5xFAD mice do not have myenteric amyloidosis, dysregulation of neuromuscular transmission or gastrointestinal dysmotility

Abstract

Background

Alterations in gastrointestinal (GI) function and the gut‐brain axis are associated with progression and pathology of Alzheimer's Disease (AD). Studies in AD animal models show that changes in the gut microbiome and inflammatory markers can contribute to AD development in the central nervous system (CNS). Amyloid‐beta (Aβ) accumulation is a major AD pathology causing synaptic dysfunction and neuronal death. Current knowledge of the pathophysiology of AD in enteric neurons is limited, and whether Aβ accumulation directly disrupts enteric neuron function is unknown.

Methods

In 6‐month‐old 5xFAD (transgenic AD) and wildtype (WT) male and female mice, GI function was assessed by colonic transit in vivo; propulsive motility and GI smooth muscle contractions ex vivo; electrochemical detection of enteric nitric oxide release in vitro, and changes in myenteric neuromuscular transmission using smooth muscle intracellular recordings. Expression of Aβ in the brain and colonic myenteric plexus in these mice was determined by immunohistochemistry staining and ELISA assay.

Key Results

At 6 months, 5xFAD mice did not show significant changes in GI motility or synaptic neurotransmission in the small intestine or colon. 5xFAD mice, but not WT mice, showed abundant Aβ accumulation in the brain. Aβ accumulation was undetectable in the colonic myenteric plexus of 5xFAD mice.

Conclusions

5xFAD AD mice are not a robust model to study amyloidosis in the gut as these mice do not mimic myenteric neuronal dysfunction in AD patients with GI dysmotility. An AD animal model with enteric amyloidosis is required for further study.

Amyloidosis in central and enteric nervous system contribute to congenic impairment and gastrointestinal dysmotility in Alzheimer's Disease (AD). 5xFAD mice, a transgenic mouse model of AD, mimic the amyloidosis in central nervous system but lacking amyloidosis in enteric nervous system.

Key points.

• Transgenic mouse models of AD carring mutated human gene, are designed to mimic the AD associated pathological changes in mice.

• Amyloidosis is one of the major cause of neurodysfunction/degeneration in AD.

• 5xFAD mouse model is one of the most popular AD mouse model to study amyloidosis associated pathological changes in central nervous system, but this model lacks the amyloidosis in enteric nervous system.

1. INTRODUCTION

Alzheimer's disease (AD) is a progressive neurodegenerative disease classically characterized by the presence of amyloid‐β (Aβ) plaques and neurofibrillary tangles. AD is associated with memory loss, short attention span, inability to learn new things, and a progressive decline in cognition. ¹ AD is considered the most common cause of dementia in the elderly. However, individuals suffering with AD also exhibit peripheral nervous system symptoms of which gut dysbiosis and constipation stand out. ² , ³ , ⁴ Several hypotheses have proposed mechanisms to explain the gut‐brain connection in AD with a focus on gut microbiome composition, disturbances in gut permeability, and systemic inflammation. ² , ⁵ , ⁶ , ⁷ The gut‐brain connection has been linked to several neurodegenerative diseases in central nervous system (CNS). ⁸ For example, the contribution of gut‐brain connection in Parkinson's disease has been well documented, where α‐synuclein neuronal aggregation is associated with GI dysmotility and dopaminergic neuronal degeneration in brain. ⁹ , ¹⁰ , ¹¹ Unlike Parkinson's disease, studies of GI dysmotility in AD are limited.

It is well established that the activity of amyloid precursor protein (APP) and Aβ accumulation are highly associated with AD pathology in the CNS causing cholinergic neurodegeneration of the basal forebrain. ¹² APP, from which Aβ is derived, is also expressed in the enteric nervous system (ENS). ¹³ The presence of Aβ accumulation in the gut and stool has been reported in patients with neuropathologically confirmed AD. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ The ENS controls GI motility, secretion, and blood flow through coordinated interactions between networks of interneurons, motorneurons, and sensory neurons that populate the GI tract from the esophagus to the anus. ¹⁹ Disruption in neurotransmission, particularly in the ENS could contribute to GI dysfunction in AD. In some animal models of AD, Aβ accumulation in the ENS causes increased inflammation, GI dysmotility, and loss of enteric neurons, ¹⁵ , ²⁰ , ²¹ , ²² , ²³ including nitrergic neurons, ²⁴ prior to the development of severe cognitive deficits. However, the fundamental effects of disruptions in enteric neuromuscular transmission, specifically in myenteric neurons in AD associated GI dysmotility, is still unknown.

