DJ-1 Gene Research Articles

DJ-1 preserves ischemic postconditioning-induced cardioprotection in STZ-induced type 1 diabetic rats: role of PTEN and DJ-1 subcellular translocation

BackgroundIschemic postconditioning (IPostC) has been reported as a promising method for protecting against myocardial ischemia-reperfusion (MI/R) injury. Our previous study found that the infarct-limiting effect of IPostC is abolished in the heart of diabetes whose cardiac expression of DJ-1 (also called PARK7, Parkinsonism associated deglycase) is reduced. However, the role and in particular the underlying mechanism of DJ-1 in the loss of sensitivity to IPostC-induced cardioprotection in diabetic hearts remains unclear.MethodsStreptozotocin-induced type 1 diabetic rats were subjected to MI/R injury by occluding the left anterior descending artery (LAD) and followed by reperfusion. IPostC was induced by three cycles of 10s of reperfusion and ischemia at the onset of reperfusion. AAV9-CMV-DJ-1, AAV9-CMV-C106S-DJ-1 or AAV9-DJ-1 siRNA were injected via tail vein to either over-express or knock-down DJ-1 three weeks before inducing MI/R.ResultsDiabetic rats subjected to MI/R exhibited larger infarct area, more severe oxidative injury concomitant with significantly reduced cardiac DJ-1 expression and increased PTEN expression as compared to non-diabetic rats. AAV9-mediated cardiac DJ-1 overexpression, but not the cardiac overexpression of DJ-1 mutant C106S, restored IPostC-induced cardioprotection and this effect was accompanied by increased cytoplasmic DJ-1 translocation toward nuclear and mitochondrial, reduced PTEN expression, and increased Nrf-2/HO-1 transcription. Our further study showed that AAV9-mediated targeted DJ-1 gene knockdown aggravated MI/R injury in diabetic hearts, and this exacerbation of MI/R injury was partially reversed by IPostC in the presence of PTEN inhibition or Nrf-2 activation.ConclusionsThese findings suggest that DJ-1 preserves the cardioprotective effect of IPostC against MI/R injury in diabetic rats through nuclear and mitochondrial DJ-1 translocation and that inhibition of cardiac PTEN and activation of Nrf-2/HO-1 may represent the major downstream mechanisms whereby DJ-1 preserves the cardioprotective effect of IPostC in diabetes.Graphical abstract

MiR-199a/b-3p inhibits HCC cell proliferation and invasion through a novel compensatory signaling pathway DJ-1\\Ras\\PI3K/AKT

Several studies have reported the effects of DJ-1 gene and miR-199a/b-3p on HCC development. However, whether miR-199a/b-3p regulates HCC progression through a novel compensatory signaling pathway involving DJ-1, Ras, and PI3K/AKT remains unknown. We used (TCGA, HPA, miRWalk and Target scan) databases, cancer and para-tissue HCC patients, dual-luciferase reporter gene analysis, proteomic imprinting, qPCR, cell proliferation, scratch, transport, and flow cytometry to detect the molecular mechanism of DJ-1 and miR-199a/b-3p co-expression in HCC cell lines. Bioinformatics analysis showed that DJ-1 was highly expressed in HCC ((P < 0.001) were closely associated with tumor stage (T), portal vein vascular invasion, OS, DSS, and PFI (P < 0.05); miR-199a/b-3p was lowly expressed in HCC (P < 0.001), which was the upstream regulator of DJ-1. Spearman coefficient r = −0.113, P = 0.031; Dual luciferase gene report verified the negative targeting relationship between them P< 0.001; Western blotting demonstrated that miR-199a/b-3p could inhibit the protein expression of DJ-1, Ras and AKT(P < 0.05); The results of CCK8, cell scratch, Transwell migration and flow cytometry showed that OE + DJ-1 increased the proliferation, migration and invasion ability of HepG2 cells, and decreased the apoptosis process, and the differences were statistically significant (P < 0.05), while miR-199a/b-3p had the opposite effect (P < 0.05).

