Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Compounds that bind to the gamma-aminobutyric acid benzodiazepine ionophore complex modulate the cellular response to oxidant stress

The γ-aminobutyric acid benzodiazepine (GABAA /BZDR) ionophore complex has been widely studied in the central nervous system (CNS) and it regulates Cl− ion movement across the plasma membrane. The complex has been found in the distal tubule and the thick ascending limb of the kidney. The goal of this study was to see if modulation of this complex by agonists or antagonists could affect the way Madin-Darby Canine Kidney (MDCK) cells responded to an oxidant stress induced by menadione. When compared to cells incubated with menadione alone, preincubation with lindane, a nonspecific GABAA antagonist, coincubation with bicuculline, a specific GABAA antagonist, and coincubation with FG7142, an inverse agonist for the BZDR, protected cells from menadione cytotoxicity. Preincubation of cells in media containing PK11195 had no effect on menadione cytotoxicity. Coincubation with flurazepam, a BZDR agonist, exacerbated menadione cytotoxicity. This suggests that modulation of the GABAA/BZDR ionophore complex within MDCK cells with agonists and antagonists can alter the cellular responsiveness to an oxidant-induced injury. These responses via agonists and antagonists may be due to alterations of Cl− ion influx during late stage necrotic cell death. ^

Dopamine β-hydroxylase deficiency impairs cellular immunity

Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions. The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response. However, the importance of this modulatory role in vivo remains uncertain. We addressed this question genetically by using mice that lack dopamine β-hydroxylase (dbh−/− mice). dbh−/− mice cannot produce norepinephrine or epinephrine, but produce dopamine instead. When housed in specific pathogen-free conditions, dbh−/− mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function. However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh−/− mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production. When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh−/− mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody. These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization.

Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions

Compelling evidence has accumulated over the last several years from our laboratory, as well as others, indicating that central hyperactive states resulting from neuronal plastic changes within the spinal cord play a critical role in hyperalgesia associated with nerve injury and inflammation. In our laboratory, chronic constriction injury of the common sciatic nerve, a rat model of neuropathic pain, has been shown to result in activation of central nervous system excitatory amino acid receptors and subsequent intracellular cascades including protein kinase C translocation and activation, nitric oxide production, and nitric oxide-activated poly(ADP ribose) synthetase activation. Similar cellular mechanisms also have been implicated in the development of tolerance to the analgesic effects of morphine. A recently observed phenomenon, the development of “dark neurons,” is associated with both chronic constriction injury and morphine tolerance. A site of action involved in both hyperalgesia and morphine tolerance is in the superficial laminae of the spinal cord dorsal horn. These observations suggest that hyperalgesia and morphine tolerance may be interrelated at the level of the superficial laminae of the dorsal horn by common neural substrates that interact at the level of excitatory amino acid receptor activation and subsequent intracellular events. The demonstration of interrelationships between neural mechanisms underlying hyperalgesia and morphine tolerance may lead to a better understanding of the neurobiology of these two phenomena in particular and pain in general. This knowledge may also provide a scientific basis for improved pain management with opiate analgesics.

The ubiquitin-proteasome system in retinal health and disease

The ubiquitin–proteasome system (UPS) is the main intracellular pathway for modulated protein turnover, playing an important role in the maintenance of cellular homeostasis. It also exerts a protein quality control through degradation of oxidized, mutant, denatured, or misfolded proteins and is involved in many biological processes where protein level regulation is necessary. This system allows the cell to modulate its protein expression pattern in response to changing physiological conditions and provides a critical protective role in health and disease. Impairments of UPS function in the central nervous system (CNS) underlie an increasing number of genetic and idiopathic diseases, many of which affect the retina. Current knowledge on the UPS composition and function in this tissue, however, is scarce and dispersed. This review focuses on UPS elements reported in the retina, including ubiquitinating and deubiquitinating enzymes (DUBs), and alternative proteasome assemblies. Known and inferred roles of protein ubiquitination, and of the related, SUMO conjugation (SUMOylation) process, in normal retinal development and adult homeostasis are addressed, including modulation of the visual cycle and response to retinal stress and injury. Additionally, the relationship between UPS dysfunction and human neurodegenerative disorders affecting the retina, including Alzheimer's, Parkinson's, and Huntington's diseases, are dealt with, together with numerous instances of retina-specific illnesses with UPS involvement, such as retinitis pigmentosa, macular degenerations, glaucoma, diabetic retinopathy (DR), and aging-related impairments. This information, though still basic and limited, constitutes a suitable framework to be expanded in incoming years and should prove orientative toward future therapy design targeting sight-affecting diseases with a UPS underlying basis.

