Article Figures & Data
Figures
- Figure 1.
Expression of immune-related genes from whole-brain lysates. Average values and SEM for the relative change in mRNA levels are shown for immune-related genes from whole-brain lysate samples from CVN-AD, mNos2−/−, WT (C57BL/6), and APPSwDI mice. A–D, Genes that were upregulated in CVN-AD mice before 36 weeks of age. E–I, Genes that were upregulated in CVN-AD mice at or after 36 weeks of age. J–L, Gene expression of chemokine receptors. mRNA expression was measured using quantitative RT-PCR and represent the 2(−delta delta C(T)) compared with WT mice. Samples were analyzed by two-way ANOVA and post hoc multiple comparison test with Bonferonni's correction. *Comparisons between CVN-AD or APPSwDI and WT. #Comparisons between CVN-AD and APPSwDI mice. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. #p < 0.05. ##p < 0.01. ###p < 0.001. ####p < 0.0001. n > 5 mice per group.
- Figure 2.
CVN-AD amyloid deposition is associated with cells expressing characteristic markers of microglia. Representative sagittal sections from a CVN-AD mouse at 6, 12, 24, and 52 weeks of age immunostained for Aβ (A–D), CD45 (E–H), Iba-1 (I–L), and CD11c (M–P). For each age, panels represent sister sections from the same mouse. For example, A, E, I, and M are sections from the same 6-week-old mouse. Scale bar, 500 μm. Q, Representative micrographs of CD11c and Iba-1 costaining in the subiculum of the hippocampus from a 24-week-old CVN-AD mouse. Scale bar, 25 μm. R, Increased CD11c immunostaining is associated with increased total brain gene expression of CD11c (Itgax). Gene expression levels (mean ± SEM) were measured using quantitative RT-PCR and represent the 2(−delta delta C(T)) compared with WT mice. Samples were analyzed by two-way ANOVA and post hoc multiple comparison test with Bonferonni's correction. *Comparisons between CVN-AD or APPSwDI and WT. #Comparisons between CVN-AD and APPSwDI mice. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. #p < 0.05. ##p < 0.01. ###p < 0.001. ####p < 0.0001. n > 5 mice per group.
- Figure 3.
CD11c+ cells from CVN-AD brains have a microglial phenotype. A, Representative flow cytometry plots from aged 48-week-old mNos2−/− and CVN-AD brains after gating on CD45+ cells. B, Quantification of total CD45+ cells and individual cell types distinguished by FACS in 48-week-old mNos2−/− and CVN-AD brains, including T lymphocytes (T), B lymphocytes (B), neutrophils (PMN), monocytes/macrophages/DCs (Mac/DC), and microglia (MG) from aged 48-week-old mNos2−/− and CVN-AD brains. *p < 0.05; ****p < 0.0001. C, Representative flow plots of CD45 and CD11c expression from aged 48-week-old mNos2−/− and CVN-AD brains after gating on all CD11b+ cells. D, Representative histogram of CD11c expression on mNos2−/− (closed gray) and CVN-AD (open black) CD11b+ CD45low cells, as well as quantitative summaries of the percentages and geometric mean fluorescence intensities of CD11c from mNos2−/− and CVN-AD CD11b+ CD45low microglia. *p < 0.01. n = 4 mice per group.
- Figure 4.
CVN-AD pathology is associated with arginase-1. A, Representative sagittal sections from CVN-AD mice at 6, 12, 24, and 52 weeks of age stained for arginase-1 in sister sections from the same mice as in Figure 1. Scale bar, 500 μm. B, Magnified view of arginase immunoreactivity in the subiculum. Representative sagittal sections from 24-week-old mNos2−/− and CVN-AD stained for arginase-1. Scale bars: top, 500 μm; bottom, 50 μm. C, Sister coronal sections from the same 52-week-old CVN-AD brain stained for Aβ, CD11c, Iba-1, and arginase-1 to show regional associations.
- Figure 5.
CVN-AD brains have decreased total l-arginine bioavailability and increased expression of arginine transporters. A, Global arginine bioavailability (arginine/(ornithine + citrulline)) for CVN-AD, mNos2−/−, WT, and APPSwDI mice. Average values (± SEM) per genotype were calculated for individual mice (n = 3–12 mice per group). Amino acid levels were measured using HILIC LC-MS/MS. **p < 0.01 (one-way ANOVA). B–E, Relative gene expression (mean ± SEM) was measured in total brain homogenates from CVN-AD, mNos2−/−, WT, and APPSwDI mice for the neutral arginine transporters Slc7a5 (LAT1) and Slc7a8 (LAT2) (B, C) and for the cationic amino acid transporters Slc7a1 (CAT1) and Slc7a2 (CAT2) (D, E). Samples were analyzed by two-way ANOVA and post hoc multiple comparison test with Bonferonni's correction. *Comparisons between CVN-AD or APPSwDI and WT. #Comparisons between CVN-AD and APPSwDI mice. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. #p < 0.05. ##p < 0.01. ###p < 0.001. ####p < 0.0001. n = 4–8 mice per group.
- Figure 6.
