Evaluation of Cytotoxic and Mutagenic Effects of the Synthetic Cathinones Mexedrone, α-PVP and α-PHP

Mexedrone, α-PVP and α-PHP are synthetic cathinones. They can be considered amphetamine-like substances with a stimulating effect. Actually, studies showing their impact on DNA are totally absent. Therefore, in order to fill this gap, aim of the present work was to evaluate their mutagenicity on TK6 cells. On the basis of cytotoxicity and cytostasis results, we selected the concentrations (35–100 µM) to be used in the further analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by flow cytometry. Mexedrone demonstrated its mutagenic potential contrary to the other two compounds; we then proceeded by repeating the analyzes in the presence of extrinsic metabolic activation in order to check if it was possible to totally exclude the mutagenic capacity for α-PVP and α-PHP. The results demonstrated instead the mutagenicity of their metabolites. We then evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the highlighted effects but the results did not show a statistically significant increase in ROS levels for any of the tested substances. Anyway, our outcomes emphasize the importance of mutagenicity evaluation for a complete assessment of the risk associated with synthetic cathinones exposure.     

Neuropsychopharmacology of Emerging Drugs of Abuse: meta- and para-Halogen-Ring-Substituted α-PVP (“flakka”) Derivatives

Changes in the molecular structure of synthetic cathinones has led to an increase in the number of novel emerging drugs in the illicit drug market at an unprecedented rate. Unfortunately, little is known about the neuropsychopharmacology of recently emerged halogen-substituted α-PVP derivatives. Thus, the aim of this study was to investigate the role of para- and meta-halogen (F-, Cl-, and Br-) substitutions on the in vitro, in silico, and in vivo effects of α-pyrrolidinopentiophenone (α-PVP) derivatives. HEK293 cells expressing the human dopamine or serotonin transporter (hDAT and hSERT) were used for the uptake inhibition and transporter affinity assays. Molecular docking was used to model the interaction mechanism against DAT. Swiss CD-1 mice were used for the horizontal locomotor activity, open field test, and conditioned place preference paradigm. All compounds demonstrated potent DA uptake inhibition and higher DAT selectivity than cocaine. Meta-substituted cathinones showed higher DAT/SERT ratios than their para- analogs, which correlates with an increased psychostimulant effect in vivo and with different meta- and para-in silico interactions at DAT. Moreover, all compounds induced rewarding and acute anxiogenic effects in mice. In conclusion, the present study demonstrates the role of meta- and para-halogen substitutions in the mechanism of action and provides the first evidence of the rewarding and anxiety-like properties of halogenated α-PVP derivatives.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

TNF-α and IL-1β Modulate Blood-Brain Barrier Permeability and Decrease Amyloid-β Peptide Efflux in a Human Blood-Brain Barrier Model

The blood-brain barrier (BBB) is a selective barrier and a functional gatekeeper for the central nervous system (CNS), essential for maintaining brain homeostasis. The BBB is composed of specialized brain endothelial cells (BECs) lining the brain capillaries. The tight junctions formed by BECs regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps and receptors. Cytokine-induced neuroinflammation, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), appear to play a role in BBB dysfunction and contribute to the progression of Alzheimer’s disease (AD) by contributing to amyloid-β (Aβ) peptide accumulation. Here, we investigated whether TNF-α and IL-1β modulate the permeability of the BBB and alter Aβ peptide transport across BECs. We used a human BBB in vitro model based on the use of brain-like endothelial cells (BLECs) obtained from endothelial cells derived from CD34+ stem cells cocultivated with brain pericytes. We demonstrated that TNF-α and IL-1β differentially induced changes in BLECs’ permeability by inducing alterations in the organization of junctional complexes as well as in transcelluar trafficking. Further, TNF-α and IL-1β act directly on BLECs by decreasing LRP1 and BCRP protein expression as well as the specific efflux of Aβ peptide. These results provide mechanisms by which CNS inflammation might modulate BBB permeability and promote Aβ peptide accumulation. A future therapeutic intervention targeting vascular inflammation at the BBB may have the therapeutic potential to slow down the progression of AD.     

