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Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-β (Aβ) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's Aβ peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the γ-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation.

Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-β precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than the WW domain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and γ-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of α-secretase in regulating nuclear translocation.

Neuroblastoma is the most common solid extracranial cancer form in childhood with an etiology that is mostly unknown. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising future anticancer drug candidate, highly malignant neuroblastoma has been reported to acquire TRAIL resistance by mechanisms that are poorly understood. Here, we show by western blot analysis, and live cell imaging using anchored FRET sensors, that the resistance to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells depends on an incomplete processing of procaspase-3, generating an immature and catalytically inactive 21 kDa fragment. We have previously shown that the naturally occurring compound curcumin can sensitize SK-N-AS cells to TRAIL. In the present study, we show that curcumin also has a similar effect on human neuroblastoma SHEP1 cells. Furthermore, we show that curcumin and TRAIL co-treatment induces complete maturation and activation of caspase-3 in both cell lines. The mechanisms behind this effect seem to be dependent on protein kinase C (PKC), since inhibition of PKC using bisindolylmaleimide XI, could also sensitize these cells to TRAIL through a similar effect on caspase-3 activation. Moreover, TRAIL co-treatment with bisindolylmaleimide XI or curcumin resulted in down-regulation of X-linked inhibitor of apoptosis protein. In conclusion, our study shows that PKC can be involved in TRAIL resistance in human neuroblastoma cells by preventing caspase-3 maturation.

Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto–Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 μg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2–4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of cancer cell lines with almost no toxicity toward normal cells. However, many neuroblastoma cells acquire resistance to TRAIL by mechanisms that are poorly understood. The objective of this study was to investigate involvement of the transcription factor NF-κB in the resistance of human neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis. We used five compounds previously reported to inhibit NF-κB activity. SN50, curcumin, oridonin, and pyrrolidine dithiocarbamate (PDTC) all sensitized cells to TRAIL-induced apoptosis. In contrast, N-alpha-tosyl-L: -phenylalanyl chloromethyl ketone (TPCK) did not affect sensitivity to TRAIL, although reporter gene assay clearly showed inhibition of NF-κB activity. In addition, neither curcumin nor oridonin had any inhibitory effect on NF-κB activity at concentrations at which sensitization to TRAIL was observed. Instead, the free radical scavenger N-acetyl-L: -cysteine (NAC) completely blocked the effect on TRAIL-induced apoptosis caused by curcumin, oridonin, and PDTC. Furthermore, exposure of SK-N-AS cells to H(2)O(2) could mimic the TRAIL-sensitizing effect of other agents. In conclusion, our results suggest that sensitization of neuroblastoma SK-N-AS cells to TRAIL-induced apoptosis is correlated with induction of reactive oxygen species (ROS) rather than inhibition of NF-κB.

The Alzheimer's amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.

Members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors have been reported to be up-regulated in Alzheimer's disease. In the present study, we have investigated the ef ...

The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.

The proinflammatory cytokine IL-1, acting via the hypothalamic IL-1 receptor type 1 (IL-1RI), activates pathways known to suppress bone formation such as the hypothalamo pituitary-adrenocortical axis and the sympathetic nervous system. In addition, peripheral IL-1 has been implicated as a mediator of the bone loss induced by sex hormone depletion and TNF. Here, we report an unexpected low bone mass (LBM) phenotype, including impairment of bone growth, in IL-1RI-deficient mice (IL-1rKO mice). Targeted overexpression of human IL-1 receptor antagonist to the central nervous system using the murine glial fibrillary acidic protein promoter (IL-1raTG mice) resulted in a similar phenotype, implying that central IL-1RI silencing is the causative process in the LBM induction. Analysis of bone remodeling indicates that the process leading to the LBM in both IL-1rKO and IL-1raTG is characterized mainly by doubling the osteoclast number. Either genetic modification does not decrease testosterone or increase corticosterone serum levels, suggesting that systems other than the gonads and hypothalamo pituitary-adrenocortical axis mediate the central IL-1RI effect on bone. We further demonstrate that WT mice express mouse IL-1ra in bone but not in the hypothalamus. Because low levels of IL-1 are present in both tissues, it is suggested that skeletal IL-1 activity is normally suppressed, whereas central IL-1 produces a constant physiologic stimulation of IL-1RI signaling. Although the pathway connecting the central IL-1RI signaling to bone remodeling remains unknown, the outburst of osteoclastogenesis in its absence suggests that normally it controls bone growth and mass by tonically restraining bone resorption.