There are over 200 transgenic animal models that have been developed to mimic the progressive pathology of familial AD (https://www.alzforum.org/research‐models/alzheimers‐disease). The 5xFAD mouse is a widely used Aβ pathogenic model whereby Aβ accumulation in the brain occurs as early as 2 months of age. In addition, neuronal loss in the CNS occurs at 3 months, and cognitive impairment is observed at 5 months of age. ²⁵ , ²⁶ Literature has reported that 5xFAD mice also present minor GI dysmotility, ²² , ²⁷ changes in enteric neuronal structure, ²⁷ altered colonic gene expressions and calcium homeostasis, increased enteric neuronal viability, ²² and GI dysbiosis. ²⁸ However, there is no study directly supporting that Aβ accumulation occurs in enteric ganglia in the myenteric plexus of 5xFAD mice and if Aβ accumulation disrupts myenteric neuromuscular transmission causing GI dysmotility. Because myenteric neurons control GI motility, we determined the expression of Aβ in the brain and colonic myenteric plexus from male and female 5xFAD and wild‐type (WT) mice by using immunostaining and an ELISA assay at 26 weeks of age. We also assessed colonic transit in vivo; propulsive motility and GI smooth muscle contractions ex vivo, electrochemical detection of colonic nitric oxide (NO) release; and changes in myenteric neuromuscular transmission using smooth muscle intracellular electrophysiological recordings. Although cholinergic neurodegeneration of the basal forebrain is a major contributor in AD brain pathology, ¹² in the current study, we determined both cholinergic and nitrergic neuromuscular transmission since the loss of enteric nitrergic neurons has been also reported in animal models of AD. ²⁴

2. METHODS AND MATERIALS

2.1. Animals

Male and female 5xFAD mice (12 of each sex) (B6SJL‐Tg [APPSwFlLon, PSEN1*M146L*L286V] 6799Vas/Mmjax, Jackson Laboratories; stock no: 34848‐JAX) and control (WT, 12 of each sex) mice (Jackson Laboratories; stock no. 000664 C57BL/6) were purchased at 8 weeks of age. Male mice were housed individually as they showed aggressive behaviors towards their littermates. Female mice were housed 3 per cage. At 26 weeks of age, all mice were euthanized by 4% isoflurane and cervical dislocation as approved and in compliance with the Panel on Euthanasia of the American Veterinary Medical Association. All procedures were approved by the Institutional Animal Use and Care Committee (IACUC) at Michigan State University (Animal use protocol #PROTO201900058).

2.2. Measurements of body weight, food intake, and fecal pellet output

To assess GI motility, we measured body weight, food intake and fecal pellet output every 4 weeks, starting at 9 weeks up to 25 weeks in all mice. After measuring body weight, mice were separated from their original cages and individually housed in a cage with access to 40 g of food and free access to water for 72 h. Total food (g) consumption was measured within 72 h.

For fecal pellet output measurements, each mouse was separated from their home cage and individually housed in cages without access to food and water for 2 h, on 3 consecutive days. Fecal pellets were assessed for number and length (mm). Wet pellet weight was assessed immediately after pellet collection, whereas dry weights were measured after 24 h desiccation at room temperature. Fecal water content was calculated by subtracting dry weight from wet weight.

2.3. Animal euthanasia and tissue collection

At 26 weeks of age, all mice were euthanized by 4% isoflurane anesthesia and cervical dislocation. The brain, small intestine, and colon were collected. Left cerebral hemispheres and a small segment of colon were immediately fixed at 4°C with Zamboni's fixative (4% formaldehyde with 5% picric acid in 0.1 M sodium phosphate buffer, pH 7.2) for 48 h. Brain tissues were immersed into 15% and 35% of sucrose in 0.1 M sodium phosphate buffer for 2–3 days, then embedded in O.C.T. and stored at −80°C for further cryostat sectioning and immunostaining. Right cerebral hemispheres and a small segment of small intestine were collected and freshly stored at −80°C for further ELISA assay.

2.4. Measurement of colonic migrating motor complex (CMMC)

The frequency and propagation velocity of CMMCs were important in evaluation of colonic GI motility. After euthanasia, the entire colon (6–8 cm) was collected from mice and the lumen was flushed with Krebs' solution. A stainless‐steel rod was inserted into the lumen, surgical ligatures were used to secure the proximal and distal ends of the colon onto the rod. The preparation was then secured in a 60 ml organ bath that contained oxygenated Krebs solution and maintained at 37°C. The colonic segment was secured to the rod ~2 cm apart with one at the end of the proximal colon and one at the start of distal colon. Both ends of the threads were attached to separate force transducers (CP122A strain gauge amplifiers, Grass Instruments, Astro‐Med, Inc. W. Warwick, RI) and were placed under an initial tension of 2 g. The colon was allowed to rest for 30 min and then CMMC frequency, duration, and propagation speed were analyzed in a 20 min window using LabChart software 8 (AD Instruments, Colorado Springs, CO).

