Abstract
Several studies suggest a relationship between anesthesia-induced tau hyperphosphorylation and the development of postoperative cognitive dysfunction. This study further characterized the effects of continuous propofol infusion on tau protein phosphorylation in rats, with or without temperature control. Propofol was administered intravenously to 8–10-week-old male Sprague-Dawley rats and infused to the loss of the righting reflex for 2 h continuously. Proteins from cortex and hippocampus were examined by western blot and immunohistochemistry. Rectal temperature was significantly decreased during propofol infusion. Propofol with hypothermia significantly increased phosphorylation of tau at AT8, AT180, Thr205, and Ser199 in cortex and hippocampus except Ser396. With temperature maintenance, propofol still induced significant elevation of AT8, Thr205, and Ser199 in cortex and hippocampus; however, increase of AT180 and Ser396 was only found in hippocampus and cortex, respectively. Differential effects of propofol with or without hypothermia on multiple tau related kinases, such as Akt/GSK3β, MAPK pathways, or phosphatase (PP2A), were demonstrated in region-specific manner. These findings indicated that propofol increased tau phosphorylation under both normothermic and hypothermic conditions, and temperature control could partially attenuate the hyperphosphorylation of tau. Further studies are warranted to determine the long-term impact of propofol on the tau pathology and cognitive functions.
INTRODUCTION
Tau is a microtubule-associated protein that is abundant in the central nervous system (CNS) and expressed mainly in axons in physiological condition. In Alzheimer’s disease (AD) or other neurodegenerative disorders, hyperphosphorylated tau constitutes a significant component of neurofibrillary lesions. Besides alpha-synucleinopathies, polyglutamine disorders, and ubiquitin disorders, tauopathy is one of the most frequent types of neurodegenerative disorders with filamentous nerve cell inclusions [1]. Extensive research has provided insights into the impact of anesthesia administration on abnormal hyperphosphorylation, aggregation, and dysfunction of tau proteins and the development of postoperative cognitive dysfunction [2 –4]. During anesthesia and the often accompanying hypothermia, the transient hyperphosphorylation of tau displays a different state to that seen in the brains of patients with AD, in which tau is polymerized into paired helical filaments admixed with straight filaments forming neurofibrillary tangles [5]. The exposure of anesthetics may potentially increase the risk of AD by accelerating tau phosphorylation either directly through kinase activation or indirectly through hypothermia-induced inhibition of phosphatases [6, 7]. Hypothermia-induced tau hyperphosphorylation may also contribute to the exacerbation of AD through environmental factors or mutations in the genome [8]. Hypothermia could be induced by many factors such as aging, hypoxia, and infection, in addition to general anesthesia, and directly can cause postoperative delirium and cognitive dysfunction [9].
Propofol is the agent of choice for total intravenous anesthesia, given its favorable pharmacokinetic and pharmacodynamic profile. This di-isopropylphenol compound induces anesthesia by modulating the inhibition of GABAA receptors [10 –12]. A single dose of propofol could directly increase tau hyperphosphorylation at different epitopes in vitro and in vivo through inhibition of several tau-related kinases without hypothermia in mice and SH-SY5Y neuronal cells [13]. However, the in vivo effects of propofol administered continuously on tau protein phosphorylation and its related kinases are not well characterized. This study tested the hypothesis that continuous infusion of propofol may have differential effects on tau phosphorylation depending on the region under examination and investigated whether it is examined under hypothermic or normothermic conditions.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley (SD) rats (8–10 weeks old weighing 270–300 g) were purchased from the Laboratory Animal Unit (LAU) of The University of Hong Kong. Rats were housed in a temperature-controlled room at 20–22°C), humidity of 50 ± 10% and were kept on a 12/12 h light/dark cycle. All animals had access to food and water ad libitum, and they underwent an acclimatization period for one week before being employed in the experiment. All the experimental protocols and animal handling procedures were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong. The laboratory animal unit of The University of Hong Kong is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC international).
