CrossTORC and WNTegration in Disease: Focus on Lymphangioleiomyomatosis

The mechanistic target of rapamycin (mTOR) and wingless-related integration site (Wnt) signal transduction networks are evolutionarily conserved mammalian growth and cellular development networks. Most cells express many of the proteins in both pathways, and this review will briefly describe only the key proteins and their intra- and extracellular crosstalk. These complex interactions will be discussed in relation to cancer development, drug resistance, and stem cell exhaustion. This review will also highlight the tumor-suppressive tuberous sclerosis complex (TSC) mutated, mTOR-hyperactive lung disease of women, lymphangioleiomyomatosis (LAM). We will summarize recent advances in the targeting of these pathways by monotherapy or combination therapy, as well as future potential treatments.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide’s active ingredient on cell adhesion and its αvβ3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvβ3, α5β1 and αllbβ3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvβ3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvβ3 binding to a synthetic polymer containing RGD; (ii) αvβ3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5β1 binding to the above polymer containing RGD; (iv) αllbβ3 binding to its ECM protein, fibrinogen and (v) αvβ3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvβ3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5β1 binding to RGD.     

Exercise Pills for Drug Addiction: Forced Moderate Endurance Exercise Inhibits Methamphetamine-Induced Hyperactivity through the Striatal Glutamatergic Signaling Pathway in Male Sprague Dawley Rats

Physical exercise reduces the extent, duration, and frequency of drug use in drug addicts during the drug initiation phase, as well as during prolonged addiction, withdrawal, and recurrence. However, information about exercise-induced neurobiological changes is limited. This study aimed to investigate the effects of forced moderate endurance exercise training on methamphetamine (METH)-induced behavior and the associated neurobiological changes. Male Sprague Dawley rats were subjected to the administration of METH (1 mg/kg/day, i.p.) and/or forced moderate endurance exercise (treadmill running, 21 m/min, 60 min/day) for 2 weeks. Over the two weeks, endurance exercise training significantly reduced METH-induced hyperactivity. METH and/or exercise treatment increased striatal dopamine (DA) levels, decreased p(Thr308)-Akt expression, and increased p(Tyr216)-GSK-3β expression. However, the phosphorylation levels of Ser9-GSK-3β were significantly increased in the exercise group. METH administration significantly increased the expression of NMDAr1, CaMKK2, MAPKs, and PP1 in the striatum, and exercise treatment significantly decreased the expression of these molecules. Therefore, it is apparent that endurance exercise inhibited the METH-induced hyperactivity due to the decrease in GSK-3β activation by the regulation of the striatal glutamate signaling pathway.     

TCR Recognition of Peptide–MHC-I: Rule Makers and Breakers

T cells are a critical part of the adaptive immune system that are able to distinguish between healthy and unhealthy cells. Upon recognition of protein fragments (peptides), activated T cells will contribute to the immune response and help clear infection. The major histocompatibility complex (MHC) molecules, or human leukocyte antigens (HLA) in humans, bind these peptides to present them to T cells that recognise them with their surface T cell receptors (TCR). This recognition event is the first step that leads to T cell activation, and in turn can dictate disease outcomes. The visualisation of TCR interaction with pMHC using structural biology has been crucial in understanding this key event, unravelling the parameters that drive this interaction and their impact on the immune response. The last five years has been the most productive within the field, wherein half of current unique TCR–pMHC-I structures to date were determined within this time. Here, we review the new insights learned from these recent TCR–pMHC-I structures and their impact on T cell activation.     

Effects of Paeonol on Anti-Neuroinflammatory Responses in Microglial Cells

Increasing studies suggest that inflammatory processes in the central nervous system mediated by microglial activation plays an important role in numerous neurodegenerative diseases. Development of planning for microglial suppression is considered a key strategy in the search for neuroprotection. Paeonol is a major phenolic component of Moutan Cortex, widely used as a nutrient supplement in Chinese medicine. In this study, we investigated the effects of paeonol on microglial cells stimulated by inflammagens. Paeonol significantly inhibited the release of nitric oxide (NO) and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with paeonol also reduced reactive oxygen species (ROS) production and inhibited an ATP-induced increased cell migratory activity. Furthermore, the inhibitory effects of neuroinflammation by paeonol were found to be regulated by phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) and glycogen synthase kinase 3 α/β (GSK 3α/β). Treatment with AMPK or GSK3 inhibitors reverse the inhibitory effect of neuroinflammation by paeonol in microglial cells. Furthermore, paeonol treatment also showed significant improvement in the rotarod performance and microglial activation in the mouse model as well. The present study is the first to report a novel inhibitory role of paeonol on neuroinflammation, and presents a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.     

