Thymoquinone ameliorates symptoms of Parkinson's disease in a 6-OHDA rat model by downregulation of miR-204-3p.

microRNAs (miRNAs) play a significant role in the pathophysiology of Parkinson's disease. In this study, we evaluated the neuroprotective effect of thymoquinone on the expression profiles of miRNA and cognitive functions in the 6-hydroxydopamine (6-OHDA)-induced Parkinson's model. Male adult Wistar albino rats (200-230 g, n  = 36) were randomly assigned to six groups: Sham, thymoquinone (10 mg/kg, p.o.), 6-OHDA, 6-OHDA + thymoquinone (10 mg/kg), 6-OHDA + thymoquinone (20 mg/kg), and 6-OHDA + thymoquinone (50 mg/kg). Behavioral changes were detected using the open field and the elevated plus maze tests. The mature 728 miRNA expressions were evaluated by miRNA microarray (GeneChip miRNA 4.0). Ten miRNAs were selected (rno-miR-212-5p, rno-miR-146b-5p, rno-miR-150-5p, rno-miR-29b-2-5p, rno-miR-126a-3p, rno-miR-187-3p, rno-miR-34a-5p, rno-miR-181d-5p, rno-miR-204-3p, and rno-miR-30c-2-3p) and confirmed by real-time PCR. Striatum samples were stained with hematoxylin-eosin to determine the effect of dopaminergic lesions. One-way ANOVA test and independent sample t -test were used for statistical analyses. rno-miR-204-3p was upregulated at 6-OHDA and downregulated at the 50 mg/kg dose of thymoquinone. In conclusion, thymoquinone at a dose of 50 mg/kg ameliorates symptoms of Parkinson's disease in a 6-OHDA rat model by downregulation of miR-204-3p. Also, the results showed that thymoquinone can improve locomotor activity and willing exploration and decreased anxiety. Therefore, thymoquinone can be used as a therapeutic agent.

PTZ-kindled rat model; evaluation of seizure, hippocampal EGR-1, and Rev-erbα gene regulation, behavioral analysis, and antioxidant capacity of Gum Arabic.

BACKGROUND: Epilepsy is a neurological disease characterized by recurrent seizures, hyperexcitable neurons and various behavioral comorbidities. The electrical charge during seizures depletes the antioxidant defense mechanism in the epileptic brain and increases the oxidative burden. Natural antioxidant compounds are potential therapeutics in the treatment of two major pathologies of epilepsy with their anticonvulsant and anxiolytic effects and can modulate these targets. Gum Arabic is one of the natural plant polysaccharides that is non-toxic and biodegradable. METHODS AND RESULTS: A total of 30 Wistar albino male rats (8-12 weeks, 350-500 g), were randomly divided into 5 groups with 6 animals in each group: 1-Control, 2-Sham (Phosphate buffer saline (PBS)), 3-PTZ, 4-Gum Arabic, 5-PTZ + Gum Arabic. PTZ was administered i.p at 35 mg/kg/day for 11 days. After 48 h, the injection was completed with 75 mg/kg PTZ. Locomotor activity, immobilization, rearing, grooming, eating, and drinking behaviors were recorded with the LABORAS behavior system for 30 min after kindling. Animals were treated with Gum Arabic (2 mg/kg/day, oral gavage) for 10 days. At the end of the period, animal behavior was recorded again. Then the hippocampus tissues were removed. Oxidative parameters (TAS and TOS), early growth response 1 (EGR1) and nuclear receptor subfamily 1 group D member 1 (Rev-erbα) gene expressions and behaviors were analyzed. CONCLUSION: Gum Arabic increased TAS levels (P = 0.000), decreased TOS levels (P = 0.000), and thus exhibited antioxidant properties by reducing oxidative stress burden. EGR1, which was upregulated in the seizure group, was downregulated after treatment (P = 0.000), and Rev-erbα was downregulated in seizure and upregulated after treatment (P = 0.000). Gum arabic may be an antiepileptic and anxiolytic therapeutic in improving epileptic seizures by reducing oxidative stress burden through EGR1 and Rev-erbα.0.

