Nano-imaging trace elements at organelle levels in substantia nigra overexpressing alpha-synuclein to model Parkinson's disease

Sub-cellular trace element quantifications of nano-heterogeneities in brain tissues offer unprecedented ways to explore at elemental level the interplay between cellular compartments in neurodegenerative pathologies. We designed a quasi-correlative method for analytical nanoimaging of the substantia nigra, based on transmission electron microscopy and synchrotron X-ray fluorescence. It combines ultrastructural identifications of cellular compartments and trace element nanoimaging near detection limits, for increased signal-to-noise ratios. Elemental composition of different organelles is compared to cytoplasmic and nuclear compartments in dopaminergic neurons of rat substantia nigra. They exhibit 150-460 ppm of Fe, with P/Zn/Fe-rich nucleoli in a P/S-depleted nuclear matrix and Ca-rich rough endoplasmic reticula. Cytoplasm analysis displays sub-micron Fe/S-rich granules, including lipofuscin. Following AAV-mediated overexpression of alpha -synuclein protein associated with Parkinson's disease, these granules shift towards higher Fe concentrations. This effect advocates for metal (Fe) dyshomeostasis in discrete cytoplasmic regions, illustrating the use of this method to explore neuronal dysfunction in brain diseases. Lemelle et al. describe the use of TEM and synchrotron X-ray fluorescence for quasi-correlative nanoimaging and sub-cellular trace element quantification of rat brain tissue. They further observe elemental (iron and sulfur) dyshomeostasis in cytoplasmic granules when overexpressing alpha -synuclein protein associated with Parkinson's disease, demonstrating the usefulness of this method to further explore dysfunctions at organelle levels in brain diseases.

FOXO3 determines the accumulation of alpha-synuclein and controls the fate of dopaminergic neurons in the substantia nigra

Ryoji Amamoto, Graham Knott, Emilda Pino, Juan-Carlos Sarria, Bernard Schneider, Lu Zheng

Parkinson's disease (PD) is characterized by the selective degeneration of neuronal populations presumably due to pathogenic interactions between aging and predisposing factors such as increased levels of a-synuclein. Here, we genetically modulate the activity of the transcription factor Forkhead box protein O-3 (FOXO3) in adult nigral dopaminergic neurons using viral vectors and explore how this determinant of longevity impacts on neuronal fate in normal and diseased conditions. We find that dopaminergic neurons are particularly vulnerable to changes in FOXO3 activity in the substantia nigra. While constitutive activation has proapoptotic effects leading to neuronal loss, inhibition of FOXO-mediated transcription by a dominant-negative competitor causes oxidative damage and is detrimental at high vector dose. To address the role of FOXO3 in PD, we modulate its activity in dopaminergic neurons overexpressing human a-synuclein. In this pathogenic condition, we find that FOXO inhibition has protective effects, suggesting that this transcription factor ultimately contributes to neuronal cell death. Nevertheless, mild FOXO3 activity also protects nigral neurons against the accumulation of human a-synuclein, albeit to a lesser extent. FOXO3 reduces the amount of a-synuclein present in the soluble protein fraction and promotes the coalescence of dense proteinase K-resistant aggregates, with an accumulation of autophagic vacuoles containing lipofuscin. Consistent with these in vivo observations, we find that FOXO3 controls autophagic flux in neuronal cells. Altogether, these results point to FOXO3 as an important determinant of neuronal survival in the substantia nigra, which may oppose a-synuclein accumulation and proteotoxicity.

Oxford University Press2014

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

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