Autophagy and LRRK2 in the Aging Brain

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.

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease.

EPFL2012

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

GTPase Activity and Neuronal Toxicity of Parkinson's Disease-Associated LRRK2 Is Regulated by ArfGAP1

Liliane Glauser, Darren Moore, Klodjan Stafa, Alzbeta Trancikova

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD-associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD-associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD.

Public Library of Science2012