Bryostatin-1 protects against amyloid- beta (Aβ) oligomer-induced neurotoxicity by activating autophagy

Abstract

The expression of PKCε, a member of the protein kinase C family, has been found to be decreased in the frontal cortex of patients with Alzheimer’s disease (AD). Bryostatin-1 is a PKCε activator and has shown benefits in AD animal models. However, how Bryo-1 protects neuronal activity and synaptic function in AD pathology is still unknown. In the present study, we first established an Aβ toxicity cell model by administering Aβ 25-35 oligomers to cultured primary hippocampal neurons. We then evaluated the protective effect of Bryo-1 on this model by administering Bryo-1 together with Aβ oligomers. Finally, we investigated the autophagic influx of primary hippocampal neurons in the presence of Aβ oligomers and Bryo-1. Imaging and electrophysiology recordings showed that Bryo-1 protected synaptic dynamics and functions against the neurotoxicity of Aβ oligomers. Furthermore, Bryo-1 was also found to rescue autophagy influx that was impaired by Aβ oligomer treatment. This discovery may explain the beneficial effects of Bryo-1 in AD animal models. In summary, we discovered a novel mechanism by which Bryo-1 protects neurons against Aβ oligomer-induced neurotoxicity. Our results may support the concept of developing PKCε activators as therapeutics for AD.

Keywords
Aβ oligomer toxicity; Bryostatin-1; Autophagy influx

INTRODUCTION

Alzheimer’s disease (AD) is one of the most common causes of dementia in elderly people and affects millions of people worldwide. The symptoms of AD include the gradual loss of memory and other cognitive abilities, which could eventually interfere with daily life and result in patient death (Yiannopoulou, Papageorgiou, 2020 ; Rajan et al., 2021 ; Scheltens et al., 2021 ). The symptoms of AD will gradually worsen over a short number of years. In the late stage of AD, patients lose the ability to start a conversation and to respond to the environment. Generally, AD patients can live 4-8 years after diagnosis (Breijyeh, Karaman, 2020 ; Bellenguez et al., 2022 ). The pathological causes of AD are still unknown, and the highest risk factor known is increasing age, as 95% of AD patients are over 65 years old (Heneka et al. , 2010 ; DeTure, Dickson, 2019 ).

To date, scientists have raised several hypotheses about the pathological mechanisms of AD, such as the amyloid hypothesis, tau pathology hypothesis, apoE4 mutation and other less validated hypotheses (Moreira et al. , 2010 ; Kumar, Singh, Ekavali, 2015 ). Although many hypotheses have been proposed, synapse loss in the frontal cortex is still the only pathological change that is correlated with the extent of dementia in both AD patients and AD animal models (Mecca et al. , 2020 ; Subramanian, Savage, Tremblay, 2020 ). Synapses connect different neurons and are the structural basis of neuronal circuits in the brain. The electrical transduction mediated through synapses is fundamental to cognitive functions (Sheng, Sabatini, Südhof, 2012 ; Wang et al. , 2019 ; Ni et al. , 2021 ; Chi et al. , 2022 ).

Each synapse consists of presynaptic and postsynaptic compartments (Palop, Mucke, 2010 ). The presynaptic compartments are the terminals of axons, while the postsynaptic compartments are dendritic spines that grow on neuronal dendrites. The dynamics of synapses, including synapse genesis, maintenance, and elimination, are currently considered to be most important for cognitive functions (Spires-Jones, Hyman, 2014 ). The functions and dynamics of dendritic spines are tightly regulated by cytoskeleton proteins, such as microtubules and their upstream regulator, Rho-GTPases (Vallejo et al. , 2021 ; Ge et al. , 2023 ; Linseman, Lu, 2023 ). Many molecular pathways underlying the dynamics of synapses have been proposed. Among these pathways, emerging evidence has shown that the PKC signaling pathway plays important roles in regulating synaptic function (Sun, Alkon, 2019 ; Emperador-Melero et al. , 2021 ; Ortiz-Sanz et al. , 2022 ).

Bryostatin 1 is a natural product derived from the marine invertebrate Bugula neritina several decades ago (Keck et al. , 2011 ). It was later proven to activate PKCε, an isotype of the PKC family, with nanomolar potency. Preclinical studies have demonstrated that the activity of PKCε in AD is dramatically decreased (Khan et al. , 2015 ), and Bryostatin 1 can activate the secretion of the neurotrophic factor BDNF and protect against amyloidβ (Aβ) oligomer-induced neuronal toxicity when administered directly into the central nervous system (Sun et al. , 2014 ; Giarratana et al. , 2020 ; Ly et al. 2020 ). Bryostatin 1 can also activate nonamyloid cleavage of amyloid precursor protein (Bellenguez , et al. , 2022 ), which leads to increased secretion of neurotrophic sAPPα (Schrott et al. , 2015 ). Furthermore, intraperitoneal administration of Bryostatin 1 in AD animal models could prevent synaptic loss (Sun et al.*, 2014 ). However, the molecular mechanisms underlying how Bryostatin 1 protects neurons against Aβ oligomer neurotoxicity are still unknown.

In the present study, we administered amyloidβ (Aβ) oligomers to cultured primary hippocampal neurons, which are the most commonly used in vitro cell model to simulate clinical AD pathology. By using this model, we confirmed that Bryostatin 1 could protect synapses from Aβ oligomer- induced toxicity. We further found that this protective effect was mediated by activating autophagy. Our findings raised the novel hypothesis that Bryostatin 1 could protect neurons from Aβ oligomer toxicity by activating autophagy.

MATERIAL AND METHODS

Reagents and Plasmids

eGFP (#176015) (Lo et al. , 2021 ), pMRX-IP-GFP- LC3-RFP (#84573) (Kaizuka et al. , 2016 ), Lamp1-RFP (#1817) (Sherer et al. , 2003 ) and pEGFP-LC3 (#21073) (Kabeya et al. , 2000 ) were all purchased from Addgene ( http://www.addgene.org/ ) Bryostatin 1 was purchased from Sigma‒Aldrich (Cat. B7431-10UG), the Aβ2535 fragment was purchased from Dalian Meilun Biology Technology Co., Ltd. The PKCε (cat. The V6473) kinase enzyme assay kit was purchased from Promega Corporation.

Primary neuronal culture and transfection

Procedures and ethics involving pregnant animals were approved by the Institutional Animal Care and Use Committee of Nantong University (IACUC approval number S20220228-023). Pregnant Sprague‒Dawley rats at embryonic Day 17 (E17) were purchased from the animal facility of Nantong University and arrived in the laboratory 1 day before dissection to adapt to the environment. A total of 50 pregnant animals were used in this study.

Cell culture and transfection were performed as previously described (Kaech, Banker, 2006 ; Sun et al. , 2013 ). In brief, prior to dissection, 35 mm cell culture dishes were pretreated with 1 ml of poly-L-lysine (0.5 mg/ml; Millipore Sigma) in a 37 °C incubator overnight and then washed with sterile water 3 times. On the second day, pregnant rats were anesthetized with 20% inhaled isoflurane (UPS level, supplied from Nantong University Medical Center) with an airflow rate of 10% volume/minute. Fifteen minutes later, the anesthesia condition was confirmed by touching the rat’s front paw. The rat was then moved into a chemical hood and euthanized by decapitation.

After rats were sacrificed, embryos were decapitated with surgical scissors and then dissected for hippocampal neuron culture according to previous literature(Sun et al. , 2013 ). During dissection, the hippocampus was collected in a 15 ml conical tube containing dissection buffer (HBSS buffer with 10 mM HEPES, pH 7.3) at room temperature. After all hippocampi were harvested, the conical tube was centrifuged, the dissection buffer was discarded, and the dissection buffer was replaced with digestion buffer (dissection buffer with 0.25% trypsin). The hippocampus was transferred into a 37 °C cell culture incubator with a conical tube for 15 mins and then rinsed with dissection buffer for 5 mins for a total of 3 times. The hippocampus was then gently triturated with a glass Pasteur pipette, and the density of neurons was quantified by using a hemocytometer. Generally, one pregnant rat will have 10-13 litters, which can provide 8x106 hippocampal neurons per litter, or 8-10 x107 hippocampal neurons in total.

