Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease methods
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Western blot
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail (Roche) on ice and boiled for 10 min. Proteins (10-50 µg) were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with anti-rabbit or anti-mouse IgG conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature, and detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies used were: Slc40A1 (Fpn1, Novus Biologicals, CO, USA)...
Immunohistochemistry
Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were cut with a vibrate. For immunohistochemistry, the slices were incubated with Fpn primary antibody (Alphadiagnosis, San Antonio, TX, USA catalog MTP11-A, 1:100) for 48 h, probed with biotin-labeled secondary antibodies (1:10,000) for 1 h at 37 °C, and detected with the DAB Kit (Zsbio, Beijing, China, Catalog ZLI-9018). The images were observed under a microscope (Olympus BX60, Tokyo, Japan). DAB-enhanced Perl's staining was used to detect iron accumulation as previously described [ ]. Sections of brain tissue were washed with PBS and incubated in...
Stereotaxic injection and drug administration
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm). Adeno-associated viruses (AAVs) (10 12 IU/ml, 2 µl) for Fpn overexpression or lentiviruses (10 9 IU/ml, 2 µl) for Fpn knockdown and the relative control viruses were bilaterally microinfused into the hippocampus via a cannula connected to a Hamilton microsyringe (Reno, NV, USA). We injected the AAV packaged full length murine Fpn cDNA into the hippocampus of APPswe/PS1dE9 mice at 9 months old. 3 months later, behavioral tests were performed in these mice at 12 months old. The infusion rate was 0.2 µl/min, and the cannula was le...
Primary neuron culture and treatment
Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they were centrifuged at 1000 × g for 5 min. The cells were triturated and plated onto a plastic culture dish and incubated at 37 °C with 5% CO 2. After 7 days in vitro, recombinant human Aβ 1-42 (Chinese Peptide Company, Hangzhou, China, Catalog AMYD-002) was resuspended in DMSO to 5 mM as described previously [ ], and 5 mM Aβ 1-42 was diluted with sterile (1 × ) PBS to 100 µM on the day before treatment. Then, Aβ 1-42 solution was incubated for 24 h at 4 ˚C. After in...
Deficiency of Fpn in excitatory neurons induced brain atrophy and cognitive impairment
We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and littermate controls (Fig. ). In Morris water maze tests, we found that the KO mice showed significantly worse learning performance during the training sessions (Fig. ). In the probe trial, the KO mice displayed reduced accuracy, prolonged latency in finding the target platform, and a shorter duration in the target quadrant (Fig. ). Additionally, Fpn fl/fl/NEXcre mice spent remarkably less freezing time in a contextual fear memory task but not in cue fear memory, indicating impaired hippocampus-related learning/memory (Fig. ). No significant differences were observed in sensitivity to electrical shocks between the two groups (Fig. ). To avoid the possible developmental deficits caused...
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Evidence quotes (5)
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail (Roche) on ice and boiled for 10 min. Proteins (10-50 µg) were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with anti-rabbit or anti-mouse IgG conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature, and detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies used were: Slc40A1 (Fpn1, Novus Biologicals, CO, USA); Slc40A1 (Fpn1, Alphadiagnosis, San Antonio, TX, USA); FTH (FTH1, ferritin heavy chain, Cell Signaling Technology, Danvers, MA, USA), Gpx4; actin (Proteintech, Wuhan, China). Detailed information about the antibodies is available in Supplementary Table.
Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were cut with a vibrate. For immunohistochemistry, the slices were incubated with Fpn primary antibody (Alphadiagnosis, San Antonio, TX, USA catalog MTP11-A, 1:100) for 48 h, probed with biotin-labeled secondary antibodies (1:10,000) for 1 h at 37 °C, and detected with the DAB Kit (Zsbio, Beijing, China, Catalog ZLI-9018). The images were observed under a microscope (Olympus BX60, Tokyo, Japan). DAB-enhanced Perl's staining was used to detect iron accumulation as previously described [ ]. Sections of brain tissue were washed with PBS and incubated in freshly prepared Perls solution (5% potassium ferrocyanide [Sigma-Aldrich, St. Louis, MO, USA]/10% hydrochloric acid) for 1 h, endogenous peroxidase activity was quenched for 20 min at room temperature in 0.3% H 2 O 2 in methanol, followed by washing with PBS five times. Then, DAB incubation...
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm). Adeno-associated viruses (AAVs) (10 12 IU/ml, 2 µl) for Fpn overexpression or lentiviruses (10 9 IU/ml, 2 µl) for Fpn knockdown and the relative control viruses were bilaterally microinfused into the hippocampus via a cannula connected to a Hamilton microsyringe (Reno, NV, USA). We injected the AAV packaged full length murine Fpn cDNA into the hippocampus of APPswe/PS1dE9 mice at 9 months old. 3 months later, behavioral tests were performed in these mice at 12 months old. The infusion rate was 0.2 µl/min, and the cannula was left in place for 10 min following completion of the infusion. AAVs for Fpn overexpression were purchased from Neuron Biotech (Shanghai, China), and lentivirus for Fpn short hairpin RNA was purchased from Genechem Co, Ltd. (Shanghai, China). Oligomeric Aβ 1-42 was injected into the mo...
Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they were centrifuged at 1000 × g for 5 min. The cells were triturated and plated onto a plastic culture dish and incubated at 37 °C with 5% CO 2. After 7 days in vitro, recombinant human Aβ 1-42 (Chinese Peptide Company, Hangzhou, China, Catalog AMYD-002) was resuspended in DMSO to 5 mM as described previously [ ], and 5 mM Aβ 1-42 was diluted with sterile (1 × ) PBS to 100 µM on the day before treatment. Then, Aβ 1-42 solution was incubated for 24 h at 4 ˚C. After incubation, the Aβ 1-42 solution was diluted into concentrations indicated for treatment with DMEM/F12. The neurons were treated with 10 µm/20 µm Aβ 1-42 as a lethal concentration for 24 h, and vehicle treatment was used for controls. Treatment with 100̴...
We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and littermate controls (Fig. ). In Morris water maze tests, we found that the KO mice showed significantly worse learning performance during the training sessions (Fig. ). In the probe trial, the KO mice displayed reduced accuracy, prolonged latency in finding the target platform, and a shorter duration in the target quadrant (Fig. ). Additionally, Fpn fl/fl/NEXcre mice spent remarkably less freezing time in a contextual fear memory task but not in cue fear memory, indicating impaired hippocampus-related learning/memory (Fig. ). No significant differences were observed in sensitivity to electrical shocks between the two groups (Fig. ). To avoid the possible developmental deficits caused by the genetic deletion of Fpn, we injected lentivirus packaged with a shRNA directed against Fpn into the hippocampus of 3-month-old C57 mice to knockdown the expression of Fpn in wild-type mice (Figs. and ). The behavior test was performed 2 months after the virus injection. Similar to the...
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