Intermittent fasting (IF) is neuroprotective across a range of insults, but the question of whether extending the interval between meals alters neurogenesis after ischemia remains unexplored. We therefore measured cell proliferation, cell death, and neurogenesis after transient middle cerebral artery occlusion (MCAO) or sham surgery (SHAM) in mice fed ad libitum (AL) or maintained on IF for 3 months. IF was associated with twofold reductions in circulating levels of the adipocyte cytokine leptin in intact mice, but also prevented further reductions in leptin after MCAO. IF/MCAO mice also exhibit infarct volumes that were less than half those of AL/MCAO mice. We observed a 30% increase in basal cell proliferation in the hippocampus and subventricular zone (SVZ) in IF/SHAM, relative to AL/SHAM mice. However, cell proliferation after MCAO was limited in IF mice, which showed twofold increases in cell proliferation relative to IF/SHAM, whereas AL/MCAO mice exhibit fivefold increases relative to AL/SHAM. Attenuation of stroke-induced neurogenesis was correlated with reductions in cell death, with AL/MCAO mice exhibiting twice the number of dying cells relative to IF/MCAO mice. These observations indicate that IF protects against neurological damage in ischemic stroke, with circulating leptin as one possible mediator.
Mitochondrial dysfunction is regarded as one of the major causes of neuronal injury in age-associated neurodegenerative diseases and stroke. Mitochondrial dysfunction leads to increased reactive oxygen species production, causing mitochondrial DNA mutations, which then results in pathological conditions. Negative conditioning of mitochondrial dysfunction via pharmacological inhibition, phytochemicals, and dietary restriction serve as an avenue for therapeutic intervention to improve mitochondrial quality and function. Here, we focus primarily on mitochondrial biology, evidence for mitochondrial dysfunction in neurodegenerative conditions such as dementia and stroke, and the possibility of using negative conditioning to restore or preserve mitochondrial function in these diseases.
Many stroke survivors suffer long‐term functional disabilities with no effective treatment option available. For that reason, we tested the potential effects of enhancing noradrenergic and serotoninergic systems in combination with delayed voluntary running on recovery after stroke in mice. To accomplish this, we used adult male mice (n=12/group) and divided them into sham, stroke, vehicle, or drug‐treated groups with either selective norepinephrine reuptake inhibitor atomoxetine (0.3, 1 mg/kg, once a day i.p.) or selective serotonin reuptake inhibitor fluoxetine (3, 10 mg/kg, once a day i.p.) starting from day 5 after stroke and continued for 12 days. For evaluation of motor function, we used grid walking and cylinder tests 3 days prior and 3, 7, 14, 28, 42 days after using ischemic stroke model (photothrombosis). At 42 days after stroke, mice brains were collected either after fixing the brain through cardiac perfusion (for infarct size measurements and immunohistochemistry experiments after brain sectioning) or as a fresh tissue from the motor cortex area (for immunoblotting experiments).The results of both motor function tests showed that all stroke‐subjected groups had comparable and substantial motor impairment on day 3 after stroke when compared to their baseline and that voluntary running group did not significantly improve throughout the days after stroke. However, administrating either atomoxetine or fluoxetine along with exercise promoted motor recovery at 42 days after stroke dose‐dependently with no significant changes seen in infarct size among groups (0.5 ± 0.1 mm 3 ). Further, immunoblotting experiment showed no significant changes amongst groups in synaptophysin, an integral membrane glycoprotein of neuronal synaptic vesicles, nor with PSD‐95, an excitatory postsynaptic density scaffolding protein, after normalization to beta actin in the ischemic hemisphere. However, the immunohistochemistry data showed that atomoxetine and fluoxetine treatment significantly decreased the expression of parvalbumin (PV), a calcium binding albumin protein associated with depressing synapses, in the boundaries of medial agranular cortex and medial frontal cortex areas of the ischemic hemisphere when compared to their corresponding area in the healthy hemisphere. Whereas no significant relative changes seen with the expression of PV showed between the healthy and ischemic hemispheres of sham, stroke, and vehicle groups. Our data showed