Primary neurons were then seeded in 35 mm cell culture dishes at low density (5x104 cells/35 mm dish) for transfection and imaging or at high density (5x105 cell/35 mm dish) for biochemical analysis. The neuronal culture medium (Neurobasal supplemented with 1x B-27 and 2 mM GlutaMAX) should be preconditioned by primary astrocytes for 24 hours prior to use. After seeding, the cell culture dishes were left in a CO2 cell culture incubator (90%-95% relative humidity and 5% CO2) at 37 °C for 4 hours, and then 1 μM AraC was added to the culture medium to inhibit potential contamination of glial cells. Primary neurons will be used for transfection or compound treatment as needed. No medium changes were needed for the first 2 weeks. Primary neurons will be regularly observed under a contrast microscope to check the cell condition. Generally, healthy primary hippocampal neurons attach to the plate within a day, and then dendrites start to grow 2 or 3 days after dissection. One week after dissection, healthy primary neurons should have intact dendrites that have grown at least 10 times longer than the size of the cell body. On the other hand, unhealthy neurons generally have discontinuous dendrites that can be easily observed under a microscope.

Transfection

Seven days after dissection, calcium transfection was performed with primary neurons according to previous literature(Sun , et al. , 2013 ). In brief, transfection medium (MEM with 1x GlutaMAX, 1 mM sodium pyruvate, 7% glucose, 1% ovalbumin and 1% N2 supplement) was preconditioned by astrocytes 24 hours prior to transfection. On the day of transfection, the cell culture medium was first changed to preconditioned transfection medium, and the CaCl2 method was used for transfection. A total of 1-4 μg of plasmid DNA were used in each 35 mm dish. The plasmids used here included eGFP, GFP- LC3-RFP, pEGFP-LC3, and Lamp1-RFP, as indicated in the reagents above. After transfection, the cells were returned to the incubator for 1 hour and then rinsed with dissection buffer 3 times, and the medium was changed back to preconditioned neuronal culture medium with 0.5 mM kynurenic acid.

Confocal imaging and image quantification

Confocal images were obtained using a Leica TCS SP8 with a 60x water objective lens (Leica Microsystems GmbH) with a sequential acquisition setting. Fourteen days after dissection (or 7 days after transfection), the transfected cells will receive 20 µM Aβ oligomer overnight with or without 10 µM Bryostatin 1 for experiments. Primary hippocampal neurons were then washed with PBS and fixed with 4% PFA. Fixed cells were then visualized with 488 nm excitation for green fluorescence and 594 nm for red fluorescence. The vesicles with green, red or yellow colors were quantified in a blinded way by 2 independent individuals using ImageJ software (version 1.50i; National Institutes of Health). The dendritic spine density and volume were also quantified independently using ImageJ software.

Western blot

Primary neurons after various treatments were lysed using protein lysis buffer (Beyotime Institute of Biotechnology) on ice for 15 min. Following centrifugation (10,000 x g, 7 min at 4 °C), the supernatant was collected, and the protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Inc.). ), reading absorbance at 560 nm by using a plate reader (BioTek Synergy HT MultiMode Microplate; BioTek instruments, Inc.). Following protein denaturation, 100 µg of total protein was loaded onto 10% or 15% SDS‒PAGE gels and then transferred onto PVDF membranes. Membranes were then blocked with 5% BSA (Beyotime Institute of Biotechnology) for 1 h at room temperature. Following blocking, the membranes were incubated with primary antibody overnight at 4 °C and then with secondary antibody for 1 h at room temperature. The primary antibodies used in the present study included rabbit anti-synapsin-1 (1:2000, Cat. #5297, Cell Signaling Technology), mouse anti-PSD95 (1:500, Cat. #36233, Cell Signaling Technology), rabbit anti-synaptotagmin-1 (1:2000, Cat #3347, Cell Signaling Technology), rabbit anti-mTOR (1:1000, Cat #2983, Cell Signaling Technology), rabbit anti-phospho mTOR (1:1000, Cat #5536, Cell Signaling Technology), rabbit anti-phospho P70S6 kinase (1:1000, Cat #9204, CST), rabbit anti-P70S6 kinase (1:2000, Cat #9202, CST), and mouse anti-βActin (1:9000, Cat #3700). The secondary antibodies used were HRP-labeled anti-rabbit IgG (1:5,000; cat. no. 7074, Cell Signaling Technology) and HRP-labeled anti-mouse IgG (1:5,000; cat. no. 7076, Cell Signaling Technology). Bands were visualized by using enhanced ECL reagent (cat. no. WBulS0500, Millipore Sigma) and developed in a dark room. The bands were semiquantified by using ImageJ software (1.50i; National Institutes of Health).

