Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase

Highlights

• Pan neuron-specific Pgk knockdown decreases ATP level and increases ROS level in the larval CNS.

• DA neuron-specific knockdown of Pgk causes locomotor defects in adult stages.

• DA neuron-specific knockdown of Pgk induces progressive DA neurons loss in the PPM1/2 cluster.

• DA neuron-specific knockdown of Pgk decreases dopamine levels in the CNS.

Abstract

Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. We herein established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons.

Introduction

Parkinson's disease (PD) is a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic (DA) neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, including autonomic dysfunction and dementia, called parkinsonism. The etiology of PD remains unknown. However, in the past 2 decades, clinical and experimental evidence on PD has collectively implicated three major pathogenic mechanisms: (i) oxidative stress and defective energy conservation, (ii) impaired protein turnover, and (iii) disrupted synaptic and endosomal vesicle and protein trafficking and recycling (Obeso et al., 2017).

The case for oxidative stress and defective energy conservation due to mitochondrial dysfunction in at least part of the pathogenesis of PD has been becoming increasingly persuasive. This is supported by several mitochondrial toxins of Complex I blockers, including rotenone and MPP + that are commonly used to create PD animal models, since they induce parkinsonism in humans and animals (Bove and Perier, 2012; Panov et al., 2005). The crucial role of mitochondria in PD was also suggested by a number of studies demonstrating that Parkin and PINK1, the genes associated with familial PD, are relevant for mitochondrial homeostasis. Moreover, another PD-related gene, DJ-1 is known to translocate from the nucleus to the mitochondrial matrix and intramembrane space under oxidative stress conditions, suggesting its role as an oxidative stress sensor (Grunewald et al., 2019; Morais et al., 2009; Zhang et al., 2005). Although the glycolysis system that is the upstream pathway of mitochondrial energy production in the cytosol has not attracted much attention in this field, recent studies reported dysregulated glycolysis in postmortem brain tissues and peripheral blood mononuclear cells from sporadic PD (Dunn et al., 2014; Liu et al., 2016) as well as in cultured cells with the ablation of Parkin (Dunn et al., 2014).

Phosphoglycerate kinase (PGK) encoded by PGK-1 (OMIM #311800), which maps to the X chromosome in humans, is a key enzyme in the glycolytic pathway catalyzing the conversion from 1.3-diphosphoglycerate to 3-phosphoglycerate. PGK deficiency (OMIM #300653) causes X-linked recessive hereditary chronic hemolytic anemia and myopathy due to insufficient energy production in red blood cells and muscles (Beutler, 2007). The disease has been known to be occasionally accompanied by levodopa-responsive parkinsonism (Morales-Briceno et al., 2019; Sotiriou et al., 2010; Virmani et al., 2014). The function of the gene has recently received growing attention in the PD field, because pharmacological enhancement of PGK activity successfully attenuated the PD phenotypes in several animal models (Cai et al., 2019). We also reported that parkinsonism was observed not only in a patient with PGK deficiency, but also in a heterozygous carrier of the disease who was originally considered to be asymptomatic (Sakaue et al., 2017). Noteworthy, PD symptoms observed in the heterozygous carrier in our case report was indistinguishable from that in typical PD (Sakaue et al., 2017). PGK-1 is located within the confirmed susceptibility locus for familial PD known as PARK12, implying that this gene might be the causative gene or one of the causative genes of PARK12. Therefore, we hypothesize that an insufficiency in PGK-1 expression may lead to the degeneration of DA neurons and confer susceptibility to PD. To provide supporting evidence for our hypothesis, we herein investigated the phenotypes of fly models with the knockdown of the Drosophila PGK-1 homologue, the Pgk gene. Drosophila has been used as a model organism to study human neurodegenerative diseases including PD (Chow and Reiter, 2017). Drosophila contains homologues of many PD-associated genes (Botella et al., 2009; Whitworth, 2011). The cell lineage of DA neurons during development has been clarified and DA neuron clusters in the central nervous system (CNS) may be easily identified in the adult stages (Botella et al., 2008). The data obtained from Drosophila Pgk knockdown flies indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons.