Constipation, deficit in colon contractions and alpha-synuclein inclusions within the colon precede motor abnormalities and neurodegeneration in the central nervous system in a mouse model of alpha-synucleinopathy
Gastrointestinal dysfunction can affect Parkinson’s disease (PD) patients long before the onset of motor symptoms. However, little is known about the relationship between gastrointestinal abnormalities and the development of PD. Contrary to other animal models, the human A53T alpha-synuclein (αS) transgenic mice, Line G2–3, develops αS-driven neurological and motor impairments after 9 months of age, displaying a long presymptomatic phase free of central nervous system (CNS) dysfunction.
To determine whether this line can be suitable to study constipation as it occurs in prodromal PD, gastrointestinal functionality was assessed in young mice through a multidisciplinary approach, based on behavioral and biochemical analysis combined with electrophysiological recordings of mouse intestinal preparations.
We found that the A53T αS mice display remarkable signs of gastrointestinal dysfunction that precede motor abnormalities and αS pathology in the CNS by at least 6 months. Young αS mice show a drastic delay in food transit along the gastrointestinal tract, of almost 2 h in 3 months old mice that increased to more than 3 h at 6 months. Such impairment was associated with abnormal formation of stools that resulted in less abundant but longer pellets excreted, suggesting a deficit in the intestinal peristalsis. In agreement with this, electrically evoked contractions of the colon, but not of the ileum, showed a reduced motor response in both longitudinal and circular muscle layers in αS mice already at 3 months of age, that was mainly due to an impaired cholinergic transmission of the underlying enteric nervous system. Interestingly, the presence of insoluble and aggregated αS was found in enteric neurons in both myenteric and submucosal plexi only in the colon of 3 months old αS mice, but not in the small intestine, and exacerbated with age, mimicking the increase in transit delay and the contraction deficit showed by behavioral and electrical recordings data.
Gastrointestinal dysfunction in A53T αS mice represents an early sign of αS-driven pathology without concomitant CNS involvement. We believe that this model can be very useful to study disease-modifying strategies that could extend the prodromal phase of PD and halt αS pathology from reaching the brain.
KeywordsAlpha-synuclein Constipation Gastrointestinal dysfunction Bowel dysmotility Non-motor symptoms Parkinson’s disease Enteric nervous system Alpha-synucleinopathy
Enteric nervous system
High molecular weight
Peripheral nervous system
Whole gut transit time
Along with typical motor dysfunctions, Parkinson's disease (PD) patients experience a variety of non-motor symptoms that involve both the central nervous system (CNS) and the peripheral nervous system (PNS), with a deep impact on their quality of life. Among them, constipation and anosmia can manifest even decades before the onset of the motor abnormalities, and thus their analysis can be potentially useful for early diagnosis and therapeutic intervention . Constipated patients display infrequent bowel movements, impairment of propulsive colonic motility, prolonged colonic transit time, reduced rectal contractions and abnormalities in motor activity of the anal sphincter . Affecting up to 80% of PD patients, constipation represents the most frequent gastrointestinal (GI) dysfunction in PD  and patients with a previous diagnosis of constipation have an increased risk of developing PD , e.g. less than 1 bowel movement per day is linked to a 2.7-fold increase in risk of PD . However, very little is known about the relationship between bowel dysfunctions and development of PD.
The pathological hallmarks of PD, Lewy bodies (LBs) and Lewy neurites, abnormal proteinaceous inclusions mainly composed of alpha-synuclein (αS) present in the brain of the patients , have been also found in the enteric nervous system (ENS) [7, 8, 9, 10], which is the network responsible for the whole innervation of the gut. In addition, several studies have implicated αS neuronal transmission and propagation within the nervous system as a mechanism of detrimental spreading of PD pathology, showing that neuron-to-neuron transfer of pathogenic αS occurs both in vitro [11, 12, 13] and in animal models [14, 15, 16, 17, 18, 19, 20] and suggesting that the accumulation of toxic αS species may originate outside the CNS and later move to the brain using anatomical connections [21, 22]. In line with these findings, detection of αS inclusions in the large intestine of human subjects has been investigated as a promising diagnostic tool in living patients, as samples can be easily obtained through biopsies . However, because of the presence of enteric phosphorylated αS aggregates in healthy controls [24, 25], variable specificity of αS antibodies employed [7, 8] and the heterogeneity in the intestinal areas analysed , the detection of αS inclusions in colonic biopsies as a reliable PD biomarker is still highly controversial [27, 28].
