Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer's disease methods
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Primary microglial isolation
Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in a centrifuge for 5 min at 800 × g at room temperature. Supernatants were discarded and cell pellets re-suspended in 6 mL of 37% stock isotonic Percoll (SIP) solution (90% Percoll + 10% 10X HBSS) per brain. The cell suspension was transferred into 15 mL conical tubes and 2 mL of 70% SIP slowly under laid. Then on top of the 37% layer, 2 mL of 30% SIP was slowly layered. The established gradient was then centrifuged for 25 min at 800 × g with zero deceleration at 20 °C. Floating myelin in the top layer was then removed and a...
Microglial Aβ42 phagocytosis assay
The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ42 required unlabeled Aβ42 (Anaspec Cat # AS-60479-01) at a 1:4 ratio. Purified Aβ42 was first linearized in 10% w / v NH 4 OH and re-lyophilized to reduce any preexisting aggregates. 0.5 mg of unlabeled lyophilized Aβ42 was then reconstituted to 150 µM by adding 1 mM NaOH, bath sonicated for 10 min followed by addition of 10X buffer (100 mM sodium phosphate, pH 7.1) to bring the final pH to 7.4. 410.64 µL of that aliquot was added to 0.1 mg of HiLyte-488 Aβ42 to achieve a total of 200 µM Aβ...
Animals
Female C57BL/6 J and female 5xFAD mice used for the studies were housed in the Department of Animal Resources at Emory University under standard conditions with no special food/water accommodations. Institutional Animal Care and Use Committee approval was obtained prior to in-vivo work and all work was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Adult mice were given intraperitoneal LPS injections (10 µg/dose × 4 daily doses) to induce acute neuroinflammation [, ]. If ≥25% of weight loss was observed, animals were euthanized. In some experiments, 5xFAD mice received i.p. doses of T0901317 (30 mg/kg) twice a week for 2 weeks.
WGCNA
The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0.8. The network was created using WGCNA::blockwiseModules() function, in a single block (maxBlockSize > 16,721). Briefly, this function calculated topologic overlap (TO) with bicor correlation function, then genes were hierarchically clustered using 1-TO (dissTOM) as the distance measure. Initial module assignments were determined by using dynamic tree-cutting, using default parameters except deepSplit = 2, mergeCutHeight =0.20, minModulesize = 40, pamStage = TRUE, and pamRespectsDendro = TRUE), but genes were allowed to...
Long-term ShK-223 treatment trial
Adult female 5xFAD mice aged 3 mo were treated intraperitoneally twice a week until 6 mo of age with PBS or ShK-223 (dose 100 µg/kg), n = 10 each and also age-matched 5 female C57BL/6 mice were included in the study. At 6 mo, neurobehavioral testing including Fear conditioning and Morris Water Maze was performed. The mice were euthanized, brains isolated and the brains were utilized for immunohistochemistry studies.
Reverse transcriptase quantitative PCR
CNS immune cells isolated from age-matched C57BL6 mice treated with four once daily intraperitoneal doses of sterile PBS, ShK-223 (100 µg/kg), LPS (20 µg/dose) or co-administered LPS + ShK-223 ( n = 3/group) were washed in PBS containing RNAase inhibitors and used for RT-qPCR using our published protocols [ ]. For total RNA extraction from cells, 1 mL Trizol was added to the cells, the pellets were homogenized in the Trizol by repetitive pipetting and incubated for at least 1 h at room temperature. Then, 0.2 mL of chloroform per 1 mL of Trizol was added and incubated for 2-3 min followed by centrifugation at 12000 x g for 15 min at 4 °C to obtain an upper colorless aqueous phase, an interphase and a lower red phenol chloroform phase. RNA in the aqueous phase was transferred to other tubes. 0.5&#...
Validation of Kv1.3 channels as specific regulators of pro-inflammatory DAM responses and neuropathology in AD mouse...
Next, we performed in-vivo studies to determine whether Kv1.3 channel inhibition by ShK-223 can inhibit pro-inflammatory DAM and augment anti-inflammatory DAM gene expression in the model of acute neuroinflammation induced by low dose systemic LPS administration [, ]. We found that ShK-223 treatment inhibited LPS-induced upregulation of proinflammatory genes (Ptgs2, Il1b) and augmented expression of anti-inflammatory DAM genes (Kcnj2, Nceh1, Timp2) without affecting LPS-induced suppression of homeostatic gene Tmem119 (Fig. ). The ability of systemic ShK-223 to modulate microglial function in this model indirectly supports its CNS bioavailability.
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Evidence quotes (7)
Adult C57BL6 mice, age-matched female 5xFAD mice were euthanized and brains were isolated following rapid cold saline cardiac perfusion and CNS immune cells were isolated as previously described [, ]. Briefly, brains were minced over a 40 µm cell strainer and single cell suspensions were washed in PBS in a centrifuge for 5 min at 800 × g at room temperature. Supernatants were discarded and cell pellets re-suspended in 6 mL of 37% stock isotonic Percoll (SIP) solution (90% Percoll + 10% 10X HBSS) per brain. The cell suspension was transferred into 15 mL conical tubes and 2 mL of 70% SIP slowly under laid. Then on top of the 37% layer, 2 mL of 30% SIP was slowly layered. The established gradient was then centrifuged for 25 min at 800 × g with zero deceleration at 20 °C. Floating myelin in the top layer was then removed and a Pasteur pipette used to carefully collect 3 mL from the 70-37% interphase without disturbing the 70% layer. Cells were then washed × 3 in 10 mL cold PBS and the pellets comprising of CNS immune cells were then re-suspended in 100 µL of the appropriate buffer.
