Accepted Manuscript Title: Microglial p53 activation is detrimental to neuronal synapses during activation-induced inflammation: Implications for neurodegeneration Author: Joseph Jebelli Claudie Hooper Jennifer M. Pocock PII: DOI: Reference:
S0304-3940(14)00720-4 http://dx.doi.org/doi:10.1016/j.neulet.2014.08.049 NSL 30801
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
18-7-2014 27-8-2014 28-8-2014
Please cite this article as: J. Jebelli, C. Hooper, J.M. Pocock, Microglial p53 activation is detrimental to neuronal synapses during activation-induced inflammation: implications for neurodegeneration, Neuroscience Letters (2014), http://dx.doi.org/10.1016/j.neulet.2014.08.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microglial p53 activation is detrimental to neuronal synapses during activation‐induced inflammation: implications for neurodegeneration Joseph Jebelli1*, Claudie Hooper2 and Jennifer M. Pocock3 1
ip t
Department of Neurology, University of Washington, 1959 NE Pacific Street, Seattle WA 98103
2
cr
Institute of Psychiatry, Kings College London, 16 De Crespigny Park, London, SE5 8AF, UK
3
Department of Neuroinflammation, University College London Institute of Neurology, 1
us
Wakefield Street, London WC1N 1PJ, UK
an
*corresponding author: Dr. Joseph Jebelli, Department of Neurology, University of Washington, 1959 NE Pacific Street, Seattle WA 98103
M
Email: [email protected] Telephone: +1(206)6168364
Ac ce p
Number of figures: 5
te
Number of pages: 17
d
Abstract word number: 192
Introduction word number: 258 Discussion word number: 575 Total Number of Words: 3750
Conflict of interest: The authors declare no competing financial interests. Acknowledgements: J.M.Pocock acknowledges funding from Aims2Cure UK and UCL Impact Award. C. Hooper acknowledges fellowship funding from the Alzheimer’s Society, UK.
Page 1 of 23
Abstract P53 is a tumour suppressor protein thought to be primarily involved in cancer biology, but recent evidence suggests it may also coordinate novel functions in the CNS, including mediation
ip t
of pathways underlying neurodegenerative disease. In microglia, the resident immune cells of
cr
the brain, p53 activity can promote an activation‐induced pro‐inflammatory phenotype [1], as well as neurodegeneration [2]. Synapse degeneration is one of the earliest pathological events
us
in many chronic neurodegenerative diseases [3,4] and may be influenced by early microglial
an
responses. Here we examined synaptic properties of neurons following modulation of p53 activity in rat microglia exposed to inflammatory stimuli. A significant reduction in the
M
expression of the neuronal synaptic markers synaptophysin and drebrin, occurred following microglial activation and was seen prior to any visible signs of neuronal cell death, including
d
neuronal cleaved caspase‐3 activation. This synaptic marker loss together with microglial
te
secretion of the inflammatory cytokines tumour necrosis factor α (TNF‐α) and interleukin 1‐β
Ac ce p
(IL‐1β) was abolished by the removal of microglia or inhibition of microglial p53 activation. These results suggest that transcriptional‐dependent p53 activities in microglia may drive a non‐cell autonomous process of synaptic degeneration in neurons during neuroinflammatory degenerative diseases. Keywords
P53, Microglia, Synapse, Neurodegeneration
Page 2 of 23
Abbreviations PFTα, pifithrin‐α; PFTμ, pifithrin‐μ; iNOS, inducible nitric oxide synthase; IL‐1β, interleukin 1β; TLR, toll‐like receptor; TNF‐α, tumour necrosis factor α; LME, leucine‐methyl‐ester; LPS,
ip t
lipopolysaccharide; PI, propidium iodide; DAPI 4’‐6‐Diamidino‐2‐phenylindole‐2HCL. MEM,
cr
minimum essential medium with Earle’s salts.
