Metallothionein induction reduces caspase-3 activity and TNFα levels with preservation of cognitive function and intact hippocampal neurons in carmustine-treated rats

Hippocampal integrity is essential for cognitive functions. On the other hand, induction of metallothionein (MT) by ZnSO4 and its role in neuroprotection has been documented. The present study aimed to explore the effect of MT induction on carmustine (BCNU)-induced hippocampal cognitive dysfunction in rats. A total of 60 male Wistar albino rats were randomly divided into four groups (15/group): The control group injected with single doses of normal saline (i.c.v) followed 24 h later by BCNU solvent (i.v). The second group administered ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v, once) then BCNU solvent (i.v) after 24 h. Third group received BCNU (20 mg/kg, i.v, once) 24 h after injection with normal saline (i.c.v). Fourth group received a single dose of ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v) then BCNU (20 mg/kg, i.v, once) after 24 h. The obtained data revealed that BCNU administration resulted in deterioration of learning and short-term memory (STM), as measured by using radial arm water maze, accompanied with decreased hippocampal glutathione reductase (GR) activity and reduced glutathione (GSH) content. Also, BCNU administration increased serum tumor necrosis factor-alpha (TNFα), hippocampal MT and malondialdehyde (MDA) contents as well as caspase-3 activity in addition to histological alterations. ZnSO4 pretreatment counteracted BCNU-induced inhibition of GR and depletion of GSH and resulted in significant reduction in the levels of MDA and TNFα as well as the activity of caspase-3. The histological features were improved in hippocampus of rats treated with ZnSO4 + BCNU compared to only BCNU-treated animals. In conclusion, MT induction halts BCNU-induced hippocampal toxicity as it prevented GR inhibition and GSH depletion and counteracted the increased levels of TNFα, MDA and caspase-3 activity with subsequent preservation of cognition

Pro-inflammatory and oxidative stress pathways which compromise sperm motility and survival may be altered by L-carnitine

The testis is an immunologically privileged organ. Sertoli cells can form a blood-testis barrier and protect sperm cells from self-immune system attacks. Spermatogenesis may be inhibited by severe illness, bacterial infections and chronic inflammatory diseases but the mechanism(s) is poorly understood. Our objective is to help in understanding such mechanism(s) to develop protective agents against temporary or permanent testicular dysfunction. Lipopolysaccaride (LPS) is used as a model of animal sepsis while L-carnitine (LCR) is used as a protective agent. A total of 60 male Swiss albino rats were divided into four groups (15/group). The control group received Saline; the 2nd group was given LCR (500 mg/kg i.p, once). The third group was treated with LPS (5 mg/kg i.p once) and the fourth group received LCR then LPS after three hours. From each group, five rats were used for histopathological examination. Biochemical parameters were assessed in the remaining ten rats. At the end of the experiment, animals were lightly anaesthetized with ether where blood samples were collected and testes were dissected on ice. Sperm count and motility were evaluated from cauda epididymis in each animal. Also, oxidative stress was evaluated by measuring testicular contents of reduced glutathione (GSH), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-HDG, the DNA adduct for oxidative damage) in testicular DNA. The pro-inflammatory mediator nitric oxide (NO) in addition to lactate dehydrogenase (LDHx) isoenzyme-x activity as an indicator for normal spermatozoal metabolism were assessed in testicular homogenate. Serum interlukin (IL)-2 level was also assessed as a marker for T-helper cell function. The obtained data revealed that LPS induced marked reductions in sperm's count and motility, obstruction in seminiferous tubules, hypospermia and dilated congested blood vessels in testicular sections concomitant with decreased testicular GSH content and LDHx activity. Moreover, the testicular levels of MDA, 8-HDG (in testicular DNA) and NO as well as serum IL-2 level were increased. Administration of LCR before LPS returned both sperm count and motility to normal levels. Also, contents of testicular GSH, MDA, 8-HDG and NO returned back to the corresponding control values. In addition, serum IL-2 level as well as histological abnormalities were markedly improved in LCR + LPS-treated rats. In conclusion, LPS increased proinflammatory and oxidative stress markers in the testis leading to a marked testicular dysfunction. L-carnitine administration ameliorates these effects by antioxidant and/or anti-inflammatory mechanisms suggesting a protective role against male infertility in severely infected or septic patients

Exosomal miR-940 maintains SRC-mediated oncogenic activity in cancer cells: a possible role for exosomal disposal of tumor suppressor miRNAs

