ions Is Accompanied by Acidification of the Cytoplasm in Cultured Rat Cerebellar Granule Neurons.

Addition of 0.07 mM zinc ions to the culture medium induced death of cerebellar granule neurons. This was preceded by a decrease in intracellular pH by 0.86±0.12 and an increase in the level of intracellular zinc registered by increasing fluorescence of FluoZin3. However, intracellular acidosis caused by acidification of the culture medium to pH 6.0 was not toxic to neurons, and even significantly increased their survival under the action of zinc ions. In general, the overload of the neurons cytoplasm with zinc ions causes acidification of the cytoplasm, which is probably associated with the activation of Zn2+/H+ exchangers and is a protective mechanism for the neurocytotoxic effect of zinc ions.

EGCG modulates PKD1 and ferroptosis to promote recovery in ST rats.

BackgroundSpinal cord injury (SCI) causes devastating loss of function and neuronal death without effective treatment. (−)-Epigallocatechin-3-gallate (EGCG) has antioxidant properties and plays an essential role in the nervous system. However, the underlying mechanism by which EGCG promotes neuronal survival and functional recovery in complete spinal cord transection (ST) remains unclear.MethodsIn the present study, we established primary cerebellar granule neurons (CGNs) and a T10 ST rat model to investigate the antioxidant effects of EGCG via its modulation of protein kinase D1 (PKD1) phosphorylation and inhibition of ferroptosis.ResultsWe revealed that EGCG significantly increased the cell survival rate of CGNs and PKD1 phosphorylation levels in comparison to the vehicle control, with a maximal effect observed at 50 µM. EGCG upregulated PKD1 phosphorylation levels and inhibited ferroptosis to reduce the cell death of CGNs under oxidative stress and to promote functional recovery and ERK phosphorylation in rats following complete ST.ConclusionTogether, these results lay the foundation for EGCG as a novel strategy for the treatment of SCI related to PKD1 phosphorylation and ferroptosis.

Neuroprotection Comparison of Rosmarinic Acid and Carnosic Acid in Primary Cultures of Cerebellar Granule Neurons.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease, are characterized by the progressive loss of neurons in specific regions of the brain and/or spinal cord. Neuronal cell loss typically occurs by either apoptotic or necrotic mechanisms. Oxidative stress and nitrosative stress, along with excitotoxicity and caspase activation, have all been implicated as major underlying causes of neuronal cell death. Diverse nutraceuticals (bioactive compounds found in common foods) have been shown to have neuroprotective effects in a variety of in vitro and in vivo disease models. In the current study, we compared the neuroprotective effects of two polyphenolic compounds, rosmarinic acid and carnosic acid, which are both found at substantial concentrations in the herb rosemary. The capacity of these compounds to rescue primary cultures of rat cerebellar granule neurons (CGNs) from a variety of stressors was investigated. Both polyphenols significantly reduced CGN death induced by the nitric oxide donor, sodium nitroprusside (nitrosative stress). Rosmarinic acid uniquely protected CGNs from glutamate-induced excitotoxicity, while only carnosic acid rescued CGNs from caspase-dependent apoptosis induced by removal of depolarizing extracellular potassium (5K apoptotic condition). Finally, we found that carnosic acid protects CGNs from 5K-induced apoptosis by activating a phosphatidylinositol 3-kinase (PI3K) pro-survival pathway. The shared and unique neuroprotective effects of these two compounds against diverse modes of neuronal cell death suggest that future preclinical studies should explore the potential complementary effects of these rosemary polyphenols on neurodegenerative disease progression.

Creatine Protects Against Cytosolic Calcium Dysregulation, Mitochondrial Depolarization and Increase of Reactive Oxygen Species Production in Rotenone-Induced Cell Death of Cerebellar Granule Neurons.

