Association of Α-Klotho with regulation of Keap1/Nrf2/Interleukin-1 pathway and AMPA receptor trafficking in the brain of suicide victims.

Suicide is a significant public health challenge worldwide. Statistical data confirm a strong relationship between suicidal behavior and depressive disorders (DDs), but the molecular mechanisms of these diseases are still poorly understood. A growing body of research suggests that the Klotho-mediated pathway may be a novel intracellular target for the development of suicide-related disorders (including DDs). To verify this hypothesis, the link between α-Klotho levels, Nrf2-related inflammatory status (IL-1α, IL-1ß, Keap1, NFκB p65), AMPA (GluA1, GluA2, p-S831-GluA1, p-S845-GluA1) receptor subunit trafficking and AMPK (AMPKα1/2; pT172-AMPKα1) signalling pathways in the brain of suicide victims as compared to controls were investigated. Commercially available enzyme-linked immunoassay (ELISA) and Western blot analysis were performed in the hippocampus (HP) and frontal cortex (FCx) of suicide victims and matched controls. Group differences were assessed using an unpaired Student's t-test. A statistically significant decrease in the level of α-Klotho (HP: p=0.001; FCx: p=0.012) with an increase in IL-1ß (HP: p=0.0108) and IL-1α (FCx: p=0.009) concentrations were shown. These alterations were associated with increased Keap1 (FCx: p=0.023) and NF-κB-p65 (HP: p=0.039; FCx: p=0.013 nuclear fraction) protein levels. Furthermore, a significant reduction in p-S831-GluA1 (HP: p=0.029; FCx=0.002) and p-S845-GluA1 (HP: p=0.0012) proteins was observed. Similarly, the level of GluA2 (HP: p=0.011; FCx: p=0.002) and in p-T172-AMPKα1 (HP: p=0.0288; FCx: p=0.0338) protein were statistically decreased. Our findings demonstrate that a reduction in α-Klotho levels in brain structures related to mood disorders (HP, FCx) correlates with suicidal behavior. Moreover, our study provides novel insights into the molecular mechanisms underlying suicide-related disorders, highlighting the role of α-Klotho, Nrf2-related inflammatory status, AMPA receptor trafficking, and AMPK signaling pathways in the pathophysiology of suicidal behavior. These results may have implications for the development of targeted interventions for individuals at risk of suicide.

Oxidative stress responses and their alterations in the Nrf2-NMDA receptor pathway in the brain of suicide victims.

Suicide is a global public health concern. There is evidence of an association between suicidal behavior and depressive disorders (DDs). An increasing number of studies have suggested that nuclear factor erythroid-derived 2-like 2 (Nrf2), a major endogenous regulator of the oxidative stress response, can be a novel target for the neurobiology of suicide-related disorders (including depression). This study aimed to investigate the relationship between oxidative stress progression, Nrf2 regulation, and N-methyl-D-aspartate receptor (NMDA) subunit composition in the hippocampus (Hp) and frontal cortex (FCx) of suicide victims (n=14) and matched controls (n=8). Furthermore, zinc and magnesium concentrations and their potency to inhibit [3H] MK-801 (radioactively labeled form of MK-801 - dizocilpine, a well-characterized NMDAR channel uncompetitive antagonist frequently used in receptor-binding assays) binding to NMDA receptor channels were measured. Our results revealed a statistically significant increase in protein carbonyl levels and thiobarbituric acid-reactive substances (TBARS) concentrations in Hp and FCx of suicide victims. Enhanced superoxide dismutase (SOD) activity (only in FCx) in suicides compared to controls was shown. These alterations were accompanied by an increase in Nrf2 protein levels in whole homogeneous tissue lysates and cytosolic fractions of Hp and FCx. Importantly, suicide victims presented a significant reduction in Nrf2 protein levels in the nuclear fraction of FCx. Finally, the observed decrease in N-methyl-D-aspartate receptor subunit 2B (GluN2B) and postsynaptic density proteins 95 (PSD-95) protein levels was associated with a statistically significant reduction in magnesium levels in the FCx of suicide victims. These results confirm for the first time that increased oxidative stress parameters are related to Nrf2 protein changes and alterations in the NMDA receptor complex in the pathophysiology of suicidal behavior.