2.5. Isometric tension recording in isolated organ bath

Longitudinal smooth muscle contractility was determined pharmacologically and electro‐physiologically using isometric tension recording in an organ bath. Following euthanasia, a 1.5 cm length of duodenum, ileum, proximal and distal colon were mounted onto a platinum foil electrode on one end and a stationary isometric force transducer on the other end with silk ligatures. The assembly was placed into a 20 ml organ bath containing oxygenated Krebs solution at 37°C and a resting tension of 1 g was applied to each preparation. To determine smooth muscle contractility for excitatory neurotransmission, bethanechol (0.1–30 μmol L⁻¹; C5256, Sigma‐Aldrich), a muscarinic receptor agonist, was cumulatively added into each organ bath at 2 min intervals to produce myogenic contractions. Each preparation was washed with Krebs' solution every 10 min and after each drug application. Enteric nerve‐evoked contractions were tested by transmural electrical stimuli (30 V, 0.8 ms pulse duration, 10 s train duration, 0.5–10 Hz) with a Grass S88 Stimulator (Grass Technologies). Tetrodotoxin (TTX, 0.3 μmol L⁻¹; 14,963, Cayman Chemical Company), a voltage‐gated Na⁺ channel inhibitor, was used to block neuromuscular transmission to reveal myogenic responses. All tissues were dried using Kimwipes and tissue weight between ligatures was weighed at the end of experiment. All drug and electrophysiological responses were calculated by subtraction of baseline from peak responses and converted into mg (contraction force)/mg (tissue weight).

2.6. Intracellular IJP recordings from circular smooth muscle cells

Myenteric neuromuscular transmission modulates GI motility. Colonic myenteric neuromuscular transmission was evaluated electro‐physiologically using sharp microelectrode intracellular recording. Following euthanasia, a 1 cm colon segment was isolated and cut along the mesenteric border, pinned flat on the dish with the mucosa facing upward, and the mucosal and submucosal layers were removed. A 1 cm² exposed circular muscle prep was transferred to a 5 ml silicone elastomer‐lined recording chamber with constant perfusion (flow rate 3 ml/min) of oxygenated 37°C Krebs' solution. The tissue was acclimated for 30 min after which microelectrodes (tip resistance, 60–120 MΩ) (borosilicate 1.0 mm × 0.5 mm fiber glass, FHC Inc.,) filled with 2 M KCl were used to impale circular smooth muscle cells. Transmural electrical stimulation (80 V, 0.5 ms pulse duration, 10 Hz train, and 100–300 ms pulse duration) was performed using a pair of Ag/AgCl wires (A‐M Systems,) connected to a Grass S88 stimulator. MRS2179 (10 μmol L⁻¹; M3808, Sigma‐Aldrich), a P2Y1 receptor antagonist, was used to block purinergic activation induced membrane hyperpolarization to reveal the nitrergic component of membrane hyperpolarization in smooth muscle cells. Resting membrane potential recordings greater than −40 mV were used for data analysis. Amplitude (mV) was measured from the traces obtained in AxoScope 10.4 (Molecular Devices,).

2.7. Continuous amperometry

To determine if enteric nitrergic neurotransmission is altered in 5xFAD mice, continuous amperometry was used to directly detect nitric oxide (NO) release from myenteric ganglia. The tissue preparation was similar to that used for IJP recordings, as described above. A modified boron‐doped diamond (BDD) microelectrode was used to make these measurements; fabrication is described in detail elsewhere. ²⁹ , ³⁰ Briefly, the BDD microelectrode was decorated with a uniform coating of Pt nanoparticles to enhance sensitivity and reduce the working potential of NO oxidation. Then, the Pt modified BDD microelectrode was dip‐coated in a thin film of Nafion® to electrostatically repel negatively charged interferents such as the nitrite anion, an oxidation product of NO. The modified microelectrode has been used to monitor NO release from the mouse colon in vitro. For amperometric recordings, the modified BDD microelectrode was carefully positioned under a low‐power microscope near a myenteric ganglia of a midcolon circular muscle preparation using a micromanipulator. The microelectrode was polarized at 0.8 V vs Ag/AgCl, a potential at which the rate of electrochemical oxidation of NO is mass transport limited and directly proportional to the number of moles of NO being oxidized. Potential was controlled using an Omni 90 potentiostat (Cypress Systems Inc.) and current‐time traces were recorded by Axoscope 10.7 (Molecular Devices). The tissue was perfused with oxygenated (95% O₂, 5% CO₂) Krebs buffer pH 7.4 containing nifedipine (1 μmol L⁻¹, blocking spontaneous longitudinal muscle contractions; N7634, Sigma‐Aldrich,) and scopolamine (1 μmol L⁻¹, to block muscarinic cholinergic receptors; S1013, Sigma‐Aldrich,) at 4 ml/min at 36–37°C. After 30 min, the tissue was electrically stimulated using two Ag/AgCl wires placed parallel to the tissue sample. Voltage pulses were delivered from an electrically isolated Grass S88 stimulator (80 V, 10 Hz, 0.5 ms pulse duration) for 1–5 stimuli. The peak current response following each stimulation was used to generate a pulse response curve in the presence and absence of the NOS inhibitor L‐nitro‐arginine (NLA, 0.1 mmol L⁻¹; N5501, Sigma‐Aldrich,) and TTX (0.5 μmol L⁻¹) to verify neurogenic NO release. The modified‐BDD microelectrode was potential cycled in 0.5 mol L⁻¹ sulfuric acid prior to each day's measurement to activate and clean the electrode surface and the Nafion® film was renewed every three measurements. Data were filtered through a 10 Hz low pass filter before peak analysis using Clampfit 10.7 software (Molecular Devices).