The activation of nuclear factor kappaB (NFkappaB) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NFkappaB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFkappaB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the kappaB site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide-PNA construct. The ability of the transport peptide-PNA-NFkappaB decoy complex to block the effect of interleukin (IL)-1beta-induced NFkappaB activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide-PNA-NFkappaB decoy (1 microM, 1 h) blocked IL-1beta-induced NFkappaB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFkappaB decoy in the absence of transport peptide-PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFkappaB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.

Interleukin-1 receptor antagonist (IL-1ra) has been shown previously to have neuroprotective effects in animal models of stroke. The effects of chronic overexpression of human soluble IL-1ra (hsIL-1ra) were studied in a mouse model of permanent focal cerebral ischemia. A transgenic mouse strain (Tg hsIL-1ra+/-) has been developed using the promoter for glial fibrillary acidic protein (GFAP) to limit the overexpression to the CNS. Analysis of the neurological scores, infarct volume and edema formation revealed no differences between Tg hsIL-1ra+/- and wild-type (WT) mice. The cerebral ischemia resulted in pronounced astrocyte proliferation and microglial activation, as well as induction of inflammatory markers in both Tg hsIL-1ra+/- and WT mice, with no major differences between the two genotypes. Interestingly, hsIL-1ra expression in astrocytes was reduced in infarcted areas as compared to non-ischemic regions and sham-operated controls. In conclusion, transgenic overexpression of hsIL1-ra was not neuroprotective in this cerebral ischemia model, possibly due to insufficient levels for protection against the extensive lesion, or an up-regulation of compensatory inflammatory signals due to the lifetime blockade of IL-1 receptors.

Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL-1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage.

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimer's disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.

A novel non-invasive marker to monitor apoptosis in the living cell is presented. A human neuroblastoma cell line expressing an integral nuclear pore membrane protein tagged with GFP (green fluorescent protein) has been established, which enables monitoring of the nuclear envelope dynamics in intact cell cultures by fluorescence microscopy. During apoptosis, but not during necrosis, the GFP fluorescence around the nuclear rim disappears at a stage preceding nucleosomal DNA fragmentation. This phenomenon can thus be used as an early and convenient marker to specifically diagnose apoptotic development in cell cultures.

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μM p -benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.

beta-Amyloid precursor protein (APP) belongs to a family of homologous beta-amyloid precursor-like proteins (APLPs) including APLP1 and APLP2. Previously it has been shown that APP is subject to regulation by retinoic acid (RA). In this paper we show that APLP1 and APLP2 mRNA expression is upregulated during RA-induced differentiation of human SH-SY5Y neuroblastoma cells. The cells were treated with RA (10 microM) for 3 and 6 days and mRNA levels were analysed by a non-radioactive Northern blot assay. RA induced a 2- to 3-fold increase in the gene expression of both APLP2 and APP, whereas the increase in APLP1 mRNA expression was significantly higher. Our results support a role for APLPs during neuronal differentiation.

Background Fe65 is a ~97kDa neuronal adaptor protein which has been shown to interact with amyloid-b precursor protein (APP) and its homologues APLP1 and APLP2. Fe65 has the ability to bind to the C-terminal YENPTY motif of APP and is believed to be involved in the regulation of APP processing. APP is a type 1-transmembrane protein which can undergo regulated intramembrane proteolysis (RIP) allowing the release of a secreted ectodomain (sAPP) and a small intracellular domain called AICD. AICD has been suggested to function in regulation of gene expression by the formation of a multimeric complex with Fe65. According to previous studies retinoic acid (RA) can up-regulate the expression of both mRNA and protein levels of APP family members in SH-SY5Y cells. Because Fe65 may be essential in APP-mediated transcriptional regulation, the extent of Fe65 protein expression and its localization were investigated in RA differentiated SH-SY5Y cells. Phosphorylation of APP at specific residues has also shown to affect the processing of APP and its binding to specific adaptor proteins, such as Fe65. To further characterize how phosphorylation affects interactions between Fe65 and APP, Fe65 constructs were designed with a tandem affinity purificationtag (TAP-tag) and several Ser/Thr/ Tyr mutant constructs of myc-tagged APP was generated. APP mutants were coexpressed in SH-SY5Y cells with Fe65-TAP in order to investigate if the mutated phosphorylation sites affect the ability of Fe65 to interact with APP or if the gain/loss of interaction with Fe65 affects the processing of APP.