Endogenous carbon monoxide (CO) as a modulating factor of molecular biological clock in the hypothalamic structures involved in light transmission in pig and wild boar hybrid during long and short day season.

Mature males of a wild boar-pig crossbreed, during the long and short day season, were used for the study which demonstrates that the chemical light carrier CO regulates the expression of biological clock genes in the hypothalamus via humoral pathways. Autologous blood with experimentally elevated concentrations of endogenous CO (using lamps with white light-emitting diodes) was infused into the ophthalmic venous sinus via the right dorsal nasal vein. Molecular biology methods: qPCR and Western Blot were used to determine the expression of genes and biological clock proteins. The results showed that elevated endogenous CO levels, through blood irradiation, induces changes in genes expression involved in the functioning of the main biological clock located in suprachiasmatic nuclei. Changes in the expression of the transcription factors Bmal1, Clock and Npas2 have a similar pattern in both structures, where a very large decrease in gene expression was shown after exposure to elevated endogenous CO levels. The changes in the gene expression of PER 1-2, CRY 1-2, and REV-ERB α-ß and ROR ß are not the same for both POA and DH hypothalamic structures, indicating that both structures respond differently to the humoral signal received. The results indicate that CO is a chemical light molecule whose production in an organism depends on the amount of light. An adequate amount of light is an essential factor for the proper functioning of the main biological clock.

Cyclooxygenase-2 inhibition affects the expression of down syndrom cell adhesion molecule via interaction with metabotropic glutamate receptor 5.

Since we previously discovered that simultaneous inhibition of cyclooxygenase-2 (COX-2; a highly inducible enzyme, crucial for the conversion of arachidonic acid to prostaglandin G2, which plays a predominant role in the CNS) by NS398 and metabotropic glutamate receptor 5 (mGluR5) through the use of its antagonist [3-((2-methyl-4-thiazolyl)ethyl)pyridine; MTEP] alters mouse behavior (e.g., affects spatial learning, and induces/intensifies the antidepressant effect), our aim was to discover the mechanism responsible for these changes. Down syndrome cell adhesion molecule (DSCAM), a member of the immunoglobulin cell adhesion molecule (Ig-CAM) superfamily, is involved in developing the central and peripheral nervous system by influencing cell adhesion mechanisms necessary for synaptic activity and plasticity. Since COX-2 has been implicated in several neuropsychiatric diseases (e.g., major depressive disorder) resulting from neuroplasticity disorders, and on the other hand, its expression is regulated by synaptic activity, we hypothesized that cognitive changes after administration of COX-2 inhibitor and mGluR5 antagonist might be a consequence of impaired DSCAM expression. Importantly, DSCAM deficiency leads to dysregulation of glutamatergic transmission and plasticity. In previous studies, we have demonstrated glutamatergic changes after NS398 and MTEP administration, further supporting the validity of our hypothesis. Due to the different effects observed in behavioral tests, this study used the prefrontal cortex (PFC) and hippocampus (HC) of C57BL/6J mice, which received NS398 and MTEP alone, or in combination, for 7 or 14 days. Among many properties, we also previously investigated the antidepressant potential of these compounds, so we used imipramine (a tricyclic antidepressant) as the reference drug. DSCAM mRNA expression was determined by qRT-PCR. Our results indicate that DSCAM expression after administration of MTEP and NS398 and imipramine along with NS398 is structure- and time-dependent.

Toxic effect of antidepressants on male reproductive system cells: evaluation of possible fertility reduction mechanism.