A Complex Interplay of DJ-1, LRRK2, and Nrf2 in the Regulation of Mitochondrial Function in Cypermethrin-Induced Parkinsonism.

Cypermethrin impairs mitochondrial function, induces redox imbalance, and leads to Parkinsonism in experimental animals. Knockdown of deglycase-1 (DJ-1) gene, which encodes a redox-sensitive antioxidant protein, aggravates cypermethrin-mediated α-synuclein overexpression and oxidative alteration of proteins. DJ-1 is also reported to be essential for maintaining stability of nuclear factor erythroid 2-related factor 2 (Nrf2), shielding cells against oxidative insult. Leucine-rich repeat kinase 2 (LRRK2), another protein associated with Parkinson's disease, is also involved in regulating mitochondrial function. However, underlying molecular mechanisms remain elusive. The study intended to explore an interaction of DJ-1, LRRK2, and Nrf2 in the regulation of mitochondrial function in cypermethrin-induced Parkinsonism. Small interfering RNA-mediated knockdown of DJ-1 and LRRK2 gene and pharmacological activation of Nrf2 were performed in rats and/or human neuroblastoma cells with or without cypermethrin. Indexes of oxidative stress, mitochondrial impairment, and Parkinsonism along with α-synuclein expression, post-translational modification, and aggregation were measured. DJ-1 gene knockdown exacerbated cypermethrin-induced increase in oxidative stress and intrinsic apoptosis and reduction in expression of mitochondrial antioxidant proteins via inhibiting nuclear translocation of Nrf2. Additionally, cypermethrin-induced oxidative stress, mitochondrial impairment, and α-synuclein expression and aggregation were found to be suppressed by LRRK2 gene knockdown, by promoting Nrf2 nuclear translocation and expression of mitochondrial antioxidant proteins. Furthermore, Nrf2 activator, sulforaphane, ameliorated cypermethrin-induced mitochondrial impairment and oxidative stress and provided protection against dopaminergic neuronal death. The findings indicate that DJ-1 and LRRK2 independently alter Nrf2-mediated changes and a complex interplay among DJ-1, LRRK2, and Nrf2 exists in the regulation of mitochondrial function in cypermethrin-induced Parkinsonism.

Effects of DJ‑1 on apoptosis and mitophagy of glomerular podocytes.

By studying the effects of DJ-1 overexpression and silencing on the morphological structure and mitophagy of glomerular podocytes, the present study aimed to identify the effects of DJ-1 on glomerular podocyte apoptosis and mitophagy. MPC5 mouse glomerular podocytes were cultured in vitro and divided into four groups: Control, DJ-1 overexpression, empty vector and DJ-1 silencing. DJ-1 gene overexpression and silencing models were prepared, the morphological structures of podocytes and mitochondria in each group were observed, and podocyte apoptosis and DJ-1/PTEN expression were subsequently detected in each group. The experimental results showed reduced volume, retracted foot processes, loosened intercellular connections, presence of dead cells, increased apoptotic rate, increased expression of PTEN, and swollen mitochondria due to the number of vacuoles and autophagosomes in podocytes in the DJ-1 silencing group. The surface areas of podocytes in the DJ-1 overexpression group were greater than those in the control group. Moreover, the structure of the foot processes was more obvious, the number of cells was greater, the intercellular connections were closer, the apoptotic rate was reduced, the expression of PTEN was decreased, the mitochondrial structure was more obvious and the mitochondrial cristae were more whole. Notably, there were no differences between the empty vector and control groups. In conclusion, these results indicated that DJ-1 may regulate podocyte apoptosis and mitophagy through the DJ-1/PTEN pathway, and could maintain the stability of the normal morphology, structure and function of glomerular podocytes.

Molecular and Physiological Determinants of Amyotrophic Lateral Sclerosis: What the DJ-1 Protein Teaches Us.