Ferroptotic Potency of ISM1 Expression in the Drug-Induced Alzheimer's Disease-Like Phenotype Under the Influence of Betulin.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by two main pathological mechanisms, mostly hyperphosphorylated tau and amyloid-ß toxicity. Although many studies focus on these basic mechanisms, ferroptosis draws attention as an important pathway responsible for neurodegeneration in AD. There is no definitive treatment for AD but alternative phytochemicals to drugs come into prominence. Betulin is usually obtained from the birch tree. It is an abundant triterpene and has a high antioxidant capacity. Isthmin-1 (ISM1) is a secreted adipokine. OBJECTIVE: In this study, we investigated the potential treatment of AD in the ferroptosis-ISM1-betulin triangle. METHODS: For this, we created an AD model with okadaic acid (200 ng/kg)) in 36 Wistar albino male rats and treated with betulin (20 mg/kg/day, i.p). We evaluated ISM1 gene expression, iron accumulation, and total oxidative metabolism parameters (TAS, TOS, OSI) in hippocampal tissue. We analyzed cognitive recovery in AD with Morris Water Maze Test and general locomotor activity, explore, and anti-anxiolytic effect with Open Field Test. RESULTS: We compared the obtained data with metabolic and genetic results. In conclusion, betulin may have a role in neuronal ferroptotic mechanisms by reducing iron accumulation by ISM1 regulation. CONCLUSIONS: Betulin may have a role in neuronal ferroptotic mechanisms by reducing iron accumulation by ISM1 regulation. Although this study suggests the corrective effect of betulin and ISM1 on cognitive gain and anxiety, it is the first study to show the total antioxidant capacity of betulin in AD.

The holistic approach to the CHRNA7 gene, hsa-miR-3158-5p, and 15q13.3 hotspot CNVs in migraineurs.

Migraine is a neurological disease characterized by severe headache attacks. Combinations of different genetic variations such as copy number variation (CNV) in a gene and microRNA (miRNA) expression can provide a holistic approach to the disease as a pathophysiological, diagnostic, and therapeutic target. CNVs, the Cholinergic Receptor Nicotinic Alpha 7 Subunit (CHRNA7) gene, and expression of gene-targeting miRNAs (hsa-miR-548e-5p and hsa-miR-3158-5p) in migraineurs (n = 102; with aura, n = 43; without aura, n = 59) and non-migraines (n = 120) aged 15-60 years, comparative, case-control study was conducted. Genetic markers were compared with biochemical parameters (BMI, WBC, Urea, GFR, ESR, CRP, HBG). All analyzes were performed by quantitative Real-Time PCR (q-PCR) and fold change was calculated with the 2-ΔΔCT method. The diagnostic power of the CHRNA7 gene, CNV, and miRNAs were analyzed with the receiver operating curve (ROC). CHRNA7 gene and hsa-miR-3158-5p are down-regulated in migraineurs and the gene is controlled by this miRNA via CNVs (p < .05). Both deletion and duplication were detected in patients with migraine for CVN numbers (p = .05). The number of CNV deletions was higher than duplications. When CHRNA7-CNV-hsa-miR-3158-5p was modeled together in the ROC analysis, the area under the curve (AUC) was 0.805, and the diagnostic power was "good". In migraineurs, the CHRNA7 gene can be controlled by hsa-miR-3158-5p via CNVs to modulate the mechanism of pain. These three genetic markers have diagnostic potential and may be used in antimigraine treatments.

Downregulatory effect of miR-342-3p on epileptogenesis in the PTZ-kindling model.

