Modulation of AMPK's activity via genetic tools and food-derived compounds as a novel approach to Parkinson's disease treatment

Aging is considered as a major risk factor for Parkinsonâs disease (PD) and can be defined as a gradual decline in the ability of biological systems to efficiently renew structural and functional components, which may lead to perturbations in energetic homeostasis. AMPK is a heterotrimeric enzyme that is both a critical sensor and regulator of the energetic status of the cell. The activity of AMPK may progressively decline with age, therefore contributing to the metabolic perturbations that underlie neurodegenerative diseases. Here, we explore the mutual interplay between Î±-synuclein (Î±-syn), a key factor in PD patholo-gy, and AMPK, using a genetic rat model based on overexpression of human Î±-syn. More specifi-cally, we explore how the expression of various genetic variants of the catalytic AMPK Î± subunit determines the effects of Î±-syn overexpression, both in vitro and in vivo. We show that overex-pression of either AMPK Î±1 and Î±2 to increase AMPK activity, have partial neuroprotective effects against human Î±-syn toxicity in the nigrostriatal system. Remarkably, chronic, low level of AMPK activity, induced via overexpression of a mutated form of AMPKÎ± (T172D AMPKÎ±1), most effec-tively prevents the loss of dopamine neurons as well as the motor impairments caused by Î±-syn accumulation. Furthermore, only T172D AMPKÎ±1 leads to the significant clearance of aggregated Î±-syn in the dopamine axonal fibers. In vitro results indicate that Î±-syn accumulation in primary cortical neurons leads to increased autophagic activity and a decline in the number of mitochondrial DNA copies. When AMPKÎ± subu-nits are overexpressed simultaneously with Î±-syn, an increased in the mitochondrial mass is ob-served, together with a rescue of the autophagic response caused by Î±-syn overexpression. Re-markably, neurons overexpressing the T172D AMPKÎ±1 variant show an intermediate phenotype, where Î±-syn-induced autophagy is still observed, although there is no more changes in mitochondrial parameters caused by Î±-syn overexpression. Finally, using the same human Î±-syn genetic model of PD, we show neuroprotective effects us-ing a diet supplemented with urolithin, a polyphenolic compound known to activate AMPK. Nigro-striatal dopaminergic neurons are significantly rescue from Î±-syn toxicity in rats fed with urolithin, underlining the importance of AMPK signaling in prospective PD therapy.

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

Identification of genetic determinants and pharmacological modulators of mitochondrial function

Pénélope Andreux

Increased life expectancy goes hand in hand with a higher incidence of age-related diseases that affect the nervous and metabolic systems, such as type 2 diabetes, cancer, Parkinson’s and Alzheimer’s disease. These complex diseases are a product of the combined actions of large networks of genes, and environmental factors, such as diet, exercise, drug use, and levels of acute and chronic stress. One of the main challenges in biomedical research is determining how these factors interact to influence human healthspan, in order to develop effective strategies for the diagnosis, prevention, and treatment of these chronic multifactorial diseases. A strategy that has emerged in the last decade is the modulation of mitochondrial function. Accumulating evidence support the involvement of mitochondrial dysfunction in the pathogenesis of type 2 diabetes, Parkinson’s disease, cancer, and to a lesser extent, Alzheimer’s disease. Mitochondrial processes are regulated by a highly dynamic and complex network of enzymes and transcription factors. One of the best characterized branches of this network is the AMPK/SIRT1 pathway and its downstream effectors. Recently, additional pathways important for the regulation of mitochondrial function have been described such as mitochondrial fusion and fission, specific autophagy of mitochondria, termed mitophagy, and mitochondrial unfolded protein response (UPRmt). These recent discoveries have extended the scope of potential therapeutic targets. This thesis describes the development of two complementary approaches to identify novel genetic and pharmacological determinants of mitochondrial function. 1. The first approach aims to identify novel genetic determinants of the regulatory networks that control metabolism in general, and mitochondrial function in particular, by using the mouse BXD genetic reference population (GRP). Chapter II summarizes the analysis of 140 metabolic traits in 42 BXD strains, and demonstrates the suitability and usefulness of the BXD GRP for the identification of genetic regulators of metabolism. 2. The second approach is focused on the development of pharmacological tests for the identification and characterization of novel drugs that modulate mitochondrial phenotypes. Chapter III presents the establishment and validation of a platform of mitochondrial assays in two species, i.e. mammalian cells and the worm C.elegans. A successful example of its use is shown in chapter IV, where the role of copper in tumorigenesis is elucidated by applying these mitochondrial assays Altogether, these two synergistic approaches pave the way that will lead to the identification of novel key factors and treatments for mitochondrial diseases and pathologies associated with mitochondrial dysfunction.

EPFL2013

The mitochondrial unfolded protein response in mammalian physiology

Johan Auwerx, Virginija Jovaisaite, Adrienne Joëlle Laurence Mottis

Mitochondria, the main site of cellular energy harvesting, are derived from proteobacteria that evolved within our cells in endosymbiosis. Mitochondria retained vestiges of their proteobacterial genome, the circular mitochondrial DNA, which encodes 13 subunits of the oxidative phosphorylation multiprotein complexes in the electron transport chain (ETC), while the remaining similar to 80 ETC components are encoded in the nuclear DNA (nDNA). A further similar to 1,400 proteins, which are essential for mitochondrial function are also encoded in nDNA. Thus, a majority of mitochondrial proteins are translated in the cytoplasm, then imported, processed, and assembled in the mitochondria. An intricate protein quality control (PQC) network, constituted of chaperones and proteases that refold or degrade defective proteins, maintains mitochondrial proteostasis and ensures the cell and organism health. The mitochondrial unfolded protein response is a relatively recently discovered PQC pathway, which senses the proteostatic disturbances specifically in the mitochondria and resolves the stress by retrograde signaling to the nucleus and consequent transcriptional activation of protective genes. This PQC system does not only transiently resolve the local stress but also can have long-lasting effects on whole body metabolism, fitness, and longevity. A delicate tuning of its activation levels might constitute a treatment of various diseases, such as metabolic diseases, cancer, and neurodegenerative disorders.