Interferon-γ increases neuronal death in response to amyloid-β1-42
© Bate et al; licensee BioMed Central Ltd. 2006
Received: 31 January 2006
Accepted: 28 March 2006
Published: 28 March 2006
Alzheimer's disease is a neurodegenerative disorder characterized by a progressive cognitive impairment, the consequence of neuronal dysfunction and ultimately the death of neurons. The amyloid hypothesis proposes that neuronal damage results from the accumulation of insoluble, hydrophobic, fibrillar peptides such as amyloid-β1-42. These peptides activate enzymes resulting in a cascade of second messengers including prostaglandins and platelet-activating factor. Apoptosis of neurons is thought to follow as a consequence of the uncontrolled release of second messengers. Biochemical, histopathological and genetic studies suggest that pro-inflammatory cytokines play a role in neurodegeneration during Alzheimer's disease. In the current study we examined the effects of interferon (IFN)-γ, tumour necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 on neurons.
Primary murine cortical or cerebellar neurons, or human SH-SY5Y neuroblastoma cells, were grown in vitro. Neurons were treated with cytokines prior to incubation with different neuronal insults. Cell survival, caspase-3 activity (a measure of apoptosis) and prostaglandin production were measured. Immunoblots were used to determine the effects of cytokines on the levels of cytoplasmic phospholipase A2 or phospholipase C γ-1.
While none of the cytokines tested were directly neurotoxic, pre-treatment with IFN-γ sensitised neurons to the toxic effects of amyloid-β1-42 or HuPrP82-146 (a neurotoxic peptide found in prion diseases). The effects of IFN-γ were seen on cortical and cerebellar neurons, and on SH-SY5Y neuroblastoma cells. However, pre-treatment with IFN-γ did not affect the sensitivity to neurons treated with staurosporine or hydrogen peroxide. Pre-treatment with IFN-γ increased the levels of cytoplasmic phospholipase A2 in SH-SY5Y cells and increased prostaglandin E2 production in response to amyloid-β1-42.
Treatment of neuronal cells with IFN-γ increased neuronal death in response to amyloid-β1-42 or HuPrP82-146. IFN-γ increased the levels of cytoplasmic phospholipase A2 in cultured neuronal cells and increased expression of cytoplasmic phospholipase A2 was associated with increased production of prostaglandin E2 in response to amyloid-β1-42 or HuPrP82-146. Such observations suggest that IFN-γ produced within the brain may increase neuronal loss in Alzheimer's disease.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment as a consequence of neuronal dysfunction and loss. The amyloid hypothesis maintains that the neuronal dysfunction and death that give rise to the clinical symptoms of AD are caused by the accumulation of fibrils consisting of amyloid-β peptides . These peptides are formed following the cleavage of the amyloid precursor protein by γ-secretases , and depositions of amyloid-β peptides are a component of the senile plaques found in diseased brains . The neuronal loss that occurs in AD has been modelled in vitro by incubating neurons with specific peptides derived from the amyloid-β protein . The neuronal injury induced by these peptides includes characteristics of apoptosis such as chromatin condensation and DNA fragmentation .
In AD, amyloid deposits containing fibrillar amyloid-β peptides frequently co-localise with inflammatory cells strongly suggesting that the deposits of amyloid-β stimulate a chronic inflammatory process . Genetic studies have identified polymorphisms in the genes of some inflammatory cytokines as risk factors for AD  suggesting that cytokine production within the brain may influence neuropathogenesis. While the effects of cytokines on astroglial cells within the brain are well reported, less is known about the direct effects of individual cytokines on neurons. In the current study we report that pre-treatment with interferon (IFN)-γ significantly increased the sensitivity of neurons to the toxic effects of amyloid-β1-42. The increased sensitivity of IFN-γ treated neurons to amyloid-β1-42 correlated with increased expression of cytoplasmic phospholipase A2 (cPLA2) in neuroblastoma cells and increased prostaglandin production in response to exogenous amyloid-β1-42. These results are consistent with prior observations that uncontrolled activation the cPLA2/cyclo-oxygenase (COX) pathway by amyloid-β1-42 leads to neuronal death .