that low duration voluntary running does not facilitate motor recovery independently after ischemic stroke in mice. But it promoted recovery of motor function only when combined with atomoxetine or fluoxetine in a dose‐dependent manner. Our data showed a significant decrease of the inhibitory interneurons PV concomitant with the recovery seen with atomoxetine and fluoxetine treatment. Our ongoing experiments include the evaluation of growth associated protein (GAP‐43) and glutamate receptors expression. Support or Funding Information Research in the Karamyan laboratory is supported by R01NS106879 Enhanced motor recovery of mice treated with atomoxetine or fluoxetine after an experimental stroke in the grid walking test (A) and the cylinder test (B). Figure 1 Atomoxetine and fluoxetine treatment significantly decreased parvalbumin expression in the ischemic hemisphere compared to the healthy hemisphere on 42 days after stroke. Figure 2
<i>Background/Aims:</i> Changes in the glucocorticoid milieu contribute to alterations in neurotropic factor expression across multiple brain regions. Insulin-resistant diabetes is often accompanied by dysregulation of adrenal steroid production in humans and animal models. Leptin receptor-deficient mice (<i>db/db)</i> show reduced expression of brain-derived neurotropic factor (BDNF) in the hippocampus and increases in circulating corticosterone levels, but the extent to which elevated corticosterone levels mediate deficits in BDNF expression has not been determined. <i>Methods:</i> Using in situ hybridization, we measured the expression of BDNF, its receptor TrkB, and neurotropin-3 (NT-3) in the hippocampus and hypothalamus of <i>db/db</i> mice and wild-type controls following adrenalectomy and low-dose corticosterone replacement (ADX+CORT) or sham operation. <i>Results:</i> Lowering corticosterone levels restored BDNF and TrkB expression in the hippocampus of <i>db/db</i> mice. However, deficits in hypothalamic BDNF expression were not reversed following ADX+CORT. There was no effect of genotype or adrenalectomy on NT-3 expression in any brain region examined. <i>Conclusion:</i> Leptin receptor-deficient mice exhibit reduced BDNF expression in the hippocampus and hypothalamus. In the <i>db/db</i> mouse hippocampus, suppression of BDNF occurs in a glucocorticoid-dependent fashion, while hypothalamic BDNF expression is reduced via glucocorticoid-independent mechanisms. Region-specific signals therefore play a role in the interaction between corticosteroids and neurotropic factor expression.
Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.
Full text Figures and data Side by side Abstract eLife assessment Introduction Materials and methods Results Discussion Data availability References Peer review Author response Article and author information Metrics Abstract Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart’s function and its vulnerability to injury and disease. eLife assessment This study provides a useful catalog of the cardiac proteome and transcriptome in response to intermittent fasting. Although mechanistic integration is limited, the technical aspects have been executed in a solid way, and sufficient evidence is provided to support the main conclusions. Future work can build on this study to expand our understanding of the relationship between dietary perturbations and cardiac function. https://doi.org/10.7554/eLife.89214.2.sa0 About eLife assessments Introduction In the developed world, the average calorie intake has steadily risen, as have associated age-related diseases (Lahey and Khan, 2018). Intermittent fasting (IF) eating patterns include frequent periods of 12–24 hr with little or no energy intake sufficient to trigger a metabolic switch from the utilization of liver-derived glucose to fatty acids and ketones. Such IF eating patterns increase health span and lifespan and delay or prevent age-associated diseases in animals (de Cabo and Mattson, 2019; Mattson et al., 2017). Rodents maintained on IF exhibit reduced resting heart rate and blood pressure, increased heart rate variability, and improved cardiovascular adaptations to stress (Mager et al., 2006; Wan et al., 2003a; Wan et al., 2003b; Wan et al., 2014). Age-related increases in oxidative stress, inflammation, and fibrosis in the heart are prevented by every-other-day (EOD) fasting (Castello et al., 2011). IF also improves health indicators and cardiovascular risk factors in human subjects (Harvie et al., 2011; Harvie et al., 2013; Stein et al., 2012; Weiss and Fontana, 2011). In a recent randomized controlled