Increasing ERK phosphorylation by inhibition of p38 activity protects against cadmium-induced apoptotic cell death through ERK/Drp1/p38 signaling axis in spermatocyte-derived GC-2spd cells.

Many studies report that cadmium chloride (CdCl2)-induces oxidative stress is associated with male reproductive damage in the testes. CdCl2 also induces mitochondrial fission by increasing dynamin-related protein 1 (Drp1) expression as well as the mitochondria-dependent apoptosis pathway by extracellular signal-regulated kinase (ERK) activation. However, it remains unclear whether mechanisms linked to the mitochondrial damage signal via CdCl2-induced mitogen-activated protein kinases (MAPK) cause damage to spermatocytes. In this study, increased intracellular and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆Ψm) depolarization, and mitochondrial fragmentation and swelling were observed at 5 µM of CdCl2 exposure, resulting in increased apoptotic cell death. Moreover, CdCl2-induced cell death is closely associated with the ERK/Drp1/p38 signaling axis. Interestingly, SB203580, a p38 inhibitor, effectively prevented CdCl2-induced apoptotic cell death by reducing ∆Ψm depolarization and intracellular and mitochondrial ROS levels. Knockdown of Drp1 expression diminished CdCl2-induced mitochondrial deformation and ROS generation and protected GC-2spd cells from apoptotic cell death. In addition, electron microscopy showed that p38 inhibition reduced CdCl2-induced mitochondrial interior damage more effectively than N-acetyl-L-cysteine (NAC), an ROS scavenger; ERK inhibition; or Drp1 knockdown. Therefore, these results demonstrate that inhibition of p38 activity prevents CdCl2-induced apoptotic GC-2spd cell death by reducing depolarization of mitochondrial membrane potential and mitochondrial ROS levels via ERK phosphorylation in a signal pathway different from the CdCl2-induced ERK/Drp1/p38 axis and suggest a therapeutic strategy for CdCl2-induced male infertility.

Peroxiredoxin I maintains luteal function by regulating unfolded protein response.

BACKGROUND: Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL. METHODS: The expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice. RESULTS: We demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice. CONCLUSION: These observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.

Peroxiredoxin 1 inhibits streptozotocin-induced Alzheimer's disease-like pathology in hippocampal neuronal cells via the blocking of Ca2+/Calpain/Cdk5-mediated mitochondrial fragmentation.

Oxidative stress plays an essential role in the progression of Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. Streptozotocin (STZ)-induced abnormal brain insulin signaling and oxidative stress play crucial roles in the progression of Alzheimer's disease (AD)-like pathology. Peroxiredoxins (Prxs) are associated with protection from neuronal death induced by oxidative stress. However, the molecular mechanisms underlying Prxs on STZ-induced progression of AD in the hippocampal neurons are not yet fully understood. Here, we evaluated whether Peroxiredoxin 1 (Prx1) affects STZ-induced AD-like pathology and cellular toxicity. Prx1 expression was increased by STZ treatment in the hippocampus cell line, HT-22 cells. We evaluated whether Prx1 affects STZ-induced HT-22 cells using overexpression. Prx1 successfully protected the forms of STZ-induced AD-like pathology, such as neuronal apoptosis, synaptic loss, and tau phosphorylation. Moreover, Prx1 suppressed the STZ-induced increase of mitochondrial dysfunction and fragmentation by down-regulating Drp1 phosphorylation and mitochondrial location. Prx1 plays a role in an upstream signal pathway of Drp1 phosphorylation, cyclin-dependent kinase 5 (Cdk5) by inhibiting the STZ-induced conversion of p35 to p25. We found that STZ-induced of intracellular Ca2+ accumulation was an important modulator of AD-like pathology progression by regulating Ca2+-mediated Calpain activation, and Prx1 down-regulated STZ-induced intracellular Ca2+ accumulation and Ca2+-mediated Calpain activation. Finally, we identified that Prx1 antioxidant capacity affected Ca2+/Calpain/Cdk5-mediated AD-like pathology progress. Therefore, these findings demonstrated that Prx1 is a key factor in STZ-induced hippocampal neuronal death through inhibition of Ca2+/Calpain/Cdk5-mediated mitochondrial dysfunction by protecting against oxidative stress.

Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

ATF6 is a critical regulator of cadmium-mediated apoptosis in spermatocytes.