2.8. Immunostaining for Aβ expression

The expression of Aβ was determined in whole brain, colonic circular muscle myenteric plexus (CMMP) in whole tissue preparation, and in coronal and transverse sections of the colon by immunostaining. Fixed brain tissues were sectioned at 10 μm thickness using a cryostat. For CMMP preparations, a 1 cm² segment was cut along the mesenteric border, pinned flat on the petri‐dish with the mucosa facing upward, and the mucosal and submucosal layers were removed. The prep was fixed overnight at 4°C with Zamboni's fixative (4% formaldehyde with 5% picric acid in 0.1 M sodium phosphate buffer, pH 7.2). The fixative was washed with 0.1 M phosphate buffer solution (84 mM Na₂HPO₄, 18 mM NaH₂PO₄, pH 7.2) and the tissue was flipped over, and the serosa and longitudinal muscle layer were dissected using fine forceps. Coronal and transverse sections of fixed segment of colon were embedded with paraffin, performed by the histology core at Michigan State University. The colonic sections were cut at 5 μm thickness and dewaxed before immune staining. All preps were incubated overnight at 4°C with primary antibodies followed by 1 h incubation at room temperature with secondary antibodies (Table S1, antibody information). All preps were examined using a Nikon C2+ upright confocal laser scanning microscope (Nikon Instruments, Inc,). Fluorophores were excited using 488 nm and 594 nm lasers and their spectra were captured using Nikon NIS‐Elements advanced research software version 4.0. Identical photomultiplier settings were used for image acquisition from all samples. Images for publication were prepared using Adobe Photoshop CS5.

2.9. ELISA assay for Aβ42 expression in ileum

Expression of Aβ42 (insoluble Aβ isoform) was evaluated in whole brain, ileum and colon from 5xFAD and WT mice by ELISA assay. The segment of ileum (~100 mg) and colon were homogenized in cell extraction buffer (FNN0011, ThermoFisher Scientific,) and the following protease inhibitors: (1 mM PMSF (36,978, ThermoFisher Scientific,) and 1X protease inhibitor cocktail (P2714,). Brain tissues were homogenized in 5 M Guanidine‐HCl/50 mM Tris, pH 8.0 with 1X protease inhibitor cocktail (P2714, Sigma,). ELISA detection of Aβ42 was performed with Mouse Aβ Elisa Kit (KMB3441, ThermoFisher Scientific,) per the manufacturer's instructions. The protein concentration of homogenized tissues was measured by protein assay (BCA1 and B9643, Sigma‐Aldrich), the expression of Aβ in tissues was finally calculated into Aβ42 pg/μg tissue protein.

2.10. Statistical analysis

In our studies, power analysis was conducted assuming a 95% confidence level (p < 0.05), a standard deviation that is 20% of the mean value, a difference in means between groups that is less than or equal to 20% of mean values and statistical power > 80%. In most of our studies, a sample size of 6 animals/group were included. Data are reported as mean ± SEM. A two‐way ANOVA followed by Bonferroni's post hoc test was used to compare the changes in body weight and fecal pellet output following age development, the concentration and frequency responses of tissue isometric force recording, and frequency responses of amperometry detection of NO release, in different groups, 5xFAD vs WT. Unpaired Student t‐tests were used for comparing two groups, such as CMMCs and IJP measurements in 5xFAD vs WT mice. Data were graphed and analyzed using GraphPad Prism 6.0 software. A p‐value of <0.05 was considered statistically significant.

3. RESULTS

3.1. 5xFAD mice did not show significant alterations in GI motility following maturation

In the beginning of this study (~9 weeks old), all male 5xFAD mice had a lower body weight when compared with aged‐matched WT mice, but this difference was not observed in female 5xFAD vs WT mice (Figure 1A). The lower body weight persisted in male 5xFAD mice (Figure 1A) until 25 weeks of age. The lower body weight in male 5xFAD mice was not associated with lower food intake, since male 5xFAD mice had similar food intake compared with WT mice (Figure 1B). All 5xFAD mice did not show significant decline in body weight and food intake until 25 weeks of age.

FIGURE 1.

Measurements of body weight (A), food intake (B), fecal pellet number (C), fecal dry weight (D), fecal water content (E), and pellet length (F) in WT and 5xFAD male and female mice from Weeks 9–25. Data are presented as mean ± SE, *p < 0.05. 5xFAD male mice showed lower body weight than WT mice persistently. All 5xFAD mice show increased pellet number than WT mice in Week 13 only. Data are presented as mean ± SE, *p < 0.05, 5xFAD vs WT.