trial, alternate day fasting improved cardiovascular markers, reduced fat mass, fat-to-lean ratio, and increased levels of the ketone β-hydroxybutyrate (BHB) (Stekovic et al., 2019). IF protects the brain and heart against ischemic injury and enhances endurance in animal models (Arumugam et al., 2010; Kim et al., 2018; Marosi et al., 2018; Mattson and Arumugam, 2018; Wan et al., 2010). Animals maintained on EOD fasting prior to experimental myocardial infarction (MI) have a reduced myocardial infarct size compared to the ad libitum (AL) fed controls (Ahmet et al., 2011; Ahmet et al., 2005; Godar et al., 2015). IF also improves survival and recovery of heart function when initiated two weeks after MI (Katare et al., 2009). Recent brain and skeletal muscle studies suggest that IF engages signaling pathways that enhance cellular stress resistance, mitochondrial biogenesis, and autophagy (Marosi et al., 2018; Mattson and Arumugam, 2018). The ketone BHB may mediate some cellular adaptations to fasting, including inducing the expression of the inhibitory neurotransmitter γ-aminobutyric acid, trophic factors, and the cytoprotective mitochondrial deacetylase sirtuin 3 (Cheng et al., 2016; Liu et al., 2019; Marosi et al., 2016; Newman and Verdin, 2017; Yudkoff et al., 2007). However, it is unknown whether similar molecular and cellular mechanisms underlie the beneficial effects of IF on the cardiovascular system. System-wide interrogation using large-scale omics technologies has proven powerful in capturing snapshots of molecular abundances in an unbiased manner. Previous studies have extensively focused on understanding the metabolic rewiring associated with different modes of fasting and feeding responses in the liver, the primary metabolic and secretory organ, and adipose tissues that functions as a fat sensor (Ng et al., 2019). Though transcriptomics remained the go-to technology for many of the earlier works in this arena, recent technological advancements have positioned mass spectrometry (MS)-based proteomics as a rational choice to simultaneously track the dynamic behavior of thousands of proteins, the prime orchestrators of cellular processes, in a global manner. Accordingly, proteotypes underlying various metabolic diseases, dietary milieu, and fasting regimens including time-restricted feeding or IF have been probed to get holistic molecular portraits of altered metabolic states (Shaik et al., 2016; Hatchwell et al., 2020). Protein post-translational modifications, especially phosphorylation, underlie several energy sensor pathways, glucose homeostasis mechanisms, and insulin secretion process, and hence deconvolution of phosphorylation networks is key to understanding signaling alterations (Sacco et al., 2019; Sacco et al., 2016). While the importance of IF in improving cardiovascular health has been extensively documented, a global investigation documenting the molecular players driven by the metabolic switch would be highly desirable. In order to understand molecular and cellular remodeling of the heart during IF, we employed advanced mass spectrometry for system-wide profiling of the proteome and phosphoproteome of heart tissues obtained from mice with daily fasting for 12 hr (IF12), 16 hr (IF16) or EOD as well as AL control feeding. We also performed transcriptome analyses using RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our results indicated that IF profoundly modifies pathways involved in metabolism, cell signaling, and epigenetic reprogramming in the heart. Of note, we observed that protein network remodeling during IF occurred in an IF duration time-dependent manner, with IF16 and EOD having the most significant effects. In addition, functional studies indicated that IF improves cardiac function compared to AL feeding. Our findings provide novel insight into the genetic and proteomic changes by which IF improves cardiac health and provide a resource for investigators in the fields of metabolism and cardiovascular disease. Materials and methods Animals and IF procedures Request a detailed protocol All in vivo experimental procedures were approved by the National University of Singapore (Ethics approval number: R15-1568) and La Trobe University (Ethics approval number: AEC21012) Animal Care and Use Committees and performed according to the guidelines set forth by the National Advisory Committee for Laboratory Animal Research (NACLAR), Singapore, and Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition) and confirmed NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering and the number