The human neuroblastoma cell line SH-SY5Y was grown in RPMI-1640 medium supplemented with 2 mM glutamine, standard antibiotics (100 U/ml Penicillin, 100 μg/ml Streptomycin) and 2% fetal calf serum (FCS). For toxicity studies cells were seeded at 3 × 104 cells per well in 48 well plates, treated with cytokines and allowed to adhere overnight before use. After 24 hours, different concentrations of peptides, staurosporine or hydrogen peroxide were added. Cell viability and/or prostaglandin E2 content were determined after a further 24 hours.
Primary neuronal cultures
Primary cortical neurons were prepared from embryonic day 15.5 mice as previously described . Neuronal progenitors were seeded at 500,000 cells per well in 48 well plates in RPMI-1640 supplemented with 2 mM glutamine, standard antibiotics and 10% FCS. After 2 hours, cultures were washed and subsequently grown in neurobasal medium containing 2 mM glutamine and B27 components (Invitrogen, Paisley, UK). Primary cerebellar neurons were prepared from the brains from newborn mice pups following dissection of the cerebellum, removal of the meninges and cell dissociation as previously described . Neuronal progenitors were plated in 10% FCS for 2 hours, and then grown in neurobasal medium containing glutamine and B27. In both these neuronal cultures, medium was supplemented with 5 mM L-leucine methyl ester to reduce the numbers of contaminating microglial cells. After 7 days, cultures were treated with cytokines for 24 hours before the addition of neurotoxins/peptides. Caspase-3 activity was measured 24 hours after the addition of neurotoxins using a flourometric immunosorbent enzyme assay kit as per the manufacturer's instructions (Roche Diagnostics, Lewes, UK). Results are expressed as fluorescent units which are proportional to caspase-3 activity. For toxicity assays medium was replaced 48 hours after the addition of neurotoxins/peptides and cell viability was determined after another 48 hours (4 days after the addition of neurotoxins/peptides).
A peptide corresponding to amino acids 1 to 42 of the amyloid-β protein (amyloid-β1-42) and a control peptide (amyloid-β42-1) were obtained from Bachem (St Helens, UK). Peptides containing amino acid residues 82 to 146 of the human PrP protein (HuPrP82-146) corresponding to a PrP fragment found in certain prion-infected human brains , a control peptide containing the same amino acids in a scrambled order (HuPrP82-146scrambled) were a gift from Professor Mario Salmona (Mario Negri Institute, Milan).
Cell viability assays
To determine cell survival, cultures were treated with WST-1 (Roche Diagnostics Ltd, Lewes, UK) for 3 hours and optical density was read on a spectrophotometer at a wavelength of 450 nm. WST-1 is cleaved to formazan by mitochondrial dehydrogenases and the amount of dye formed correlates to the number of metabolically active cells. Percentage cell survival in cultures was calculated by reference to untreated cells incubated with WST-1 (100%).
SH-SY5Y neuroblastoma cells were lysed in an extraction buffer containing 10 mM Tris-HCl, pH 7.8, 100 mM sodium chloride, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and 2 mM phenylmethylsulphonylflouride at 1 × 106 cells per ml. Protein content was determined using a BCA kit (Pierce, Cramlington UK) and protein concentrations standardised. 20 μl samples were analysed via PAGE or blotted onto a PVDF membrane. Where appropriate, dilutions of lysates were made prior to blotting. Blots were probed with monoclonal antibodies (mabs) to cPLA2 or phospholipase C (PLC)γ-1 (Upstate, Milton Keynes, UK) and developed with an anti-mouse IgG-alkaline phosphatase conjugate followed by BCIP/NBT (Sigma).
Prostaglandin E2 assay
Analysis of total prostaglandin E2 levels was performed using an enzyme-immunoassay kit Amersham Biotech (Amersham, UK).
Recombinant murine TNFα, IL-6, IL-1β, IFN-γ were supplied from (R&D systems, Abingdon, UK). Human IFN- was obtained from (Sigma, Poole, UK).
Comparison of treatment effects were carried out using one and two way analysis of variance techniques as appropriate. Post hoc comparisons of means were performed as necessary.