Elevated levels of free fatty acids (FFAs) in plasma and increased incidence of chronic systemic inflammation are associated with obesity. In the brain, activated microglia are believed to play different roles during inflammation that may either be neuroprotective or promote neurodegeneration. Here, we have investigated the effects of FFAs on microglial response to inflammatory stimuli. Our results indicate that the saturated FFA palmitate on its own induces alternative activation of BV-2 microglia cells. Further, pre-exposure to palmitate changed the response of microglia to lipopolysaccharide (LPS). We show that palmitate affects the mRNA levels of the pro-inflammatory cytokines interleukin-1β and interleukin-6. The transcription factor CCAAT/enhancer-binding protein δ is also affected by pre-exposure to palmitate. Furthermore, the phagocytic activity of microglia was investigated using fluorescent beads. By analyzing the bead uptake by fluorescence-activated cell sorting, we found that palmitate alone, as well as together with LPS, stimulated the phagocytic activity of microglia. Taken together, our results suggest that exposure of microglia to increased levels of free fatty acids may alter the consequences of classical inflammatory stimuli.

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

Background Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-β peptide as a causative agent in AD. Results Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-β peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed. Conclusions Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-β peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

Here, we show that O-GlcNAcase and O-GlcNAc transferase regulate the level of APP that is immunoprecipitated with O-GlcNAc antibody in human neuroblastoma SH-SY5Y cells. We also show that O-GlcNAcylation increases a-secretase processing, resulting in increased levels of the neuroprotective sAPPa fragment and decreased Ab secretion. The proteolytic processing of the APP homologue, APLP2, remained unchanged in response to increased O-GlcNAcylation. Furthermore, the effect of O-GlcNacylation on APP processing seems to be specific for neuron-like cells, since the levels of sAPPa from the human embryonic kidney cell-line HEK293 remains unchanged in response to inhibition of O-GlcNAcase, whereas the neuroblastoma cell-line SK-N-AS show increased sAPPa levels, but not to the same extent as in SH-SY5Y cells.

Background The transcription factors CCAAT/enhancer binding proteins (C/EBP) α, β and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB). In general, C/EBPα is down-regulated, whereas both C/EBPβ and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-β (Aβ) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aβ deposits with the aim of defining new therapeutic targets. Methods Here we have investigated the effects of Aβ on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aβ deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay. Results We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aβ inhibit up-regulation of C/EBPδ expression in interleukin-1β-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPβ was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aβ deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1β-induced C/EBPδ DNA binding activity to both C/EBP and κB sites is abolished after exposure to Aβ. Conclusions These data suggest that both expression and function of C/EBPδ are dysregulated in Alzheimer's disease. C/EBPδ seems to be differently regulated in response to different conformations of Aβ. We propose that Aβ induces an imbalance between NF-κB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.

Background: Apoptosis is an evolutionary conserved cellular process important for normal development, maintenance of tissue homeostasis and an effective immune system. Cysteine-aspartic proteases, ...

Members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors have been reported to be up-regulated in Alzheimer's disease. In the present study, we have investigated the effects of amyloid-beta (Abeta) peptides on C/EBPbeta and C/EBPdelta, previously shown to be induced by inflammatory stimuli in glial cells. Surprisingly, electrophoretic mobility shift assay showed that both Abeta(1-42) and Abeta(25-35) blocked C/EBP activation induced by the inflammatory cytokine interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS) in mixed primary glial cell cultures from rat. Abeta also blocked IL-1beta- or LPS-induced C/EBP protein levels. The most prominent effects were observed on DNA binding activity and protein levels of C/EBPdelta. Our results demonstrate a dysregulation of C/EBP when glial cells are activated in the presence of Alzheimer Abeta peptides.