Electrophysiology recording

Whole-cell recordings were performed in voltage- clamp mode using a MultiClamp 700B amplifier (Molecular Devices, LCC) at 50 kHz of sample frequency. Recording signals were numberlized by using a Digidata 1440 digitizer (Molecular Devices, LCC). Patch pipettes were made with 3-5 MΩa resistance when filled with a standard intracellular solution (95 mM K-gluconate, 50 mM KCl, 10 mM HEPES, 4 mM Mg-ATP, 0.3 NaGTP, 10 mM phosphocreatine; pH 7.2, 300 mOsm). Miniature excitatory postsynaptic current (mEPSC) was measured in rat hippocampal neurons following Aβ oligomer treatment at room temperature in artificial cerebrospinal fluid (126 mM NaCl, 2.5 mM KCl, 10 mM glucose, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2 and 26 mM NaHCO3) with 0.5 μM tetrodotoxin (Sigma‒Aldrich; Merck KGaA).

In vitro PKCε kinase activity

PKCε kinase activity was measured using the PKCε Kinase Enzyme System (cat. no., V4037; Promega Corporation) according to the manufacturer’s instructions. Briefly, high-density cortical neurons with different treatments were lysed with RIPA buffer, and 100 µg of total protein from each group was then incubated with 5 µM ATP and 0.1 µg/µl substrate for 60 min at room temperature. A total of 25 ng of purified PKCε was used as the positive control. Following incubation, ADP-Glo was added to the system and incubated at room temperature for another 40 min. Luminescence signals were detected using a microplate reader (BioTek Synergy HT MultiMode microplate reader).

Statistical analysis

GraphPad Prism Software Version 8.0 (GraphPad Software, Inc.) was used for results analysis. All data are presented as the mean ± SD. Significant differences were determined using one-way ANOVA with Tukey’s post hoc test (for studies with 3 groups) or two-way ANOVA with Tukey’s post hoc test. All electrophysiological data were analyzed using pCLAMP 11 (Molecular Devices). P<0.05 was considered to indicate a statistically significant difference.

RESULTS:

Bryostatin 1 activated PKCε activity in primary neurons after Aβ oligomer treatment

To investigate the effects of Bryostatin 1 on neurons, we first tested whether Bryostatin 1 regulates PKCε activity. By using in vitro kinase activity assays, we administered Bryostatin 1 in ascending doses and found that Bryostatin 1 activated recombinant PKCε in a dose- dependent manner with an IC50 of 5.89 µM ( Figure 1A ). We next investigated whether Bryostatin 1 could activate PKCε activity in primary neurons after Aβ oligomer treatment. In this study, we treated primary neurons with 20 µM Aβ oligomers overnight and then administered 10 µM Bryostatin 1 for 2 hours as a rescue treatment. We then lysed cells to perform kinase activity assays and western blots to investigate PKCε activity. The results from the in vitro kinase activity assay showed that Aβ oligomer treatment could significantly decrease PKCε activity, while Bryostatin 1 could rescue PKCε activity back to 80% of the level of the control group ( Figure 1B ). Furthermore, western blots also showed that although the total protein of PKCε did not change significantly after Aβ oligomer treatment, the phosphorylation level of the PKCε S729 site was significantly decreased, which indicated that the activity of PKCε was suppressed. Furthermore, 2 hours of Bryostatin 1 treatment significantly recovered the phosphorylation level at the S729 site, which indicated that Bryostatin 1 could promote PKCε activity and was consistent with the results from the kinase activity assay ( Figure 1C, D ).

FIGURE 1
FIGURE 1 - Bryostatin 1 could increase PKCε activity. PKCε kinase activity was detected in vitro (A) or in hippocampal neurons (B). The results are represented as luminescence units (Lo et al. , 2021 ). n=3. (C) western blots from hippocampal neurons treated with Aβ oligomers alone or with Bryostatin 1. (D) Quantification of the ratio of phosphorylated PKCε to total PKCε. n=3, * p < 0.05. p- PKCε: phosphorylated PKCε at site S729; t- PKCε: total PKCε.