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease which causes the progressive degeneration of cortical and spinal motoneurons, leading to death a few years after the first symptom onset. ALS is mainly a sporadic disorder, and its causative mechanisms are mostly unclear. About 5-10% of cases have a genetic inheritance, and the study of ALS-associated genes has been fundamental in defining the pathological pathways likely also involved in the sporadic forms of the disease. Mutations affecting the DJ-1 gene appear to explain a subset of familial ALS forms. DJ-1 is involved in multiple molecular mechanisms, acting primarily as a protective agent against oxidative stress. Here, we focus on the involvement of DJ-1 in interconnected cellular functions related to mitochondrial homeostasis, reactive oxygen species (ROS) levels, energy metabolism, and hypoxia response, in both physiological and pathological conditions. We discuss the possibility that impairments in one of these pathways may affect the others, contributing to a pathological background in which additional environmental or genetic factors may act in favor of the onset and/or progression of ALS. These pathways may represent potential therapeutic targets to reduce the likelihood of developing ALS and/or slow disease progression.

Investigation of codon alternation patterns in two neurodegenerative diseases through numerical representation and codon classification

Several evidences claim similarities between two neuro-degenerative diseases viz.Parkinson's disease and Glaucoma. But proper pathogenetic factors specially in bio-molecular level are still not cleared. Hence, the prime objectives of the study here is to investigate evolutionary relationships among the genes involved in the diseases and to understand the pattern of mutations responsible in the two disease pathogenesis. Reported research work established that the strengths of nucleotides (according to number of hydrogen bonds they have) have a deep impact on protein formation. Moreover, depending upon the position the virulence of mutation differs. Effective mutation analysis can help to predict the fate of the diseased individual which can be validated later by in-vitro experiments. In this present study, PARKIN, PINK1, and DJ1 genes are taken for Parkinson's disease and CYP1B1 and MYOC for Glaucoma. Importance has been given to the nucleotides existing at first and second positions of a codon. We followed Rumer's classification of doublets based on the strength of nucleotides and Duplij’s numeric interpretation of the strength of each nucleotide as “determinative degree” to classify the 64 codons and corresponding amino acids into three major classes viz. strong, transitional, and weak. The determinative degrees of all 64 codons and corresponding 20 amino acids are calculated to define their strengths. The complete analysis is carried out based on this classification scheme. Further we proposed an alignment-free method SSADDA (Sequence Similarity Analysis using Determinative Degree of Amino acid) based on determinative degree of amino acid and applied to the wild type amino acid sequences to measure proximity among themselves. The methodology gives us a more microscopic view of the existing genetic code table.

Upregulation of DJ-1 in Dopaminergic Neurons by a Physically-Modified Saline: Implications for Parkinson’s Disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder in human and loss-of-functions DJ-1 mutations are associated with a familial form of early onset PD. Functionally, DJ-1 (PARK7), a neuroprotective protein, is known to support mitochondria and protect cells from oxidative stress. Mechanisms and agents by which the level of DJ-1 could be increased in the CNS are poorly described. RNS60 is a bioactive aqueous solution created by exposing normal saline to Taylor-Couette-Poiseuille flow under high oxygen pressure. Recently we have described neuroprotective, immunomodulatory and promyelinogenic properties of RNS60. Here we delineate that RNS60 is also capable of increasing the level of DJ-1 in mouse MN9D neuronal cells and primary dopaminergic neurons, highlighting another new neuroprotective effect of RNS60. While investigating the mechanism we found the presence of cAMP response element (CRE) in DJ-1 gene promoter and stimulation of CREB activation in neuronal cells by RNS60. Accordingly, RNS60 treatment increased the recruitment of CREB to the DJ-1 gene promoter in neuronal cells. Interestingly, RNS60 treatment also induced the enrollment of CREB-binding protein (CBP), but not the other histone acetyl transferase p300, to the promoter of DJ-1 gene. Moreover, knockdown of CREB by siRNA led to the inhibition of RNS60-mediated DJ-1 upregulation, indicating an important role of CREB in DJ-1 upregulation by RNS60. Together, these results indicate that RNS60 upregulates DJ-1 in neuronal cells via CREB-CBP pathway. It may be of benefit for PD and other neurodegenerative disorders.

Searching for Biomarkers in the Blood of Patients at Risk of Developing Parkinson's Disease at the Prodromal Stage.

