Gait analysis showed 2-month-old A30P mice are pre-symptomatic (psA30P mice)
Gait analysis has been used to show onset of obvious PD manifestations in mice . We analyzed ten gait parameters of A30P and wild type (WT) mice using the CatWalk XT system. Animal footprints were automatically captured by the video camera of the system and were further categorized into individual paws like right forepaw (RF), right hindpaw (RH), left forepaw (LF), and left hindpaw (LH) via the software (Fig. 1a). To characterize pre-symptomatic PD or phenotype-manifested PD in mice, we assessed the gait in two age groups: 2 months old and 12–13 months old. Body mass was not different between 2-month-old A30P and WT mice, but 12–13-month-old A30P mice were significantly heavier than age-matched WT mice (Supplementary Fig. 1).
As expected, the older A30P mice showed significant motor impairments in all tested parameters compared to age-matched controls: maximum contact area, terminal dual stance, body speed, body speed variation, run maximum variation (p ≤ 0.05, Fig. 1b and Supplementary Fig. 2), base of support on front paws (Fig. 1c), run duration (Fig. 1d), average speed (Fig. 1e) and cadence (Fig. 1f). In addition, the older mouse group displayed significantly extended stands (p ≤ 0.05, Fig. 1g–j) and markedly changed footprint patterns from all paws (Fig. 1k-n). In contrast, younger A30P mice showed no significant changes in all investigated gait parameters compared with age-matched WT mice, indicating that these mice have a pre-symptomatic stage of the disease.
We also measured gut length in these mice. Gut length did not differ between A30P and WT mice in both age groups, but both intestines were significantly longer in 12–13-month-old A30P and WT mice compared with 2-month-old A30P and WT mice (Supplementary Fig. 3). Based on these findings, we defined the non-motor-impaired 2-month-old mice as the pre-symptomatic A30P mouse group (psA30P), and used these mice for further investigations in the gut.
Altered GI motility in psA30P mice
To investigate the hypothesis that the gut is the first site of PD manifestation, we compared GI motility patterns in the small intestine (SI) and the large intestine (LI) of psA30P mice and age-matched WT animals in isolated gut segments. We measured GI movement using several parameters, including changes in amplitude (∆ amplitude), number of contractions, mean interval (Fig. 2a), and velocity of motions (Fig. 2b).
Both SI and LI had an isochronous motility pattern in WT mice (SI: Fig. 2c,d; LI: Fig. 2j,k), whereas psA30P mice showed a discontinuous outline with extended passive interphases (SI: Fig. 2e,f; LI: Fig. 2l,m).
In the SI, more contractions were observed in WT mice (40.7 ± 2.6 contractions/min) than in psA30P mice (35.7 ± 3.6 contractions/min; p ≤ 0.05, Fig. 2g). Furthermore, the movements of WT mice were significantly faster (14.7 ± 2.8 mm/s) than those of psA30P mice (9.2 ± 2.2 mm/s; p ≤ 0.05, Fig. 2h). The mean interval was higher in psA30P mice because of extended and variable interphases of (0.50 ± 0.1 s) compared with WT mice (0.39 ± 0.7 s), but this difference was not significant (Fig. 2i).
In the LI, WT mice exhibited 9.5 ± 2.4 contractions (Fig. 2n) with an appropriate velocity of 0.9 ± 0.4 mm/s (Fig. 2o) over a 10-min period. The number (5.3 ± 2.6 contractions per 10 min; p ≤ 0.05, Fig. 2n) and velocity of contractions (0.3 ± 0.1 mm/s; p ≤ 0.01, Fig. 2o) were significantly lower in psA30P mice. In addition, the mean interval was significantly shorter in WT mice (69.1 ± 10.8 s) compared within psA30P mice (104.4 ± 33.4 s; p ≤ 0.05, Fig. 2p).
The reduced gut motility in psA30P mice can be visualized in the video recordings (Supplementary Videos 1–4); these videos show retarded activity, slower contractions, and longer static phases in psA30P mice compared with WT mice (Fig. 2).
Dysregulated protein expressions in the ENS of psA30P mice
To investigate the role of the ENS in PD pathogenesis, we dissected the myenteric plexus (MP) of the SI and the LI in psA30P and WT mice. Whole protein was isolated from the gut tissue, separated by high-performance liquid chromatography (HPLC), and further analyzed by mass spectroscopy.