Rotenone is a neurotoxin that is an active component of many pesticides which has been shown to induce Parkinsonism in animal models. We show that the cytotoxicity of exposure to nanomolar concentrations of rotenone in cultures of mature cerebellar granule neurons (CGN) in serum-free medium is not due to phagocytosis by glial contamination. A concentration as low as 5.65 ± 0.51nM of rotenone was enough to trigger 50% cell death of mature CGN in culture after 12h. The addition of serum proteins to the culture medium attenuated rotenone neurotoxicity, and this can account at least in part for the requirement of higher rotenone concentrations to elicit neuronal cytotoxicity reported in previous works. Creatine partial protection against CGN death promoted by 5nM rotenone correlated with creatine protection against rotenone-induced mitochondrial depolarization and oxidative stress. Furthermore, creatine largely attenuated the early dysregulation of cytosolic Ca2+ concentration after acute rotenone treatment. Noteworthy, our results also revealed that the sustained alteration of Ca2+ homeostasis induced by rotenone takes place at the onset of the enhancement of intracellular oxidative stress and before mitochondrial depolarization, pointing out that cytosolic Ca2+ dysregulation is a very early event in the rotenone toxicity to CGN.

RIP2 Gates TRAF6 Interaction with Death Receptor p75NTR to Regulate Cerebellar Granule Neuron Survival

Cerebellar granule neurons (CGNs) undergo programmed cell death during the first postnatal week of mouse development, coincident with sustained expression of the death receptor p75NTR. Although ablation of p75NTR does not affect CGN cell death, deletion of the downstream effector RIP2 significantly increases CGN apoptosis, resulting in reduced adult CGN number and impaired behaviors associated with cerebellar function. Remarkably, CGN death is restored to basal levels when p75NTR is deleted in RIP2-deficient mice. We find that RIP2 gates the signaling output of p75NTR by competing with TRAF6 for binding to the receptor intracellular domain. In CGNs lacking RIP2, more TRAF6 is associated with p75NTR, leading to increased JNK-dependent apoptosis. In agreement with this, pharmacological inhibition or genetic ablation of TRAF6 restores cell death levels in CGNs lacking RIP2. These results reveal an unexpected mechanism controlling CGN number and highlight how competitive interactions govern the logic of death receptor function.

Reactive Oxygen Species Evoked by Potassium Deprivation and Staurosporine Inactivate Akt and Induce the Expression of TXNIP in Cerebellar Granule Neurons.

The reactive oxygen species (ROS) play a critical role in neuronal apoptosis; however, the mechanisms are not well understood. It has been shown that thioredoxin-interacting protein (TXNIP) overexpression renders cells more susceptible to oxidative stress and promotes apoptosis and that the activation of PI3K/Akt pathway leads to a downregulation of TXNIP. Here, we evaluated the role of ROS in the regulation of Akt activity and the subsequent regulation of the TXNIP expression in a model of apoptotic death of cerebellar granule neurons (CGN). We observed that two apoptotic conditions that generate ROS at short times led to an increase in the expression of TXNIP in a time-dependent manner; antioxidants significantly reduced this expression. Also, H2O2 caused an increase in TXNIP expression. Moreover, apoptotic conditions induced inactivation of Akt in a time-dependent manner similar to TXNIP expression and H2O2 treatment led to Akt inactivation. Besides, the pharmacological inhibition of Akt increases TXNIP expression and induces CGN cell death. Together, these results suggest that ROS promote neuronal apoptosis through the Akt-TXNIP signaling pathway, supporting the idea that the PI3K/Akt pathway regulates the TXNIP expression. This study highlights the potential importance of this mechanism in neuronal death.

Microglia mediate diesel exhaust particle-induced cerebellar neuronal toxicity through neuroinflammatory mechanisms

