Isoflurane increases Tau-PS262 levels in brain tissues of WT mice.

<p>Isoflurane anesthesia (lanes 2, 4 and 6) does not increase PS262 levels as compared to the control condition (lanes 1, 3 and 5) in the brain tissues of WT mice at six hours after the isoflurane anesthesia. <i>b.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, P = 0.782, N.S.) does not increase PS262 levels as compared to the control condition (white bar). <i>c.</i> Isoflurane anesthesia (lanes 4 to 6) increases Tau-PS262 levels as compared to the control condition (lanes 1 to 3) in the brain tissues of WT mice at 12 hours after the isoflurane anesthesia. <i>d.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, *P = 0.049) increases Tau-PS262 levels as compared to the control condition (white bar). <i>e.</i> Isoflurane anesthesia (lanes 2, 4 and 6) increases Tau-PS262 levels as compared to the control condition (lanes 1, 3 and 5) in the brain tissues of WT mice at 24 hours after the isoflurane anesthesia. <i>f.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, *P = 0.019) increases Tau-PS262 levels as compared to the control condition (white bar). <i>g.</i> Isoflurane anesthesia (lanes 4–6 and lanes 10–12) does not increase levels of total tau as compared to the control condition (lanes 1–3 and lanes 7–9) in the brain tissues of WT mice at 12 and 24 hours after the isoflurane anesthesia. <i>h.</i> Isoflurane anesthesia (lane 5) leads to a more visible band in the Western blot analysis (at about 55 kDa) as compared to control condition (lane 4) in WT mice brain tissues, whereas tau knockout (KO) mice brain tissues (lanes 2 and 3, the negative controls) do not show such band. We have averaged results from three to six independent experiments. WT, wild-type. N = 3–6.</p

Z-VAD and L-685,458 attenuate the isoflurane-induced increase in Tau-PS262 levels in AD Tg mice primary neurons.

<p><i>a.</i> Treatment with isoflurane plus Z-VAD (lanes 4 to 6) leads to reductions in Tau-PS262 levels as compared to treatment with isoflurane plus DMSO (lanes 1 to 3) in AD Tg mice primary neurons. <i>b.</i> Quantification of the Western blot shows that treatment with isoflurane plus Z-VAD (black bar, **P = 0.0056) leads to reductions in Tau-PS262 levels as compared to isoflurane plus DMSO (white bar). <i>c.</i> Treatment with isoflurane plus L-685,458 (lanes 4 to 6) leads to reductions in Tau-PS262 levels as compared to treatment with isoflurane plus DMSO (lanes 1 to 3) in AD Tg mice primary neurons. <i>d.</i> Quantification of the Western blot shows that treatment with isoflurane plus L-685,458 (black bar, *P = 0.0254) leads to reductions in Tau-PS262 levels as compared to isoflurane plus DMSO (white bar). <i>e.</i> Z-VAD (lanes 4 to 6) alone does not significantly affect Tau-PS262 levels as compared to DMSO (lanes 1 to 3) in AD Tg mice primary neurons. <i>f.</i> Quantification of the Western blot shows that Z-VAD (black bar, P = 0.92, N.S.) does not significantly alter Tau-PS262 levels as compared to DMSO (white bar) in AD Tg mice primary neurons. <i>g.</i> L-685,458 (lanes 4 to 6) alone does not significantly affect Tau-PS262 levels as compared to DMSO (lanes 1 to 3) in AD Tg mice primary neurons. <i>h.</i> Quantification of the Western blot shows that L-685,458 (black bar, P = 0.78, N.S.) does not significantly alter Tau-PS262 levels as compared to DMSO (white bar) in AD Tg mice primary neurons. We have averaged the results from 6 to 12 independent experiments. AD, Alzheimer’s disease; Tg, transgenic; Z-VAD, Cardobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone; DMSO, dimethyl sulfoxide. N = 6−12.</p

Z-VAD and L-685,458 attenuate the isoflurane-induced increase in Tau-PS262 levels in WT mice primary neurons.

