Kathy Steece-Collier

The role of α-synuclein in Parkinson's disease: insights from animal models

The abnormal accumulations of fibrillar α-synuclein in Lewy bodies and the mutations in the gene for α-synuclein in familial forms of Parkinsons disease have led to the belief that this protein has a central role in a group of neurodegenerative diseases known as the synucleinopathies. Our understanding of the biology of α-synuclein has increased significantly since its discovery in 1997, and recently developed animal models of the synucleinopathies have contributed to this understanding. The information gleaned from animal models has the potential to provide a framework for continuing the development of rational therapeutic strategies.

Pathophysiology of L-dopa-induced motor and non-motor complications in Parkinson's disease.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa (L-dopa) therapy for Parkinsons disease (PD). L-dopa-induced dyskinesia (LID) are ultimately experienced by the vast majority of patients. In addition, psychiatric conditions often manifested as compulsive behaviours, are emerging as a serious problem in the management of L-dopa therapy. The present review attempts to provide an overview of our current understanding of dyskinesia and other L-dopa-induced dysfunctions, a field that dramatically evolved in the past twenty years. In view of the extensive literature on LID, there appeared a critical need to re-frame the concepts, to highlight the most suitable models, to review the central nervous system (CNS) circuitry that may be involved, and to propose a pathophysiological framework was timely and necessary. An updated review to clarify our understanding of LID and other L-dopa-related side effects was therefore timely and necessary. This review should help in the development of novel therapeutic strategies aimed at preventing the generation of dyskinetic symptoms.

Failure of proteasome inhibitor administration to provide a model of Parkinson's disease in rats and monkeys

McNaught and colleagues 1 reported recently that systemic administration of proteasome inhibitors PSI (Z‐Ileu‐Glu(OtBu)‐Ala‐Leu‐CHO) or epoxomicin recapitulated many of the degenerative changes seen in Parkinsons disease including loss of striatal dopamine and cell loss in the substantia nigra, locus ceruleus, dorsal motor nucleus of the X cranial nerve, and nucleus basalis of Meynert. Intracytoplasmic inclusions resembling Lewy bodies were also described. All experiments administering PSI to rats using identical procedures and multiple attempts failed to induce any of the previously described changes. Furthermore, administration of PSI or epoxomicin to monkeys in an attempt to extend the model to a primate species failed. Currently, systemic proteasome inhibition is not a reliable model for Parkinsons disease. Ann Neurol 2006;60:264–268

Chronic levodopa impairs morphological development of grafted embryonic dopamine neurons

Degeneration and plasticity of dopamine (DA) neurons may be influenced by their own neurotransmitter and/or metabolic by-products. Substances with known neurotoxic properties, such as hydrogen peroxide and 6-hydroxydopamine, are produced during oxidation of DA. Additionally, DA can directly regulate neurite outgrowth in both invertebrate and vertebrate species. We have begun to investigate the influence of increased local transmitter concentrations on the morphological plasticity of neurons by examining the effect of chronic levodopa, a drug that increases DA synthesis, on grafted embryonic nigral DA neurons in a rat model of experimental parkinsonism and in monolayer cell cultures. Results from our in vivo investigation show that although chronic levodopa does not significantly affect the number of surviving grafted cells, morphological development of these embryonic DA neurons appears impaired. Levodopa administered chronically to fetal DA neurons in culture results in a decreased number of surviving neurons as well as a reduction in neurite outgrowth with increasing concentration of levodopa ranging from 10(-8) to 10(-4) M. This information provides further evidence to support the hypothesis that excess DA or its metabolites can influence the survival and growth of DA neurons. These results may be important in the design of pharmacotherapy for Parkinsons disease and the combination of drug and neural grafting therapies in this disorder.