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1β

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP₃ generation, which results in intracellular Ca²⁺ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca²⁺ release-activated Ca²⁺ (CRAC) current and, in association with TRPC1, the store-operated Ca²⁺ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca²⁺ channel (ARC/LRC), store-independent Ca²⁺ influx activated by the secretory pathway Ca²⁺-ATPase (SPCA2) and the small conductance Ca²⁺-activated K⁺ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca²⁺ signals triggered by each variant, and their downstream modulatory effect within the cell.     

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions

The α₂δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α₂δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α₂δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α₂δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α₂δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α₂δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α₂δ-1-3 isoforms (α₂δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α₂δ proteins. In contrast to this redundant presynaptic function, α₂δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α₁-α₂δ protein–protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α₂δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α₂δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α₂δ and Cachd1.     

Structural Fluctuations of the Human Proteasome α7 Homo-Tetradecamer Double Ring Imply the Proteasomal α-Ring Assembly Mechanism

The 20S proteasome, which is composed of layered α and β heptameric rings, is the core complex of the eukaryotic proteasome involved in proteolysis. The α7 subunit is a component of the α ring, and it self-assembles into a homo-tetradecamer consisting of two layers of α7 heptameric rings. However, the structure of the α7 double ring in solution has not been fully elucidated. We applied cryo-electron microscopy to delineate the structure of the α7 double ring in solution, revealing a structure different from the previously reported crystallographic model. The D7-symmetrical double ring was stacked with a 15° clockwise twist and a separation of 3 Å between the two rings. Two more conformations, dislocated and fully open, were also identified. Our observations suggest that the α7 double-ring structure fluctuates considerably in solution, allowing for the insertion of homologous α subunits, finally converting to the hetero-heptameric α rings in the 20S proteasome.     

Pathogenic Impact of α-Synuclein Phosphorylation and Its Kinases in α-Synucleinopathies

α-Synuclein is a protein with a molecular weight of 14.5 kDa and consists of 140 amino acids encoded by the SNCA gene. Missense mutations and gene duplications in the SNCA gene cause hereditary Parkinson’s disease. Highly phosphorylated and abnormally aggregated α-synuclein is a major component of Lewy bodies found in neuronal cells of patients with sporadic Parkinson’s disease, dementia with Lewy bodies, and glial cytoplasmic inclusion bodies in oligodendrocytes with multiple system atrophy. Aggregated α-synuclein is cytotoxic and plays a central role in the pathogenesis of the above-mentioned synucleinopathies. In a healthy brain, most α-synuclein is unphosphorylated; however, more than 90% of abnormally aggregated α-synuclein in Lewy bodies of patients with Parkinson’s disease is phosphorylated at Ser129, which is presumed to be of pathological significance. Several kinases catalyze Ser129 phosphorylation, but the role of phosphorylation enzymes in disease pathogenesis and their relationship to cellular toxicity from phosphorylation are not fully understood in α-synucleinopathy. Consequently, this review focuses on the pathogenic impact of α-synuclein phosphorylation and its kinases during the neurodegeneration process in α-synucleinopathy.     

Protein Kinase C (Pkc)-δ Mediates Arginine-Induced Glucagon Secretion in Pancreatic α-Cells

The pathophysiology of type 2 diabetes involves insulin and glucagon. Protein kinase C (Pkc)-δ, a serine–threonine kinase, is ubiquitously expressed and involved in regulating cell death and proliferation. However, the role of Pkcδ in regulating glucagon secretion in pancreatic α-cells remains unclear. Therefore, this study aimed to elucidate the physiological role of Pkcδ in glucagon secretion from pancreatic α-cells. Glucagon secretions were investigated in Pkcδ-knockdown InR1G9 cells and pancreatic α-cell-specific Pkcδ-knockout (αPkcδKO) mice. Knockdown of Pkcδ in the glucagon-secreting cell line InR1G9 cells reduced glucagon secretion. The basic amino acid arginine enhances glucagon secretion via voltage-dependent calcium channels (VDCC). Furthermore, we showed that arginine increased Pkcδ phosphorylation at Thr⁵⁰⁵, which is critical for Pkcδ activation. Interestingly, the knockdown of Pkcδ in InR1G9 cells reduced arginine-induced glucagon secretion. Moreover, arginine-induced glucagon secretions were decreased in αPkcδKO mice and islets from αPkcδKO mice. Pkcδ is essential for arginine-induced glucagon secretion in pancreatic α-cells. Therefore, this study may contribute to the elucidation of the molecular mechanism of amino acid-induced glucagon secretion and the development of novel antidiabetic drugs targeting Pkcδ and glucagon.     

