Neuroinflammatory activation of glia is considered a pathological hallmark of Parkinson’s disease (PD) and is seen in both human PD patients and in animal models of PD; however, the relative contributions of these cell types to the progression of disease is not fully understood. The transcription factor, nuclear factor kappa B (NFκB), is an important regulator of inflammatory gene expression in glia and is activated by multiple cellular stress signals through the kinase complex, IKK2. We therefore sought to determine the role of NFκB in modulating inflammatory activation of astrocytes in a model of PD by generating a conditional knockout mouse (hGfapcre/Ikbk2F/F) in which IKK2 is specifically deleted in astrocytes. Measurements of IKK2 revealed a 70% deletion rate of IKK2 specific to astrocytes, as compared to littermate controls (Ikbk2F/F). Use of this mouse in a subacute, progressive model of PD through exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) revealed significant protection in exposed mice to direct and progressive loss of dopaminergic neurons. hGfapcre/Ikbk2F/F mice were also protected against MPTPp-induced loss in motor activity, loss of striatal proteins, and genomic alterations in nigral NFκB gene expression, but were not protected from loss of striatal catacholamines. Neuroprotection in hGfapcre/Ikbk2F/F mice was associated with inhibition of MPTPp-induced astrocytic expression of inflammatory genes and protection against nitrostative stress and apoptosis in neurons. These data indicate that deletion of IKK2 within astrocytes is neuroprotective in the MPTPp model of PD and suggests that reactive astrocytes directly contribute to both the initiation and potentiation of dopaminergic pathology.
Significance Statement
The innate immune response of the central nervous system, known as neuroinflammation, is vital in protection against insult, but when unchecked, can exacerbate neurodegenerative conditions such as Parkinson’s disease (PD). Astrocytes and microglia are implicated in the progression of PD, therefore, we sought to create a mouse model whereby a major inflammatory pathway known as Nuclear Factor kappa Beta (NFκB) was deleted only in astrocytes. After confirming specificity of deletion to astrocytes, mice and littermate controls were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp), an established chemical model of PD. Mice with astrocytic deletion of NFκB were protected against loss of dopaminergic neurons, striatal proteins, and loss of locomotor parameters correlated with reduced glia activation, nitrosative stress and neuronal apoptosis.
Introduction
Activation of glial cells is implicated in the progression of neuronal loss in neurodegenerative disorders such as Parkinson’s disease (PD) through the upregulation and release of pro-inflammatory and pro-oxidant factors that exert toxic effects on surrounding neurons (Hirsch and Hunot, 2009). In PD, neuroinflammation is seen in both human patients and in animal models, characterized by the presence of reactive astrocytes and microglia in affected brain regions that closely associate with protein aggregates (Damier et al., 1993; Nagatsu and Sawada, 2005; Ouchi et al., 2009). Furthermore, several lines of evidence point to reactive glia being important participants in disease pathology, because upregulation of glial-derived factors such as tumor necrosis factor alpha (TNF) and inducible nitric oxide synthase 2 (NOS2) occurs prior to loss of dopaminergic neurons (Sugama et al., 2003; Hirsch and Hunot, 2009; Saijo et al., 2009; Miller et al., 2011) and alterations in familial PD genes can affect production of inflammatory cytokines (Dzamko et al., 2015). Experimental models in which these and other inflammatory factors are genetically or pharmacologically inhibited markedly protect against neurodegeneration (Dehmer et al., 2000; 2003; McCarty, 2006; Mondal et al., 2012). Although reactive gliosis is associated in neuronal injury in PD (McCarty, 2006; Chen et al., 2009), glial responses can also be neuroprotective often linked with an A1 (neurotoxic) versus A2 (neuroprotective) phenotype (Frank-Cannon et al., 2009; Neal and Richardson, 2018; Liddelow et al., 2017) revealing a time-dependent and state-dependent action. Thus better models are needed to determine the exact contributions of each glial subtypes to disease pathology.