Anesthesia exposure
Twenty-four male adult SD rats were randomly assigned to three groups (n = 7-8). Control rats received no treatment (CON). The other rats were subjected to propofol treatment (10 mg ml–1, B. Braun, Germany) under either temperature maintained at 35°C (Pnormo) using a heating pad or had no temperature control (Phypo). The rats received 2 ml kg–1 of propofol for induction and loss of righting reflex was scored within 2 min after infusion [14]. Then, the infusion rates were maintained at 40 mg h–1 and 15 mg h–1 in Pnormo and Phypo group, respectively. The drug was administered using a micro-pump (ALC-IP600LB, Shanghai Alcott Biotech, China) for 2 h continuously through a 24-gauge tail vein cannula. The infusion rate was adjusted every 15 min as required, and rectal temperature was monitored by an animal body temperature thermometer (BW-TH5, USA).
Brain protein extraction
Following propofol exposure, rats were allowed to recover from anesthesia for 1 h at room temperature before being sacrificed by CO2 asphyxiation, which is in accordance with the guidelines of the American Veterinary Medical Association to allow the rats to enter a coma and then the head was cut for euthanization. The rats were then transcardially perfused with cold 0.9% saline and the brains were quickly removed and dissected on ice. Afterwards, the hippocampal and cortical tissues were dissected from the left hemisphere, immediately frozen in liquid nitrogen and stored at –80°C until analysis. The right hemispheres were fixed with 4% paraformaldehyde for 72 h, and then dehydrated in serial ethanol and embedded in paraffin for immunofluorescence. Proteins extracts were prepared by mechanically homogenizing the hippocampal and cortical tissues in ice-cold lysis buffer containing protease and phosphatase inhibitors purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). All homogenates were incubated in ice for 30 min, and total lysate was collected after centrifuging at 14,000 g for 30 min at 4°C and total protein content was determined in the supernatant by using the bicinchoninate assay (Bio-Rad, USA).
SDS-PAGE and western blot analysis
Brain tissues were subjected to western blot analysis as described previously [15]. The expressions of phosphorylated tau and total tau as well as the tau kinases and phosphatases were determined by SDS-PAGE coupled with western blot analysis. Total lysate were subjected to SDS-10% polyacrylamide gels electrophoresis and transferred onto PVDF membranes. Non-specific binding sites were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. The membranes were then incubated overnight at 4°C with primary antibodies for detecting phosphorylation of tau, total tau, specific tau kinase, or phosphatase. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (DAKO, Denmark) for 1.5 to 2 h at room temperature. The immunoreactive band signal intensity was subsequently visualized by chemiluminescence (ECL or ECL-plus, Amersham GE Healthcare, UK). All immunoblots were normalized for gel loading with β-actin (1 : 10,000 dilution, Cell signaling Technology, USA) or GAPDH (1 : 3000 dilution, Cell signaling Technology, USA) antibodies. The intensities of chemiluminescent bands were measured by Quantity One® ; software (Bio-Rad). Phosphorylated tau levels were adjusted to total tau level after normalized to GAPDH or β-actin, and the levels of different kinases were presented as the ratio of phosphokinase to total kinase, after being normalized by internal control.
Antibodies used in this study were directed at tau phosphorylated at the following epitopes: AT8 (pSer202/Thr205, 1 : 1000 dilution, Thermo Fisher Scientific), AT180 (pThr231/Ser235, 1 : 1000 dilution, Thermo Fisher Scientific), pS396 (pSer396, 1 : 3000 dilution, Invitrogen), pS199 (pSer199, 1 : 3000 dilution, Biosource), pT205 (pThr205, 1 : 3000 dilution, Invitrogen), and total tau (polyclonal rabbit anti-human tau, 1 : 30,000 dilution, DAKO, Denmark, USA).
