Use of lentiviral vectors for modelling and treating Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the abnormal appearance of intracellular inclusions named Lewy bodies (LBs). Over the past few years, the discovery of genes involved in hereditary forms of the disease led to new insights into the pathogenesis of PD. Mutations in the α-synuclein gene have been associated with autosomal dominant PD, and mutations in parkin with autosomal recessive-juvenile parkinsonism (AR-JP). α-synuclein has emerged as a key protein in the pathogenesis of PD, as it appears to be the major structural component of LBs and its accumulation seems to play a prominent role in sporadic PD. To date, genetic PD models based on α-synuclein conventional transgenesis in rodents have been unsuccessful in recapitulating the main features of the human pathology, especially the loss of nigral dopamine neurons. The lack of clear dopaminergic cellular degeneration in these transgenic animal models is likely to be attributed to insufficient expression levels of α-synuclein in the substantia nigra. Injection of viral vectors constitutes an alternative approach for the development of genetic models, as viral vectors allow high gene expression levels in a localized brain region and can be applied in mammalian species other than mice. In the present study, the viral-mediated expression of different α-synuclein forms (normal or mutated) was explored in the substantia nigra of rats. Following the full characterization of this new genetic model, several neuroprotective strategies based on the delivery of potential neuroprotective factors such as the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor), the E3 ligase parkin or the chaperone Hsp104 were evaluated in this α-synuclein rat model. As HIV-1-derived lentiviral vectors can efficiently transduce neurons in the brain, these retroviral vectors were used to overexpress normal or mutated forms of α-synuclein in the substantia nigra of adult rats. In contrast to α-synuclein transgenic mice, the lentiviral-based model developed a progressive and selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum in animals expressing either wild-type or mutant forms of human α-synuclein. The neuronal degeneration correlated with the appearance of ?α-synuclein-positive inclusions. This viral-based model thus constitutes an excellent genetic model for testing molecules that can interfere with the neurodegenerative process induced by α-synuclein. Delivery of GDNF, a potent neuroprotective factor for the survival of dopaminergic neurons, is currently the most promising strategy for the treatment of PD. The neuroprotective properties of GDNF were therefore evaluated in the lentiviral-based model expressing mutated human α-synuclein. Although a robust expression of GDNF was observed in the whole nigrostriatal pathway due to retrograde and/or anterograde transport, nigral GDNF delivery did not prevent the α-synuclein-induced neurodegeneration. As loss of function of parkin leads to dopaminergic cell loss in Autosomal Recessive-Juvenile Parkinsonism (AR-JP) patients, parkin may represent a critical survival factor for dopaminergic neurons. The neuroprotective role of the E3 ubiquitin ligase parkin was assessed in the viral-based model overexpressing mutated human α-synuclein. Co-expression of parkin with α-synuclein prevented the nigrostriatal degeneration and increased the number of hyperphosphorylated α-synuclein inclusions. These results suggest that parkin may also play a key role in the formation of aggregates. Abnormal folding and aggregation of α-synuclein is now recognized as a critical issue in the pathology of PD. Chaperones can block or reverse the incorrect folding of misfolded proteins, and increase the clearance of aggregates. To enhance the elimination of aggregated α-synuclein and to understand the importance of inclusions in the α-synuclein pathogenesis, we expressed a protein disaggregase, the yeast chaperone Hsp104, in the lentiviral-based model of PD. This chaperone can facilitate the clearance of misfolded proteins from an aggregate state. Expression of Hsp104 reduced the dopaminergic cell loss induced by lentiviralmediated expression of PD-linked mutated α-synuclein. This neuroprotective effect correlated with a reduction in the formation of hyperphosphorylated α-synuclein inclusions, indicating that enhancing clearance of protein aggregates through the disaggregase activity of Hsp104 may constitute a novel therapeutic strategy for PD and other diseases caused by misfolded protein accumulation. The present thesis demonstrates that lentiviral vectors expressing human α-synuclein constitute a powerful and flexible tool to mimic the neuropathological features of PD. Although not effective in the lentiviral-based model of PD, GDNF may still represent a promising molecule to promote the sprouting of dopaminergic axons. On the contrary, the E3 ligase parkin and the chaperone Hsp104 have demonstrated potent neuroprotective properties against the accumulation of the toxic mutated α-synuclein. Therapeutic approaches aiming at increasing parkin or chaperone levels in the brain may therefore open new perspectives for the treatment of PD.

