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

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.

PGC-1 alpha activity in nigral dopamine neurons determines vulnerability to alpha-synuclein

Wojciech Janusz Bobela, Graham Knott, Bernard Schneider, Lu Zheng

Introduction: Mitochondrial dysfunction and oxidative stress are critical factors in the pathogenesis of age-dependent neurodegenerative diseases. PGC-1 alpha, a master regulator of mitochondrial biogenesis and cellular antioxidant defense, has emerged as a possible therapeutic target for Parkinson's disease, with important roles in the function and survival of dopaminergic neurons in the substantia nigra. The objective of this study is to determine if the loss of PGC-1 alpha activity contributes to alpha-synuclein-induced degeneration. Results: We explore the vulnerability of PGC-1 alpha null mice to the accumulation of human alpha-synuclein in nigral neurons, and assess the neuroprotective effect of AAV-mediated PGC-1 alpha expression in this experimental model. Using neuronal cultures derived from these mice, mitochondrial respiration and production of reactive oxygen species are assessed in conditions of human alpha-synuclein overexpression. We find ultrastructural evidence for abnormal mitochondria and fragmented endoplasmic reticulum in the nigral dopaminergic neurons of PGC-1 alpha null mice. Furthermore, PGC-1 alpha null nigral neurons are more prone to degenerate following overexpression of human alpha-synuclein, an effect more apparent in male mice. PGC-1 alpha overexpression restores mitochondrial morphology, oxidative stress detoxification and basal respiration, which is consistent with the observed neuroprotection against alpha-synuclein toxicity in male PGC-1 alpha null mice. Conclusions: Altogether, our results highlight an important role for PGC-1 alpha in controlling the mitochondrial function of nigral neurons accumulating alpha-synuclein, which may be critical for gender-dependent vulnerability to Parkinson's disease.

Biomed Central Ltd2015

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

Over-expression of alpha-synuclein in human neural progenitors leads to specific changes in fate and differentiation

Patrick Aebischer, Bernard Schneider

Missense mutations and extra copies of the alpha-Synuclein gene result in Parkinson disease (PD). Human stem and progenitor cells can be expanded from embryonic tissues and provide a source of non-transformed neural cells to explore the effects of these pathogenic mutations specifically in human nervous tissue. We over-expressed the wild type, A53T and A30P forms of alpha-synuclein in expanded populations of progenitors derived from the human fetal cortex. The protein localized in the nucleus and around microvesicles. Only the A53T form was acutely toxic, suggesting a unique vulnerability of these progenitors to this mutation. Interestingly, constitutive over-expression of wild-type alpha-synuclein progressively impaired the innate ability of progenitors to switch toward gliogenesis at later passages. To explore the effect of alpha-synuclein on neuronal subtypes selectively affected in PD, such as dopaminergic neurons, alpha-synuclein and its mutations were also over-expressed in terminally differentiating neuroectodermal cultures derived from human embryonic stem cells (hESC). Alpha-synuclein induced acute cytotoxicity and reduced the number of neurons expressing either tyrosine hydroxylase or gamma-aminobutyric acid over time. Consistent with the selective vulnerability of ventral midbrain dopaminergic neurons, alpha-synuclein cytotoxicity appeared most pronounced following FGF8/SHH specification and was decreased by inhibition of dopamine synthesis. Together, these data show that alpha-synuclein over-expressed in human neural embryonic cells results in patterns of degeneration that in some cases match features of Parkinson Disease. Thus, neural cells derived from hESC provide a useful model system to understand the development of alpha-synuclein-related pathologies and allow therapeutic drug screening.