us
1. Introduction
P53, a tumour suppressor gene encoding a 393 amino acid, 53 kilo Dalton (kDa) protein
an
product, belongs to a family of highly homologous proteins [5,6] commanding crucial and
M
multifaceted functions in cell‐cycle control, apoptosis, and maintenance of genetic stability [7]. In microglia, the resident immunocompetent cells of the CNS, p53 mediates microglial
d
activation and the subsequent pro‐inflammatory phenotypes seen in many neurodegenerative
Ac ce p
unclear.
te
diseases [1,2,8,9], but the mechanisms of p53‐driven microglial‐evoked neurotoxicity remain
Degeneration of synapses is an early pathological event in many chronic neurodegenerative diseases [3,5] and activation of toll like receptors (TLR) is implicated in innate immunity and neuroinflammatory processes within the CNS [10]. Moreover, p53 activity has recently been linked to TLR signaling in diverse mammalian cell types [11,12]. To determine whether microglial p53 activation influences neuronal synapse integrity, we measured the protein expression levels of two key synaptic proteins; synaptophysin, and drebrin in the presence of TLR‐stimulated stimulated microglia or following exposure to microglial conditioned medium (MGCM). Incubation of neurons with lipopolysaccharide (LPS)‐stimulated
Page 3 of 23
MGCM or by direct activation of microglia in the neuronal‐glial cultures resulted in reduced expression of synaptophysin and drebrin, prior to the triggering of neuronal death cascades. These effects were driven by the specific microglial activation of p53 and correlated with
ip t
increased phosphorylated‐p53 expression in microglia. Selective elimination of microglia
cr
abolished the disruption of neuronal synaptic marker expression and microglial p53 inhibition abolished inflammatory cytokine production. Overall, these results indicate that transcriptional‐
an
synaptic degeneration in neurons during inflammation.
us
dependent p53 activities in microglia may be responsible for a non‐cell autonomous process of
2.1 Cell culture preparation and treatment
M
2. Materials and Methods
d
Primary cultured rat microglia and cerebellar granule cell neuronal/glial (CGCs) cultures were
te
prepared from 5‐day‐old Sprague Dawley rats as previously described [13], in accordance with
Ac ce p
the United Kingdom Animals (Scientific Procedures) Act, 1986. Where indicated, microglia or CGCs were treated directly with pifithrin‐α (PFTα) (Sigma, P4359) (10μM), pifithrin‐μ (PFTμ) (Sigma, P0122) (5μM), and lipopolysacharide (LPS, 1μg/ml, (Sigma) for 24 or 48 h with cells treated with PFTα or PFTμ 1 h before treatment with LPS. Microglial conditioned medium (MGCM) was collected and aliquots snap‐frozen until required. MGCM was subsequently used at a 1:1 ratio with CGC medium.
Page 4 of 23
2.2 Specific depletion of microglia from neuronal cultures Microglia in CGC cultures were removed by treatment with 25mM leucine‐methyl‐ester (LME), as previously described [14,15]. Briefly, cells were exposed to LME for 1 h, followed by washing,
were treated identically except for the omission of LME.
us
2.3 Assessment of apoptosis and cell death in neuronal cultures
cr
ip t
replacement of original medium, and resting for 24 h before further treatment. Control cultures
an
Apoptosis and cell death were quantified on live, non‐fixed CGCs to determine the effects of MGCM on neuronal survival after 24 or 48 h of treatment, using propidium iodide (PI, for cell
M
death) and Hoechst 33342 (for apoptosis) as previously described [16]. Cells were observed with a Zeiss Axioskop 2 fluorescence microscope plus 40x Neofluar objective (Zeiss,
d
Oberkochen, Germany). Hoechst fluorescence was captured with a DAPI filter set and PI
te
fluorescence with a rhodamine filter set. Cell counts were performed on at least three cell fields
Ac ce p
per coverslip, three coverslips per treatment from three independent experiments.