of animals used. All sections of the manuscript were performed in accordance with ARRIVE guidelines. Animals were anaesthetized in 1–2% isoflurane, and anesthesia was confirmed by abolished pain reflexes. Animals were euthanized by cervical dislocation, and the chest was then opened to extract the heart. C57BL/6 male mice were purchased at 2 months of age (InVivos, Singapore; ARC, Australia) and housed in the animal facility at the National University of Singapore and La Trobe University. The animals were exposed to light from 07:00-19:00 (12 hr light: 12 hr dark cycle) and were fed with pellets for rodents which consisted of 58%, 24%, and 18% of calories originating from carbohydrate, protein, and fat, respectively (Teklad Global 18% Protein rodent diet #2918; Envigo, Madison, WI, USA). Water was provided ad libitum to all dietary groups. At the age of 3 months, the mice were randomly assigned to the four different dietary intervention groups: IF12 (N=20), IF16 (N=60), EOD (Every Other Day fasting) (N=20), and AL group as a control (N=60). The mice in the IF12, IF16, and EOD groups fasted for 6 months daily 12 hr (19:00-07:00), 16 hr (15:00-07:00), or 24 hr (a whole day fasting followed by an entire day feeding). The AL group was supplied with food pellets ad libitum. The body weight was regularly measured. Blood glucose and ketone levels using FreeStyle Optium Neo system with FreeStyle Optium blood glucose and ketone test strips (Abbott Laboratories, Berkshire, UK) were measured in the morning before IF animals received food (N=20 in each experimental group). After 6 months, animals were anesthetized and euthanized. EOD mice were euthanized on a food-deprivation day. All mice were euthanized under isoflurane anesthesia between 7 a.m. and noon. All mice were perfused with cold PBS. The chest cavity was rapidly opened, and the heart was removed and rinsed in two washes of ice-cold saline. Major blood vessels and connective tissue were removed, and the heart was blotted dry, and weighed. The heart samples were collected and kept at –80 °C until further use. Sample preparation for proteome analyses Request a detailed protocol Frozen tissue samples from all groups (Number of animals in AL = 4, IF12=5, IF16=5, EOD = 4) were crushed in liquid nitrogen prior to lysis with 6 M urea (Sigma-Aldrich), 2 M thiourea (Invitrogen), and 20 mM HEPES. The lysate was collected after centrifugation at 20,000 g for 20 min at 15 °C, and protein measurement was made using Pierce 660 nm Protein Assay Reagent (Thermo Scientific) according to the manufacturer’s protocol. Fifty μg of protein was used to prepare for each TMT label. For the reference channel of 126-TMT label, a total of 50 μg protein pooled from the 4 ALs was used. The reduction was performed with a final concentration 5 mM dithiothreitol (Sigma-Aldrich) incubation for 30 min. This was followed by alkylation with a final concentration of 10 mM iodoacetamide (Sigma-Aldrich) for 30 min incubation in the dark. Digestion was carried out using lysyl endopeptidase digestion (Wako Chemicals GmbH) with enzyme: protein = 1:100 at 37 °C overnight. The urea in the lysis buffer was then diluted to a final concentration of 1 M with 50 mM ammonium bicarbonate followed by trypsin digestion (Promega) with enzyme: protein = 1:50 at 37 °C for 8 hr. Digested peptides were desalted using 1 ml Empore C18 cartridges (3 M) before isobaric labeling. Sample preparation for phosphoproteome analysis Request a detailed protocol A total of three animals from each group were used for phosphoproteome analysis. Immobilized metal affinity chromatography (IMAC) was employed for the selective enrichment of phosphorylated peptides. Briefly, Ni-NTA agarose conjugates (Qiagen) were rinsed thrice with MilliQ water and incubated with 100 mM EDTA pH 8.0 for 30 min at RT to strip the nickel. Residual nickel was rinsed thrice with MilliQ water, followed by incubation with 100 mM iron chloride solution for 30–45 min. The resin was then washed thrice with MilliQ water followed by 80% acetonitrile / 0.1% trifluoroacetic acid. The tryptic peptides were then enriched using the iron chloride treated IMAC beads. Tryptic peptides were reconstituted in 50% acetonitrile/0.1% trifluoroacetic acid, followed by 1:1 dilution with 99.9% acetonitrile/0.1% trifluoroacetic acid. The peptides were incubated with 10 μL of IMAC beads for 30 min with end-over-end rotation, and the beads were subsequently loaded onto self-packed C18 stage tips (Rappsilber et al., 2007) pretreated with 200 μl methanol, washed with 50% acetonitrile/0.1% formic