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Parkinson’s disease (PD) is the most common movement disorder and the second most common neurodegenerative disorder, with a prevalence of 1 to 2 individuals per 1000 at any age, increasing to 1% of the population above 60 years. The pathological hallmarks of PD are neuronal proteinaceous inclusions called Lewy bodies and neurites – rich in alpha-synuclein (αS) – and death of dopaminergic neurons in the pars compacta of the substantia nigra, which can only be detected in patients’ brains post mortem. Diagnosis is mainly based on the observation of the typical motor symptoms (tremor, rigidity, bradykinesia) caused by these molecular alterations. Although PD is considered a movement disorder, patients also suffer from a variety of non motor alterations, some of which (e.g. constipation) can occur even decades before the onset of the motor ones. Moreover, Lewy pathology has been found in patients not only in the central (CNS), but also in the peripheral and enteric nervous systems (ENS), suggesting a correlation between αS inclusions in these sites and non motor symptoms, and a possible spreading of the pathology from the periphery to the brain. This study is focused on gastrointestinal (GI) dysfunctions and specifically constipation, for which the relationship with PD development is still poorly understood. For this research we used a transgenic (Tg) mouse model over-expressing human A53T αS under the control of the murine PrP promoter (line G2-3), one of the first genetic model developed to study α-synucleinopathies (which include PD). These mice develop neurological abnormalities after 9 months of age that manifest with motor symptoms which become progressively more severe culminating into a fatal paralysis within 14-21 days. Diseased mice show an accumulation of intracellular, phosphorylated and ubiquitinated αS inclusions, neuroinflammation and neuronal degeneration in the CNS. For the purpose of this study, sick αS Tg mice at 12-14 months, presymptomatic mice at 1, 2, 3, 6, 9 and 12 (if still healthy) months, and age-matched nTg littermate controls were used. Presymptomatic αS Tg mice displayed a drastic delay in GI transit time of almost 2 hours from 3 months old that increased with age, reaching more than 3 hours delay at 6 months. Such delay was associated with abnormal formation of stools for αS Tgs, that resulted in less abundant but longer pellet excreted, although normal for dry and wet weight. After that we recorded the contractile activity from longitudinal and circular muscle preparations of colon and ileum, to verify the intestinal function. In line with our previous observations, electrically evoked contractions of the colon, but not of the ileum, showed a reduced response in both muscle layers in αS Tg mice already at 3 months of age, mainly due to an impaired cholinergic transmission of the ENS. Furthermore, molecular analyses were carried out to check on αS enteric distribution. Interestingly, insoluble and aggregated αS was found in enteric neurons in both myenteric and submucosal plexi only in the colon and not in the small intestine of 3 months old Tg mice, and exacerbated with age, mimicking the increase in transit delay and contraction deficits showed in behavioral and electrical recording experiments. Following this GI characterization of PrP A53T αS Tg mice, we designed a disease modifying therapy to be carried out with an antisense oligonucleotide (ASO) against αS, in presymptomatic animals. After an in vitro evaluation, the selected ASO was administred to 10 weeks old Tg mice for 7 days, through osmotic pumps or rectal administration. Very surprisingly, Tg mice which received the ASO displayed a significant reduction in their GI transit time compared to the values before starting the treatment and to the PBS control group, for both administration routes. Together with the improvement in constipation, ASO treatment induced a reduction, although not significant, of the total level of αS in the distal colon for both delivery methods. This research demonstrates for the first time that the PrP human A53T αS Tg mice line G2-3 is a unique model to investigate GI dysfunction in prodromal PD, thanks to the net spatio-temporal separation of αS-driven pathologies in the ENS and in the CNS. Moreover, the promising results obtained in this model by using an ASO peripherally support the correlation between GI behavior and αS levels and the hypothesis that lowering the total level of αS can be a successful disease modifying therapy against PD.

Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP- 1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology. Bowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP-1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology.

Introduction. Gastrointestinal dysfunctions represent one of the most common non-motor symptoms of Parkinson's disease (PD). Several lines of evidence suggest that enteric accumula- tion of α-synuclein (αS) inclusions (a hallmark of PD) and colonic inflammation could contribute to bowel motor dysfunctions since the earliest stages of PD. However, current knowledge does not allow to establish a clear relationship between altered mucosal permeabil- ity, enteric inflammation, bowel dysmotility and PD pathology. This study examined concom- itantly enteric αS accumulation, mucosal permeability, bowel inflammation and in vitro colonic motor activity in a transgenic model of PD, before onset of neurodegeneration in the CNS. Methods. PrP human A53T αS transgenic (Tg) mice, Line G2-3 develop a progres- sive PD-like neurological and motor deficiency after 9 months of age, accompanied by neuronal degeneration and pathological accumulation of aggregated αS in the central nervous system (CNS). Animals were sacrificed at the age of 3, 6 and 9 months, in order to evaluate the presence and timing of enteric αS inclusions, alterations of intestinal mucosal barrier and colonic inflammation since the very early phases of the disease, before the overt develop- ment of CNS dysfunction. Blood samples were collected to evaluate circulating lipopolysac- charide (LPS) levels (an indirect index of intestinal permeability) by ELISA assay; then distal colon was excised and processed for: 1) expression and aggregation of αS (western blot); 2) tissue tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) (ELISA). Colonic longitu- dinal and circular muscle preparations were set up in organ baths with Krebs solution, and connected to isometric transducers to record contractions elicited by electrical stimulation (ES, 10 Hz, 0.5 ms, 30 mA). Results: An accumulation of insoluble and aggregated αS was present in the enteric neurons of the distal colon in Tg mice since 3 months of age, as compared with controls. In addition, at 3, 6 and 9 months of age Tg mice displayed a significant elevation of circulating LPS along with increased colonic TNF and IL-1β levels. Electrically evoked contractions in longitudinal and circular muscle preparations were impaired in Tg mice at 3, 6 and 9 months of age, as compared with controls. Conclusion. The present findings suggest that a concomitance of enteric αS accumulation, altered mucosal permeability, bowel inflammation and impaired colonic motor activity represent early events in PD, occurring before the onset of CNS neurodegeneration. These changes could contribute to the pathogenesis of intestinal motor dysfunctions known to be associated with PD in humans.