BACKGROUND: Epileptogenesis is a process that results in neurons firing abnormally, causing seizures. Increasing evidence has shown that miRNAs expressed in the epileptic hippocampus are involved in epileptogenesis. We demonstrated the expression changes of miRNAs that may be effective in epileptogenesis in silico analysis in the kindling model created with Pentylenetetrazole (PTZ). Thus, we aimed to identify the target genes responsible for epileptogenesis. METHODS AND RESULTS: Fifteen male Wistar-albino rats (200-230 g) were randomly divided into two groups control (n = 6) and PTZ (n = 9). The control group received 0.5 ml saline, and the PTZ group (35 mg/kg i.p.) intraperitoneally (i.p.) (11 times, every other day) to induce tonic-clonic seizures. Seizures were observed and scored 30 min after PTZ injection. After the last dose of PTZ (75 mg/kg) administration, the hippocampus tissues of the rats were removed by anesthesia. Analysis of miRNAs was performed with the Affymetrix gene chip miRNA sequence (728 miRNA) and confirmed by the Real-Time Polymerase Chain Reaction (Real-Time PCR) method (29 miRNAs). We evaluated the expression change of the target gene of miRNA, whose expression change was detected using in silico analysis, by q-RT PCR. Eight miRNAs with changes in expression were detected. Of these miRNAs, miR-342-p was downregulated in the PTZ group and was statistically significant (p < 0.005). Ultimately, we determined that the target gene of miR-342-p is a metabotropic glutamate receptor 2 (GRM2) and that GRM2 expression is upregulated. CONCLUSIONS: Downregulation of miR-342-3p in the PTZ kindling model may result in the upregulation of GRM2.

Pentylenetetrazole-induced kindling rat model: miR-182 and miR-27b-3p mediated neuroprotective effect of thymoquinone in the hippocampus.

OBJECTIVES: Epilepsy is a neurological disease that pathologically affects brain functions. The epileptic hippocampus has modified microRNA(miRNA) levels. Therefore, we aimed to evaluate the neuroprotective effect of thymoquinone (TQ) in PTZ-induced epilepsy and to demonstrate the overlap between miRNA and mRNA expression profiles. METHODS: Male adult Wistar albino rats (200-230 g, n = 20) were divided into three groups as control (n = 6), PTZ (n = 7), and TQ + PTZ (n = 7). The PTZ kindling model was created by injecting PTZ in sub convulsive doses to rats on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, and 24 of the study into animals. Clonic and tonic seizures were induced by injecting a convulsive dose of PTZ on day 26 of the study. Rats in the TQ+PTZ group were treated by oral gavage with a 20 mg/kg TQ 2 h before each PTZ injection. The rats in the control group were treated with 0.5 ml saline. Seizure severity was evaluated with the Racine scale. The genes and signaling pathways targeted by miRNAs were determined by bioinformatics analysis. RESULTS: In the rat hippocampus, mature 728 miRNAs were analyzed by microarray and the nine miRNA were verified by quantitative Real-Time PCR. rno-miR-182 and rno-miR-27b-3p were up-regulated in the PTZ group and down-regulated in the TQ + PTZ group. DISCUSSION: In the PTZ kindling epilepsy model, the expression of these two miRNAs was regulated by TQ and exerted a neuroprotective effect by controlling the activities of target genes.

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE.

Genome editing technologies include techniques used for desired genetic modifications and allow the insertion, modification or deletion of specific DNA fragments. Recent advances in genome biology offer unprecedented promise for interdisciplinary collaboration and applications in gene editing. New genome editing technologies enable specific and efficient genome modifications. The sources that inspire these modifications and already exist in the genome are DNA degradation enzymes and DNA repair pathways. Six of these recent technologies are the clustered regularly interspaced short palindromic repeats (CRISPR), leveraging endogenous ADAR for programmable editing of RNA (LEAPER), recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing (RESTORE), chemistry-based artificial restriction DNA cutter (ARCUT), single homology arm donor mediated intron-targeting integration (SATI), RNA editing for specific C-to-U exchange (RESCUE). These technologies are widely used from various biomedical researches to clinics, agriculture, and allow you to rearrange genomic sequences, create cell lines and animal models to solve human diseases. This review emphasizes the characteristics, superiority, limitations, also whether each technology can be used in different biological systems and the potential application of these systems in the treatment of several human diseases.