TRANSFER OF HOST-DERIVED ALPHA SYNUCLEIN TO GRAFTED DOPAMINERGIC NEURONS IN RAT

Multiple laboratories have recently demonstrated that long-term dopaminergic transplants form Lewy bodies in patients with Parkinsons disease. Debate has arisen as to whether these Lewy bodies form from the transfer of α synuclein from the host to the graft or whether they form from intrinsic responses of the graft from being placed into what was, or became, an inflammatory focus. To test whether the former hypothesis was possible, we grafted fetal rat ventral mesencephalon into the dopamine depleted striatum of rats that had previously received 6-hydroxydopamine lesions. One month after the transplant, rats received viral over expression of human α synuclein (AAV2/6-α synuclein) or green fluorescent protein (AAV2/6-GFP) into the striatum rostral to the grafts. Care was taken to make sure that the AAV injections were sufficiently distal to the graft so no cells would be directly transfected. All rats were sacrificed five weeks after the virus injections. Double label immunohistochemistry combined with confocal microscopy revealed that a small number of grafted tyrosine hydroxylase (TH) neurons (5.7% ± 1.5% (mean ± SEM) of grafted dopamine cells) expressed host derived α synuclein but none of the grafted cells expressed host-derived GFP. The α synuclein in a few of these cells was misfolded and failed to be digested with proteinase K. These data indicate that it is possible for host derived α synuclein to transfer to grafted neurons supporting the concept that this is one possible mechanism by which grafted dopamine neurons form Lewy bodies in Parkinsons disease patients.

Etiology of Parkinson's disease: Genetics and environment revisited

Idiopathic Parkinsons disease (PD) is the second most common neurodegenerative disorder and affects more than 1 million Americans over the age of 55. The disease selectively affects dopaminergic neurons of the substantia nigra pars compacta, culminating in their demise. After ≈50% of the dopamine neurons and 75–80% of striatal dopamine is lost, patients start to exhibit the classical symptoms of PD including bradykinesia, postural reflex impairment, resting tremor, and rigidity (1, 2). Although there are several treatments that are effective for a number of years, their usefulness wanes over time and is accompanied by unacceptable side effects. New therapies are clearly needed for this disorder. Despite many years of focused research, the causes of this disease remain to be elucidated. Understanding the cause of PD is critical as that knowledge could lead to directed research that will develop new and potent therapies. The relative contributions of genetic versus environmental factors regarding the cause of PD have been hotly debated. In an attempt to define a cause for this disease, early epidemiological studies examining twins suggested an absence of genetic factors (3). However, these studies were not definitive and could never account for differences in disease progression between twin pairs that could account for discordance in diagnosis. The discovery that the protoxin n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in both humans and nonhumans further strengthened the hypothesis that PD had an environmental etiology. Other environmental toxins have been shown to induce a parkinsonian state as well, supporting this view (4). However, the recent discovery of inherited forms of PD shifted the emphasis back to genetic factors. Among the different genetic forms of PD, mutations in the gene encoding for α-synuclein have received the most attention. Mutations in this gene cause rare forms of PD. Furthermore, α-synuclein is also present in the Lewy bodies that are the pathoneumonic feature of PD. The outstanding and comprehensive paper by Dauer et al. (5) published in this issue of PNAS examines the nigrostriatal system of α-synuclein knockout mice in response to dopaminergic neurotoxins and re-emphasizes the contributory roles of both genetics and environment in the manifestation of experimental PD. In this study, the authors conclusively demonstrate that targeted disruption of the α-synuclein gene confers specific resistance of dopaminergic neurons to the toxic effects of MPP+ or MPTP in vitro and in vivo. This resistance to degeneration is not mediated through the dopamine transporter but rather through the inability of the toxin to inhibit complex 1 activity in the absence of α-synuclein. Although not the first to do so (e.g., ref. 6), this landmark study provides important evidence that α-synuclein may be critical to dopamine neuronal viability.