The ability of acutely isolated CNS mononuclear cells from wild type, LPS-treated and age-matched 5xFAD mice to phagocytose fluorescent-labeled Aβ fibrils ex-vivo was assessed by a flow cytometric assay. Fluorescent-labeled Aβ42 fibrils were prepared by co-incubating unlabeled and Hilyte488-labeled Aβ42 required unlabeled Aβ42 (Anaspec Cat # AS-60479-01) at a 1:4 ratio. Purified Aβ42 was first linearized in 10% w / v NH 4 OH and re-lyophilized to reduce any preexisting aggregates. 0.5 mg of unlabeled lyophilized Aβ42 was then reconstituted to 150 µM by adding 1 mM NaOH, bath sonicated for 10 min followed by addition of 10X buffer (100 mM sodium phosphate, pH 7.1) to bring the final pH to 7.4. 410.64 µL of that aliquot was added to 0.1 mg of HiLyte-488 Aβ42 to achieve a total of 200 µM Aβ42 solution. The sample was incubated in dark at room temperature for 5-7 days before being transferred to 4 °C. Phagocytosis of fluorescent-labeled Aβ42 fibrils (fAβ42-HiLyte488) was performed by incubating acutely isolated CNS immune cells (pretreated ex-vivo with S...
Female C57BL/6 J and female 5xFAD mice used for the studies were housed in the Department of Animal Resources at Emory University under standard conditions with no special food/water accommodations. Institutional Animal Care and Use Committee approval was obtained prior to in-vivo work and all work was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Adult mice were given intraperitoneal LPS injections (10 µg/dose × 4 daily doses) to induce acute neuroinflammation [, ]. If ≥25% of weight loss was observed, animals were euthanized. In some experiments, 5xFAD mice received i.p. doses of T0901317 (30 mg/kg) twice a week for 2 weeks.
The R package WGCNA was used to construct a co-expression network using normalized and batch-corrected data of the 43 microarray-measured samples just described using a previously published approach [ ]. A threshold power of 10 was chosen since it was the smallest threshold that resulted in a scale-free R 2 fit of 0.8. The network was created using WGCNA::blockwiseModules() function, in a single block (maxBlockSize > 16,721). Briefly, this function calculated topologic overlap (TO) with bicor correlation function, then genes were hierarchically clustered using 1-TO (dissTOM) as the distance measure. Initial module assignments were determined by using dynamic tree-cutting, using default parameters except deepSplit = 2, mergeCutHeight =0.20, minModulesize = 40, pamStage = TRUE, and pamRespectsDendro = TRUE), but genes were allowed to be reassigned to modules with better correlation if p < 0.05 for those correlations. The network type was signed, so that anticorrelated genes were not assigned to the same module. Resulting 19 modules or groups of co-expressed genes ranging in size from 2165 genes (turquoise) to 75 ge...
Adult female 5xFAD mice aged 3 mo were treated intraperitoneally twice a week until 6 mo of age with PBS or ShK-223 (dose 100 µg/kg), n = 10 each and also age-matched 5 female C57BL/6 mice were included in the study. At 6 mo, neurobehavioral testing including Fear conditioning and Morris Water Maze was performed. The mice were euthanized, brains isolated and the brains were utilized for immunohistochemistry studies.
CNS immune cells isolated from age-matched C57BL6 mice treated with four once daily intraperitoneal doses of sterile PBS, ShK-223 (100 µg/kg), LPS (20 µg/dose) or co-administered LPS + ShK-223 ( n = 3/group) were washed in PBS containing RNAase inhibitors and used for RT-qPCR using our published protocols [ ]. For total RNA extraction from cells, 1 mL Trizol was added to the cells, the pellets were homogenized in the Trizol by repetitive pipetting and incubated for at least 1 h at room temperature. Then, 0.2 mL of chloroform per 1 mL of Trizol was added and incubated for 2-3 min followed by centrifugation at 12000 x g for 15 min at 4 °C to obtain an upper colorless aqueous phase, an interphase and a lower red phenol chloroform phase. RNA in the aqueous phase was transferred to other tubes. 0.5 mL of 100% iso-propranolol was added to the aqueous phase per 1 mL of Trizol used and the mixture was incubated for 10 min and centrifuged at 12000 x g for 10 min at 4 °C. The pellet was washed with 1 mL of 75% ethanol per 1 mL of Trizol used (initial homogeniz...
Next, we performed in-vivo studies to determine whether Kv1.3 channel inhibition by ShK-223 can inhibit pro-inflammatory DAM and augment anti-inflammatory DAM gene expression in the model of acute neuroinflammation induced by low dose systemic LPS administration [, ]. We found that ShK-223 treatment inhibited LPS-induced upregulation of proinflammatory genes (Ptgs2, Il1b) and augmented expression of anti-inflammatory DAM genes (Kcnj2, Nceh1, Timp2) without affecting LPS-induced suppression of homeostatic gene Tmem119 (Fig. ). The ability of systemic ShK-223 to modulate microglial function in this model indirectly supports its CNS bioavailability.
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