The MP protein expression profile was different between psA30P and corresponding WT mice in the SI (Fig. 3a) and LI (Fig. 3b). In total, 1,044 proteins were detected by mass spectroscopy; in 7% of proteins, expression was significantly altered in the SI (Fig. 3a, Supplementary Fig. 4a, Supplementary Table 2) and almost twice as many proteins (14%) in the LI (Fig. 3b, Supplementary Fig. 4b and Supplementary Table 2), indicating a more serious manifestation of PD in the LI. Proteins with significant changes in expression were analyzed and visualized by
Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Each dot represents a single protein, while clustered proteins or proteins in the direct neighborhood indicate a similar function (Fig. 3c and d; Supplementary Fig. 4).
We used a Venn diagram to illustrate the distribution of dysregulated proteins in the SI (74 proteins) and the LI (147 proteins); 13 proteins were differentially expressed in both tissues (Fig. 3e). Categorization into groups by the STRING database yielded i.a. six different functional clusters: actin and microtubule organization, vesicle transport, calcium binding, ubiquitination, and response to oxidative stress (Fig. 3f). Proteins in these groups are known to be involved in PD pathogenesis in mice
and humans, mainly affecting the CNS (references in Table 1a). This functional classification emphasized that more proteins are dysregulated in the LI than in the SI. Therefore, we focused all further investigations on the LI. We selected three proteins with altered expression for detailed examinations: neurofilament light chain (Nefl), vesicle-associated membrane protein 2 (Vamp2) and calbindin 2 (Calb2). We chose these proteins because they have already been implicated in PD pathology. For example, Nefl and Calb2 expression is altered in the brain of humans with PD as well as in PD models, but nothing is known about how these proteins are affected in the gut during early stages of PD. In addition, both, Nefl and Calb2 can co-localize with α-synuclein. Several studies have shown a role for Vamp2 in neurodegeneration, but no direct link to PD pathogenesis has been described to date, although Vamp2 does bind to α-synuclein (Table 1b).
Differential Nefl, Calb2, and Vamp2 expression in the ENS of psA30P mice
To evaluate the expression of Nefl, Calb2, and Vamp2 in the LI during pre-symptomatic early-onset PD, we performed immunohistochemically stains on whole-mounts of the LI from psA30P and WT mice (Fig. 4). Based on our proteomic data and because of their pivotal role in the ENS, we decided to see whether these proteins can serve as biomarkers for early PD. Preliminary assessments of the LI whole-mounts showed a significantly smaller ganglionic area in psA30P mice than in WT mice (Supplementary Fig. 5).
Nefl staining revealed a large number of myenteric ganglia that were evenly distributed over the underlying circular muscle layer. Nefl-positive cells were also frequently encountered in the interconnecting strands running parallel to the circular muscle layer. These fibers had a thinner and smoother morphology than those running longitudinally. In general, Nefl expression was weaker in psA30P mice (8.4 ± 0.8%) than in WT mice (18.6 ± 3.9%; p ≤ 0.001, Fig. 4a). Fiber density was especially reduced in the interconnecting strands. Consistently, there were significantly fewer synaptic vesicles in the muscle layers of psA30P mice (6.4 ± 1.6%) compared within WT animals (18.3 ± 4.2%; p ≤ 0.01, Fig. 4b). Calb2 showed prominent immunoreactivity in the cytoplasm of neuronal soma and in axonal projections in both psA30P and WT mice. However, there were significantly more Calb2-positive cells in WT mice (8.8 ± 1.5%) than in psA30P mice (4.2 ± 0.86%; p ≤ 0.001, Fig. 4 c). Calb2 is expressed not only in the MP, but also in the submucosal plexus, which is located in the submucosal layer. Therefore, we imaged the whole gut in 3-D to include both plexuses, and confirmed the reduced Calb2 expression in psA30P mice (Fig. 4d).
Differential Nefl, Calb2, and Vamp2 expression in primary ENS cells from an in vitro PD model
To verify the dose-dependent toxicity of the aggregated mutant A30P α-synuclein protein on primary MP cells, we performed a live-dead assay as a pilot experiment with different incubation times (not shown). Based on the results, we chose a concentration of 0.5 µM A30P α-synuclein and an incubation time of 5 days for further experiments (Supplementary Fig. 6a). To ascertain the cellular effects of A30P α-synuclein on MP cells, we isolated and cultivated MP cells and performed immunocytochemistry for protein gene product (PGP) 9.5, class III beta tubulin (Tuj1), Nefl, Calb2, and Vamp2. The total number of neurons was calculated as the number of PGP9.5-positive neurons, and was reduced after exposure to A30P α-synuclein (WT: 27.5 ± 3.4 PGP9.5-positive cells; A30P: 17.9 ± 2.5 PGP9.5-positive cells; p ≤ 0.001, Fig. 4e). There were also significantly fewer Nefl-positive cells after exposure to A30P α-synuclein (WT: 0.08 ± 0.02 ratio Nefl/PGP9.5; A30P: 0.06 ± 0.01 ratio Nefl/PGP9.5; p ≤ 0.05, Fig. 4f and Supplementary Fig. 6b). Examination of synaptic vesicles revealed significantly fewer Vamp2-positive signals in A30P α-synuclein-treated cells per neuronal area (WT: 53.7 ± 11.1%; A30P: 24.7 ± 5.4%; p ≤ 0.01, Fig. 4g). Conversely, we found nearly twice as many Calb2-positive neurons in α-synuclein-treated cells compared with unchallenged cultures (ratio Calb2/PGP9.5 WT: 0.09 ± 0.01; A30P: 0.2 ± 0.06, p ≤ 0.01, Fig. 4h and Supplementary Fig. 6c).
