Journal: Alzheimer's & Dementia

Article Title: Fibrillar tau alters cerebral endothelial cell metabolism, vascular inflammatory activation, and barrier function in vitro and in vivo

doi: 10.1002/alz.70077

Figure Lengend Snippet: Protofibrillar tau induces loss of barrier resistance independently of cell death. A, Transmission electron microscope analysis of tau aggregates after negative staining with 1% uranyl acetate, reveals primarily protofibrillar species (scale bars 600 nm). B, One hour of treatment with AcidSensor‐labeled protofibrillar tau demonstrates early internalization in hCMECs. C, Left panel TEER (at 4000 Hz) measurement of hCMECs with the ECIS Zθ system illustrates barrier formation at about 48 hours after plating. Middle panel: TEER of hCMECs monolayers treated with 5 or 25 nM fibrillar tau, measured over time for 48 hours, indicates progressive loss of barrier resistance induced by protofibrillar tau species. Right panel: TEER of hCMECs treated with fibrillization vehicle, protofibrillar or monomeric tau (25 nM) for 48 hours (right). Data are represented as change in resistance normalized to the untreated control, n = 3 experiments with two replicates per condition. D, Apoptosis, measured as DNA fragmentation, of hCMECs treated for 24 hours with 5 or 25 nM tau. E, LDH release for hCMECs treated for 24 hours with 5 or 25 nM tau. F, Caspase 3/7 fluorescence in hCMECs treated with aggregated tau for 24 hours, or H 2 O 2 as a positive control (left), with quantification of caspase positive cells (right). **Statistical significance established by one‐way ANOVA (Cell Death ELISA, LDH); Tukey post test. Two‐way ANOVA (ECIS). Significant P values are reported in the graphs. ANOVA, analysis of variance; hCMEC, human cerebral microvascular endothelial cell; LDH, lactate dehydrogenase; TEER, trans‐endothelial electrical resistance.

Article Snippet: Immortalized human cerebral microvascular endothelial cells (hCMECs/D3), shown to maintain characteristics of the BBB, were obtained from Babette Weksler (Cornell University).

Techniques: Transmission Assay, Microscopy, Negative Staining, Labeling, Control, Fluorescence, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Alzheimer's & Dementia

Article Title: Fibrillar tau alters cerebral endothelial cell metabolism, vascular inflammatory activation, and barrier function in vitro and in vivo

doi: 10.1002/alz.70077

Figure Lengend Snippet: Metabolic alterations induced by protofibrillar tau in association with pro‐inflammatory endothelial cell activation. A, Representative trace for glycolysis stress test ECAR measurement using a Seahorse XF96 extracellular flux analyzer in hCMECs treated with aggregated tau for 24 hours. B, ECAR analysis indicates increased glycolysis (top) and glycolytic capacity (bottom) in the presence of 5 or 25 nM tau for 24 hours. C, Representative ECAR trace for glycolysis stress test in hCMECs treated with aggregated tau for 3 hours. D, ECAR indicates increased glycolysis (top) and glycolytic capacity (bottom) after 3 hours of protofibrillar tau challenge. E, Expression of adhesion markers VCAM‐1 and ICAM‐1 at 24 hours post treatment with 5 and 25 nM tau, as determined by western blot analysis. F, Expression of adhesion markers VCAM‐1 and ICAM‐1 at 3 hours post treatment with 5 and 25 nM tau. G, Increased IL‐4, IL‐10, TNF‐α, and IFN‐γ release after 25 nM tau treatment for 3 hours, determined by MSD proinflammatory panel analysis. H, Increased levels of IL‐4 and increasing trends of IL‐6 released in the media of hCMECs treated with 25 nM protofibrillar tau for 24 hours. I, Treatment with α‐IL‐4 does not decrease VCAM‐1 expression in hCMECs at 24 hours post‐tau treatment. J, TEER measurements during co‐treatment with tau protofibrils (25 nM) and a neutralizing antibody against IL‐4 (α‐IL‐4) only demonstrate a partial recovery against tau‐mediated loss of barrier resistance. Statistical significance established by one‐way analysis of variance; (WB), Seahorse (SH), and Tukey post hoc test, multiple unpaired t test (MSD). Significant P values are reported in the graphs. ECAR, extracellular acidification rate; hCMEC, human cerebral microvascular endothelial cell; ICAM‐1, intercellular adhesion molecule 1; IFN‐γ, interferon gamma; IL, interleukin; MSD, Meso Scale Discovery; TEER, trans‐endothelial electrical resistance; TNF‐α, tumor necrosis factor alpha; VCAM‐1, vascular cell adhesion molecule 1; WB, western blot.