Depression is acknowledged as a major public health problem. Pharmacological treatment may cause adverse drug reactions and sexual side effects. At the same time, the knowledge of the molecular mechanisms associated with antidepressant-mediated toxicity to reproductive cells is fragmentary. The aim of this study was the multilevel evaluation of the potential toxicity of several antidepressants or antipsychotic drugs (amitriptyline, 10 µM; escitalopram, 30 µM; fluoxetine, 5 µM; imipramine, 20 µM; mirtazapine, 150 µM; olanzapine, 40 µM; reboxetine, 30 µM; venlafaxine, 250 µM) on the cells of the spermatogenesis pathway. Effects of various drugs were monitored by several methods including mitochondrial activity MTT test, fluorescent staining, real-time PCR, morphology analysis, immunofluorescence, and Western blots. Obtained results suggest the concentration- and the time-dependent cytotoxic effect. The molecular mechanism of cytotoxic effect is mediated by disturbances in the redox balance (increased production of reactive oxygen species and reactive nitrogen species), failure of enzymatic and non-enzymatic cell protection mechanisms (glutathione system, nuclear factor-κB and fibroblast growth factor 2-mediated pathways), and impairment of mitochondrial functions. In addition, we provide for the first time, to our knowledge, evidence that antidepressant treatment may contribute to spindle apparatus assembly defects and organelle distribution during cell division in vitro (alterations in the levels of small C terminal domain phosphatase-1 and -3, NuMa, and calnexin protein levels). This study sheds new light on the pathomechanisms of antidepressants action and their associated toxicity towards the reproductive system, emerging issues linked with animal or human reproductive health, and treatment of mood disorders.

The role of zinc deficiency-induced changes in the phospholipid-protein balance of blood serum in animal depression model by Raman, FTIR and UV-vis spectroscopy.

Depression is a serious mental illness. To study the mechanisms of diseases and search for new, more effective therapies, animal models are used. Unfortunately, none of the available models does reflect all symptoms of depression. Zinc deficiency is proposed as a new animal model of depression. However, it has not been yet validated in a detailed manner. Recently, spectroscopic techniques are increasingly being used both in clinical and preclinical studies. Here we examined the effect of zinc deficiency and amitryptyline treatment on the phospholipid - protein balance in the blood serum of rats using Raman, Fourier Transform Infra Red (FTIR) and UV-vis technique. Male Sprague Dawley rats were fed with a zinc ample diet (ZnA, 50mg Zn/kg) or a zinc deficient diet (ZnD, 3mg Zn/kg) for 4 weeks. Then amitriptyline administration (AMI, 10mg/kg, i.p.) was started. After injecting the drug for 2-weeks, blood samples were collected and analyzed. It was found that zinc deficiency decreases both the level of phospholipids and proteins and also causes structural changes in their structures. In the ZnD group amitriptyline treatment influenced the protein level and structure. UV-vis spectroscopy combined with the second derivative calculated from the FTIR spectra provided information that the proteins in blood serum of rat fed with a low Zn diet regain their intact structure after amitriptyline medication. Simultaneously, the antidepressant therapy did not have any effect on the level of phospholipids in this group of rats. Additionally, our results show, that amitriptyline administration can change the structure of phospholipids in rats subjected to zinc ample diet. This altered structure of phospholipids was identified as shortening of carbon chains. Our findings indicate that the decreased level of zinc may be the cause of depressive disorders, as it leads to changes in the phospholipid-protein balance necessary for the proper functioning of the body. This study also shows possible new applications of spectroscopic techniques in the diagnosis of affective disorders, and maybe even identifies markers of depressive disorders.

Qualitative and quantitative changes in phospholipids and proteins investigated by spectroscopic techniques in animal depression model.

Depression becomes nowadays a high mortality civilization disease with one of the major causes being chronic stress. Raman, Fourier Transform Infra Red (FTIR) and Ultraviolet-Visible (UV-vis) spectroscopies were used to determine the changes in the quantity and structure of phospholipids and proteins in the blood serum of rats subjected to chronic mild stress, which is a common animal depression model. Moreover, the efficiency of the imipramine treatment was evaluated. It was found that chronic mild stress not only damages the structure of the phospholipids and proteins, but also decreases their level in the blood serum. A 5weeks imipramine treatment did increase slightly the quantity of proteins, leaving the damaged phospholipids unchanged. Structural information from phospholipids and proteins was obtained by UV-vis spectroscopy combined with the second derivative of the FTIR spectra. Indeed, the structure of proteins in blood serum of stressed rats was normalized after imipramine therapy, while the impaired structure of phospholipids remained unaffected. These findings strongly suggest that the depression factor, which is chronic mild stress, may induce permanent (irreversible) damages into the phospholipid structure identified as shortened carbon chains. This study shows a possible new application of spectroscopic techniques in the diagnosis and therapy monitoring of depression.

