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• W2754683485 abstract "Anti-Aβ clinical trials are currently under way to determine whether preventing amyloid deposition will be beneficial in arresting progression of Alzheimer disease. Both clinical and preclinical studies suggest that antiamyloid strategies are only effective if started at early stages of the disease process in a primary prevention strategy. Because this approach will be difficult to deploy, strategies for secondary prevention aimed at later stages of disease are also needed. In this study, we asked whether combining innate immune activation in the brain with concurrent Aβ suppression could enhance plaque clearance and could improve pathologic outcomes in mice with moderate amyloid pathologic disorder. Starting at 5 months of age, tet-off amyloid precursor protein transgenic mice were treated with doxycycline (dox) to suppress further amyloid precursor protein/Aβ production, and at the same time mice were intracranially injected with adeno-associated virus 1 expressing murine IL-6 (AAV1-mIL-6). Three months later, mice treated with the combination of Aβ suppression and AAV1-mIL-6 showed significantly less plaque pathologic disorder than dox or AAV1-mIL-6 only groups. The combination of AAV1-mIL-6 + dox treatment lowered total plaque burden by >60% versus untreated controls. Treatment with either dox or AAV1-mIL-6 alone was less effective than the combination. Our results suggest a synergistic mechanism by which the up-regulation of mIL-6 was able to improve plaque clearance in the setting of Aβ suppression. Anti-Aβ clinical trials are currently under way to determine whether preventing amyloid deposition will be beneficial in arresting progression of Alzheimer disease. Both clinical and preclinical studies suggest that antiamyloid strategies are only effective if started at early stages of the disease process in a primary prevention strategy. Because this approach will be difficult to deploy, strategies for secondary prevention aimed at later stages of disease are also needed. In this study, we asked whether combining innate immune activation in the brain with concurrent Aβ suppression could enhance plaque clearance and could improve pathologic outcomes in mice with moderate amyloid pathologic disorder. Starting at 5 months of age, tet-off amyloid precursor protein transgenic mice were treated with doxycycline (dox) to suppress further amyloid precursor protein/Aβ production, and at the same time mice were intracranially injected with adeno-associated virus 1 expressing murine IL-6 (AAV1-mIL-6). Three months later, mice treated with the combination of Aβ suppression and AAV1-mIL-6 showed significantly less plaque pathologic disorder than dox or AAV1-mIL-6 only groups. The combination of AAV1-mIL-6 + dox treatment lowered total plaque burden by >60% versus untreated controls. Treatment with either dox or AAV1-mIL-6 alone was less effective than the combination. Our results suggest a synergistic mechanism by which the up-regulation of mIL-6 was able to improve plaque clearance in the setting of Aβ suppression. The amyloid hypothesis of Alzheimer disease (AD) postulates that Aβ accumulation in the brain triggers a pathologic cascade that leads to neurodegeneration and progressive cognitive impairment.1Hardy J. Selkoe D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2002; 297: 353-356Crossref PubMed Scopus (10949) Google Scholar, 2Golde T.E. The Abeta hypothesis: leading us to rationally-designed therapeutic strategies for the treatment or prevention of Alzheimer disease.Brain Pathol. 2005; 15: 84-87Crossref PubMed Scopus (98) Google Scholar, 3Karran E. De Strooper B. The amyloid cascade hypothesis: are we poised for success or failure?.J Neurochem. 2016; 139 Suppl 2: 237-252Crossref PubMed Scopus (244) Google Scholar Recent biomarker and amyloid positron emission tomography imaging studies suggest that brain Aβ amyloidosis may begin several decades before overt neurodegeneration and cognitive deficits.4Jack Jr., C.R. Knopman D.S. Jagust W.J. Shaw L.M. Aisen P.S. Weiner M.W. Petersen R.C. Trojanowski J.Q. Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade.Lancet Neurol. 2010; 9: 119-128Abstract Full Text Full Text PDF PubMed Scopus (3180) Google Scholar, 5Jack Jr., C.R. Knopman D.S. Jagust W.J. Petersen R.C. Weiner M.W. Aisen P.S. Shaw L.M. Vemuri P. Wiste H.J. Weigand S.D. Lesnick T.G. Pankratz V.S. Donohue M.C. Trojanowski J.Q. Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers.Lancet Neurol. 2013; 12: 207-216Abstract Full Text Full Text PDF PubMed Scopus (2635) Google Scholar From these observations, significant time and effort has been devoted in the development of anti-Aβ therapies. Numerous studies of anti-Aβ immunotherapy have shown efficacy in preventing amyloid deposition in AD mouse models (reviewed in Golde et al6Golde T.E. Petrucelli L. Lewis J. Targeting Abeta and tau in Alzheimer's disease, an early interim report.Exp Neurol. 2010; 223: 252-266Crossref PubMed Scopus (76) Google Scholar, 7Golde T.E. Schneider L.S. Koo E.H. Anti-abeta therapeutics in Alzheimer's disease: the need for a paradigm shift.Neuron. 2011; 69: 203-213Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar); however, when advanced to human clinical trials, these approaches have largely failed to stabilize or improve cognitive function.7Golde T.E. Schneider L.S. Koo E.H. Anti-abeta therapeutics in Alzheimer's disease: the need for a paradigm shift.Neuron. 2011; 69: 203-213Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 8Salloway S. Sperling R. Fox N.C. Blennow K. Klunk W. Raskind M. Sabbagh M. Honig L.S. Porsteinsson A.P. Ferris S. Reichert M. Ketter N. Nejadnik B. Guenzler V. Miloslavsky M. Wang D. Lu Y. Lull J. Tudor I.C. Liu E. Grundman M. Yuen E. Black R. Brashear H.R. Bapineuzumab 301 and 302 Clinical Trial InvestigatorsTwo phase 3 trials of bapineuzumab in mild-to-moderate Alzheimer's disease.N Engl J Med. 2014; 370: 322-333Crossref PubMed Scopus (565) Google Scholar, 9Salloway S. Sperling R. Brashear H.R. Phase 3 trials of solanezumab and bapineuzumab for Alzheimer's disease.N Engl J Med. 2014; 370: 1460Crossref PubMed Scopus (1292) Google Scholar, 10Arrighi H.M. Barakos J. Barkhof F. Tampieri D. Jack Jr., C. Melancon D. Morris K. Ketter N. Liu E. Brashear H.R. Amyloid-related imaging abnormalities-haemosiderin (ARIA-H) in patients with Alzheimer's disease treated with bapineuzumab: a historical, prospective secondary analysis.J Neurol Neurosurg Psychiatry. 2016; 87: 106-112PubMed Google Scholar, 11De Strooper B. Chavez Gutierrez L. Learning by failing: ideas and concepts to tackle gamma-secretases in Alzheimer's disease and beyond.Annu Rev Pharmacol Toxicol. 2015; 55: 419-437Crossref PubMed Scopus (97) Google Scholar These failures have led to the assumption that by the time patients are clinically diagnosed with AD, it may be too late for anti-Aβ therapies to be effective.3Karran E. De Strooper B. The amyloid cascade hypothesis: are we poised for success or failure?.J Neurochem. 2016; 139 Suppl 2: 237-252Crossref PubMed Scopus (244) Google Scholar, 7Golde T.E. Schneider L.S. Koo E.H. Anti-abeta therapeutics in Alzheimer's disease: the need for a paradigm shift.Neuron. 2011; 69: 203-213Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar Given that timing is critical, prevention trials are now under way in genetically identified at-risk patients (APOE4 and PSEN1 E280A carriers) and in cognitive normal subjects with preexisting amyloid pathologic disorder (A4 study12Sperling R.A. Rentz D.M. Johnson K.A. Karlawish J. Donohue M. Salmon D.P. Aisen P. The A4 study: stopping AD before symptoms begin?.Sci Transl Med. 2014; 6: 228fs13Crossref PubMed Scopus (498) Google Scholar). Promising results were recently reported in a phase Ib trial of anti-Aβ antibody aducanumab in patients with mild disease, showing reductions of amyloid signal in the brain and some cognitive benefit.13Sevigny J. Chiao P. Bussiere T. Weinreb P.H. Williams L. Maier M. Dunstan R. Salloway S. Chen T. Ling Y. O'Gorman J. Qian F. Arastu M. Li M. Chollate S. Brennan M.S. Quintero-Monzon O. Scannevin R.H. Arnold H.M. Engber T. Rhodes K. Ferrero J. Hang Y. Mikulskis A. Grimm J. Hock C. Nitsch R.M. Sandrock A. The antibody aducanumab reduces Abeta plaques in Alzheimer's disease.Nature. 