Focal not widespread grafts induce novel dyskinetic behavior in parkinsonian rats

Dyskinesias are a common consequence of dopaminergic therapy in patients with Parkinsons disease. Little is known about the influence of cellular replacement strategies upon drug-induced dyskinesias. In the current study, we employed parkinsonian rats to test whether the distribution of dopamine neuron grafts could differentially alter striatal circuitry and levodopa-induced dyskinesias. Specifically, we compared behavioral and neurochemical consequences of dopamine reinnervation restricted to a focal region of the striatum to innervation encompassing the majority of the striatum by distributing the same number of cells into single locus or multiple locations. Both the single-site and widespread grafts reduced pregraft dyskinesias and normalized FosB/DeltaFosB in the dorsal two-thirds of the lateral striatum. However, single-site DA graft recipients developed a robust, novel forelimb-facial stereotypy and upregulated FosB/DeltaFosB expression in the ventrolateral striatum, an area associated with movements of tongue and forelimbs. The onset of forelimb-facial stereotypy correlated with measures of increased graft function.

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

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinsons 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.

Chronic levodopa impairs the recovery of dopamine agonist-induced rotational behavior following neural grafting

SummaryThe effect of chronic levodopa treatment on the function of embryonic mesencephalic tissue grafts was assessed in rats by monitoring rotational behavior elicited by dopamine (DA) agonists before and after neural grafting. Rats were given unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway and baseline measures of rotational behavior induced by D1 receptor stimulation, D2 receptor stimulation, or amphetamine were determined. Subsequently, DA grafts were implanted into the lesioned striatum and chronic regimens of either saline or levodopa began one day after neural grafting and were continued for 7 weeks. Rotational behavior elicited by the D1 agonist, SKF 38393, was completely attenuated throughout the six week-period following the commencement of levodopa treatment, regardless of the absence or presence of a DA graft. Conversely, rotational behavior elicited by the D2 agonist, quinpirole, was significantly elevated in ungrafted animals receiving chronic levodopa. Grafted animals receiving chronic levodopa did not show a significant reduction in rotational behavior, whereas grafted animals receiving chronic saline showed a significant 67% reduction in quinpirole-induced rotational behavior. Amphetamine-induced rotational behavior was reduced in both levodopa and saline treated grafted animals, however grafted animals receiving chronic levodopa treatment showed a reduction of rotational behavior that was uncharacteristic and less compensatory than that observed in grafted animals receiving chronic saline treatment. Morphology of grafts indicate that there were areas of impaired neurite outgrowth of TH-positive fibers in animals treated with levodopa. The results of the present study suggest that the impaired recovery in quinpirole and amphetamine-induced rotational behavior in grafted animals receiving chronic levodopa treatment may be related to (1) impaired graft function, (2) an alteration in pre- and postsynaptic mechanisms in the host DAergic system, or (3) a combined effect of (1) and (2).

Embryonic mesencephalic grafts increase levodopa-induced forelimb hyperkinesia in parkinsonian rats.

Recent observations from clinical trials of neural grafting for Parkinsons disease (PD) have demonstrated that grafted dopamine neurons can worsen dyskinesias in some graft recipients. This deleterious side effect reveals a new challenge for neural transplantation, that of elucidating mechanisms underlying these postgraft dyskinesias. One problem facing this challenge is the availability of a cost‐effective and reliable animal model in which to pursue initial investigations. In the current study, we investigated the interaction of an embryonic ventral mesencephalic (VM) dopamine (DA) neuron graft on levodopa (LD)‐induced dyskinetic movements in unilaterally 6‐hydroxydopamine‐lesioned rats. Rats were administered LD (levodopa‐carbidopa, 50:5 mg/kg) twice daily for 6 weeks after either a sham graft or VM DA graft. Although a single solid graft of embryonic DA neurons can prevent progression of some lesioned‐induced behavioral abnormalities such as LD‐induced rotation and dystonia, it significantly increases hyperkinetic movements of the contralateral forelimb. This differential effect of grafted neurons on abnormal behavioral profiles is reminiscent of that reported in grafted patients with PD. Data from this study illustrate important similarities between this model of parkinsonism and PD in human patients that make it suitable for initial preclinical investigations into possible mechanisms underlying postgraft aggravation of dyskinetic movements.