Elsevier

Neurobiology of Disease

Volume 82, October 2015, Pages 185-199
Neurobiology of Disease

Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration

https://doi.org/10.1016/j.nbd.2015.06.003Get rights and content

Highlights

  • Striatal injection of fibrillized mouse α-syn triggers a neurodegenerative cascade in the rat.

  • Ipsilateral α-syn accumulations induce bilateral degeneration in the nigra.

  • α-syn accumulations result in significant disruption of ultrasonic vocalizations.

  • Pattern of α-syn accumulation suggests only retrograde transmission.

  • Rat PFF model reproduces important features of the human sporadic PD condition.

Abstract

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson's disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague–Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

Introduction

Parkinson's disease (PD) pathology is characterized by the formation of intraneuronal Lewy body (LB) and Lewy neurite (LN) inclusions which are composed primarily of misfolded, fibrillar α-synuclein (α-syn) (Goedert, 2001). α-syn is a 140-amino acid protein that adopts a α-helical structure when bound to membranes; however, it can easily fold into a β-sheet-rich structure that polymerizes into fibrils and aggregates (Stefanis, 2012). Duplication, triplication or genetic mutations in the α-syn gene (A53T, A30P, E46K, G51D, etc.) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD (Kiely et al., 2013, Polymeropoulos et al., 1997, Kruger et al., 1998, Zarranz et al., 2004). Moreover, these mutations affect α-syn amyloid fibril formation in vitro by either accelerating the formation of fibrils (Conway et al., 1998) or by making them unstable and easier to propagate than wild-type (WT) fibrils (Yonetani et al., 2009). Taken together, these data suggest that the rate of fibril formation and propagation (gradual, systematic spread) is an important factor involved in the disease process.

Mounting evidence suggests that pathological forms of α-syn self-propagate and spread progressively throughout interconnected brain regions, thereby correlating to the staging of clinical parkinsonian symptoms (Braak et al., 2001, Muller et al., 2005). This concept evolved from a hypothetical caudo-rostral progression pattern of pathological α-syn accumulations in post-mortem tissue from select cases of PD both prior to and after the onset of motor deficits (Braak et al., 2001, Braak et al., 2003, Ulusoy et al., 2013, Bancher et al., 1993). Until recently, this hypothesis was widely criticized since the transmission of α-syn had not been demonstrated. However, the discovery of LB pathology in fetal grafts from PD patients at autopsy strongly suggests that host-to-graft transmission occurs within the human brain (Chu and Kordower, 2010, Li et al., 2008); an observation that was reproduced in grafting and cell culture models (Kordower et al., 2011, Hansen et al., 2011). The mechanism whereby pathological α-syn is taken up and transported to adjacent cells is unknown; yet, based on the current evidence it is hypothesized that pathological α-syn species spread from cell-to-cell as a result of the templated misfolding and/or aggregation of nascent or properly configured α-syn, which is subsequently transferred to neighboring cells (Volpicelli-Daley et al., 2011, Freundt et al., 2012, Holmes et al., 2013). Further evidence in support of this transcellular spread of pathological α-syn comes from the initial observation that intrastriatal injection of synthetic α-syn preformed fibrils (PFF) into WT mice induces α-syn pathology that propagates throughout anatomically interconnected regions (Luk et al., 2012). Moreover, endogenous α-syn is required for pathological transmissibility, as fibril injection into α-syn knockout mice produces no LB/LN pathology. Several other groups have also demonstrated in mice and non-human primates that pathological α-syn can be transferred to the central nervous system (CNS) and induce a similar pattern of PD-like α-syn pathology (Freundt et al., 2012, Recasens et al., 2014, Masuda-Suzukake et al., 2013, Pan-Montojo et al., 2012).

