Elsevier

Neurobiology of Disease

Volume 36, Issue 1, October 2009, Pages 96-102
Neurobiology of Disease

Chronic rotenone exposure reproduces Parkinson's disease gastrointestinal neuropathology

https://doi.org/10.1016/j.nbd.2009.06.017Get rights and content

Abstract

Gastrointestinal disorders, particularly severe constipation and delayed gastric emptying, are core symptoms of Parkinson's disease that affect most patients. However, the neuropathological substrate and physiological basis for this dysfunction are poorly defined. To begin to explore these phenomena in laboratory models of PD, rats were treated with either vehicle or rotenone (2.0 mg/kg, i.p.; 5 days/week) for 6-weeks. Myenteric plexus α-synuclein aggregate pathology and neuron loss were assessed 3-days and 6-months after the last rotenone injection. Gastrointestinal motility was assessed at 3-days, 1-month and 6-months after the last rotenone injection. Rotenone treatment caused an acute reduction in α-synuclein-immunoreactivity, but this was followed 6 months later by a robust increase in aggregate pathology and cytoplasmic inclusions that were similar in appearance to enteric Lewy-bodies in idiopathic PD. Rotenone-treated rats also had a moderate but permanent loss of small intestine myenteric neurons and an associated modest slowing of gastrointestinal motility 6-months after treatment. Our results suggest that a circumscribed exposure to an environmental toxicant can cause the delayed appearance of parkinsonian α-synuclein pathology in the enteric nervous system and an associated functional deficit in gastrointestinal motility. The rotenone model may therefore, provide a means to investigate pathogenic mechanisms and to test new therapeutic interventions into gastrointestinal dysfunction in PD.

Introduction

Idiopathic Parkinson's disease (PD) is a multi-system, complex disorder with an uncertain etiology, that affects selected neuronal populations throughout the central and peripheral nervous systems (Bonifati, 2004, Hirsch, 1999, Schapira, 1998, Sherer, 2002a). Although PD is a prototypical movement disorder, many non-motor symptoms occur in the disease, including cognitive dysfunction, sleep disorders, psychiatric symptoms and, most commonly, gastrointestinal (GI) dysfunction (Poewe, 2008). Symptoms such as dysphagia, nausea and distension as a result of impaired gastric emptying, and bowel dysfunction, including both reduced bowel movement frequency and difficulty defecating, are among the most common non-motor symptoms of PD (Cersosimo and Benarroch, 2008, Natale, 2008, Pfeiffer, 1998, Poewe, 2008). As such, GI dysfunction is now often considered a cardinal PD symptom that can dominate the clinical picture for some patients.

Little is known about the GI dysfunction of PD. Interestingly, GI problems may precede the onset of classical motor symptoms by many years, and their occurrence in otherwise healthy people is associated with an increased risk of developing PD (Abbott et al., 2001). A pathological hallmark of PD in the brain is the presence of Lewy-bodies and Lewy-neurites, which are cytoplasmic inclusions of insoluble, aggregated proteins, including α-synuclein (Spillantini et al., 1997). Lewy-bodies and Lewy-neurites are also found in neurons of the myenteric and submucosal plexuses (Braak, 2006, Lebouvier, 2008, Wakabayashi, 1988). Lewy-body pathology in the enteric nervous system (ENS) represents an early event in the disease, as they have been observed in both clinically diagnosed cases with advanced pathology, and in non-symptomatic subjects with PD-related brain lesions limited to the lower brainstem (Braak et al., 2006). In fact, according to the hypothesis proposed by Braak, the presence of Lewy-bodies in the GI tract may represent one of the earliest manifestations of the disease. It is still unclear if there is overt enteric neuron loss in PD. Attempts at quantifying enteric neuron loss in PD have been limited. In one report, a majority of PD patients showed reduced dopamine neurons in the colon myenteric and submucosal plexuses with little difference in other neuronal subtypes (Singaram et al., 1995). Similarly, another group failed to find overt neuronal loss in the colon submucosal plexus in colonic biopsies taken from a few patients (Lebouvier et al., 2008). It is clear that detailed post-mortem studies of ENS innervation throughout the GI tract are lacking.

Few studies have explored GI dysfunction in laboratory models of PD. Systemic administration of the selective dopaminergic neurotoxin MPTP causes a loss of enteric dopamine neurons in mice and non-human primates (Anderson, 2007, Chaumette, 2009, Tian, 2008), and interestingly, has been associated with an increased number of neurons per ganglia in the MPTP-treated monkey (Chaumette et al., 2009). Functionally, in mice, MPTP causes an increase in stool frequency and colonic relaxation defects in mice which disappeared within one week after MPTP cessation (Anderson, 2007, Tian, 2008). These deficits are not consistent with the permanent slowing of GI motility that occurs in PD. Further, although inhibitory enteric dopamine neurons may be affected in PD, it is unlikely that they the primary or exclusive neuropathological target of the disease. Enteric DA neurons represent a small proportion of total neurons and their inhibitory role in GI muscle contraction suggests they do not play a role in PD-related gastroparesis or slowing of motility. That is, loss of inhibition of contraction would be expected to speed rather than slow motility — and this is what was found with MPTP treatment. Further, neuropathology is observed in non-dopaminergic neurons (Wakabayashi et al., 1990). Thus, it is likely that other, non-dopaminergic neuronal populations are primarily affected in the disease. An accurate disease model should reflect this.