2.4 Cell lysis and western blotting
Cells were harvested and cytosolic fractions resolved on 10% SDS gels, and transferred to PVDF membranes using standard protocols [11]. Membranes were incubated with mouse monoclonal anti‐p53 (Cell Signalling 1C12; 1:1000), rabbit polyclonal anti‐phospho‐p53 (Ser15) (Cell Signalling 9284; 1:1000), mouse monoclonal anti‐synaptophysin (Abcam ab8049; 1:500), rabbit polyclonal anti‐drebrin (Millipore AB10140; 1:5000) overnight at 4oC. Washed membranes were incubated with an appropriate HRP‐conjugated secondary antibody and blots developed using
Page 5 of 23
ECL. Membranes were reprobed with mouse anti‐β‐actin (Sigma A5441, 1:2000) as a loading control. All experiments were performed at least three times, quantified by densitometric analysis and normalised for β‐actin expression.
ip t
cr
2.5 Immunocytochemistry
Cells fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, in mM; 140 NaCl, 5
us
KCl, 25 Na2HPO4, 2.9 Na2HPO4.2H2O, 11 glucose, 0.2% bovine serum albumin, pH7.4) were
an
permeabilised with 0.1% triton‐X for 10 min at room temperature. Following washing and blocking, cells were incubated with rabbit monoclonal anti‐cleaved caspase‐3 (1:1000), mouse
M
monoclonal anti‐ED1 (Abcam ab31630, 1:500) or mouse monoclonal anti‐NeuN (Millipore MAB377; 1:500) overnight at 4oC, counterstained with the appropriate fluorescent tagged
d
secondary antibody and the nuclear stain 4’‐6‐Diamidino‐2‐phenylindole‐2HCL (DAPI). Cells
te
were viewed in a Zeiss Axioskop fluorescence microscope (Carl Zeiss Ltd, Welwyn Garden City,
Ac ce p
UK). Negative controls consisted of cells treated in the same way but with primary antibody omitted or with isotype controls.
2.6 Enzyme‐linked immunosorbent assay (ELISA) Tumour necrosis factor alpha (TNFα) and interleukin 1β (IL‐1β) concentrations in MGCM were quantified using Quantikine Rat TNFα/IL‐1β immunoassay kits according to the manufacturer’s instructions (R&D Systems, Abingdon, UK). MGCM cytokine concentrations were determined against a standard curve of TNFα/IL‐1β and analysed from three individual coverslips of microglia in three independent experiments with each sample assayed in duplicate.
Page 6 of 23
2.8 Statistical analysis To directly compare two treatments, two‐tailed paired Student’s t‐tests were performed. P values
S0304-3940(14)00720-4 http://dx.doi.org/doi:10.1016/j.neulet.2014.08.049 NSL 30801
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
18-7-2014 27-8-2014 28-8-2014
Please cite this article as: J. Jebelli, C. Hooper, J.M. Pocock, Microglial p53 activation is detrimental to neuronal synapses during activation-induced inflammation: implications for neurodegeneration, Neuroscience Letters (2014), http://dx.doi.org/10.1016/j.neulet.2014.08.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microglial p53 activation is detrimental to neuronal synapses during activation‐induced inflammation: implications for neurodegeneration Joseph Jebelli1*, Claudie Hooper2 and Jennifer M. Pocock3 1
ip t
Department of Neurology, University of Washington, 1959 NE Pacific Street, Seattle WA 98103
2
cr
Institute of Psychiatry, Kings College London, 16 De Crespigny Park, London, SE5 8AF, UK
3
Department of Neuroinflammation, University College London Institute of Neurology, 1
us
Wakefield Street, London WC1N 1PJ, UK
an
*corresponding author: Dr. Joseph Jebelli, Department of Neurology, University of Washington, 1959 NE Pacific Street, Seattle WA 98103
M
Email: [email protected] Telephone: +1(206)6168364
Ac ce p
Number of figures: 5
te
Number of pages: 17
d
Abstract word number: 192
Introduction word number: 258 Discussion word number: 575 Total Number of Words: 3750
Conflict of interest: The authors declare no competing financial interests. Acknowledgements: J.M.Pocock acknowledges funding from Aims2Cure UK and UCL Impact Award. C. Hooper acknowledges fellowship funding from the Alzheimer’s Society, UK.
