Rashmi Chandra,1 Annie Hiniker,2 Yien-Ming Kuo,3 Robert L. Nussbaum,3,4 and Rodger A. Liddle1,5
First published June 15, 2017 – More info
Parkinson’s disease (PD) is a progressive neurodegenerative disease with devastating clinical manifestations. In PD, neuronal death is associated with intracellular aggregates of the neuronal protein α-synuclein known as Lewy bodies. Although the cause of sporadic PD is not well understood, abundant clinical and pathological evidence show that misfolded α-synuclein is found in enteric nerves before it appears in the brain. This suggests a model in which PD pathology originates in the gut and spreads to the central nervous system via cell-to-cell prion-like propagation, such that transfer of misfolded α-synuclein initiates misfolding of native α-synuclein in recipient cells. We recently discovered that enteroendocrine cells (EECs), which are part of the gut epithelium and directly face the gut lumen, also possess many neuron-like properties and connect to enteric nerves. In this report, we demonstrate that α-synuclein is expressed in the EEC line, STC-1, and native EECs of mouse and human intestine. Furthermore, α-synuclein–containing EECs directly connect to α-synuclein–containing nerves, forming a neural circuit between the gut and the nervous system in which toxins or other environmental influences in the gut lumen could affect α-synuclein folding in the EECs, thereby beginning a process by which misfolded α-synuclein could propagate from the gut epithelium to the brain.
Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by motor disturbances, including resting tremor, rigidity, and slow movements, as well as gastrointestinal symptoms, such as constipation and gastroparesis. The pathological hallmarks of PD are cytoplasmic inclusions known as Lewy bodies (within the cell body) and Lewy neurites (in axons) of the brain and enteric nervous system (1). These inclusions are associated with degeneration of dopaminergic neurons in the substantia nigra pars compacta (2, 3), which produces the distinctive disorders of movement and vagal nerve dysfunction. The major component of Lewy pathology is aggregated α-synuclein, a synaptic protein with the propensity to misfold and aggregate (4). Misfolded α-synuclein plays a critical role in PD pathogenesis, and recent evidence supports a model in which propagation of Lewy pathology occurs via cell-to-cell transmission of misfolded α-synuclein onto recipient cells (5–9). Misfolded α-synuclein recruits native α-synuclein in the recipient cell and acts as a template or nidus for the development of aggregates that eventually lead to formation of Lewy bodies and ultimately PD (1, 8, 10).
Although the pathogenesis of PD is incompletely understood, Braak and colleagues suggest that the pathological process begins in the enteric nervous system (11, 12). Both clinical and experimental data support such a model. Clinically, PD patients frequently experience gastrointestinal symptoms many years before motor deficits develop (13, 14), and α-synuclein aggregates appear in enteric nerves before they are found in the brain (12, 15, 16). α-Synuclein immunoreactive inclusions have been found in neurons of the submucosal plexus, whose axons project to the mucosa (15, 17, 18). Moreover, it has been reported recently that bilateral vagotomy reduces the risk of PD (19).
Experimentally, direct transmission of α-synuclein from the gut to the brain was demonstrated in a key experiment in which α-synuclein injected into the intestine was transported via the vagus nerve to the dorsal motor nucleus of the vagus in the brainstem (20, 21). This finding is consistent with the original observation that Lewy pathology appears in the projection neurons of the dorsal motor nucleus of the vagus in the early stages of PD (12). Experimentally, the vagal route of α-synuclein transport has also been documented following exposure to the environmental toxin rotenone (22) as well as following direct injection of adenoassociated viral vectors overexpressing human α-synuclein into the vagus nerve (23). A route from intrinsic enteric neurons to the vagus nerve is supported by the observation that both myenteric neurons and preganglionic vagal nerves express α-synuclein (24). These findings are consistent with the hypothesis that α-synuclein aggregation begins in the gut and spreads to the central nervous system via the vagus nerve.
Recently, it has been shown that individuals with PD have an altered gut microbial composition (25–28), raising the possibility that gut microbes affect PD pathogenesis. In a mouse model of PD, gut microbiota promoted α-synuclein aggregation and the development of motor disturbances (29). Moreover, colonization of mice with microbiota from PD-affected individuals enhanced physical deterioration (29), further emphasizing the role of the gut in PD. However, the mechanism by which gut microbes affect the progression of PD is not well understood.
Furthermore, an environmental basis for PD has been suspected for over 50 years, and a number of studies have shown an increased incidence of PD in individuals exposed to pesticides and herbicides (30–32). Enteric nerves are limited by the intestinal epithelium and do not extend into the intestinal lumen. Therefore, nerves of the gut do not have direct contact with luminal contents. Thus, even though data suggest that PD pathology begins in the gut, the mechanism by which luminal environmental toxins might induce changes in enteric neurons is unknown (12–16, 20).