GI function has also been explored in transgenic mice overexpressing wild-type α-synuclein driven by the Thy-1 promoter (Wang et al., 2008). These mice had delayed colonic transit and impaired stress-induced motility. Histopathological data from the ENS have yet to be reported from these mice, but the initial findings suggest that genetic models may also provide important information regarding GI dysfunction in PD. Together, these studies highlight a pressing need to continue to develop and characterize models that reproduce PD-related GI dysfunction.

To date, there has not been a good experimental model that reproduces both the GI pathology and dysfunction of PD. Chronic systemic exposure to rotenone recapitulates key pathological and clinical hallmarks of PD in the central nervous system and is a common in vivo and in vitro laboratory model of the disease (Betarbet, 2000, Sherer, 2002b, Sherer, 2003). Rotenone is a lipophilic compound that easily crosses biological membranes. Following systemic administration, rotenone evenly distributes throughout the body and gains access to all cells, including those within the GI tract (Greenamyre et al., 1992). It is a potent mitochondrial complex I inhibitor that hinders ATP production and promotes reactive oxygen species formation. Oxidative stress and ROS play roles in PD pathogenesis (Hald and Lotharius, 2005, Jenner, 2003, Onyango, 2008, Schapira, 1995, Zhang, 2000), and may also contribute to ENS pathology and GI symptoms (Braak et al., 2003b).

Recently, Greene et al. (2009) showed that chronic rotenone treatment (22–28 days) impaired gastric emptying in a subset of rats, and transiently decreased stool frequency and impaired longitudinal muscle contraction in response to electrical stimulation, indicative of ENS defects. No loss of ENS neurons was observed in this study and α-synuclein pathology was not examined. These initial findings suggest that rotenone administration may more accurately recapitulate parkinsonian gastroparesis and slowing of motility than does MPTP. Therefore, identifying the pathological and functional defects in the rotenone model may enhance our understanding of the nature of GI disorders in PD.

The present studies investigate the immediate and long-term effects of chronic rotenone on gastrointestinal function and PD-related ENS pathology. Here we show that chronic rotenone exposure recapitulates the primary pathological hallmark of PD in the ENS, an increase in α-synuclein-positive protein aggregates that are reminiscent of enteric Lewy-bodies described by Braak and Del Tredici (2008). Rotenone also caused a moderate loss of small intestine myenteric neurons. These pathological changes were associated with a modest slowing of GI motility.

Section snippets

Animals

Male Lewis rats (n = 23) age 3–4 months were obtained from Hilltop Laboratories (Scottdale, PA). Animals were individually housed and maintained in a temperature (22 ± 1 °C) and light controlled (12L:12D) room. Food and water were provided ad libitum. The University of Pittsburgh Institutional Animal Care and Use Committee approved all experiments described herein.

Rotenone administration

Rats were injected with either rotenone (2.0 mg/kg, 1.0 ml/kg, i.p.) dissolved in dimethyl sulfoxide (DMSO) and diluted in Miglyol 812N

Progressive increase in formic acid-resistant α-synuclein-immunoreactivity

Lewy-bodies and Lewy-neurites are pathological hallmarks of Parkinson's disease and, similar to the brain, they are found in enteric neurons in the gut. To determine whether rotenone-induced oxidative stress causes α-synuclein pathology, the small intestine myenteric plexus (duodenum, jejunum and ileum) of control and rotenone-treated rats was examined for α-synuclein-positive protein inclusions with immunohistochemical methods used to identify Lewy-bodies in human tissue (formic acid or

Discussion

Rotenone, a naturally-occurring pesticide and inhibitor of mitochondrial complex I, has been used to model PD and, with chronic administration, it recapitulates key behavioral and pathological aspects of the disease, including intracellular aggregates of α-synuclein similar to Lewy-bodies. Here, we have shown that chronic low-grade exposure to rotenone (at a dose that does not lesion nigrostriatal dopamine neurons or produce motor impairments) reproduces an essential ‘non-motor’ aspect of the

Acknowledgments

This work was funded by a grant from the Picower Foundation, the American Parkinson Disease Association (APDA) Center for Advanced Research at the University of Pittsburgh and a Postdoctoral Fellowship from the APDA (to R.E.D.).

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