In addition to the well-established effects of air pollution on the cardiovascular and respiratory systems, emerging evidence has implicated it in inducing negative effects on the central nervous system. Diesel exhaust particulate matter (DEP), a major component of air pollution, is a complex mixture of numerous toxicants. Limited studies have shown that DEP-induced dopaminergic neuron dysfunction is mediated by microglia, the resident immune cells of the brain. Here we show that mouse microglia similarly mediate primary cerebellar granule neuron (CGN) death in vitro. While DEP (0, 25, 50, 100μg/2cm2) had no effect on CGN viability after 24h of treatment, in the presence of primary cortical microglia neuronal cell death increased by 2–3-fold after co-treatment with DEP, suggesting that microglia are important contributors to DEP-induced CGN neurotoxicity. DEP (50μg/2cm2) treatment of primary microglia for 24h resulted in morphological changes indicative of microglia activation, suggesting that DEP may induce the release of cytotoxic factors. Microglia-conditioned medium after 24h treatment with DEP, was also toxic to CGNs. DEP caused a significant increase in reactive oxygen species in microglia, however, antioxidants failed to protect neurons from DEP/microglia-induced toxicity. DEP increased mRNA levels of the pro-inflammatory cytokines IL-6 and IL1-β, and the release of IL-6. The antibiotic minocycline (50μM) and the peroxisome proliferator-activated receptor-γ agonist pioglitazone (50μM) attenuated DEP-induced CGN death in the co-culture system. Microglia and CGNs from male mice appeared to be somewhat more susceptible to DEP neurotoxicity than cells from female mice possibly because of lower paraoxonase-2 expression. Together, these results suggest that microglia-induced neuroinflammation may play a critical role in modulating the effect of DEP on neuronal viability. .

The critical role of lipid rafts nanodomains in the cross-talk between calcium and reactive oxygen and nitrogen species in cerebellar granule neurons apoptosis by extracellular potassium deprivation

Trabajo financiado por: Ministerio de Economia y Competitividad. Becas BFU2011-30178, BFU2014-53641-P (I+D+i) Junta de Extremadura y Fondeos FEDER. Beca GR15139 Portuguese Fundacao para a Ciencia e a Tecnologia. Beca predoctoral para Sofia Isabel Almeida Fortalezas Fundacao para a Ciencia e Tecnologia. Beca postdoctoral para Alejandro Khalil Samhan Arias

ROS produced by NOX2 control in vitro development of cerebellar granule neurons development.

Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H2O2) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development.

Mitochondrial calcium uniporter protein MCU is involved in oxidative stress-induced cell death.

Mitochondrial calcium uniporter (MCU) is a conserved Ca2+ transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stress-induced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca2+ uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca2+ uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.

Neuron-type-specific signaling by the p75NTR death receptor is regulated by differential proteolytic cleavage.

Signaling by the p75 neurotrophin receptor (p75(NTR), also known as NGFR) is often referred to as cell-context dependent, but neuron-type-specific signaling by p75(NTR) has not been systematically investigated. Here, we report that p75(NTR) signals very differently in hippocampal neurons (HCNs) and cerebellar granule neurons (CGNs), and we present evidence indicating that this is partly controlled by differential proteolytic cleavage. Nerve growth factor (NGF) induced caspase-3 activity and cell death in HCNs but not in CGNs, whereas it stimulated NFκB activity in CGNs but not in HCNs. HCNs and CGNs displayed different patterns of p75(NTR) proteolytic cleavage. Whereas the p75(NTR) carboxy terminal fragment (CTF) was more abundant than the intracellular domain (ICD) in HCNs, CGNs exhibited fully processed ICD with very little CTF. Pharmacological or genetic blockade of p75(NTR) cleavage by γ-secretase abolished NGF-induced upregulation of NFκB activity and enabled induction of CGN death, phenocopying the functional profile of HCNs. Thus, the activities of multifunctional receptors, such as p75(NTR), can be tuned into narrower activity profiles by cell-type-specific differences in intracellular processes, such as proteolytic cleavage, leading to very different biological outcomes.

Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3'-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress-induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

Endogenous XIAP, but not other members of the inhibitory apoptosis protein family modulates cerebellar granule neurons survival

