Viral vectors, animal models and new therapies for Parkinson's disease

The involvement of alpha-synuclein in familial forms of Parkinson's disease suggests a potential causative role in the pathogenesis. We have explored the possibility of generating animal models of Parkinson's disease by overexpressing alpha-synuclein in the nigrostriatal pathway using viral vectors. Both lentiviral and adeno-associated vectors efficiently transduce dopaminergic neurons in the substantia nigra, and transgenic expression of alpha-synuclein leads to the progressive loss of neurons positive for dopaminergic markers, with the formation of intraneuronal alpha-synuclein aggregates. With a high tropism for nigral dopaminergic neurons, adeno-associated vectors allow for the monitoring of dopaminergic function using spontaneous and drug-induced behaviour. We propose that virus-based rodent alpha-synuclein models provide a valuable approach for the preclinical testing of therapeutics.     

New animal models of Parkinson's disease

Background: Parkinson's disease is a progressive neurodegenerative disorder mainly characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta and the presence, in the affected brain regions, of protein inclusions named Lewy Bodies. Despite the fact that numerous mutations causing hereditary forms of Parkinson's disease have been identified in the last decade, current transgenic animal models do not adequately reproduce cardinal features of the human disease. Altogether, the animal models derived of human mutations indicate that the nigrostriatal degenerative process results from the combination of several mechanisms that implicate mitochondrial dysfunction, oxidative damage, and protein degradation impairment.Methods and Results: We performed a literature search between 2008 and 2010.Discussion: The absence of adequate in vivo experimental models of Parkinson's disease has severe repercussions for therapeutic intervention success for this incurable neurodegenerative disorder. The present nonexhaustive review looks at invertebrate and mammalian models of Parkinson's disease generated in the last three years.     

Recombinant adeno-associated viral vectors bring gene therapy for Parkinson's disease closer to reality

The recombinant adeno-associated viral (rAAV) vector is a powerful tool for delivering therapeutic genes into mammalian brains. In rodents and non-human primates, a substantial number of striatal neurons can be transduced with high titer rAAV vectors by simple stereotaxic injection. Efficient and long-term expression of genes for dopamine (DA)-synthesizing enzymes in the striatum restored local DA production and achieved behavioral recovery in animal models of Parkinson's disease (PD). Moreover, sustained expression of a glial cell line-derived neurotrophic factor gene in the striatum rescued nigral neurons and led to functional recovery in a rat model of PD, even when treatment was delayed until after the onset of progressive degeneration. These results suggest that gene therapy using rAAV vectors may become a novel and feasible treatment for PD.     

Evaluation of animal models of Parkinson's disease for neuroprotective strategies

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic nigral neurons and striatal dopamine. Despite the advances of modern therapy to treat the symptoms of PD, most of the patients will eventually experience debilitating disability. The need for neuroprotective strategies that will slow or stop the progression of the disease is clear. The progress in the understanding of the cause and pathogenesis of PD is providing clues for the development of disease-modifying strategies. In that regard, animal models of PD and non-human primate models in particular, are essential for the preclinical evaluation and testing of candidate therapies. However, the diversity of models and different outcome measures used by investigators make it challenging to compare results between neuroprotective agents. In this review we will discuss methods for the selection, development and assessment of animal models of PD, the role of non-human primates and the concept of "multiple models/multiple endpoints" to predict the success in the clinic of neuroprotective strategies.     

A tale on animal models of Parkinson's disease

Parkinson's disease is a neurodegenerative disorder whose cardinal manifestations are due primarily to a profound deficit in brain dopamine. Since the 1980s, several therapeutic strategies have been discovered to treat the symptoms of this neurological disorder, but as of yet, none halts or retards the neurodegenerative process. In an attempt to shed light on the neurobiology of Parkinson's disease, a number of experimental models have been developed, especially during the last 25 years. They come essentially in 3 flavors: pharmacological (eg, reserpine), toxic (eg, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine), and genetic (eg, transgenic synuclein mice). These models can also be recast as etiologic, pathogenic, and symptomatic/pathophysiologic, as each may contribute to our understanding of the cause, the mechanisms, and the treatment of Parkinson's disease. In this review, we will discuss the question of Parkinson's disease models, starting from the period when this journal was born to today. During this journey of 25 years, we will discuss both the significant contributions of the Parkinson's disease models and hurdles that remain to be overcome to one day cure this neurological disease. © 2011 Movement Disorder Society     

