C-terminal mechano-growth factor induces heme oxygenase-1-mediated neuroprotection of SH-SY5Y cells via the protein kinase Cϵ/Nrf2 pathway

Recently, a variant of insulin‐like growth factor‐1, mechano‐growth factor (MGF), has been discovered whose 24‐amino‐acid carboxy end is protective in models of stroke, nerve injury, and amyotrophic lateral sclerosis, suggesting broad‐spectrum neuroprotective properties. Moreover, we recently demonstrated in vitro and in vivo that a modified protease‐resistant 24‐amino‐acid MGF derivative (MGF24) protects dopaminergic neurons from oxidative stress‐induced apoptosis via induction of the stress response protein heme oxygenase‐1. However, the underlying mechanism by which MGF24 up‐regulates heme oxygenase‐1 expression is unknown. In this study, we demonstrate that MGF24‐induced heme oxygenase‐1 up‐regulation is dependent on activation of protein kinase Cϵ and NF‐E2‐related factor‐2 (Nrf2). MGF24 induces nuclear translocation of Nrf2, and siRNA knockdown of Nrf2 or of heme oxygenase‐1 prevents MGF24‐induced heme oxygenase‐1 up‐regulation and neuroprotection of SH‐SY5Y cells against 6‐hydroxydopamine‐induced cell death. Pharmacological inhibition of ERK, p38 MAPK, PI3K/Akt, or PKC signaling revealed that only PKC inhibition by GF109203X prevents MGF24's ability to protect against 6‐hydroxydopamine‐induced cell death. GF109203X also prevented MGF24‐induced Nrf2 nuclear translocation and heme oxygenase‐1 up‐regulation. siRNA knockdown of protein kinase Cϵ blocks MGF24‐induced Nfr2 nuclear translocation, heme oxygenase‐1 expression, and neuroprotection. Taken together, these results demonstrate that PKC activity is needed for MGF24's activation of Nrf2, which in turn increases heme oxygenase‐1 expression, a critical event in mediating MGF24's neuroprotection against 6‐hydroxydopamine‐induced apoptosis. Published 2011 Wiley‐Liss, Inc.     

Insights into the pathogenesis of ATP1A1‐related CMT disease using patient‐specific iPSCs

The development of patient‐specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot‐Marie‐Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post‐mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post‐mitotic neurons within 6‐8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post‐mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient‐derived iPSCs into post‐mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1‐CMT disease.     

Impact of melatonin receptor‐signaling on Zeitgeber time‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)‐mediated signaling. Adult melatonin‐proficient C3H/HeN mice and melatonin‐proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12‐h light, 12‐h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT‐dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin‐proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT‐dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24‐h light‐dark cycle. Apoptosis showed ZT‐dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT‐dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT‐dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2‐mediated signaling appears to be crucial for generation and timing of ZT‐dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.     

Lack of Oestrogenic Inhibition of the Nuclear Factor‐κB Pathway in Somatolactotroph Tumour Cells

Activation of nuclear factor (NF)‐κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)‐α by inhibiting NF‐κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF‐κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17β‐oestradiol (E₂) (10^?9 m ) increased the nuclear concentration of NF‐κB/p105, p65 and p50 in GH3 cells. However, E₂ did not modify the expression of Bcl‐xL, a NF‐κB target gene. TNF‐α induced apoptosis of GH3 cells incubated in either the presence or absence of E₂. Inhibition of the NF‐kB pathway using BAY 11‐7082 (BAY) (5 μm ) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)‐positive GH3 cells. BAY also increased TNF‐α‐induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c‐Jun N‐terminal protein kinase pathway, SP600125 (10 μm ). We also analysed the role of the NF‐κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL‐positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF‐κB pathway and that the pro‐apoptotic effect of TNF‐α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF‐κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF‐κB pathway could interfere with pituitary tumour progression.     

Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture

The actions and interactions of the neurotransmitter glutamate and the intracellular messengers calcium, cyclic AMP, and protein kinase C (PKC) in the regulation of neurite outgrowth and cell survival were examined in hippocampal pyramidal-like neurons in isolated cell culture. Low, subtoxic levels of glutamate (10-100 microM) caused the regression of dendrites but not axons; millimolar levels caused cell death. Calcium ionophore A23187 (50-100 nM) and the PKC activator phorbol-12-myristate-13-acetate (PMA; 10-50 nM) caused the regression of both axons and dendrites, whereas the adenylate cyclase activator forskolin enhanced outgrowth rates in both axons and dendrites. The effects of glutamate, A23187, PMA, and forskolin on outgrowth were mediated locally at the growth cones; dendrites were more sensitive than axons to each of these agents. High levels of A23187 (1 microM) or PMA (100 nM) significantly reduced cell survival. Co2+ and trifluoperazine each significantly reduced glutamate-induced dendritic regression and neurotoxicity suggesting that calcium influx and/or PKC activation mediated glutamate's actions. Fura-2 measurements showed that glutamate caused a rapid rise in intracellular calcium levels; this rise was prevented by Co2+. PMA and forskolin did not alter intracellular calcium levels, nor did these agents affect glutamate-induced calcium rises. Taken together, the results indicate that parallel intracellular messenger pathways that influence neurite outgrowth and cell survival are operative in hippocampal neurons; these messengers may play roles in the formation and modification of neuronal circuitry.     

Cellular and subcellular localization of estrogen and progestin receptor immunoreactivities in the mouse hippocampus

Estrogen receptor‐α (ERα), estrogen receptor‐β (ERβ), and progestin receptor (PR) immunoreactivities are localized to extranuclear sites in the rat hippocampal formation. Because rats and mice respond differently to estradiol treatment at a cellular level, the present study examined the distribution of ovarian hormone receptors in the dorsal hippocampal formation of mice. For this, antibodies to ERα, ERβ, and PR were localized by light and electron immunomicroscopy in male and female mice across the estrous cycle. Light microscopic examination of the mouse hippocampal formation showed sparse nuclear ERα and PR immunoreactivity (‐ir) most prominently in the CA1 region and diffuse ERβ‐ir primarily in the CA1 pyramidal cell layer as well as in a few interneurons. Ultrastructural analysis additionally revealed discrete extranuclear ERα‐, ERβ‐, and PR‐ir in neuronal and glial profiles throughout the hippocampal formation. Although extranuclear profiles were detected in all animal groups examined, the amount and types of profiles varied with sex and estrous cycle phase. ERα‐ir was highest in diestrus females, particularly in dendritic spines, axons, and glia. Similarly, ERβ‐ir was highest in estrus and diestrus females, mainly in dendritic spines and glia. Conversely, PR‐ir was highest during proestrus, mostly in axons. Except for very low levels of extranuclear ERβ‐ir in mossy fiber terminals in mice, the labeling patterns in the mice for all three antibodies were similar to the ultrastructural labeling found previously in rats, suggesting that regulation of these receptors is well conserved across the two species. J. Comp. Neurol. 518:2729–2743, 2010. © 2010 Wiley‐Liss, Inc.     

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)

Cumulative evidence indicates that aberrant re‐expression of many cell cycle‐related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer’s disease (AD) pathogenesis. Evidence of cell cycle activation in post‐mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway‐specific RT²Profiler? PCR Arrays, we detected changes in a number of cell cycle‐related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5′‐bromo‐2′‐deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin‐dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle‐related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.     