Inflammatory signaling through the canonical (NFκB) pathway involves activation of inhibitory kappa alpha kinase beta (IKKβ/IKK2) and translocation of p65/p50 dimers to the nucleus to stimulate inflammatory gene expression through binding to cis-acting promoter elements (Karin, 1999; Bonizzi and Karin, 2004; Karin, 2005), which is critical for production of inflammatory mediators in glial cells (Glass et al., 2010). Activation of NFκB is also directly associated with PD, as noted studies describe upregulation and nuclear translocation of NFκB/p65 in both neurons (Hunot et al., 1997) and glia of the PD brain (Ghosh et al., 2007) and in experimental animal models (Saijo et al., 2009). Studies have demonstrated that globally suppressing NFκB can be protective in neurotoxin-based models of PD (Dehmer et al., 2003; Ghosh et al., 2007; Saijo et al., 2009; Mondal et al., 2012; De Miranda et al., 2013), but these studies do not ascertain the specific cellular mechanisms of this neuroprotection and are difficult to translate to clinical applications due to detrimental effects from complete functional loss of NFκB (Grilli and Memo, 1999; Herrmann et al., 2005). Additionally, neuronal NFκB activation appears to protect neurons against degeneration (Mettang et al., 2018) while glial NFκB activations promotes neurodegeneration (Mattson and Camandola, 2001; Brambilla et al., 2009; Zhang et al., 2017).
Previous studies in our laboratory utilizing a transgenic reporter mouse expressing green fluorescent protein under the control of NFκB enhancer elements reported activation of the pathway in astrocytes prior to overt loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc; Miller et al., 2011). Other neurodegenerative models including multiple sclerosis, traumatic spinal cord injury, and Huntington’s disease have also shown that selective deletion of the NFκB pathway to be neuroprotective (Brambilla, 2005; van Loo et al., 2006; Brambilla et al., 2009) while constitutive activation to lead to high levels of basal inflammation{Oeckl:2012dd, Lian:2012iv}. Based on these data, we postulated that cell-specific inhibition of inflammatory NFκB signaling in astrocytes would suppress a reactive phenotype and protect dopaminergic neurons in the MPTPp model of PD. As the phosphorylation of the IκB inhibitor complex by IKKβ/IKK2 is an important regulatory step in the classical inflammatory pathway of NFκB (Karin, 1999), we sought to test this hypothesis by utilizing Cre-loxP technology to delete IKKβ/IKK2 selectively in astrocytes to determine the role of this signaling factor in injury to dopaminergic neurons.
Materials and Methods
Animals and Genotyping
Astrocyte specific Ikbk2-deficient mice (hGfapcre/Ikbk2F/F) were generated by breeding Ikbk2-floxed mice (Li et al., 2003); C57Bl/6 background; provided by Dr. Michael Karin at University of California San Diego) with hGfapcre transgenic mice (Zhuo et al., 2001); FVB-Tg(GFAP-CRE)25Mes/J; FVB background; Jackson Laboratories, Bar Harbor, ME) expressing Cre under the control of the human glial fibrillary acidic protein (GFAP) promoter. Mice were bred to homozygosity for the floxed-Ikbk2 allele and both male and female littermates from the 4th generation aged to 5 months were utilized in expression and treatment studies. Using animals older than 5 months was limited due to spontaneous development of inflammatory and neoplastic skin lesions{Kirkley:2017wj}. Littermates lacking hGfapcre (known as Ikbk2F/F) were utilized as controls.
PCR genotyping on ear tags was performed using the primers
5’-GTC ATT TCC ACA GCC CTG TGA-3’ and 5’-CCT TGT CCT ATA GAA GCA CAA C-3’, that amplifies both the Ikbk2+ (220-bp) and Ikbk2F (310-bp) alleles and primers 5’-ACT CCT TCA TAA AGC CCT CG-3’ and 5’-ATC ACT CGT TGC ATC GAC CG-3’, that amplifies the hGfapcre allele (190-bp). Animals were housed in microisolater cages (2-3 animals per cage), kept on a 12-h light/dark cycle, and had access to both chow and water ad libitum. All procedures were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and with the approval by the Institutional Animal Care and Use Committee (IACUC) of Colorado State University.
