Effects of Amylin Against Amyloid-β-Induced Tauopathy and Synapse Loss in Primary Neurons

Reagent preparation

HFIP treated human Aβ_1-42 was purchased from AnaSpec (Cat# AS-64129-05). Aβ_1-42 was suspended in sterile filtered DMSO to a concentration of 2.2 mM. This was sonicated in a water bath on high for 1 min and then was diluted to a concentration of 1 mM Aβ with Neurobasal Medium (final concentrations of 90% Neurobasal Medium and 10% DMSO) and rotated for 24 h at 4°C [16]. After 24 h, different concentrations of this amyloid peptide solution diluted in control medium were incubated with primary neurons for different durations.

Human amylin was purchased from AnaSpec (Fremont, CA) and pramlintide was purchased from Amylin Pharmaceutical (San Diego, CA). They were resolved in water to a concentration of 256 μM and diluted in control solution to working concentration before being used [14].

Mice and experimental treatments

All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Boston University Animal Care and Use Committee.

CD1 pregnant mice were purchased from Charles River laboratories International, Inc. All mice were maintained in a microisolator of the animal facility at Boston University School of Medicine, MA, USA. An AD mouse model, 5XFAD mice, which are amyloid precursor protein (APP)/presenilin 1 (PS1) double transgenic mice with five familial AD mutations [17] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Female 5XFAD mice aged 4 months were used for the experiments. The mice were treated with intraperitoneal (i.p.) injections of amylin 200 μg/kg versus 800 μg/kg versus phosphate-buffered saline (PBS) once daily for 6 weeks. Experimental groups (n = 14–16 per cohort) were matched for age and weight.

Primary neuronal cultures

For primary mouse neuronal cultures, primary cultures were prepared from cortical tissue obtained from the brains of postnatal 0–1 day (P0-P1) CD1 mouse pups as described by Beaudoin III et al. [18] with some modifications. Briefly, early postnatal mouse cortex was dissected, incubated in 0.25% trypsin (Gibco, 15090-046) with DNase I Solution (1:100; Stemcell, 7900) for 15 min, and dispersed by trituration in Neurobasal medium (Invitrogen, 21103-049) with 2% B-27 supplement (Gibco, 17504-044), 2 mM L-Glutamine (Invitrogen, 25030-081), 0.3% Glucose (Sigma, G6152), and 5% FBS (fetal bovine serum). Dissociated cells were plated onto 24-well plates precoated with 0.1 mg/mL poly-D-Lysine (Sigma, P-9155) at a density of 1×10⁴ cells/cm² for immunofluorescence assay or 6-well plates precoated with 0.1 mg/mL poly-D-Lysine at a density of 2×10⁵ cells/cm² for western blot assay. Cells were maintained in Neurobasal medium with 2% B-27 supplement, 2 mM L-glutamine, and 0.3% Glucose and incubated in 5% CO₂ at 37°C. The medium was changed twice a week. Experiments were carried out at 14 days in vitro (DIV). On Day 14 of culture, cells were pre-incubated with different concentrations of amylin peptides for 1 h, followed by the addition of different concentrations of aggregated Aβ_1-42.

Rat cortical neurons were cultured, and the experiments were conducted as described [19, 20]. Pregnant female rats at gestational day 15 were sacrificed by cervical dislocation (Rats Wistar; Janvier), and the fetuses were removed from the uterus. The cells from the cortex were seeded at a density of 30,000 cells/well in 96 well-plates pre-coated with poly-L-lysine (10 μg/ml, Greiner, 655930) and cultured at 37°C in a humidified air (95%)/CO₂ (5%) atmosphere (6 wells/condition, 1 culture). On Day 12 of culture, cells were pre-incubated with different concentrations of different amylin peptides for 1 h, followed by the addition of aggregated Aβ_1-42 (10 μM).