Differential Effects of High-Fish Oil and High-Lard Diets on Cells and Cytokines Involved in the Inflammatory Process in Rat Insulin-Sensitive Tissues

Dietary fat sources may differentially affect the development of inflammation in insulin-sensitive tissues during chronic overfeeding. Considering the anti-inflammatory properties of ω-3 fatty acids, this study aimed to compare the effects of chronic high-fish oil and high-lard diets on obesity-related inflammation by evaluating serum and tissue adipokine levels and histological features in insulin-sensitive tissues (white adipose tissue, skeletal muscle and liver). As expected, a high-lard diet induced systemic and peripheral inflammation and insulin resistance. Conversely, compared with a high-lard diet, a high-fish oil diet resulted in a lower degree of systemic inflammation and insulin resistance that were associated with a lower adipocyte diameter as well as lower immunoreactivity for transforming growth factor β 1 (TGFβ1) in white adipose tissue. A high-fish oil diet also resulted in a lower ectopic lipid depot, inflammation degree and insulin resistance in the skeletal muscle and liver. Moreover, a high-fish oil diet attenuated hepatic stellate cell activation and fibrogenesis in the liver, as indicated by the smooth muscle α-actin (α-SMA) and TGFβ1 levels. The replacement of lard (saturated fatty acids) with fish oil (ω-3 fatty acids) in chronic high-fat feeding attenuated the development of systemic and tissue inflammation.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

Anticonvulsant Effects of Topiramate and Lacosamide on Pilocarpine-Induced Status Epilepticus in Rats: A Role of Reactive Oxygen Species and Inflammation

Background: Status epilepticus (SE) is a neurological disorder characterized by a prolonged epileptic activity followed by subsequent epileptogenic processes. The aim of the present study was to evaluate the early effects of topiramate (TPM) and lacosamide (LCM) treatment on oxidative stress and inflammatory damage in a model of pilocarpine-induced SE. Methods: Male Wistar rats were randomly divided into six groups and the two antiepileptic drugs (AEDs), TPM (40 and 80 mg/kg, i.p.) and LCM (10 and 30 mg/kg, i.p.), were injected three times repeatedly after pilocarpine administration. Rats were sacrificed 24 h post-SE and several parameters of oxidative stress and inflammatory response have been explored in the hippocampus. Results: The two drugs TPM and LCM, in both doses used, succeeded in attenuating the number of motor seizures compared to the SE-veh group 30 min after administration. Pilocarpine-induced SE decreased the superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels while increasing the catalase (CAT) activity, malondialdehyde (MDA), and IL-1β levels compared to the control group. Groups with SE did not affect the TNF-α levels. The treatment with a higher dose of 30 mg/kg LCM restored to control level the SOD activity in the SE group. The two AEDs, in both doses applied, also normalized the CAT activity and MDA levels to control values. In conclusion, we suggest that the antioxidant effect of TPM and LCM might contribute to their anticonvulsant effect against pilocarpine-induced SE, whereas their weak anti-inflammatory effect in the hippocampus is a consequence of reduced SE severity.     

ATP13A2 Regulates Cellular α-Synuclein Multimerization,Membrane Association,and Externalization

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.     

The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.     

