Unveiling the regulatory of miR-101-3p on ZNF746 in a Parkinson's disease cell model: Implications for therapeutic targeting.

In this study, we explored the regulatory role of microRNA miR-101-3p on the zinc finger protein 746 (ZNF746), also known as PARIS, which is implicated in both sporadic and familial forms of Parkinson's disease. In a Parkinson's disease cell model, utilizing SH-SY5Y cells treated with 1-methyl-4-phenylpyridine (MPP+), we observed that miR-101-3p was downregulated, while ZNF746 was upregulated. To investigate the direct impact of miR-101-3p on ZNF746, our team conducted overexpression experiments, successfully reversing ZNF746's expression at both the mRNA and protein levels, as confirmed through quantitative PCR and western blotting. We also performed luciferase assays, providing compelling evidence that ZNF746 is a direct target of miR-101-3p. Additionally, we noted that miR-101-3p overexpression resulted in increased expression of PGC1α, a gene targeted by ZNF746. Functionally, we assessed the implications of miR-101-3p overexpression through MTS assays and flow cytometry, revealing significant promotion of cell viability, inhibition of ROS production, and reduced apoptosis in the Parkinson's disease cell model. In conclusion, this study highlights the role of miR-101-3p in regulating ZNF746 expression and suggests its potential as a therapeutic target for Parkinson's disease. These findings provide valuable molecular insights that could pave the way for innovative treatment strategies in combating this debilitating neurodegenerative disorder.

Compound heterozygous p. Arg949Trp and p. Gly970Ala mutations deteriorated the function of PEX1p: A study on PEX1 in a patient with Zellweger syndrome.

The peroxisome is responsible for a variety of vital pathways in primary metabolism, including the very long-chain fatty-acid oxidation and plasmalogen lipid biosynthesis. Autosomal recessive disorder of the Zellweger spectrum (ZSD) is a major subset of peroxisome biogenesis disorders (PBDs) that can be caused by mutations in any of the 14 PEX genes. Zellweger syndrome (ZS) is the foremost common and severe phenotype within the heterogeneous ZSD. However, missense mutations encode proteins with residual functions, which are associated with phenotypes that are milder than ZS. Mutations in the PEX1 gene are among the most prevalent. PEX1 and PEX6 proteins, belonging to the AAA family of ATPases, form a hexameric complex, which is associated with peroxisome membranes and essential for peroxisome biology. In this study, a two-month-old Iranian boy with hypotonia, poor feeding, and difficulty in breathing was diagnosed with Zellweger syndrome. The parents of the patient were second cousins and healthy and no similar cases were observed in the parents' family. The PEX1 gene was sequenced in the patient and his parents. The compound heterozygous mutations, p. Arg949Trp and p. Gly970Ala, were identified in the patient, while the parents were heterozygous for these alleles. Sequence analysis of the mutant PEX1 D2 domain revealed that mutation p. Arg949Trp precisely occurred in a conserved arginine residue (P4 Arg), which hinders the substrate processing of the complex. Several database records have reported mutation p. Arg949Trp(R949W) but its clinical significance is given as uncertain. We report here a novel mutation, p. Gly970Ala, which is not recorded before and may prevent proper interaction of PEX1 and PEX6 proteins. In summary, the clinical findings and peroxisome profile of the patient suggested that compound heterozygosity for these two missense mutations resulted in a nonfunctional PEX1/PEX6 complex causing the severe ZS phenotype.

Correction: A compound downregulation of SRRM2 and miR-27a-3p with upregulation of miR-27b-3p in PBMCs of Parkinson's patients is associated with the early stage onset of disease.

[This corrects the article DOI: 10.1371/journal.pone.0240855.].

A compound downregulation of SRRM2 and miR-27a-3p with upregulation of miR-27b-3p in PBMCs of Parkinson's patients is associated with the early stage onset of disease.

