Berberine Alleviates Amyloid β-Induced Mitochondrial Dysfunction and Synaptic Loss

Synaptic structural and functional damage is a typical pathological feature of Alzheimer's disease (AD). Normal axonal mitochondrial function and transportation are vital to synaptic function and plasticity because they are necessary for maintaining cellular energy supply and regulating calcium and redox signalling as well as synaptic transmission and vesicle release. Amyloid-β (Aβ) accumulation is another pathological hallmark of AD that mediates synaptic loss and dysfunction by targeting mitochondria. Therefore, it is important to develop strategies to protect against synaptic mitochondrial damage induced by Aβ. The present study examined the beneficial effects of berberine, a natural isoquinoline alkaloid extracted from the traditional medicinal plant Coptis chinensis, on Aβ-induced mitochondrial and synaptic damage in primary cultured hippocampal neurons. We demonstrate that berberine alleviates axonal mitochondrial abnormalities by preserving the mitochondrial membrane potential and preventing decreases in ATP, increasing axonal mitochondrial density and length, and improving mitochondrial motility and trafficking in cultured hippocampal neurons. Although the underlying protective mechanism remains to be elucidated, the data suggest that the effects of berberine were in part related to its potent antioxidant activity. These findings highlight the neuroprotective and specifically mitoprotective effects of berberine treatment under conditions of Aβ enrichment.


Introduction
Synaptic dysfunction is an early event in the pathogenesis of Alzheimer's disease (AD), and memory and cognitive loss is more strongly correlated with synaptic dysfunction than with the development of senile plaques, neurofibrillary tangles, or gliosis [1][2][3]. Synapses are the basic functional unit of signal transduction in the central nervous system, forming connections and transmitting electrical and chemical signals between neurons. Accordingly, synapses are sites of high energy demand [4]. Adequate mitochondrial function is critical to meeting the high energy requirements of the synapse. Synaptic mitochondria are synthesized in neuronal soma, transported to axons and dendrites, and distributed among synapses to support synaptic function and modulate calcium homeostasis [5,6]. Recent studies also indicate that the appropriate intracellular distribution and trafficking of mitochondria are essential for normal neuronal functions including neurotransmission, synaptic plasticity, and axonal outgrowth [7][8][9]. Importantly, abnormalities in mitochondrial function play an important role in AD [6].
Amyloid β (Aβ) is an important pathogenic peptide that is associated with AD and directly disturbs mitochondrial function [10,11]. Aβ is transported into the mitochondria via the receptor for advanced glycation end products, the translocase of the outer mitochondrial membrane, or endoplasmic reticulum-mitochondrial crosstalk [10,[12][13][14]. Mitochondrial Aβ accumulation impairs mitochondrial respiration, decreases ATP production and the mitochondrial membrane potential, and increases calcium influx, cytochrome c release, and oxidative stress [15,16]. Furthermore, the interaction of Aβ with proteins such as alcohol dehydrogenase and cyclophilin D exacerbates Aβ-induced mitochondrial and neuronal stress [17][18][19][20]. Recent studies indicate that brief exposure of cultured hippocampal neurons to relatively low concentrations of Aβ is sufficient to mediate the rapid (within 10 min) and severe impairment of mitochondrial transport in the absence of apparent cell death or significant morphological changes [21]. Taken together, these studies suggest that mitochondria are a direct site for Aβmediated cellular perturbation and that overt mitochondrial dysfunction occurs in an Aβ-rich environment.
Berberine is a natural isoquinoline alkaloid derived from the traditional medicinal plant Coptis chinensis that has numerous pharmacological properties including antimicrobial, antioxidant, anti-inflammatory, antidiarrheal, antidiabetic, antidyslipidemic, and antitumour activities [22,23]. Recent studies have indicated that berberine treatment significantly improves memory and cognitive dysfunction in different animal models of AD [24,25]. Due to its ability to cross the blood-brain barrier [26], berberine exerts beneficial neuroprotective effects against homocysteic acid, calyculin A, 6-hydroxydopamine, streptozotocin, and mercury-induced neurodegeneration as demonstrated through in vivo and in vitro studies [27][28][29][30][31]. Furthermore, berberine treatment inhibits Aβ 25-35 -induced cytotoxicity and apoptosis by suppressing the release of cytochrome C, apoptotic protein expression, and caspase activity in primary cultured hippocampal neurons [32]. Yet whether berberine has protective effects against Aβ-induced mitochondrial dysfunction and synaptic loss in neurons remains unclear. Therefore, we investigated the effects of berberine on Aβ oligomerinduced axonal mitochondrial dysfunction using an in vitro hippocampal neuron-cultured model.