Association of Irisin/FNDC5 with ERRα and PGC-1α Expression in NSCLC

The rapid growth and division of cancer cells are associated with mitochondrial biogenesis or switching to glycolysis. ERRα, PGC-1α and irisin/FNDC5 are some of the proteins that can influence these processes. The aim of this study was to determine the correlation of these proteins in non-small cell lung cancer (NSCLC) and to investigate their association with clinicopathological parameters. Immunohistochemistry reactions were performed on tissue microarrays (860 NSCLC, 140 non-malignant lung tissue). The normal fibroblast cell line (IMR-90) and lung cancer cell lines (NCI-H1703 and NCI-H522) were used as co-cultures. The mRNA levels of FNDC5 and ESRRA (encoding ERRα) were assessed in IMR-90 cells after co-culture with lung cancer cells. We observed a decreased level of ERRα with an increase in tumor size (T), stages of the disease, and lymph node metastases (N). In the adenocarcinoma (AC) subtype, patients with a higher ERRα expression had significantly longer overall survival. A moderate positive correlation was observed between FNDC5 mRNA and ESRRA mRNA in NSCLCs. The expression of FNDC5 mRNA in IMR-90 cells increased after 24 h, and ESRRA gene expression increased after 48 h of co-culture. The ERRα receptor with PGC-1α participates in the control of FNDC5/irisin expression. Normal fibroblasts revealed an upregulation of the FNDC5 and ESRRA genes under the influence of lung cancer cells.     

Synthesis of Oxidized 3β,3′β-Disteryl Ethers and Search after High-Temperature Treatment of Sterol-Rich Samples

It was proven that sterols subjected to high-temperature treatment can be concatenated, which results in polymeric structures, e.g., 3β,3′β-disteryl ethers. However, it was also proven that due to increased temperature in oxygen-containing conditions, sterols can undergo various oxidation reactions. This study aimed to prove the existence and perform quantitative analysis of oxidized 3β,3′β-disteryl ethers, which could form during high-temperature treatment of sterol-rich samples. Samples were heated at 180, 200 and 220 °C for 0.5 to 4 h. Quantitative analyses of the oxidized 3β,3′β-disteryl ethers were performed with liquid extraction, solid-phase extraction and liquid chromatography coupled with mass spectrometry. Additionally, to perform this analysis, the appropriate standards of all oxidized 3β,3′β-disteryl ethers were prepared. Eighteen various oxidized 3β,3′β-disteryl ethers (derivatives of 3β,3′β-dicholesteryl ether, 3β,3′β-disitosteryl ether and 3β,3′β-distigmasteryl ether) were prepared. Additionally, the influence of metal compounds on the mechanism of ether formation at high temperatures was investigated.     

Effect of Overexpression of γ-Tocopherol Methyltransferase on α-Tocopherol and Fatty Acid Accumulation and Tolerance to Salt Stress during Seed Germination in Brassica napus L.

