Enhanced activity of glycolytic enzymes in Drosophila and human cell models of Parkinson's disease based on DJ-1 deficiency

Abstract

Parkinson's disease (PD) is a neurodenerative debilitating disorder characterized by progressive disturbances in motor, autonomic and psychiatric functions. The pathological hallmark of PD is the loss of dopaminergic neurons in the substantia nigra pars compacta, which causes striatal dopamine deficiency. Although most PD cases are sporadic (iPD), approximately 5-10% of all patients suffer from monogenic PD forms caused by highly penetrant rare mutations segregating with the disease in families (fPD). One of the genes linked to monogenic PD is DJ-1. Mutations in DJ-1 cause autosomal recessive early-onset forms of fPD; however, it has been shown that an over-oxidized and inactive form of the DJ-1 protein is found in the brains of iPD individuals. Valuable insights into potential PD pathogenic mechanisms involving DJ-1 have been obtained from studies in cell and animal PD models based on DJ-1 deficiency such as Drosophila. Flies mutant for the DJ-1β gene, the Drosophila ortholog of human DJ-1, exhibited disease-related phenotypes such as motor defects, increased reactive oxygen species production and high levels of protein carbonylation. In the present study, we show that loss of DJ-1β function significantly increased the activities of several regulatory glycolytic enzymes. Similar results were obtained in DJ-1-deficient SH-SY5Y neuroblastoma cells, thus suggesting that loss of DJ-1 function in both PD models produces an enhancement of glycolysis. Our results also show that FDA-approved compounds such as meclizine and dimethyl fumarate, which have different clinical applications, are able to attenuate PD-related phenotypes in both models.Moreover, we found that they could exert their beneficial effect by increasing glycolysis through the activation of key glycolytic enzymes. Taken together, these results are consistent with the idea that increasing glycolysis could be a potential disease-modifying strategy for PD, as recently suggested. Besides, they also support further evaluation and potential repurposing of meclizine and dimethyl fumarate as modulators of energy metabolism for neuroprotection in PD.

A High-Throughput Chemical Screen in DJ-1β Mutant Flies Identifies Zaprinast as a Potential Parkinson's Disease Treatment

“…All cell culture materials were purchased from Biowest. Viability of cells treated with candidate compounds, the GPR35 antagonist CID2745687 and DMSO as vehicle was evaluated using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2-5diphenyltetrazolium bromide) assay, as previously described [22]. To determine whether CID2745687 was able to interfere with the neuroprotective effect of zaprinast, cells were pretreated for 2 h with different concentrations of the compound before the addition of zaprinast.…”

“…To determine whether CID2745687 was able to interfere with the neuroprotective effect of zaprinast, cells were pretreated for 2 h with different concentrations of the compound before the addition of zaprinast. Next, viability assays were carried out as described [22]…”

“…Protein carbonylation and H 2 O 2 levels were measured in 5-day-old DJ-1β mutant flies that were treated with the vehicle (0.1% DMSO) as control or treated with 10 μM zaprinast. Protein carbonyl groups were measured in fly extracts using 2,4-dinitrophenyl hydrazine derivatization in 96-well plates (Greiner 96-well plate, polypropylene) as previously described [22]. H 2 O 2 levels were measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) in fly extracts as previously described [34].…”

“…Protein extraction and Western blots of pLKO.1 and DJ-1-deficient SH-SY5Y cells treated with 1 µM zaprinast and vehicle (0.1% DMSO) under OS conditions were carried out as previously described [22]. The primary antibodies used were anti-Akt, anti-phospho-Akt (Ser473), anti-JNK, and anti-phospho-JNK (Thr183/Tyr185) (1:1000, Cell Signaling).…”

“…Mutations in one such gene, DJ-1 (also known as PARK7), associate with an early-onset recessive form of Parkinsonism [18]. DJ-1 was initially described as an oncogene, but additional functions for the DJ-1 protein include roles as an antioxidant via freeradical scavenging, a transcriptional regulator of antioxidant genes, a redox-dependent molecular chaperone, a modulator of mitochondrial function, a deglycase, and as a factor involved in proteolysis and metabolism [19][20][21][22]. Also, a study encountered an over-oxidized and inactive form of the DJ-1 protein in brains of sporadic PD patients [23], strongly suggesting that results obtained in animal and cell models of familial PD based on loss of DJ-1 function may also have relevance to the sporadic form of the disease [24].…”