Changes in melatonin synthesis parameters after carbon monoxide concentration increase in the cavernous sinus.

Previous studies indicate that the gaseous messenger carbon monoxide (CO) is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight. This study was designed to determine whether the increased concentration of CO in ophthalmic venous blood affects the synthesis of melatonin and therefore, whether CO released from the eye under normal lighting conditions can be a carrier of light intensity information. Thirty six mature male wild boar and pig crossbreeds (n = 36) were studied. We measured the difference in the scotophase melatonin pathway response in terms of mean concentration of increased melatonin levels after 48 hours infusion of autologous blood plasma with an experimentally induced approximately 3-fold increase in the concentration of CO into the ophthalmic venous sinus. We demonstrated in this crossbreed a marked variation in the duration and amplitude of nocturnal melatonin peak in response to increased concentration of CO in ophthalmic venous blood. During the winter this treatment limited the nocturnal melatonin rise. During the summer this same experimental treatment enhanced the nocturnal melatonin rise. Changes in melatonin levels were always associated with parallel changes in AANAT protein levels. This work demonstrates that non-physiological changes in CO concentration in ophthalmic venous blood can have an acute impact on the systemic melatonin level. These results support humoral phototransduction as a mechanism for some of bright light's effects in animal chronobiology and treatment of winter seasonal affective disorder.

The involvement of NMDA and AMPA receptors in the mechanism of antidepressant-like action of zinc in the forced swim test.

Antidepressant-like activity of zinc in the forced swim test (FST) was demonstrated previously. Enhancement of such activity by joint administration of zinc and antidepressants was also shown. However, mechanisms involved in this activity have not yet been established. The present study examined the involvement of the NMDA and AMPA receptors in zinc activity in the FST in mice and rats. Additionally, the influence of zinc on both glutamate and aspartate release in the rat brain was also determined. Zinc-induced antidepressant-like activity in the FST in both mice and rats was antagonized by N-methyl-D-aspartic acid (NMDA, 75 mg/kg, i.p.) administration. Moreover, low and ineffective doses of NMDA antagonists (CGP 37849, L-701,324, D-cycloserine, and MK-801) administered together with ineffective doses of zinc exhibit a significant reduction of immobility time in the FST. Additionally, we have demonstrated the reduction of immobility time by AMPA receptor potentiator, CX 614. The antidepressant-like activity of both CX 614 and zinc in the FST was abolished by NBQX (an antagonist of AMPA receptor, 10 mg/kg, i.p.), while the combined treatment of sub-effective doses of zinc and CX 614 significantly reduces the immobility time in the FST. The present study also demonstrated that zinc administration potentiated a veratridine-evoked glutamate and aspartate release in the rat's prefrontal cortex and hippocampus. The present study further suggests the antidepressant properties of zinc and indicates the involvement of the NMDA and AMPA glutamatergic receptors in this activity.

Intraperitoneal zinc administration increases extracellular zinc in the rat prefrontal cortex.

Single administration of zinc evokes pharmacological behavioral effects in rodents, while no brain zinc alterations were detected. The aim of the present study was to examine the effect of a single zinc hydroaspartate intraperitoneal (ip) administration on the extracellular (synaptic) zinc concentration in the rat prefrontal cortex. We used anodic stripping voltammetric (ASV) method of zinc determination in microdialysate, which assays the extracellular zinc concentration. We report that acute (65 mg/kg) zinc hydroaspartate administration (ip) increases the extracellular zinc by 48% in the rat prefrontal cortex. These data for the first time demonstrate: 1) utility of ASV zinc detection in brain microdialysates and 2) that single ip zinc administration increases brain (cortical) extracellular zinc pool. The results indicate zinc-induced fast brain penetration and may explain its rapid pharmacological effects.