Inhibition of protein kinase C promotes differentiation of neuroblastoma × glioma NG108-15 hybrid cells

Differentiation of neuroblastoma × glioma NG108‐15 hybrid cells can be induced by different means, but the mechanisms involved are unclear. Our aim was to characterize the role of protein kinase C (PKC) in this process. The PKCs present in NG108‐15 cells, i.e. PKCα, PKCδ, PKCε and PKCζ, were inhibited using a cocktail of Go6983 and Ro318220 or were downregulated by treatment with phorbol 12‐myristate 13‐acetate (PMA). In high‐glucose Dulbecco’s modified Eagle medium, neuritogenesis was induced by 24 h treatment with a cocktail of Go6983 and Ro318220 or by 48 h treatment with PMA, the latter process thus requiring a longer treatment. However, when cells treated with PMA for only 24 h were placed in extracellular standard salts solution, e.g. Locke’s buffer, for 3 h, morphological and functional differentiation occurred, with rounding of the cell body, actin polymerization subjacent to the plasma membrane and an increase in voltage‐sensitive Ca²⁺ channel activity in the absence of cell death. This rapid differentiation was not due to autophagy, growth arrest or increased cyclic AMP response element binding protein phosphorylation, but coincided with combined activation of p38 mitogen‐activated protein kinase (MAPK) and inhibition of extracellular signal‐regulated kinase (ERK) and Akt, as confirmed by the effects of selective inhibitors. Furthermore, PKC activation blocked thapsigargin‐induced neuritogenesis, whereas PKC downregulation did not. These results show that PKC downregulation promotes differentiation and this effect is accelerated by exposure to Locke’s buffer. Although this experimental paradigm cannot be related to the in vivo situation and disease, it implies that combined inhibition of Akt and p44/p42 ERK and activation of p38 MAPK promotes differentiation.     

Orexin‐A increases the firing activity of hippocampal CA1 neurons through orexin‐1 receptors

Orexins including two peptides, orexin‐A and orexin‐B, are produced in the posterior lateral hypothalamus. Much evidence has indicated that central orexinergic systems play numerous functions including energy metabolism, feeding behavior, sleep/wakefulness, and neuroendocrine and sympathetic activation. Morphological studies have shown that the hippocampal CA1 regions receive orexinergic innervation originating from the hypothalamus. Positive orexin‐1 (OX₁) receptors are detected in the CA1 regions. Previous behavioral studies have shown that microinjection of OX₁ receptor antagonist into the hippocampus impairs acquisition and consolidation of spatial memory. However, up to now, little has been known about the direct electrophysiological effects of orexin‐A on hippocampal CA1 neurons. Employing multibarrel single‐unit extracellular recordings, the present study showed that micropressure administration of orexin‐A significantly increased the spontaneous firing rate from 2.96 ± 0.85 to 8.45 ± 1.86 Hz (P < 0.001) in 15 out of the 23 hippocampal CA1 neurons in male rats. Furthermore, application of the specific OX₁ receptor antagonist SB‐334867 alone significantly decreased the firing rate from 4.02 ± 1.08 to 2.11 ± 0.58 Hz in 7 out of the 17 neurons (P < 0.05), suggesting that endogenous orexinergic systems modulate the firing activity of CA1 neurons. Coapplication of SB‐334867 completely blocked orexin‐A–induced excitation of hippocampal CA1 neurons. The PLC pathway may be involved in activation of OX₁ receptor–induced excitation of CA1 neurons. Taken together, the present study's results suggest that orexin‐A produces excitatory effects on hippocampal neurons via OX₁ receptors. © 2016 Wiley Periodicals, Inc.     

Expression of cell division markers in the hippocampus in Alzheimer’s disease and other neurodegenerative conditions

Recent studies, showing that cell cycle-related nuclear proteins p105 and Ki-67 are associated with Alzheimer’s disease (AD)-related cytoskeletal pathology, suggested that these proteins, in addition to their functions in regulating the cell cycle, may have more specialised functions in the adult nervous system. In order to test this hypothesis we studied the expression of the cell cycle-related proteins Ki-67, pCNA and p53 in the hippocampi of 33 subjects, including some with AD or other neurodegenerative disorders and some with no neurological disease. By immunohistochemistry we found nuclear expression of Ki-67 in all subregions of the hippocampus, with the highest levels in the dentate gyrus. Both neurons and glial cells expressed this protein. The proportion of cells positive for Ki-67 and the distribution pattern varied considerably depending on the pathological diagnosis. Neuronal nuclear expression of Ki-67 was increased in AD but was also elevated in young Down’s syndrome subjects and in those with Pick’s disease. Expression of this protein was therefore not AD-specific. We did not find nuclear pCNA or p53 expressed in our patient groups. Contrary to previous studies AD-type neurofibrillary tangles were not labelled with any of the cell cycle markers used. The presence of nuclear Ki-67 expression indicates that some hippocampal neurons are not in the quiescent G₀ phase but have re-entered the cell cycle. The absence of nuclear pCNA or p53 suggests that the cycle is arrested in G₁. The significance of our findings and their relationship to the production of neurodegenerative cell death via an apoptotic mechanism are discussed. Received: 10 June 1996 / Revised, accepted: 31 July 1996     

