We have documented profound, early ENS dysfunction in two transgenic lines of mice carrying insertions of a human PAC containing the entire human SNCA
gene. The PAC was engineered to contain either the A53T or the A30P mutation previously shown to cause autosomal dominant PD in families and the mice were crossed to mice with a knock-out mutation in the endogenous Snca
gene so that the only α-synuclein in these mice was a human mutant protein. An artificial chromosome was chosen for expressing α-synuclein in these models because it contained the entire human SNCA
gene with its normal exon and intron structure as well as 35 kb of upstream sequences. Although the regulatory elements controlling SNCA
expression have not been completely defined, there are clearly upstream promoter elements as well as regulatory sequences in the first intron (45
). Thus, expression in these transgenic mice would likely be driven and controlled by endogenous gene regulatory elements. We found that transcript and protein expression were both markedly increased in the ENS of the colon in these mice over what was seen in endogenous mouse GI tract. The ENS dysfunction was reflected in reduced total fecal mass and fecal water content per unit time, increased time required to expel a bead from the colon, and increased WGTT in both lines carrying the PD-associated mutations, A53T and A30P, but not from an equally overexpressed wild-type SNCA
transgene. Given that these lines were independently generated and expressed different PD-associated mutations, the ENS dysfunction must be the direct result of overexpression of the mutant α-synucleins and not a non-specific effect of differences in genetic background, position effects from the transgene insertions or mutations at insertion sites.
ENS dysfunction could already be seen as early as 3 months of age, reached its maximum severity by 6 months of age and then persisted to 18 months. The ENS dysfunction occurred at a young age, before there was any evidence of olfactory or dysfunction in the autonomic innervation of the heart and before there were pathological changes in the brain stem. The lack of autonomic dysfunction, as seen in 24-h heart rate variability measurements and the absence of pathological changes in the brain stem, including the dorsal motor nucleus of the vagus [one of the first structures of the CNS affected by Lewy body and Lewy neurite pathology in idiopathic PD (2
)], suggest that the ENS dysfunction in the mice is an intrinsic
defect caused by mutant α-synuclein expression and aggregation in the ENS rather than to an extrinsic
effect of abnormal central innervation of the gut.
Wang et al
) previously reported colonic dysfunction in 12-month-old male transgenic mice in which wild-type α-synuclein expression was driven by the heterologous Thy-1 promoter (Thy1-αSynuclein). These mice exhibited decreased fecal output that increased to control levels in response to corticotropin releasing factor. However, they saw no significant change in colonic motility (measured by bead expulsion) and observed what seemed to be a trend towards increased fecal output of the Thy1-α-synuclein mice when they were placed in a novel environment. Our studies showed abnormalities only when the mutant human α-synuclein was expressed, and not the human protein, despite the fact that levels of transcript and protein expression in the mice with wild-type constructs were at least as great, if not greater. Finally, we observed that not only was colonic motility abnormal, but WGTT was also markedly prolonged. The effect on WGTT was seen regardless of the sex of the mouse and is indicative of widespread ENS abnormalities.
One interesting observation is that colonic propulsion in mice is strikingly different depending on gender. Gender differences in anorectal function have not been reported previously in mice but are quite consistent with observations made in humans. Human males have a greater sphincter length at rest and with squeezing, and the mean maximum squeeze pressure required for defecating are significantly greater in males than in females (40
Increased gastrointestinal transit time, constipation and difficulty with defecation are common in the course of sporadic PD and occur well before the movement disorder appears (7
). The gastrointestinal dysfunction is unlikely to be a secondary consequence of the movement disorder itself since patients with parkinsonism due to lacunar infarcts in the caudate, putamen or globus pallidus do not have the same gastrointestinal difficulties (8
). The gastrointestinal dysfunction also does not result from the medications used to treat the movement disorder since gastrointestinal dysfunction is common in newly diagnosed PD patients prior to the initiation of drug treatment (48
). Decreased bowel movement (BM) frequency in early adulthood is strongly associated with the development of Lewy body pathology indicative of PD later in life. In the longitudinal Honolulu Asia Aging Study, 245 men without PD were followed and later died and came to autopsy (11
). Those with <1 or 1 BM/day had 4.3-fold and 2.2-fold increased odds, respectively, of incidental LB on autopsy compared than in those with >1 BM/day. A large body of evidence supports the conclusion that ENS dysfunction is an early, intrinsic component of the PD phenotype.
In light of the findings reported here, it would be interesting to know whether the familial form of PD due to α-synuclein mutations also starts in the ENS. Although detailed clinical descriptions of affected individuals from the families with the A53T or A30P mutations have been published (50
), the authors of these studies only provide clinical data for motor and cognitive abnormalities and do not comment on the presence or absence of gastrointestinal dysfunction.