acid and equilibrated with 1% formic acid. Following loading onto the stage tip, the IMAC beads were washed with 80% acetonitrile/0.1% trifluoroacetic acid and 1% formic acid. The phosphopeptides were eluted from the IMAC beads onto C18 membranes using 500 mM dibasic sodium phosphate (pH 7.0), followed by washing using 1% formic acid. The phosphopeptides were stored on the stage tip until they were ready to be analysed by liquid chromatography-mass spectrometry (LC-MS). The phosphopeptides were eluted from the C18 membranes with 50% acetonitrile/0.1% formic acid, dried using a speed vac, and reconstituted in 0.1% formic acid for LC-MS analysis. Isobaric labeling Request a detailed protocol All samples were run in a total of two sets of TMT10plex Isobaric Label Reagent Set (Thermo Scientific) and were assigned to each TMT set (9 samples to 9 plexes and an internal reference sample made up of a mixture of the ALs) by randomization. Isobaric labelling was performed according to the manufacturer’s protocol. Briefly, the TMT label was reconstituted in anhydrous acetonitrile (Sigma-Aldrich) before mixing with respective peptides and incubated for 1 hr. The reaction was quenched using the final 5% hydroxylamine (Merck) in triethylammonium hydrogen carbonate buffer (Fluka) for 15 min. Labeled peptides were mixed before being vacuum-dried and fractionated using basic reverse phase chromatography (Shimadzu NexeraXR high performance liquid chromatography). Fractions were combined orthogonally into 15 fractions and dried using a vacuum centrifuge. Liquid chromatography and mass spectrometry Request a detailed protocol For proteome analysis, peptides were reconstituted in 0.1% formic acid for analysis on Thermo Easy nLC 1000 that was connected to Q Exactive Plus Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). The trap column used was C18 Acclaim PepMap 100 of 5 μm, 100 A, 100 μm I.D. x 2 cm, and the analytical column was PepMap RSLC C18, 2 μm, 100 A, 75 μm I.D. x 50 cm. The LC solvent A comprised of 0.1 M formic acid in 2% acetonitrile, and LC solvent B comprised 0.1 M formic acid in 95% acetonitrile. The gradient was as follows: 5–50% solvent B in 180 min; 50–100% solvent B in 10 min; 100% solvent B for 10 min at the flow rate of 200 nL/min. The mass spectrometer was set in the data dependent acquisition mode. Full scan MS spectra (m/z 380–1600) were acquired with a resolution of R=140,000 at an AGC target of 3e6 and a maximum injection time of 60ms. The top 20 most intense peptide ions in each MS scan were sequentially isolated to an ACG target value of 1e5 with a resolution of 35,000 and fragmented using a normalized collision energy of 30 at MS2 level with fixed first mass at 110 and an isolation window of 1.2 m/z. For phosphoproteome, reconstituted peptides were analysed using an EASY-nLC 1000 (Proxeon, Thermo Fisher Scientific) attached to a Q-Exactive (Thermo Fisher Scientific). Peptides were enriched using a C18 precolumn and separated on a 50 cm analytical column (EASY-Spray Columns, Thermo Fisher Scientific) at 50 °C using a 265 min gradient ranging from 0 to 40% acetonitrile/0.1% formic acid, followed by a 10 min gradient ranging from 40 to 80% acetonitrile/0.1% formic acid and maintained for 10 min at 80% acetonitrile/0.1% formic acid. Survey full scan MS spectra (m/z 310–2000) were collected with a resolution of r=70,000,, an AGC target of 3e6, and a maximum injection time of 10ms. Twenty of the most intense peptide ions in each survey scan with an intensity threshold of 10,000, underfill ratio of 1%, and a charge state ≥2 were sequentially isolated with a window of 2 Th to a target value of 50,000 with a maximum injection time of 50ms. These were fragmented in the high energy collision cell by dissociation, using a normalized collision energy of 25%. The MS/MS was acquired with a starting mass of m/z 100 and a resolution of 17,500 and dynamic exclusion of duration of 15 s. Total RNA extraction and quality control for transcriptome analyses Request a detailed protocol EZ-10 Total RNA Mini-Prep Isolation kit (Bio Basic, Canada) was used according to the kit’s instructions to extract total RNA from the heart samples. To assess the quality of the extracted total RNA, agarose gel electrophoresis and Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA) were used. All RNA samples (N=5 in each experimental group) had high quality with integrity numbers >6.9 for total RNA. cDNA library preparation and RNA sequencing Request a detailed protocol Poly-T oligo-attached magnetic beads were used to purify the mRNA from total