The changes in the expression and activation of specific tau kinase or phosphatase were determined using the following antibodies: glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (Ser9), Akt, phospho-Akt (Ser473), p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK) 1/2), phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204), stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK), phospho-SAPK-JNK (Thr183/Tyr185) (1 : 1000 dilution, all purchased from Cell Signaling Technology, USA), PP2A-C (1 : 3000, Millipore, Temecula, CA, USA), and phospho-PP2A (Tyr307) (1 : 1000, Epitomics, CA, USA).
Western blot detection of Aβ
For the Aβ oligomerization assessment, whole protein lysate were extracted from hippocampal and cortical tissues, and finally 10 μl of the mixture of the sample and loading buffer was loaded and resolved in each lane on 4-12% SDS-sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE), transferred to PVDF membrane (Bio-Rad), probed with the anti-Human Amyloid Beta Protein 6E10 (1 : 1000, Signet, USA). β-Amyloid1-42 (ChinaPeptides, China) was used as the positive control for evaluating the effect of propofol with or without hypothermia on Aβ oligomerization. The antibody anti-α-tubulin (1 : 40,000 dilution, Invitrogen) was used for gel loading control.
Immunofluorescence
Paraffin blocks were prepared from the brain samples and 5- μm-thick coronal slices were made. In brief, sections were deparaffinated in xylene and rehydrated through serial ethanol, then treated with 0.01 M citrate buffer (pH 6.0) with 0.1% Tween-20 at 90°C for 15 min for antigen retrieval. After washing with phosphate-buffered saline (PBS), sections were blocked with 10% normal goat serum for 1 h at room temperature, and then incubated with primary antibodies at 4°C overnight. The following antibodies were used: AT8 (1 : 200 dilution), AT180 (1 : 200 dilution), or total tau (1 : 400 dilution). Sections were then incubated with Alexa Fluor 488 conjugated anti-mouse or rabbit IgG (1 : 400 dilution, Invitrogen) second antibodies for 2 h after washing with PBS at room temperature. Finally, sections were co-stained with 5 μM DAPI for 10 min at room temperature, and then mounted on glass slides with Mounting Medium (DAKO). Immunolabeled tissues were observed under a laser scanning confocal fluorescent microscope (Carl Zeiss LSM 700, Germany) equipped with ZEN light software.
Statistical analysis
The data of animal rectal temperature was analyzed by Two-Way analysis of variance (ANOVA) followed by Bonferroni’spost hoc tests, using the statistic software Prism 6 (GraphPad). Normalized band intensities in western blot were analyzed by One-Way ANOVA followed by Turkey’spost hoc test. All data were expressed as mean ± standard derivation (SD), and p < 0.05 was considered as statistically significant.
RESULTS
Rectal temperature declined during propofol infusion
A significant decrease in rectal temperature was observed during continuous propofol infusion. From similar rectal temperatures at baseline between two anesthesia groups (35.06 ± 0.39°C for Phypo versus 35.06 ± 0.20°C for Pnormo, F1,13 = 0.01663, p > 0.05), it kept around the basal level in Pnormo group throughout the whole infusion duration. However, in Phypo group, 15 mg hr-1 propofol infusion led to severe hypothermia, it dropped very rapidly to reach 33.65 ± 0.37°C 15 min after induction significantly (F1,13 = 4.458, p < 0.001) (30 min, 32.93 ± 0.74°C; 60 min, 31.74 ± 0.57°C), 30.03 ± 1.11°C (F1,13 = 17.39, p < 0.0001, Fig. 1) after 2 h of propofol infusion. Correspondingly, the dose required for maintaining loss of righting reflex in Phypo group was significantly decreased. Therefore, without active warming measures, propofol can induce profound hypothermia in rats. However, normothermia is quickly reestablished upon recovery from anesthesia (data not shown).