Braak and his co-workers have reported that many individuals without any signs of PD at the time of death have Lewy bodies in the ENS, as well as in the olfactory bulbs and the dorsal motor nucleus of the vagus, but not in the substantia nigra or other areas of the CNS typically affected in PD (2
). The Lewy bodies in the ENS found at autopsy in individuals who lack the motor symptoms of PD are often in VIP-containing neurons of the myenteric and submucosal plexuses (16
). Assuming that incidental Lewy body pathology in asymptomatic individuals represents the earliest stages of PD, Braak et al
. proposed a staging scheme in which a presymptomatic phase, characterized by ENS, brainstem and olfactory bulb pathology, is succeeded by a symptomatic phase when the movement disorder becomes manifest, characterized by pathology in the midbrain, including substantia nigra and ending with a cortical phase, with widespread cortical Lewy bodies. Because of the limitations of autopsy studies, the scheme was not able to establish the temporal relationship between the enteric Lewy bodies and other early pathological changes in the olfactory bulb or brainstem. Although Braak's staging schema has been criticized by some for being oversimplified and tainted with ascertainment bias (55
), it has also received additional support (57
). At a minimum, it has drawn the attention of researchers to the early, non-motor signs of the disease and away from an exclusive focus on dopaminergic neuronal loss and the parkinsonian movement disorder.
There is ample evidence that the movement disorder seen in PD results from loss of dopaminergic neurons in the substantia nigra and other regions of the CNS (58
). There also appears to be a role for dopaminergic dysfunction in ENS function in PD as well. Human PD patients with abnormal colonic motility reportedly show reduced ENS dopamine levels and a loss of dopaminergic neurons in the colon (53
). Studies in rodents also indicate that dopaminergic neurons are important for colonic motility. Dopaminergic neurons are present as intrinsic components of both the submucosal and myenteric plexus of rodent ENS and changes in dopaminergic function are associated with marked changes in colonic motility (59
). We saw overexpression of α-synuclein in dopaminergic neuronal cell bodies but not in the extrinsic sympathetic noradrenergic projections to the bowel. It seems unlikely, however, that the ENS dysfunction in the mutant α-synuclein transgenic mice is due entirely to a defect in dopaminergic function since the overexpressed protein was also seen in other types of neuron. Mice treated acutely with MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) had a 40% reduction in colonic dopaminergic neurons but only a transiently abnormal colonic motility and no gastric or small intestine motility deficits (61
). In the small intestine of normal rats, only a small fraction of neurons are α-synuclein-immunoreactive and, depending on which part of the stomach or small intestine is being studied, the expression is predominantly coincident with nitrergic and cholinergic, but not TH-immunoreactive, neurons (62
). Thus, it is likely that a number of different neurotransmitter-defined neurons, in addition to those that contain dopamine are impaired to cause the ENS dysfunction reported here. Careful studies with dopamine and other neurotransmitter agonists and antagonists may help to determine which neurons are most responsible for the mutant α-synuclein-induced motor abnormalities of the bowel.
Along with robust signs of ENS dysfunction, the dbl-PAC-Tg(SNCAA53T
mice, also exhibited reproducible abnormalities in motor function detected by rotarod and open field measurements as early as 6 months of age. The movement abnormalities occurred in the absence of changes typical of PD in the CNS such as Lewy body pathology, neurodegeneration or even changes in striatal dopamine levels. As reviewed by Chesselet (27
), Meredith et al
), Terzioglu and Galter (29
) and Melrose et al
), most transgenic rodent models have little or no CNS pathology and none fully recapitulate PD, although, in some cases, rare protein aggregates with Lewy-body-like pathology occur. In a few transgenic models, massive non-physiological overexpression of α-synuclein in cells, such as the motor neurons of the anterior horn of the spinal cord, which usually express only low levels of α-synuclein in comparison to other parts of the CNS, results in lower motor neuron degeneration that is not characteristic of human PD (33
). We conclude that even modest overexpression of the mutant A53T α-synuclein can subtly interfere with normal neuronal function in both the CNS and ENS prior to the development of frank neurodegeneration. Motor dysfunction was, however, not seen in the dbl-PAC-Tg(SNCAA30P
despite expression levels of transcript and protein equivalent to the dbl-PAC-Tg(SNCAA53T
mice. Why mice overexpressing A53T and not A30P α-synuclein showed abnormalities on rotarod and open field testing is not known. One possibility is that the A30P mutation may be a less severe mutation than the A53T mutation. For example, the average age of onset of the motor abnormalities of PD was 45.6 ± 13.5 years in the large Italian–American kindred with the A53T mutation described by Duvoisin, Golbe and their colleagues and 59.7 ± 10.8 years in the German family with the A30P mutation reported by Krüger et al
). This difference just reaches statistical significance (P
= 0.04, two-tailed t
Compared with the ENS, α-synuclein protein expression in the brain was only modestly increased despite very substantial increases in transcript levels. These findings suggest there are mechanisms by which the brain can regulate α-synuclein levels despite increased steady-state human SNCA transcript. Such regulation could be at the level of protein translation or degradation and remains to be elucidated.
Our results support the view that, just as has been suggested for sporadic PD, familial PD due to abnormalities in α-synuclein, manifests early, perhaps even first, in the ENS. The early onset of robust ENS dysfunction in the PAC transgenic mice described here is a convenient mammalian model system with simple assays in which to study interventions designed to reverse the deleterious effects of α-synuclein overexpression. They also could be viewed as a sensitized model in which to investigate the role of additional factors in the pathogenesis of PD. Braak et al
) and others (64
) have suggested that the gastrointestinal tract is a point of entry for a second pathogenic hit that goes on to affect the CNS. The double transgenic mice expressing mutant α-synucleins offer an opportunity to investigate the hypothesis that the early ENS dysfunction is not only an early marker of the disease, but also which, when triggered, facilitates the entry of deleterious factors that cause progression and spread to the CNS.