Influence of human amylin on the membrane stability of rat primary hippocampal neurons

hAmylin induced neuronal loss in hippocampal tissue in vivo

Previous studies have demonstrated that, similar to oligomeric β-amyloid, hAmylin is toxic to several kinds of cells in vitro [6, 27–29]. To determine the effects of hAmylin in vivo, we injected 5 μL of hAmylin (10 μM) into the right lateral ventricles of adult mice (Figure 1A). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect neuronal apoptosis induced by hAmylin. Twenty-four hours after the injection, the proportion of apoptotic cells in the hippocampal dentate gyrus was significantly greater in the hAmylin group than in the control group (***p < 0.001, Figure 1B and 1C). Transmembrane protein 199 (TMEM199)⁺ cells (microglia) and ionized calcium binding adaptor molecule 1 (Iba1)⁺ cells (microglia and macrophages) were not observed (Supplementary Figure 1A and 1B), indicating that microglial migration was not involved and that neuronal cells were probably the main targets of hAmylin.

Figure 1. Immunostaining of hippocampal cells in brain slices from adult Kunming mice. (A) The right lateral ventricle was injected with 5 μL of hAmylin (10 μM). (B, C) Twenty-four hours after the intraventricular injection, TUNEL⁺ cells (markers of apoptosis) in the dentate gyrus of the hippocampus increased significantly.

Aggregation of hAmylin on the surface of neurons

Next, we fluorescently labeled the N-terminus of hAmylin with fluorescein amidite (FAM), and investigated whether the peptide entered neurons. Consistent with our previous findings [14], FAM-hAmylin easily aggregated when it was incubated with primary cultured neurons at 37 °C for 30 min. Many fluorescent aggregates were observed under a microscope, and FAM-hAmylin aggregation slowly increased with increasing incubation times (Figure 2A and 2B). However, after the culture dishes were washed with fresh medium and shaken, most of the fluorescence representing aggregated hAmylin disappeared (Figure 2C), indicating that the aggregation process of hAmylin oligomers was irreversible, but the aggregates of hAmylin probably attached to the surface of the cell membrane instead of entering the neurons.

Figure 2. (A, B) FAM-labeled hAmylin aggregates on the surface of a primary culture of hippocampal neurons, after 30 min of incubation. The fluorescence area of FAM-hAmylin aggregates increased slightly with time (A: typical image; B: fluorescence area change). (C) After the culture dish had been washed and shaken with fresh medium without FAM-hAmylin, the fluorescence of aggregated hAmylin was significantly reduced.

Long-term effects of hAmylin on the morphology of hippocampal neurons

Since hAmylin was able to aggregate on the surface of neurons, we used a live-cell imaging system to measure the effects of 10 μM hAmylin on neuronal survival over a long period of incubation. We used cell rupture as an indication of cell death (Figure 3A). Only 77.78% of neurons survived after being incubated with hAmylin for 12 h, while a significantly greater proportion of neurons survived in the control group (***p < 0.001, Figure 3B). We also used cellular immunofluorescence to detect the effects of hAmylin incubation on measures of cell morphology (Figure 3C and 3D), including cell size, neurite length, neurite number and synapse number (Figure 3E–3H, respectively). After the neurons had been incubated with 10 μM hAmylin for 4 h, their synapse numbers (***p < 0.001) and neurite lengths (*p < 0.05) were significantly reduced. After 9 h of incubation, the cell sizes (***p < 0.001) and neurite numbers (***p < 0.001) were significantly reduced.

Figure 3. Long-term effects of 10 μM hAmylin on the morphology of hippocampal neurons. (A) Typical images of cultured hippocampal neurons were captured in fresh medium with or without 10 μM hAmylin at different time points. (B) The survival percentages in the control (n = 108) and 10 μM hAmylin (n = 126) groups. ***p < 0.001 versus control group (Gehan-Breslow-Wilcoxon test). (C, D) MAP2 (a neuronal marker) (C) and PSD95 (a synapse marker) (D) were used to detect morphological changes in neurons at different time points of hAmylin incubation. (E–H) Cell size (E), neurite length (G), neurite number (F) and synapse number (H) were measured. After the neurons had been incubated for 4 h with 10 μM hAmylin, their synapse numbers (p < 0.001) and neurite lengths (p < 0.05) were significantly reduced. After 9 h of incubation, the cell sizes (p < 0.001) and neurite numbers (p < 0.01) were significantly reduced.