Rat hippocampal neurons were cultured, and the experiments were conducted as described by Callizot et al. [21]. Briefly, pregnant female rats at gestational day 17 were killed by cervical dislocation (Rats Wistar; Janvier). The fetuses were removed from the uterus, and the hippocampi were dissociated by trypsinization for 20 min at 37°C (Trypsin EDTA 1X; PanBiotech, P10-023100). After the reaction was stopped, cells were then mechanically dissociated by 3 passages through a 10-ml pipette and then centrifuged at 515× g for 10 min at 4°C. The supernatant was discarded, and the cells of the pellet were re-suspended in a defined culture medium consisting of neurobasal medium (Nb, Invitrogen, 21103) supplemented with 2% B27 (Invitrogen, 17504), 2 mM L-glutamine (Panbiotech, P04-80100), 2% PS solution and 10 ng/ml brain-derived neurotrophic factor (BDNF, PanBiotech, CB-1115002). Viable cells were counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 20,000 cells/well in 96 well-plates pre-coated with poly-D-lysine (10 μg/ml, Greiner, 655930) and cultured at 37°C in a humidified air (95%)/CO₂ (5%) atmosphere (6 wells/condition, 1 culture). After 17 days of culture, the cells were pre-incubated with test peptides for 1 h before the application of Aβ_1-42 (0.6 μM).

Immunocytochemistry

Primary mouse neurons cultured in coverslips were washed using 1XPBS and fixed with 4% PFA for 15 min. Then neurons were permeabilized with 0.05% Triton X-100 and non-specific sites were blocked with 10% donkey serum for 1 h at room temperature. Later neurons were incubated with primary antibody. After washing, anti-mouse antibody conjugated with Alexa Fluor® 488 (Life Technology, A21202; 1:1000) and anti-rabbit antibody conjugated with Alexa Fluor® 594 (Life Technology, A21207; 1:1000) staining was performed for 1 h at room temperature. Neurons were counterstained with DAPI (4’, 6-Diamidino-2-Phenylindole, Dihydrochloride) for nuclear staining. The stained neurons were observed under the fluorescence microscopy (Carl Zeiss, Germany).

For primary rat neurons, cells were fixed with a cold solution of ethanol (95%, Sigma, 32221)/acetic acid (5%, Sigma, 33209) for 5 min. Cells were then permeabilized, and non-specific sites were blocked with a solution of PBS (PanBiotech, P04-36500) containing 0.1% saponin (Sigma Aldrich, S7900) and 1% FCS for 15 min. Cells were then incubated with primary antibody overnight at 4°C. The antibody stainings for different proteins were revealed with an Alexa Fluor 488 goat anti-mouse antibody (Molecular probe, A11001; 1:400) for 1 h. The nuclei of cells were labeled by a fluorescent marker (Hoechst solution, SIGMA, B1155). Twenty pictures per well were taken using InCell AnalyzerTM 2000 (GE Healthcare) with 20x magnification.

To study the effects of amylin on tauopathy formation in primary neurons, mouse neurons were incubated with mouse monoclonal antibody anti-PHF-1 (1:200) [22, 23] or mouse monoclonal antibody anti-CP13 (1:200) [24] overnight at 4°C. PHF-1 antibody recognizes Tau phosphorylated at Ser396 and Ser404 and CP13 antibody recognizes Tau phosphorylated at Ser202. A co-staining was done using either chicken polyclonal microtubule-associated protein 2 (MAP-2) (Abcam, ab5392; 1:500) or rabbit monoclonal antibody anti-NeuN (Abcam, ab177487; 1:300) primary antibody. Rat neurons were exposed to 2.5 μM of Aβ_1-42 in a defined medium [21] for 16 h. BDNF (50 ng/ml) was used as positive control. Cells were incubated with a mouse monoclonal primary against human hyperphosphorylated tau paired helical filament (Fisher scientific, MN1060; 1:400) overnight in the same solution. This antibody recognizes PHF-tau phosphorylated at Ser212 and Thr214. A co-staining was done using a chicken polyclonal microtubule-associated protein 2 (MAP-2) primary antibody (Abcam, ab5392; 1:400). The area and intensity of phosphorylated tau was analyzed by ImageJ software (http://rsbweb.nih.gov/ij/) as described [25, 26] and the ratio with the number of cell bodies was calculated.