Programmed cell death plays a critical role during cerebellar development. In particular, it has been shown in vivo and in vitro that developing cerebellar granule neurons (CGN) die apoptotically. Apoptosis involves a series of morphological changes and the activation of caspases. Inhibitor of apoptosis proteins (IAPs) is implicated in negative regulation of caspase activation and apoptotic cell death. Although apoptotic death of CGN has been extensively studied, there is no information about the role of IAPs in the developing cerebellum. Here, we studied the participation of some members of IAPs in the survival of the developing rat CGN in culture and under physiological conditions. Under these conditions, we found a differential expression pattern of cIAP-1, cIAP-2, XIAP and survivin during cerebellar development in an age-dependent manner, highlighting the significant increase of XIAP levels. We also detected an interaction between XIAP and caspase 3 at postnatal day (P) 12 and 16. On the other hand, we found a significant decrease of XIAP levels in cultured CGN maintained in chronic potassium deprivation, an apoptotic condition, suggesting a possible relationship between XIAP levels and neuronal viability. Under these conditions, we also detected the interaction of XIAP with active caspase-3. The down-regulation of XIAP in CGN cultured under survival conditions (chronic potassium depolarization) induced a reduction of cell viability and an increment of apoptotic cells. These findings support the idea that IAPs could be involved in the survival of CGN and that XIAP might be critical for neuronal survival in cerebellar development and during chronic depolarization in cultured CGN through a mechanism involving caspase inhibition.

Novel TASK channels inhibitors derived from dihydropyrrolo[2,1-a]isoquinoline

TASK channels belong to the family of K+ channels with 4 transmembrane segments and 2 pore domains (4TM/2P) per subunit. These channels have been related to apoptosis in cerebellar granule neurons (CGN), as well as cancer in other tissues. TASK current is regulated by hormones, neurotransmitters, anesthetics and divalent cations, which are not selective. Recently, there has been found some organic compounds that inhibit TASK current selectively. In order to find other modulators, we report here a group of five dihydropyrrolo[2,1-a]isoquinolines (DPIs), four of them with putative anticancer activity, that were evaluated on TASK-1 and TASK-3 channels. The compounds 1, 2 and 3 showed IC50 < 320 μM on TASK-1 and TASK-3, intermediate activity on TASK-1/TASK-3 heterodimer, moderate effect over hslo and TREK-1 (500 μM), and practically not inhibition on Shaker-IR, herg and IRK2.1 potassium channels, when they were expressed heterologously in Xenopus laevis oocytes. In rat CGN, 500 μM of these three compounds induced a decrement by >39% of the TASK-carried leak current. Finally, only compound 1 showed significant protection (∼36%) against apoptotic death of CGN induced by K+ deprivation. These results suggest that DPI compounds could be potential candidates for designing new selective inhibitors of TASK channels.

Mechanisms of penitrem-induced cerebellar granule neuron death in vitro: Possible involvement of GABAA receptors and oxidative processes

The fungal neurotoxin penitrem A has previously been found to cause neurological disorders in animals and humans after ingestion of contaminated food and/or feed. It penetrates the blood–brain-barrier and causes cerebellar pathology in rats, including mild effects on granule neurons.The aim of the current study was to investigate the potential toxicity of penitrem A in rat cerebellar granule neurons in vitro, and to examine the involvement of the GABAA, AMPA and NMDA receptors, intracellular signalling pathways as well as the role of oxidative stress in penitrem A-induced neuronal death. Cerebellar granule cells were exposed to penitrem A, alone or together with different pharmacological agents, before cell survival was assessed with the MTT assay or formation of reactive oxygen species (ROS) was investigated with the DCF assay.Penitrem A caused a time- and concentration-dependent reduction in cell survival, as well as a concentration-dependent increase in ROS production. Co-incubation with diazepam, GABA, BAPTA-AM, vitamin E, SP600125 and cyclosporine A significantly reduced cell death. Our results show that penitrem A is toxic to cerebellar granule neurons in vitro. Further, ROS production and the GABAA receptor are likely to be involved in the induction of neuronal death following penitrem A exposure. A disruption of calcium homeostasis and activation of the JNK pathway may also play a role in penitrem A neurotoxicity.