CVN-AD memory deficits and pathology are reversed by an inhibitor of arginine utilization. A, RAWM assessment of spatial memory acquisition and recall in CVN-AD mice treated with putrescine (put.) alone or putrescine and DFMO (10 mg/kg) by oral gavage for 3 d/week for 14 weeks. All mice were naive to the behavioral procedure and tested on the final 2 d of treatment. Day 1 depicts 5 trial groups for the acquisition phase (learning), and day 2 depicts successive trials for memory recall. Data represent the average number of errors (± SEM) made finding the escape platform for each group of trials. Open circles represent CVN-AD mice treated with putrescine and vehicle. Closed circles represent CVN-AD mice treated with DFMO and putrescine. Data were analyzed by two-way ANOVA and post hoc multiple comparison test with Bonferonni's correction. ***p < 0.001. n = 7 or 8 mice per group. B, Soluble and insoluble Aβ40 and Aβ42 peptides in total brain homogenates from CVN-AD mice treated with vehicle containing putrescine only or putrescine plus DFMO as measured by ELISA. Data represent average levels (± SEM) of Aβ40 or Aβ42 peptides. ***p < 0.001 for DFMO-treated compared with putrescine/vehicle-treated using an unpaired Student's t test. n = 7 or 8 mice/group. C, DFMO treatment alters mRNA levels of immune genes. Average mRNA expression levels (± SEM) for Itgax, Pdcd1 (programmed death receptor 1), Arg1, and Ssat were measured in total brain lysates from CVN-AD mice treated with putrescine in vehicle or putrescine and DFMO. Gene expression levels were measured using quantitative RT-PCR and represent the 2(−delta delta C(T)) with untreated mice as the comparator. Significance between untreated and treated was determined using the unpaired Student's t test. *p < 0.05. **p < 0.01. n = 7 or 8 mice per group. D, Representative sagittal sections from CVN-AD mice treated with either putrescine alone or putrescine and DFMO stained for Aβ or CD11c. For each treatment group, panels represent sister sections from the same mouse. Scale bar, 500 μm.
- Figure 7.
Simplified schematic of arginine catabolism and the actions of DFMO to block arginine utilization. NOHA, N-hydroxyarginine. Gray text indicates a reduction of this protein/product in CVN-AD mice.
Tables
- Table 1.
Gene expression in CVN-AD CD11c+ microglia compared with CVN-AD CD11c− microglia, C57BL/6 microglia, and mNos2−/− microgliaa
Gene Name Fold change p value Reported actions References Mamdc MAM domain containing protein 2 515 9.7 × 10−7 Glycosaminoglycan binding, linked to negative regulation of synapses Pettem et al., 2013 Spp1 Secreted phosphoprotein 1; osteopontin 515 5.9 × 10−6 Increased in CSF of AD patients; enhances immunosuppression Comi et al., 2010; Sangaletti et al., 2014 Gpnmb Glycoprotein-transmembrane nmb; osteoactivin, Fe65-like1 442 1.5 × 10−6 Tissue repair, M2 state; phagocytic vesicle processing Duffield, 2010 Wfdc17 Whey acidic protein four disulfide core domain 17; AMWAP 136 4.1 × 10−5 Overexpression increases Arg-1, reduces IL-6, IL-1β Karlstetter et al., 2010 Itgax Integrin alpha X (complement component 3 receptor 4 subunit), CD11c 124 5.8 × 10−6 Leukocyte-specific integrin; associated with dendritic cells; phagocytosis of complement-coated particles; found on microglia in AD Akiyama and McGeer, 1990; Tooyama et al., 1990; Becher and Antel, 1996; Butovsky et al., 2007 Gp49a Glycoprotein 49a; Lirb4 107 2.8 × 10−4 Member of inhibitory Ig superfamily; increased IL-4; suppresses LPS; inhibits Fc-gamma-mediated phagocytosis Arm et al., 1997; McCormick et al., 1999 Gng12 Guanine nucleotide binding protein 12 64 1.7 × 10−4 Inhibits LPS-mediated pro-inflammation Larson et al., 2010 Pdcd1 Programmed cell death 1; PD-L1, CD279 51 4.1 × 10−6 Immunoglobin superfamily; shifts microglia to M2 phenotype; regulates Arg-1 activity Yao et al., 2014 Apbb2 Amyloid precursor protein binding protein 2 25.8 2.4 × 10−4 Adaptor protein binds to cytoplasmic domain of APP; polymorphisms associated with dementia in aged population; involved in ECM synthesis by Macs Wright et al., 2005; Grupe et al., 2006; Golanska et al., 2013 TIMP2 Tissue inhibitor of metalloprotease-2 20.7 3.2 × 10−4 Blocks metalloprotease activity; associated with M2 phenotype Ridnour et al., 2007; Wilcock et al., 2011a Igf1 Insulin-like growth factor 1; 2; somatomedin c 27.4 2.0 × 10−5 Found in human microglia, protects from IL-1- and IFN-γ-mediated damage; promotes Aβ clearance Trueba-Sáiz et al., 2013 Igf2 8.0 2.1 × 10−3 Apoe Apolipoprotein E 20.1 3.6 × 10−4 Strongest single gene risk factor for AD, suppresses proinflammatory cytokines Saunders et al., 1996 CD200r Cell surface glycoprotein CD200 receptor 2, OX2R 13.8 3.3 × 10−3 Inhibitory immune receptor found on microglia, marker for M2 activation in human MG, less so in mice Denieffe et al., 2013; Walker and Lue, 2013 Klf6 Kruppel-like factor 6 0.12 4.9 × 10−6 Suppression is linked to M2 phenotype Date et al., 2014 Apobec3 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 0.18 4.1 × 10−4 Promotes antiviral immunity through production of neutralizing antibody Santiago et al., 2008 Ifngr1 Interferon-γ receptor 1; CD119 0.19 9.1 × 10−6 Encodes ligand binding domain for IFN-γ; downregulated by TLR2; IFN-β Curry et al., 2004; Kearney et al., 2013 Siglech Siglec-H 0.42 2.6 × 10−4 DAP12 signaling molecule, microglial “sensome,” decreased with aging Hickman et al., 2013 ↵aSelect genes that were significantly upregulated or downregulated in CVN-AD CD11c+ microglia compared with CVN-AD CD11c− microglia, C57BL/6 microglia, and mNos2−/− microglia, including fold change in expression, p value, and reported actions with references.