A Study Investigating the Role of 2 Candidate SNPs in Bax and Bcl-2 Genes in Alzheimer's Disease.

OBJECTIVE: The proto-oncogene Bax (Bcl-2-associated X protein) and related protein Bcl-2 (B-cell chronic lymphocytic leukemia/lymphoma-2) genes are triggers of apoptosis in Alzheimer's disease (AD). The balance of these proteins has an important role in the death or life of a neuronal cell, and the functional polymorphisms in genes expressing these proteins have been found to promote apoptosis. To investigate the role of Bax and Bcl-2 genes in AD, we examined the presence of the 2 polymorphisms in peripheral blood. To our knowledge, this is the first clinical association study of these 2 functional SNPs using the peripheral blood of patients with AD. METHODS: Bax (rs4645878) and Bcl-2 (rs2279115) in Alzheimer's patients (N = 132) and healthy controls (N = 109), aged 65 to 85 years, were analyzed by qPCR (Quantitative Polymerase Chain Reaction) using TaqMan probe technology. Statistical analyses were done using SPSS, 11.5. The differences between groups were analyzed using an independent-samples t test. The relationships between genotypes and alleles were analyzed using chi-square or likelihood ratio test. The Hardy-Weinberg balance was checked for the patient and control groups. A p-value of less than 0.05 was taken as significant. RESULTS: Sporadic AD patients and non-demented age matched control subjects were genotyped in this case-control study. No statistically significant relationship was found between the patients and controls for allele or genotype frequencies (p>0.05). CONCLUSION: Our data suggest that these two polymorphisms do not contribute to AD in the population from the Mersin region of the Eastern Mediterranean. Further studies with larger sample sizes must be conducted to ascertain the association between the 2 polymorphisms.

Childhood Obesity Risk in Relationship to Perilipin 1 (PLIN1) Gene Regulation by Circulating microRNAs.

Childhood obesity is a growing public health burden in many countries. The lipid perilipin 1 (PLIN1) gene is involved in regulation of lipolysis, and thus represents a viable candidate mechanism for obesity genetics research in children. In addition, the regulation of candidate gene expression by circulating microRNAs (miRNAs) offers a new research venue for diagnostic innovation. We report new findings on associations among circulating miRNAs, regulation of the PLIN1 gene, and susceptibility to childhood obesity. In a sample of 135 unrelated subjects, 35 children with obesity (between ages 3 and 13) and 100 healthy controls (between ages 4 and 16), we examined the expression levels of four candidate miRNAs (hsa-miR-4777-3p, hsa-miR-642b-3p, hsa-miR-3671-1, and hsa-miR-551b-2) targeting the PLIN1 as measured by real-time polymerase chain reaction in whole blood samples. We found that the full genetic model, including the four candidate miRNAs and the PLIN1 gene, explained a statistically significant 12.7% of the variance in childhood obesity risk (p = 0.0034). The four miRNAs together explained 10.1% of the risk (p = 0.008). The percentage of variation in childhood obesity risk explained by hsa-miR-642b-3p and age was 19%. In accordance with biological polarity of the observed association, for example, hsa-miR-642b-3p was upregulated, while the PLIN1 expression decreased in obese participants compared to healthy controls. To the best of our knowledge, this is the first clinical association study of these candidate miRNAs targeting the PLIN1 in childhood obesity. These data offer new molecular leads for future clinical biomarker and diagnostic discovery for childhood obesity.

The impact of synapsin III gene on the neurometabolite level alterations after single-dose methylphenidate in attention-deficit hyperactivity disorder patients.

