Elsevier

Neurobiology of Disease

Volume 47, Issue 2, August 2012, Pages 258-267
Neurobiology of Disease

Alpha-synuclein overexpressing transgenic mice show internal organ pathology and autonomic deficits

https://doi.org/10.1016/j.nbd.2012.04.009Get rights and content

Abstract

While studying transgenic mice that overexpress human wildtype alpha-synuclein (Thy1-ASO, ASO) for typical brain alpha-synucleinopathy and central nervous system neuropathology, we observed progressive functional changes in the gastrointestinal and other peripheral organs. A more systematic study revealed that the gastrointestinal tract in ASO mice showed severe distension and blockage of the large intestine by 9–12 months of age. Functional assessments demonstrated a reduction in fecal water content and fecal pellet output, and increased whole gut transit time, in ASO mice compared to wildtype littermates, indicative of constipation, a symptom commonly reported by Parkinson's disease (PD) patients. Food intake was increased and body weight was decreased in 12 month old ASO mice, suggestive of metabolic abnormalities. Post-mortem histological analyses showed that human alpha-synuclein protein was robustly expressed in axonal fibers and in occasional cell bodies of the enteric nervous system, and in the heart of ASO mice. Accumulation of proteinase-K insoluble alpha-synuclein, reminiscent of neurodegenerative processes in PD was also observed. The functional and pathological changes we document here in ASO mice could relate to the autonomic deficits also seen in idiopathic and alpha-synuclein-mediated genetic forms of PD. These experimental data provide a foundation for therapeutic modeling of autonomic changes in PD and related alpha-synucleinopathies.

Highlights

► Thy1-α-synuclein (ASO) transgenic mice exhibit CNS and PNS deficits. ► ASO mice show progressive gastrointestinal functional deficits, indicative of constipation. ► Human α-synuclein is overexpressed in neurons in gastrointestinal and heart tissue. ► PNS pathology includes axonal swellings and accumulation of insoluble α-synuclein.

Introduction

The non-motor symptomatology of Parkinson's disease (PD) includes autonomic dysfunction, hyposmia, and sleep abnormalities (Chaudhuri et al., 2006). Such symptoms commonly experienced by PD patients are often detected before clinical motor symptoms, progressively become very disabling and significantly impact the quality of life for patients (Savica et al., 2010). Dysfunction of gastrointestinal and cardiovascular systems are significant autonomic features of PD, and post-mortem studies have identified PD neuropathology throughout the autonomic nervous system including the gastrointestinal tract, heart and sympathetic ganglia (Beach et al., 2010, Goldstein, 2010).

PD has multiple etiologies, including mitochondrial dysfunction and oxidative stress, and genes involved in such pathways have been identified (e.g. DJ-1, PINK1) (Cookson and Bandmann, 2010). Toxin-based and genetic approaches to induce mitochondrial dysfunction and oxidative stress in animals have been widely used to model central nervous system (CNS) dysfunction and the selective loss of midbrain dopamine neurons (Cannon and Greenamyre, 2010, Magen and Chesselet, 2010). These models have also indicated involvement of the peripheral nervous system (PNS). MPTP, a complex I inhibitor selective for the dopaminergic system, alters gastrointestinal dopaminergic transmission and related function, and reduces dopamine neurons in the enteric nervous system (Anderson et al., 2007, Chaumette et al., 2009, Natale et al., 2010, Tian et al., 2008). Systemic administration of rotenone, a general mitochondrial complex I inhibitor, in rats, induces clear gastrointestinal dysfunction and neuropathology in the enteric nervous system, similar to PD (Drolet et al., 2009, Greene et al., 2009). These toxin-based models demonstrate that mechanisms involved in CNS dysfunction in PD can also elicit functional deficits and pathology in the PNS. While such models depend on external agents that induce, for example, oxidative stress, a major genetic and pathologic component of PD is associated with the synaptic protein, alpha-synuclein. Mutations in, or multiplication of SNCA leads to early onset PD and diffuse Lewy body disease, and polymorphisms in the regulatory elements of SNCA can predispose to PD (Cookson and Bandmann, 2010). In both sporadic and familial PD, alpha-synuclein is a component of proteinacious Lewy body inclusions and neurites found throughout the CNS and also in the PNS. Alpha-synuclein is a highly expressed presynaptic protein, involved in SNARE protein assembly (Burre et al., 2010), and with an important role in neurotransmitter release (Nemani et al., 2010). Overexpression of either wildtype or mutant alpha-synuclein, or knockdown of alpha-synuclein together with beta and gamma synucleins, causes neuronal dysfunction and degeneration (Burre et al., 2010, Chung et al., 2009, Greten-Harrison et al., 2010, Nemani et al., 2010, Ulusoy et al., 2010). A variety of transgenic mouse models of alpha-synucleinopathy exist and although CNS deficits have been the focus of studies in such models (Chesselet, 2008, Chesselet et al., 2008, Dawson et al., 2010, Fleming et al., 2004, Fleming et al., 2008, McLean et al., 2012, Rockenstein et al., 2002, Song et al., 2004, Watson et al., 2009), more recently, functional deficits in cardiac and gastrointestinal systems have also been described (Fleming et al., 2007, Fleming et al., 2009, Kuo et al., 2010, Wang et al., 2008).