Enteroendocrine cells (EECs) are chemosensory cells that are dispersed throughout the mucosal lining of the intestine and oriented with their apical surface open to the lumen of the intestine, so that they can sense luminal contents, such as ingested nutrients or gut microbes. Traditionally, EECs were viewed exclusively as hormone-producing cells of the gastrointestinal tract; however, we recently discovered that EECs also connect to neurons (33, 34). Their location places EECs at the interface between gut contents and the nervous system and provides a direct route for substances in the gut to affect neural function. In addition, EECs are electrically excitable and possess many neuronal features, including neurotrophin receptors, presynaptic and postsynaptic proteins, small clear secretory vesicles, neurofilaments, and basal processes known as neuropods (35). A functional synaptic connection between EECs and enteric nerves was established using rabies viral tracing, in which a modified rabies virus placed into the lumen of the intestine infected EECs and was transmitted into enteric nerves (33). These neuronal features suggest that EECs are sensory cells in the gut. Impressed by their neuron-like properties, we sought to determine if EECs express α-synuclein. If they do, their location at the interface of the gut lumen and the nervous system suggests that EECs could be a target for the induction of abnormal α-synuclein and initiation of the prion-like cascade leading to PD.
α-Synuclein is expressed in enteroendocrine STC-1 cells. STC-1 cells were derived from the duodenum of transgenic mice that expressed SV40 large T antigen downstream from a rat insulin promoter (36); they are widely accepted as a model of native EECs (37). STC-1 cells express several gastrointestinal hormones, such as cholecystokinin (CCK) and peptide YY (PYY), whose secretion is regulated in a manner similar to native EECs (38–40). The inability to culture EECs or obtain sufficient numbers from intestinal tissue for in vitro assays makes STC-1 cells attractive for evaluating properties of EECs. Expression of α-synuclein in STC-1 cells was analyzed by real-time PCR, immunoblotting, and immunofluorescence. Figure 1A shows the relative quantitation of Gapdh, α-synuclein (Snca), Cck, and Pyy in STC-1 cells compared with the SH-SY5Y neuroblastoma cell line. HeLa cells were used as the comparator and β-actin (Actb) transcript level was used to normalize RNA abundance. The relative amount of Snca mRNA in STC-1 cells (~15-fold) was comparable in magnitude to that present in SH-SY5Y cells (~34-fold), whereas STC-1 cells expressed a much higher amount of the Cck transcript (1.5 × 105–fold versus ~1-fold). The Pyy transcript was also expressed at a higher level in STC-1 cells (~150-fold), although SH-SY5Y cells appeared to express some PYY transcript (~8-fold) relative to HeLa cells. The relative amount of Gapdh/GAPDH mRNA was similar between the 3 cell lines examined.
α-Synuclein protein is expressed in STC-1 cells.Figure 1
α-Synuclein protein is expressed in STC-1 cells. (A) Relative quantitation of Gapdh, α-synuclein (Snca), cholecystokinin (Cck), and peptide YY (Pyy) mRNAs in STC-1 and SH-SY5Y cells showing that α-synuclein mRNA is present in STC-1 cells. β-Actin (Actb) was used as the normalizer, and RNA isolated from HeLa cells served as a comparator. Data represent mean ± SEM of 3 individual experiments. (B) Coomassie Blue–stained 4%–12% gradient SDS-PAGE gel and corresponding immunoblot of STC-1 cells, A53T mouse brain, and Snca-/- mouse brain extracts with α-synuclein monoclonal antibody. A nonspecific band (asterisk) was detected in A53T and Snca-/- mouse brain extracts. The molecular weights of protein bands present in the ladder are indicated in kDa. (C) 3D Z-stack image of STC-1 cells showing α-synuclein (red) in the cytoplasm. Cytoskeletal staining is shown with β-tubulin (green). Scale bar: 20 μm.
To examine α-synuclein protein levels, a cellular extract of STC-1 cells was electrophoresed, along with whole-brain lysate from A53T mice and α-synuclein–knockout (Snca-/-)mice. A53T transgenic mice contain 4 copies of the human α-synuclein gene carrying the A53T mutation on a Snca-/- background (41) (Figure 1B). Using an α-synuclein antibody that has been extensively characterized (41), α-synuclein was found to be present in both STC-1 cell and A53T mouse brain extracts but not in brain extracts of Snca-/- mice (41). A faint nonspecific band was observed in both brain samples and has previously been noted with this antibody (Y.-M. Kuo and R.L. Nussbaum, unpublished observations) (42). We also examined the cellular localization of α-synuclein in STC-1 cells by immunofluorescence. A general low level of α-synuclein immunofluorescence was present in the entire cytoplasm (Figure 1C). No immunofluorescence was detected in the absence of primary antibodies (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.92295DS1).