In this study, we examined the mechanisms of cadmium exposure-induced endoplasmic reticulum (ER) stress response and apoptosis in spermatocytes. Responses to cadmium toxicity were investigated using spermatocytes overexpressing p50ATF6, ATF4, and spliced XBP1s, belonging to the 3 unfolded protein response pathways. The ER stress and apoptosis response to cadmium were most strongly stimulated through the activating transcription factor 6 (ATF6) pathway; in contrast, siRNA-induced inhibition of protein expression could reduce apoptosis under stressful conditions. An in vivo experiment using mice confirmed that upregulation of p50ATF6 in the testis increased apoptosis in response to cadmium exposure. Further, when confirming the correlation between ER stress and MAPK in cadmium toxicity, p38 MAPK phosphorylation was strongly regulated by p50ATF6; p-p38 also mediated the activity of p50ATF6. Overall, these findings suggest that modulating the activity of p38 MAPK and p50ATF6 in cadmium exposure-induced toxicity can be considered a potential strategy to treat infertility.

Colorimetric Nanoparticle-Embedded Hydrogels for a Biosensing Platform.

Hydrogels containing colorimetric nanoparticles have been used for ion sensing, glucose detection, and microbial metabolite analyses. In particular, the rapid chemical reaction owing to both the hydrogel form of water retention and the sensitive color change of nanoparticles enables the rapid detection of target substances. Despite this advantage, the poor dispersibility of nanoparticles and the mechanical strength of nanoparticle-hydrogel complexes have limited their application. In this study, we demonstrate a milliliter agarose gel containing homogeneously synthesized polyaniline nanoparticles (PAni-NPs), referred to as PAni-NP-hydrogel complexes (PNHCs). To fabricate the optimal PNHC, we tested various pH solvents based on distilled water and phosphate-buffered saline and studied the colorimetric response of the PNHC with thickness. The colorimetric response of the prepared PNHC to the changes in the pH of the solution demonstrated excellent linearity, suggesting the possibility of using PNHC as a pH sensor. In addition, it was verified that the PNHC could detect minute pH changes caused by the cancer cell metabolites without cytotoxicity. Furthermore, the PNHC can be stably maintained outside water for approximately 12 h without deformation, indicating that it can be used as a disposable patch-type wearable biosensing platform.

XperCT-guided Intra-cisterna Magna Injection of Streptozotocin for Establishing an Alzheimer's Disease Model Using the Cynomolgus Monkey (Macaca fascicularis).

Till date, researchers have been developing animal models of Alzheimer's disease (AD) in various species to understand the pathological characterization and molecular mechanistic pathways associated with this condition in humans to identify potential therapeutic treatments. A widely recognized AD model that mimics the pathology of human AD involves the intracerebroventricular (ICV) injection with streptozotocin (STZ). However, ICV injection as an invasive approach has several limitations related to complicated surgical procedures. Therefore, in the present study, we created a customized stereotaxic frame using the XperCT-guided system for injecting STZ in cynomolgus monkeys, aiming to establish an AD model. The anatomical structures surrounding the cisterna magna (CM) were confirmed using CT/MRI fusion images of monkey brain with XperCT, the c-arm cone beam computed tomography. XperCT was used to determine the appropriate direction in which the needle tip should be inserted within the CM region. Cerebrospinal fluid (CSF) was collected to confirm the accurate target site when STZ was injected into the CM. Cynomolgus monkeys were administered STZ dissolved in artificial CSF once every week for 4 weeks via intracisterna magna (ICM) injection using XperCT-guided stereotactic system. The molecular mechanisms underlying the progression of STZ-induced AD pathology were analyzed two weeks after the final injection. The monkeys subjected to XperCT-based STZ injection via the ICM route showed features of AD pathology, including markedly enhanced neuronal loss, synaptic impairment, and tau phosphorylation in the hippocampus. These findings suggest a new approach for the construction of neurodegenerative disease models and development of therapeutic strategies.

Determination of the Unilaterally Damaged Region May Depend on the Asymmetry of Carotid Blood Flow Velocity in Hemiparkinsonian Monkey: A Pilot Study.

The hemiparkinsonian nonhuman primate model induced by unilateral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the carotid artery is used to study Parkinson's disease. However, there have been no studies that the contralateral distribution of MPTP via the cerebral collateral circulation is provided by both the circle of Willis (CoW) and connections of the carotid artery. To investigate whether MPTP-induced unilaterally damaged regions were determined by asymmetrical cerebral blood flow, the differential asymmetric damage of striatal subregions, and examined structural asymmetries in a circle of Willis, and blood flow velocity of the common carotid artery were observed in three monkeys that were infused with MPTP through the left internal carotid artery. Lower flow velocity in the ipsilateral common carotid artery and a higher ratio of ipsilateral middle cerebral artery diameter to anterior cerebral artery diameter resulted in unilateral damage. Additionally, the unilateral damaged monkey observed the apomorphine-induced contralateral rotation behavior and the temporary increase of plasma RANTES. Contrastively, higher flow velocity in the ipsilateral common carotid artery was observed in the bilateral damaged monkey. It is suggested that asymmetry of blood flow velocity and structural asymmetry of the circle of Willis should be taken into consideration when establishing more efficient hemiparkinsonian nonhuman primate models.