To investigate the effects of amylin on synapse formation, rat hippocampal cells were pretreated with amylin or pramlintide for 1 h, and then treated with 0.6 μM of Aβ_1-42 in the presence of test peptide(s). One culture was used per condition. After 48 h of the treatment, cells were fixed, permeabilized, and blocked. Cells were then incubated with a mouse monoclonal primary antibody against post-synaptic density 95 kDa (PSD95) (Abcam, ab2723, 1:100) and a rabbit polyclonal primary antibody against Synaptophysin (Sigma, ab7837, 1:100) overnight at 4°C. The nuclei of cells were labeled by a fluorescent marker (Hoechst solution, Sigma, B1155). The results are expressed in μm² per field [21].

To study the effects of amylin on neurite outgrowth and neuronal survival, cells were treated by adding 10 μM of Aβ_1-42 in a defined medium in the presence of test peptides for 24 h. Cells were incubated with a mouse monoclonal primary microtubule-associated protein 2 antibody (MAP-2, Sigma M4403; 1:400) for 2 h in the same solution. This antibody specifically stains the cell bodies and neurites of neurons [21].

All of the images were taken in the same conditions and there were three replications.

Tissue processing

Animals were anesthetized deeply and perfused with ice-cold PBS, then followed by brain dissection. The brains were separated into left and right hemispheres. From one of the hemispheres, the cerebral cortex tissues were separately dissected, flash-frozen in dry ice and stored at –80°C until biochemical analysis were performed as described below. The other hemisphere was fixed in 4% paraformaldehyde/PBS solution for 24 h and transferred to 30% sucrose/PBS solution until tissue dropped to the bottom of the container. And then the brain was frozen in OCT media for cryosectioning, cut into 30-μm coronal sections, and stored at –20°C in 30% glycerol/30% ethylene glycol/PBS solution until used.

Western blots

Western blot assays were performed as described before [12]. Sarkosyl-insoluble fractionation was used to separate the soluble and insoluble fractions of brain extracts from 5XFAD mice. Briefly, primary neurons or brain tissues were lysed in the radioimmunoprecipitation assay buffer (Thermo Scientific, PI89900) mixed with protease inhibitor cocktail (Thermo Scientific, 78440; 1:400) and homogenized with a sonicator. Protein concentration of each sample was determined by the BCA protein assay kit (Thermo Scientific, 23227). Protein (50 μg) was electrophoresed on 4%–20% Tris-Glycine Mini Gels (Thermo Scientific, XP04200BOX) and transferred to the PVDF transfer membrane (Thermo Scientific, 88520). The membrane was blocked by 5% non-fat dry milk in Tris Buffered Saline with Tween-20 (TBST) (American Bioanalytical, Inc., AB14330-04000) and then incubated with the primary antibody diluted in the blocking buffer overnight at 4°C. Anti-PSD95 (Cell signaling, 3450T; 1:1000), anti-Synaptophysin (Santa Cruz Biotech, sc-17750; 1:1000), anti-actin (Santa Cruz Biotech, sc-81178, 1:500), PHF-1 (1:300), CP13 (1:500), and total Tau (Santa Cruz, sc-32274; 1:1000) were used. After washing, the blots were incubated with m-IgGκ BP-HRP (anti-mouse IgG-HRP; Santa Cruz Biotech, sc-516102; 1:2000) or mouse anti-rabbit IgG-HRP (Santa Cruz Biotech, sc-2357; 1:2000) for 1 h at room temperature. Immunoblots were visualized with the ECLtrademark Blotting Reagents (GE Healthcare, RPN2109) according to the manufacturer’s instruction. The molecular weight was identified by Precision Plus Proteintrademark Dual Color Standards (Bio-Rad, #1610374). The control protein bands, β-actin for mouse proteins and neurofilament for rat proteins, were used in western blots.

Immunohistochemistry of mouse brains

Immunohistochemistry was used to characterize the mouse brain pathology (at least 10 animals were used in each treatment group). 30-μm brain sections were prepared as described above. After air drying and washing with PBS, quenching of endogenous peroxidase activity was performed by incubating the sections for 30 min in 0.3% H₂O₂ in methanol followed by PBS washes.