Pre-treatment with IFN-γ reduces the survival of cortical neurons incubated with amyloid-β1-42
Treatment with IFN-γ reduces neuronal survival following incubation with amyloid-β1-42. SH-SY5Y cells or primary neuronal cell cultures were pre-treated with IFN-γ (1 ng/ml) for 24 hours prior to the addition of amyloid-β peptides as shown. Cell survival was determined using the WST-1 test after 24 hours (cell lines) or 4 days (primary neuronal cultures). Each value is the mean percentage cell survival ± SD from triplicate experiments repeated 3 times (9 observations). ** = Neuronal survival significantly less than untreated neurons incubated with amyloid-β1-42 (p < 0.05).
% Neuronal Survival
62 ± 4
33 ± 9**
101 ± 4
98 ± 7
68 ± 7
36 ± 7**
98 ± 3
96 ± 5
79 ± 4
58 ± 11**
101 ± 8
102 ± 8
IFN-γ treated neurons show increased sensitivity to HuPrP82-146. Neurons treated with 1 ng/ml IFN-γ for 24 hours prior to the addition of neurotoxins as shown. Cell survival was determined 24 hours later using the WST-1 test. Each value is the mean percentage cell survival ± SD from triplicate experiments repeated 3 times (9 observations). ** = Neuronal survival significantly less than untreated neurons incubated with HuPrP82-146 (p < 0.05).
Neuronal survival (%)
48 ± 6
12 ± 7**
64 ± 5
22 ± 8**
92 ± 9
41 ± 9**
37 ± 4
40 ± 6
78 ± 6
72 ± 8
92 ± 3
95 ± 4
Hydrogen peroxide (μM)
22 ± 5
18 ± 7
58 ± 7
55 ± 6
87 ± 6
89 ± 9
IFN-γ raises cytoplasmic PLA2 levels in neurons
Pre-treatment with IFN-γ increases prostaglandin E2 production in response to amyloid-β or HuPrP82-146. SH-SY5Y neuroblastoma cells were pre-treated for 24 hours with 1 ng/ml cytokines as shown prior to the addition of amyloid-β or PrP peptides. Cells were lysed 24 hours later and total prostaglandin E2 levels were measured. Each value given represents the mean ± SD from triplicate experiments repeated twice (6 observations). ** = Prostglandin E2 levels significantly higher than untreated neurons incubated with amyloid-β1-42 (p < 0.05).
Prostaglandin E2 (pg/ml)
241 ± 62
187 ± 40
443 ± 112**
66 ± 38
303 ± 55**
228 ± 54
202 ± 46
218 ± 66
210 ± 48
55 ± 35
255 ± 82
214 ± 33
Reports that activated microglial cells are found in close association with damaged neurons in AD raise the possibility that glial-derived cytokines are involved in neuropathogenesis. In the current studies the survival of either primary neuronal cultures (cortical or cerebellar neurons) or SH-SY5Y neuroblastoma cells was not affected by incubation with high concentrations of recombinant cytokines (up to 10 ng/ml). However, while none of the cytokines were directly neurotoxic, pre-treatment with IFN-γ significantly reduced the survival of neurons that incubated with amyloid-β1-42. This effect of IFN-γ was dose-dependent and was observed at concentrations previously reported in the cerebral cortex of APP(SWE) transgenic mice .
Pre-treatment with IFN-γ also increased the sensitivity of neurons to HuPrP82-146, a neurotoxic peptide found in prion diseases . However, neurons pre-treated with IFN-γ did not demonstrate increased sensitivity to all neurotoxins: there was no change in the neurotoxicity of staurosporine, a drug that causes programmed cell death in neurons via activation of the ceramide pathway , or of hydrogen peroxide which causes oxidation of cellular membranes. These observations strengthen the hypothesis that IFN-γ treatment selectively increases the expression of proteins involved in specific apoptotic pathways. Previous reports showed that amyloid-β peptides activate PLA2 , that PLA2 inhibitors protect against the amyloid-β1-42 induced neurotoxicity , and more specifically, that the cPLA2 isoform is required for induced neurotoxicity . The current study showed that IFN-γ increased expression of cPLA2 in neurons, a result consistent with previous observations that IFN-γ increases gene expression of cPLA2 in epithelial cells . The activation of cPLA2 results in the release of arachidonic acid which is subsequently metabolised by the COXs to prostaglandins and in the present study the increased expression of cPLA2 in IFN-γ treated neurons was associated with significantly greater amounts of prostaglandin E2 produced following the addition of amyloid-β1-42 or HuPrP82-146. IFN-γ treatment increased cPLA2 levels without affecting levels of PLCγ-1, further evidence that IFN-γ selectively increases expression of specific pathways.