Preparation of Oligomeric Aβ.
Lyophilized human Aβ  peptide was gently dissolved in 100% HFIP at a concentration of 1 mg/mL and quickly aliquoted into 0.1 mg stock solutions. The stock solutions were stored at room temperature and protected from light for 2-24 h before removing HFIP by evaporation, leaving a thin transparent film of peptide on the internal surface of the tube. Then, HFIP-treated peptide was dissolved in anhydrous DMSO at 5 mM, sonicated in a water bath for 10 min, and diluted to 100 μM in PBS (pH 7.4). Diluted peptides were then incubated for 24 h at 4°C to obtain oligomeric Aβ 1-42 [33].

Primary Hippocampal Neuron Culture and Drug
Treatment. One-day-old male C57BL/6 mice (Vital River Laboratory Animal Technology, Beijing, China) were used in this study. The mice were sacrificed by decapitation, and primary neuronal cultures were prepared from the hippocampi. Briefly, hippocampi were dissected and collected in ice-cold D-Hanks solution, then treated with 0.05% (v/v) trypsin for 20 min at 37°C. FBS was added to terminate the digestion. The suspension was centrifuged at 800 × g for 10 min and resuspended in Neurobasal-A medium supplemented with 2% (v/v) B27. Cells were plated onto poly-Dlysine (10 μg/mL) precoated 96-well plates or glass-bottom dishes with 4 chambers (cellVIEW, Greiner, Germany). The cells were cultured at 37°C under a humidified atmosphere of 5% CO 2 until use. Half of the initial medium was removed on day 1 and replaced with fresh medium. Neurons were used for experiments after 14 days in vitro. In the cell viability assay, neurons were treated with different doses of berberine (0, 0.375, 0.75, 1.5, or 3 μM) or oligomeric Aβ 1-42 (0, 0.1, 0.2, 0.5, 1, 2, or 5 μM) for 24 h before the incubation of MTT. In the mitochondrial membrane potential detection, intracellular ATP assay, ROS, and MDA measurement, neurons were preincubated in the absence or presence of berberine (0.1, 0.3, or 1 μM) for 1 h before the addition of oligomeric Aβ 1-42 (0.5 μM) for 24 h to assess the protective effects of berberine on neurons treated with Aβ 1-42 aggregates. In the axonal mitochondrial density and length measurement and axonal mitochondrial trafficking recording experiment, neurons were preincubated in the absence or presence of berberine (1 μM) for 1 h before the addition of oligomeric Aβ 1-42 (0.5 μM) for 24 h.

Cell Viability Assay.
To explore the cytotoxicity of berberine and oligomeric Aβ 1-42 and to examine the effect of berberine on cell death induced by oligomeric Aβ 1-42 , cell viability was evaluated using the MTT colorimetric assay. Cells were incubated with MTT (0.5 mg/mL final concentration) dissolved in fresh complete medium for 4 h at 37°C, and metabolically active cells were visualized by the formation of formazan. Formazan crystals were dissolved in DMSO, and absorbance values were measured on a Multiskan MK3 microplate reader (Thermo Labsystems, Waltham, MA) using a reference wavelength of 630 nm and a test wavelength of 490 nm.
2.5. Mitochondrial Membrane Potential. Mitochondrial membrane potentials in cultured primary hippocampal neurons were determined using the JC-1 mitochondrial transmembrane potential detection kit (Cell Technology, Fremont, CA). Neurons cultured in glass-bottom dishes with 4 chambers were incubated with 1× JC-1 reagent for 20 min at 37°C under a humidified atmosphere of 5% CO 2 . Cells were then washed with assay buffer and examined using a laser-scanning confocal microscope (TCS-SPE, Leica, Germany). JC-1 monomers (green, excitation 485 nm, emission 535 nm) and JC-1 aggregates (red, excitation 550 nm, emission 600 nm) were measured. The ratio of red to green fluorescence was used to measure the mitochondrial membrane potential.
2.6. Intracellular ATP Assay. ATP levels were measured using the HS II ATP Bioluminescence Assay kit (Roche, Basel, Switzerland) in accordance with the manufacturer's specifications. Briefly, treated hippocampal neurons were rapidly washed with cold PBS, scraped into a dilution buffer, and transferred to microcentrifuge tubes. An equivalent volume of the cell lysis reagent was added to each tube, and tubes were incubated for 5 min at room temperature. After transferring the lysates to a black microtiter plate, a luciferase reagent was added to each sample by automated injection and measurement was started after a 1 s delay using a Flx800 fluorescence microplate reader (BioTek, VT). A standard curve was generated on the same plate.