Parkinson's disease (PD) is diagnosed when motor symptoms emerges, which almost 70% of dopamine neurons are lost. Therefore, early diagnosis of PD is crucial to prevent the progress of disease. Blood-based biomarkers, which are minimally invasive, potentially used for diagnosis of PD, including miRNAs. The aim of this study was to assess whether SRRM2 and miR-27a/b-3p could act as early diagnostic biomarkers for PD. Total RNAs from PBMCs of 30 PD's patients and 14 healthy age and gender matched subjects was extracted. The expression levels of respective genes were assessed. Data were presented applying a two-tailed unpaired t-test and one-way ANOVA. We observed significant down-regulation of SRRM2 (p = 0.0002) and miR-27a-3p (p = 0.0001), and up-regulation of miR-27b-3p (p = 0.02) in PBMCs of Parkinson's patients. Down-regulation of miR-27a-3p is associated with increasing disease severity, whereas the up-regulation of miR-27b-3p was observed mostly at HY-1 and disease duration between 3-5 years. There was a negative correlation between SRRM2 and miR-27b-3p expressions, and miR-27a-3p positively was correlated with miR-27b-3p. Based on functional enrichment analysis, SRRM2 and miR-27a/b-3p acted on common functional pathways. miR-27a/b-3p could potentially predict the progression and severity of PD. Although both miRs had no similarity on expression, a positive correlation between both miRs was identified, supporting their potential role as biomarkers in clinical PD stages. Of note that SRRM2 and miR-27a-3p were able to distinguish PD patients from healthy individuals. Functional analysis of the similarity between genes associated with SRRM2 and miR-27a/b-3p indicates common functional pathways and their dysfunction correlates with molecular etiopathology mechanisms of PD onset.

Could analysis of testis-specific genes, as biomarkers in seminal plasma, predict presence of focal spermatogenesis in non-obstructive azoospermia?

Cell-free seminal mRNA (cfs-mRNA) exists in the human ejaculate that is proposed as a potential noninvasive procedure to prognosis pathophysiological conditions. This study applied cfs-mRNA of ESX1, ZMYND15 and its target haploid genes (TNP1 and PRM1) to identify the presence of spermatozoa in men with azoospermia. This study included 35 semen samples from 16 normozoospermic and 19 nonobstructive azoospermic individuals. Expression levels of target genes were determined by real-time reverse transcription-polymerase chain reaction using ΔΔCt method. The expression level of these genes (ZMYND15, TNP1 and PRM1) was significantly decreased in semen samples of nonobstructive azoospermia compared to normozoospermia. Similarly, the expression level of TNP1 and PRM1 was significantly decreased in the sample with negative sperm (SR-) versus positive sperm retrieval (SR+). The expression level of these genes may have the potential for prediction of successful sperm retrieval with high sensitivity and specificity according to the receiver operating characteristics (ROC) curve analysis.

Expression Profiling of Blood microRNAs 885, 361, and 17 in the Patients with the Parkinson's disease: Integrating Interaction Data to Uncover the Possible Triggering Age-Related Mechanisms.

MicroRNAs (miRNAs) have been reported to contribute to the pathophysiology of the Parkinson's disease (PD), an age related-neurodegenerative disorder. The aim of present study was to compare the expression profiles of a new set of candidate miRNAs related to aging and cellular senescence in peripheral blood mononuclear cells (PBMCs) obtained from the PD patients with healthy controls and then in the early and advanced stages of the PD patients with their controls to clarify whether their expression was correlated with the disease severity. We have also proposed a consensus-based strategy to interpret the miRNAs expression data to gain a better insight into the molecular regulatory alterations during the incidence of PD. We evaluated the miRNA expression levels in the PBMCs obtained from 36 patients with PD and 16 healthy controls by the reverse transcription-quantitative real-time PCR and their performance to discriminate the PD patients from the healthy subjects assessed using the receiver operating characteristic curve analysis. Also, we applied our consensus and integration approach to construct a deregulated miRNA-based network in PD with the respective targets and transcription factors, and the enriched gene ontology and pathways using the enrichment analysis approach were obtained. There was a significant overexpression of miR-885 and miR-17 and the downregulation of miR-361 in the PD patients compared to the controls. The blood expression of miR-885 and miR-17 tended to increase along with the disease severity. On the other hand, the lower levels of miR-361 in the early stages of the PD patients, as compared to controls, and its higher levels in the advanced stages of PD patients, as compared to the early stages of the PD patients, were observed. Combination of all three miRNAs showed an appropriate value of AUC (0.985) to discriminate the PD patients from the healthy subjects. Also, the deregulated miRNAs were linked to the known PD pathways and the candidate related target genes were presented. We revealed 3 candidate biomarkers related to aging and cellular senescence for the first time in the patients with PD. Our in-silico analysis identified candidate target genes and TFs, including those related to neurodegeneration and PD. Overall, our findings provided novel insights into the probable age-regulatory mechanisms underlying PD and a rationale to further clarify the role of the identified miRNAs in the PD pathogenesis.