Exosomes have emerged as important mediators of diverse biological functions including tumor suppression, tumor progression, invasion, immune escape and cell-to-cell communication, through the release of molecules such as mRNAs, miRNAs, and proteins. Here, we identified differentially expressed exosomal miRNAs between normal epithelial ovarian cell line and both resistant and sensitive ovarian cancer (OC) cell lines. We found miR-940 as abundant in exosomes from SKOV3-IP1, HeyA8, and HeyA8-MDR cells. The high expression of miR-940 is associated with better survival in patients with ovarian serous cystadenocarcinoma. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, and migration and triggered G0/G1 cell cycle arrest and apoptosis in OC cells. Overexpression of miR-940 also inhibited tumor cell growth in vivo. We showed that proto-oncogene tyrosine-protein kinase (SRC) is directly targeted by miR-940 and that miR-940 inhibited SRC expression at mRNA and protein levels. Following this inhibition, the expression of proteins downstream of SRC, such as FAK, paxillin and Akt was also reduced. Collectively, our results suggest that OC cells secrete the tumor-suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype

Metallothionein Induction Reduces Caspase-3 Activity and Tnfα Levels with Preservation of Cognitive Function and Intact Hippocampal Neurons in Carmustine-Treated Rats

Hippocampal integrity is essential for cognitive functions. On the other hand, induction of metallothionein (MT) by ZnSO4 and its role in neuroprotection has been documented. The present study aimed to explore the effect of MT induction on carmustine (BCNU)-induced hippocampal cognitive dysfunction in rats. A total of 60 male Wistar albino rats were randomly divided into four groups (15/group): The control group injected with single doses of normal saline (i.c.v) followed 24 h later by BCNU solvent (i.v). The second group administered ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v, once) then BCNU solvent (i.v) after 24 h. Third group received BCNU (20 mg/kg, i.v, once) 24 h after injection with normal saline (i.c.v). Fourth group received a single dose of ZnSO4 (0.1 µmol/10 µl normal saline, i.c.v) then BCNU (20 mg/kg, i.v, once) after 24 h. The obtained data revealed that BCNU administration resulted in deterioration of learning and short-term memory (STM), as measured by using radial arm water maze, accompanied with decreased hippocampal glutathione reductase (GR) activity and reduced glutathione (GSH) content. Also, BCNU administration increased serum tumor necrosis factor-alpha (TNFα), hippocampal MT and malondialdehyde (MDA) contents as well as caspase-3 activity in addition to histological alterations. ZnSO4 pretreatment counteracted BCNU-induced inhibition of GR and depletion of GSH and resulted in significant reduction in the levels of MDA and TNFα as well as the activity of caspase-3. The histological features were improved in hippocampus of rats treated with ZnSO4 + BCNU compared to only BCNU-treated animals. In conclusion, MT induction halts BCNU-induced hippocampal toxicity as it prevented GR inhibition and GSH depletion and counteracted the increased levels of TNFα, MDA and caspase-3 activity with subsequent preservation of cognition

Exosomes: From Garbage Bins to Promising Therapeutic Targets

Intercellular communication via cell-released vesicles is a very important process for both normal and tumor cells. Cell communication may involve exosomes, small vesicles of endocytic origin that are released by all types of cells and are found in abundance in body fluids, including blood, saliva, urine, and breast milk. Exosomes have been shown to carry lipids, proteins, mRNAs, non-coding RNAs, and even DNA out of cells. They are more than simply molecular garbage bins, however, in that the molecules they carry can be taken up by other cells. Thus, exosomes transfer biological information to neighboring cells and through this cell-to-cell communication are involved not only in physiological functions such as cell-to-cell communication, but also in the pathogenesis of some diseases, including tumors and neurodegenerative conditions. Our increasing understanding of why cells release exosomes and their role in intercellular communication has revealed the very complex and sophisticated contribution of exosomes to health and disease. The aim of this review is to reveal the emerging roles of exosomes in normal and pathological conditions and describe the controversial biological role of exosomes, as it is now understood, in carcinogenesis. We also summarize what is known about exosome biogenesis, composition, functions, and pathways and discuss the potential clinical applications of exosomes, especially as biomarkers and novel therapeutic agents

Complex formation between α7 nAChR and NMDAR in the adult 3xTg-AD mouse brain.