Primary Glial and Neuronal Cultures
Mixed glial cultures from whole brain (excluding cerebellum and brain stem) of postnatal day 1 (neonatal) and 3-month old (adult) Ikbk2F/F and hGfapcre/Ikbk2F/F mice were isolated using a modification of a previously described method (Aschner and Kimelberg, 1991; Carbone et al., 2008; Moreno et al., 2008). Briefly, mice were euthanized by decapitation under isoflurane anesthesia and whole brains were rapidly dissected out and placed into ice-cold minimum essential medium with L-glutamine (MEM; Gibco). Meninges were removed, and tissues completely digested with dispase (1.5U/ml) with each animal extracted separately. Dissociated cells from individual animals were plated onto 100-mm tissue culture plates and kept in MEM supplemented with 10% heat-inactivated FBS (Sigma) and penicillin (0.002 mg/ml), streptomycin (0.002 mg/ml), and neomycin (0.001 mg/ml) antibiotic mixture (PSN). Media was changed every 4-5 days and cells were maintained at 37°C and 5% CO2 in humidified chambers until confluency was reached (~14-18 days). Microglia were purified from astrocytes via column-free magnetic separation using the EasySep mouse CD11b positive selection kit (Stemcell technologies, Vancouver, Canada) according to manufacturer instructions and as described (Gordon et al., 2011; Kirkley et al., 2017) and determined to be 97% pure via flow cytometry.
Primary striatal neurons were extracted in a similar fashion as the mixed glial cultures except performed in neurobasal medium. Primary neuronal cultures were seeded onto poly(L-lysine)-coated 22mm glass coverslips at 4 x 105 cells/well and maintained in neurobasal media supplemented with 2mM L-glutamine, B27 supplement, and PSN antibiotic mixture (Gibco, Waltham, MA). Neuronal culture media was changed every 2 days with purity ascertained via cell morphology and immunolabeling with the neuron specific marker microtubule associated factor 2 (MAP2).
Flow Cytometry
The percent of glia in astrocyte cultures were determined by immunophenotyping using direct labeling with anti-GLAST-PE (Miltenyi Biotec, San Diego, CA), anti-Cd11b-FITC (BD Biosciences, San Jose, CA) followed by flow cytometric analysis as described (Kirkley et al., 2017). Briefly, purified cells were counted using a Bio-Rad TC10 automated cell counter, and 1 × 106 cells/mL were resuspended in 100 μL of incubation buffer (PBS with 0.05% bovine serum albumin). Purified cells were labeled using the mouse anti-GLAST-PE (20 μg/mL) and mouse anti-CD11b-FITC (10 μg/mL) at room temperature for 1 h. After labeling, the cells were washed twice in incubation buffer and resuspended at a final volume of 500 μL of PBS and stored at 37 °C until analysis. Flow cytometry was performed on a Beckman Coulter CyAn ADP flow cytometer operated with Summit software for data collection at Colorado State University’s Flow Cytometry Core Facility. All further data analysis was done utilizing FlowJo software (version 10.1; FlowJo, Ashland, OR).
Evaluation of Genomic Deletion of Ikbk2F/F via qPCR
Genomic DNA was isolated from neonatal and adult astrocyte cultures and neonatal microglia and neuronal cultures utilizing a DNeasy kit (Qiagen, Valencia, CA) with purity and concentration confirmed using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). 100 ng of DNA was mixed with Sybrgreen (Bio-Rad, Hercules, CA) and primers (10μM) 5’-AAG ATG GGC AAA CTG TGA TGT G-3’ and 5’-CAT ACA GGC ATC CTG CAG AAC A-3’ to amplify the Ikbk2F allele or 5’-ATG GCC TTG CAT GAG GAT ACA CCA-3’ and 5’-GAG TCT CAG TCT TCA ACT CCC TGT-3’ to amplify the Nos2 promoter which was utilized as a control. Percent expression of Ikbk2F in astrocytes, microglia, and neurons from hGfapcre/Ikbk2F/F mice was determined based on comparison of Ikbk2F signal from Ikbk2F/F littermate controls, defined at 100%, after normalization to Nos2 promoter signal.