Bryostatin 1 protected synapse function in primary culture against Aβ oligomer toxicity

Previous reports have shown that PKCε can regulate synapse dynamics. Considering that the first toxic effect of Aβ oligomers on neurons is synaptic function damage, we next planned to investigate whether Bryostatin 1 could rescue Aβ oligomer-induced synaptic dysfunction. We employed primary hippocampal neurons in this study and transfected them with the eGFP construct for visualization. Seven days after transfection, we administered Aβ oligomers at 20 µM with or without 10 µM Bryostatin 1 for 24 hours and then directly counted the dendrite spine number under a confocal microscope. The results showed that Aβ oligomer treatment significantly increased the total number of protrusions in the dendrites, while the number of mature dendritic spines decreased. The average width of the spine head was also decreased. This result indicated that the dendritic spines were damaged by Aβ oligomer treatment, and this impairment was rescued by Bryostatin 1 treatment ( Figure 2A-D ).

FIGURE 2
FIGURE 2 - The PKCε activator Bryostatin 1 rescues Aβ oligomer-induced dendritic spine morphology impairments. (A) Primary hippocampal neurons were transfected with GFP at DIV7 and then treated with Aβ oligomers alone or in combination with Bryostatin 1 at DIV 17. Cells were visualized under confocal microscopy. Scale bar = 30 μm. (B) The number of protrusions from each group was quantified. n=1,200 protrusions from at least 25 neurons. (C-D) Quantification of spine length and width from (A). *P<0.05, **P<0.01 and ***P<0.001.

We also conducted electrophysiology recordings to measure synaptic functions. The results showed that the frequency of mEPSCs was significantly reduced after Aβ oligomer treatment but was fully rescued by Bryostatin 1 ( Figure 3 ). This result further confirmed that Bryostatin 1 could rescue the dysfunction of synapses caused by Aβ oligomer treatment.

FIGURE 3
FIGURE 3 - The PKCε activator Bryostatin 1 rescues Aβ oligomer-induced synapse dysfunction. (A) mEPSCs were recorded in primary hippocampal neurons following Aβ oligomer treatment alone or in combination with Bryostatin 1. The (B) current amplitude and (C) current frequency were quantified. n=25 neurons. *P<0.05.

Finally, we performed western blots to investigate biomarkers that both represent presynaptic and postsynaptic compartments. The results here were consistent with the morphology and functional results, as Synapsin-1, Synaptotagmin-1, and PSD-95 were all decreased in the Aβ oligomer groups and recovered in the Bryostatin 1 group ( Figure 4 ).

FIGURE 4
FIGURE 4 - Bryostatin 1 protects synaptic markers. (A) Primary hippocampal neurons were treated with Aβ oligomers alone or in combination with Bryostatin 1, and Western blotting was then performed to measure synaptic markers. (B-D) Quantification of A. n=5, *P<0.05, **P<0.01 and ***P<0.001.

Bryostatin 1 activated the mTOR signaling pathway

Previous reports have shown that PKCε activates the mTOR signaling pathway in cardiomyocytes under hypoxic conditions (Moschella et al. , 2013 ). Activation of the mTOR pathway could enhance the resistance of cells to stressful environments. Based on the above results, we hypothesized that the activation of the PKCε pathway could then activate the mTOR pathway in neurons to resist Aβ oligomer toxicity. To investigate

this hypothesis, we performed western blots to measure the phosphorylation level of mTOR components and its downstream target, S6K1 (p70 S6 kinase). In this study, we administered 20 µM Aβ oligomers with or without 10 µM Bryostatin 1 for 24 hours and then lysed the cells for western blotting. The results showed that Bryostatin 1 could significantly increase the phosphorylation level of S6K1 at the T389 and T421/424 sites and increase the phosphorylation of mTOR1. However, Aβ oligomer treatment alone did not change those protein biomarkers significantly ( Figure 5 ).