Effects of hAmylin on the cell membrane integrity

It is well known that macromolecules such as primary antibodies cannot pass through the cell membrane unless it has been permeabilized, for instance, by Triton X-100. We took advantage of this property of antibodies in immunofluorescence experiments to evaluate the integrity of the cell membrane and to determine whether hAmylin damaged the neuronal membrane. Intracellular fluorescence (marked by microtubule-associated protein 2 (MAP2), a neuronal marker) was detected when neurons were incubated with Triton X-100, but was almost invisible when Triton X-100 was replaced with phosphate-buffered saline (PBS). However, when 10 μM hAmylin was used (for 1 min or 30 min) instead of Triton X-100, intracellular MAP2 fluorescence was still observed (Figure 4A and 4B). The percentage of positive cells was 31.01% for 1-min incubation and 35.15% for 30-min incubation (Figure 4C). Intracellular fluorescence of an astrocytic marker (S100) was not clearly detected in astrocytes when 10 μM hAmylin was used instead of Triton X-100 (incubation for 1 min) (Figure 4D). However, when the incubation time with hAmylin was prolonged to 30 min, intracellular fluorescence was observed in 34.65% of astrocytes (Figure 4D–4F). These results indicated that hAmylin disrupts the cell membrane integrity in a Triton-like manner, but requires different amounts of time to destroy different types of cell membranes.

Figure 4. The permeabilization effects of hAmylin on neurons and astrocytes. (A, B) Neuron-specific fluorescence (MAP2) was observed in neurons after their incubation with Triton X-100. When Triton X-100 was replaced with PBS, intracellular fluorescence was almost invisible. However, when 10 μM hAmylin was used instead of Triton X-100 (incubation for 1 min or 30 min), intracellular fluorescence could still be observed in neurons. (C) The percentage of positive cells was 31.01% for 1 min incubation and 35.15% for 30 min incubation. (D–F) In contrast, astrocyte-specific fluorescence (S100) was not clearly observed when 10 μM hAmylin was used instead of Triton X-100 (incubation for 1 min). However, when the incubation time was prolonged to 30 min, intracellular fluorescence could be observed (D, E) in 34.65% of astrocytes (F). ***p < 0.001 versus PBS group (chi-square test).

Next, scanning electron microscopy was used to further examine the effects of hAmylin on the stability of the cell membrane. After the cells had been incubated with hAmylin (10 μM, 1 h), the integrity of the plasma membrane was destroyed in both neurons and astrocytes (Figure 5A). Several pores were visible on the surface of the cell membrane, and as the cell structure collapsed, the contours of the nucleus became apparent.

Figure 5. Scanning electron microscopy images of primary cultured hippocampal cells with or without hAmylin (10 μM, 1 h). (A) The plasma membrane was smooth and integral for primary cultured neurons and astrocytes without amylin incubation. After the cells had been treated with hAmylin (10 μM), significant plasma membrane damage was observed on the surface of primary cultured neurons and astrocytes. (B) Intracellular ROS generation induced by 10 μM hAmylin was measured in hippocampal neurons labeled with DCFH-DA dye. Representative traces are shown of the effects of 1 μM hAmylin, 10 μM hAmylin, 10 μM hAmylin + 1 μM CsA and 10 μM hAmylin + free Ca²⁺ on ROS generation. (C) Significant ROS generation was induced by 10 μM hAmylin, and was not inhibited by 1 μM CsA or free extracellular Ca²⁺. (D) Changes in neuronal DCFH-DA fluorescence before and after 10 μM hAmylin incubation. (E) The reduction in the mtΔΨ induced by 10 μM hAmylin was measured in hippocampal neurons labeled with JC-1 dye. Representative traces are shown of the effects of 1 μM hAmylin, 10 μM hAmylin, 10 μM hAmylin + 1 μM CsA and 10 μM hAmylin + free Ca²⁺ on the mtΔΨ. (F) Significant mtΔΨ reduction was induced by 10 μM hAmylin and inhibited by 1 μM CsA, but not by free extracellular Ca²⁺. (G) Changes in neuronal JC-1 fluorescence before and after 10 μM hAmylin incubation. ***p < 0.001 versus 10 μM hAmylin (one-way ANOVA followed by Bonferroni’s post hoc test).

Effects of hAmylin on ROS generation and the mtΔΨ in neurons

ROS generation and changes in oxidation status are important contributors to the membrane impairment caused by β-amyloid proteins in neurons [30–33]. Like β-amyloid, hAmylin enhanced the generation of ROS in neurons when it was applied at a high concentration (10 μM, ***p < 0.001), but not when it was applied at a low concentration (1 μM). In addition, the removal of extracellular calcium had no effect on the ROS generation induced by a high concentration of hAmylin (Figure 5B–5D).