OBJECTIVE: To investigate the neurometabolite level changes according to synapsin III gene rs133945G>A and rs133946C>G polymorphisms by using magnetic resonance spectroscopy (MRS) in patients with attention-deficit hyperactivity disorder (ADHD). METHODS: Fifty-seven adults diagnosed with ADHD were recruited for the study. The participants were examined by single-voxel (1)H MRS when medication naïve and 30 minutes after oral administration of 10 mg methylphenidate (Mph). Those who had been on a stimulant discontinued the medication 48 hours before MRS imaging. Spectra were taken from the anterior cingulate cortex, dorsolateral prefrontal cortex, striatum, and cerebellum, and N-acetylaspartate (NAA), choline, and creatine levels were examined. For genotyping of the synapsin III gene polymorphisms, DNA was isolated from peripheral blood leukocytes. The effects of age, sex, and ADHD subtypes were controlled in the analyses. RESULTS: After a single dose of Mph, choline levels increased significantly in the striatum of rs133945G>A polymorphism-GG genotypes (P=0.020) and NAA levels rose in the anterior cingulate cortex of rs133946C>G polymorphism-CG genotypes (P=0.014). Both rs133945G>A and rs133946C>G polymorphisms were found to statistically significantly affect the alteration of NAA levels in response to Mph in dorsolateral prefrontal cortex with two-way repeated measure of analysis of variance. Post hoc comparisons revealed a significant difference between CG and GG genotypes of rs133946C>G polymorphisms after Bonferroni adjustment (P=0.016). CONCLUSION: Synapsin III gene polymorphisms may be affecting the changes in neurometabolite levels in response to Mph in adult ADHD patients. Future studies are needed to confirm our findings.

SNP Variation in MicroRNA Biogenesis Pathway Genes as a New Innovation Strategy for Alzheimer Disease Diagnostics: A Study of 10 Candidate Genes in an Understudied Population From the Eastern Mediterranean.

Alzheimer disease (AD) is a common complex neurodegenerative disorder accounting for nearly 50% to 70% of dementias worldwide. Yet the current diagnostic options for AD are limited. New diagnostic innovation strategies focusing on novel molecules and pathways are sorely needed. In this connection, microRNAs (miRNAs) are conserved small noncoding RNAs that regulate posttranscriptional gene expression and are vital for neuronal development and its functional sustainability. Conceivably, biological pathways responsible for the biogenesis of miRNAs represent a veritable set of upstream candidate genes that can be potentially associated with the AD pathophysiology. Notably, whereas functional single-nucleotide polymorphisms (SNPs) in miRNA biogenesis pathway genes have been studied in other complex diseases, surprisingly, virtually no such study has been conducted on their relevance in AD. Moreover, novel diagnostics identified in easily accessible peripheral tissues such as the whole blood samples represent the initial entry or gateway points on the biomarker discovery critical path for AD. To the best of our knowledge, we report here the first association study of functional SNPs, as measured by real-time PCR in 10 "upstream" candidate genes critically situated on the miRNA biogenesis pathway, in a large sample of AD patients (N=172) and healthy controls (N=109) in a hitherto understudied world population from the Mersin region of the Eastern Mediterranean. We observed a significant association between 2 candidate genes and AD, TARBP2 rs784567 genotype and AD (χ=6.292, P=0.043), and a trend for RNASEN rs10719 genotype (χ=4.528, P=0.104) and allele (P=0.035). Functional SNP variations in the other 8 candidate genes (DGCR8, XPO5, RAN, DICER1, AGO1, AGO2, GEMIN3, and GEMIN4) did not associate with AD in our sample. Given the putative biological importance of miRNA biogenesis pathways, these emerging data can provide a new foundation to stimulate future debate and genetic investigations of AD, focusing on new molecular mechanisms such as miRNA biogenesis, particularly in accessible peripheral tissues for novel molecular diagnostics for dementia.