Axonal Mitochondrial Density and Length Measurement.
The operational procedure was the same as described previously [34]. Briefly, hippocampal neurons cultured in 4-well glass-bottom dishes were incubated with 100 nM Mito-Tracker Red (Thermo Fisher Scientific), fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 10% goat serum. Then, neurons were incubated with anti-Tau antibody (1 : 500, Abcam, UK) and secondary antibody (Alexa Fluor 488, Abcam). The mitochondria were colored red fluorescence while axons were colored green. Images of the mitochondria with axons were collected using a confocal microscope and analysed using ImageJ (National Institutes of Health, Bethesda, MD).

Axonal Mitochondrial Trafficking
Recording. The operational procedure was the same as described previously [34]. Briefly, neurons were incubated with MitoTracker Green (Thermo Fisher Scientific). And time-lapse images of axonal mitochondria were captured with a heated 37°C, 5% CO 2 controlled stage for a total of 5 min. Stacks were processed using ImageJ software with a Kymograph plug-in under maximum intensity projection. A mitochondrion was considered stationary if the displacement was less than 2 μm during the entire recording period. Mitochondrial movements (direction and velocity) were determined from the corresponding kymographs using ImageJ.

Reactive Oxygen Species (ROS) Measurement.
Neurons were loaded with dichlorodihydrofluorescein diacetate (DCFH-DA) to detect ROS. After incubation with berberine or Aβ 1-42 , cultured hippocampal neurons were washed with PBS and incubated with 10 μM DCFH-DA for 30 min at 37°C under a humidified atmosphere of 5% CO 2 . Images were collected using a confocal microscope and analysed using ImageJ.
2.10. Malondialdehyde (MDA) Measurement. The contents of MDA were measured using the malondialdehyde (MDA) assay kit (Nanjing Jiancheng, Nanjing, China) in accordance with the manufacturer's specifications. In brief, treated hippocampal neurons were rapidly washed with cold PBS, scraped into a dilution buffer, and transferred to microcentrifuge tubes. An equivalent volume of a cell lysis reagent was added to each tube, and tubes were incubated for 5 min at room temperature. The protein concentrations of lysates samples were determined by the BCA method, and then, samples were diluted with the assay buffer solution in the kit for the determination of MDA. The mixture was incubated for 40 min at 95°C, and absorbance was read optical density (OD) at 532 nm.
2.12. Statistical Analysis. The data were analysed using SPSS version 20.0 (IBM, Armonk, NY) and are shown as the mean ± standard error of the mean of 4 independent experiments. Significant differences between values were determined using a one-way analysis of variance followed by least significant difference post hoc tests. The threshold for the statistical significance was p < 0 05.

Berberine Prevents Aβ 1-42 Cytotoxicity in Primary
Cultured Hippocampal Neurons. The conventional MTT reduction assay was used to examine cell viability. Cells were treated with berberine or oligomeric Aβ 1-42 at the indicated concentrations for 24 h. Neurons treated with oligomeric Aβ 1-42 showed a significant, dose-dependent decline in viability, while treatment with berberine at concentrations up to 1.5 μM had no effect on cell viability (Figures 1(a) and  1(b)). Notably, pretreatment with berberine (0.1, 0.3, and 1 μM) for 1 h before the addition of oligomeric Aβ 1-42 (0.5 μM) rescued cell viability in a concentration-dependent manner; pretreatment with 1 μM berberine increased cell viability by 9.1% compared to cells incubated with oligomeric Aβ 1-42 alone (p < 0 05, Figure 1(c)).