We first noticed evidence for autonomic dysfunction in transgenic mice that overexpress human wildtype alpha-synuclein on the Thy1 promoter (ASO, Thy1-ASO) by their extreme sensitivity to the alpha2-adrenergic agonist, xylazine, used as a routine anesthetic agent (Hallett et al., unpublished data). We also observed at post-mortem examination, clear gut and bladder distension in older (> 12 months) ASO mice. Based on these observations we investigated gastrointestinal functional deficits, and neuropathology of the gastrointestinal system in ASO mice.

Section snippets

Animals

All animal procedures were performed in accordance with the guidelines of the National Institute of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) at McLean Hospital, Harvard Medical School. Animals were housed according to standard conditions, in a dark/light cycle of 12 h, with ad libetum access to food and water. Transgenic mice overexpressing human wildtype alpha-synuclein under the Thy1 promoter have been previously described (Rockenstein et al., 2002).

Assessment of CNS functional deficits and pathology in ASO mice

To verify involvement of the CNS in ASO mice, behavioral analyses of sensorimotor function, using the challenging beam traversal test, and post-mortem analysis to examine proteinase-K resistant alpha-synuclein immunostaining in the brain, was performed in a cohort of 12–15 month old ASO and WT littermate mice, as previously described (Fernagut et al., 2007, Fleming et al., 2004). In the beam traversal test (Figs. 1A, B), ASO mice had a significantly increased number of footslips off the beam,

Discussion

In summary, we show that the Thy1-ASO transgenic model of alpha-synuclein overexpression displays pathological changes in peripheral organ systems, including the gastrointestinal and cardiac systems, and functional autonomic deficits, similar to the pathology and debilitating clinical symptoms observed in patients with genetic and sporadic forms of PD. Specifically, the severely distended large intestine, decreased fecal water content and increased whole gut transit time in ASO mice are

Conclusion

These results emphasize that deficits and pathology in peripheral neuronal systems caused by the overexpression of human wildtype alpha-synuclein are similar to those that occur both in age-related sporadic PD and alpha-synuclein-mediated genetic forms of the disease. In addition, our work demonstrates that overexpression of wildtype alpha-synuclein on the Thy1 promoter is sufficient to cause autonomic deficits and systemic pathology outside of the CNS. ASO mice offer a useful platform for the

Acknowledgments

This work was conducted at McLean Hospital and was supported by funds to O.I. from the National Institutes of Health/National Institute of Neurological Disorders and Stroke P50 (Grant NS39793), The Consolidated Anti-Aging Foundation, The Poul Hansen Family and the Harold and Ronna Cooper Family. We thank Dr. Oliver Cooper for experimental advice, and Tana Brown, Sarah Izen and Yvette Leung for technical assistance.

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