Rapeseed (Brassica napus L.) is an important oil crop and a major source of tocopherols, also known as vitamin E, in human nutrition. Enhancing the quality and composition of fatty acids (FAs) and tocopherols in seeds has long been a target for rapeseed breeding. The gene γ-Tocopherol methyltransferase (γ-TMT) encodes an enzyme catalysing the conversion of γ-tocopherol to α-tocopherol, which has the highest biological activity. However, the genetic basis of γ-TMT in B. napus seeds remains unclear. In the present study, BnaC02.TMT.a, one paralogue of Brassica napus γ-TMT, was isolated from the B. napus cultivar “Zhongshuang11” by nested PCR, and two homozygous transgenic overexpression lines were further characterised. Our results demonstrated that the overexpression of BnaC02.TMT.a mediated an increase in the α- and total tocopherol content in transgenic B. napus seeds. Interestingly, the FA composition was also altered in the transgenic plants; a reduction in the levels of oleic acid and an increase in the levels of linoleic acid and linolenic acid were observed. Consistently, BnaC02.TMT.a promoted the expression of BnFAD2 and BnFAD3, which are involved in the biosynthesis of polyunsaturated fatty acids during seed development. In addition, BnaC02.TMT.a enhanced the tolerance to salt stress by scavenging reactive oxygen species (ROS) during seed germination in B. napus. Our results suggest that BnaC02.TMT.a could affect the tocopherol content and FA composition and play a positive role in regulating the rapeseed response to salt stress by modulating the ROS scavenging system. This study broadens our understanding of the function of the Bnγ-TMT gene and provides a novel strategy for genetic engineering in rapeseed breeding.     

Exogenous Integrin αIIbβ3 Inhibitors Revisited: Past,Present and Future Applications

The integrin αIIbβ3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new αIIbβ3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects.     

CK1δ-Derived Peptides as Novel Tools Inhibiting the Interactions between CK1δ and APP695 to Modulate the Pathogenic Metabolism of APP

Alzheimer’s disease (AD) is the major cause of dementia, and affected individuals suffer from severe cognitive, mental, and functional impairment. Histologically, AD brains are basically characterized by the presence of amyloid plaques and neurofibrillary tangles. Previous reports demonstrated that protein kinase CK1δ influences the metabolism of amyloid precursor protein (APP) by inducing the generation of amyloid-β (Aβ), finally contributing to the formation of amyloid plaques and neuronal cell death. We therefore considered CK1δ as a promising therapeutic target and suggested an innovative strategy for the treatment of AD based on peptide therapeutics specifically modulating the interaction between CK1δ and APP. Initially, CK1δ-derived peptides manipulating the interactions between CK1δ and APP695 were identified by interaction and phosphorylation analysis in vitro. Selected peptides subsequently proved their potential to penetrate cells without inducing cytotoxic effects. Finally, for at least two of the tested CK1δ-derived peptides, a reduction in Aβ levels and amyloid plaque formation could be successfully demonstrated in a complex cell culture model for AD. Consequently, the presented results provide new insights into the interactions of CK1δ and APP695 while also serving as a promising starting point for further development of novel and highly innovative pharmacological tools for the treatment of AD.     

Evaluation of the Anti-Atopic Dermatitis Effects of α-Boswellic Acid on Tnf-α/Ifn-γ-Stimulated HaCat Cells and DNCB-Induced BALB/c Mice

Boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and anti-tumor efficacy. Atopic dermatitis is a chronic, non-infectious inflammatory skin disease. However, the effects of α-boswellic acid on atopic dermatitis have not been studied. Therefore, in this study we examined the expression level of pro-inflammatory cytokines, histopathological analysis, and physiological data from BALB/c mice with atopic-like dermatitis induced by 2,4-dinitrochlorobenzene and TNF-α/IFN-γ-stimulated HaCaT cells to better understand the agent’s anti-atopic dermatitis efficacy. First, we found that α-boswellic reduced the epidermal thickening, mast cell numbers, and dermal infiltration of 2,4-dinitrochlorobenzene-induced atopic-like dermatitis in BALB/c mice. Furthermore, we also found that α-boswellic acid can restore transepidermal water loss and skin reddening in mice. In human keratinocytes inflamed by TNF-α/IFN-γ, α-boswellic acid inhibited MAP kinase activation and showed a reduction in NF-κB nuclear translocation. Finally, α-boswellic acid can reduce the expression level of cytokines (IL-1β, IL-6, and IL-8) following the stimulation of TNF-α/IFN-γ in HaCaT cells. Taken together, our study suggests that α-boswellic acids are a potential component for the development of anti-atopic dermatitis drugs.