LRRK2 Parkinson's disease: from animal models to cellular mechanisms

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) play a major role in the development of Parkinson's disease. The most frequently defined mutations of LRRK2 are located in the central catalytic region of the LRRK2 protein, suggesting that dysregulations of its enzymatic activities contribute to PD pathogenesis. Herein, we review recent progress in research concerning how LRRK2 mutations affect cellular pathways and lead to neuronal degeneration. We also summarize recent evidence revealing the endogenous function of LRRK2 protein within cells. These concepts can be used to further understand disease pathophysiology and serve as a platform to develop therapeutic strategies for the treatment of Parkinson's disease.     

How to judge animal models of Parkinson's disease in terms of neuroprotection

Ideally, animal models of Parkinson's should reproduce the clinical manifestation of the disease, a loss of some but not all dopaminergic neurons, a loss of some non dopaminergic neurons and alpha-synuclein positive inclusions resembling Lewy bodies. There are at least three ways to develop animal models of PD. The first two are based on the etiology of the disease and consist in 1) reproducing in animals the mutations seen in inherited forms of PD; 2) intoxicating animals with putative environmental toxins causing PD. The last method currently used, which is not exclusive of the first two, is to try to reproduce the molecular or biochemical changes seen post-mortem in the brain of patients with PD. In this review we discuss the advantages and the drawbacks in term of neuroprotection of the currently used models.     

Genetically engineered animal models of Parkinson's disease: From worm to rodent

Parkinson's disease (PD) is a progressive neurological disorder characterised by aberrant accumulation of insoluble proteins, including alpha‐synuclein, and a loss of dopaminergic neurons in the substantia nigra. The extended neurodegeneration leads to a drop of striatal dopamine levels responsible for disabling motor and non‐motor impairments. Although the causes of the disease remain unclear, it is well accepted among the scientific community that the disorder may also have a genetic component. For that reason, the number of genetically engineered animal models has greatly increased over the past two decades, ranging from invertebrates to more complex organisms such as mice and rats. This trend is growing as new genetic variants associated with the disease are discovered. The EU Joint Programme – Neurodegenerative Disease Research (JPND) has promoted the creation of an online database aiming at summarising the different features of experimental models of Parkinson's disease. This review discusses available genetic models of PD and the extent to which they adequately mirror the human pathology and reflects on future development and uses of genetically engineered experimental models for the study of PD.     

Gene therapy for Parkinson's disease: studies in animal models]

Recent developments in viral vectors capable of providing high levels of long-term transgene expression in the brain have led to the pursuit of two strategies in gene therapy for the treatment of Parkinson's disease (PD). One is the local production of dopamine in the striatum achieved by inducing the expression of dopamine-synthesizing enzymes. Three enzymes are necessary for efficient dopamine synthesis: tyrosine hydroxylase (TH) converts tyrosine to L-DOPA, aromatic L-amino acid decarboxylase (AADC) then converts L-DOPA to dopamine, and guanosine triphosphate cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of TH co-factor tetrahydrobiopterine. We have previously demonstrated that transduction with separate adeno-associated virus (AAV) vectors expressing TH, AADC, and GCH is effective in reducing motor abnormalities in 6-hydroxydopamine-lesioned rats and in MPTP-treated monkeys. Behavioral recovery persisted for at least 18 months after intrastriatal injection in parkinsonian rats. In MPTP monkeys, the amelioration of motor abnormalities was remarkable on the contralateral side, accompanied by robust transgene expression and elevated dopamine synthesis in the AAV-injected putamen. The second strategy entails the expression of neurotrophic factors or brain vesicular monoamine transporter in the striatum or the substantia nigra to slow the degeneration of dopamine neurons. Gene therapy using viral vectors offers a promising approach in the treatment of PD patients.     