Article Snippet: Immortalized human cerebral microvascular endothelial cells (hCMECs/D3), shown to maintain characteristics of the BBB, were obtained from Babette Weksler (Cornell University).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Alzheimer's & Dementia

Article Title: Fibrillar tau alters cerebral endothelial cell metabolism, vascular inflammatory activation, and barrier function in vitro and in vivo

doi: 10.1002/alz.70077

Figure Lengend Snippet: Modulation of glycolytic metabolism reverts fibrillar tau‐mediated loss of barrier resistance. A, HA decreases tau‐mediated IL‐4 production in hCMECs 24 hours after treatment. B, Western blot analysis of hCMECs co‐treated with tau and HA reveals a decrease in VCAM‐1 expression in the presence of the glycolysis inhibitor. C, TEER measurement after co‐treatment with protofibrillar tau and 100 nM HA indicates that blocking glycolysis prevents tau‐mediated loss of barrier resistance. D, 2‐deoxyglucose partially rescues tau‐mediated loss of barrier resistance after 3 hours of tau treatment. Statistical significance established by one‐way ANOVA (WB), and Tukey post hoc test, two‐way ANOVA (Electrical Cell Impedance Sensing‐Zθ). Significant P values are reported in the graphs. ANOVA, analysis of variance; hCMEC, human cerebral microvascular endothelial cell; HA, heptelidic acid; IL, interleukin; TEER, trans‐endothelial electrical resistance; VCAM‐1, vascular cell adhesion molecule 1; WB, western blot

Article Snippet: Immortalized human cerebral microvascular endothelial cells (hCMECs/D3), shown to maintain characteristics of the BBB, were obtained from Babette Weksler (Cornell University).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Alzheimer's & Dementia

Article Title: Fibrillar tau alters cerebral endothelial cell metabolism, vascular inflammatory activation, and barrier function in vitro and in vivo

doi: 10.1002/alz.70077

Figure Lengend Snippet: Mitochondrial OXPHOS alterations and endothelial cell death after longer treatment with protofibrillar tau. A, DNA fragmentation in hCMECs is increased 48 hours post‐treatment with protofibrillar tau. B, Adhesion molecule VCAM‐1 is decreased 48 hours post‐treatment with 25 nM tau but not 5 nM tau, as determined by western blot analysis. C, Extracellular acidification rate demonstrate no changes in glycolytic rates at 48 hours. D, Representative trace for OCR (mitochondrial stress test) measured using a Seahorse XF96 Extracellular flux Analyzer. E, OCR measurement during mitochondrial stress test indicates increased mitochondrial basal respiration (left), maximal respiration (middle), and ATP production rate (right), with 5 nM protofibrillar tau. F, Immunocytochemical assessment of mitochondrial membrane potential using MitoTracker Red CM‐H2XROS. G, Increased expression of mitochondrial superoxide dismutase SOD2 with 5 nM tau, determined by western blot analysis. H, DNA fragmentation in hCMECs after 72 hours of challenge with protofibrillar tau. I, LDH release from hCMECs after 72 hours of challenge with protofibrillar tau. J, Basal respiration rate and ATP production for hCMECs after 72 hours of challenge with protofibrillar tau. K, Glycolytic rates for hCMECs after 72 hours of treatment with protofibrillar tau. Statistical significance was established by one‐way analysis of variance; (WB, SH, LDH, and CDE) and Tukey post hoc test. Significant P values are reported in the graphs. ATP, adenosine triphosphate; hCMEC, human cerebral microvascular endothelial cell; LDH, lactate dehydrogenase; OCR, oxygen consumption rate; OXPHOS, oxidative hyperphosphorylation; SH, Seahorse; SOD2, superoxide dismutase 2; VCAM‐1, vascular cell adhesion molecule 1; WB, western blot.

Article Snippet: Immortalized human cerebral microvascular endothelial cells (hCMECs/D3), shown to maintain characteristics of the BBB, were obtained from Babette Weksler (Cornell University).

Techniques: Western Blot, Membrane, Expressing

Journal: Alzheimer's & Dementia

Article Title: Fibrillar tau alters cerebral endothelial cell metabolism, vascular inflammatory activation, and barrier function in vitro and in vivo

doi: 10.1002/alz.70077

Figure Lengend Snippet: Human‐derived fibrillar tau induces loss of barrier resistance and pro‐inflammatory EC activation. A, TEER trace (at 4000 Hz) of hCMECs treated with 25 nM aggregated 1N4R tau or 25 nM human‐derived tau fibrils. B, Representative trace for glycolysis stress test ECAR for hCMECs treated with human‐derived fibrillar tau for 24 hours. C, ECAR analysis in hCMECs treated with 25 nM human‐derived tau fibrils reveals increased glycolytic rates. D, Western blot analysis of VCAM‐1 expression for hCMECs treated with human fibrillar tau (25 nM) for 24 hours. E, Immunocytochemical assessment of protofibrillar 1N4R tau and human‐derived tau fibrils on hCMEC monolayers after 24 hours of treatment. Statistical significance established by Student t test (WB, SH) and two‐way analysis of variance (Electrical Cell Impedance Sensing‐Zθ). Significant P values are reported in the graphs. EC, endothelial cell; ECAR, extracellular acidification rate; hCMEC, human cerebral microvascular endothelial cell; SH, Seahorse; TEER, trans‐endothelial electrical resistance; VCAM‐1, vascular cell adhesion molecule 1; WB, western blot.

Article Snippet: Immortalized human cerebral microvascular endothelial cells (hCMECs/D3), shown to maintain characteristics of the BBB, were obtained from Babette Weksler (Cornell University).

Techniques: Derivative Assay, Activation Assay, Western Blot, Expressing