On the other hand, in terms of animal models, until now, there has been a lack of a suitable paradigm that would allow the study of constipation as it occurs in the prodromal phase of PD, without overt motor dysfunction. Indeed, the vast majority of current genetic or chemically induced models present GI deficits and αS accumulation in the gut concomitant with impaired motor activity or αS pathology in the CNS [29, 30, 31, 32, 33], making it difficult to understand the progressive nature of constipation during the PD prodromal phase and how it relates to CNS neuropathology. Here, we show that the PrP human A53T αS transgenic (Tg) mice, line G2–3, , one of the first genetic model developed to study α-synucleinopathy, displays GI dysfunction with simultaneous accumulation of αS inclusions in the ENS, months before the typical onset of motor abnormalities, neuronal degeneration and formation of αS positive inclusions in the CNS. In this model, constipation represents an early sign of αS-driven pathology that is temporally unrelated to CNS involvement. Because of this, we believe that the A53T αS G2–3 line can be particularly useful to study the contribution and the influence of constipation on αS-driven CNS neuropathology.
Tg mice expressing human A53T αS under the control of the mouse prion protein (PrP) promoter [line G2–3 (A53T)] have been described previously [34, 35]. This model develops neurological abnormalities as early as 9 months of age (with an average peak at ~ 13 months) that manifest initially with reduced locomotion, wobbling, lack of balance and weakness of the hind limbs. This disease phenotype becomes progressively more severe, culminating in a fatal paralysis within 14–21 days from the onset of the first symptoms. Because of the high variability in time of onset (between 9 to 16 months), Tg mice are closely monitored after 9 months of age with the understanding that once the first symptoms appear, that mouse is committed to develop the full phenotype. Diseased mice show an accumulation of intracellular, phosphorylated (serine 129) and ubiquitinated αS inclusions, neuroinflammation and neuronal degeneration in CNS . For the purpose of this study, sick Tg mice at 12–14 months, presymptomatic mice at 3, 6, 9 and 12 (if still healthy) months, and age-matched nTg littermate controls were used. All animal studies were approved by and complied in full with the national and international laws for laboratory animal welfare and experimentation (EEC council directive 86/609, 12 December 1987 and Directive 2010/63/EU, 22 September 2010).
Behavioral analysis was carried out on groups of 20–30 mice, whereas for the gait test, food intake and glucose blood level groups consisted of ~ 10 mice each. Both male and female animals were used. Each trial was performed 1 to 3 times per animal on non-consecutive days.
The gait test was assessed in mice after o/n starvation. Each animal’s paw was painted with blue washable paint and the mouse was allowed to walk onto a white paper strip, at the end of which a piece of mouse chow was placed as a food reward. The footprints were circled and allowed to dry. Stance length, sway distance and stride length were measured for each mouse.
Whole gut transit time
Whole gut transit time (WGTT) was assessed in mice after oral gavage of a 0.05 mL chocolate milk containing 4% of Brilliant Blue food dye. Post-gavage, the animals were observed until the time of excretion of the first blue stool, which was recorded for each mouse.
Stool collection assays were performed between 9:00 AM and 11:00 AM on each day. Each animal was removed from its home cage and placed in a clean plastic cage without food or water for 1 h. Stools were collected immediately after expulsion and placed in sealed tubes. At the end of the trial, the stools were counted and weighed (total weight), then dried o/n at 65 °C and weighed again to provide dry weight. Water content was calculated as a difference between total weight and dry weight.
Each animal was removed from its home cage and single-housed for 24 h with free access to food and water. Food was weighed before and after the trial and food intake was calculated as a difference between the two amounts.