Measurement of IKK2 via Western Blot
Purified neonatal astrocyte cultures from Ikbk2F/F and hGfapcre/Ikbk2F/F and mice were lysed using a RIPA lysis buffer supplemented with complete protease inhibitor (Roche, Indianapolis, IN). Protein was quantified using a BCA Assay (Pierce, Rockford, IL) and 50μg of protein was separated using gel electrophoresis in a 10% SDS polyacrylamide gel followed by a wet transfer to a polyvinylidene fluoride membrane (Pall Corporation, Pensacola, FL). Membranes were blocked in 5% non-fat dry milk in tris-buffered saline containing 0.2% tween-20 then incubated with primary rabbit anti-IKK2 (1:500; Cell Signaling, Danvers, MA) overnight followed by incubation in horseradish peroxidase-conjugated secondary antibody (1:5000, Santa Cruz, Dallas, TX) for one hour. Chemiluminescent detection was performed and analyzed using the ChemiDoc XRX imaging system (Bio-Rad). Membranes were reprobed with β-actin as a control with all densitometric analysis normalized to β-actin signal using Fiji software.
Immunofluorescence of IKK2
Purified astrocytes, microglia, and neurons from Ikbk2F/F and hGfapcre/Ikbk2F/F mice were plated at a density of 1 x 104 cells on 12mm poly-D-lysine coated glass coverslips and allowed to adhere for 48 hours. Cells were fixed using methanol, washed in PBS, and then blocked in 1% bovine serum albumin (w/v) in PBS for one hour. Cells were incubated overnight at 4°C in primary antibodies for IKK2 (1:50; Imgenex, San Diego, CA) and for cell specific markers GFAP (1:500; Sigma, St. Louis, MO), ionized binding adaptor protein-1 (IBA1; 1:250; Wako, Osaka, Japan), or MAP2 (1:100; Abcam, Cambridge, MA). After rinsing in PBS, cells were incubated in for one hour at room temperature in AlexaFluor-488 (IKK2) and AlexaFluor-647 (cell markers) conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA) and then mounted in medium containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to detect cell nuclei.
In vivo assessment of IKK2 was performed on free-floating 40μm brain sections obtained from the SN of Ikbk2F/F and hGfapcre/Ikbk2F/F mice (procedure detailed below) using primary antibodies for IKK2 (1:50; Imgenex) and MAP2 (1:100; Abcam) and Alexafluor-488 and Alexa-fluor-647 conjugated secondary antibodies (1:500; Invitrogen). Sections were mounted in medium containing DAPI. Images were acquired using a 40x air plan apochromatic objectives on a Zeiss Axiovert 200M inverted fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a Hamamatsu ORCA-ER-cooled charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan). Mean fluorescence intensity of IKK2, reported with subtraction of mean background fluorescence, was determined by utilizing Slidebook software (Intelligent Imaging Innovations Inc., Denver, CO) with 10-15 individual fields examined per animal with at least 3 animals utilized per genotype.
MPTP Treatment Regiment
Ikbk2F/F and hGfapcre/Ikbk2F/F male and female littermates aged to 5 months were divided between treatment groups and then exposed to a subacute PD lesioning model utilizing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTPp) as described previously (Miller et al., 2011; De Miranda et al., 2013). In brief, mice were injected every other day with probenecid (250 mg/kg i.p. prepared in 5% sodium bicarbonate; Sigma) and with saline or MPTP (20 mg/kg s.c. prepared in saline as free base, Sigma) for 7 days receiving a total of 4 injections. Mice were euthanized under deep isoflurane anesthesia at either 7 days (MPTPp7d) or 14 days (MPTPp14d) after their initial injection (Figure 3A). For all groups, a minimum of 7 mice was utilized with per parameter (stereology versus neurochemistry) with a minimum of 10 mice used for neurobehavioral data.