Molecular mechanisms of levodopa action in animal models of Parkinson's disease

Parkinson's disease is a progressive neurodegenerative movement disorder, affecting mainly the elderly. One of the most important hallmarks of Parkinson's disease is the loss of neuronal cell bodies containing neuromelanin in the substantia nigra zona compacta, and subsequently, loss of dopamine terminals in basal ganglia nuclei of the brain. The discovery by Hornykiewicz and co-workers that levodopa could successfully treat Parkinson's disease in humans was one of the most important events of medicine in the 20th century. Since loss of nigrostriatal dopaminergic function is the basic underlying pathophysiology of this disease, drugs that enhance dopaminergic function in the striatum, including the exogenous precursor levodopa, remain the most effective symptomatic agents in the treatment of Parkinson's disease. However, there are some areas of controversy about levodopa-evoked motor complications (dyskinesias, on-off phenomena) as well as neuroprotective or neurotoxic activity of this drug, etc. In this article the authors try to clarify the molecular mechanisms involved in levodopa action, such as volume transmission - a crucial process for successful levodopa therapy, evidence that serotoninergic neurons may accumulate levodopa and convert it into dopamine as well as some aspects of neuroprotective action of levodopa.     

Deleterious effects of minocycline in animal models of Parkinson's disease and Huntington's disease

Minocycline has been shown to exert anti-inflammatory effects underlying its putative neuroprotective properties in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease and in the R6/2 mouse model of Huntington's disease (HD). However, contradictory results have recently been reported. We report deleterious effects of minocycline in two phenotypic (toxic) models of Parkinson's disease and HD in monkey and mouse. Of seven MPTP-intoxicated female cynomolgus monkeys (0.2 mg/kg, i.v. until day 15), three received minocycline (200 mg b.i.d.). While placebo-MPTP-treated animals displayed mild parkinsonism at day 15, the minocycline/MPTP-treated animals tended to be more affected (P = 0.057) and showed a greater loss of putaminal dopaminergic nerve endings (P < 0.0001). In the 3-nitropropionic acid (3-NP) mouse model of HD, minocycline (45 mg/kg i.p.) was administered 30 min before each i.p. injection of 3-NP (b.i.d., cumulated dose, 360 mg/kg in 5 days). Mice receiving minocycline exhibited a worsening of the mean motor score with a slower recovery slope, more impaired general activity and significantly deteriorated performances on the rotarod, pole test and beam-traversing tasks. The histopathological outcome demonstrated that minocycline-treated mice presented significantly more severe neuronal cell loss in the dorsal striatum. The effect of minocycline vs. 3-NP was also investigated on hippocampal and cortical cell cultures. minocycline blocked 3-NP-induced neurotoxicity at certain doses (1 mm cortical neurons) but not at higher doses (10 mm). Thus, minocycline may have variable and even deleterious effects in different species and models according to the mode of administration and dose.     

Viral vectors for gene therapy in Parkinson's disease

The ability of transplanted neurons from aborted foetuses to produce some therapeutic benefit in Parkinson's disease makes this disease an obvious target for the development of gene therapy procedures which involve delivering the same factors as are provided by the foetal neurons but using a reagent which could be produced in large amounts in a standardised manner. This approach could involve both the delivery of the gene encoding tyrosine hydroxylase to boost dopamine production or the delivery of genes encoding neurotrophic factors such as GDNF to promote the survival of dopaminergic neurons. A variety of different viral and non-viral methods for achieving such gene delivery has been described. These are discussed together with the particular advantages of herpes simplex virus-based vectors which have the potential to deliver multiple therapeutic genes in a single virus vector.     

Early synaptic dysfunction in Parkinson's disease: Insights from animal models

The appearance of motor manifestations in Parkinson's disease (PD) is invariably linked to degeneration of nigral dopaminergic neurons of the substantia nigra pars compacta. Traditional views on PD neuropathology have been grounded in the assumption that the prime event of neurodegeneration involves neuronal cell bodies with the accumulation of metabolic products. However, this view has recently been challenged by both clinical and experimental evidence. Neuropathological studies in human brain samples and both in vivo and in vitro models support the hypothesis that nigrostriatal synapses may indeed be affected at the earliest stages of the neurodegenerative process. The mechanisms leading to either structural or functional synaptic dysfunction are starting to be elucidated and include dysregulation of axonal transport, impairment of the exocytosis and endocytosis machinery, altered intracellular trafficking, and loss of corticostriatal synaptic plasticity. The aim of this review is to try to integrate different lines of evidence from both pathogenic and genetic animal models that, to different extents, suggest that early synaptic impairment may represent the key event in PD pathogenesis. Understanding the molecular and cellular events underlying such synaptopathy is a fundamental step toward developing specific biomarkers of early dopaminergic dysfunction and, more importantly, designing novel therapies targeting the synaptic apparatus of selective, vulnerable synapses. © 2016 International Parkinson and Movement Disorder Society     