Propofol induced tau phosphorylation under both hypothermic and normothermic conditions
The effects of propofol on tau protein phosphorylation at different sites of the hippocampus and cortex were quantified by western blot analysis. There was no difference in total tau protein expression in these two brain regions. However, significant increases of tau protein phosphorylation were found in hippocampal regions after 2 h of propofol intravenous infusion. When compared with CON, tau hyperphosphorylation occurred at the AT8, AT180, Thr205, and Ser199 not only in Pnormo group but also in Phypo group significantly (p < 0.05). When compared with Pnormo, there were significantly higher hyperphosphorylation of AT8, AT180, and Thr205 in Phypo (p < 0.05). However, there was no change in tau phosphorylation at all for the Ser396 epitope (Fig. 2A).
For the cortex, significant increase tau phosphorylation for AT8, AT180, Thr205, and Ser199 was shown in Phypo group compared with control (p < 0.05). Nevertheless, tau hyperphosphorylation at AT8, Thr205, and Ser199 was also observed in the cortical tissue of Pnormo group when compare with control. Furthermore, the hyperphosphorylation of tau at AT8, AT180 and Thr205 were significantly less in Pnormo group than Phypo group (p < 0.05, p < 0.01, p < 0.05, respectively). In fact, for AT180, there was no difference between Pnormo and control. Although there was less phosphorylation for Ser199 in Pnormo, it did not reach significance when compared with Phypo. Interestingly, there was a slight increase in phosphorylation of tau protein at Ser396 in Pnormo group (Fig. 2B).
Aβ1–42 was also detected for investigating the effect of continuous propofol infusion on Aβ production and AβPP processing. Not surprisingly that there was no Aβ expression in young SD rat brain with or without propofol infusion (Supplementary Fig. 1), even though the protective effect of propofol on attenuating the isoflurane or sevoflurane induced oligomerization of Aβ1–42 [16].
Propofol induced tau phosphorylation in the rat CA3 hippocampal and cortical neurons
Immunofluorescence staining was performed to confirm the findings of the western blot analysis as well as to evaluate the distribution of tau phosphorylation in cortical and hippocampal regions. As AT8 and AT180 showed a distinct pattern in both Pnormo or Phypo groups in western blot analysis, these two epitopes were chosen for further investigation by using immunofluorescence staining. Similarly, there was no difference in immunofluorescent intensity for total tau among three groups in two brain regions. As shown in Fig. 3, increased green fluorescence was observed in AT8 and AT180 in the CA3 area of hippocampus in both Pnormo and Phypo groups after continuously infusion of propofol for 2 h, consistent with that seen in the western blot analysis. In the cortex, fluorescent immunoreactivity in AT8 was markedly increased in both Pnormo and Phypo groups, while that in AT180 showed observable increase in Phypo group only. These results are also consistent with that in western blot analysis.
Effect of propofol and hypothermia on kinases
The balance between protein phosphorylation and dephosphorylation induced by different tau kinases may ultimately determine whether abnormal hyperphosphorylation of tau occurs [17 –19]. Therefore, the direct impact of propofol on specific kinases commonly involved in the regulation of tau phosphorylation was further examined. These included glycogen synthase kinase-3β (GSK3β), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK).
The Akt/GSK3β signaling pathway is one of the identified signal transduction pathways crucial in the pathogenesis of AD as its activation promotes tau phosphorylation [19 –21]. The role of this pathway in propofol-induced tau phosphorylation in rats was investigated by western blot analysis. In the hippocampus, a significant increase of p-GSK3β (Ser9) was found in both propofol groups compared with control. However, there was also a significant increase in Phypo group hypothermia compared with Pnormo group (p < 0.01). In addition, a significant increase in Akt phosphorylation, an upstream kinase of GSK3β was observed in Phypo group (p < 0.01 versus CON) (Fig. 4A). In the cortex, when compared with CON group, p-GSK3β (Ser9) was only increased in Pnormo group (p < 0.05 versus CON) and p-Akt was only increased in the Phypo group (p < 0.01) (Fig. 4B).