RNA. Then, a fragmentation buffer was added to the mRNA. First-strand cDNA was subsequently synthesized using random hexamer primer and M-MuLV reverse transcriptase (RNase H-; New England BioLabs, Ipswich, MA, USA). Next, second-strand cRNA synthesis was performed using DNA polymerase I and RNase H. AMPure XP beads (Beckman Colter Life Sciences, Indianapolis, IN, USA) were used to purify the double-stranded cDNA. By using exonuclease/polymerase activities, the remaining overhangs of the purified double-stranded cDNA were transformed into blunt ends. The 3’ ends of the DNA fragments were adenylated, followed by ligation of the NEBNext adaptor with a hairpin loop structure to prepare for hybridization. The library fragments were purified with the AMPure XP system to select cDNA with the base pair length of 150–200. Lastly, the library was amplified by polymerase chain reaction (PCR), and the PCR products were purified using AMPure XP beads. The HiSeq 2500 platform (Illumina, San Diego, CA, USA) was used to conduct the high-throughput sequencing. Transcriptome data mapping and differential expression analyses Request a detailed protocol The RNA sequencing results from the HiSeq system were output as quality and color space fasta files. The files were mapped to the Ensembl-released mouse genome sequence and annotation (Mus musculus; GRCm38/mm10). Bowtie v.2.0.6 Indexes were used to build the reference genome, and TopHat v.2.0.9 with a mismatch parameter limited to 2 was used to align the paired-end clean reads to the reference genome. A total of 48.5 million clean reads were generated per sample, and 43.7 million reads (90.1% of total clean reads) per sample were mapped to the reference genome. HTSeq v.0.6.1 was used for the quantification of gene expression levels to count the read numbers mapped of each gene. Then FPKMs of each gene were calculated based on the length of the gene and reads count mapped to the same gene. DESeq R Package v.1.10.1 was used to perform differential expression analysis. To control the false discovery rate, the p-values resulting from the analysis were adjusted to q-values using Benjamini and Hochberg’s approach. Genes with q-value <0.05 found by DESeq were assigned as differentially expressed. Heat map generation and functional enrichment analyses Request a detailed protocol R and the R package heatmap3 were used along with the log2 fold change output from EdgeR v.3.2.4 to create heatmaps of DEGs. To estimate the possible biologic functional changes derived from the gene expression changes, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. GO enrichment analysis was implemented by the GOseq R package, in which gene length bias was corrected. For KEGG pathway enrichment analysis, we used KEGG Orthology Based Annotation System (KOBAS) software to test the statistical enrichment. GO terms and KEGG pathways with a q-value less than 0.05 were considered significantly enriched by DEGs. Proteome and phosphoproteome data processing Request a detailed protocol For 10plex-TMT labeled proteome analysis, raw MS data were processed using Proteome Discoverer 2.2 (Thermo Scientific). Database search was performed using the integrated Sequest HT search engine against the Uniprot mouse FASTA database (release January 2018) for tryptic peptides with a maximum of two missed cleavage sites, MS and MS/MS mass tolerance of 10 ppm and 0.02 Da, respectively. Searches included cysteine carbamidomethylation and TMT-modifications at peptide N-termini and lysine residues as fixed modifications and protein N-terminal acetylation and methionine oxidation as dynamic modifications. Peptide and protein identifications were performed at a false discovery rate (FDR)<0.01. For phosphoproteome analysis, raw MS data were processed using MaxQuant version 1.6.0.1 (Tyanova et al., 2016a). The Andromeda search engine was employed to search against the UniProt mouse FASTA database. For identification of proteins, peptides, and modifications, the FDR was set at 0.01, allowing a maximum of two missed cleavages as well as an initial mass tolerance of 4.5 ppm for precursor ions and 20 ppm for fragment ions. The search was performed with phosphorylation of sites serine, threonine, and tyrosine (STY), oxidation of methionine, and acetylation of protein N-term as variable modification, cysteine carbamidomethylation as fixed modification, and trypsin as cleaving enzyme. Label-free quantitation (LFQ) was performed using the MaxLFQ algorithm as implemented in MaxQuant with minimum ratio count set to 1 and match between run feature enabled to