The implementation of acute versus chronic animal models for treatment discovery in Parkinson's disease

Animal models for neuropsychiatric disorders are implemented for the purpose of investigating a single or multiple aspects of a specific disease entity. In Parkinson's disease (PD) several models have been utilised to study the biochemical and behavioural consequences of dopamine (DA) neurone degeneration with the intent of further understanding the aetiology of this disease and improving its treatment. While the bilateral 6-hydroxydopamine (6-OHDA) model has been used to produce a broad spectrum of neurochemical and behavioural deficits characterising DA degeneration in humans, this model is traumatic, labour intensive and is associated with high mortality due to the acute effects of the neurotoxin. Consequently, the unilateral 6-OHDA model was developed and implemented. In this model damage to the ascending DA system is produced on one side of the brain thereby inducing postural rotation. This movement is exaggerated by activating the remaining DA systems with apomorphine or amphetamine thereby making it more quantifiable. In view of the less traumatic effects on homeostasis and relative ease with which this model can be implemented it has been used routinely for the purpose of screening potential anti-Parkinsonian drugs for clinical use. However, like any model, the use of the unilateral rotation model has its limitations. It is proposed that the process of exaggerating DA function using this paradigm limits the discovery of potential anti-PD drugs to those which are effective in counteracting an exaggerated DA response. This factor may account for the high incidence of unwanted side effects including involuntary movement, tardive dyskinaesia (TD) and psychosis which are commonly observed in DA replacement therapy. Secondly, this approach limits potential drug candidates to those acting exclusively on brain DA systems. This too is a problem in the sense that PD is known to be a disease involving numerous systems in the human brain and potential therapies acting via other neurochemical systems are being excluded when this model is used exclusively. The object of the present paper is to report the discovery of a non-DA drug possessing potent anti-Parkinsonian qualities which were revealed using the bilateral 6-OHDA model of PD as a screening tool. When the same drug was retested in the traditional unilateral screening model no effect was observed, while more advanced models confirmed its efficacy. These results illustrate that the implementation of appropriate models for revealing new treatment strategies for PD should be broadly based so that single treatment entities are not exclusively pursued for diseases whose aetiologies are multifaceted. Premature extrapolation of findings from a single, early stage model to its clinical counterpart can be detrimental to advancing new treatment strategies, induce false hope, and increase morbidity in PD patients.     

Assessing neuroprotection in Parkinson's disease: from the animal models to molecular neuroimaging in vivo

An important goal in Parkinson's Disease research is to identify neuroprotective therapy, and the interaction between basic science and clinical research is needed to discover drugs that can slow or halt the disorder progression. At present there is not a perfect animal model of PD to test neuroprotective strategies, however the models that portray the basic characteristics needed are toxin-induced and gene-based models. The first group comprehends 6-OHDA e MPTP and recently rotenone, paraquat and epoxomicin treated animals that shows some of human disease characteristics. Gene-based models are various and, even if with limits, they seem suitable models to test neuroprotection in PD since they present replicable lesions, a predictable pattern of neurodegeneration and a well-characterized behavior, biochemistry and morphology to assist in the understanding of induced changes. In clinical trials researchers have first used as marker of disease progression clinical scores and motor tasks which are limited by the potential symptomatic effect of tested drugs and are not useful in the pre-clinical phases of PD. Recently has emerged the important role of neuroimaging (Dopamine Transporter SPECT, 18FDopa-PET) as surrogate biomarker of PD progression. Even if there are still concerns about the influence of regulatory effects of tested drugs, neuroimaging features could represent a good outcome measure to evaluate PD progression and putative neuroprotective effect of pharmacological and non-pharmacological manipulations.     