FIGURE 5
FIGURE 5 - The PKCε activator Bryostatin 1 regulates the mTOR signaling pathway. (A) Primary hippocampal neurons were treated with either Aβ oligomers alone or in combination with Bryostatin 1, and Western blotting was then performed to measure key proteins in the mTOR signaling pathway. (B-D) Quantification of A. n=5, *P<0.05, **P<0.01 and ***P<0.001. p-mTOR: phosphorylated mTOR. T-mTOR: total mTOR. pT421-S6K1: S6K1 phosphorylated at T421; pT389-S6K1: S6K1 phosphorylated at T389.

Bryostatin 1 could enhance autophagic influx.

Enhanced autophagy influx is one of the most important events downstream of mTOR activation. Considering that abnormal autophagic processes were previously discovered in AD patients (Nixon, Yang, 2011 ), we next wanted to investigate whether Bryostatin 1 could rescue the autophagic influx damage caused by Aβ oligomers. For this purpose, we transfected a GFP:RFP:LC3 plasmid into primary hippocampal neurons and then treated them with Aβ oligomers and Bryostatin 1 for 24 hours. The results showed that Aβ oligomer treatment could significantly impair autophagic influx. For instance, large numbers of yellow vesicles were observed. Furthermore, Bryostatin 1 treatment rescued the deficiency of LC3 degradation ( Figure 6 ), as fewer yellow vesicles but more individual green or red vesicles were observed in this group.

FIGURE 6
FIGURE 6 - The PKCε activator Bryostatin 1 rescues Aβ oligomers from impaired autophagy function. (A) Primary hippocampal neurons were transfected with GFL-RFP-LC3 plasmid at DIV7 and then treated with Aβ oligomers alone or combined with Bryostatin 1 at DIV 17. Cells were visualized under confocal microscopy. Scale bar = 30 μm. The numbers of green vesicles only (B), red vesicles only (C) and yellow vesicles (D) were quantified. *P<0.05, and ***P<0.001. n =120 neurons

Next, we cotransfected GFP-LC3 and RFP-lamp1 plasmids into primary neurons and then administered Aβ oligomers and Bryostatin 1 overnight. The results here also showed that although LC3 cannot be correctly degraded in lysosomes after Aβ oligomer treatment, Bryostatin 1 cotreatment could efficiently rescue this degradation ( Figure 7 ).

FIGURE 7
FIGURE 7 - The PKCε activator Bryostatin 1 rescues Aβ oligomers from impaired lysosome function. (A) Primary hippocampal neurons were transfected with GFL-LC3 and RFP-LAMP1 plasmids at DIV7 and then treated with Aβ oligomers alone or combined with Bryostatin 1 at DIV 17. Cells were visualized under confocal microscopy. Scale bar = 30 μm. The numbers of green vesicles only (B), red vesicles only (C) and yellow vesicles (D) were quantified. *P<0.05, **P<0.01, and ***P<0.001. n=120 neurons

DISCUSSION:

In the present study, we discovered that Bryostatin 1 could protect cultured primary neurons against Aβ oligomer-induced toxicity by elevating autophagic influx. For instance, we first found that Bryostatin 1 could protect synaptic function and synapse morphology against Aβ oligomers and then found that Bryostatin 1 could activate the mTOR pathway, which is a key pathway regulating cell survival under stress conditions. Finally, we found that Bryo1 rescued autophagic influx, which was impaired by Aβ oligomers. Overall, advanced from previous reports, our results demonstrated a novel mechanism by which Bryostatin 1 protects neurons from Aβ oligomer toxicity and cognitive functions in an AD animal model.

Bryostatin 1 has been found to elevate PKCε activity in many different cell types(Pohjolainen et al. , 2020 ; Erin et al. , 2021 ; La Cognata et al. , 2023 ). Furthermore, our study demonstrated that Bryostatin 1 could activate PKCε in primary neurons. PKC family members have many important roles in maintaining basic cellular functions, such as cell survival and cell movements. A previous study showed that PKCε could activate the mTOR signaling pathway under stress conditions (Huang et al. , 2016 ; Pakri Mohamed et al. , 2018 ; Hanim et al. , 2020 ), which facilitates cell survival by activating many basic cell metabolism pathways. Our study is the first to show that PKCε could also activate the mTOR pathway under Aβ oligomer toxicity in primary neurons, which indicates that mTOR may be developed as a novel therapeutic target for AD treatment.