transfer peptide identifications across MS runs (match time window = 2, alignment time window = 20) (Cox et al., 2014). A minimum Andromeda score of 40 was used for phosphopeptide identification. Proteome and phosphoproteome data analyses Request a detailed protocol For TMT-based proteome analysis, the common internal reference, pooled AL sample, at the 126 TMT-reporter channel of each TMT set was used for normalization across the two sets. The pooled ad libitum was included as an internal reference for the TMT sets as our focus was on inferring proteome abundance changes and associated biological pathways across the different IF regimens in comparison with the control ad libitum group. Protein abundances across all the channels were first normalized for equal total protein intensities before obtaining log2 ratios of each protein against the normalized intensity of the respective reference channel. After combining the two TMT sets, only those proteins that were quantified in at least three biological replicates within each group were retained for subsequent analysis. For differential expression analysis of proteins regulated with IF, the replicates from the two TMT sets were grouped, and one-way analysis of variance (ANOVA) was performed with followed by Dunnett’s post hoc tests at adjusted p-value <0.01 to test for pairwise significance testing against AL control group. Protein annotations for gene ontology (GO) terms (biological process, cellular component, and molecular function) and KEGG pathways were obtained, and functional enrichment analysis was performed using Fisher’s exact test (FDR ≤0.05) for the modulated clusters. In addition, 1-D annotation enrichment analysis as implemented in Perseus was performed for testing the preference of protein expression values to be higher or lower in comparison to the global distribution for specific functional groups (Tyanova et al., 2016b). The FDR was controlled at a threshold of 0.05, and for each functional group, a score was assigned, with scores those higher than zero and close to 1 confirming to positive enrichment, and those lesser than zero and close to –1 representing negative enrichment (Subramanian et al., 2005). To construct functional networks over the different IF regimens, gene-set enrichment analysis (GSEA) was performed on the proteome data using all curated mouse GO biological processes and pathways. Pairwise comparisons of the different groups were performed, and the statistical significance of the enrichment score was assessed by a permutation test. Based on the similarity between enriched gene sets (P-value <0.01), the EnrichmentMap plugin, as implemented in Cytoscape, was used to visualize all overlapping gene set clusters (Merico et al., 2010). All heat maps corresponding to functional categories were visualized using an internal log-transformed reference normalized abundance value for each protein (Plubell et al., 2017). Transcription factor enrichment analysis for significant proteins in each IF group was performed using the Chip Enrichment Analysis (ChEA) implemented in the Enrichr web tool (Kuleshov et al., 2016; Lachmann et al., 2010). For phosphoproteome analysis, the label-free intensities of all identified phosphopeptides were log-transformed, and only those phosphopeptides quantified in at least two biological replicates of each group were retained for all subsequent analyses. Using the Perseus software environment, missing values were imputed to represent intensities of low abundant proteins based on random numbers drawn from a normal distribution in each sample (Tyanova et al., 2016b). Phosphorylation sites differentially regulated between the groups were identified using one-way ANOVA followed by Dunnett’s post hoc test at an adjusted p-value ≤0.05. Biological processes and KEGG pathways for differentially altered phosphoproteins were derived from DAVID bioinformatics resources (Huang et al., 2009). For upstream kinase enrichment analysis, kinase substrate motifs were extracted from the human protein reference database (HPRD), and enrichment analysis was performed using Fisher’s exact test for altered phosphosite clusters across the groups. Comparison of proteome and transcriptome data Request a detailed protocol For the evaluation of the proteome against the transcriptome, only those transcripts with corresponding protein quantification were used. Correlation analysis was performed using FPKM as a proxy for mRNA abundance and normalized protein abundance derived from TMT experiments for protein quantification. Data were log-transformed prior to visualization. For comparison of transcriptome and proteome changes across different IF groups, log-transformed fold changes relative to AL group were used as input to perform two dimensional (2-D) functional enrichment analyses as implemented in Perseus (Tyanova et al., 2016b). The respective annotation matrix of KEGG pathways and GO terms (biological process) of both the transcriptome and proteome were compared for significant bias to larger or smaller expression values in comparison to overall distribution. The FDR was controlled at a 0.05 threshold and position score was assigned to denote enrichment at higher (0