Nervous and muscle system development in Phascolion strombus (Sipuncula)

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Harnessing the power of Drosophila model to obtain new insights on MYC and ABCC1 cellular functions: from neuroblastoma to autism spectrum disorder

MYCN amplification is a genetic hallmark of the childhood tumour neuroblastoma. MYCN-MAX dimers activate the expression of genes promoting cell proliferation. Moreover, MYCN seems to transcriptionally repress cell differentiation even in absence of MAX. We adopted the Drosophila eye as model to investigate the effect of high MYC to MAX expression ratio on cells. We found that dMyc overexpression in eye cell precursors inhibits cell differentiation and induces the ectopic expression of Antennapedia (the wing Hox gene). The further increase of MYC/MAX ratio results in an eye-to-wing homeotic transformation. Notably, dMyc overexpression phenotype is suppressed by low levels of transcriptional co-repressors and MYCN associates to the promoter of Deformed (the eye Hox gene) in proximity to repressive sites. Hence, we envisage that, in presence of high MYC/MAX ratio, the “free MYC” might inhibit Deformed expression, leading in turn to the ectopic expression of Antennapedia. This suggests that MYCN might reinforce its oncogenic role by affecting the physiological homeotic program. Furthermore, poor neuroblastoma outcome associates with a high level of the MRP1 protein, encoded by the ABCC1 gene and known to promote drug efflux in cancer cells. Intriguingly, this correlation persists regardless of chemotherapy and ABCC1 overexpression enhances neuroblastoma cell motility. We found that Drosophila dMRP contributes to the adhesion between the dorsal and ventral epithelia of the wing by inhibiting the function of integrin receptors, well known regulators of cell adhesion and migration. Besides, integrins play a crucial role during synaptogenesis and ABCC1 locus is included in a copy number variable region of the human genome (16p13.11) involved in neuropsychiatric diseases. Interestingly, we found that the altered dMRP/MRP1 level affects nervous system development in Drosophila embryos. These preliminary findings point out novel ABCC1 functions possibly defining ABCC1 contribution to neuroblastoma and to the pathogenicity of 16p13.11 deletion/duplication

Plasticidade neural: relações com o comportamento e abordagens experimentais

Behavioral adaptiveness to different situations as well as behavioral individuality result from the interrelations between environmental sitmuli and the responses of an organism.These kind of interrelationships also shape the neural circuits as well as characterize the plasticity and the neural individuality of the organism. Studies on neural plasticity may analyze changes in neural circuitry after environmental manipulations or changes in behavior after lesions in the nervous system. Issues on neural plasticity and recovery of function refer both to physiology and behavior as well as to the subjacent mechanisms related to morphology, biochemistry and genetics. They may be approached at the systemic, behavioral, cellular and molecular levels. This work intends to characterize these kinds of studies pointing to their relations with the analyis of behavior and learning.The analysis of how the environmental-organismic interrelationships affect the neural substrates of behavior is pointed as a very stimulating area for investigation.

Histomorfometria das meninges encefálicas de ratos Wistar em diferentes faixas etárias