In AD and prion diseases much of the neuronal death occurs though apoptosis . Although neurons incubated with fibrillar PrP/amyloid-β peptides in vitro show signs of apoptosis, the precise mechanisms that activate neuronal apoptosis remain unknown. In the present study both amyloid-β1-42 and HuPrP82-146 increased neuronal caspase-3 activity, a marker of apoptosis that is increased in AD . IFN-γ has been implicated in the pathogenesis of AD and IFN-responsive mRNAs have been found in Creutzfeldt-Jakob disease . IFN-γ can be produced in the brain by glial cells and IFN-γ immunoreactivity and IFN-γ-gene expression have been detected in human sensory neurons . Thus, these results indicate that IFN-γ has the potential to increase neuronal loss in AD or prion diseases, consistent with a previous report that the induction of IFNs hastens the progression of experimental prion diseases in mice .
We report that pre-treatment with IFN-γ increased the levels of cPLA2 in SH-SY5Y neuroblastoma cells without affecting total cellular protein concentrations, or the levels of PLCγ-1. The increased levels of cPLA2 were associated with increased prostaglandin E2 production in response to amyloid-β1-42 or HuPrP82-146. More importantly, pre-treatment with IFN-γ resulted in reduced neuronal survival following the addition of amyloid-β1-42 or HuPrP82-146. Such results are consistent with previous observations that cPLA2 is involved in neurodegeneration in AD or prion diseases and indicate that IFN-γ may hasten neuronal loss in these diseases.
cytoplasmic phospholipase A2
flourometric immunosorbent enzyme assay
tumour necrosis factor
This work was supported by a grant from the European Commission (QLK3-CT-2001-00283) and the EU FP6 – Network of Excellence "Neuroprion".
- Hardy J, Selkoe DJ: The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002, 297: 353-356. 10.1126/science.1072994.View ArticlePubMedGoogle Scholar
- Esler WP, Wolfe MS: A portrait of Alzheimer secretases – new features and familiar faces. Science. 2001, 293: 1449-1454. 10.1126/science.1064638.View ArticlePubMedGoogle Scholar
- Selkoe DJ: Translating cell biology into therapeutic advances in Alzheimer's disease. Nature. 1999, 399: A23-A31. 10.1038/19866.View ArticlePubMedGoogle Scholar
- Yankner BA, Dawes LR, Fisher S, Villa-Komaroff L, Oster-Granite ML, Neve RL: Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science. 1989, 245: 417-420.View ArticlePubMedGoogle Scholar
- Forloni G, Chiesa R, Smiroldo S, Verga L, Salmona M, Tagliavini F, Angeretti N: Apoptosis mediated neurotoxicity induced by chronic application of beta amyloid fragment 25–35. Neuroreport. 1993, 4: 523-526.View ArticlePubMedGoogle Scholar
- Akiyama H, Arai T, Kondo H, Tanno E, Haga C, Ikeda K: Cell mediators of inflammation in the Alzheimer disease brain. Alzheimer Dis Assoc Disord. 2000, 14 (Suppl 1): S47-S53.View ArticlePubMedGoogle Scholar
- Papassotiropoulos A, Bagli M, Jessen F, Bayer TA, Maier W, Rao ML, Heun RA: A genetic variation of the inflammatory cytokine interleukin-6 delays the initial onset and reduces the risk for sporadic Alzheimer's disease. Ann Neurol. 1999, 45: 666-668. 10.1002/1531-8249(199905)45:5<666::AID-ANA18>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Bate C, Salmona M, Williams A: The role of platelet activating factor in prion and amyloid-β neurotoxicity. Neuroreport. 2004, 15: 509-513. 10.1097/00001756-200403010-00025.View ArticlePubMedGoogle Scholar