2016; 537: 50-56Crossref PubMed Scopus (1617) Google Scholar Despite this success, clinical failures continue to beset the AD field, because Merck & Co (Kenilworth, NJ) recently halted their trial of β-site amyloid precursor protein enzyme (BACE)1 inhibitor verubecestat in mild-to-moderate AD for lack of efficacy.14Mullard A. BACE inhibitor bust in Alzheimer trial.Nat Rev Drug Discov. 2017; 16: 155Google Scholar Clearly, more aggressive or novel treatment strategies are needed for patients with moderate or advanced disease.15Sala Frigerio C. De Strooper B. Alzheimer's disease mechanisms and emerging roads to novel therapeutics.Annu Rev Neurosci. 2016; 39: 57-79Crossref PubMed Scopus (82) Google Scholar We had previously demonstrated that innate immune activation in the brain by overexpression of mIL-6 and other cytokines can significantly attenuate amyloid deposition in amyloid precursor protein (APP) mouse models.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar, 17Chakrabarty P. Ceballos-Diaz C. Beccard A. Janus C. Dickson D. Golde T.E. Das P. IFN-gamma promotes complement expression and attenuates amyloid plaque deposition in amyloid beta precursor protein transgenic mice.J Immunol. 2010; 184: 5333-5343Crossref PubMed Scopus (148) Google Scholar, 18Chakrabarty P. Herring A. Ceballos-Diaz C. Das P. Golde T.E. Hippocampal expression of murine TNFalpha results in attenuation of amyloid deposition in vivo.Mol Neurodegener. 2011; 6: 16Crossref PubMed Scopus (93) Google Scholar These studies suggested that cytokine up-regulation enhanced microglial-mediated Aβ clearance to attenuate amyloid deposition only when initiated in young APP mice before amyloid deposition. Immune activation was far less effective in reducing pathologic disorder when tested after plaque onset.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar We hypothesized that a combination approach may offer improved efficacy at these stages when considerable pathologic disorder already exists. Here, we tested whether combining immune activation with Aβ reduction would be more effective than either treatment alone in mice harboring moderate preexisting plaque load. We took advantage of the tet-off APP transgenic line to genetically suppress APP/Aβ production mid-life and simultaneously induced microglial activation with the use of adeno-associated virus (AAV)1-mediated murine (m)IL-6 overexpression. After 3 months of treatment, mice were harvested for histopathologic analysis to determine how the combination approach compared with Aβ suppression or AAV-mIL-6 treatment alone. APP transgenic mice, under the control of the tet-off promoter (tet off-APPswe/ind, line 102) were used for these studies.19Jankowsky J.L. Slunt H.H. Gonzales V. Savonenko A.V. Wen J.C. Jenkins N.A. Copeland N.G. Younkin L.H. Lester H.A. Younkin S.G. Borchelt D.R. Persistent amyloidosis following suppression of Abeta production in a transgenic model of Alzheimer disease.PLoS Med. 2005; 2: e355Crossref PubMed Scopus (180) Google Scholar The tet off-APPswe/ind mice were mated to mice expressing tetracycline transactivator (tTA) under the control of the Ca2+/calmodulin-dependent kinase II α promoter–tTA line B.20Mayford M. Bach M.E. Huang Y.Y. Wang L. Hawkins R.D. Kandel E.R. Control of memory formation through regulated expression of a CaMKII transgene.Science. 