Page 1 of 23
Abstract P53 is a tumour suppressor protein thought to be primarily involved in cancer biology, but recent evidence suggests it may also coordinate novel functions in the CNS, including mediation
ip t
of pathways underlying neurodegenerative disease. In microglia, the resident immune cells of
cr
the brain, p53 activity can promote an activation‐induced pro‐inflammatory phenotype [1], as well as neurodegeneration [2]. Synapse degeneration is one of the earliest pathological events
us
in many chronic neurodegenerative diseases [3,4] and may be influenced by early microglial
an
responses. Here we examined synaptic properties of neurons following modulation of p53 activity in rat microglia exposed to inflammatory stimuli. A significant reduction in the
M
expression of the neuronal synaptic markers synaptophysin and drebrin, occurred following microglial activation and was seen prior to any visible signs of neuronal cell death, including
d
neuronal cleaved caspase‐3 activation. This synaptic marker loss together with microglial
te
secretion of the inflammatory cytokines tumour necrosis factor α (TNF‐α) and interleukin 1‐β
Ac ce p
(IL‐1β) was abolished by the removal of microglia or inhibition of microglial p53 activation. These results suggest that transcriptional‐dependent p53 activities in microglia may drive a non‐cell autonomous process of synaptic degeneration in neurons during neuroinflammatory degenerative diseases. Keywords
P53, Microglia, Synapse, Neurodegeneration
Page 2 of 23
Abbreviations PFTα, pifithrin‐α; PFTμ, pifithrin‐μ; iNOS, inducible nitric oxide synthase; IL‐1β, interleukin 1β; TLR, toll‐like receptor; TNF‐α, tumour necrosis factor α; LME, leucine‐methyl‐ester; LPS,
ip t
lipopolysaccharide; PI, propidium iodide; DAPI 4’‐6‐Diamidino‐2‐phenylindole‐2HCL. MEM,
cr
minimum essential medium with Earle’s salts.
us
1. Introduction
P53, a tumour suppressor gene encoding a 393 amino acid, 53 kilo Dalton (kDa) protein
an
product, belongs to a family of highly homologous proteins [5,6] commanding crucial and
M
multifaceted functions in cell‐cycle control, apoptosis, and maintenance of genetic stability [7]. In microglia, the resident immunocompetent cells of the CNS, p53 mediates microglial
d
activation and the subsequent pro‐inflammatory phenotypes seen in many neurodegenerative
Ac ce p
unclear.
te
diseases [1,2,8,9], but the mechanisms of p53‐driven microglial‐evoked neurotoxicity remain
Degeneration of synapses is an early pathological event in many chronic neurodegenerative diseases [3,5] and activation of toll like receptors (TLR) is implicated in innate immunity and neuroinflammatory processes within the CNS [10]. Moreover, p53 activity has recently been linked to TLR signaling in diverse mammalian cell types [11,12]. To determine whether microglial p53 activation influences neuronal synapse integrity, we measured the protein expression levels of two key synaptic proteins; synaptophysin, and drebrin in the presence of TLR‐stimulated stimulated microglia or following exposure to microglial conditioned medium (MGCM). Incubation of neurons with lipopolysaccharide (LPS)‐stimulated
Page 3 of 23
MGCM or by direct activation of microglia in the neuronal‐glial cultures resulted in reduced expression of synaptophysin and drebrin, prior to the triggering of neuronal death cascades. These effects were driven by the specific microglial activation of p53 and correlated with
ip t
increased phosphorylated‐p53 expression in microglia. Selective elimination of microglia
cr
abolished the disruption of neuronal synaptic marker expression and microglial p53 inhibition abolished inflammatory cytokine production. Overall, these results indicate that transcriptional‐
an
synaptic degeneration in neurons during inflammation.
us
dependent p53 activities in microglia may be responsible for a non‐cell autonomous process of
2.1 Cell culture preparation and treatment
M
2. Materials and Methods
d
Primary cultured rat microglia and cerebellar granule cell neuronal/glial (CGCs) cultures were
te
prepared from 5‐day‐old Sprague Dawley rats as previously described [13], in accordance with
Ac ce p
the United Kingdom Animals (Scientific Procedures) Act, 1986. Where indicated, microglia or CGCs were treated directly with pifithrin‐α (PFTα) (Sigma, P4359) (10μM), pifithrin‐μ (PFTμ) (Sigma, P0122) (5μM), and lipopolysacharide (LPS, 1μg/ml, (Sigma) for 24 or 48 h with cells treated with PFTα or PFTμ 1 h before treatment with LPS. Microglial conditioned medium (MGCM) was collected and aliquots snap‐frozen until required. MGCM was subsequently used at a 1:1 ratio with CGC medium.