Substance P (SP) and neurokinin A (NKA), two coexisting neuropeptides of the tachykinin family, stimulated the basal (5 mM K+) and evoked (40 mM K+) release of [3H]5-HT (5-hydroxytryptamine) from tissue slices of the rat cerebral cortex. Spantide ([DArg1,-DTrp7,9,Leu11]SP; 10(-5) M) inhibited the effects of SP but potentiated the effects of NKA. The effects of SP and NKA appear to be exerted at distinct receptors but involve a common post-receptor mechanism as no full additivity of the SP- and NKA-mediated stimulation of [3H]5-HT could be observed. The effects of the 3 tachykinins, SP, NKA and NKB, are compared with respect to stimulation of the release of [3H]5-HT.

Retinoic acid stimulates alpha-secretase processing of amyloid precursor protein (APP) and decreases beta-secretase cleavage that leads to amyloid-beta formation. Here, we investigated the effect of retinoic acid on the two putative alpha-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the beta-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of alpha-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.

Background: Neuronal and synaptic loss can be observed in several neurologic disorders, like Alzheimer’s disease (AD). The mechanism behind cell death in AD has been intensively studied and apoptos ...

Background: Accumulating evidence supports the importance of inflammation in neurodegenerative disorders like Alzheimer’s disease (AD). Epidemiological studies have revealed that patients taking an ...

Inhibition of nuclear factor (NF)-kappaB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer's disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-kappaB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer beta-amyloid peptide in the presence of the inflammatory cytokine interleukin (IL)-1beta. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-kappaB binding activity and IL-6 mRNA expression, respectively.

An increasing body of evidence suggests that beta-amyloid (Abeta) and activated glial cells play a crucial part in the pathogenesis of Alzheimer's disease (AD). Activated glial cells surrounding the senile plaques, formed by Abeta peptides, have been proposed to promote neurodegeneration by producing putatively toxic factors, including the inflammatory cytokine interleukin-1beta (IL-1beta). Elevated levels of both IL-1beta and activated nuclear factor kappaB (NF-kappaB), a key transcription factor regulating a wide variety of inflammatory genes, have been found in the brains of AD patients. In this study, we have investigated the ability of the Abeta(25-35) peptide and IL-1beta, either alone or together, in activating NF-kappaB in glial cells. Mixed primary glial cells from rat were treated with IL-1beta and/or Abeta(25-35), and NF-kappaB binding activity was analyzed by electophoretic mobility shift assay. We observed that the induction of NF-kappaB binding activity induced by either IL-1beta or Abeta(25-35) showed a peak at 30 min, and significantly declined after 2 h. The induced NF-kappaB activation persisted after 24 h and even seemed to increase in cells treated with Abeta(25-35). The activation of NF-kappaB by Abeta(25-35) was shown to be dose-dependent. In addition, Abeta(25-35) potentiated the effect of IL-1beta in a dose-dependent manner when co-stimulating the cells. The potentiating effect of Abeta(25-35) on IL-1beta-induced NF-kappaB binding activity was observed after 30 min, 2 h and 24 h, and did not significantly differ over time. A possible explanation is that when glial cells are stimulated by inflammatory factors in the presence of Abeta peptides or senile plaques, the NF-kappaB negative feedback regulation is no longer functional.

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.

During apoptosis (also called programmed cell death), the chromatin condenses and the DNA is cleaved into oligonucleosomal fragments. Caspases are believed to play a major role in nuclear apoptosis. However, the relation between dismantling of nuclear pores, disruption of the nucleocytoplasmic barrier, and nuclear entry of caspases is unclear. We have analyzed nuclear import of the green fluorescent protein fused to a nuclear localization signal (GFP-NLS) in tissue culture cells undergoing apoptosis. Decreased nuclear accumulation of GFP-NLS could be detected at the onset of nuclear apoptosis manifested as dramatic condensation and redistribution of chromatin toward the nuclear periphery. At this step, dismantling of nuclear pores was already evident as indicated by proteolysis of the nuclear pore membrane protein POM121. Thus, disruption of nuclear compartmentalization correlated with early signs of nuclear pore damage. Both these events clearly preceded massive DNA fragmentation, detected by TUNEL assay. Furthermore, we show that in apoptotic cells, POM121 is specifically cleaved at aspartate-531 in its large C-terminal portion by a caspase-3-dependent mechanism. Cleavage of the C-terminal portion of POM121, which is adjoining the nuclear pore complex, is likely to disrupt interactions with other nuclear pore proteins affecting the stability of the pore complex. A temporal correlation of apoptotic events supports a model where caspase-dependent disassembly of nuclear pores and disruption of the nucleocytoplasmic barrier paves the way for nuclear entry of caspases and subsequent activation of CAD-mediated DNA fragmentation.