Intestinal EECs express α-synuclein. The presence of α-synuclein in STC-1 cells suggested that this protein is expressed in EECs of the intestine. To evaluate this possibility, we purified GFP-positive CCK cells from the duodenums of CCK-GFP mice using fluorescence-activated cell sorting and quantitated gene expression by real-time PCR as described previously (43, 44). Cck gene expression was almost 2,000-fold greater than Actb in GFP-positive cells and Snca was over 150-fold increased over the control gene Actb (Figure 2A), indicating that α-synuclein mRNA is greatly enriched in CCK cells. In this experiment, α-synuclein RNA expression was compared between GFP-positive CCK cells and GFP-negative mucosal cells that contained non–CCK-GFP EECs. Since α-synuclein is expressed in non–CCK EECs (see data below), it is likely that this relative quantitation of Snca gene in CCK-GFP cells is an underestimation of the actual abundance of Snca transcript in CCK cells.
α-Synuclein is present in mouse duodenal CCK cells.Figure 2
α-Synuclein is present in mouse duodenal CCK cells. (A) Relative quantitation of cholecystokinin (Cck), α-synuclein (Snca), and β-actin (Actb) mRNAs in FAC-sorted CCK-GFP cells from mouse duodenum. Gapdh was used as the normalizer, and RNA isolated from CCK-GFP–negative cells served as a comparator. Data represent mean ± SEM of 3 individual experiments. (B) A frozen section (10-μm thickness) of A53T mouse duodenum was fixed in a mixture of methanol and acetone and stained for α-synuclein and CCK. The image shows a small section of the villus with a CCK cell. Nuclei (blue channel) have been removed from the image on the right for an uninterrupted view. α-Synuclein (red) is present in the cytoplasm of the CCK cell (green), which has been rendered 50% transparent. α-Synuclein is also expressed in enteric nerves of the villus. This CCK cell is present in close juxtaposition to enteric nerves. Scale bar: 5 μm.
Characterizing α-synuclein in mice has been challenging, due, in part, to low endogenous levels of protein; thus, new genetic models have been used to enhance α-synuclein expression and assess its function. Therefore, as a first step, we examined α-synuclein expression in A53T mice. Our goal was to determine if α-synuclein expressed from the human promoter in A53T transgenic mice could be visualized in EECs. Figure 2B shows α-synuclein immunofluorescence in the villus of the A53T mouse duodenum. α-Synuclein–positive enteric nerves were also present in the crypt region (Supplemental Figure 2). The CCK cell also expressed α-synuclein (Figure 2B, right), and the basolateral surface of this cell rested on an α-synuclein–containing nerve. No fluorescence in EECs was observed in the absence of CCK primary antibody (Supplemental Figure 3). In wild-type (CCK-GFP) mice, α-synuclein staining was detected within some CCK cells but could not easily be visualized in the enteric nerves (Supplemental Figure 4). The striking difference in immunofluorescence intensity between A53T and wild-type mouse intestine could be attributed to higher levels of α-synuclein in A53T mice (41). It is also possible that the human protein is recognized better by the primary antibody used in these experiments. No α-synuclein staining was detected in the small intestines of Snca-/- mice (Supplemental Figure 5), suggesting that the staining observed in A53T mice was antigen specific (41).
Importantly, α-synuclein staining was also present in EECs in human duodenum, as shown in Figure 3 (a CCK cell present in the center of the image on the left is shown at higher magnification on the right). A small amount of α-synuclein was visible inside the CCK cell, and this cell was adjacent to the terminus of an α-synuclein–positive enteric nerve. However, using this technique, not all CCK cells were positive for α-synuclein and only a minority of cells were found in contact with enteric nerves. Approximately 71% of CCK cells contained intracellular α-synuclein, while 33% of the cells were apposed to α-synuclein in nerves or glia of A53T;CCK-GFP mice (Table 1).
α-Synuclein expression in CCK cells and enteric nerves of human duodenum.Figure 3
α-Synuclein expression in CCK cells and enteric nerves of human duodenum. Paraffin-embedded section (5-μm thickness) of human duodenum showing a cross section of villi. α-Synuclein (red) staining is visible in enteric nerves located between the villi. One of the cholecystokinin (CCK) cells (boxed) is shown at higher magnification on the right. α-Synuclein is present inside the CCK cell, and this cell is present in close proximity to an α-synuclein–containing enteric nerve. Scale bar: 20 μm (left); 5 μm (right).
