The prodromal phase of Parkinson’s disease: a mouse model study focused on alpha-synuclein pathology and dysfunction in the colon

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.