The cytokine interleukin-1 (IL-1) is produced by peripheral immune cells as well as glia and neurons within the brain; it plays a major role in immune to brain communication and in modulation of neural, neuroendocrine, and behavioral systems during illness. Although previous studies demonstrated that excess levels of IL-1 impaired memory processes and neural plasticity, it has been suggested that physiological levels of IL-1 are involved in hippocampal-dependent memory and long-term potentiation (LTP). To examine this hypothesis, we studied IL-1 receptor type I knockout (IL-1rKO) mice in several paradigms of memory function and hippocampal plasticity. In the spatial version of the water maze test, IL-1rKO mice displayed significantly longer latency to reach a hidden platform, compared with wild-type controls. Furthermore, IL-1rKO exhibited diminished contextual fear conditioning. In contrast, IL-1rKO mice were similar to control animals in hippocampal-independent memory tasks; i.e., their performance in the visually guided task of the water maze and the auditory-cued fear conditioning was normal. Electrophysiologically, anesthetized IL-1rKO mice exhibited enhanced paired-pulse inhibition in response to perforant path stimulation and no LTP in the dentate gyrus. In vitro, decreased paired-pulse responses, as well as a complete absence of LTP, were observed in the CA1 region of hippocampal slices taken from IL-1rKO mice compared with WT controls. These results suggest that IL-1 contributes to the regulation of memory processes as well as short- and long-term plasticity within the hippocampus. These findings have important implications to several conditions in humans, which are associated with long-term defects in IL-1 signaling, such as mutations in the IL-1 receptor accessory protein-like gene, which are involved in a frequent form of X-linked mental retardation.

To examine the role of IL-1 in the regulation of the hypothalamo-pituitary-adrenal (HPA) axis, mice with knockout of the IL-1 receptor type I (IL-1rKO) were exposed to psychological and metabolic stressors. When exposed to mild stressors (auditory stress or a low dose of 2-deoxyglucose), IL-1rKO mice displayed a significantly diminished corticosterone secretion, compared with wild-type (WT) controls. In response to more severe stressors (60-min restraint or a high dose of 2-deoxyglucose), both groups exhibited a similar increase in corticosterone secretion. To examine the role of IL-1 in HPA axis feedback regulation, serum ACTH levels were measured after adrenalectomy (ADX) in IL-1rKO mice and in mice with transgenic overexpression of IL-1 receptor antagonist within the brain (IL-1raTG). As expected, WT controls exhibited ADXinduced ACTH hypersecretion, whereas IL-1rKO and IL1raTG mice showed no increase in ACTH levels, suggesting that brain IL-1 has a critical role in ADX-associated ACTH hypersecretion. Similarly, WT mice that were chronically exposed to IL-1ra in utero displayed a diminished ADX-induced ACTH hypersecretion, compared with vehicle-treated controls, suggesting a developmental role of IL-1 in HPA axis regulation. In conclusion, our results suggest that endogenous IL-1 plays a critical role in HPA axis activation after stress and ADX. (Endocrinology 144: 4453– 4458, 2003)

The cytokine interleukin-1 (IL-1) has been implicated in modulation of pain perception under various inflammatory conditions. The present study examined the hypothesis that IL-1 signaling is also involved in pain sensitivity under normal, non-inflammatory states, using three mouse models of impaired IL-1 signaling: targeted deletion of the IL-1 receptor type I or the IL-1 receptor accessory protein, and transgenic over-expression of IL-1 receptor antagonist within the brain and spinal cord. Thermal and mechanical pain sensitivity was assessed using the paw-flick, hot-plate, and von Frey tests. All mutant strains displayed significantly lower pain sensitivity, compared with their respective wild-type control strains, and with their parent strains (C57BL/6, CBA and 129), in all tests. In contrast, mice with targeted deletion of the p55 or p75 TNF receptor, or of interleukin-18, displayed normal or higher pain sensitivity compared to their respective controls. To differentiate between developmental vs. on-going effects of IL-1, mice were chronically treated with IL-1 receptor antagonist (IL-1ra) via osmotic micropumps, either in adulthood or prenatally (throughout the last 2 weeks of gestation). Adult mice that were treated with IL-1ra either in adulthood or in utero, displayed lower pain sensitivity, similar to mice with impaired IL-1 signaling. These findings suggest that basal pain sensitivity is genetically, developmentally and tonically influenced by IL-1 signaling.