ERK and JNK are two of the MAPKs bearing strong relationship with the pathogenesis of AD, and also contributed to the phosphorylation of tau [22]. In the hippocampus, significant increase of p-ERK1/2 in Pnormo group (p < 0.001 versus CON) was found while no change occurred in Phypo group. In contrast, no difference in the expression of p-ERK1/2 was found in the cortex among all three groups. On the other hand, significant increase of p-JNK was found in both Pnormo and Phypo group in hippocampal and cortical regions compared with the CON group (Fig. 4A, B).
Hyperphosphorylation of tau protein could be one of results from inactivation of protein dephosphorylation, and PP2A family represents a major fraction of cellular Ser/Thr phosphatase activity in the brain. Therefore, tau protein phosphatase catalytic subunit was further examined after continuous administration of propofol. Differential expression pattern was observed in hippocampal and cortical tissues. In the hippocampus, significant decrease in p-PP2A was observed in both Pnormo and Phypo groups (p < 0.05 versus CON), with lower level in Phypo group (p < 0.001 versus Pnormo). Surprisingly, propofol significantly increased PP2A-C under normothermia and hypothermia (p < 0.001 versus CON), with lower level in Phypo group (p < 0.001 versus Pnormo). In the cortex, the only observable change was a significant increase of p-PP2A in Phypo group (p < 0.001 versus CON or Pnormo). For PP2A, there was also a significant increase in Phypo group, while a significant decrease in Pnormo group (p < 0.01 versus CON).
These results demonstrated that propofol might have differential impact on the MAPK pathways and phosphatase of tau protein in hippocampus and cortex under hypothermic and normothermic conditions. The above results are summarized in Table 1.
DISCUSSION
The intravenous agent propofol has both beneficial and detrimental effects upon the CNS and individual neurons. While it enhances the inhibition of the presynaptic and postsynaptic transmission [12 , 24] which could partially explain its neuroprotective effects in acute mechanical injury [25], it also exerts neurotoxic effects, such as that seen in human stem cell-derived neurons [26] and in the developing brain through multiple mechanisms such as disrupting blood-brain barrier permeability [27]. Moreover it has been shown that propofol intensifies endocrine responses to stress [28], induces apoptosis of neurons and oligodendrocytes [29] and delays physical and neurological reflexes [30, 31] to cause persistent learning deficits and neurobehavioral abnormalities. Similarly, propofol could impair endogenous neurogenesis and functional recovery after acute traumatic brain injury in SD rats [32]. However, much less is known regarding the effect of continuous propofol infusion on the development of neurodegenerative changes, in particular, on tau protein phosphorylation.
This study investigated the effects of propofol on tau phosphorylation under normothermic and hypothermic conditions in different brain regions. It was important to perform the experiments under these separate conditions as some earlier findings regarding anesthesia and tau hyperphosphorylation was attributed to anesthesia-induced hypothermia. Furthermore, instead of examining the effects of only a single dose of propofol as seen in some of the earlier studies, we titrated the dose to provide “natural anesthesia” [33] as judged by the loss of righting reflex [14] which is an acceptable standard for maintenance dosage of anesthesia in vivo. This justifiably resulted in a significant difference in dosage between the two groups, which in part can be explained by the effects that hypothermia has on the elimination process [34, 35]. The use of rats instead of mice in vivo models made it more feasible to induce and maintain anesthesia intravenously, which enabled better control of blood levels of propofol, as well as avoiding an excessively deep anesthesia associated with bolus effect of intraperitoneal induction in the initial period.