1996; 274: 1678-1683Crossref PubMed Scopus (1101) Google Scholar Male APP/tTA double-transgenic mice were then mated with wild-type Friend leukemia virus B female mice to generate experimental mice for these studies (n = 5 to 8 females per group). All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Doxycycline (dox) treatment was started at 5 months of age and continued for 3 months until harvest at 8 months of age. Dox was formulated in mouse chow (Purina 5001 chow; Bio-Serv, Flemington, NJ) at a concentration of 50 mg/kg. AAV1 expressing mIL-6 or enhanced green fluorescent protein (EGFP) under the control of the cytomegalovirus enhancer/chicken β-actin promoter was generated and injected in both sides of the hippocampus as described previously.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar For stereotactic injections, mice (n = 5 to 8 females per group) were anesthetized with 1.5% isoflurane in 1% oxygen and secured into a Kopf apparatus (Model 900 Small Animal Stereotactic Instrument; David Kopf Instruments, Tujunga, CA). The coordinates for hippocampal injection were −1.7 caudal, −1.6 lateral, and −1.2 ventral from the bregma. A UMP2 Microsyringe Injector and Micro4 Controller were used to inject 2 μL of AAV (AAV1-mIL6 at 7.10E+12 viral particles/hippocampus and AAV1-EGFP at 6.3E+12 viral particles/hippocampus) at a constant rate over a 10-minute period. After an additional 10 minutes were allowed, the needle was raised slowly, the scalp incision was closed aseptically, and mice were revived under a heating lamp. For measurements of Aβ levels in mice, frozen left hemi brains were sequentially extracted in Tris-buffered saline (TBS), TBS buffer containing 1% Triton X-100 (TBSx), and 5 mol/L guanidine in 50 mm Tris-HCl, pH 8.0, as described previously.21Chakrabarty P. Li A. Ceballos-Diaz C. Eddy J.A. Funk C.C. Moore B. DiNunno N. Rosario A.M. Cruz P.E. Verbeeck C. Sacino A. Nix S. Janus C. Price N.D. Das P. Golde T.E. IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior.Neuron. 2015; 85: 519-533Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar Aβ levels from forebrain lysates (cortex + hippocampus combined) of APP/tTA mice were measured biochemically using human Aβ end-specific sandwich enzyme-linked immunosorbent assay (ELISA) as described previously.21Chakrabarty P. Li A. Ceballos-Diaz C. Eddy J.A. Funk C.C. Moore B. DiNunno N. Rosario A.M. Cruz P.E. Verbeeck C. Sacino A. Nix S. Janus C. Price N.D. Das P. Golde T.E. IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior.Neuron. 2015; 85: 519-533Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar IL-6 cytokine levels were analyzed from TBS-solubilized forebrain lysates (cortex + hippocampus combined) using BD OptiEIA ELISA kits (BD Biosciences Pharmingen, San Diego, CA). To detect APP levels, TBSx-extracted protein samples were separated on Bis-Tris 12% XT gels (Bio-Rad, Hercules, CA), then probed with CT20 antibody (dilution 1:1000; Mayo Clinic, Jacksonville, FL) as previously described.21Chakrabarty P. Li A. Ceballos-Diaz C. Eddy J.A. Funk C.C. Moore B. DiNunno N. Rosario A.M. Cruz P.E. Verbeeck C. Sacino A. Nix S. Janus C. Price N.D. Das P. Golde T.E. IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior.Neuron. 2015; 85: 519-533Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar Relative band intensity was quantified using ImageJ software version 1.43 (NIH, Bethesda, MD). After tissue harvesting, the right hemi brain was fixed in 4% paraformaldehyde for 24 hours, paraffin embedded, and 5-μm sections were used for immunostaining using the following antibodies as described before21Chakrabarty P. Li A. Ceballos-Diaz C. Eddy J.A. Funk C.C. Moore B. DiNunno N. Rosario A.M. Cruz P.E. Verbeeck C. Sacino A. Nix S. Janus C. Price N.D. Das P. Golde T.E. IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior.Neuron. 