Slides were preincubated in blocking solution (5% [vol/vol] goat serum, Sigma, St Louis, MO, USA) and 1% bovine serum albumin in TBST for 1 h at room temperature, followed by incubation in Mouse on Mouse Blocking Reagent (Vector Labs, Inc., MKB-2213) for 1 h. Primary antibodies against different components were incubated individually with the slides overnight. For Aβ, mouse mAb 6E10 (Biolegend, SIG-39320; 1:2000) was used. The secondary antibodies used were biotinylated mouse antibodies (Vector Labs, Inc.; 1:4000) for immunohistochemistry. Immunobinding of primary antibodies was detected by biotin-conjugated secondary antibodies and a Vectastain ABC kit (Vector Labs, Inc.) using DAB (3,3^′-diaminobenzidine; Vector Labs, Inc.) as a substrate for peroxidase and counter-staining with hematoxylin. The end products were visualized as eight-bit RBG images using Image J software at a total magnification of×10.

End products were visualized as 8-bit RBG images using NIS Elements BR 3.2 software (http://www.nikoninstruments.com/Products/Software/NIS-Elements-Br-Microscope-Imaging-Software) at a total magnification of 40x. For analysis of the amyloid burden, ImageJ software (http://rsbweb.nih.gov/ij/) was used. After adjusting for threshold, ImageJ was used to count all plaques and to measure the area taken up by plaques, average size of the plaques, and the percent of total brain area occupied by plaques. The intensity×size values of the plaques were measured by analyzing average raw gray levels of the plaques in the image, normalizing them to the background of the slide not taken up by brain, and multiplying by the average size of the plaques in the image. The brain area (cortex or hippocampus) was outlined using the edit plane function. The number of plaques in the outlined structure was also recorded. Data were pooled from three independent readers who were blind to the treatment for all three sections and averaged. The outcomes of amyloid burden in the brain between treatment and controls were compared by using one-way ANOVA.

Morris water maze test

Morris water maze test with spatial learning and memory performance [27] was used with 5XFAD mice (at least 10 animals were used in each treatment group) after the completion of treatment. All mice underwent reference memory training with a hidden platform in one quadrant of the pool for 10 days with four trials per day. After the last trial of day 10, the platform was removed, and each mouse received one 60 s swim probe trial. Escape latency (seconds) was recorded and analyzed using an HVS image video tracking system.

Statistical analysis

Statistical analyses were performed using the GraphPad Prism 8 (version 8.1.0, GraphPad data analysis software, USA). For Morris water maze test, two-way between-subjects ANOVA with Tukey’s post hoc testing was performed. For other experiments, one-way ANOVA with Tukey’s post hoc testing was used for multiple comparisons. For western blots (Fig. 5E), there are four replications. For all other cell experiments, there are three replications. All values are expressed as the mean±SE. In all cases, p < 0.05 was considered significant.

Aβ or amylin alone versus mixed for cellular tauopathy

To test the effect of either Aβ or amylin alone on the cellular tauopathy, we first added different concentrations of Aβ_1-42 (Fig. 2A) or amylin (Fig. 2B) alone to the primary mouse cortical neurons. Either peptide alone with elevating concentrations increased cellular tau phosphorylation (pTau) detected by PHF-1 antibody (Ser396/Ser404) immunostaining. Aβ_1-42 (from 10 nM to 1 μM) significantly increased the expression of pTau in a dose-dependent manner and the maximum amount of Aβ_1-42 (2.5 μM) induced less pTau than 1 μM Aβ_1-42 due to some neuronal death. Increasing concentration of human amylin alone also induced the pTau levels in primary neurons. The data demonstrated that both Aβ and amylin alone could induce the cellular tauopathy when the concentration is high.