Tissue Processing and Sectioning
At day 7 and 14, mice were euthanized, and tissues obtained as reported previously (Miller et al., 2011). Briefly, animals were terminated under isoflurane anesthesia and transcardially perfused with 20 mM cacodylate-phosphate buffered saline (cPBS) containing 10U/ml heparin, followed by 4% paraformaldehyde in cPBS. Brains were carefully removed from the skull and placed within 4% formaldehyde in cPBS overnight then cryoprotected in 15% sucrose (w/v cPBS) then 30% sucrose (w/v cPBS). Brains were stored in 30% sucrose at 4°C until sectioning. Coronal 40μm sections through the entire length of the striatum (ST) and substantia nigra (SN) were collected using a freezing sliding microtome (Microm HM450; Thermoscientific, Waltham, MA). Sections were stored free floating at -20°C in cryoprotectant (30% w/v sucrose, 30% v/v ethylene glycol; 0.05M phosphate buffer) until staining.
Stereological Counts of Tyrosine Hydroxylase (TH) Positive Neurons
Stereological assessment of TH-positive dopaminergic neurons within the SN (Miller et al., 2011). In brief, free floating serial sections were obtained by systematic sampling of every third tissue from sections encompassing the entire length of the SNpc and immunolabeled using primary rabbit anti-TH antibody (1:500 overnight, Chemicon, Temecula, CA) and AlexaFluor-555 conjugated secondary antibody (1:500 for 3 hours; Invitrogen). Slides were mounted in DAPI containing medium and stored at 4°C until imaging.
Slides were imaged using a 40x air plan aprochromat objective on a Zeiss Axiovert 200M inverted fluorescence microscope (Carl Zeiss) with stereological counts of TH-positive cells performed using Slidebook software (v5.0, Intelligent Imaging Innovations, Denver, CO). The boundary of the SNpc was determined using 10x magnification montaging and numbers of TH-positive cells determined via assessment of uniform (40x) randomly placed counting frames (100μm x 100μm) using an optical dissector of 30μm with 5μm upper and lower guard zones. Representative montage images were generated for each treatment group with use of BX51 microscope (Olympus, Center Valley, PA, USA) equipped with a Hammatsu ORCA-Flash4.0 digital CMOS camera, ProScan III stage controller (Prior, Rockland, MA USA) and CellSens Dimension software (version 1.12, Olympus, Center Valley, PA, USA). Representative images were processed using Fiji (National Institutes of Health freeware) with shade correction, standard background subtraction and contrast enhancement. Original hues were altered when indicated to limit the use of red-green combinations and for consistency.
Behavioral Assessment
Open field activity parameters were assessed using the Versamax behavior chambers with an infrared beam grid detection array (Accuscan Instruments, Inc., Columbus, OH). Mice were monitored for 10 minutes under low ambient light in the presence of white noise. Stride length was assessed via video recording mice freely walking across a plexiglass track (5 cm x 1m) with 3 recordings obtained per mouse per assessment. Animals were pre-conditioned one day prior to their first treatment and then assessed at day 0 (first day of treatment) to establish baseline, day 7, and day 14. Several behavioral parameters were collected and analyzed using Versadat Software (Accuscan Instruments, Inc.) including total distance traveled, number of movements, time spent moving, time spent in the margin, and the number of rearing movements. Stride length was calculated using the Tracker Video Modeling software (Tracker v.4.85 for MacOS X). These parameters have been previously shown to assess basal ganglia function (Liu et al., 2006; Moreno et al., 2009; Miller et al., 2011; Streifel et al., 2012) and was reported as change from baseline (day 0) assessment.
HPLC Analysis of Striatal Dopamine and Metabolites
Mice were terminated at day 7 and day 14 under deep isoflurane anesthesia and quickly decapitated followed by rapid removal of the ST using a brain matrix block for reference. The tissue was flash-frozen in liquid nitrogen and stored at -80°C until analysis. Samples were coded for unbiased analysis and sent to the Neurochemistry Core Laboratory at Vanderbilt University’s Center for Molecular Neuroscience Research (Nashville, TN). High Performance Liquid Chromatography with electrochemical detection was used to determine the concentrations of dopamine (DA) and dopamine-related metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum of control and MPTPp treated mice as detailed in (Perez and Palmiter, 2005).