Estimation of α-synuclein–expressing EECs
To determine if α-synuclein is expressed in other EECs, we examined PYY-containing cells of the mouse and human intestine. As shown in Figures 4 and 5, α-synuclein is prominent in mouse and human PYY cells. The top row of images of Figure 4 show 4 GFP-positive PYY cells present in the colon of a PYY-GFP mouse. These cells expressed α-synuclein, and in one of the cells, the α-synuclein protein extended into the neuropod of the PYY cell. Enteric nerves were not visible in these sections, possibly because immunofluorescence of α-synuclein was very weak in the enteric nervous system of wild-type mice (45). The image on the bottom of Figure 4 shows a PYY-positive cell in the small intestine of an A53T mouse that is located in close juxtaposition to an α-synuclein–containing enteric nerve, which runs in the lamina propria of the villus. α-Synuclein is also expressed in PYY cells of the human colon. The left side of Figure 5 shows two cells that express PYY and α-synuclein. The right side of Figure 5 shows colocalization of PYY and α-synuclein in 3 cells of the human colon. Approximately 37% of PYY cells in the colon contain intracellular synuclein, and 32% of these cells are in contact with α-synuclein in nerves or glia (Table 1).
α-Synuclein is expressed in PYY cells of mouse colon.Figure 4
α-Synuclein is expressed in PYY cells of mouse colon. Top row: Frozen sections (10-μm thickness) of PYY-GFP mouse colon were fixed with formalin and stained for GFP (green) and α-synuclein (red). α-Synuclein is present in the cytoplasm of PYY-GFP cells and extends into the neuropod of one of the cells (arrow). Bottom row: Paraffin-embedded section (5-μm thickness) of A53T mouse intestine showing a PYY cell (green) in close proximity to a α-synuclein–stained (red) nerve in the lamina propria. Scale bar: 10 μm. PYY, peptide YY.
α-Synuclein is expressed in PYY cells of human colonic crypts.Figure 5
α-Synuclein is expressed in PYY cells of human colonic crypts. Paraffin-embedded section (5-μm thickness) of human colon showing PYY cells (green) that contain α-synuclein (red). The merged image on the right shows colocalization of these two proteins in yellow. Scale bar: 10 μm. PYY, peptide YY.
Chromogranin A is located in secretory vesicles of endocrine cells and has been used as a marker of most EECs. We identified α-synuclein in chromogranin A–positive cells (Supplemental Figure 6) in the intestine. α-Synuclein staining was also observed in rare mucosal cells that did not stain with one of the three EEC markers (CCK, PYY, and chromogranin A) used in this study. Figure 6 shows a CCK-GFP cell located near an α-synuclein–positive but GFP-negative mucosal cell. The CCK cell expresses α-synuclein in the basolateral region and is located near a nerve identified with the pan-neuronal marker, protein gene product 9.5 (PGP9.5), that also contains α-synuclein. Notably, both the CCK and non-CCK α-synuclein–positive mucosal cells contained intracellular PGP9.5. Since only one EEC marker was evaluated in any given experiment, we cannot rule out the possibility that α-synuclein is expressed in an occasional non-EEC intestinal cell; however, the CCK and non-CCK cells possess the characteristic flask shape typical of EECs, and their α-synuclein and PGP9.5 expression imply neuronal properties.
Identification of α-synuclein in cholecystokinin-positive and -negative celFigure 6
Identification of α-synuclein in cholecystokinin-positive and -negative cells of the intestinal mucosa. Frozen sections (16-μm thickness) of A53T;CCK-GFP mouse duodenum showing a CCK-GFP (green) mucosal cell adjacent to an α-synuclein–positive (turquoise) cell that does not contain CCK. Protein gene product 9.5 (PGP9.5) (red) is identified in both mucosal cells (thin arrows), which lie in close proximity to a PGP9.5-positive neuron. α-Synuclein within the CCK cell (thick arrow) lies in close proximity to extracellular α-synuclein. Colocalization of α-synuclein (turquoise) and PGP9.5 (red) within the cell and in the nerve appears white. Note the similar flask shape of both the CCK-positive and -negative α-synuclein–containing mucosal cells, which is typical for enteroendocrine cells. Scale bar: 10 μm. CCK, cholecystokinin.