2015; 85: 519-533Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar: pan-Aβ antibody 33.1.1 (dilution 1:1500; Mayo Clinic), Aβ 1-40 specific monoclonal antibody (mab) 13.1.1 (dilution 1:500, Mayo Clinic), glial fibrillary acidic protein (GFAP; dilution 1:1000; Sigma-Aldrich, St. Louis, MO), Iba-1 (dilution 1:1000; Wako, Richmond, VA), and EGFP (dilution 1:1000; Invitrogen, Carlsbad, CA). Target antigen retrieval was performed by steaming the sections for 30 minutes in deionized water. Endogenous peroxidase was blocked for 5 minutes with 0.03% hydrogen peroxide. Sections were then blocked with 5% normal goat serum for 20 minutes. Subsequently, sections were incubated 1 hour at room temperature in primary antibodies, then incubated in secondary antibodies (Envision-Plus–labeled polymer-horseradish peroxidase; Dako, Carpentaria, CA), for 30 minutes at room temperature. Peroxidase labeling was visualized with the chromogen solution 3, 3′-diaminobenzidine. Sections immunostained for microglia (Iba1) and astrocytes (GFAP) were further stained with 1% Congo red (Sigma-Aldrich) to visualize amyloid plaques. Additional sections were also stained with 1% Thioflavin S (Thio-S, practical grade; Sigma-Aldrich) to visualize amyloid plaques. Immunostained sections were captured using the Scanscope XT image scanner (Aperio Technologies, Vista, CA). Aβ plaque burden in the cortex and hippocampus was calculated using the Positive Pixel Count Program (Imagescope software, version 12.3; Aperio Technologies) in sections immunostained with pan-Aβ 33.1.1 or Aβ 1-40–specific mab 13.1.1 antibody. At least three sections per mouse brain (n = 5 to 8 mice per group) were used to calculate the average plaque burden in the hippocampus and cortex for each sample. All of the above analyses were performed in a blinded manner (C.V., and A.C.). Thio-S quantitation (mean area and integrated density in the cortex and hippocampus) was performed using ImageJ. Total RNA from mice hippocampus was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Total RNA was dissolved in nuclease-free water and stored at −80°C. Reverse transcription was performed using Superscript III (Invitrogen) with the use of Mastercycler pro, and the reaction mix was subjected to quantitative RT-PCR with the use of iQ SYBR Green Supermix (Bio-Rad) to detect the amplification products. Relative quantification of mRNA expression was calculated by the ΔCT method after adjusting the levels to the corresponding internal glyceraldehyde-3-phosphate dehydrogenase control for each sample. The sequences of primers used to amplify target genes by quantitative RT-PCR were as follows: Gapdh [5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse)], Aif1 [5′-CTTGAAGCGAATGCTGGAGAA-3′ (forward) and 5′-GGCAGCTCGGAGATAGCTTT-3′ (reverse)], Itgam [5′-GTGTGACTACAGCACAAGCCG-3′ (forward) and 5′-CCCAAGGACATATTCACAGCCT-3′ (reverse)], Cx3cr1 [5′-ACCGGTACCTTGCCATCGT-3′ (forward) and 5′-ACACCGTGCTGCACTGTCC-3′ (reverse)], Trem2 [5′-GCCTTCCTGAAGAAGCGGAA-3′ (forward) and 5′-GAGTGATGGTGACGGTTCCA-3′ (reverse)], Hexb [5′-ACTCCAAGATTATGGCCTCGAGCA-3′ (forward) and 5′-GCTATTCCACGGCTGACCATTCT-3′ (reverse)], and Gfap [5′-ACCAGCTTACGGCCAACAG-3′ (forward) and 5′-CCAGCGATTCAACCTTTCTCT-3′ (reverse)]. Two-way analysis of variance with Tukey's multiple-comparison post hoc test or 2-tailed t-test was used for statistical comparison (GraphPad Prism 6 Software; GraphPad, San Diego, CA). All treatments were started at 5 months of age to ensure that APP/tTA mice would already have developed a moderate amyloid load throughout the cortex before any intervention. A schematic for the experimental design, including all treatment groups, is provided in Figure 1A. Mice used for combination treatment were fed with dox (50 mg/kg in chow) to suppress further expression of transgenic APP/Aβ by approximately 50% versus untreated (no dox) mice (Figure 1, B and C). At the same age, combination treatment mice were administered recombinant AAV1-mIL-6 by stereotaxic intracranial injection to target the hippocampus to promote innate immune activation. When AAV1-IL-6 vector is injected into the mouse brain at postnatal day 0, this approach produces widespread expression of mIL-6 in the mouse brain.