Western Blot Analysis of Striatal Proteins
Flash-frozen striatal tissue from Ikbk2F/F and hGfapcre/Ikbk2F/F mice was collected at day 7 and day 14, flash frozen in liquid nitrogen and stored at -80°C until homogenized using a glass pestle and grinder followed by low power sonication in RIPA lysis buffer supplemented with complete protease inhibitor. Protein was quantified using a BCA Assay (Pierce, Rockford, IL) and 20μg of protein was separated using gel electrophoresis in a 10% SDS polyacrylamide gel followed by semi-dry transfer to a polyvinylidene fluoride membrane. Membranes were blocked in 5% non-fat dry milk in tris-buffered saline containing 0.2% tween-20 then incubated with rabbit anti-TH (1:500; Millipore, Burlington, MA), rabbit anti-VMAT2 (1:500, Millipore), or rabbit anti-DAT (1:100, Santa Cruz) overnight followed by incubation in horseradish peroxidase-conjugated secondary antibody (1:5000, Santa Cruz) for one hour. Chemiluminescent detection was performed and analyzed using the ChemiDoc XRX imaging system. Membranes were reprobed with β-actin as a control with all densitometric analysis normalized to β-actin signal using Fiji.
In Vivo Immunofluorescence
Free floating 40 μM coronal sections of SN or ST described above were mounted on positively charged microscope slides and immunolabeled for TH (1:500), MAP2 (1:100, Abcam), 3-nitrotyrosine (3-NT; 1:100, Abcam), GFAP (1:500, Dako), IBA1 (1:100, Wako), TNF (1:100, Cell signaling), and NOS2 (1:100, BD Bioscience) with Alexa Fluor 488, 555, and 647 secondary antibodies (1:500; Invitrogen,). Three serial sections were utilized per brain region for each immunolabeling combination. Slides were imaged using a 10x or 40x air aprochromat objective on a Zeiss Axiovert 200M inverted fluorescence microscope (Carl Zeiss) for quantitative analysis and BX51 microscope (Olympus) equipped with a Hammatsu ORCA-Flash4.0 digital CMOS camera, ProScan III stage controller (Prior) and CellSens Dimension software (version 1.12, Olympus) for representative images. All representative images were processed using Fiji with standard background subtraction and contrast enhancement. Montages had further processing of shade correction to account for stitched images. Original hues were altered when indicated to limit the use of red-green combinations and for consistency.
Quantitative analysis of TH intensity in the ST was performed on 10x magnification montages. Randomized images were segmented manually to highlight the ST and an average TH fluorescence intensity generated. Average TH fluorescence intensity was normalized via division by the mean of the average TH intensity of the saline Ikbk2F/F processed on the same day to account for inconsistencies in staining or imaging during batch processing.
Generation of total neuronal counts in the SNpc was performed on sections immunolabeled for TH and MAP2. As used for TH cell counts described above, the boundary of the SNpc was determined via 10x magnification montaging and then quantified on random 40x counting frames (100μM x 100μM) using Slidebook software. For assessment of gliosis and gliosis expression of TNF and NOS2, slides were imaged using a 40x air plan aprochromat objective with analysis determined using Slidebook software. The development of random 40x counting frames (150μM x 150μM) was similar to methods used for stereological counting for TH described above. Images were randomized, and images obtained per tissue segmented for the protein of interest. Once segmented, Slidebook generated an object count based on parameters of objects greater than 300 μM (GFAP) or 200μM (IBA1). These object counts were summed for a total count per tissue in gliosis analysis and per frame for assessment of TNF and NOS2 intensity. Furthermore, in TNF and NOS2 analysis in GFAP+ cells, the total sum intensity in only GFAP+ cells were calculated and normalized to the total GFAP+ cell count for that image.
qPCR Array Analysis
RNA from the SN of Ikbk2F/F and hGfapcre/Ikbk2F/F mice collected at euthanasia, flash frozen in liquid nitrogen, and stored at -80 °C until homogenized and lysed using a Qiashredder (Qiagen) and then purified using the RNeasy kit (Qiagen). RNA was quantified and converted to cDNA as described above with 250 ng per sample amplified using RT2 profiler PCR array for NFkB signaling pathway genes (Qiagen PAMM-025z). Gene expression fold change was analyzed using SAbiosciences software with genes divided for biological gene otology using DAVID Bioinformatics Resources 6.8 (Da Wei Huang et al., 2009; Huang et al., 2009). Calculation of false discovery rate (FDR) was performed using significance analysis of microarray (SAM) version 5.0 from Stanford University (Chu et al., 2001).
