Role of the FoxO3a Transcription Factor in alpha-synuclein Induced Neurodegeneration

Increased expression of α-synuclein and point mutations in its amino acid sequence play a causative role in familial forms of Parkinson's disease (PD). In addition, circumstances affecting the level of α-synuclein expression significantly increase the risk of developing sporadic PD. This is further corroborated by the abundance of α-synuclein in protein aggregates called Lewy bodies (LBs), a histological hallmark of the disease. Aging constitutes the most prominent risk factor in the pathogenesis of PD and other synucleinopathies. Protective stress response and repair pathways gradually decline over time, a process likely to contribute to the appearance of neurodegenerative disease. Studies in lower organisms, such as the yeast and the worm Caenorhabditis elegans, have highlighted the role of the evolutionarily conserved transcription factor FoxO3a in the aging process, increasing life span and reducing molecular processes that possibly underlie neurodegeneration in higher organisms. This transcription factor is mainly controlled by insulin/Insulin-like growth factor (IGF) signaling (IIS) and impacts a broad range of disease-related responses involving neuronal apoptosis and adaptation to stress. We have hypothesized that FoxO3a activity may regulate the response of dopaminergic neurons to PD-related stress such as reactive oxygen species (ROS) and α-synuclein proteotoxicity. To address this question, AAV2/6 vectors encoding various forms of FoxO3a were used to modulate the activity of this transcription factor in the adult rat substantia nigra pars compacta (SNpc). We determined a viral dose where expression of the wild-type form of FoxO3a did not impair the survival of nigral neurons and their striatal projections. In contrast, a constitutively active FoxO3a mutant (FoxOm), with an Akt-independent nuclear import, led to a significant loss of dopaminergic cell bodies, consistent with the pro-apoptotic activity of FoxO3a. Expression of a dominant-negative competitor composed of the FoxO DNA-binding domain (FoxODBD) did not significantly impair the survival of nigral neurons. However, at a high viral dose, FoxO inhibition led to neuronal accumulation of 4-hydroxynonenal (HNE), an indicator of chronic oxidative stress, and to a loss of neurons positive for dopaminergic markers. Altogether, these results confirm in nigral neurons the critical role of FoxO3a activity at the intersection between apoptotic response and neuronal resistance to oxidative stress. Next, we hypothesized that FoxO3a may regulate the response of dopaminergic neurons to α-synuclein proteotoxicity. To investigate the effect of FoxO3a on α-synuclein accumulation, we co-injected AAV2/6 vectors modulating FoxO3a activity with a wild-type human α-synuclein vector. When compared to control and FoxOm, both wild-type FoxO3a and FoxODBD significantly reduced the α-synuclein-induced loss of neurons positive for dopaminergic markers. Importantly, the injection of vectors activating FoxO3a (wild-type and FoxOm) led to an overall reduction in soluble α-synuclein monomeric forms, concomitant with the formation of dense proteinase K-resistant aggregates found in the neuronal cell soma. With the dominant negative FoxODBD, we observed an accumulation of α-synuclein, with a diffuse distribution of proteinase K-resistant immunoreactivity in both axon and neuronal soma. These data point to FoxO3a as an important regulator of neuronal survival in the SNpc, which can oppose α-synuclein accumulation and proteotoxicity. The role of FoxO3a in α-synuclein toxicity may provide some clues about the role of the aging process in PD neurodegeneration.

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

Autophagy and LRRK2 in the Aging Brain

Salvatore Novello

Autophagy is a highly conserved process by which long-lived macromolecules, protein aggregates and dysfunctional/damaged organelles are delivered to lysosomes for degradation. Autophagy plays a crucial role in regulating protein quality control and cell homeostasis in response to energetic needs and environmental challenges. Indeed, activation of autophagy increases the life-span of living organisms, and impairment of autophagy is associated with several human disorders, among which neurodegenerative disorders of aging, such as Parkinson's disease. These disorders are characterized by the accumulation of aggregates of aberrant or misfolded proteins that are toxic for neurons. Since aging is associated with impaired autophagy, autophagy inducers have been viewed as a strategy to counteract the age-related physiological decline in brain functions and emergence of neurodegenerative disorders. Parkinson's disease is a hypokinetic, multisystemic disorder characterized by age-related, progressive degeneration of central and peripheral neuronal populations, associated with intraneuronal accumulation of proteinaceous aggregates mainly composed by the presynaptic protein alpha-synuclein. alpha-synuclein is a substrate of macroautophagy and chaperone-mediated autophagy (two major forms of autophagy), thus impairment of its clearance might favor the process of alpha-synuclein seeding and spreading that trigger and sustain the progression of this disorder. Genetic factors causing Parkinson's disease have been identified, among which mutations in the LRRK2 gene, which encodes for a multidomain protein encompassing central GTPase and kinase domains, surrounded by protein-protein interaction domains. Six LRRK2 mutations have been pathogenically linked to Parkinson's disease, the most frequent being the G2019S in the kinase domain. LRRK2-associated Parkinson's disease is clinically and neuropathologically similar to idiopathic Parkinson's disease, also showing age-dependency and incomplete penetrance. Several mechanisms have been proposed through which LRRK2 mutations can lead to Parkinson's disease. The present article will focus on the evidence that LRRK2 and its mutants are associated with autophagy dysregulation. Studies in cell lines and neurons in vitro and in LRRK2 knock-out, knock-in, kinase-dead and transgenic animals in vivo will be reviewed. The role of aging in LRRK2-induced synucleinopathy will be discussed. Possible mechanisms underlying the LRRK2-mediated control over autophagy will be analyzed, and the contribution of autophagy dysregulation to the neurotoxic actions of LRRK2 will be examined.

FRONTIERS MEDIA SA2019

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.