Glucose levels were assessed using reactive stripes (OneTouch Verio, LifeScan Italia, Milan, Italy) with a single drop sample of blood taken from the tail of each animal. Glucose level was analyzed after o/n food removal and after the mice were allowed free access to chow for 1 h.
Recording of contractile activity from longitudinal and circular muscle preparations of colon and ileum
Contractile activity of colonic or ileal longitudinal and circular smooth muscle was recorded as previously described [36, 37]. After sacrifice, the colon and the ileum were removed and placed in cold Krebs solution. Longitudinal and circular muscle strips of the intestine were set up in organ baths containing Krebs solution at 37 °C, bubbled with 95% O2 + 5% CO2. The strips were connected to isometric force transducers (2Biological Instruments, Besozzo, Italy). Mechanical activity was recorded as a measure of tension using a BIOPAC MP150 system (2Biological Instruments). A pair of coaxial platinum electrodes was positioned at a distance of 10 mm from the longitudinal axis of each preparation to deliver transmural electrical stimulation (ES) by a BM-ST6 stimulator (Biomedica Mangoni, Pisa, Italy). ES were applied as 10-s single trains consisting of square wave pulses (0.5 ms, 30 mA). To measure muscle contractility of the colon or the ileum, electrically evoked motor responses were recorded from tissue preparations maintained in standard Krebs solution. To measure the neurogenic contribution to muscle contraction, electrically evoked motor responses were recorded after selective stimulation of the nitrergic, cholinergic or NK1-mediated tachykinergic pathways from colonic preparations maintained in Krebs solution containing respectively: guanethidine (10 μM), L-732,138 (10 μM), GR159897 (1 μM), SB218795 (1 μM) and atropine (1 μM) in order to inhibit the noradrenergic, NK-mediated tachykinergic and cholinergic pathways, while recording the nitrergic signal; L-NAME (100 μM), guanethidine (10 μM), GR159897 (1 μM), SB218795 (1 μM) and atropine (1 μM) in order to prevent the recruitment of the nitrergic, noradrenergic, NK2 and NK3-mediated tachykinergic and cholinergic systems, while recording the NK1-mediated tachykinergic pathway; L-NAME, guanethidine, L-732,138, GR159897, SB218795, in order to record the cholinergic response while inhibiting the nitrergic, noradrenergic and tachykinergic signals. To evaluate the myogenic contribution to the total contractile activity of the colon, muscle response was evoked by direct pharmacological activation of muscarinic receptors located on smooth muscle cells. For this purpose, colonic preparations were maintained in Krebs solution containing tetrodotoxin (1 μM) and stimulated with carbachol (10 μM). The tension developed by each preparation (grams) was normalized by the wet tissue weight (g/g tissue). All the chemical compounds were purchased from Sigma Aldrich (St. Louis, Missouri USA).
Tissue collection and western blot analysis
For biochemical analysis the mouse intestine was cut into 6 segments corresponding to duodenum (D), jejunum (J), proximal ileum (PI), distal ileum (DI), proximal colon (PC) and distal colon (DC), flushed of fecal contents, opened longitudinally, scraped for removing the epithelium and minced. Cold phosphate-buffered saline (PBS), with proteases and phosphatases inhibitors, was used for this procedure. All samples were frozen on dry ice immediately after collection and stored at − 80 °C until use. Frozen intestine segments were homogenized using a Potter-Elvehjem Grinder homogenizer on ice in 20% (w/v) TNE lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA) with proteases and phosphatases inhibitors. An equal volume of TNE buffer containing 2% of NP-40 was added to initial homogenates that were then centrifuged at 10,000 x g at 4 °C in order to collect NP-40 soluble and insoluble fractions. Pellets were then washed one time with TNE buffer with 1% of NP-40 and resuspended in 10% of the original homogenization volume in TNE containing 1% NP-40, 1% SDS, 0.1% DOC. NP-40 insoluble fractions were then sonicated and boiled for 5 min at 95 °C. Protein amount was determined with BCA. Immunoblot analyses were performed as previously described . Briefly, lysates were run on a 4–20% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA) and then transferred onto nitrocellulose membrane at 200 mA, o/n at 4 °C, using carbonate transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, 20% MeOH). Transfer efficiency was controlled by Ponceau staining. Unspecific binding sites were blocked by 30 min membranes incubation with 5% nonfat dry milk (Bio-Rad) in 1X PBS containing 0.01% Tween-20 (PBS-T) at RT. Membranes were then incubated with the specific primary antibody dissolved in 2.5% nonfat dry milk in PBS-T, o/n at 4 °C. The following primary antibodies were used: Syn-1 (BD Biosciences, NJ, USA), pser129-αS (EP1536Y, Abcam), GAPDH (Thermofisher, Carlsbad, CA, USA), α-Tubulin (Sigma-Aldrich). Membranes were washed with PBS-T and incubated for 1 h at RT with the appropriate horseradish peroxidase-conjugated secondary antibody in 2.5% nonfat milk in PBS-T. Only for pser129-αS detection, 1X Tris-buffered saline containing 0.01% Tween-20 instead of PBS-T was used for the entire procedure. The chemiluminescent signals were visualized using a CCD-based Bio-Rad Molecular Imager ChemiDoc System. Band intensities were analyzed using Quantity One software (Bio-Rad).