Parkinson's disease (PD) is diagnosed many years after its onset, under a significant degradation of the nigrostriatal dopaminergic system, responsible for the regulation of motor function. This explains the low effectiveness of the treatment of patients. Therefore, one of the highest priorities in neurology is the development of the early (preclinical) diagnosis of PD. The aim of this study was to search for changes in the blood of patients at risk of developing PD, which are considered potential diagnostic biomarkers. Out of 1835 patients, 26 patients were included in the risk group and 20 patients in the control group. The primary criteria for inclusion in a risk group were the impairment of sleep behavior disorder and sense of smell, and the secondary criteria were neurological and mental disorders. In patients at risk and in controls, the composition of plasma and the expression of genes of interest in lymphocytes were assessed by 27 indicators. The main changes that we found in plasma include a decrease in the concentrations of l-3,4-dihydroxyphenylalanine (L-DOPA) and urates, as well as the expressions of some types of microRNA, and an increase in the total oxidative status. In turn, in the lymphocytes of patients at risk, an increase in the expression of the DA D3 receptor gene and the lymphocyte activation gene 3 (LAG3), as well as a decrease in the expression of the Protein deglycase DJ-1 gene (PARK7), were observed. The blood changes we found in patients at risk are considered candidates for diagnostic biomarkers at the prodromal stage of PD.

Analysis of the structural dynamics of the mutations in the kinase domain of PINK1 protein associated with Parkinson’s disease

Parkinson’s disease (PD) is a very common neurodegenerative disorder and is considered to be one of the most severe disorders worldwide. Mutations in some PD causing genes are responsible for the early onset of the disease. Pathogenic variants in parkin, PINK1 and DJ1 genes can cause early-onset of PD. Many PINK1 gene mutations have been reported, but not all variants are pathogenic. The gene product of PINK1, also known as PINK1 protein, has 581 amino acid residues in it. Several different mutations are present throughout the kinase domain of PINK1 protein. In this work, we used in silico approaches to analyze the different types of mutations that are distributed in the kinase domain of the PINK1 protein. Based on our results, we categorized the mutations as high, moderate and low pathogenic variants. Furthermore, we performed molecular dynamics simulations of the pathogenic PINK1 variants to decipher their possible impacts on the structure and made a comparison with the wild type PINK1. In conclusion, we suggested the possible mechanistic roles of the pathogenic variants of PINK1 kinase domain that can affect its function. These pathogenic variants are the causative agents of early onset of PD called autosomal recessive Parkinson disease.

Interval training and Crataegus persica ameliorate diabetic nephropathy via miR-126/Nrf-2 mediated inhibition of stress oxidative in rats with diabetes after myocardial ischemia-reperfusion injury

Myocardial disorders are the most common cause of renal failure and mortality in diabetic patients, but the molecular mechanism of this process is not yet clear. The reduction of nuclear Erythroid2-related factor-2 (Nrf-2) and positive regulators of Nrf-2 proteins, such as DJ-1 and microRNA-126 (miR-126), after hypoxia and the promotion of reactive oxygen species, might be an intervention indicator in renal failure after myocardial ischemia-reperfusion. Therefore, this study evaluates the renoprotective effect of exercise training and Crataegus persica extract (CE) on myocardial ischemia-reperfusion-induced kidney injury in diabetic rats. Fifty rats were divided into five groups: healthy sedentary control (Con), sedentary diabetic (D), interval trained diabetic (TD), diabetic plus Crataegus persica extract treatment (CD), and interval trained diabetic plus Crataegus persica extract treatment (TCD) groups. The rats in the exercise groups were subjected to moderate-intensity interval training five days per week for ten weeks. The rats in CD and TCD groups received 300 mg/kg of Crataegus persica through gavage for ten weeks. Then, the subjects underwent 30 min of myocardial ischemia and subsequently reperfusion for 24 h. At the end of the experiment, insulin sensitivity, oxidative stress, renal function, histopathology of the kidney, Nrf-2, miR-126, and DJ-1 gene expression levels were evaluated. The results show that the treatments decreased elevated levels of renal oxidative stress, glomerular filtration rate, insulin sensitivity, and pathological score in diabetic rats. Also, the expression of Nrf-2 and miR-126, unlike DJ-1, decreased in diabetic rats due to interval training. Due to the results, diabetes aggravates acute myocardial ischemia-reperfusion-induced kidney injury, while moderate-intensity interval training and Crataegus persica treatment simultaneously ameliorate myocardial ischemia-reperfusion-induced renal injury via miR-126/Nrf-2 pathway and improve insulin sensitivity and renal function in type 1 diabetic rats.