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar Here, a control AAV1 was used to deliver EGFP by stereotaxic injection into the adult hippocampus to demonstrate that this approach expresses the gene of interest in the hippocampus and overlying cortical layers (Figure 1D). that the stereotaxic hippocampal injection of AAV1-mIL-6 results were confirmed in more than threefold elevation of mIL-6 protein in the brain as measured by ELISA from TBS-solubilized forebrain lysates (cortex + hippocampus combined): 3435.8 ± 812 mg/mL in the AAV-IL-6–injected mice versus 1105.4 ± 213 mg/mL in untreated controls. Animals were harvested at 8 months of age after 3 months of differential treatment. We first evaluated the impact of combining Aβ suppression (dox treatment) with AAV1-mIL-6 on amyloid deposition in the brains of APP/tTA mice. Mice treated with AAV1-mIL-6 + dox showed significant attenuation of amyloid deposition in the cortex (11.7% surface area) (Figure 2, E and H) versus untreated controls (29.9% surface area) (Figure 2, B and H). APP/tTA mice treated only with dox for the same length of time had a smaller reduction in amyloid deposition (23.1% surface area) (Figure 2, A and H), whereas treatment with AAV1-mIL-6 alone (mIL6-only group) had minimal effect on amyloid deposition (27.4% surface area) (Figure 2, F and H). For comparison, we included two additional control groups: AAV1-EGFP + dox (18.5% surface area) (Figure 2C) and AAV1-EGFP only (28.3% surface area) (Figure 2D). Because AAV1-mIL-6 was targeted to the hippocampus, we also assessed Aβ plaque burden in the hippocampus. Once again, the combination of AAV1-mIL-6 significantly reduced amyloid deposition (11.5% surface area) (Figure 2, E and I) versus untreated controls (34.7% surface area) (Figure 2, B and I). Hippocampal amyloid burden in the other experimental groups were as follows (Figure 2, A–G and I): dox only (24.0% surface area), AAV1-mIL6 only (40.0% surface area), AAV1-EGFP + dox (18.4% surface area), and AAV1-EGFP only (37.1% surface area). ELISA Aβ measurement from guanidine-solubilized forebrain lysates (cortex + hippocampus combined) showed similar results: the concentration of Aβ42 was significantly reduced relative to untreated controls only in mice receiving AAV1-mIL-6 + dox (Table 1). The concentration of Aβ40 also decreased in the AAV1-mIL-6 + dox group but did not reach statistical significance (Table 1).Table 1Aβ ELISA Showed a Significant Decrease of Guanidine-Extracted Aβ42 in the Combination AAV1-mIL-6 + Dox Group versus Control GroupsAβ speciesPretreatedUntreatedmIL-6 onlyDox onlymIL-6 + doxAβ42, pmol/g2301 ± 3024973 ± 4024327 ± 3943947 ± 2802764 ± 377∗P < 0.05.Aβ40, pmol/g2094 ± 2853773 ± 3623628 ± 2752668 ± 2572325 ± 299Data are expressed as means ± SEM.AAV1-mIL-6, adeno-associated virus 1 expressing murine IL-6; Dox, doxycycline; ELISA, enzyme-linked immunosorbent assay.∗ P < 0.05. Open table in a new tab Data are expressed as means ± SEM. AAV1-mIL-6, adeno-associated virus 1 expressing murine IL-6; Dox, doxycycline; ELISA, enzyme-linked immunosorbent assay. To further assess the efficacy of combination treatment, we compared amyloid plaque burden in the brains of untreated controls with either single modality (dox only and AAV1-mIL-6 only) versus combination treatment (AAV1-mIL-6 + dox) (Figure 2J). Immunohistologic analysis of pan-Aβ staining showed that at the start of the experiment, 5-month-old APP/tTA mice harbored a moderate degree of preexisting amyloid in the cortex (9.1% surface area). Left untreated, the area of cortical Aβ staining in APP/tTA mice harvested at 8 months of age increased by approximately threefold (29.9% surface area). Relative to the approximately 30% burden in untreated mice, administration of AAV1-mIL-6 produced only a small approximately 8% decrease in amyloid plaque area. Similarly, in the dox-only group, amyloid plaque burden decreased by just approximately 21%. In contrast, treatment with AAV1-mIL-6 + dox decreased plaque area by approximately 60% versus