α-Synuclein is expressed in the enteric nervous system. We also examined α-synuclein in submucosal enteric nerves using PGP9.5 immunostaining. Previous studies have shown that α-synuclein and PGP9.5 colocalize in cutaneous autonomic nerves (46). Figure 7 shows that α-synuclein is present in PGP9.5-positive nerves. However, the distribution of α-synuclein in the nerves is not continuous but appears to be localized to distinct zones or domains. This observation was also apparent in Figure 2B and Figure 3, where the α-synuclein staining is not continuous, but patchy, although a pattern of fluidity can be constructed. Higher magnification of the α-synuclein–positive mucosal cell revealed numerous microscopic processes that extend from the surface of the cell and appear to make contact with the PGP9.5-positive enteric nerve. The images in Figure 7 clearly demonstrate that α-synuclein is present in mucosal cells (e.g., EECs) and suggest that α-synuclein has the potential to migrate from specific mucosal cells (EECs) into enteric nerves not only via endocytosis of extracellular α-synuclein (47, 48) but also through direct physical contact between mucosal cells and enteric nerves.
α-Synuclein–containing mucosal cell is in contact with an enteric nerve.Figure 7
α-Synuclein–containing mucosal cell is in contact with an enteric nerve. Left: Paraffin-embedded section of human duodenum (5-μm thickness) showing colocalization (yellow) of α-synuclein in certain regions of protein gene product 9.5–positive (PGP9.5-positive) enteric nerves (red). Right: A high-magnification image of the α-synuclein–positive cell (green) exhibiting numerous fiber-like cellular processes, which encircle the PGP9.5-positive nerve (red). Scale bar: 20 μm (left); 5 μm (right).
Previous studies have shown that α-synuclein and tyrosine hydroxylase (TH) are coexpressed in the brain and dopaminergic cell lines and that α-synuclein may play a role in regulating dopamine synthesis (49, 50). TH is also known to be expressed in neurons of the enteric nervous system, and TH mRNA was previously detected in intestinal PYY cells by real-time PCR (33, 51). Interestingly, EECs, like PD-affected neurons, are TH positive. Figure 8 shows that TH protein is detected by immunofluorescence in α-synuclein–positive mucosal cells of the human colon.
TH is expressed in α-synuclein–expressing mucosal cells.Figure 8
TH is expressed in α-synuclein–expressing mucosal cells. Paraffin-embedded section (5-μm thickness) of human colon showing colocalization of TH (green) and α-synuclein (red). Not all α-synuclein–containing mucosal cells are positive for TH. Scale bar: 20 μm. TH, tyrosine hydroxylase.
Glial fibrillary acidic protein (GFAP) is a common marker for identification of glia. To determine if glia also express α-synuclein, we examined the expression of GFAP in the human duodenum (Figure 9). Glial processes were not very abundant in the human duodenal sections used for this work, and the immunofluorescence was not bright; however, α-synuclein was present in certain regions of GFAP-positive glial processes. Supplemental Figure 7 shows that there is some colocalization of α-synuclein and GFAP in frozen sections of the perfused mouse duodenum. In other regions of the tissue, GFAP staining appeared to surround α-synuclein–positive nerves. The pattern of α-synuclein expression was similar to that described above for enteric nerves, where it was not distributed evenly in the entire glial process but visible in small areas or domains. The factors that cause the segregation and maintenance of α-synuclein in these domains remain to be established.
α-Synuclein colocalizes with glial marker GFAP.Figure 9
α-Synuclein colocalizes with glial marker GFAP. Paraffin-embedded sections (5-μm thickness) of human duodenum showing colocalization of GFAP (green) and α-synuclein in some mucosal cells (asterisk) and in certain regions of fine glial processes (arrows). Scale bar: 10 μm. GFAP, glial fibrillary acidic protein.
In summary, here we demonstrate that α-synuclein is expressed in the EEC line STC-1 as well as in mouse and human EECs of the duodenum and colon. α-Synuclein is also expressed in enteric nerves and to a lesser extent in enteric glia. Some EECs are in direct contact with α-synuclein–containing nerves, which could lead to the transmission of aggregated α-synuclein from EECs to the enteric nervous system (Figure 10).
Hypothetical pathway for pathogenic migration of α-synuclein in the gut.Figure 10
Hypothetical pathway for pathogenic migration of α-synuclein in the gut. The apical surface of enteroendocrine cells (EECs) is exposed to the lumen and thus is in contact with ingested toxins and metabolites produced by gut microbes. The basolateral surface of EECs is in contact with enteric nerves and glia. We propose that toxin uptake by EEC can cause aggregation of α-synuclein inside these cells and this aggregated protein can migrate to enteric nerves, thereby initiating a pathogenic cascade leading to α-synucleinopathies.