For immunofluorescence analysis, mice were perfused with 4% paraformaldehyde (PFA) in PBS 1X after 1 mL intraperitoneal injection of 2% w/v Tribromoethanol. The colon was collected and flushed of fecal contents, post-fixed o/n in 4% PFA/PBS at 4 °C and stored in maintenance solution (30% sucrose, 0.1% NaN2 in PBS) at 4 °C. A segment of 2 cm of distal colon was embedded in Tissue-Tek® OCT (Sakura, The Netherlands), cut at the cryostat in serial 12 μm sections and mounted on SuperFrost Plus glass slide (Thermofisher). Slides were then washed with PBS and let dry for ~ 3 h at 37 °C. Sections were then incubated with blocking solution [3.5% fat dry milk, 0.3% Triton X-100 (Tx-100), 6% normal goat serum (NGS) in PBS] for 1 h at RT and then incubated with primary antibody o/n at RT in blocking buffer. The following antibodies were used: pser129-αS and LB509 (Abcam), β-3-Tubulin and Syn204 (Cell Signaling Technology, Danvers, MA, USA), ChAT and Tyrosine Hydroxylase (TH) (Millipore, Burlington, MA, USA). On the next day, the sections were washed twice in PBS and incubated with Alexa Fluor secondary antibodies (ThermoFisher) in PBS containing 1.5% NGS, 0.3% Tx-100 for 1 h at RT. Sections were counterstained with Dapi and mounted on a glass slide using Fluormount (Sigma-Aldrich).
All values are expressed as the mean ± S.E.M. Differences between means were evaluated by two-way ANOVA, followed by Fisher LSD post-hoc test (Prism, Graph Pad Software, San Diego, CA).
Constipation in Prp A53T αS Tg mice is already present at 3 months of age in absence of overt motor dysfunction and accumulation of αS CNS inclusions
Presymptomatic αS Tg mice show a reduced electrically evoked motor response of the colonic muscle layers since 3 months of age
Accumulation of αS soluble and insoluble HMW species in the colon increases over time in presymptomatic αS Tg mice
Toxic αS in presymptomatic and diseased αS Tg mice is found in cholinergic and dopaminergic enteric neurons of the colon
In this paper we show that the Prp A53T αS Tg mice, line G2–3, can be an extremely valuable model, and probably the only one, to the best of our knowledge, to study constipation as it occurs in PD prodromal phase, that is, in absence of overt motor abnormalities and CNS neurodegeneration. Together with typical motor symptoms upon which diagnosis is still mainly made, PD patients experience a variety of non-motor dysfunctions that affect both the CNS and the PNS . Among these, GI dysfunction and specifically constipation represent an important feature of the prodromal phase of the pathology, due to the fact that GI-related symptoms can appear even decades prior the onset of the motor signs . The relevance of studying constipation is also attributable to the fact that 80% of PD patients experience it and that after being diagnosed with constipation, there is almost a 3-fold increased risk of developing PD . Nevertheless, whether there is a direct pathogenic correlation between GI dysfunction and PD still remains an open question, caused in part by the absence of appropriate animal models that recapitulate the chronic and progressive development of PD pathological stages. Indeed, all the αS Tg mouse lines analyzed for constipation so far showed GI deficit only concurrent with CNS neuropathology [30, 31, 32] while when considering the pharmacologically induced PD models only a small study, based on chronic injections of rotenone at low dosage in rats, showed mild GI abnormalities in absence of CNS αS pathology . Nevertheless, because of the low amount of rotenone used for this purpose, brain pathology and motor symptoms never occurred in these mice. By contrast, in the G2–3 line constipation and GI dysfunction precede motor abnormalities and neurodegeneration in the CNS by at least 6 months. Indeed these mice from an