Our data indicate that continuously infusion of propofol for 2 h under both normothermic and hypothermic conditions induce tau hyperphosphorylation in both hippocampal and cortical regions. Firstly, significant hypothermia was generated after propofol infusion immediately, and continued until the end of anesthesia. Therefore, continuous hypothermia induced by propofol infusion constructed a hypothermic condition. More importantly, we have demonstrated the effects of propofol on the phosphorylation of tau depend not only on the phosphorylation site but also regional specific in the brain. In western blot analysis, our data demonstrated an increase of phosphorylation of tau at AT8, Ser199, and Thr205 in both hippocampus and cortex under either normothermic or hypothermic conditions. However, under normothermic condition, significant increase of phosphorylation of tau at AT180 and Ser396 was only found in the hippocampus and cortex, respectively. Furthermore, under hypothermic condition, there was no significant change in phosphorylation of tau at Ser396 in either hippocampal or cortical region. These results suggest a different vulnerability between neurons from the two regions and the different responses of AT180 and Ser396 phosphorylation may be an indicator of the effects of propofol or hypothermia. A site-specific effect of tau phosphorylation on microtubule assembly activity and self-aggregation has been reported [36]. Phosphorylation of tau at the C-terminus region, including Ser396 and Ser404 sites, increases its microtubule assembly activity, whereas phosphorylation at the Proline-rich regions, such as Ser199, Ser202, Thr205 and Thr231 (one of AT180 epitopes) inhibits its activity. Moreover, phosphorylation at C-terminus region may promote tau self-aggregation more efficiently than tau phosphorylation at other regions [36, 37]. Of note, our findings here may implicate that the direct impact on tau phosphorylation at different phosphoepitopes by exposure to propofol could promote the dissociation of tau from microtubules and not increase self-aggregation of tau in the hippocampus. However, phosphorylation of tau at Thr231 has been proposed to be an early event in tau pathology and is strongly associated with future development of AD in patients with mild cognitive impairment [37, 38]. One has speculated that a decrease in the affinity of tau for microtubules may lead to an increase in the free pool of tau, and may be followed by phosphorylation of sites that make tau more fibrillogenic [38, 39]. Further investigation is required to clarify the contribution of propofol on the specific sites to the alterations in biological activities of tau.
In addition, we have made a further comparison on the effect of propofol under normothermic and hypothermic conditions. Hypothermia [39, 40] or anesthetics [15 , 41–44] has been reported to elicit an independent effect on tau phosphorylation. Further, a reversed effect on tau hyperphosphorylation and partial recovery in cognitive impairment has been shown after single injection of propofol with temperature control [45]. However, our results indicated temperature maintenance does not avoid hyperphosphorylation of tau after exposure to propofol infusion. Nevertheless, our results demonstrate that hypothermia may exert a synergistic effect on tau phosphorylation at AT8, Ser199, Thr205, and AT180, which implicate that temperature maintenance may be one of the critical factors in mitigating the anesthetic induced effect. Further investigation is required to clarify the impact of propofol and change of temperature on cognitive impairment.
Tau hyperphosphorylation is a result of an imbalanced regulation of protein kinases and protein phosphatases, which contribute to the regulation of biological activity of tau by controlling its phosphorylation level [37, 46]. Tau proteins contain phosphorylation sites for a large number of protein kinases, which include GSK3β, MAPK/ERK1/2, and SAPK/JNK [46]. In this study, we examined the expression and/or activation in three of these tau kinases. Activation of Akt kinase, an upstream of GSK3β kinase, has been correlated to AD pathogenesis [47]. From our results, propofol-induced hypothermia triggered the activation of Akt without affecting GSK3β activity in the cortex. In contrast, an increase of Akt activity together with the increase in p-GSK3β was found in the hippocampus. These results suggest that Akt is not the sole upstream regulator of GSK3β. GSK3β is constitutively active in all cells and plays a pivotal role in tau phosphorylation in neurons and glial cells [48 –50]. Its activity is primarily regulated through inhibition by activated Akt that in turn does so by phosphorylation of GSK3β at its Ser9 site [49]. We demonstrated an increase in the inhibition of GSK3β activity in the hippocampal region following propofol exposure with or without temperature maintenance. However, in the cortical region, a decrease in GSK3β activity was only found under normothermic conditions. In AD brains, increase in GSK3β activity has been suggested to be involved in excessive tau phosphorylation [49]. However, the opposite results found here suggest that propofol-elicited effects on tau phosphorylation at AT8, Ser199, Thr205, and AT180 may not be primarily mediated by the activation of GSK3β, but rather through other kinase or phosphatases activity. Indeed, the phosphorylation sites AT8, Ser199 and Thr205 are located in the proline-rich region that has been suggested the major phosphorylation sites by Dyrk1A [36, 51].