Fecal pellet number, dry weight, fecal water content, and pellet length in all mice are shown in Figure 1C–F. In both male and female 5xFAD mice, we observed a very transient increase in fecal pellet number compared with WT mice at the early stages of age progression (Figure 1C). Overall, compared with the WT mice, all 5xFAD mice did not show significant changes in fecal pellet dry weight (Figure 1D), water content (Figure 1E), and pellet length (Figure 1H) during the age progression study.

3.2. 5xFAD mice did not show significant changes in longitudinal smooth muscle contractility in response to excitatory neuromuscular transmission ex vivo

Myogenic contractions in the duodenum, ileum, proximal and distal colon were induced by using the muscarinic cholinergic agonist, bethanechol (0.1–30 μmol L⁻¹, Figure 2). Overall, there were no significant changes in concentration‐dependent post‐junctional cholinergic muscular reactivity in 5xFAD mice across the four regions of the GI tract (Figure 2A–D). Only female 5xFAD mice showed an increased colonic reactivity at very high concentration of bethanechol compared with WT mice (Figure 2D).

FIGURE 2.

Concentration response curves for bethanechol in small intestinal and colonic longitudinal smooth muscle from WT and 5xFAD male and female mice. Bethanechol induced contractions in the (A) duodenum, (B) ileum, (C) proximal colon, and (D) distal colon. Bethanechol‐induced contraction force was converted to mg (force)/mg (tissue weight). Data are mean ± SE; * p < 0.05, 5xFAD vs WT.

Next, we measured muscle contractions induced by electrical stimulation at frequencies of 0.5, 1, 3, 5, and 10 Hz (Figure 3). There were no significant differences in frequency response curves in the four regions of the GI tract between WT and 5xFAD mice (Figure 3). TTX (0.3 μmol L⁻¹) was used to block neurogenic neuromuscular transmission via nerve stimulation. After TTX treatment, frequency response curves for nerve stimulation were significantly reduced in all tissues and there were no significant differences between WT and 5xFAD male or female mice. Overall, there were no changes in excitatory neuromuscular transmission in 5xFAD mice.

FIGURE 3.

Frequency responses curves for electrical nerve stimulation in small intestinal and colonic longitudinal smooth muscle from WT and 5xFAD male and female mice, before and after application of TTX (0.3 μmol L⁻¹). Longitudinal smooth muscle contractions were recorded after electrical stimulation in male mice using the (A) duodenum, (B) ileum, (C) proximal colon, and (D) distal colon; and in female mice using the (E) duodenum, (F) ileum, (G) proximal colon, and (H) distal colon. Contraction force was converted to mg (force)/mg (tissue weight). Data are presented as mean ± SE

3.3. There were no changes in colonic propulsion or function of the neuromuscular junction ex vivo

Colonic propulsion was evaluated by measurement of the CMMC (Figure 4). Compared with male WT mice, male 5xFAD mice showed an increase in total number of CMMCs with shorter durations in between each CMMC (Figure 4A,B) but without changes in propagation speed (Figure 4C). However, female mice had similar measurements and there were no significant changes in colonic propulsion in female 5xFAD and WT mice (Figure 4A–C).

FIGURE 4.

Colonic migrating motor complexes (CMMCs) recorded from the colon of WT and 5xFAD male and female mice. (A), Total number of CMMCs; (B), CMMC duration; and (C), CMMC propagation speed were measured. 5xFAD male mice showed increases in number of CMMC and decreased CMMC duration than WT mice, but there were no significant changes in propagation speed. Data are presented as mean ± SE, *p < 0.05, 5xFAD vs WT.

Using 5xFAD mice, we also explored whether there was dysfunction at the colonic neuromuscular junction. IJPs were recorded from colonic circular smooth muscle cells (Figure 5). IJPs consist of a fast purinergic hyperpolarization of membrane potential followed by a slower nitrergic hyperpolarization before the membrane potential returns to baseline (~‐45 mV) (Figure 5A). Using train durations of 100–300 ms, we did not observe significant differences in IJP amplitude in 5xFAD mice vs. WT mice (Figure 5B–D). When the purinergic component of the IJP was blocked by MRS2179 (10 μmol L⁻¹, a P2Y1 receptor inhibitor), IJP amplitudes were significantly reduced in all mice, the nitrergic component of the IJP (leftover of IJP from purinergic blockade) was also similar in 5xFAD and WT mice (Figure 5B–D). Overall, there were no significant changes in colonic inhibitory neurotransmission in all 5xFAD mice.

FIGURE 5.