<p>Affinity purification performed with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) using frontal cortical tissue lysates from adult 3xTg-AD mice (76–84 weeks old) and age- and sex-matched WT mice. (<b>A</b>) A representative example of a western blot illustrating GluN1, α7 nAChR and GABA_AR α1 protein levels in total lysates (Input) and pulled down (Pull Down) samples from WT and 3xTg-AD mouse cortical homogenates. <b>(B-C)</b> Quantification of total GluN1 (B) and total α7 (C) in lysates from WT and 3xTg-AD mouse cortical homogenates (both normalized to stain-free gel). <b>(D-E)</b> Quantification of α7 pulled-down (normalized to stain-free gel) (D), and of GluN1 pulled-down with α7 (normalized to the pulled-down α7) (E). In B-E, the control group (WT) is set to 1, and values are shown as mean ± SEM. **p < 0.01 indicates statistical significant difference from WT group in unpaired <i>t</i>-tests, n = 8 (WT) and n = 8 (3xTg-AD).</p

Probing the putative α7 nAChR/NMDAR complex in human and murine cortex and hippocampus: Different degrees of complex formation in healthy and Alzheimer brain tissue

<div><p>α7 nicotinic acetylcholine receptors (nAChRs) and <i>N</i>-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer’s disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-β_1–42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.</p></div

Complex formation between α7 nAChR and NMDAR in murine and human cortex and hippocampus.

<p><b>(A-B)</b> Affinity purification with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) on homogenates from murine (A) and human (B) cortical (CTX) and hippocampal (Hippo) tissues. Total lysates (Input) and pulled-down (Pull Down) samples were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits. The gels in A and B are representative for different experiments using tissues from 4 different mouse hippocampi, 4 different mouse cortices, 2 different human hippocampi and 2 different human cortices. (<b>C</b>) Total lysates (Input) and pulled-down (Pull Down) samples from WT and α7 KO mouse cortical homogenates were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits and β-actin. (<b>D</b>) Total lysates (Input) and pulled-down (Pull Down) samples from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM) were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, GluN2A, α7 nAChR and GABA_AR α1 subunits. (<b>E</b>) Quantification of α7 pulled-down and GluN1 and GluN2A pulled-down (normalized to the pulled-down α7) from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM). Values are given as mean ± SEM (n = 3–4, i.e. 3–4 different mouse cortices, the experiment was performed once). *p <0.05 and **p < 0.01 indicate statistically significant difference from the vehicle-treated group in Kruskal-Wallis test with Dunn’s multiple comparison test. ^#p <0.05 indicates statistically significant difference between GluN1/α7 Pulled-down ratios between α7-pep2 (50 μM) or α7-pep1 (50 μM) in unpaired <i>t</i>-tests.</p

Complex formation between α7 nAChR and NMDAR in murine and human cortex and hippocampus.

<p><b>(A-B)</b> Affinity purification with agarose beads covalently coupled with α-bungarotoxin (BGT) or BSA (Ctrl) on homogenates from murine (A) and human (B) cortical (CTX) and hippocampal (Hippo) tissues. Total lysates (Input) and pulled-down (Pull Down) samples were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits. The gels in A and B are representative for different experiments using tissues from 4 different mouse hippocampi, 4 different mouse cortices, 2 different human hippocampi and 2 different human cortices. (<b>C</b>) Total lysates (Input) and pulled-down (Pull Down) samples from WT and α7 KO mouse cortical homogenates were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, α7 nAChR and GABA_AR α1 subunits and β-actin. (<b>D</b>) Total lysates (Input) and pulled-down (Pull Down) samples from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM) were submitted to gel electrophoresis and Western blotting followed by detection using antibodies for GluN1, GluN2A, α7 nAChR and GABA_AR α1 subunits. (<b>E</b>) Quantification of α7 pulled-down and GluN1 and GluN2A pulled-down (normalized to the pulled-down α7) from mouse cortical homogenates pretreated with buffer or buffer supplemented with α7-pep2 (10 μM and 50 μM) or α7-pep1 (10 μM and 50 μM). Values are given as mean ± SEM (n = 3–4, i.e. 3–4 different mouse cortices, the experiment was performed once). *p <0.05 and **p < 0.01 indicate statistically significant difference from the vehicle-treated group in Kruskal-Wallis test with Dunn’s multiple comparison test. ^#p <0.05 indicates statistically significant difference between GluN1/α7 Pulled-down ratios between α7-pep2 (50 μM) or α7-pep1 (50 μM) in unpaired <i>t</i>-tests.</p

Clinicopathologic data of patients and human brain materials.

<p>Clinicopathologic data of patients and human brain materials.</p