The effects of chronic exposure to excitatory amino acids (EAAs) were examined in cultured cerebellar granule cells (CGCs) from wild type (WT) and interleukin-1 receptor type I (IL-1RI)-deficient mice. After 8 days in culture, the cells were exposed to 100 microM glutamate or 300 microM N-methyl-D-aspartate (NMDA) for 24 h. Analysis of cell viability, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and phase-contrast microscopy revealed that CGCs from IL-1RI-deficient mice were more vulnerable to EAAs as compared to the WT controls. The results indicate that IL-1RI signalling is important for neuronal survival. The effect of glutamate on the CGCs from IL-1RI-deficient mice was decreased by the non-competitive NMDA-receptor antagonist MK-801, supporting the involvement of NMDA receptors in the glutamate-induced excitotoxicity. J Neuroimmunol

The acute inflammatory response following traumatic brain injury (TBI) has been shown to play an important role in the development of secondary tissue damage. The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFalpha), are induced early after brain injury and have been implicated in the delayed damage. The IL-1 receptor antagonist (IL-1ra) has been shown to modulate the proinflammatory cytokine cascade by blocking the binding of IL-1 to its signaling receptor. In this study, we investigated the effect of transgenic overexpression of IL-1ra on the cytokine expression and neurological damage in a closed head injury (CHI) model of TBI. The neurological recovery, as analyzed by neurological severity score (NSS), was significantly higher in transgenic mice overexpressing the human secreted form of IL-1ra in astrocytes, directed by the murine glial fibrillary acidic protein promoter, as compared to wild-type mice. Analysis of tissue levels of cytokines by ELISA showed increased levels of TNFalpha in the cerebral cortex from the wild type mice 1 h after injury. After 4 h significant increases in the levels of IL-1beta and IL-6 were observed in the wild type mice. In the transgenic mice, on the other hand, no effect on TNFalpha levels was observed and no significant increases in IL-1beta and IL-6 levels could be detected until 6 h after injury. Thus, it can be concluded that blockage of IL-1 signaling by elevated levels of IL-1ra has a neuroprotective effect, in agreement with previous reports, and that central overexpression of IL-1ra results in delayed proinflammatory cytokine induction and improved neurological recovery after traumatic brain injury.

Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation.The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated.The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.

The main constituent of the Alzheimer amyloid plaques is the amyloid beta (Abeta) peptide shown to activate glial cells in vitro. Activated glial cells are believed to contribute to neurotoxicity through production of inflammatory cytokines, such as interleukin-1 (IL-1), chemokines and neurotoxic substances. The IL-1 system has been proposed to play a role in neurodegenerative processes and can in turn induce expression of other cytokines such as IL-6. Recently, association of IL-1 and IL-6 gene polymorphism with Alzheimer's disease was reported, suggesting that these cytokines may play an important role in the development of the disease. In this study, rat primary mixed glial cells were treated with IL-1beta, Abeta(1-42) or Abeta(25-35). As expected the different treatments all resulted in activation of the transcription factor NFkappaB observed by electrophoretic mobility shift assay. Significant increases in IL-1beta and IL-6 mRNA levels, as analysed by reverse transcriptase-polymerase chain reaction (RT-PCR), were detected after the different treatments. In addition, increased secretion of IL-6 was detected by ELISA after 96 h exposure in response to IL-1beta, Abeta(1-42) or Abeta(25-35). When cells were exposed to both IL-1beta and Abeta(25-35) additive effects were observed. This supports that the effect of Abeta can be potentiated by concurrent exposure to inflammatory cytokines and that the IL-1 system is not necessary for Abeta effects on IL-6 expression in agreement with previous studies.