Inhibitory junction potentials (IJP) in the colon from WT and 5xFAD male and female mice. (A), Representative IJP recording from colonic circular smooth muscle of a WT mouse with or without MRS2179, a P2Y1 antagonist at 300 ms stimulation duration. Black arrow indicates the IJP amplitude measurement with Krebs buffer, gray arrow indicates the IJP measurement after MRS2179 application. (B)–(D), measurements of IJP at 100 ms, 200 ms, and 300 ms stimulation duration. 5xFAD male and female mice do not show significant differences in IJP amplitude (mV) at 100 ms, 200 ms, and 300 ms duration compared with WT mice. MRS2179 significantly decreased IJP amplitude but no significant differences were seen between WT and 5xFAD mice. Data are presented as mean ± SE, #p < 0.05, control vs MRS2179

3.4. 5xFAD mice did not show significant changes in NO release from colonic myenteric nitrergic neurons

Inhibitory neurotransmission plays a key role in control of colonic motility, and loss of nitrergic myenteric neurons in AβPP/PS1 AD mice has been reported previously. ²⁴ We determined colonic myenteric nitrergic neuronal function directly by measuring electrically stimulated NO release from myenteric ganglia using amperometry. NO oxidation current response curves were similar in 5xFAD vs WT in male and female mice, although all female mice had a smaller peak current than male mice (Figure 6A–C). A post‐hoc test following two‐way ANOVA revealed that these sex differences were only statistically significant for the 5xFAD mice at 3 pulses. In addition, peak oxidation currents were nearly completely inhibited by L‐NNA (NOS inhibitor) and TTX in all mice, demonstrating that the responses were neurogenic NO release from myenteric ganglia (Figure S1A–E). Overall, we did not observe significant changes in colonic nitrergic inhibitory neurotransmission in 5xFAD mice compared with WT mice.

FIGURE 6.

Detection of NO release from colonic myenteric ganglia in WT and 5xFAD male and female mice using continuous amperometry. NO release was directly measured using transmural electrical stimulation and continuous amperometry. A frequency‐response curve was generated by plotting the peak current response normalized to the electrode's electrochemically active area against the number of electrical stimuli delivered. (A) Peak NO current responses curves from male and female mice. (B) and (C), Representative current‐time recordings from male and female mice. Increasing the number of stimuli caused increased current until a peak response is observed at 3 pulses. This response then plateaus and decreases slightly with increasing number of stimuli. The black bar indicates the onset of stimulation and the corresponding stimulation artifact. Data are presented as mean ± SE

3.5. Aβ accumulation was undetectable in small intestinal and colonic myenteric plexus in 5xFAD mice

We used two anti‐Aβ antibodies with different epitopes in our study, from ThermoFisher and Cell Signaling (Table S1). The ThermoFisher antibody (71–5800) is a 30 amino acid synthetic polyclonal peptide derived from the full length (1–43 amino acid) Aβ peptide (https://www.thermofisher.com/antibody/product/beta‐Amyloid‐Antibody‐Polyclonal/71‐5800). All 5xFAD mice at 6 months of age showed strong Aβ immunoclusters in whole brain slices, without visible immunoclusters in the brain of WT mice (Figure 7). However, we did not detect any visible positive Aβ immune staining in the colonic myenteric plexus (CMP) in whole mount tissue preparations (Figure 7, whole), or in colonic coronal and transverse sections from all 5xFAD mice (Figure 7, section). The Cell Signaling antibody detects several isoforms of Aβ (Aβ‐37, −38, −39, −40, and − 42) (https://www.cellsignal.com/products/primary‐antibodies/b‐amyloid‐d54d2‐xp‐rabbit‐mab/8243). With the Cell Signaling antibody, our data were similar to the ThermoFisher antibody results, where the antibody detected very strong Aβ‐immunoclusters in the brain from all 5xFAD mice. However, we could not detect any visible positive Aβ immune staining in the myenteric plexus from these mice too (data not shown). More specifically, the expression of insoluble Aβ₄₂ (by ELISA assay) in the brains from 5xFAD mice was significantly increased when compared with brains from WT mice, Aβ₄₂ expression was undetectable in brains from WT mice and the ileum and clone tissues from all 5xFAD mice (Figure 8).

FIGURE 7.

Detection of Aβ expression in parasagittal cortex sections and colonic myenteric ganglia in WT and 5xFAD male and female mice. Row one and two, representative images from brain sections; row three to six, representative images from colonic circular myenteric plexus (CMP); row three and four, representative images from whole tissue preparations (whole); row five and six, colonic cross sections (section). Circle markers indicate the colonic myenteric ganglia area between circular and longitudinal smooth muscle layers. NeuN (green), a neuronal nuclear protein marker antibody, indicates the neurons in brain slices; HuC/D (green), a neuronal nuclear protein marker antibody, indicates ENS neurons in GI; Aβ (red), anti‐Aβ antibody from ThermoFisher, indicates the expression of Aβ in brain and myenteric ganglia. Confocal images were acquired at 10x and 20x. n = 6 in each group of mice

FIGURE 8.