<p><i>a.</i> Treatment with isoflurane plus Z-VAD (lanes 6 to 9) leads to reductions in Tau-PS262 levels as compared to treatment with isoflurane plus DMSO (lanes 1 to 5) in WT mice primary neurons. <i>b.</i> Quantification of the Western blot shows that treatment with isoflurane plus Z-VAD (black bar, **P = 0.0057) leads to a reduction in Tau-PS262 levels as compared to isoflurane plus DMSO (white bar). <i>c.</i> Treatment with isoflurane plus L-685,458 (lanes 4 to 6) leads to reductions in Tau-PS262 levels as compared to treatment with isoflurane plus DMSO (lanes 1 to 3) in WT mice primary neurons. <i>d.</i> Quantification of the Western blot shows that treatment with isoflurane plus L-685,458 (black bar, *P = 0.0209) leads to reductions in Tau-PS262 levels as compared to isoflurane plus DMSO (white bar). <i>e.</i> Z-VAD (lanes 4 to 6) alone does not significantly affect Tau-PS262 levels as compared to DMSO (lanes 1 to 3) in WT mice primary neurons. <i>f.</i> Quantification of the Western blot shows that Z-VAD (black bar, P = 0.16, N.S.) does not significantly alter Tau-PS262 levels as compared to DMSO (white bar) in WT mice primary neurons. <i>g.</i> L-685,458 (lanes 4 to 6) alone does not significantly affect Tau-PS262 levels as compared to DMSO (lanes 1 to 3) in WT mice primary neurons. <i>h.</i> Quantification of the Western blot shows that L-685,458 (black bar, P = 0.219, N.S.) does not significantly alter Tau-PS262 levels as compared to DMSO (white bar) in WT mice primary neurons. We have averaged the results from 6 to 12 independent experiments. WT, wild-type; Z-VAD, Cardobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone; DMSO, Dimethyl sulfoxide. N = 6−12.</p

Isoflurane increases Tau-PS262 levels in WT and AD Tg mice primary neurons.

<p><i>a.</i> Isoflurane treatment (lanes 4 and 5) increases Tau-PS262 levels as compared to the control condition (lanes 1 to 3) in WT mice primary neurons. <i>b.</i> Quantification of the Western blot shows that isoflurane treatment (black bar, *P = 0.0162) increases Tau-PS262 levels as compared to the control condition (white bar). <i>c.</i> Isoflurane treatment (lane 4) increases Tau-PS262 levels as compared to the control condition (lanes 1 to 3) in AD Tg mice primary neurons. <i>d.</i> Quantification of the Western blot shows that isoflurane treatment (black bar, *P = 0.014) increases Tau-PS262 levels as compared to the control condition (white bar) in AD Tg mice primary neurons. <i>e.</i> The baseline levels of Tau-PS262 levels in AD Tg mice primary neurons (lanes 7 to 9) are higher than those in WT mice primary neurons (lanes 1 to 3); and isoflurane treatment (lanes 10 to 12) induces a greater increase in Tau-PS262 levels in AD Tg mice primary neurons than in WT mice primary neurons (lanes 4 to 6). <i>f.</i> Quantification of the Western blot shows that there is a higher baseline Tau-PS262 levels in AD Tg mice primary neurons (gray bar) than WT mice primary neurons (white bar): **P = 0.0014; and isoflurane induces a greater increase in Tau-PS262 levels in AD Tg mice primary neurons (net bar) than in WT mice primary neurons (black bar): **P = 0.0038. We have averaged results from 6 to 12 independent experiments. WT, wild-type, AD, Alzheimer’s disease, Tg, transgenic. N = 6–12.</p

Isoflurane increases Tau-PS262 levels in brain tissues of AD Tg mice.

<p><i>a.</i> Isoflurane anesthesia (lanes 2 and 4) increases Tau-PS262 levels as compared to the control condition (lanes 1 and 3) in the brain tissues of AD Tg mice at six hours after the isoflurane anesthesia. <i>b.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, **P = 0.0069) increases Tau-PS262 levels as compared to the control condition (white bar). <i>c.</i> Isoflurane anesthesia (lanes 2 and 4) increases Tau-PS262 levels as compared to the control condition (lanes 1 and 3) in brain tissues of AD Tg mice at 12 hours after the isoflurane anesthesia. <i>d.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, **P = 0.0016) increases Tau-PS262 levels as compared to the control condition (white bar). <i>e.</i> Isoflurane anesthesia (lanes 2, 4 and 6) increases Tau-PS262 levels as compared to the control condition (lanes 1, 3 and 5) in brain tissues of AD Tg mice at 24 hours after the isoflurane anesthesia. <i>f.</i> Quantification of the Western blot shows that isoflurane anesthesia (black bar, *P = 0.044) increases Tau-PS262 levels as compared to the control condition (white bar). We have averaged results from three independent experiments. AD, Alzheimer’s disease, Tg, transgenic. N = 3.</p

Sevoflurane alters the relative proportions of diffuse-type versus cluster-type filopodia in DIV9 developing mouse hippocampal neurons.