Although the beta2-integrins have been implicated in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury, the relative contributions of the alpha-subunits to the pathogenesis of ischemic stroke remains unclear. The objective of this study was to determine whether and how genetic deficiency of either lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 (Mac-1) alters the blood cell-endothelial cell interactions, tissue injury, and organ dysfunction in the mouse brain exposed to focal I/R. Middle cerebral artery occlusion was induced for 1 h (followed by either 4 or 24 h of reperfusion) in wild-type mice and in mice with null mutations for either LFA-1 or Mac-1. Neurological deficit and infarct volume were monitored for 24 h after reperfusion. Platelet- and leukocyte-vessel wall adhesive interactions were monitored in cortical venules by intravital microscopy. Mice with null mutations for LFA-1 or Mac-1 exhibited significant reductions in infarct volume. This was associated with a significant improvement in the I/R-induced neurological deficit. Leukocyte adhesion in cerebral venules did not differ between wild-type and mutant mice at 4 h after reperfusion. However, after 24 h of reperfusion, leukocyte adhesion was reduced in both LFA-1- and Mac-1-deficient mice compared with their wild-type counterparts. Platelet adhesion was also reduced at both 4 and 24 h after reperfusion in the LFA-1- and Mac-1-deficient mice. These findings indicate that both alpha-subunits of the beta2-integrins contribute to the brain injury and blood cell-vessel wall interactions that are associated with transient focal cerebral ischemia.
Mitochondrial dysfunction and oxidative stress are frequently observed in the early stages of Alzheimer's disease (AD). Studies have shown that presenilin-1 (PS1), the catalytic subunit of γ-secretase whose mutation is linked to familial AD (FAD), localizes to the mitochondrial membrane and regulates its homeostasis. Thus, we investigated how five PS1 mutations (A431E, E280A, H163R, M146V, and Δexon9) observed in FAD affect mitochondrial functions. Methods: We used H4 glioblastoma cell lines genetically engineered to inducibly express either the wild-type PS1 or one of the five PS1 mutants in order to examine mitochondrial morphology, dynamics, membrane potential, ATP production, mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), oxidative stress, and bioenergetics. Furthermore, we used brains of PS1M146V knock-in mice, 3xTg-AD mice, and human AD patients in order to investigate the role of PS1 in regulating MAMs formation. Results: Each PS1 mutant exhibited slightly different mitochondrial dysfunction. Δexon9 mutant induced mitochondrial fragmentation while A431E, E280A, H163R, and M146V mutants increased MAMs formation. A431E, E280A, M146V, and Δexon9 mutants also induced mitochondrial ROS production. A431E mutant impaired both complex I and peroxidase activity while M146V mutant only impaired peroxidase activity. All PS1 mutants compromised mitochondrial membrane potential and cellular ATP levels were reduced by A431E, M146V, and Δexon9 mutants. Through comparative profiling of hippocampal gene expression in PS1M146V knock-in mice, we found that PS1M146V upregulates Atlastin 2 (ATL2) expression level, which increases ER-mitochondria contacts. Down-regulation of ATL2 after PS1 mutant induction rescued abnormally elevated ER-mitochondria interactions back to the normal level. Moreover, ATL2 expression levels were significantly elevated in the brains of 3xTg-AD mice and AD patients. Conclusions: Overall, our findings suggest that each of the five FAD-linked PS1 mutations has a deleterious effect on mitochondrial functions in a variety of ways. The adverse effects of PS1 mutations on mitochondria may contribute to MAMs formation and oxidative stress resulting in an accelerated age of disease onset in people harboring mutant PS1.
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