In this study, we transfected a GFP-RFP-LC3 construct into primary neurons to investigate autophagic influx. LC3 is a biomarker that is expressed inside autophagosomes. In healthy neurons, the autophagosome content is under high acidification, which quenches the fluorescence of GFP but retains the fluorescence of RFP because RFP has a lower PKa value to maintain the correct protein structure in an acidic environment (Kaizuka ,et al. , 2016 ). However, when the autophagic process is impaired, the acidification process in autophagosomes is ultimately impaired through unveiled mechanisms. At this moment, both GFP and RFP can be observed in those autophagosomes. In our study, we found that after Aβ oligomer treatment, a large number of yellow vesicles were observed. These yellow vesicles represent the colocalization of GFP and RFP fluorescence and indicate the impairment of autophagosome function. However, after cotreatment with Bryostatin 1, the number of yellow vesicles was reduced significantly, and more RFP-only vesicles reappeared, which indicated that the impairment of autophagosomes was restored by Bryostatin 1.

We next transfected GFP-LC3 and RFP-LAMP1 into cultured primary hippocampal neurons to visualize both autophagosomes and lysosomes. In healthy control cells, autophagosomes merge into lysosomes and were quickly degraded. Therefore, green fluorescence and red fluorescence alone vesicles should be observed, which represent the autophygosome and lysosomes, respectively. However, if the degradation function of lysosomes is impaired, which is also common in AD pathology, GFP-labeled autophagosomes will accumulate in RFP-labeled lysosomes and thus show a yellow color. In our study, we found that Aβ oligomer treatment could block the degradation of LC3 in lysosomes, as shown by increased numbers of yellow vesicles. Furthermore, Bryostatin 1 treatment restored the function of lysosomes, as shown by fewer yellow vesicles. This result also supported the hypothesis that restoring lysosomal function may be a novel therapeutic target for AD treatment.

Autophagic function has been recently shown to be closely correlated with AD pathological processes, and autophagic influx was also found to be impaired in the frontal cortex of AD patients (Nixon, Yang, 2011 ; Ihara, Morishima-Kawashima, Nixon, 2012 ; Li, Liu, Sun, 2017 ). For instance, Nixson’s group reported that abnormally enlarged autophagosomes accumulated in axons and dendrites in the brains of AD patients. These autophagosomes are degraded rapidly in lysosomes and are invisible in healthy people. Our study was the first to show that Bryostatin 1 could restore Aβ oligomer-induced impairment of autophagic influx in primary hippocampal neurons. This restoration was initiated through activation of the mTOR pathway, as shown by the increased phosphorylation levels of mTOR and S6K1. Interestingly, although Aβ oligomers could inhibit autophagic influx in primary neurons in our study, our results also indicated that this inhibition was not through the mTOR signaling pathway, as phosphorylation of mTOR and S6K1 was not changed significantly after Aβ oligomer treatment. We presume that Aβ oligomers might block autophagy through other mechanisms, such as vesicle trafficking or the fusion of autophagosomes and lysosomes.

The dynamics of synapses play key roles in cognitive function maintenance. Once formed, synapses will grow or be eliminated under tight regulations to correctly form electric impulses. Recent studies have shown that autophagy regulates the turnover of some key receptors that are expressed in dendrite spines, such as NMDA and AMPA receptors (Li, Liu, Sun, 2017 ). When autophagic influx is impaired, these receptors cannot be internalized and thus are sustainably expressed on the postsynaptic compartment, eventually leading to abnormal hyperactive neuronal activities. Our study showed that as fast as 24 hours of incubation with Aβ oligomers, autophagic influx and synaptic function were compromised in cultured primary neurons. Furthermore, Bryostatin 1 could rescue Aβ oligomer toxicity by restoring autophagic influx. We hypothesized that the synaptic protection of Bryostatin 1 may be mediated by regulating the ratio of postsynaptic NMDA and AMPA receptors.

In summary, our current study may identify novel therapeutic targets for AD treatment, and we also demonstrated a novel mechanism by which Bryostatin 1 protects against Aβ oligomer toxicity.

ACKNOWLEDGEMENTS

The present study was supported by The Science and Technology Project Fund of Nantong city (grant no. Jc2019097)

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Publication Dates

• Publication in this collection
  09 Aug 2024
• Date of issue
  2024

History

• Received
  18 Apr 2023
• Accepted
  19 Oct 2023