ZIF-8 nanoparticles induce neurobehavioral disorders through the regulation of ROS-mediated oxidative stress in zebrafish embryos

Zeolite imidazolate framework-8 (ZIF-8) is a nanomaterial of metal-organic frameworks (MOFs), which have various applications in drug delivery and water pollution remediation. However, little is known about its developmental neurotoxicity in aquatic organisms, especially on the low-level exposure. In the present study, we investigated the toxic effects of ZIF-8 NPs on the neuron development, behavioral traits, oxidative stress and gene expression in zebrafish embryos. Firstly, our results showed that ZIF-8 induced significantly embryonic malformations and abnormal development of nervous system in zebrafish embryos with a concentration-dependent manner. Meanwhile, the locomotor behavior was obviously inhibited while the anxiety behavior was greatly increased after ZIF-8 exposure. Secondly, the levels of ROS and antioxidant enzyme activities (CAT, SOD and MDA) together with AChE and ATPase were substantially increased in the ZIF-8 exposed groups. At the molecular level, ZIF-8 NPs could down-regulate the expression profiles of neural development-related genes (gap43, synapsin 2a and neurogenin 1) and PD-like related genes (dj-1, dynactin and parkin), but up-regulate the expression levels of neuro-inflammatory genes (nox-1, glip1a and glip1b) in larval zebrafish. In addition, we further explored the molecular mechanism of neurotoxicity induced by ZIF-8 with pharmacological experiments. The results showed that specific inhibition of ROS-mediated oxidative stress by the astaxanthin could reverse the expression patterns of ATPase, AChE and neurodevelopmental genes. Moreover, astaxanthin can partially rescue the ZIF-8-modulated locomotor behavior. Taken together, our results demonstrated that ZIF-8 had the potential to cause neurotoxicity in zebrafish embryos. These informations presented in this study will help to elucidate the molecular mechanisms of ZIF-8 nanoparticles exposure in zebrafish, which providing a scientific evaluation of its safety to aquatic ecosystems.

A Study on Curcumol Influencing Proliferation and Apoptosis of Hepatocellular Carcinoma Cells through DJ-1/PTEN/PI3K/AKT Pathway.

Objective To study the mechanism of curcumol affecting the proliferation and apoptosis of liver cancer cells through the DJ-1/PTEN/PI3K/AKT pathway. Method HepG2 cells were cultured in vitro, treated with curcumol at concentrations of 10, 30, and 100 μg/mL, and DMSO was used as a control. The levels of cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. RT-PCR and western blot were used to detect PTEN, p-AKT, DJ-1, and PI3K gene and protein expression changes. Result (1) Compared with the DMSO blank control group, the proliferation level of liver cancer cells in the 10 μg/mL curcumol group decreased, and the proportion of apoptosis increased (p <0.05). (2) Compared with the blank control group and the 10 and 30 μg/mL concentration groups, the proliferation level of liver cancer cells in the 100 μg/mL curcumol group was significantly reduced, and the proportion of cell apoptosis was significantly increased (p < 0.05). (3) Curcumol can significantly increase the expression of PTEN gene and protein in liver cancer cells and reduce the expression of DJ-1 and PI3K genes and protein in liver cancer cells (p < 0.05). Conclusion Curcumol can regulate DJ-1, PTEN, PI3K, and AKT signal transduction pathways, inhibit cell proliferation, and cause a significant increase in the proportion of cell apoptosis, and the pharmacodynamic effect of curcumol is dependent on the time and dose of action.