O desenvolvimento do sistema nervoso é bastante complexo, existindo poucos estudos sobre a organização dos envoltórios cerebrais relacionados ao crescimento encefálico. Utilizando como modelo experimental o rato, analisaram-se os diferentes aspectos estruturais e morfométricos da paquimeninge e leptomeninge durante o processo de envelhecimento. Foram utilizados quatro grupos de ratos em diferentes faixas etárias e analisadas as meninges em microscopias de luz e eletrônica. Verificamos que o grupo de ratos adultos apresentou uma maior área de fibras colágenas tanto do tipo I e quanto do tipo III, em relação aos outros grupos. Encontramos também que as fibras colágenas do tipo III em todos os grupos analisados ocupam uma maior área quando comparados com as fibras do tipo I. Os resultados revelam que a coloração de Weigert Oxona, que mostra fibras elásticas, elaunínicas e oxitalânicas, apresentou uma diferença estatisticamente maior de fibras quando comparados com as colorações de Weigert e Verhoeff, que mostra fíbras elaunínicas e elásticas, respectivamente. Os resultados ultra-estruturais demonstraram a presença de muitos fibroblastos e mitocôndrias tanto na paquimeninge como nas leptomeninges dos grupos de ratos neonatos e adultos, indicativo de alta atividade celular e conseqüentemente, intensa formação de tecido conjuntivo. Como as fibras colágenas do tipo III atuam na manutenção da estrutura de tecidos delicados e expansíveis, o estudo mostra que as funções das meninges encefálicas não estão relacionadas apenas com a resistência a trações e tensões a que estão sujeitas o encéfalo. Mas também a função relacionada com a distensibilidade dos vasos meníngeos e cerebrais de acordo com a necessidade do aporte sanguíneo em diversas funções específicas regionais do tecido nervoso.

Preliminary biocompatibility investigation of magnetic albumin nanosphere designed as a potential versatile drug delivery system

Background: The magnetic albumin nanosphere (MAN), encapsulating maghemite nanoparticles, was designed as a magnetic drug delivery system (MDDS) able to perform a variety of biomedical applications. It is noteworthy that MAN was efficient in treating Ehrlich's tumors by the magnetohyperthermia procedure. Methods and materials: In this study, several nanotoxicity tests were systematically carried out in mice from 30 minutes until 30 days after MAN injection to investigate their biocompatibility status. Cytometry analysis, viability tests, micronucleus assay, and histological analysis were performed. Results: Cytometry analysis and viability tests revealed MAN promotes only slight and temporary alterations in the frequency of both leukocyte populations and viable peritoneal cells, respectively. Micronucleus assay showed absolutely no genotoxicity or cytotoxicity effects and histological analysis showed no alterations or even nanoparticle clusters in several investigated organs but, interestingly, revealed the presence of MAN clusters in the central nervous system (CNS). Conclusion: The results showed that MAN has desirable in vivo biocompatibility, presenting potential for use as a MDDS, especially in CNS disease therapy.

Central nitrergic system regulation of neuroendocrine secretion, fluid intake and blood pressure induced by angiotensin-II

Background: Nitric oxide (NO) synthesis has been described in several circumventricular and hypothalamic structures in the central nervous system that are implicated in mediating central angiotensin-II (ANG-II) actions during water deprivation and hypovolemia. Neuroendocrine and cardiovascular responses, drinking behavior, and urinary excretions were examined following central angiotensinergic stimulation in awake freely-moving rats pretreated with intracerebroventricular injections of N omega-nitro-L-arginine methyl ester (L-NAME, 40 mu g), an inhibitor of NO synthase, and L-arginine (20 ug), a precursor of NO. Results: Injections of L-NAME or ANG-II produced an increase in plasma vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) levels, an increase in water and sodium intake, mean arterial blood pressure and sodium excretion, and a reduction of urinary volume. L-NAME pretreatment enhanced the ANG-II response, while L-arginine attenuated VP and OT release, thirst, appetite for sodium, antidiuresis, and natriuresis, as well as pressor responses induced by ANG-II. Discussion and conclusion: Thus, the central nitrergic system participates in the angiotensinergic responses evoked by water deprivation and hypovolemia to refrain neurohypophysial secretion, hydromineral balance, and blood pressure homeostasis.

GSTP1 Ile105Val polymorphism in astrocytomas and glioblastomas

Glutathione S-transferases (GSTs) constitute a superfamily of ubiquitous multifunctional enzymes that are involved in the cellular detoxification of a large number of endogenous and exogenous chemical agents that have electrophilic functional groups. People who have deficiencies in this family of genes are at increased risk of developing some types of tumors. We examined GSTP1 Ile105Val polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the Val allele of the GSTP1 Ile105Val polymorphism had an increased risk of tumor development (odds ratio = 8.60; 95% confidence interval = 4.74-17.87; P < 0.001). Overall survival of patients did not differ significantly. We suggest that GSTP1 Ile105Val polymorphisms are involved in susceptibility to developing astrocytomas and glioblastomas.

Differential expression of E-cadherin gene in human neuroepithelial tumors

Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.

Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

Background: Protein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours. Results: We demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM) lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats. Conclusion: Rotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.