Effects of β-Phenylethylamine on Psychomotor,Rewarding, and Reinforcing Behaviors and Affective State: The Role of Dopamine D1 Receptors

Beta-phenylethylamine (β-PEA) is a well-known and widespread endogenous neuroactive trace amine found throughout the central nervous system in humans. In this study, we demonstrated the effects of β-PEA on psychomotor, rewarding, and reinforcing behaviors and affective state using the open-field test, conditioned place preference (CPP), self-administration, and ultrasonic vocalizations (USVs) paradigms. We also investigated the role of the dopamine (DA) D1 receptor in the behavioral effects of β-PEA in rodents. Using enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting, we also determined the DA concentration and the DA-related protein levels in the dorsal striatum of mice administered with acute β-PEA. The results showed that acute β-PEA increased stereotypic behaviors such as circling and head-twitching responses in mice. In the CPP experiment, β-PEA increased place preference in mice. In the self-administration test, β-PEA significantly enhanced self-administration during a 2 h session under fixed ratio (FR) schedules (FR1 and FR3) and produced a higher breakpoint during a 6 h session under progressive ratio schedules of reinforcement in rats. In addition, acute β-PEA increased 50-kHz USV calls in rats. Furthermore, acute β-PEA administration increased DA concentration and p-DAT and TH expression in the dorsal striatum of mice. Finally, pretreatment with SCH23390, a DA D1 receptor antagonist, attenuated β-PEA-induced circling behavior and β-PEA-taking behavior in rodents. Taken together, these findings suggest that β-PEA has rewarding and reinforcing effects and psychoactive properties, which induce psychomotor behaviors and a positive affective state by activating the DA D1 receptor in the dorsal striatum.     

The Influence of Interaction between Cadmium with 17β-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone on Viability and p-Glycoprotein in Ovarian Cancer Cell Line

Occupational and environmental exposure to xenoestrogens, a subgroup of endocrine disruptors (EDCs), can affect the endocrine system and increase the risk of cancer, primarily the hormone-dependent kind. This type of cancer includes ovarian cancer, which is the leading cause of death from gynecological tumors. The aim of this study was to assess the role of 17β-estradiol and its metabolites: 2-MeOE2, 16α-OHE1 in exposure to the metalloestrogen cadmium. The effect of interactions of cadmium with estrogens on the viability of cells in malignant ovarian cancer cells SKOV-3 was investigated, both in simultaneous action and in the pre-incubation model. There are no known interactions between estrogens and cadmium in ovarian cancer cells. Due to the frequent occurrence of multidrug resistance (MDR) in ovarian cancer, the effects of estrogens and cadmium on MDR in SKOV-3, measured as P-glycoprotein (P-gp), were assessed. An interaction study showed that E2 had an antagonistic effect on cadmium-induced cell damage, while 2-MeOE2 showed less of a protective effect in combination with CdCl₂ than E2. There were two types of interaction: toxic synergism and beneficial antagonism. E2 and cadmium increased P-gp expression in SKOV-3 cells, while 2-MeOE2 decreased P-gp expression to a potentially beneficial effect on MDR prevention. The obtained results constitute an interesting starting point for further research in the field of interactions between estrogens and xenoestrogens in ovarian cancer.     

Alterations in Tau Protein Level and Phosphorylation State in the Brain of the Autistic-Like Rats Induced by Prenatal Exposure to Valproic Acid

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by deficient social interaction and communication besides repetitive, stereotyped behaviours. A characteristic feature of ASD is altered dendritic spine density and morphology associated with synaptic plasticity disturbances. Since microtubules (MTs) regulate dendritic spine morphology and play an important role in spine development and plasticity the aim of the present study was to investigate the alterations in the content of neuronal α/β-tubulin and Tau protein level as well as phosphorylation state in the valproic acid (VPA)-induced rat model of autism. Our results indicated that maternal exposure to VPA induces: (1) decrease the level of α/β-tubulin along with Tau accumulation in the hippocampus and cerebral cortex; (2) excessive Tau phosphorylation and activation of Tau-kinases: CDK5, ERK1/2, and p70S6K in the cerebral cortex; (3) up-regulation of mTOR kinase-dependent signalling in the hippocampus and cerebral cortex of adolescent rat offspring. Moreover, immunohistochemical staining showed histopathological changes in neurons (chromatolysis) in both analysed brain structures of rats prenatally exposed to VPA. The observed changes in Tau protein together with an excessive decrease in α/β-tubulin level may suggest destabilization and thus dysfunction of the MT cytoskeleton network, which in consequence may lead to the disturbance in synaptic plasticity and the development of autistic-like behaviours.     

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.     

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.