ERK and JNK have also been reported to be involved in phosphorylation of tau, and are more effective at phosphorylating the Ser202/Thr205 sites of the AT8 epitope [51]. Therefore, these two pathways that are involved in the pathophysiological process of AD were further investigated. Our data showed an increase in p-JNK levels in hippocampal and cortical regions under both hypothermic and normothermic conditions whereas the up-regulation of ERK1/2 activity was observed in cortex under normothermia only. These results suggest that tau hyperphosphorylation shown in hippocampus may be due to increased activities of JNK signaling. In fact, in a recent study of cigarette smoking-induced AD neuropathology in rats, increased activities of JNK were also found in mediating tau phosphorylation by cigarette smoke [52]. Most importantly, these results indicate the anesthetic alone may directly trigger activation of ERK1/2 and JNK in the hippocampus. Activation of ERK1/2 has been suggested that they are not related to phosphorylation of tau under physiological conditions, and their participation in tau hyperphosphorylation under pathologic conditions has not been excluded [53]. Taken together, our results demonstrate a direct effect of propofol on the activity of tau kinases and in turn the phosphorylation of tau. The dysregulation of these events may lead to deleterious consequence in cognitive functions as the tau pathology has been suggested involving in impairment of hippocampal synaptic transmission and association with concomitant memory impairment [4]. Imbalance in MAPK pathway after anesthesia has been demonstrated using dexmedetomidine [15]. This suggests that continuous exposure to propofol may perturb MAPK, and hence cognitive function.
Protein phosphatases of the type 2A family (PP2A), as a major tau phosphatase, expression and/or activity have been found significantly decreased in AD. In healthy neurons, PP2A is inactive in the nucleus and active in the cytoplasm as indicated, preventing phosphorylation of cytoplasmic tau [54]. However, Wang et al. found that PP2A and GSK3β regulated each other and control tau phosphorylation both directly and indirectly to each other. In our study, the consequences of propofol on PP2A expression in brain were more complicated. In the hippocampus, we demonstrated that significant decrease in the inhibition of PP2A but increase in PP2A-C at the same time. Even though propofol with hypothermia showed significant lower expression. Actually, the increase of PP2Ac is an indicator of PP2A inhibition, and autoregulation of PP2A translation would be switched on to maintain steady protein level when the activity changed [55]. It suggests that GSK3β and/or PP2A are/is the major mechanism underlying hyperphosphorylation of tau protein triggered by propofol in hippocampus; and hypothermia could intensify this level through the same mechanism(s). In cortex, protein phosphatase does not seem to be the only signaling machinery to trigger tau phosphorylation during anesthesia with or without hypothermia, even though there were increased levels of PP2A expression and phosphorylation. Hyperphosphorylation of tau occurred during general anesthesia produced by continuous propofol infusion may be due to the different efficiencies of tau-related kinases and protein phosphatases toward differentsites.
In conclusion, our findings demonstrate a site and neuronal specific in phosphorylation of tau after exposure to propofol for 2 hours continuously in an in vivo model. Most importantly, the presence of hypothermia during anesthesia exposure appears to exacerbate the degree of phosphorylation of tau. However, although it may exert a synergistic effect on tau phosphorylation, hypothermia may not be an absolute requisite for the development of tau pathology following anesthesia exposure, as changes in level of phosphorylation of tau were also found during normothermic conditions. Propofol may be administrated for a few hours in the intraoperative period, to days as in the case in postoperative sedation in intensive care units [13]. Therefore, the effect of propofol on phosphorylation of tau should not be underestimated. As our results show a differential effect of propofol on different sites of tau phosphorylation in different brain regions, this may provide new insight into the anesthetics induced site-specific tau phosphorylation and the effects on the sites required for the abnormal hyperphosphorylation of tau in AD.