Development of new treatments for Parkinson's disease in transgenic animal models: a role for beta-synuclein

Neuronal death in Parkinson's disease (PD), one of the most common neurodegenerative disorders in the adult and aging population is probably caused by misfolding of synaptic proteins such as alpha-synuclein. Although, some treatments are currently available to control some of the symptoms of PD, none of these approaches directly addresses the mechanisms of disease. With the advent of new experimental animal models for this disorder, the potential for development and discovery of new treatment has been significantly bolstered. Among them, overexpression of alpha-synuclein results in motor deficits. dopaminergic loss and formation of inclusion bodies. Co-expression of mutant amyloid precursor protein, accelerates alpha-synuclein aggregation and enhances the neurodegenerative pathology in these mice, providing a unique model where to investigate the interactions between Abeta1-42 and alpha-synuclein and to develop treatments for combined Alzheimer's disease and PD. Development of anti-parkinsonian treatments based on these models includes: (i) anti-aggregation or pro-degradation compounds, (ii) neuroprotective compounds, and (iii) neurotrophic agents. Among them, we characterized beta-synuclein, the non-amyloidogenic homologue of alpha-synuclein, as an inhibitor of aggregation of alpha-synuclein. Our results raise the intriguing possibility that beta-synuclein might be a natural negative regulator of alpha-synuclein aggregation, and that a similar class of endogenous factors might regulate the aggregation state of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of beta-synuclein might also provide a novel strategy for the treatment of neurodegenerative disorders.     

The effect of chronic treatment of deprenyl in animal models of Parkinson's disease

Recent studies have demonstrated that deprenyl can delay the progression of parkinsonian syndrome. Deprenyl selectively and irreversibly inhibits the activity of monoamine oxidase-type B (MAO-B), and subsequent enzyme activity requires de novo synthesis of MAO-B. In this study we investigated (1); the effects of deprenyl on striatal dopamine (DA) levels in MPTP-lesioned and undamaged mice and (2); the effect of deprenyl on L-DOPA induced rotational movements in a hemiparkinsonian model monkey. In MPTP-lesioned mice, deprenyl increased the striatal DA levels by 123%, 78% immediately and one week after termination of deprenyl treatment respectively, while by 31%, 24% in MPTP-undamaged mice, respectively. Four weeks after termination of treatment, and the striatal DA was not significant. It shows that deprenyl is effective in elevation of striatal DA at least one week after termination of treatment, and the degree of increase of striatal DA following deprenyl treatment is greater in MPTP-treated mice than in undamaged mice. Deprenyl seemed to be effective longer in a hemiparkinsonian monkey than in rodents. The result suggests that less frequent administration of deprenyl may be effective in controlling side effects of L-DOPA without reducing therapeutic efficacy.     

Synergistic antidyskinetic effects of topiramate and amantadine in animal models of Parkinson's disease

L-Dopa-induced dyskinesia in patients with Parkinson's disease can be alleviated by amantadine, an antagonist at N-methyl-D-aspartate glutamate receptors. The antiepileptic drug topiramate, which blocks α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, has also been shown to reduce dyskinesia. The purpose of this study was to examine the behavioral pharmacology of topiramate alone and in combination with amantadine in animal models of PD and L-dopa-induced dyskinesia. The effects of topiramate (5-20 mg/kg) and amantadine (5-20 mg/kg) on abnormal involuntary movements (the rat homologue of dyskinesia) and Rotarod performance were assessed alone and in combination in the 6-hydroxydopamine-lesioned rat following chronic L-dopa treatment. Dyskinesia, parkinsonian disability, and "on-time" were assessed in the MPTP-lesioned nonhuman primate following administration of topiramate (5-20 mg/kg) and amantadine (0.1-1.0 mg/kg) alone and in combination. Topiramate and amantadine dose-dependently reduced dyskinesia in the 6-hydroxydopamine-lesioned rat, whereas topiramate reduced Rotarod performance; there was no effect on parkinsonian disability in the MPTP-lesioned nonhuman primate, in which both drugs reduced dyskinesia. Topiramate and amantadine exhibited differential antidyskinetic effects on dyskinesia elicited by the dopamine D1 receptor agonist SKF 38393 (2 mg/kg). Subthreshold doses of both drugs in combination had a synergistic effect on dyskinesia in the 6-hydroxydopamine-lesioned rat, with no worsening of motor performance; this effect was confirmed in the MPTP-lesioned nonhuman primate, with a selective reduction in "bad on-time." These data confirm the antidyskinetic potential of topiramate and suggest that combination with low-dose amantadine may allow better reduction of dyskinesia with no adverse motor effects.