Exploring the pathological interaction between LRRK2 and alpha-synuclein

Alessandra Musso

Parkinson’s disease (PD) is the most common age-related neurodegenerative movement disorder that affects 1-2% of the general population over the age of 65 and rising to 4-5% over 80 years of age. It is estimated that 6.3 million people worldwide have Parkinson’s disease and 1.2 million in the European Union (EU) alone. As the disease progresses, patients frequently develop a mask-like facial expression, festinating gait and cognitive impairment and the final clinical symptoms are muscle rigidity, bradykinesia, resting tremor and postural instability in addition to non-motor problems that result in a marked reduction in quality of life. The motor symptoms of PD arise from the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) leading to a deficiency of the neurotransmitter dopamine (DA) in the striatum. While dopamine replacement therapy is initially effective in treating the motor symptoms of PD, there is no known cure or neuroprotective therapy for this debilitating disease. Although the pathology underlying the disease is well defined, it is still unclear why DAergic neurons degenerate and die. PD is generally considered as a sporadic disease of unknown etiology, however in the last two decades it has become clear that heritability plays an important role in the pathogenesis of PD. Of the different genes associated with familial disease, mutations in two genes encoding for α-synuclein and leucine-rich repeat kinase 2 (LRRK2) proteins cause autosomal dominant PD. One of the neuropathological hallmarks of idiopathic and some familial forms of PD are Lewy bodies, which are intracytoplasmic inclusions that are enriched with fibrillar α- synuclein protein. Notably, LRRK2 mutation carriers with PD predominantly develop Lewy body pathology suggesting that LRRK2 may lie upstream of α-synuclein aggregation. These observations and other in vivo studies have suggested a common pathogenic pathway with LRRK2 potentially regulating the aggregation and neuronal toxicity of α-synuclein. However, the relationship between LRRK2 and α-synuclein in the mammalian brain is presently unclear and requires further evaluation, especially within midbrain dopaminergic neurons that degenerate in PD. The aim of this thesis work was to delineate a potential pathogenic interplay between these two PD-related gene products in mediating neurodegeneration in vivo. Understanding how and where the functional pathways of LRRK2 and α-synuclein converge will provide important insight into the common mechanisms underlying neurodegeneration in PD.[...]

EPFL2014

Alpha-Synuclein Effects at the ER-to-Golgi Level and Potential Biomarkers in Rat Models of the Early Phase of Parkinson's Disease

Philippe Guillaume Coune

While Parkinson's disease has been described nearly 200 years ago, the mechanisms leading to the degeneration of selectively vulnerable populations of neurons, such as dopaminergic neurons in the substantia nigra, remain mostly unknown. Our poor understanding of the disease etiology has dramatically hampered the rational development of therapies interfering with the processes underlying neuronal degeneration. Current therapeutic approaches provide symptomatic relief, but fail to slow down the course of Parkinson's disease. In addition, the diagnosis of Parkinson's disease relies primarily on the clinical assessment of motor symptoms that become detectable only when a large part of the nigral dopaminergic neurons have already degenerated. There is therefore a strong need to better understand the pathological processes underlying early stages of Parkinson's disease to improve the tools for diagnosis as well as to develop effective disease-modifying therapies. Towards these goals, it is important to develop, analyze and validate animal models, which faithfully replicate the human disease. In particular, the induced degeneration of nigral dopaminergic neurons in the adult mammalian brain provides an experimental tool to evaluate in vivo how the disease process leads to motor symptoms. Although neurotoxins such as 6-hydroxydopamine produce a selective degeneration of nigral dopaminergic neurons, their mode of action is clearly different from what takes places in the human pathology. During the last decade, the identification of genes linked to familial inheritance of Parkinson's disease led to the development of novel animal models based on the actual genetic cause of these rare forms of the disease. Alpha-synuclein, an abundant brain protein encoded by a gene involved in autosomal dominant forms of Parkinson's disease, is now considered a primary actor in the pathogenesis of this neurodegenerative disorder. Our laboratory has developed a rat model of Parkinson's disease based on the over-expression of human α-synuclein in the rat substantia nigra using adeno-associated viral vectors. In this model, α-synuclein over-expression progressively leads to a moderate loss of nigral dopaminergic neurons. Remarkably, animals develop motor deficits that are partly due to impaired neurotransmitter release in the remaining dopaminergic axons. This model system, where some nigral dopaminergic neurons survive in presence of overabundant human α-synuclein, can be used to explore early pathologic mechanisms that undermine the nigrostriatal system prior to outright neurodegeneration. The first part of this thesis investigates the detrimental effects of α-synuclein accumulation on the secretory pathway at the level of the endoplasmic reticulum (ER) and Golgi apparatus in the rat substantia nigra. We found that a significant proportion of the nigral neurons expressing α-synuclein display a pathological fragmentation of the Golgi apparatus. This effect has been explored by co-expressing the small GTPase Rab1A, a positive regulator the ER-to-Golgi vesicular trafficking. Interestingly, Rab1A co-expression does not prevent nigrostriatal degeneration but significantly corrects the motor deficits caused by α-synuclein. These results show that enhancing the ER-to-Golgi vesicular trafficking partially protects dopaminergic neurons from the α-synuclein-induced pathology in vivo, by restoring Golgi morphology in the remaining neurons and significantly preserving motor behavior. In order to further investigate the possible implication of the ER in the stress caused by α-synuclein, we evaluated the effects on the pathology induced by A53T α-synuclein of Salubrinal, an inductor of the PERK pathway of the unfolded protein response. Similarly to Rab1A, the Salubrinal treatment robustly reduced Golgi fragmentation and delayed the appearance of motor symptoms, without preventing nigrostriatal degeneration. Taken together, these results indicate that α-synuclein over-expression impairs the ER/Golgi system in rat dopaminergic neurons and thereby leads to neuronal dysfunction in the surviving nigral neurons. The second part of this thesis explores potential biomarkers for early stages of Parkinson's disease using ultra high field 1H magnetic resonance spectroscopy. The technology of magnetic resonance spectroscopy allows for the non-invasive quantification of abundant brain metabolites including metabolic markers and neurotransmitters. We measured metabolite levels in the striatum following nigral injection of an adeno-associated viral vector to express human α-synuclein and induce mild degeneration. The observed changes were compared to similar measurements made following intoxication with 6-hydroxydopamine to induce near complete nigro-striatal denervation. Expectedly, the level of the metabolic marker N-acetyl aspartate considered to reflect neuronal degeneration was significantly decreased only in the 6-hydroxydopamine model and failed to detect the mild neuronal loss induced by α-synuclein. On the other hand, we found an increase in striatal GABA concentration clearly detectable in both models. This result indicates that the measurement of GABA levels by magnetic resonance spectroscopy can be used as a sensitive marker for the striatal effects of α-synuclein accumulation. The increase in GABA levels observed in conditions of mild degeneration suggests that the functional deficits induced by human α-synuclein can have measurable consequences on neurotransmitter levels in the nigrostriatal system before extensive neuronal loss. Therefore, assessments of the neurotransmitter levels by magnetic resonance spectroscopy in the basal ganglia could be a promising approach for the early detection of Parkinson's disease. Overall, this thesis work shows that neurodegeneration is not the only actor in the α-synuclein-induced phenotype. We demonstrate that modifiers of α-synuclein toxicity at the ER/Golgi level mainly improve motor deficits without any effect on α-synuclein-induced degeneration. In addition, the over-expression of α-synuclein has detectable effects on the homeostasis of striatal neurotransmitters. If confirmed in human patients, these results may improve our understanding of the processes leading to the initial motor symptoms and open new avenues for early diagnosis of Parkinson's disease.