Discussion
Synapses are the basic structural foundation of signal transduction in the central nervous system, and synaptic plasticity in the brain is thought to be the cellular substrate of learning and memory. Severe synaptic structural and functional damage, which is a typical pathological features of AD, causes memory and cognitive dysfunction [34]. Mitochondria support synaptic function by maintaining the cellular energy supply, regulating calcium and redox signalling, and regulating synaptic transmission and vesicle release. Accordingly, multiple lines of evidence indicate that synaptic function and plasticity depend on the mitochondria [35]. Aβ 1-42 is a neurotoxic peptide that induces synaptic loss by disrupting the mitochondrial membrane potential, decreasing ATP generation, enhancing intracellular ROS production, and activating mitophagy [15,34,36,37]. Berberine is a natural isoquinoline alkaloid that has an array of neuroprotective properties; yet a majority of researches has focused on its beneficial effects on neurodegenerative diseases based on its antioxidant activity [23]. In the present study, we add to a previous literature by characterising berberine as a mitoprotective agent that prevents synaptic loss associated with Aβ toxicity.
The △ΨM is an important indicator of cellular health. △ΨM is generated by the proton gradient (complexes I, III, and IV) and serves as an essential component of energy storage during oxidative phosphorylation. △ΨM forms the transmembrane potential that is used by ATP synthase to generate ATP. Normally, cellular △ΨM and ATP levels are relatively stable and only undergo transient changes due to physiological activity. Yet chronic disruptions of △ΨM and ATP production compromise cell viability, leading to various pathological consequences [38]. In this study, we report for the first time the ability of berberine to ameliorate the Aβ 1-42 -induced impairment of △ΨM and ATP generation in primary cultured hippocampal neurons in a dose-dependent manner.
Because mitochondria are synthesized perinuclearly, they must be trafficked from the soma to distal synapses via mitochondrial transport and constantly reconfigure to meet synaptic needs. Moreover, mitochondrial morphology is also dynamic and can be regulated through fusion and fission. It was reported that an elongated morphology conferred bioenergetic advantages for ATP generation and dispersal [39]. Because of the technical limitations on observing mitochondrial trafficking in vivo, we performed studies in a primary cultured hippocampal neuronal model as reported previously [6]. We selected axonal processes for the quantitative analysis of mitochondrial length, density, distribution, and mobility because of their known morphological and dynamic characteristics [6,34,40]. Berberine pretreatment rescued Aβ-induced axonal mitochondrial fragmentation and abnormal trafficking, indicating that treatment supported energy homeostasis and prevented Aβ-induced mitochondrial morphological as well as functional impairment.
The exact mechanism underlying the protective effects of berberine is unclear. Various studies have established the antioxidant activity of berberine in disorders such as diabetes, high cholesterol, and CNS disorders. Berberine induces antioxidant defences by quenching superoxide anion and nitric oxide, increasing levels of non-enzymatic antioxidants and the activities of antioxidant enzymes, and upregulating Nrf2 (defends against ROS damage), GFAP, GLP-1, and other protective molecules [22,23,[41][42][43][44]. Therefore, we examined ROS levels in primary cultured hippocampal neurons, as mitochondrial dysfunction is an important source of oxidative stress. We found that Aβ 1-42 -induced oxidative stress was decreased by berberine pretreatment, consistent with previously published data. Furthermore, berberine also has a protective effect against synaptic loss instigated by oligomer Aβ 1-42 . This study supported the opinion that berberine could be a potential therapeutic agent for the treatment of AD, based on its effect of alleviating Aβ-induced axonal mitochondrial abnormalities and synaptic loss. However, there remain some limitations of this study requiring further investigation. One limitation is that the definite mechanism underlying protection of berberine against Aβ-induced axonal mitochondrial abnormalities remains unclear and needs to be elucidated. The mitoprotective effects of berberine may be related to its antioxidant activity which has been proven in present study, whereas antioxidants may not be the only effect and berberine may still have other potential protective mechanisms. Another limitation of this study is that protective effects of berberine against neuronal Aβ toxicity were examined only in vitro. We need to further verify the experimental results in vivo, in order to make the neuronal protective effect more credible.

Conclusions
We demonstrate that berberine protected against Aβinduced axonal mitochondrial abnormalities in primary cultured hippocampal neurons by preserving the mitochondrial membrane potential and ATP generation, increasing axonal mitochondrial density and length, and improving mitochondrial motility and trafficking, ultimately preventing synaptic loss. Although the underlying mechanism of these effects is unclear, the data suggest that the protective effects of berberine may be related to its antioxidant activity. Future research should investigate berberine as a potential therapeutic agent in the context of AD.

Data Availability
All data included in this study are available upon request by contact with the corresponding author.

Conflicts of Interest
The authors declare that there is no conflict of interest associated with this manuscript.