Restoration of intestinal function in an MPTP model of Parkinson's Disease

Abstract

Patients with Parkinson's disease often experience non-motor symptoms including constipation, which manifest prior to the onset of debilitating motor signs. Understanding the causes of these non-motor deficits and developing disease modifying therapeutic strategies has the potential to prevent disease progression. Specific neuronal subpopulations were reduced within the myenteric plexus of mice 21 days after intoxication by the intraperitoneal administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and was associated with a reduction in stool frequency, indicative of intestinal dysfunction. Oral administration of the divalent copper complex, Cu(II)(atsm), which has been shown to be neuroprotective and restore motor performance to MPTP lesioned mice, improved stool frequency and was correlated with restoration of neuronal subpopulations in the myenteric plexus of MPTP lesioned mice. Restoration of intestinal function was associated with reduced enteric glial cell reactivity and reduction of markers of inflammation. Therapeutics that have been shown to be neuroprotective in the central nervous system, such as Cu(II)(atsm), therefore also provide symptom relief and are disease modifying in the intestinal tract, suggesting that there is a common cause of Parkinson's disease pathogenesis in the enteric nervous system and central nervous system.

Figure 1. Cu^II(atsm) restores gastrointestinal function and neuronal loss following MPTP lesioning.

Following MPTP lesioning mice were treated with SSV (open bars) or Cu^II(atsm) (closed bars) for 21 days. Stool frequency (a) that was significantly reduced at 18 days compared to 4 days following MPTP lesioning was restored by Cu^II(astm) treatment in mice 18 days after post lesioning. 2way ANOVA with Bonferroni’s multiple comparisons test; ***p < 0.001 *p < 0.05; n = 20; mean ± sem. Neuronal populations (b) were quantified in the myenteric plexus of MPTP lesioned mice treated with SSV (open bars) or Cu^II(astm) (closed bars). The number of neurons per field of view is expressed as a percentage of the average number of neurons in saline lesioned/SSV treated mice (control). The number of tyrosine hydroxylase (TH) and neurofilament M (NFM) immunoreactive neurons was significantly reduced in MPTP lesioned mice. The number of neuronal nitric oxide synthase (nNOS) immunoreactive neurons was not affected by MPTP lesioning. The number of NFM-IR and NOS-IR neurons was significantly increased by Cu^II(atsm) treatment, whereas treatment had no significant affect on the number TH-IR neurons. *p < 0.05, **p < 0.01 two-tailed t-test, n ≥ 5; mean ± sem.

Figure 2. The characteristics and localisation of α-synuclein does not correlate with the therapeutic effects of Cu^II(atsm) treatment of MPTP lesioned mice.

The SDS-insoluble α -synuclein (a) present in the brain (Br) or ileum (Il) of hA53T transgenic mice and PBS-insoluble α -synuclein (b) present in the brain or ileums of hA53T transgenic (TG), α-synuclein knock-out (KO) or wild-type (WT) mice following lesioning with saline (SAL) or MPTP was detected by western immunoblot analysis of homogenates normalised for equivalent protein. α -synuclein immunoreactivity was not detected in wholemounts (c–j) prepared from the myenteric plexus of KO (c) but was present in hA53T TG (i) mice and saline lesioned (d–f) and MPTP lesioned (g,h,j) C57Bl6 mice, treated with SSV (d,g,h) or Cu^II(atsm) (e,f,j). α-synuclein was observed in cell bodies (arrow heads in d,d’,h,h’) and as aggregates (arrows in e,g). Insets (d,f,h,j) are shown magnified x2.5 (d’,f’,h’,j’). Scale bar 100 μm.

Figure 3. Glial cell immunoreactivity correlates with loss of nNOS-IR in MPTP lesioned mice.

GFAP-IR and nNOS-IR cells were detected in whole mounts prepared from the myenteric plexus of saline or MPTP lesioned C57Bl6 mice treated with SSV or Cu^II(atsm). In areas of glial cell distortion (arrows) there was a corresponding absence of nNOS-IR. Insets are shown as merged images of GFAP-IR (red) and nNOS-IR (blue) magnified x2.5. Note strongly immunoreactive thickened glial cell process in the MPTP/SSV image. Scale bar 100 μm.

Figure 4. The expression of pro-inflammatory molecules correlates with therapeutic effects of Cu^II(atsm) treatment of MPTP lesioned mice.

The expression of pro-inflammatory (MCP-1, TNF, iNOS) and anti-inflammatory (HO-1, TGFb) genes and the metallothionen gene (MT-1) were assessed in mRNA isolated from segments of ileum prepared from saline and MPTP lesioned mice treated with SSV or Cu^II(atsm). The cycle threshold is shown relative to β-tubulin. *p < 0.05 two-tailed t-test, n = 5–6, mean ± SEM.