Through observations from clinical and post-mortem studies and numerous in vitro and in vivo investigations, significant strides have been made toward understanding both the etiology and the pathogenesis of PD. Despite the advancement of the field, major gaps still exist regarding our understanding of its molecular and cellular underpinnings. As a consequence, researchers rely heavily on experimental models to gain greater insight into the cause and pathogenesis of PD. While genetic studies have lead to the development of multiple transgenic PD models (Antony et al., 2011), none exhibit the typical degeneration of substantia nigra (SN) dopaminergic neurons that is inherent to the disease process. In fact, a majority of traditional models do not recapitulate many of the characteristic features of the human condition. For example, the commonly utilized toxin models (6-OHDA, MPTP, etc.) are excellent for examining symptomatic therapies or studying the end stages of PD; however, these models exhibit acute neuronal loss and lack LB/LN pathology. To better understand the role α-syn plays in PD, viral vector-mediated overexpression of WT or mutant human α-syn has been targeted to the nigrostriatal system of naïve mice or rats. While this form of α-syn overexpression results in aggregation and nigral degeneration, these events occur at an accelerated pace (1–2 months) and neuropathology is limited to the nigrostriatal circuitry (Koprich et al., 2010, Gombash et al., 2013) and therefore does not model the widespread pathological α-syn accumulation observed in the brains of PD subjects. More recently, viral vectors were used to express human α-syn in the rat vagus nerve, resulting in more widespread caudo-rostral pathology, but not neurodegeneration (Ulusoy et al., 2013). However, the α-syn PFF mouse model (Luk et al., 2012) offers a novel platform in which the time-dependent accumulation and propagation of widespread LB-like α-syn pathology and subsequent death of nigrostriatal dopamine neurons more closely model the human condition.

Using the rat as a model system offers distinct advantages over mice. Rats have more complex motor behaviors than mice and exhibit fine motor coordination, behaviors that have been extensively characterized (Whishaw et al., 2001). Additionally, rats are more similar to humans in their genetics and pharmacokinetics than mice (Gibbs et al., 2004, Lin, 1995), suggesting that results of therapeutics tested in rats would provide greater predictive validity than testing in mice. The greater synaptic complexity observed in the rat brain indicates that neural plasticity may be better modeled in the rat rather than mice (Whishaw et al., 2001). On a practical level, the larger brain and body size of the rat allows researchers to collect more tissue and fluid samples, enables neurosurgical interventions and in vivo electrophysiology, facilitates intrathecal drug delivery and can allow for neuroimaging procedures that prove more difficult in mice. With these advantages in mind, the aim of the current study was to determine whether exogenous PFF formed by recombinant WT full length mouse α-syn injected into the striatum of naive rats result in the widespread α-syn pathology and nigrostriatal degeneration previously observed following PFF injections into WT mice (Luk et al., 2012). Further, we evaluated the effects of intrastriatal α-syn PFF injections on ultrasonic vocalizations (USVs), which we previously found to be affected in response to viral vector induced nigrostriatal α-syn overexpression (Gombash et al., 2013).

Section snippets

Animals

Adult male Sprague–Dawley rats (200–225 g; Harlan Laboratory, Indianapolis, IN) were utilized in all experiments. Studies were conducted at Michigan State University (MSU). Rats at MSU were housed in the Van Andel Research Institute vivarium. The animal facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care and complied with all Federal animal care and use guidelines. The Institutional Animal Care and Use Committee approved all protocols.

Experiment 1

Unilateral, intrastriatal injection of synthetic mouse α-syn PFF results in widespread endogenous α-syn aggregation throughout the rat brain

To determine whether PFF assembled from recombinant mouse α-syn initiate the pathological conversion of endogenous α-syn when injected into rat, we administered a unilateral injection of either α-syn PFFs or recombinant α-syn solution into the left dorsal striatum at one or two sites (Fig. 1). At the site of the striatal injection at 30 d pi hyper-phosphorylated α-syn (detected by α-syn phosphorylated at serine 129 (pSyn) immunohistochemistry) appeared as accumulations in neuritic processes (

Discussion

We show here the successful establishment and characterization of a rat α-synucleinopathy model that recapitulates many features of sporadic PD including progressive spread and bilateral death of nigral neurons across a protracted time course. This model is based on direct injection of the PFFs form of α-syn that triggers the pathological conversion of endogenous α-syn, which is associated with progressive PD-like neurodegeneration. Our time-course study demonstrates that at early time-points

Relevant conflicts of interest/financial disclosures

All funding sources provided unrestricted support and had no role in the oversight or review of the research data or reporting.

Funding

This work was supported by the Saint Mary's Foundation and the Morris K. Udall Centers of Excellence for Parkinson's Disease Research at Michigan State University (NS 058830) and the University of Pennsylvania (NS 053488).

Acknowledgments

The authors acknowledge Dr. Michelle Ciucci for her expert advice and statistical help with the USV analyses.

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