We next examined if amylin had synergistic or antagonizing effects for Aβ inducing cellular pTau expression by the parallel addition of increasing amounts of amylin and Aβ_1-42 to primary neurons (Fig. 2C and Supplementary Figure 1). Amylin had a dose-dependent antagonizing effect against Aβ toxicity, e.g., reduced the levels of tau phosphorylation, when it was up to the concentration of 2.56 μM or 1:2.56 ratio with Aβ, but only the ratio of 1 μM Aβ: 2.56 μM amylin reached statistical significance. However, when the higher amylin concentration, 10.2 μM, was added with 4:1 ratio with Aβ (2.5 μM), amylin lost the antagonizing effect and tended to have a synergistic effect with Aβ to increase pTau expression.

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Fig.2

Either Aβ or amylin alone could induce cellular tau phosphorylation in primary neurons. Mouse cortical neurons were grown and treated with Aβ_1-42 alone (A), or human amylin alone (B), or Aβ_1-42 plus amylin (C). The neurons were fixed, incubated with the pTau antibody, PHF-1. When the concentration of either peptide was increased, both Aβ_1-42 (A) and human amylin (B) alone induced cellular tau phosphorylation (pTau) (Scale bars: 50 μm). The maximum amount of 2.5 μM Aβ_1-42 induced less pTau than 1 μM Aβ_1-42 due to some neuronal death. The level of tau phosphorylation was normalized by DAPI to allow comparisons among different experimental groups. Values are expressed relative to the controls (untreated mouse primary neurons), which were set as 100%. Values are the mean±SE from all experiments. For A and B, compared to the control condition, differences with statistical significance are shown with ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. For C, compared to the condition of Aβ_1-42 alone, the differences of condition of amylin plus Aβ_1-42 are shown with statistical significance ^#p < 0.05; ^# #p < 0.01; the differences between the conditions with amylin alone and amylin plus Aβ_1-42 are shown with statistical significance ^∧p < 0.05; ^∧∧p < 0.01. Each experiment was repeated three times and the representative images are shown.

Dose-dependent and U-shape effects of amylin-type peptides for reducing Aβ-induced tauopathy

The data in Fig. 2 suggested that that imbalance of Aβ and amylin could be critical for cellular tauopathy formation. To address this, we first preincubated the primary neurons with a fixed level of amylin (2.56 μM), and then added the increasing concentrations of Aβ_1-42. Interestingly, in the presence of the fixed amount of amylin, increasing amounts of Aβ_1-42, unlike Aβ_1-42 alone, did not increase, but decrease, the cellular pTau levels (Fig. 3). The best ratio of Aβ_1-42 to amylin against pTau expression was 1:2.56 in mouse cortical neurons.

Next, we tested the effects of increasing concentrations of amylin but with a fixed amount of Aβ_1-42 (1 μM) on the cellular tauopathy detected by two different antibodies, PHF-1 antibody (Fig. 4A) and CP13 (Supplementary Figure 2). In the presence of the fixed amount of Aβ_1-42, preincubating with increasing concentrations of amylin to the primary neurons reversed Aβ-induced tau phosphorylation with a dose-dependent U shape pattern. A lower concentration of amylin, from 2.56 nM (10 ng/ml) to 2.56 μM (10 μg/ml), was beneficial against Aβ-induced pTau in primary neurons (Figs. 2–4). The lowest effective concentration of amylin was 2.56 nM (10 ng/ml) and was also the EC50 of amylin to inhibit Aβ_1-42-induced tauopathy, and the maximum effective concentration of amylin to reduce pTau was 2.56 μM. However, when the concentrations was higher than 2.56 μM, in the presence of Aβ, amylin became less effective or not effective as well as caused neurotoxicity in a U-shape pattern. Western blot results confirmed that 2.56 μM amylin, but not a higher concentration than this, significantly decreased the cellular pTau levels induced by 1 μM Aβ_1-42 in primary neurons (Fig. 4B; Supplementary Figure 3).

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Fig.3

Preincubation with amylin dramatically reduced Aβ-induced cellular tau phosphorylation in primary neurons regardless of increasing amounts of Aβ. Mouse cortical neurons were grown and pretreated without and with amylin (2.56 μM) for 1 h before adding increasing amounts of aggregated Aβ_1-42 for 16 h. The neurons were fixed, and incubated with the pTau antibody, PHF-1, to detect cellular tauopathy. The neurons were also immunostained by MAP2 antibody and DAPI (Scale bars: 50 μm). The differences between the absence and the presence of amylin for each concentration of Aβ_1-42 are shown with statistical significance ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. The differences between the condition with amylin alone and amylin plus Aβ_1-42 are shown with statistical significance ^#p < 0.05; ^# #p < 0.01. The experiment was repeated three times and the representative images are shown.

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Fig.4

Amylin’s protective effects against the pathological process of tauopathy in cortical neurons are a dose-dependent, U shaped pattern. Mouse cortical neurons were grown and pretreated without and with increasing concentrations of amylin for 1 h before adding the fixed amount of aggregated Aβ_1-42 (1 μM) for 16 h. The neurons were fixed, incubated with the pTau antibody, PHF-1, to detect cellular tauopathy and quantitated. The neurons were immunostained by NeuN antibody and DAPI as well (Scale bars: 50 μm). An indicated (arrow) PHF-1 positive-neuron is showed in a higher-magnification inset in the right panel (Scale bars: 10 μm) (A). Western blots were conducted to reveal the expressions of pTau (PHF-1 antibody) in neurons in the conditions of control, Aβ alone, Aβ plus amylin (B). To confirm the result, we also used rat fetal cortical neurons and conducted the immunohistochemistry (C) as described for mouse cortical neurons except adding aggregated Aβ_1-42 (2.5 μM-best concentration to induce tauopathy in rat neurons). Western blot (D) was conducted to confirm the immunohistochemistry in (C). The same concentrations of amylin and pramlintide were added to the rat neurons for 1 h before adding aggregated Aβ_1-42 in the immunostainning experiment for pTau expression (E). The neurons were fixed, incubated with different antibodies to reveal the pathological component of pTau, and quantified, and the representative photographs are shown. # represents the comparisons between the control condition of treatment (PBS) and the condition with adding aggregated Aβ (p < 0.001). Compared to the condition with aggregated Aβ, differences in the preincubation of either amylin or pramlintide are shown with statistical significance *p<0.05; **p<0.01; ***p<0.001. Each experiment was repeated three times and the representative images and the western blot gels are shown.

To confirm that the balance of Aβ and amylin is critical for amylin’s beneficial effects in neurons and imbalance of these two peptides causes cellular toxicity, we additionally used rat cortical neuron cultures to repeat the same experiment. Although the concentrations of Aβ to induce the maximum tauopathy and the concentrations of amylin to antagonize the pathology formation were different in the mouse and rat cultures, adding amylin had dose-dependent effects antagonizing the cellular tauopathy induced by Aβ_1-42 with a similar U-shape in both cultures (Fig. 4C). The U-shape effects of amylin suggest that at more than 10.24 μM, amylin lost its ability to antagonize Aβ-induced pTau in neurons. Similar to amylin, adding pramlintide, an amylin analog, also reversed the tau phosphorylation caused by Aβ_1-42 in these neurons (Fig. 4E). Taken together, there were two factors determining the amylin’s effects against Aβ-induced tauopathy: 1) the amount of amylin and 2) the ratio between Aβ and amylin (Figs. 2, 3, and 4A). The best ratio of Aβ_1-42 to amylin against pTau expression in neurons was 1:2.56 in the mouse cortical cultures, while the best ratio for the rat neurons was 1:1 (Fig. 4C).

Dose-dependent effects of amylin peptides for reversing synapse loss and neurite network damage caused by Aβ

Since synaptic damage is the key step in AD pathological process to cause cognitive decline, we predicted that adding amylin would enhance synapses formation in the presence of Aβ. Neuronal synapses were evaluated by measuring the number of cellular sites showing colocalization of PSD95 and Synaptophysin (yellow color in Fig. 5) in primary rat hippocampal neurons. While significant synapse loss (40% decrease in the number of synapses) was observed after 48 h of incubation with Aβ_1-42 (0.6 μM) [21], preincubation with amylin resulted in an effect to reverse the synapse loss caused by Aβ_1-42 (Fig. 5A). Interestingly, these effects tended to be presented or were presented as dose-dependent, inverted U-shape to enhance synapse formation in both rat and mouse neurons (Fig. 5B, E; Supplementary Figure 4). Pramlintide also effectively reversed the synaptic damage induced by Aβ (Fig. 5A, C). Western blots showed that amylin significantly increased the expression level of PSD95 in a dose dependent way with an inverted U-shape, but did not affect Synaptophysin expression, in both rat and mouse neurons (Fig. 5E).