- Bate C, Reid S, Williams A: Killing of prion-damaged neurones by microglia. Neuroreport. 2001, 12: 2589-2594. 10.1097/00001756-200108080-00059.View ArticlePubMedGoogle Scholar
- Salmona M, Morbin M, Massignan T, Colombo L, Mazzoleni G, Capobianco R, Diomede L, Thaler F, Mollica L, Musco G, Kourie JJ, Bugiani O, Sharma D, Inouye H, Kirschner DA, Forloni G, Tagliavini F: Structural properties of Gerstmann-Straussler-Scheinker disease amyloid protein. J Biol Chem. 2003, 278: 48146-48153. 10.1074/jbc.M307295200.View ArticlePubMedGoogle Scholar
- Wiesner DA, Dawson G: Staurosporine induces programmed cell death in embryonic neurons and activation of the ceramide pathway. J Neurochem. 1996, 66: 1418-1425.View ArticlePubMedGoogle Scholar
- Cohen GM: Caspases: the executioners of apoptosis. Biochem J. 1997, 326 (Pt 1): 1-16.PubMed CentralView ArticlePubMedGoogle Scholar
- Kriem B, Sponne I, Fifre A, Malaplate-Armand C, Lozac'h-Pillot K, Koziel V, Yen-Potin FT, Bihain B, Oster T, Olivier JL, Pillot T: Cytosolic phospholipase A2 mediates neuronal apoptosis induced by soluble oligomers of the amyloid-β peptide. The FASEB Journal. 2004, 04-1807fje.Google Scholar
- Abbas N, Bednar I, Mix E, Marie S, Paterson D, Ljungberg A, Morris C, Winblad B, Nordberg A, Zhu J: Up-regulation of the inflammatory cytokines IFN-gamma and IL-12 and down-regulation of IL-4 in cerebral cortex regions of APP(SWE) transgenic mice. J Neuroimmunol. 2002, 126: 50-57. 10.1016/S0165-5728(02)00050-4.View ArticlePubMedGoogle Scholar
- Lehtonen JY, Holopainen JM, Kinnunen PK: Activation of phospholipase A2 by amyloid beta-peptides in vitro. Biochemistry. 1996, 35: 9407-9414. 10.1021/bi960148o.View ArticlePubMedGoogle Scholar
- Bate C, Reid S, Williams A: Phospholipase A2 inhibitors or platelet activating factor antagonists prevent prion replication. J Biol Chem. 2004, 279: 36405-36411. 10.1074/jbc.M404086200.View ArticlePubMedGoogle Scholar
- Lindbom J, Ljungman AG, Lindahl M, Tagesson C: Increased gene expression of novel cytosolic and secretory phospholipase A(2) types in human airway epithelial cells induced by tumor necrosis factor-alpha and IFN-gamma. J Interferon Cytokine Res. 2002, 22: 947-955. 10.1089/10799900260286650.View ArticlePubMedGoogle Scholar
- Blasko I, Stampfer-Kountchev M, Robatscher P, Veerhuis R, Eikelenboom P, Grubeck-Loebenstein B: How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes. Aging Cell. 2004, 3: 169-176. 10.1111/j.1474-9728.2004.00101.x.View ArticlePubMedGoogle Scholar
- Baker CA, Lu ZY, Manuelidis L: Early induction of interferon-responsive mRNAs in Creutzfeldt-Jakob disease. J Neurovirol. 2004, 10: 29-40. 10.1080/13550280490261761.View ArticlePubMedGoogle Scholar
- Neumann H, Schmidt H, Wilharm E, Behrens L, Wekerle H: Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation. J Exp Med. 1997, 186: 2023-2031. 10.1084/jem.186.12.2023.PubMed CentralView ArticlePubMedGoogle Scholar
- Allen LB, Cochran KW: Acceleration of scrapie in mice by target-organ treatment with interferon inducers. Ann N Y Acad Sci. 1977, 284: 676-680.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.