Page 4 of 23
2.2 Specific depletion of microglia from neuronal cultures Microglia in CGC cultures were removed by treatment with 25mM leucine‐methyl‐ester (LME), as previously described [14,15]. Briefly, cells were exposed to LME for 1 h, followed by washing,
were treated identically except for the omission of LME.
us
2.3 Assessment of apoptosis and cell death in neuronal cultures
cr
ip t
replacement of original medium, and resting for 24 h before further treatment. Control cultures
an
Apoptosis and cell death were quantified on live, non‐fixed CGCs to determine the effects of MGCM on neuronal survival after 24 or 48 h of treatment, using propidium iodide (PI, for cell
M
death) and Hoechst 33342 (for apoptosis) as previously described [16]. Cells were observed with a Zeiss Axioskop 2 fluorescence microscope plus 40x Neofluar objective (Zeiss,
d
Oberkochen, Germany). Hoechst fluorescence was captured with a DAPI filter set and PI
te
fluorescence with a rhodamine filter set. Cell counts were performed on at least three cell fields
Ac ce p
per coverslip, three coverslips per treatment from three independent experiments.
2.4 Cell lysis and western blotting
Cells were harvested and cytosolic fractions resolved on 10% SDS gels, and transferred to PVDF membranes using standard protocols [11]. Membranes were incubated with mouse monoclonal anti‐p53 (Cell Signalling 1C12; 1:1000), rabbit polyclonal anti‐phospho‐p53 (Ser15) (Cell Signalling 9284; 1:1000), mouse monoclonal anti‐synaptophysin (Abcam ab8049; 1:500), rabbit polyclonal anti‐drebrin (Millipore AB10140; 1:5000) overnight at 4oC. Washed membranes were incubated with an appropriate HRP‐conjugated secondary antibody and blots developed using
Page 5 of 23
ECL. Membranes were reprobed with mouse anti‐β‐actin (Sigma A5441, 1:2000) as a loading control. All experiments were performed at least three times, quantified by densitometric analysis and normalised for β‐actin expression.
ip t
cr
2.5 Immunocytochemistry
Cells fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, in mM; 140 NaCl, 5
us
KCl, 25 Na2HPO4, 2.9 Na2HPO4.2H2O, 11 glucose, 0.2% bovine serum albumin, pH7.4) were
an
permeabilised with 0.1% triton‐X for 10 min at room temperature. Following washing and blocking, cells were incubated with rabbit monoclonal anti‐cleaved caspase‐3 (1:1000), mouse
M
monoclonal anti‐ED1 (Abcam ab31630, 1:500) or mouse monoclonal anti‐NeuN (Millipore MAB377; 1:500) overnight at 4oC, counterstained with the appropriate fluorescent tagged
d
secondary antibody and the nuclear stain 4’‐6‐Diamidino‐2‐phenylindole‐2HCL (DAPI). Cells
te
were viewed in a Zeiss Axioskop fluorescence microscope (Carl Zeiss Ltd, Welwyn Garden City,
Ac ce p
UK). Negative controls consisted of cells treated in the same way but with primary antibody omitted or with isotype controls.
2.6 Enzyme‐linked immunosorbent assay (ELISA) Tumour necrosis factor alpha (TNFα) and interleukin 1β (IL‐1β) concentrations in MGCM were quantified using Quantikine Rat TNFα/IL‐1β immunoassay kits according to the manufacturer’s instructions (R&D Systems, Abingdon, UK). MGCM cytokine concentrations were determined against a standard curve of TNFα/IL‐1β and analysed from three individual coverslips of microglia in three independent experiments with each sample assayed in duplicate.
Page 6 of 23
2.8 Statistical analysis To directly compare two treatments, two‐tailed paired Student’s t‐tests were performed. P values