3-Pyridinylboronic Acid Ameliorates Rotenone-Induced Oxidative Stress Through Nrf2 Target Genes in Zebrafish Embryos.

Parkinson's disease (PD) is one of the most common forms of neurodegenerative diseases and research on potential therapeutic agents for PD continues. Rotenone is a neurotoxin that can pass the blood-brain barrier and is used to generate PD models in experimental animals. Boron is a microelement necessary for neural activity in the brain. Antioxidant, non-cytotoxic, anti-genotoxic, anti-carcinogenic effects of boric acid, the salt compound of boron has been reported before. Boronic acids have been approved for treatment by FDA and are included in drug discovery studies and pyridine boronic acids are a subclass of heterocyclic boronic acids used in drug design and discovery as substituted pyridines based on crystal engineering principles. The aim of our study was to determine the effect of 3-pyridinylboronic acid in rotenone-exposed zebrafish embryos, focusing on oxidant-antioxidant parameters and gene expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes gclm, gclc, hmox1a, nqo1, and PD related genes, brain-derived neurotrophic factor, dj1, and tnfα. Zebrafish embryos were exposed to Rotenone (10 μg/l); Low Dose 3-Pyridinylboronic acid (100 μM); High Dose 3-Pyridinylboronic acid (200 μM); Rotenone + Low Dose-3-Pyridinylboronic acid (10 μg/l + 100 μM); Rotenone + High Dose-3-Pyridinylboronic acid (10 μg/l + 200 μM) in well plates for 96 h post-fertilization (hpf). Our study showed for the first time that 3-pyridinylboronic acid, as a novel sub-class of the heterocyclic boronic acid compound, improved locomotor activities, ameliorated oxidant-antioxidant status by decreasing LPO and NO levels, and normalized the expressions of bdnf, dj1, tnf⍺ and Nrf2 target genes hmox1a and nqo1 in rotenone exposed zebrafish embryos. On the other hand, it caused the deterioration of the oxidant-antioxidant balance in the control group through increased lipid peroxidation, nitric oxide levels, and decreased antioxidant enzymes. We believe that these results should be interpreted in the context of the dose-toxicity and benefit-harm relationship of the effects of 3-pyridinylboronic.

CDNA Cloning and Partial Characterization of the DJ-1 Gene from Tribolium castaneum.

The DJ-1 gene is highly conserved across a wide variety of organisms and it plays a role in anti-oxidative stress mechanisms in cells. The red flour beetle, Tribolium castaneum, is widely used as a model insect species because it is easy to evaluate gene function in this species using RNA interference (RNAi). The T. castaneum DJ-1 (TcDJ-1) sequence is annotated in the T. castaneum genome database; however, the function and characteristics of the TcDJ-1 gene have not been elucidated. Here, we investigated the cDNA sequence of TcDJ-1 and partially characterized its function. First, we examined the TcDJ-1 amino acid sequence and found that it was highly conserved with sequences from other species. TcDJ-1 mRNA expression was higher in the early pupal and adult developmental stages. We evaluated oxidant tolerance in TcDJ-1 knockdown adults using paraquat and found that adults with TcDJ-1 knockdown exhibited increased sensitivity to paraquat. Our findings show that TcDJ-1 has an antioxidant function, as observed for DJ-1 from other insects. Therefore, these results suggest that TcDJ-1 protects against oxidative stress during metamorphosis.

Clinical and molecular genetic findings of hereditary Parkinson's patients from Turkey