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Fig.5

Amylin reversed Aβ-induced synapse loss in primary neurons. Rat fetal hippocampal neurons were grown and treated with aggregated Aβ_1-42 (0.6 μM), causing loss of synapses, defined by co-localization of PSD95 and Synaptophysin, in these neurons. The cultures were treated with control medium or medium containing different concentrations of amylin or pramlintide before adding Aβ_1-42. The neurons were then fixed, incubated with different antibodies to reveal PSD95 and Synaptophysin, and quantified. The experiment was repeated three times and the representative images are shown. The photos of controls, Aβ, Aβ+amylin (10.24 μM) and Aβ+pramlintide (5.06 μM) are shown (A) and quantified (C); Aβ+different concentrations of amylin were quantified (B). Western blots were conducted to reveal the expressions of PSD95 and Synaptophysin in primary rat hippocampal neurons. The experiment was repeated three times and the representative western blot gels are shown (D). In addition, western blots of mouse cortical neurons in the conditions of control, Aβ alone, and Aβ plus amylin were conducted and quantitated to detect the expressions of PSD95 and Synaptophysin (E). The experiment was repeated four times and the representative western blot gels are shown. # represents the condition of synapse loss after adding aggregated Aβ and compared with that in the control condition (p < 0.001). Compared to the incubation condition with aggregated Aβ alone, differences with statistical significance are shown with ^*p < 0.05; ^**p < 0.01.

As expected, Aβ_1-42 elicited a 50% loss of neurite length in primary cortical cultures (Fig. 6). Preincubation with amylin resulted in a dose-dependent effect to rescue the deficit in neurite length outgrowth caused by Aβ_1-42, and the concentration of amylin that completely reversed the toxicity of Aβ_1-42 was 5.12 μM (Fig. 6B). Again, this effect of amylin presented as an inverted U-shape. Adding pramlintide to the primary cortical culture also showed a trend in similar effect on neurite outgrowth (data not shown).

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Fig.6

Amylin type peptides’ effects on Aβ-induced neurite damage in primary cortical neurons. Rat fetal cortical neurons were grown and treated with aggregated Aβ_1-42, causing reduced neurite outgrowth in these neurons. The neurons were fixed, incubated with the antibodies to reveal MAP2 and nuclei, and quantified for the neurite length. The representative photos of controls, Aβ, and Aβ plus amylin (2.56 μM) are shown (A). The average length of neurites in rat neurons which pretreated without and with increasing concentrations of amylin for 1 h before adding the fixed amount of aggregated Aβ_1-42 (10 μM) for 24 h were quantified (B). # represents the condition of incubation after adding aggregated Aβ and compared with that in the control condition (p < 0.001). Compared to the incubation condition with aggregated Aβ alone, differences with statistical significance are shown with ^*p < 0.05; ^**p < 0.01. The experiment was repeated three times and the representative images are shown.

Peripheral amylin treatment with a low versus a high dose reduces AD pathology in the brain of an AD mouse model

We conducted an in vivo experiment to investigate whether amylin treatment with different doses would show different results in reducing AD pathology in the brain. 5XFAD mice aged 4 months, which is an age when there are prominent Aβ amyloid deposits in the brain, were used. The mice were given a 6-week course of daily i.p. injections of PBS saline versus different doses of human amylin (200 μg/kg versus 800 μg/kg) (n = 14–16 per group) (Fig. 7). Figure 7A and Supplementary Figure 5 shows that immunostaining of amyloid plaques in the cortex regions in the treatment of PBS versus low dose versus high dose of amylin. Quantitatively, compared to PBS treatment, amylin treatment with either 200 μg/kg or 800 μg/kg significantly reduced the amyloid pathology in the cortex, e.g., the average size of amyloid plaques, the total amyloid area, and the amounts of Aβ (p < 0.05) (Fig. 7B). While amylin treatment with 200 μg/kg dose reduced the percentage area of amyloid burden and the amount of Aβ_1-42 determined by ELISA in the brain, the higher dose of amylin (800 μg/kg) only showed the trend and did not reach statistical significance (Supplementary Figure 6). A dose of amylin 400 μg/kg had the effects between 200 μg/kg and 800 μg/kg on amyloid burden in the brain (data not shown).

