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

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.

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1

Bernard Schneider, Pamela Solange Valdés Undurraga

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD.

National Academy of Sciences2014

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

Christophe Lo Bianco

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.

EPFL2005

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

Patrick Aebischer, Bernard Schneider, Romain Zufferey

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.

2008

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

Christophe Lo Bianco

EPFL2005