untreated controls, showing that combination treatment was significantly more effective in attenuating amyloid plaque deposition than either Aβ suppression or AAV1-mIL-6 treatment alone. We then evaluated whether combination treatment affected dense-core amyloid plaques in the APP/tTA mice with the use of Thio-S histological examination and Aβ40 immunostaining to detect this subset of Aβ deposits.22Moore B.D. Chakrabarty P. Levites Y. Kukar T.L. Baine A.M. Moroni T. Ladd T.B. Das P. Dickson D.W. Golde T.E. Overlapping profiles of Abeta peptides in the Alzheimer's disease and pathological aging brains.Alzheimers Res Ther. 2012; 4: 18Crossref PubMed Scopus (79) Google Scholar APP/tTA mice treated with AAV1-mIL-6 + dox showed significant reductions in both measures of dense-core plaque burden (Figure 3) versus untreated controls. Comparable reductions in dense-core plaque load were also seen in the dox-only group and, to a lesser extent, in the mIL-6-only group (Figure 3), suggesting that combination therapy did not promote the clearance of fibrillar plaques beyond the effect attained by Aβ suppression alone. We previously demonstrated that mIL-6 activates both astrocytes and microglia in the cortex and hippocampus of AAV1-mIL-6–injected APP mice.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar mIL-6 overexpression promotes microglial up-regulation of phagocytic markers such as CD11b, suggesting a role for activated microglia in Aβ clearance.16Chakrabarty P. Jansen-West K. Beccard A. Ceballos-Diaz C. Levites Y. Verbeeck C. Zubair A.C. Dickson D. Golde T.E. Das P. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition.FASEB J. 2010; 24: 548-559Crossref PubMed Scopus (246) Google Scholar Consistent with this earlier finding, treatment with AAV1-mIL-6 in the combination group significantly increased the density of Iba-1–positive microglia and GFAP-positive astrocytes. Throughout the hippocampus and cortex, both cell types displayed morphologic changes such as hypertrophy consistent with immune activation in mice given AAV1-mIL6 + dox (Figure 4, A and B). Whereas in dox only and AAV1-EGFP + dox control groups, Iba-1–positive microglia and GFAP-positive astrocytes were predominantly clustered around plaques (Figure 4, A and B). Quantitative real-time PCR confirmed that Aif1 mRNA encoding Iba1 was increased approximately threefold in the mIL-6 + dox group versus dox only and AAV1-EGFP + dox control mice (Figure 4C). Moreover, Itgam mRNA encoding the microglial pattern recognition and professional phagocytic receptor CD11b23Ehlers M.R. CR3: a general purpose adhesion-recognition receptor essential for innate immunity.Microbes Infect. 2000; 2: 289-294Crossref PubMed Scopus (258) Google Scholar was increased >20-fold, whereas the fractalkine receptor Cx3cr1 mRNA was increased >10-fold in the mIL-6 mice + dox group versus the dox-only and AAV1-EGFP + dox control mice (Figure 4C). mRNA for the lysosomal β-hexosaminidase Hexb and the triggering receptor expressed on myeloid cells 2 Trem224Zhang Y. Chen K. Sloan S.A. Bennett M.L. Scholze A.R. O'Keeffe S. Phatnani H.P. Guarnieri P. Caneda C. Ruderisch N. Deng S. Liddelow S.A. Zhang C. Daneman R. Maniatis T. Barres B.A. Wu J.Q. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.J Neurosci. 2014; 34: 11929-11947Crossref PubMed Scopus (2920) Google Scholar, 25Butovsky O. Jedrychowski M.P. Moore C.S. Cialic R. Lanser A.J. Gabriely G. Koeglsperger T. Dake B. Wu P.M. Doykan C.E. Fanek Z. Liu L. Chen Z. Rothstein J.D. Ransohoff R.M. Gygi S.P. Antel J.P. Weiner H.L. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia.Nat Neurosci. 2014; 17: 131-143Crossref PubMed Scopus (1576) Google Scholar were not significantly altered (Figure 4C). The failure of multiple recent anti-Aβ clinical trials in patients with mild-to-moderate AD has prompted discussion of future trials to test preventative treatments. Despite the disapp" @default.
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