Aβ‐42 expression in tissues from WT and 5xFAD male and female mice. ELISA assay was used to determine expression of Aβ42 from the brain, ileum, and colon tissues from these mice. Expression of Aβ42 in the ileum and colon from all 5xFAD mice was undetectable (n = 4–6 in each group)

4. DISCUSSION

Patients with neuropathologically confirmed AD also exhibit peripheral nervous system symptoms such as gut dysbiosis and constipation. ² , ³ , ⁴ In AD patients, Aβ accumulation in the gut wall and stool have been reported, ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ which strongly supports the contribution of amyloidosis in GI dysmotility of AD patients. Some mouse models of AD also revealed amyloidosis in the GI wall and moderation of GI dysmotility, alterations of microbiome composition, disturbances in gut permeability, changes GI structure and systemic inflammation. ¹⁵ , ²⁰ , ²¹ , ²² , ²³ , ²⁷ However, Aβ accumulation associated with enteric neuronal dysfunction, specifically myenteric neurons in GI dysmotility, has not been studied mechanistically, even though Aβ accumulation was directly detected by positive immune staining in the myenteric ganglia in AD mouse models of AβPP/PS1 (APPswe, PSNE1dE9) ¹⁵ and TgCRND8,. ²³ These two studies directly showed that amyloidosis occurred in the myenteric ganglia. Our goal was to identify a novel mechanism for myenteric neuronal dysfunction in GI dysmotility of AD. We used an APP overexpression mouse model that produces robust levels of Aβ and Aβ₄₂ leading to synaptic dysfunction and neuronal cell death in the brain causing cognitive deficits. The 5xFAD mouse model is one of the most popular models used in AD studies. These mice show rapidly accumulating and robust intraneuronal Aβ₄₂ levels as early as 1.5 months of age. ²⁵ Aβ₄₂ favors formation of insoluble fibrils in AD and has a strong genetic link to early‐onset familial AD. However, reported GI dysmotility in 5xFAD mice was very limited, ²² , ²⁷ 5xFAD mice had only either a shorter colonic propulsion in ex‐vivo ²² or a shorter transition time in vivo at age of 21 and 40 weeks ²⁷ in previous reports. These authors did not provide any other studies supporting if 5xFAD mice showed the signs of constipation (a shorter colonic propulsion with less fecal pellet output), or the diarrhea (a shorter transition time with more fecal pellet output) et al, these experiments may be more physiologically relevant in measurement of mouse GI function. Although we did not perform similar experiments as reported by these authors, we have included multiple in vivo and ex vivo studies, which are more comprehensive in assessing GI function. Overall GI function should not be assessed using only one experiment in animal models.

In addition, amyloidosis associated GI dysfunction in 5xFAD mice cannot be concluded without the support of amyloidosis in ENS. In previous studies in 5xFAD mice, ²² , ²⁷ the authors did not provide any direct evidence showing the occurrence of amyloidosis in GI from 5xFAD mice. More importantly, the authors have analyzed the expression of the most common AD‐linked genes in different regions of the GI tract, but overall they did not find significant changes in these gene expressions in 5xFAD mice compared with WT mice, even until at 40 weeks of age. ²⁷ In their studies, even the expression of APP and PSEN1/2 genes in entire GI tract were very comparable between 5xFAD vs WT mice, ²⁷ these data against that overexpressions of APP/PSEN gene have been employed in GI from 5xFAD mice, but the data are strongly supporting our observations that 5xFAD mice do not have amyloidosis in colonic ENS, since 5xFAD mice should be with the overexpression of three APP and two PSEN1 genes.

It has been also reported that there were changes in enteric neuronal structure/function and altered cellular viability in enteric neurons in 5xFAD mice. ²² , ²⁷ However, these authors determined the neuronal variability, structure/function and other biomarkes in cultured enteric neurons, not the primary enteric neurons. They also used recombinant human Aβ to induce neuronal amyloidosis. Therefore, the results from these studies are incomparable with our studies. Although 5xFAD mice were also used to study GI dysbiosis, ²⁸ however, there was no direct evidence supporting the amyloidosis in myenteric ganglia in these mice too.

In our studies, at the age of 6 months, 5xFAD mice have developed some cognitive impairments in behavioral tests (we did not include the data in this submission). In clinical relevance, the appearance of GI symptoms is much earlier than the symptoms of cognitive impairment in AD patients. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ We expected that the development of enteric amyloidosis will be earlier than in brain. Gene expressional studies ²⁷ also did not support that extending the time point will increase the chance of Aβ expression in GI tract, because of lacking the changes of AD‐linked gene expression in GI tract from 5xFAD mice even at age of 40 weeks.

Taken together, we believe that the amyloidosis associated myenteric neuronal dysfunction in GI dysmotility has not been reported in 5xFAD AD model.