IL-1β and its endogenous receptor antagonist (IL-1Ra) are rapidly induced by seizures in the rodent hippocampus. Exogenously applied IL-1β prolongs seizures in an IL-1R type I-mediated manner. This effect depends onN-methyl-d-aspartate receptor activation. We report here that intrahippocampal application of recombinant IL-1Ra or its selective endogenous overexpression in astrocytes under the control of glial acidic fibrillary protein promoter potently inhibits motor and electroencephalographic seizures induced by bicuculline methiodide in mice. Accordingly, transgenic mice show a reduced seizure-relatedc-fosmRNA expression in various forebrain areas compared with their wild-type littermates. Recombinant IL-1Ra was ineffective in mice deficient in IL-1R type I, havingper sea delayed onset to generalized convulsions. These results demonstrate that IL-1Ra mediates potent anticonvulsant effects acting on IL-1R type I and suggest that the balance between brain IL-1β and IL-1Ra represents a crucial mechanism to control seizure generalization.

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels.

betaA25-35, a neurotoxic fragment of the Alzheimer beta-amyloid peptide (betaA), acts as a strong inducer of pro-inflammatory cytokines, such as interleukin (IL)-1 and IL-6, in glial cells. Since IL-1 is known to induce expression of both IL-1 and IL-6, we have investigated to what extent the induction of IL-1alpha and IL-6 by betaA25-35, is dependent on the IL-1 receptor type I (IL-1RI), the only known signalling IL-1 receptor. Primary astroglial cell cultures prepared from wild-type and IL-1RI-deficient mice were incubated in the presence of betaA25-35 (100 microM) for 19 h, followed by analysis of mRNA levels of IL-1alpha and IL-6. Cell cultures treated with betaA25-35 showed a significant increase in mRNA levels for IL-1alpha and IL-6 and in addition increased levels of IL-1alpha immunoreactivity. A supersensitive IL-1alpha response was observed in astroglial cell cultures lacking the IL-1 RI as compared to betaA25-35 treated cell cultures from wild-type mice. In contrast the betaA25-35-induced increase of IL-6 was lower in the absence of IL-1RI. In conclusion, these results suggest that a functional IL-1 signal transduction is not necessary for induction of mRNA levels of IL-1alpha and IL-6 in astroglial cell cultures treated with betaA25-35, but that induction of IL-6 involves at least two distinct mechanisms, one of which occurs via activation of the IL-1RI.

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 microM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate alpha-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na+, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na+ by Li+ in the perfusion buffer. We conclude that the co-transport of Na+, in the Na(+)-dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na+/K(+)-ATPases.

INTRODUCTION. Previously it was reported that during apoptosis the GFP (green fluorescent protein) fluorescence disappeared from a neuroblastoma cell line overexpressing the integral nuclear pore membrane protein POM121 tagged with GFP (1). The Alzheimerassociated peptide beta-amyloid, which forms senile plaques in the brain of Alzheimer patients, has been shown to have neurotoxic properties and to induce apoptosis. In this study we have used human SH-SY5Y neuroblastoma cells transfected with POM121-GFP to investigate the proapoptotic properties of beta-amyloid peptides. METHOD. Transfected SH-SY5Y cells (1), grown on cover slips, were treated with 1 µM retinoic acid (RA) in serum-free medium for 3 days. The cover slips were mounted on a chamber containing beta-amyloid peptide in serum-free medium and the time-lapse experiments were immediately started. GFP fluorescence was analyzed by fluorescence microscopy in parallel with phase contrast microscopy analysis of the morphological appearance. RESULTS. The transfected neuroblastoma cells were differentiated into post mitotic cells in the presence of RA, which also induced neurite outgrowth. Morphological effects on the cells were evident already within one hour after exposure to the beta-amyloid (25-35) peptide (25 µM) and the fluorescence was almost completely abolished within 4 hours (see Fig. 1). The results indicate that the transfected neuroblastoma cells are as sensitive as primary cultures of neuronal cells to induction of apoptosis by beta-amyloid peptides, and that monitoring the disappearance of the nuclear rim fluorescence is a sensitive method to detect apoptosis within hours after exposure.