early age (3 months old), in which the CNS is free of αS-driven neuropathology, manifest significant GI dysfunction, as shown by a robust increase in food transit time in the GI tract and simultaneous abnormalities in stool formation, which results in expulsion of elongated but less frequent pellets. Because of the similarities in stool consistency, food intake ration and body and stool weight between Tgs and age-matched nTg littermates, which allow us to exclude episodes of diarrhoea or chow malabsorption, the transit delay seen in young Tg mice could indicate a GI motility dysfunction in the process of propagation and expulsion of stools. Consistent with this, we found a decreased capacity of contraction of the longitudinal and circular muscle layers of the colon in young Tg mice that was mainly dictated by a reduced cholinergic transmission. Indeed, while the colon muscles in Tg mice can still contract normally when directly stimulated, it is the neuronal pathway supplying the underlying muscles that was found to be deficient. In addition, at 6 months of age, this GI abnormal behavior progressively worsens, reaching a delay in gut transit time of more than 3 h that was paralleled by a further decrease in colon contractility, still in absence of any CNS neurodegeneration.
Most of the neurotransmitters active in the CNS are also present in the ENS, with few differences, and contribute to the unrolling of all the diverse functions of the GI apparatus . Specifically, the motor activity of the bowel is mainly regulated by the cholinergic and tachykinergic systems for contraction while the nitrergic system is the main regulator of gut muscle relaxation in mice [39, 44]. When considering only the recruitment of postganglionic cholinergic motor neurons, electrically evoked contractions resulted decreased in αS Tg compared to nTg mice in both muscle layers (Fig. 3b). On the other hand, when stimulating specifically the nitrergic or the tachykinergic system, the muscle responses were unchanged between the two groups, at all ages, confirming that GI delay in food transit was mainly ascribable to a deficit in cholinergic-evoked muscle contractions and not to an excess of the relaxant response. In line with this view, a more accurate immunohistochemistry analysis, found toxic αS accumulated in enteric neurons, including cholinergic and dopaminergic kinds, of both myenteric and submucosal plexi in presymptomatic and diseased animals. Notably, in the same section, not all the neurons stained for αS aggregates, indicating that accumulation of toxic αS is a progressive process where not all the neuronal populations present the same susceptibility to αS insults, especially in the gut where neuronal biodiversity is remarkable . Aside from the renowned central dopaminergic dysfunction linked to the typical motor features of PD , other neuronal systems that can be related to both motor and non-motor symptoms, within and outside the CNS, can become affected in PD. In particular, the cholinergic system has been implicated in PD progression in relation to the occurrence of typical motor symptoms such as postural instability and gait disturbances , but also to non-motor symptoms such as dementia and cognitive deficits [46, 47], REM sleep behavior disorder , hyposmia , and possibly dysphagia , which may all manifest in preclinical PD. Interestingly, selective accumulation of LBs in cholinergic neurons in the nucleus basalis of Meynert, in the basal forebrain, has been described to occur concomitantly with dopaminergic neuronal loss in substantia nigra . In addition, dopamine exerts a negligible control of intestinal motility at the level of the lower digestive tract in humans , strongly suggesting the implication of other neurotransmitters during the development of GI dysfunction in PD.