<p>(A) 3% sevoflurane for four hours on DIV7 caused an increase in diffuse-type and a decrease in cluster-type filopodia two days later compared to control unexposed hippocampal neurons. (B) representative fluorescent image of F-actin containing diffuse-type filopodia (arrows) in sevoflurane-exposed neurons. (C) representative fluorescent image of F-actin containing cluster-type filopodia (arrows) in control neurons. Phalloidin staining of F-actin was performed as described in Methods.(**: the difference between sevoflurane-exposed and unexposed neurons). N = 233 protrusions, similar results were obtained in two experiments. Calibration bars (B,C) are 5 microns in length.</p

Direct Tracking of Amyloid and Tau Dynamics in Neuroblastoma Cells Using Nanoplasmonic Fiber Tip Probes

Amyloid plaques and neurofibrillary tangles are the pathological hallmarks of Alzheimer’s disease. However, there has been a long-standing discussion on the dynamic relations between Aβ and tau proteins, partially due to the lack of a tool to track protein dynamics in individual live neurons at the early stage of Aβ generation and tau phosphorylation. Here, we developed nanoplasmonic fiber tip probe (nFTP) technology to simultaneously monitor Aβ42 generation and tau phosphorylation (at serine 262) in living, single neuroblastoma cells over 12 h. We observed that Aβ42 generation, under clinically relevant anesthetic treatment, preceded tau phosphorylation, which then facilitated Aβ42 generation. This observation is also supported by measuring proteins in cell lysates using the ultrasensitive label-free photonic crystal nanosensors. nFTP therefore provides an advanced method to investigate protein expression and post-translational modification in live cells and determine outcomes of intervention of Alzheimer’s disease and other neurodegenerative disorders

Dual F-actin (phalloidin) and drebrin immunoreactivity in control or sevoflurane-exposed DIV9 hippocampal neurons.

<p>(A-C) Control neurons stained with (A) phalloidin, (B) drebrin A/E polyclonal antibodies, or (C) in merged phalloidin/drebrin images demonstrate clustering of F-actin, and drebrin-IR in the protruding stalk or at the base of filopodial processes (arrows). (D-F) Sevoflurane-exposed neurons stained with (D) phalloidin, (E) drebrin antibodies, or (F) in merged phalloidin/drebrin images demonstrate clustering of F-actin, and drebrin IR predominantly in the submembranous regions of dendritic shafts (arrows) and much less co-clustering in filopodia stalks. Close-up views in merged image from control (G) neuron demonstrates the characteristic co-localization of drebrin-IR and drebrin-IR/phalloidin to the bases and mid-stalk filopodial locations (arrows, G); a merged image from sevoflurane-exposed (H) neuron reveals the absence of drebrin-IR in filopodial stalks, and thread-like, drebrin-IR projections emerging alongside drebrin-IR negative filopodia (arrowheads, H). Calibration bars are 5 microns (A-F) and 2.5 microns (G,H) in length.</p

Recovery of filopodia length 2 days after sevoflurane exposure on DIV7.

<p>(A) Two days after acute sevoflurane exposure, filopodia length recovered to mean levels observed in control unexposed DIV9 hippocampal neurons. (B) Two days after acute sevoflurane exposure, thin spine length exceeded the mean level in control unexposed DIV9 hippocampal neurons. (*: the difference between sevoflurane-exposed and unexposed neurons.) N = 76 protrusions, similar results were obtained in three experiments.</p

The Rho kinase inhibitor, Y27632, prevents sevoflurane-induced filopodial shortening in DIV7 immature hippocampal neurons.

<p>(A) 3% sevoflurane for four hours on DIV7 caused length-shortening in filopodia (solid bars) compared to control unexposed neurons (open bars). The length-shortening was completely prevented by co-incubating sevoflurane-exposed neurons with the selective Rho kinase inhibitor Y27632 (diagonal bars). Y 27632 alone (gray bars) increased filopodia length compared to control neurons. (**: the difference between sevoflurane-exposed and unexposed neurons; ^^ or the difference between sevoflurane-exposed in the presence or absence of Y27632; ## or the difference between control neurons in the presence or absence of Y27632. There was no significant interaction between Y27632 and sevoflurane. N = 318 protrusions, similar results were obtained in two experiments. Representative phalloidin images in (B) control, (C) sevoflurane-exposed, (D) Y27632-treated, or (E) Y2632 plus sevoflurane-exposed neurons. Calibration bars (B-E) are 5 microns in length.</p