Neuronal Viability
Primary astrocytes from Ikbk2F/F and hGfapcre/Ikbk2F/F mice and neurons from wild type C57/Bl6J mice were cultured in appropriate medium as described in detail above. Neurons were seeded directly onto poly(L-lysine) coated 12-mm glass coverslips at 1 x 105 cells/well. At confluency, astrocytes were treated with 10 μM MPTP and 10 pg/mL TNF and 1 ng/mL interferon-gamma (IFNγ) for 8 hours; an established protocol known to elicit neuroinflammatory activation in astrocytes (Carbone et al., 2008). Medium was removed, and astrocytes washed 3 times with PBS to prevent carryover of treatment to neurons and then placed in neurobasal media supplemented with 2mM L-glutamine, B27 supplement, and PSN antibiotic mixture for 24 hours. After 24 hours, the medium was removed, spun down to remove any cellular debris and placed on cultured neurons for an additional 24 hours. After 24 hours, neurons were assessed for cellular death via live-cell fluorescence imaging. Caspase activity was determined using CellEvent caspase-3/7 green detection reagent (Thermofisher) according to manufacturer’s instructions, overall cell death with 3 μM propidium iodide (PI; Sigma), and nuclei using 2μM Hoechst 33342 (Thermofisher). Using a 20x Plan apochromat air objective, 10–12 fields per treatment were blindly captured and assessed via blind cell counts using Slidebook software. For each genotype and treatment, there was a minimum of 3 biological replicates with 3-4 repetitions of the experiment.
P65 translocation
Primary astrocytes from Ikbk2F/F and hGfapcre/Ikbk2F/F mice were seeded directly onto 12mm glass coverslips at 5 x 104 cells/well and treated with saline or 10 μM MPTP and 10 pg/mL TNF and 1 ng/mL IFNγ for 1 hour. Cells were rinsed with PBS and fixed using cold-methanol as described above. Cells were immunolabeled for p65 (polyclonal 1:100, Cell Signaling) and GFAP (monoclonal 1:500, Cell Signaling) with Alexa Fluor 568 and 488 secondary antibodies (Invitrogen), respectively. Using a 40x aprochromat air objective, 10-12 fields per treatment were blindly captured and assessed for positive nuclear p65 per GFAP+ cell per field using Slidebook software. For each genotype and treatment, there was a minimum of 3 biological replicates with 3-4 repetitions of the experiment.
Inflammatory Gene Expression in Cultured Astrocytes
Confluent primary astrocytes cultures from Ikbk2F/F and hGfapcre/Ikbk2F/F mice were treated with saline or 10 μM MPTP and 10 pg/mL TNF and 1 ng/mL IFNγ for 8 hours. Cells were then rinsed with cold PBS and RNA was isolated from glia utilizing the RNeasy Mini Kit (QIAGEN, Valencia, CA) with purity and concentration confirmed using a NanoDrop ND-1000 spectrophotometer. Five hundred ng of RNA were used as a template for reverse transcriptase reactions using the iScript RT kit (Bio-Rad) cDNA was mixed with SYBR Green (Bio-Rad) with primer pairs for Tnf, Nos2, interleukin 1-beta (Il-1β), interleukin 6 (Il-6), chemokine like ligand 2 and 5 (Ccl2 and Ccl5) as published previously (Kirkley et al., 2017).
Statistical Analyses
All statistical analyses were performed using Prism software (version 6.0; Graphpad Software, Inc., San Diego, CA) with a Student’s t-test utilized for comparison of two means, whereas a two-way analysis of variance (ANOVA) followed by a Tukey-Kramer multiple comparison post-hoc test was used for comparison of three or more means. Independent variables for two-way ANOVA were defined as genotype (versus Ikbk2F/F versus hGfapcre/Ikbk2F/F) and treatment (saline versus MPTPp). Statistical significance was defined at a p-value less than 0.05 and indicated by asterisks.