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

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.

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

Impact of phosphorylation of alpha-synuclein in Parkinson's disease

Samareh Azeredo da Silveira Lajaunias

Parkinson's disease (PD) is characterized by the degeneration of dopamineric neurons of the substantia nigra pars compacta projecting to the striatum, and by the deposition of cytoplasmic protein inclusions. It is further defined by the persistent presence of the α-synuclein (α-syn) protein in these inclusions. Moreover, mutations of α-syn as well as a triplication of the α-syn gene have been identified as a cause of inherited PD. These rare familial cases indicate that modifications in the sequence and in the level of expression have a tremendous impact on the protein's properties. In both familial and sporadic PD, post-translational modifications of α-syn have been reported. In particular, phosphorylation of α-syn Serine at position 129 is found to be a major modification, tightly associated with pathogenesis in human PD. Whether it be through changes in the protein's folding properties or through aberrant interactions with other proteins and lipids, these modifications transform α-syn into toxic species. In the present work, we unilaterally injected one hemisphere of the substantia nigra of rats with recombinant adeno-associated vectors (rAAV) mediating the expression of human wild-type α-syn or of human α-syn bearing the familial A30P mutation. In both cases, we engineered the human α-syn gene to replace its Serine 129 either with an Alanine (S129A), in order to prevent the attachment of a phosphate, or with an Aspartate (S129D) with the objective of mimicking the negative charge of a bound phosphate at this site. In our experimental design, rAAV-mediated human α-syn expression levels in the substantia nigra were roughly equivalent to those of the rat endogenous protein. Eight weeks after injection, we measured a significant, dose-dependent decline in the number of dopaminergic neurons injected with the wild-type form or with A30P, as compared to the non-injected side. Thus a single amino-acid modification at position 129 caused marked differences in the patterns of toxicity. Notably, expression of the S129A variant dramatically increased the pathology in rats, while expression of the S129D appeared to protect against neuronal loss. This response was the exact opposite of that obtained following expression of the S129A and S129D variants in transgenic Drosophila. We then showed that the enhanced dopaminergic loss triggered by S129A was accompanied by extensive presence of α-syn- and β-pleated sheet-positive, proteinase K-resistant small aggregates. Immuno-electron microscopy revealed that these electro-dense cytoplasmic aggregates were frequently associated with lysosomes and with a phagosome-like glassy body with vesicular content. α-syn S129A was also extensively present in intracytoplasmic tubulo-membranous systems, in the outer membrane of mitochondria, in the nucleus, and was associated with neurofilaments within neuritic fibers. The nuclei of cells exhibiting these aggregates were lobulated, and aggregates were particularly present in the indented regions of the nucleus. In addition to the morphological characteristics of apoptosis, such as nucleus condensation and lobulation, markers of apoptotic pathways were activated. Strikingly, despite the absence of dopaminergic cells loss, aggregates were present also in the S129D-injected brains, although to a much lesser extent, and apoptotic markers were also activated in these rats. The present study did not report any substantial differences that can be attributed to the expression of wild-type α-syn versus that of the A30P mutant, whether these were mutated at Serine 129 or not. However, the divergence of our results from those of the Drosophila raises the question of the adequacy of the fly as a model for PD, in view of its nervous system and its lack of any endogenous homologue to α-syn. The fact that cellular mechanisms of death were triggered by the expression of all variants, despite their significant differences in terms of induced nigral degeneration, indicates that discrepancies between variants mostly reside in the dynamics of the pathogenesis and/or in the toxic potential of the aggregated proteins. Therefore this work also brings new perspective to the current efforts to modulate kinases/phosphatases activity directed towards α-syn as a therapeutic tool.

EPFL2008

Conditional expression of Parkinson's disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration

Shi-Yan Caroline Foo, Liliane Glauser, Meghna Kannan, Graham Knott, Darren Moore, Elpida Tsika

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2 years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD. (C) 2014 Elsevier Inc All rights reserved.

Elsevier2014