The Role of Zinc in the Modulation of Neuronal Proliferation and Apoptosis

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G₀/G₁ phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.     

The theta‐related firing activity of parvalbumin‐positive neurons in the medial septum‐diagonal band of broca complex and their response to 5‐HT1A Receptor stimulation in a rat model of Parkinson's disease

The parvalbumin (PV)‐positive neurons in the medial septum‐diagonal band of Broca complex (MS‐DB) play an important role in the generation of hippocampal theta rhythm involved in cognitive functions. These neurons in this region express a high density of 5‐HT_1A receptors which regulate the neuronal activity and consequently affect the theta rhythm. In this study, we examined changes in the theta‐related firing activity of PV‐positive neurons in the MS‐DB, their response to 5‐HT_1A receptor stimulation and the corresponding hippocampal theta rhythm, and the density of PV‐positive neurons and their co‐localization with 5‐HT_1A receptors in rats with 6‐hydroxydopamine lesions of the substantia nigra pars compacta (SNc). The lesion of the SNc decreased the rhythmically bursting activity of PV‐positive neurons and the peak frequency of hippocampal theta rhythm. Systemic administration of 5‐HT_1A receptor agonist 8‐OH‐DPAT (0.5–128 µg/kg, i.v.) inhibited the firing rate of PV‐positive neurons and disrupted rhythmically bursting activity of the neurons and the theta rhythm in sham‐operated and the lesioned rats, respectively. The cumulative doses producing inhibition and disruption in the lesioned rats were higher than that of sham‐operated rats. Furthermore, local application of 8‐OH‐DPAT (0.005 μg) in the MS‐DB also inhibited the firing rate of PV‐positive neurons and disrupted their rhythmically bursting activity in sham‐operated rats, while having no effect on PV‐positive neurons in the lesioned rats. The lesion of the SNc decreased the density of PV‐positive neurons in the MS‐DB, and percentage of PV‐positive neurons expressing 5‐HT_1A receptors. These results indicate that the lesion of the SNc leads to suppression of PV‐positive neurons in the MS‐DB and hippocampal theta rhythm. Furthermore, the lesion decreases the response of these neurons to 5‐HT_1A receptor stimulation, which attributes to dysfunction and/or down‐regulation of 5‐HT_1A receptor expression on these neurons. These changes may be involved in cognitive impairments of Parkinson's disease. © 2013 Wiley Periodicals, Inc.     

Protein kinase CK2 enhances Mcl‐1 gene expression through the serum response factor‐mediated pathway in the rat hippocampus

The protein kinase CK2 (casein kinase 2) is a ubiquitous serine/threonine protein kinase that suppresses apoptosis. CK2 is composed of catalytic and regulatory subunits, and CK2‐dependent phosphorylation is a global mechanism in the inhibition of caspase signaling pathways. The serum response factor (SRF) is an important regulator of cell growth and differentiation. Although CK2 has been shown to phosphorylate SRF in vitro, the biological relevance of this interaction remains largely unclear. We observed increased SRF phosphorylation and increased Mcl‐1 gene expression in hippocampal CA1 neurons following transfection with a plasmid expressing the wild‐type CK2α (CK2αWT) protein, whereas transfection with a plasmid expressing a catalytically inactive mutant of CK2α (CK2α156A) reduced Mcl‐1 gene expression. Cotransfection with a plasmid expressing the inactive SRF99A mutant inhibited the CK2αWT‐induced upregulation of Mcl‐1 gene expression. The expression of either the CK2α156A or the SRF99A mutant also inhibited the glutamate‐induced upregulation of Mcl‐1 protein expression in PC12 cells. Our results suggest that CK2‐mediated signaling represents a cellular mechanism that may aid in the development of alternative therapeutic strategies to attenuate apoptosis in hippocampal neurons. © 2013 Wiley Periodicals, Inc.     