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol-induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol-induced increase in intracellular calcium [Ca(2+)](i) causes the death of CGN. Alcohol induced a dose-dependent (200-800 mg/dL) neuronal death within 24 h. Ratiometric Ca(2+) imaging with Fura-2 revealed that alcohol causes a rapid (1-2 min), dose-dependent increase in [Ca(2+)](i), which persisted for the duration of the experiment (5 or 7 min). The alcohol-induced increase in [Ca(2+)](i) was observed in Ca(2+) -free media, suggesting intracellular Ca(2+) release. Pre-treatment of CGN cultures with an inhibitor (2-APB) of the inositol-triphosphate receptor (IP(3) R), which regulates Ca(2+) release from the endoplasmic reticulum (ER), blocked both the alcohol-induced rise in [Ca(2+)](i) and the neuronal death caused by alcohol. Similarly, pre-treatment with BAPTA/AM, a Ca(2+) -chelator, also inhibited the alcohol-induced surge in [Ca(2+) ](i) and prevented neuronal death. In conclusion, alcohol disrupts [Ca(2+)](i) homeostasis in CGN by releasing Ca(2+) from intracellular stores, resulting in a sustained increase in [Ca(2+)](i). This sustained increase in [Ca(2+)](i) may be a key determinant in the mechanism underlying alcohol-induced neuronal death.

Vacuolar H+-ATPase (V-ATPase) Promotes Vacuolar Membrane Permeabilization and Nonapoptotic Death in Stressed Yeast

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

Opposing roles for E2F1 in survival and death of cerebellar granule neurons

The transcription factor E2F1 is upregulated when cerebellar granular neurons (CGNs) undergo apoptosis under potassium deprivation. In this study, we examined the effects of E2F1 upregulation on the survival and death of CGNs isolated from C57 mice and Sprague-Dawley (SD) rats. Plasmid- and adenovirus-mediated expression of E2F1 dose-dependently induced apoptosis in mouse CGNs but unexpectedly failed to induce apoptosis in rat CGNs. Caspase 3, a marker for neuronal apoptosis, was significantly activated by ectopic E2F1 expression in mouse CGNs but not in rat CGNs. Furthermore, overexpression of E2F1 significantly promoted apoptotic progression in mouse CGNs following potassium deprivation but attenuated apoptosis in rat CGNs, whereas E2F1 lacking DNA binding ability (E2F1-M132) lost its pro-apoptotic role in mouse CGNs and anti-apoptotic role in rat CGNs. Together, our results demonstrated that upregulation of E2F1 by potassium deprivation promotes apoptosis in C57 mouse CGNs but antagonizes apoptosis in SD rat CGNs, suggesting opposing roles for E2F1 in regulating CGN fate.

BIT/SHPS-1 Promotes Antiapoptotic Effect of BDNF on Low Potassium-Induced Cell Death of Cultured Cerebellar Granule Neurons

Brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is a neuronal adhesion molecule that is highly expressed in cerebellar granule neurons (CGNs); however its function in CGNs remains unclear. Our previous studies indicated that BIT/SHPS-1 is able to modulate the antiapoptotic effect of brain-derived neurotrophic factor (BDNF) on CNS neurons by cell type-specific mechanisms. In this article, we have studied the role of BIT/SHPS-1 in the antiapoptotic function of BDNF on low potassium (LK)-induced cell death of cultured CGNs which is an in vitro model system of neuronal apoptosis during brain development. Cultured rat CGNs were transduced with wild-type rat BIT/SHPS-1 (BIT/SHPS-1(WT)), its 4F-mutant (BIT/SHPS-1(4F), in which all cytoplasmic tyrosine residues were substituted with phenylalanine), or nuclear localization signal-attached beta-galactosidase (NLS-LacZ, as control)-expressing adenoviruses. Expression of BIT/SHPS-1(WT) and BIT/SHPS-1(4F) alone did not affect steady-state cell viability. Tyrosine phosphorylation of BIT/SHPS-1 was only detected in BIT/SHPS-1(WT)-expressing cultures in the presence and the absence of BDNF. When subjected to LK in the presence of BDNF, BIT/SHPS-1(WT)- and BIT/SHPS-1(4F)-expressing cultures showed a significant resistance to cell death, while the control virus-transfected culture did not. In addition, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, attenuated the antiapoptotic effect of BDNF on BIT/SHPS-1(WT)-, and BIT/SHPS-1(4F)-expressing cultures. These results demonstrated that in both tyrosine phosphorylation-independent and PI3-K-dependent manners, BIT/SHPS-1 promotes the antiapoptotic effect of BDNF on the LK-induced cell death of CGNs.