For in vitro studies, myenteric plexus (MP) cells isolated from C57B6/J mice (postnatal day 2) were treated with 0.5 µM A30P α-synuclein for 5 days. This significantly reduced the number of protein gene product (PGP) 9.5-positive neurons (e). In addition, exposure to A30P α-synuclein significantly reduced the number of Nefl-positive cells in relation to total neuron number (f) and significantly reduced the expression of Vamp2 (g) compared with controls. The number of Calb2-positive cells significantly increased in relation to total neuron number (h). Nefl and Calb2 expression are indicated as a ratio to PGP9.5 and were normalized to the control. Vamp2 expression was only measured in neuronal areas. Non-treated cells (control = CO) are shown in yellow, A30P α-synuclein-treated cells are shown in green. Quantitative data were generated with ImageJ and are expressed as means ± SD from four to six independent experiments (N = 90 images per condition) using GraphPad Prism 8. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 using Student's t and Cohen’s d (Supplementary Table 7d).
Dysregulated miRNA expression in the ENS of psA30P mice
To measure miRNA expression in psA30P mice, a NanoString nCounter® mouse expression assay was performed (Supplementary Table 3). Volcano plots showed that 166 miRNAs were robustly expressed (more than 100 counts) in the MP of psA30P mice and 210 miRNAs were in the mesencephalon of psA30P mice compared with WT mice (Supplementary Tables 4 and 5, Fig. 5a,b). This dysregulation was statistically significant in 45 miRNAs in the MP (Fig. 5a), and in only eight miRNAs in the mesencephalon (Fig. 5b). Three miRNAs were differentially expressed in both tissues, and the fold changes in expression of these miRNAs were distinctly higher in the LI than in the mesencephalon (Fig. 5c).
The top ten regulated miRNAs with the highest fold change and the lowest p-value were miRNA-19a, 126-3p, 126-5p, 136-5p, 146a-5p, 210-3p, 301a-3p, 338-3p, 377-3p, and 1937c (Supplementary Figure 7 and Fig. 5a). Significant changes in expression were observed in miRNAs that target Nefl, Vamp2, and Calb2, supporting our idea that these proteins are potential biomarkers for early-stage PD. Detailed examination of protein-miRNA clustering using mirWALK 3.0  revealed that Nefl (Fig. 5d, pink framed boxes) and Vamp2 (Fig. 5d, blue framed boxes) are both targeted by ten miRNAs, and Calb2 (Fig. 5d, green framed boxes) is clustering with four miRNAs. All miRNAs were significantly upregulated. Two miRNAs, miR-22-3p and miR-30c-5p, target our three selected proteins, Nefl, Vamp2 and Calb2.
miRNA dysregulation is consistent in mice and men
To determine the functional impact of miRNA dysregulation on PD-associated protein networks, we performed an IPA-based network analysis using all 45 upregulated miRNAs and all 49 altered proteins from our defined functional groups (Fig. 6). The majority of dysregulated miRNAs were involved in the same pathways as the proteins identified in our proteome analyses, such as protein ubiquitination, calcium signaling, cell and oxidative stress, synaptic transmission, and cytoskeleton assembly. These data revealed 77 protein-miRNA interactions, involving 25 proteins and 31 miRNAs, in which the proteins were downregulated and the respective miRNAs were upregulated, as well as eight protein-protein interactions. Nefl, Calb2, and Vamp2 clustered extensively with several miRNAs. Thirty-one miRNAs from all dysregulated miRNAs in psA30P mice have been shown to be dysregulated in human PD tissues and body fluids (Supplementary Table 6a), and 13 have been implicated in PD in mouse, rat, and in vitro models (Supplementary Table 6b). However, these miRNAs were only investigated during clinical stages of PD and in the CNS, and not during pre-symptomatic stages and in the gut or MP like in the present study. In addition, we detected 10 significantly dysregulated miRNAs in the MP of the LI in psA30P mice, which, to best of our knowledge, have not been associated with PD to date (Supplementary Table 6c).