IntroductionThe majority of Parkinson's disease (PD) ensue late-onset with a complex spectrum of environmental and genetic risk factors. Awareness of genetic causes in patients with PD is essential for genetic counseling and future genotype-oriented therapeutic developments. MethodsLarge pathogenic changes in eight PD-related genes and small pathogenic sequence variants in 22 PD-related genes were investigated simultaneously in 82 PD patients from 79 families where clinical evaluations were performed. The phenotypic characteristics of the patients with molecular changes were examined for genotype-phenotype relations. ResultsPathogenic variants in SNCA, PRKN, DJ-1, FBXO7, and GBA genes were determined in 25 patients from 24 families (24/79, 30%). Associated variants were found in PRKN in 14, SNCA in three, FBXO7 in two, and DJ-1 in one patient. A novel homozygous deletion (c.491delT, p.(V164Dfs*13) (SCV001733595)) leading to protein truncation in the PRKN gene was identified in two patients from the same family. Furthermore, heterozygous GBA gene variants were detected in five patients from different families. ConclusionIt has been shown that the most common cause of genetically transmitted PD is the PRKN gene, while LRRK2 does not play an essential role in this selected population. It has been suggested that even if the autosomal recessive inheritance is expected, genes with autosomal dominant effects such as SNCA should not be overlooked and suggested for investigation. Our study is also the first for evaluating the pathogenic GBA variants’ frequency in PD patients from Turkey.

Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype

Whether direct manipulation of Parkinson’s disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6–10 months), and thus provides a practical transgenic monkey model for future PD studies.

DJ-1 Protein and Its Role in the Development of Parkinson's Disease: Studies on Experimental Models.

DJ-1, also known as Parkinson's disease protein 7, is a multifunctional protein ubiquitously expressed in cells and tissues. Interacting with proteins of various intracellular compartments, DJ-1 plays an important role in maintaining different cellular functions. Mutant DJ-1 forms containing amino acid substitutions (especially L166P), typical of Parkinson's disease, are characterized by impaired dimerization, stability, and folding. DJ-1 exhibits several types of catalytic activity; however, in the enzyme classification it exists as protein deglycase (EC 3.5.1.124). Apparently, in different cell compartments DJ-1 exhibits catalytic and non-catalytic functions, and their ratio still remains unknown. Oxidative stress promotes dissociation of cytoplasmic DJ-1 dimers into monomers, which are translocated to the nucleus, where this protein acts as a coactivator of various signaling pathways, preventing cell death. In mitochondria, DJ-1 is found in the synthasome, where it interacts with the β ATP synthase subunit. Downregulation of the DJ-1 gene under conditions of experimental PD increases sensitivity of the cells to neurotoxins, and introduction of the recombinant DJ-1 protein attenuates manifestation of this pathology. The thirteen-membered fragment of the DJ-1 amino acid sequence attached to the heptapeptide of the TAT protein penetrating into the cells exhibited neuroprotective properties in various PD models both in cell cultures and after administration to animals. Low molecular weight DJ-1 ligands also demonstrate therapeutic potential, providing neuroprotective effects seen during their incubation with cells and administration to animals.

DJ-1 alleviates oxidative stress injury by activating the Nrf2 pathway in rats with cerebral ischemia-reperfusion injury

To investigate the antioxidant effect of DJ-1 (Park7) in rats with cerebral ischemia/reperfusion (IR) injury and its potential mechanism. A total of 108 SD rats were randomly divided into sham-operated group, middle cerebral artery occlusion (MCAO) group, Scramble group, DJ-1 siRNA group, negative control (NC) group and DJ-1 overexpression group. Except for those in the sham group, all the rats were subjected to MCAO to establish models of cerebral IR injury. In DJ-1 siRNA and DJ-1 overexpression group, a DJ-1 siRNA and an adeno-associated virus vector carrying DJ-1 gene was injected into the lateral ventricle of the rats, respectively. In each group, neurological scores and brain water content were determined after the operation, and pathological changes of the brain tissue and neuronal injury in the cortical infarction area were assessed using HE and Nissl staining. Oxidative stress in the brain tissues was analyzed by detecting superoxide dismutase (SOD) and malondialdehyde (MDA). The expression levels of DJ-1, Nrf2, Ho-1 and NQO1 in the brain tissue were detected with Western blotting, and the expression and nucleation of Nrf2 was determined by immunofluorescence staining. Compared with those in MCAO group, the neurological scores (P < 0.001) and brain water content (P < 0.001) were significantly increased in DJ-1 siRNA group. Intracerebral injection of DJ-1 siRNA following MCAO obviously aggravated neuron injury in cerebral ischemia region, further reduced SOD activity and increased MDA content (P < 0.001), and significantly lowered the expression levels of Nrf2 and its downstream proteins HO-1 and NQO1 (P < 0.001). Intracerebral injection of the adenoviral vector for DJ-1 (P=0.003) overexpression significantly upregulated the levels of Nrf2 (P=0.006) and its downstream proteins HO-1 (P=0.004) and NQO1 (P=0.014). As an important neuroprotective factor, DJ-1 alleviates oxidative stress induced by cerebral IR injury in rats by activating the Nrf2 pathway.