ROS, the natural byproducts of normal respiratory metabolism, are mainly generated by mitochondria. Mitochondria are also particularly vulnerable to oxidative stress. ROS can activate the mitochondrial permeability transition pore (mPTP), which can depolarize the mtΔΨ. JC-1 dye was used to investigate the effects of hAmylin on the mtΔΨ in neurons. The mtΔΨ was significantly reduced by a high concentration of hAmylin (10 μM), but not by a low concentration (1 μM). Although the reduction of the mtΔΨ induced by a high concentration of hAmylin was not suppressed by the removal of extracellular calcium, it was significantly inhibited (***p < 0.001) by the administration of 1 μM cyclosporin A (CsA, an inhibitor of mPTP opening; Figure 5E–5G). However, CsA did not inhibit the increases in [Ca²⁺]_i (Supplementary Figure 1C and 1D) and ROS levels (Figure 5B and 5C) in response to hAmylin.

Animals and primary hippocampal cultures

Pregnant adult Sprague-Dawley rats (RRID: MGI:5651135) and adult Kunming laboratory mice (RRID: MGI:5651867) were purchased from the Hebei Laboratory Animal Center. All animal care and experimental procedures complied with the regulations of the Animal Care and Management Committee of the Second Hospital of Hebei Medical University (permit No. HMUSHC-130318) and the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for subsequent experiments [51, 52].

Primary cultures of hippocampal neurons and astrocytes were prepared from Sprague-Dawley rats according to previously described methods [53].

Main chemicals

Dulbecco’s modified Eagle’s medium, fetal bovine serum, fura-2-acetoxy-methyl ester, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glucose and PBS were obtained from Invitrogen (USA). Penicillin and streptomycin, NaCl, KCl, MgCl₂, CaCl₂ and glucose were purchased from Sigma (USA). CsA, JC-1, an In Situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110), anti-TMEM119 (ab209064), anti-Iba1 (ab153696), anti-S100 (ab868), anti-MAP2 (ab11267) and anti-PSD95 (ab18258) were obtained from Abcam (USA). Donkey anti-mouse IgG (Cat#A-21202, Alexa Fluor 488), donkey anti-rabbit IgG (Cat#A-21206, Alexa Fluor 488) and donkey anti-rabbit IgG (Cat#A-21207, Alexa Fluor 594) were purchased from Thermo Fisher (USA). Finally, 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) was purchased from Beyotime (China).

hAmylin (Tocris, UK) was dissolved to 500 μM in sterile water and immediately diluted with HEPES buffer (see calcium imaging methods) to a final concentration of 1 or 10 μM at room temperature. FAM-hAmylin was purchased from Shanghai Science Peptide Biological Technology Co., Ltd. (China). The sequence of hAmylin is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (Modifications: Tyr-37 = C-terminal amide, Disulfide bridge between 2 - 7).

Lateral ventricle injection

Six adult male Kunming mice were randomly divided into two groups. The mice were subjected to isoflurane inhalation anesthesia (initial concentration 3%, maintenance concentration 1.5%). Their teeth were buckled onto a tooth bar and their noses were fastened with a clip. Ear bars were inserted into the external ear canals of both ears so that the scales of the two bars were consistent. After the head was fixed, the height of the body was adjusted so that the body and head were horizontal. The head skin was disinfected with iodine volt cotton swabs. A midline incision was made at the top of the head to expose the bregma, and a marker was used to denote a point 1 mm to the right and 0.22 mm to the rear of the bregma at a needle depth of 2.3 mm. An electric skull drill was used to drill a small hole with a diameter of about 0.5 mm at the marked location, and a fixed micro-injector was slowly inserted to the specified depth. Then, 5 μL of 10 μM hAmylin was injected with a micro-pump at a speed of 1 μL/min. The mice in the control group were injected with saline. After the injection, the mice were monitored for 5 min to ensure that the entire drug dose in the micro-injector had entered the lateral ventricle. The micro-injector was extracted over a period of 5 min, and the incision was sutured. The mice were sacrificed 24 h after the injection, and three brain slices near the pinhole were collected. To avoid left and right confusion, we only retained right brain tissue during sampling.

Immunochemistry

Brains were collected after the mice had been perfused with 0.9% NaCl followed by 4% paraformaldehyde. Immunofluorescence analysis of the hippocampus was conducted according to previously described methods [54]. Cerebral sections were cut from frozen blocks with a sliding microtome at a thickness of 30 μm. Fixed brain slices were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature and then blocked with 10% donkey serum for 30 min. The slices were incubated at 4°C overnight with the following primary antibodies: anti-TMEM119 (1:200), anti-Iba1 (1:200), anti-S100 (1:200), anti-MAP2 (1:200) and anti-PSD95 (1:200). After being washed with PBS three times, the slices were incubated for 1 h at 37 °C with secondary antibodies (1:100), and then were washed in PBS for 5 min three times. Cell nuclei were visualized with 4’,6-diamidino-2-phenylindole (DAPI) in PBS for 10 min at room temperature. A laser scanning confocal microscope (Olympus BX61+DP71, Japan) was used to observe immunofluorescence.