Macrophage peroxiredoxin 5 deficiency promotes lung cancer progression via ROS-dependent M2-like polarization.

Strategies for cancer treatment have traditionally focused on suppressing cancer cell behavior, but many recent studies have demonstrated that regulating the tumor microenvironment (TME) can also inhibit disease progression. Macrophages are major TME components, and the direction of phenotype polarization is known to regulate tumor behavior, with M2-like polarization promoting progression. It is also known that reactive oxygen species (ROS) in macrophages drive M2 polarization, and M2 polarization promote lung cancer progression. Lung cancer patients with lower expression of the antioxidant enzyme peroxiredoxin 5 (Prx5) demonstrate poorer survival. This study revealed that Prx5 deficiency in macrophages induced M2 macrophage polarization by lung cancer. We report that injection of lung cancer cells produced larger tumors in Prx5-deficit mice than wild-type mice independent of cancer cell Prx5 expression. Through co-culture with lung cancer cell lines, Prx5-deficient macrophages exhibited M2 polarization, and reduced expression levels of the M1-associated inflammatory factors iNOS, TNFα, and Il-1ß. Moreover, these Prx5-deficient macrophages promoted the proliferation and migration of co-cultured lung cancer cells. Conversely, suppression of ROS generation by N-acetyl cysteine (NAC) inhibited the M2-like polarization of Prx5-deficient macrophages, increased expression levels of inflammatory factors, inhibited the proliferation and migration of co-cultured lung cancer cells, and suppressed tumor growth in mice. These findings suggest that blocking the M2 polarization of macrophages may promote lung cancer regression.

Peroxiredoxin 5 ameliorates obesity-induced non-alcoholic fatty liver disease through the regulation of oxidative stress and AMP-activated protein kinase signaling.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease globally. NAFLD-which can develop into liver fibrosis, nonalcoholic steatohepatosis, cirrhosis, and hepatocellular carcinoma-is defined as an excess accumulation of fat caused by abnormal lipid metabolism and excessive reactive oxygen species (ROS) generation in hepatocytes. Recently, we reported that Peroxiredoxin 5 (Prx5) plays an essential role in regulating adipogenesis and suggested the need to further investigation on the potential curative effects of Prx5 on obesity-induced fatty liver disease. In the present study, we focused on the role of Prx5 in fatty liver disease. We found that Prx5 overexpression significantly suppressed cytosolic and mitochondrial ROS generation. Additionally, Prx5 regulated the AMP-activated protein kinase pathway and lipogenic gene (sterol regulatory element binding protein-1 and FAS) expression; it also inhibited lipid accumulation, resulting in the amelioration of free fatty acid-induced hepatic steatosis. Silence of Prx5 triggered de novo lipogenesis and abnormal lipid accumulation in HepG2 cells. Concordantly, Prx5 knockout mice exhibited a high susceptibility to obesity-induced hepatic steatosis. Liver sections of Prx5-deletion mice fed on a high-fat diet displayed Oil Red O-stained dots and small leaky shapes due to immoderate fat deposition. Collectively, our findings suggest that Prx5 functions as a protective regulator in fatty liver disease and that it may be a valuable therapeutic target for the management of obesity-related metabolic diseases.

Peroxiredoxin 5 regulates adipogenesis-attenuating oxidative stress in obese mouse models induced by a high-fat diet.

Elevated levels of reactive oxygen species (ROS) are a hallmark of obesity. Peroxiredoxin 5 (Prx5), which is a cysteine-dependent peroxidase enzyme, has an intensive ROS scavenging activity because it is located in the cytosol and mitochondria. Therefore, we focused on the role of Prx5 in regulating mitochondrial ROS and adipogenesis. We demonstrated that Prx5 expression was upregulated during adipogenesis and Prx5 overexpression suppressed adipogenesis by regulating cytosolic and mitochondrial ROS generation. Silencing Prx5 promoted preadipocytes to differentiate into adipocytes accumulating lipids by activating adipogenic protein expression. Prx5-deletion mice fed on a high-fat diet (HFD) exhibited significant increase in body weight, enormous fat pads, and adipocyte hypertrophy in comparison to wild type mice. Prx5 deletion also remarkably induced adipogenesis-related gene expression in white adipose tissue. These phenotypic changes in Prx5-deletion mice were accompanied with lipid metabolic disorders, such as excessive lipid accumulation in the liver, severe hepatic steatosis, and high levels of triglyceride in the serum. These results demonstrated that Prx5 deletion increased the susceptibility to HFD-induced obesity and several of its associated metabolic disorders. In conclusion, we suggest that Prx5 inhibits adipogenesis by modulating ROS generation and adipogenic gene expression, implying that Prx5 may serve as a potential strategy to prevent and treat obesity.