EPFL2011

DJ-1 as a neuroprotective protein in models of Parkinson's disease

Katarzyna Piorkowska-Stanco

Parkinson's disease (PD) is a common progressive neurodegenerative disorder characterized clinically by the combination of motor symptoms like bradykinesia, tremor, rigidity and postural instability. The pathology of PD is related to the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNpc) leading to the deficiency of dopamine in the striatal projection areas of these neurons. The pathogenesis of PD is not fully elucidated yet, however there is strong evidence that protein aggregation, mitochondrial dysfunction and oxidative stress play a primary role in the etiology of the disease. Most of PD cases are sporadic, however, several genes have been characterized to cause familial forms of the disease. Among them, mutations in DJ-1 were identified in families with autosomal-recessive early-onset PD and account for 2% of inherited PD cases. The DJ-1 protein was identified as an important player in cellular defense against oxidative stress as it possibly acts as an oxidative stress-induced chaperone and inhibits the formation of inclusions of α-synuclein, another protein related to PD. Alpha-synuclein mutations cause an autosomal dominant form of the disease. Moreover, this protein is a main component of Lewy Bodies, a pathological hallmark of PD. The goal of the experiments during my thesis was to investigate the potential protective effect of DJ-1 in toxin-based [6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)] as well as genetic models of PD in vivo. Towards this goal, the DJ-1 protein was over-expressed in rodent SNpc through the injections of recombinant adeno-associated viral vector (rAAV). In the first study, the DJ-1 over-expression led to significant protection of dopaminergic cells and spontaneous motor behavior in 6-OHDA-lesioned animals. However, this effect of DJ-1 was not confirmed when DJ-1 was over-expressed in the brains of mice lesioned with MPTP. In the third part of the study, the nigral over-expression of mutated a-synuclein (A30P mutation found in familial PD cases) was chosen as the genetic model of PD, already developed in our lab. Interestingly, significant preservation of spontaneous behavior was obtained when a-synuclein (A30P) was co-injected with DJ-1. However, this was accompanied by a modest protection of dopaminergic cells, suggesting the implication of other, subtle mechanisms regulating DJ-1-related basal ganglia output. Although the DJ-1 protection of dopaminergic cells was not always achieved, the effect of DJ-1 on the spontaneous behavior implies the interesting capacity of this protein. Our results suggest that DJ-1 is one of key factors playing role in maintenance of dopaminergic function. For a possible clinical application, DJ-1 may have to be linked with another neuroprotecting factor to assure a stronger protection effect.

EPFL2007