Embryos derived from couples with consanguineous marriages with globozoospermia should be screened for gender or DPY19L2 deletion.

Globozoospermia or round-headed spermatozoa are a rare type of infertility which accounts for <0.1% of male infertility. Several genes are associated with this disease, including DPY19L2, SPATA16, PICK1 and CCIN that DPY19L2 accounts for 75% of globozoospermia. Isfahan Fertility and Infertility Center (IFIC) is a referral centre for globozoospermia, and individuals with globozoospermia are routinely screened for DPY19L2 deletion. In the present study, we have screened six couples with globozoospermia and consanguineous marriages. Genomic DNA both female and male partners were screened for DPY19L2 deletion for exons 1, 11 and 22 as exons most prone to non-homologous recombination. In addition, qPCR was carried out on genomic samples of their partners to determine whether they are heterozygous for DPY19L2 deletion. The results revealed that one female was heterozygous for DPY19L2 deletion. Therefore, this couple decided to undergo intracytoplasmic sperm injection and gender selection and two XX embryos were transferred for this couple and two healthy girls were born. In conclusion, we advise for the couples with DPY19L2-globozoospermia and consanguineous marriages to be screened for DPY19L2 deletion in the hope of reducing occurrence of globozoospermia in future progeny.

Downregulation of miR-130a, antagonized doxorubicin-induced cardiotoxicity via increasing the PPARγ expression in mESCs-derived cardiac cells.

Doxorubicin (Dox) is a widely used powerful chemotherapeutic component for cancer treatment. However, its clinical application has been hampered due to doxorubicin-induced cardiomyopathy upon the cessation of chemotherapy. Previous studies revealed that PPARγ plays a crucial protective role in cardiomyocytes. Modulation of miRNA expression is an applicable approach for prohibition of toxicity induction. Therefore, the aim of present study is uprising of PPARγ transcript levels via manipulation of miRNAs to limit Dox-induced cardiotoxicity in mESCs-derived cardiac cells, as in vitro model cell to provide a simple direct approach for further clinical therapies. Based on bioinformatics data mining, eventually miR-130a was selected to target PPARγ. This miRNA is highly expressed in heart. The expression of miR-130a increases sharply upon Dox treatment while specific antagomiR-130a reverses Dox-induced reduced expression of PPARγ, cellular apoptosis, and inflammation. Our data strongly suggest that antagomiR-130a limits Dox-induced cellular toxicity via PPARγ upregulation and may have clinical relevance to limit in vivo Dox toxicity.

Expression of ZMYND15 in Testes of Azoospermic Men and Association With Sperm Retrieval.

OBJECTIVE: To compare the expression levels of ZMYND15 and its target haploid genes (TNP1, PRM1, and SPEM1) in testicular samples of non-obstructive azoospermia (NOA) vs obstructive azoospermia (OA). The levels of these transcripts were also compared in azoospermic samples with positive and negative sperm retrieval (SR). METHOD: This study included 63 testis biopsy samples from 16 OA and 47 NOA individuals. Expression levels of these target genes were determined by real-time reverse transcription-polymerase chain reaction using ΔΔCt method. RESULT: The expression level of ZMYND15 and its target genes were significantly lower in testicular samples of NOA compared with OA. Similarly, the expression levels of these transcripts were lower in samples with negative vs positive SR. CONCLUSION: Expression level of ZMYND15 may have potential for prediction of successful SR with sensitivity of 90% and specificity of 60% for total population and sensitivity of 100% and specificity of 75% for NOA, according to the receiver operating characteristics curve.

