Alpha-synuclein overexpressing transgenic mice show internal organ pathology and autonomic deficits
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.
References (61)
Loss of enteric dopaminergic neurons and associated changes in colon motility in an MPTP mouse model of Parkinson's disease
Exp. Neurol.
(2007)Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology
Neurosci. Lett.
(2006)- et al.
Neurotoxic in vivo models of Parkinson's disease recent advances
Prog. Brain Res.
(2010) Non-motor symptoms of Parkinson's disease: diagnosis and management
Lancet Neurol.
(2006)In vivo alpha-synuclein overexpression in rodents: a useful model of Parkinson's disease?
Exp. Neurol.
(2008)Strengths and limitations of genetic mouse models of Parkinson's disease
Parkinsonism Relat. Disord.
(2008)Genetic animal models of Parkinson's disease
Neuron
(2010)Chronic rotenone exposure reproduces Parkinson's disease gastrointestinal neuropathology
Neurobiol. Dis.
(2009)Delayed gastric emptying and enteric nervous system dysfunction in the rotenone model of Parkinson's disease
Exp. Neurol.
(2009)Synuclein deposition and non-motor symptoms in Parkinson disease
J. Neurol. Sci.
(2011)
Loss of transmitter-associated enzyme staining following axotomy does not indicate death of brainstem cholinergic neurons
Brain Res.
Genetic mouse models of Parkinson's disease The state of the art
Prog. Brain Res.
Transcript expression levels of full-length alpha-synuclein and its three alternatively spliced variants in Parkinson's disease brain regions and in a transgenic mouse model of alpha-synuclein overexpression
Mol. Cell. Neurosci.
Pathology of the sympathetic nervous system corresponding to the decreased cardiac uptake in 123I-metaiodobenzylguanidine (MIBG) scintigraphy in a patient with Parkinson disease
J. Neurol. Sci.
MPTP-induced parkinsonism extends to a subclass of TH-positive neurons in the gut
Brain Res.
Increased expression of alpha-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis
Neuron
Gastrointestinal dysfunction in Parkinson's disease
Parkinsonism Relat. Disord.
Alpha-synuclein immunopositive aggregates in the myenteric plexus of the aging Fischer 344 rat
Exp. Neurol.
Enhanced substantia nigra mitochondrial pathology in human alpha-synuclein transgenic mice after treatment with MPTP
Exp. Neurol.
Alteration of dopaminergic markers in gastrointestinal tract of different rodent models of Parkinson's disease
Neuroscience
Viral vector-mediated overexpression of alpha-synuclein as a progressive model of Parkinson's disease
Prog. Brain Res.
Alterations in corticostriatal synaptic plasticity in mice overexpressing human alpha-synuclein
Neuroscience
Cardiovascular autonomic dysfunction in Parkinson's disease
J. Neurol. Sci.
Frequency of bowel movements and the future risk of Parkinson's disease
Neurology
Environmental, life-style, and physical precursors of clinical Parkinson's disease: recent findings from the Honolulu-Asia Aging Study
J. Neurol.
Multi-organ distribution of phosphorylated alpha-synuclein histopathology in subjects with Lewy body disorders
Acta Neuropathol.
Alpha-synuclein immunopositive Parkinson's disease-related inclusion bodies in lower brain stem nuclei
Acta Neuropathol.
Impaired cardiac uptake of meta-[123I]iodobenzylguanidine in Parkinson's disease with autonomic failure
Acta Neurol. Scand.
Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro
Science
Neurochemical plasticity in the enteric nervous system of a primate animal model of experimental Parkinsonism
Neurogastroenterol. Motil.
Cited by (84)
Neurology and the gut: Autonomic neuropathy and dysautonomia
2023, Handbook of Gastrointestinal Motility and Disorders of Gut-Brain Interactions, Second EditionRole of rodent models in advancing precision medicine for Parkinson's disease
2023, Handbook of Clinical NeurologyPreclinical models of disease and multimorbidity with focus upon cardiovascular disease and dementia
2020, Mechanisms of Ageing and DevelopmentMice deficient in GM1 manifest both motor and non-motor symptoms of Parkinson's disease; successful treatment with synthetic GM1 ganglioside
2020, Experimental NeurologyCitation Excerpt :For Immunohistochemical (IHC) studies, animals were subjected to transcardial perfusion with BSS beginning 2 h after the final E. coli-GM1 injection; this was followed by 4% paraformaldehyde in phosphate buffered saline (PBS), and tissues (brain, colon, heart) were stored in the same fixing solution at 4 °C pending IHC. Coronal cryosections (25 μm) of brains were stained for tyrosine hydroxylase (TH) and aSyn as previously described (Wu et al. 2012; Hadaczek et al. 2015), employing antibodies shown useful for those substances (Hallett et al. 2012). For phosphorylated RET (pRET) we used the method of Hadaczek et al. (2015).