Frequency of mutations in PRKN, PINK1, and DJ1 in Patients With Early-Onset Parkinson Disease from neighboring countries in Central Europe

IntroductionApproximately 10% of patients with Parkinson disease (PD) present with early-onset disease (EOPD), defined as diagnosis before 50 years of age. Genetic factors are known to contribute to EOPD, with most commonly observed mutations in PRKN, PINK1, and DJ1 genes. The aim of our study was to analyze the frequency of PRKN, PINK1, and DJ1 mutations in an EOPD series from 4 neighboring European countries: Czech Republic, Germany, Poland, and Ukraine. MethodsDiagnosis of PD was made based on UK Brain Bank diagnostic criteria in departments experienced in movement disorders (1 from Czech Republic, 1 from Germany, 9 from Poland, and 3 from Ukraine). EOPD was defined as onset at or before 50 years of age. Of the 541 patients recruited to the study, 11 were Czech, 38 German, 476 Polish, and 16 Ukrainian. All cohorts were fully screened with Sanger sequencing for PRKN, PINK1, and DJ1 and multiplex ligation-dependent probe amplification for exon dosage. ResultsPRKN homozygous or double heterozygous mutations were identified in 17 patients: 1 Czech (9.1%), 1 German (2.6%), 14 Polish (2.9%), and 1 Ukrainian (6.3%). PINK1 homozygous mutations were only identified in 3 Polish patients (0.6%). There were no homozygous or compound heterozygous DJ1 mutations in analyzed subpopulations. One novel variant in PRKN was identified in the Ukrainian series. ConclusionIn the analyzed cohorts, mutations in the genes PRKN, PINK1, and DJ1 are not frequently observed.

A Flavonoid-Rich Extract of Mandarin Juice Counteracts 6-OHDA-Induced Oxidative Stress in SH-SY5Y Cells and Modulates Parkinson-Related Genes.

Parkinson’s disease (PD) is a degenerative disorder of the nervous system due to unceasing impairment of dopaminergic neurons situated in the substantia nigra. At present, anti-PD drugs acting on dopamine receptors are mainly symptomatic and have only very limited neuroprotective effects, whereas drugs slowing down neurodegeneration of dopaminergic neurons and deterioration of clinical symptoms are not yet available. Given that, the development of more valuable pharmacological strategies is highly demanded. Comprehensive research on innovative neuroprotective drugs has proven that anti-inflammatory and antioxidant molecules from food sources may prevent and/or counteract neurodegenerative diseases, such as PD. The present study was aimed at the evaluation the protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y human neuroblastoma cell death. Treatment of differentiated SH-SY5Y cells with 6-OHDA brought cell death, and specifically, apoptosis, which was significantly inhibited by the preincubation with MJe through caspase 3 blockage and the modulation of p53, Bax, and Bcl-2 genes. In addition, it showed antioxidant properties in abiotic models as well as in vitro, where it reduced both reactive oxygen and nitrogen species induced by 6-OHDA, along with restored mitochondrial membrane potential, and prevented the oxidative DNA damage evoked by 6-OHDA. Furthermore, MJe restored the impaired balance of SNCA, LRRK2, PINK1, parkin, and DJ-1 gene levels, PD-related factors, caused by 6-OHDA oxidative stress. Overall, these results indicate that MJe exerts neuroprotective effects against 6-OHDA-induced cell death in SH-SY5Y cells by mechanisms involving both the specific interaction with intracellular pathways and its antioxidant capability. Our study suggests a novel possible strategy to prevent and/or ameliorate neurodegenerative diseases, such as PD.