Ameliorating the Effect of Pioglitazone on LPS-Induced Inflammation of Human Oligodendrocyte Progenitor Cells.

Oligodendrocyte progenitor cells (OPCs) are appropriate model cells for studying the progress of neurodegenerative disorders and evaluation of pharmacological efficacies of small molecules for treatment of these disorders. Here, we focused on the therapeutic role of Pioglitazone, which is a selective agonist of peroxisome proliferator-activated receptor gamma (PPARγ), a respective nuclear receptor in inflammatory responses. Human embryonic stem cell-derived OPCs were pretreated by Pioglitazone at differing concentrations. Pretreated OPCs were further examined after induction of inflammation by LPS. Interestingly, Pioglitazone reversed the inflammatory conditions and enhanced OPC viability. Data showed that Pioglitazone reduced Nitric Oxide (NO) production. Moreover, Pioglitazone enhanced cell viability through distinct mechanisms including reduction of apoptosis and regulation of cell cycle markers. This study demonstrated that NO induces apoptosis through FOXO1 and degradation of ß-catenin, while the presence of Pioglitazone inhibited these effects in rescuing human OPCs from apoptosis. Also, Pioglitazone did not show a significant influence on mRNA levels of TLR2, TRL4, and TNFα. Furthermore, simultaneous treatment of Pioglitazone with CHIR, a GSKß inhibitor, facilitated anti-apoptotic responses of OPCs. Taken together, therapy with Pioglitazone represents a novel potential drug in alleviating the loss of OPCs in neurodegenerative conditions.

Overexpression of Annexin A1 Suppresses Pro-Inflammatory Factors in PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium.

OBJECTIVE: Annexin A1 (ANXA1) is suggested to have anti-inflammatory function. However, the precise function of ANXA1 has remained unclear. In this study, we therefore examined the potency of ANXA1 in regulating reactive oxygen species (ROS) production and suppressing pro-inflammatory responses in PC12 cells induced by 1-methyl-4-phenylpyridinium (MPP+). MATERIALS AND METHODS: In this experimental study, cDNA of ANXA1 was cloned and inserted to the PGL268 pEpi-FGM18F vector to produce a recombinant PGL/ANXA1 vector for transfection into the PC12 cells. ANXA1 transfected cells were then treated with MPP+. Apoptosis and the content of pro-inflammatory factors including ROS, Interlukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) were assessed by flow-cytometry, real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ANXA1-transfected cells and the data were compared with those obtained from mock and control cells. RESULTS: Data revealed that overexpression of ANXA1 is associated with decreased levels of ROS and expression level of IL-6 and iNOS transcripts, and NF-κB protein in MPP+ treated PC12 cells. CONCLUSION: ANXA1 may be considered as an agent for prevention of neurodegenerative or inflammatory conditions.

PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2.

TLR4 is transmembrane pattern-recognition receptor that initiates signals in response to diverse pathogen-associated molecular patterns especially LPS. Recently, there have been an increasing number of studies about the role of TLRs in the pathogenesis of several disorders as well as the therapeutic potential of TLR intervention in such diseases. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a ligand-activated transcription factor with numerous biological effects. PPARγ has been shown to exert a potential anti-inflammatory effect through suppression of TLR4-mediated inflammation. Therefore, PPARγ agonists may have a potential to combat inflammatory conditions in pathologic states. The current study aims to show the decrease of inflammation by overexpression of PPARγ in a cell reporter model. To reach this goal, recombinant pBudCE4.1 (+) containing encoding sequences of human TLR4 and MD2 was constructed and used to transfect HEK cells. Subsequently, inflammation was induced by LPS treatment as control group. In the treatment group, overexpression of PPARγ prior to inflammation was performed and the expression of inflammatory markers was assessed in this condition. The expression of inflammatory markers (TNFα and iNOS) was defined by quantitative real time PCR and the amount of phosphorylated NF-κB was measured by western blot. Data indicated expression of TNFα and iNOS increased in LPS induced inflammation of stably transformed HEK cells with MD2 and TLR4. In this cell reporter model overexpression of PPARγ dramatically prevented LPS-induced inflammation through the blocking of TLR4/NF-κB signaling. PPARγ was shown to negatively regulate TLR4 activity and therefore exerts its anti-inflammatory action against LPS induced inflammation.