For the primary cultured hippocampal cell analysis, neurons or astrocytes were applied to coverslips, washed with PBS three times and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. The subsequent steps of immunochemistry were the same as those described above for the brain slices. In the fluorescence experiments to assess whether hAmylin disrupted the integrity of the cell membrane, we incubated the cells with 10 μM hAmylin or PBS instead of Triton X-100 (1 min or 30 min), but the other steps were the same.

TUNEL staining was performed according to the In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit instructions. The proportion of apoptotic cells was calculated as the number of TUNEL⁺ cells / the number of DAPI⁺ cells × 100%.

Scanning electron microscopy

The specimen was fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for more than 4 h, and then was washed three times in phosphate buffer. Next, the specimen was post-fixed with 1% OsO₄ in phosphate buffer (pH 7.0) for 1 h, and was subsequently washed three times in phosphate buffer. The specimen was dehydrated in a graded series of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 min at each step. Then, it was transferred to a mixture of ethanol and isoamyl acetate (v:v = 1:1) for about 30 min, and transferred to pure isoamyl acetate for about 1 h. The specimen was then dehydrated in a Hitachi Model HCP-2 critical point dryer with liquid CO₂. The dehydrated specimen was coated with gold-palladium and observed with an S-3500N scanning electron microscope (Hitachi, Japan).

Live cell imaging

To investigate the long-term effects of hAmylin on neurons, we used a large BL incubator (PeCon, Germany) with a Leica microscope (Leica, #11600198) to image living cells at 37 °C with 5% CO₂. Neurons treated with FAM-labeled hAmylin were imaged every 30 min for 12 h. In the cell survival experiment with 10 μM hAmylin, images were taken every 10 min for 12 h.

Calcium imaging

Calcium imaging was performed as described previously [14]. Fura-2-acetoxymethyl ester (2 μM) was loaded into cultured rat hippocampal neurons for 20 min at 37 °C. After the loading step, the hippocampal neurons were washed with HEPES buffer to remove the extracellular dye, the cells were observed through an inverted microscope (Leica DMI3000), and images were captured with a ratiometric imaging system (Metafluor, CA) at room temperature. The calcium signals were generated using 340 and 380 nm excitation (monochromator, Polychrome V, TILL Photonics, NY, USA) and imaged with a cooled electron multiplying charge-coupled device camera (Andor, Germany). The HEPES buffer contained 145 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose and 10 mM HEPES (adjusted to pH 7.4 with NaOH). The 340/380-nm fluorescence intensity ratio was calculated as previously described [14]. All agents for calcium imaging were dissolved in HEPES buffer. The drugs were applied locally to the cells through an eight-channel pressure-controlled drug application system (ALA Scientific, USA).

Measurement of ROS generation

As described in our previously published paper [53], we assessed the intracellular ROS generation induced by the drug intervention by measuring fluorescence intensity using DCFH-DA. Hippocampal neurons were loaded with DCFH-DA in the dark at 37 °C for 20 min, and then were washed and perfused with HEPES buffer according to the same method used for calcium imaging. ROS signals were excited at 488 nm on a Leica DMI3000B microscope (Leica Microsystems Inc.) equipped with a ratiometric imaging system, and images were recorded at 1-s intervals using a cooled electron multiplying charge-coupled device camera (Andor). ROS generation, as indicated by the fluorescence intensity at 488 nm in cells loaded with DCFH-DA, was calculated as follows: ROS generation (488 nm) = [(P488 nm – B488 nm) / B488 nm] × 100%, where P488 nm is the peak fluorescence intensity after the intervention, and B488 nm is the baseline fluorescence intensity before the intervention.

Measurement of the mitochondrial membrane potential

The mtΔΨ was measured as previously described [53]. JC-1 dye (10 μg/mL, 20 min, 37 °C) was loaded into hippocampal neurons, and images were captured on a Leica DMi8 two-photon confocal laser scanning microscope (Leica Microsystems Inc., Germany). Wavelengths of 488 and 519 nm were used for the mtΔΨ signals, and the ratio of 519/488 nm represented the change in the mtΔΨ.

Statistical analysis

Data are presented as the mean ± standard deviation. A Gehan-Breslow-Wilcoxon test was used to analyze the neuronal survival curve, and a chi-square test was used to evaluate differences in the percentage of positive cells between the groups. One-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was used to compare the ROS levels and mtΔΨ of the groups. Differences between pre- and post-treatment values in the same cell type were assessed with a paired t test. A p value less than 0.05 was considered statistically significant. Data were analyzed with GraphPad Prism software (v. 5.00, USA) and SPSS software (v. 20.0, USA). The sample sizes were based on previous international research experience and published papers. No randomization methods were used in this study. The experimenters were not blinded to the conditions of the study. Our experiments did not involve any missing data, lost data or excluded data.