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Fig.7

Peripheral amylin treatment with a low versus a high dose reduces AD pathology and improves learning and memory in an AD mouse model. Amylin treatment of 5XFAD mice reduces the AD pathology and improves their learning and memory. At 4 months of age, 5XFAD mice were treated by i.p. injection of PBS or amylin (200 μg/kg versus 800 μg/kg) daily for 6 weeks (n = 14–16 per group). A) Dense-cored Aβ plaque burden is significantly reduced by the amylin 200 μg/kg treatment in the cortex and less so by the amylin 800 μg/kg treatment. The photos of cerebral cortex are shown (Scale bars: 200 μm). B) Amylin treated (200 μg/kg versus 800 μg/kg) mice had a significant reduction in Aβ amyloid burden in cortex quantitated by average plaque size, total amyloid area. Again, the dose of 800 μg/kg was less effective than 200 μg/kg. C) Protein extracts from the brains were fractionated into soluble fraction by TBS extraction and insoluble fraction by the sarkosyl-extraction followed by western blots with the PFH-1 antibody against pTau and quantitated. Total tau and actin were detected by western blots and are shown. The amylin treatment with 800 μg/kg was more effective to reduce tauopathy in the brain than the dose of 200 μg/kg. D) Total protein from the brains was extracted followed by western blots with the antibody against PSD95 and the one against Synaptophysin. While the amylin treatment with the dose of 200 μg/kg, but not the dose of 800 μg/kg, effectively increased the expression of PSD95, both amylin doses did not influence the expression of Synaptophysin. E) The amylin treated 5XFAD mice illustrated improved cognition compared to the PBS treatment by showing shortened times in Morris water maze test in finding the hidden platform at day 7 and 8 (D7-8), in memory at day 11 (D11) after the completion of training and skipping two days, and in the probe trial. Mean±SE was used with *p<0.05.

We next investigated whether amylin treatment could influence the other key component, tauopathy, in the brain of 5XFAD mice. Western blots with PHF-1 antibody demonstrated that amylin treatments with both doses reduced the levels of pTau in the soluble and insoluble brain fractions, while the higher the dose, the better the effects (Fig. 7C, Supplementary Figure 7). We examined the effects of amylin on synaptic proteins. Consistent with the in vitro data in neurons (Fig. 5), the in vivo data show that compared to PBS treatment, amylin treatment with the dose of 200 μg/kg, but not 800 μg/kg, increased the level of PSD95 expression (p = 0.00005) (Fig. 7D). Again, the treatment did not influence the expression of Synaptophysin in the brain. The results indicated that amylin treatment with a high dose tended to be less effective to reduce AD pathology.

Peripheral amylin treatment with different doses improved learning and memory

The Morris water maze test was conducted to investigate the effects of amylin with different doses on learning and memory. While both amylin treatments with either 200 μg/kg or 800 μg/kg equally improved learning and memory in these 5XFAD mice, the dose of 200 μg/kg was more statistically significant than the dose of 800 μg/kg to improve cognition in the AD mouse models (Fig. 7E). When compared to PBS treatment, the 200 μg/kg amylin treatment reduced the time for acquisition during maze training at day 7 (p = 0.02) and day 8 (p = 0.04), and shortened the time for memory retention (p = 0.02) and for probe trial (p = 0.01) two days after maze training in these 5XFAD mice. Similarly, but less significant in p values, the 800 μg/kg amylin treatment only showed statistical significance for acquisition at day 8. The amylin treatments regardless of the doses used had no such effect in wild type mice (data not shown).