Cardiac differentiation of mouse embryonic stem cells is influenced by a PPAR γ/PGC-1α-FNDC5 pathway during the stage of cardiac precursor cell formation.

Peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α) up-regulation induces FNDC5 expression in muscle and consequently causes browning of white adipose tissue (WAT). In addition to skeletal muscle, FNDC5 is mainly expressed in heart and brain tissues. Here, we demonstrate that FNDC5 expression increased during the process of cardiac differentiation of mouse embryonic stem cells (mESCs) similar to PGC-1α and PPARα. To testify the correlation between PGC-1α and FNDC5 in cardiac cell differentiation of mESCs, we utilized specific PPARγ agonist and antagonist in two stages of cardiac differentiation, during and post-cardiac precursor cells (CPCs) formation. Our results indicated that a reduction in PGC-1α expression, via treatment with GW9662 during CPCs formation stage, down-regulated FNDC5 transcript levels as well as mitochondrial markers which negatively influenced on the whole process of cardiac differentiation efficiency. On the other hand, increase PGC-1α expression during CPCs formation stage via rosiglitazone treatment increase FNDC5 and mitochondrial markers transcript levels which enhanced cardiac differentiation efficiency. Importantly, such alteration in PGC-1α expression at post-CPCs formation stage did not affect overall cardiac differentiation rate as expression of FNDC5 and mitochondrial markers were not significantly changed. We concluded that PPARγ agonist and antagonist induced up and down-regulation of PGC-1α and subsequently modulated the process of CPCs formation through an alteration in FNDC5 and mitochondrial markers expression.

Acute course of deferoxamine promoted neuronal differentiation of neural progenitor cells through suppression of Wnt/ß-catenin pathway: a novel efficient protocol for neuronal differentiation.

Neural progenitor cells (NPCs) are feasible therapeutically model cells in regenerative medicine. However, a number of obstacles oppose their applications including insufficiency in differentiation protocols. These complications should be overwhelmed to obtain a significant clinical application. Deferoxamine (DFO), as a small molecule with a clinically high-affinity to chelate intracellular Iron, has been granted orphan drug status for treatment of traumatic spinal cord injury, while its neuroprotective function is not well understood. The aim of the present study is evaluating whether DFO could modulate neuronal differentiation process of NPCs. A varies concentrations of DFO were used to promote neuronal differentiation of mouse and human NPCs with different serum condition as an extracellular source of Iron. Several neural markers were assessed by RT-qPCR and Western analysis. Meanwhile ß-catenin content was evaluated as key member of Wnt pathway. The maximal neuronal differentiation rate was observed when treating cells were treated with acute dosage of DFO (100 µM) for 6h in serum free condition. This treatment produced a significant increase in expression of neuronal markers and resulted in dramatically decrease in expression of glial markers. The protein content of ß-catenin was also decreased by this treatment. Despite of chronic concentration of DFO, which reduced the size of EBs apparently due to G1/S arrest of cell cycle as known features of DFO. Application of acute courses of DFO increased neuronal differentiation rate of NPCs in serum free conditions. We concluded that suppression of Wnt/ß-catenin pathway was induced through chelating of intracellular Iron due to DFO treatment. These findings help to understand therapeutic benefit of DFO as a neuroprotective agent.