β‐Catenin expression in human neural cell lines following exposure to cytokines and growth factors

β‐Catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between β‐catenin and the mutant presenilin‐1 induces neuronal apoptosis, and that β‐catenin levels are decreased in the brains of patients with Alzheimer’s disease (AD). Since activated microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor‐regulated expression of β‐catenin in human neural cell lines, including NTera2 teratocarcinoma‐derived differentiated neurons (NTera2‐N), IMR‐32 neuroblastoma, SKN‐SH neuroblastoma and U‐373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain‐derived neurotrophic factor (BDNF), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, IL‐6, interferon (IFN)‐γ, transforming growth factor (TGF)‐β1, dibutyryl cyclic adenosine 3′,5′‐cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12‐myristate 13‐acetate (PMA). β‐Catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, β‐catenin protein levels were reduced markedly in NTera2‐N cells by exposure to dbcAMP, EGF or bFGF, and in U‐373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF‐α, IL‐1β, IL‐6, IFN‐γ or TGF‐β1. These results indicate that β‐catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type‐specific manner.     

Nuclear factor-kappaB-dependent cyclin D1 induction and DNA replication associated with N-methyl-D-aspartate receptor-mediated apoptosis in rat striatum

Cell cycle reentry has been found during apoptosis of postmitotic neurons under certain pathological conditions. To evaluate whether nuclear factor-kappaB (NF-kappaB) activation promotes cell cycle entry and neuronal apoptosis, we studied the relation among NF-kappaB-mediated cyclin induction, bromodeoxyuridine (BrdU) incorporation, and apoptosis initiation in rat striatal neurons following excitotoxic insult. Intrastriatally injected N-methyl-D-aspartate receptor agonist quinolinic acid (QA, 60 nmol) elicited a rise in cyclin D1 mRNA and protein levels (P<0.05). QA-induced NF-kappaB activation occurred in striatal neurons and nonneuronal cells and partially colocalized with elevated cyclin D1 immunoreactivity and TUNEL-positive nuclei. QA triggered DNA replication as evidenced by BrdU incorporation; some striatal BrdU-positive cells were identified as neurons by colocalization with NeuN. Blockade of NF-kappaB nuclear translocation with the recombinant peptide NF-kappaB SN50 attenuated the QA-induced elevation in cyclin D1 and BrdU incorporation. QA-induced internucleosomal DNA fragmentation was blunted by G(1)/S-phase cell cycle inhibitors. These findings suggest that NF-kappaB activation stimulates cyclin D1 expression and triggers DNA replication in striatal neurons. Excitotoxin-induced neuronal apoptosis may thus result from, at least partially, a failed cell cycle attempt.     

Protein kinase C stabilizes X‐linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser87 to suppress apoptotic cell death

Background: Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X‐linked inhibitor of apoptosis protein (XIAP). Methods: Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti‐XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH‐SY5Y cells treated with PKC activator, phorbol 12‐myristate 13‐acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide‐induced apoptosis. Results: Recombinant XIAP was phosphorylated at Ser⁸⁷ by PKC in vitro and treatment of XIAP‐transfected SH‐SY5Y cells with a PKC activator, phorbol 12‐myristate 13‐acetate (PMA) induced phosphorylation of XIAP at Ser⁸⁷. Pulse chase experiments revealed that, when phosphorylated at Ser⁸⁷, wild‐type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 µg/ml etoposide. Conclusion: The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser⁸⁷ and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.