4.1. Aβ accumulation and GI dysmotility were absent in 5xFAD mice

When we attempted to recapture amyloidosis in the colonic myenteric ganglia, we did not find visible Aβ immunostaining in colonic myenteric ganglia in 5xFAD mice at 26 weeks of age (Figure 7). Aβ immunoclusters were visualized in brain slices from all 5xFAD mice, indicating that the experimental timeline is sufficient to develop amyloidosis in these mice. To confirm our observations, we used two anti‐Aβ antibodies with different ectopic sequences from different companies. The results were very similar in both antibodies, 5xFAD mice did not show any visible Aβ accumulation in myenteric ganglia. In addition, Aβ42, an insoluble Aβ isoform, was also undetectable in the ileum and colon but was detected in brain tissues from 5xFAD mice using an ELISA assay (Figure 8). This study did not support amyloidosis in the GI wall in 5xFAD mice as well, as has been reported in other studies. ²⁰ , ²² Likewise, we did not observe significant GI dysmotility or colonic myenteric neuronal dysfunction in 5xFAD mice too (Figure 1, 2, 3, 4, 5). GI motility was broadly evaluated through GI transit in vivo (fecal pellet output), GI neuromuscular contractility ex vivo, and also the contribution of colonic myenteric neuromuscular transmission. None of these studies support the presence of myenteric dysfunction associated GI dysmotility in 5xFAD mice. Although 5xFAD male mice showed an increase in total number of CMMCs with shorter durations in between each CMMC (Figure 4A,B) but without changes in propagation speed. These mice did not have any significant GI dysmotility in vivo (Figure 1), as we did not detect any signs of decline in body weight and food intake, constipation, or diarrhea from these mice. In addition, we did not detect the presence of nitrergic neurodegeneration in colonic myenteric ganglia (Figure 6, Figure S1).

4.2. Animal models of amyloidosis‐associated myenteric neuronal dysfunction in GI dysmotility

APP, a precursor to Aβ, is expressed in the ENS. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Therefore, we anticipated an accelerated drive toward intraneuronal Aβ₄₂ generation throughout the ENS. However, amyloidosis was absent in myenteric ganglia in 5xFAD mice. Amyloidosis in myenteric ganglia was detected directly in other AD animal models such as the AβPP/PS1 (APPswe, PSNE1dE9) ¹⁵ and TgCRND8 ²³ models. We cannot completely explain why the amyloidosis is absent in the ENS of 5xFAD mice, but the model facilitates studies of amyloidosis phenotypes in CNS. Recently, we used similar anti‐Aβ antibodies and identified amyloidosis in colonic myenteric ganglia from AβPP/PS1 (APPswe, PSNE1dE9) mice, which indicates that amyloidosis in the ENS depends on the animal model of AD, and amyloidosis in the ENS is technically detectable, and the antibodies used in the current study are efficient to detect amyloidosis in GI too. 5xFAD mice overexpress five human mutated genes and use the Thy1 transgene cassette as a promoter, ²⁵ but AβPP/PS1 and TgCRND8 models overexpress only two or three human mutated genes and use the prion transgene cassette as a promoter. ³¹ , ³² , ³³ It is well known that the prion promotor is more efficient for gene transfer, and gives the highest level of transgene expression in contrast to other promotors. ³⁴ In addition, neurons in the ENS and CNS are derived from different embryonic precursors (i.e., neural crest vs neural tube), ³⁵ and these neurons do not share similar molecular signaling pathways in neuronal development. ³⁶ Therefore, the absence of amyloidosis phenotype in the ENS is not surprising. These data also indicate that the 5xFAD mouse model may not be suitable for studies of amyloidosis‐associated myenteric neuronal dysfunction and GI dysmotility in AD. Other animal models, such as the AβPP/PS1 mouse, may be appropriate for this study. We have to be aware that all AD animal models are developed to mimic the amyloidosis and tau pathology in CNS. Currently, there is no reliable AD animal model to specifically show the amyloidosis and tau pathology in ENS.

Finally, we emphasize that we only determined the amyloidosis phenotypes in colonic myenteric ganglia in these 5xFAD mice. We did not examine amyloidosis in other GI areas. Therefore, we cannot exclude amyloidosis‐associated alterations of the microbiome, mucosal permeability, gene expression, and inflammatory markers in the GI tract of these mice, as these alterations have been reported by other investigators. ²² , ²⁷ , ²⁸

5. CONCLUSIONS

In conclusion, although 5xFAD mouse model is reliable for studies of the role of amyloidosis in CNS pathology, 5xFAD mice may be inadequate to study amyloidosis in the ENS and GI dysmotility in AD. This model lacks amyloidosis in myenteric ganglia and associated GI dysmotility. Further work using animal models with promoters that target enteric specific neurons are needed to elucidate the gut‐brain connection in AD.

AUTHORS CONTRIBUTIONS

NKY and MM performed the studies in vivo. HX performed the experiments using organ bath. SH performed amperometry measurements. NKY performed the measurements of CMMCs and IJP. MM, HX, and RF performed immunostaining and ELISA experiments. NKY, MM, SH, RF, and HX performed data analysis. GS and JG participated in research design and supervised the study. Manuscript was written by NKY and HX under the supervision of JG.

CONFLICT OF INTEREST

The authors have no competing interests.

Supporting information

Appendix S1

Yelleswarapu NK, Masino M, Henderson S, et al. 5xFAD mice do not have myenteric amyloidosis, dysregulation of neuromuscular transmission or gastrointestinal dysmotility. Neurogastroenterology & Motility. 2022;34:e14439. doi: 10.1111/nmo.14439