Moreover, in the present study we demonstrated that the bowel dysfunction shown by the behavioral and electrical recordings data in Tg mice is supported by a robust biochemical basis since immunoblot analysis of the whole intestinal tract revealed that young presymptomatic Tg mice predominantly express αS transgene only in the colon with consequent selective accumulation in this region of insoluble and phosphorylated HMW aggregates already at 3 months of age. Remarkably, while the distribution of αS transgene expression did not change with age, including after CNS pathology onset, the amount of insoluble aggregates increased over time, reaching a plateau at 6 months of age, temporally matching the increased delay in food transit and reduction of gut motility and providing at the same time a strong molecular basis to these behavioral and functional GI abnormalities. This strong correlation between presence of αS expression and LB-like αS aggregates in the colon and GI dysfunction is also confirmed by the absence of bowel dysmotility in the ileum of Tg mice, where αS expression is minimal. Thus GI αS inclusions, colonic neuronal deficit and GI dysmotility are tightly connected to αS overexpression in the gut in this mouse line and represent an early sign of αS-driven dysfunction, without CNS involvement. In PD patients, αS and LBs have been found in enteric neurons of both submucosal and myenteric plexi along the whole GI tract including the colon [8, 25]. An early investigation led by Beach and coworkers found a rostrocaudal gradient of accumulation of pser129-αS within the ENS with a higher incidence of LBs in the lower esophagus and submandibular gland and with less extent in the colon and rectum . Although this observation has to be confirmed in a larger population, involvement of the vagal nerve, which directly innervates the stomach, the small intestine and the ascending colon, as the main dissemination route of toxic αS along the gut-brain axis, has been suggested, based also on the finding of LBs presence in the dorsal motor nucleus of the vagal nerve in prodromal PD . However, the high incidence of aggregated αS in all the segments of the spinal cord suggests that other nerves might be implicated in αS propagation . In addition, several studies have indicated LBs presence in human colon biopsies as a possible diagnostic method for preclinical PD [10, 24, 27]. Thus, while expression of human αS transgene in the gastric segment of the GI tract remains to be investigated in our mouse model, the early accumulation of toxic αS in the large intestine is sufficient in our mice to recapitulate distinctive features of GI dysfunction of human PD, making the G2–3 line an optimal paradigm to study constipation in premotor PD. In addition, and more surprisingly detergent-stable, soluble oligomers of αS were also found in presymptomatic 3 months old Tg mice and their accumulation increased over time, a striking difference with the CNS, where αS soluble oligomers are only found at low level in adult (> 9 months old) or diseased mice and mainly associated with the microsomal vesicles fraction [13, 40]. Furthermore, level of pser129-αS in the soluble fraction was also abundant, suggesting that αS pathobiology may differ between the brain and the gut, where modified, HMW αS remains soluble for a longer period of time, instead of being confined in insoluble LBs. While their toxicity remains to be investigated, increased solubility of αS toxic species may facilitate their spreading and tissue propagation.
Constipation in the PrP human A53T αSTg mouse model, line G2–3, represents an early sign of αS pathology that manifests at least 6 months prior to motor symptoms and neuronal degeneration in the CNS. This net spatio-temporal separation of the two αS-driven pathologies makes the line G2–3 a unique tool to investigate the gut-brain connection in PD progression and to study GI dysfunction in prodromal PD.
We thank Prof. Simona Capsoni from University of Ferrara for her suggestions on behavioral analysis and Dr. Francesco Tenore from JHU/APL and Dr. Jonathan Yu from Johns Hopkins School of Medicine for linguistic editing.
This work has been funded by the Italian Ministry of University and Research through the Career Reintegration grant scheme (RLM Program for Young Researcher) and from Scuola Normale Superiore.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
LR and EC performed behavioral and biochemical experiments and analyzed the data; CP, LB and MF performed electrical recordings and analyzed the data; LR and EC wrote the paper and interpreted the results; EC designed the research; CB edited the paper and provided helpful discussion on GI physiology; AC reviewed the manuscript. All authors read and approved the final manuscript.
All animal studies were approved and complied in full by the national and international laws for laboratory animal welfare and experimentation (EEC council directive 86/609, 12 December 1987 and Directive 2010/63/EU, 22 September 2010).
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All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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