The neuronal ceroid lipofuscinosis-related protein CLN8 regulates endo-lysosomal dynamics and dendritic morphology.

BACKGROUND INFORMATION: The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known neuronal ceroid lipofuscinoses (NCLs), a group of inherited diseases characterised by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterised. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyse structural, dynamic and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. RESULTS: CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalisation in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. CONCLUSIONS: Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. SIGNIFICANCE: This is, to our knowledge, the first characterisation of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease.

De novo tetrahydrobiopterin biosynthesis is impaired in the inflammed striatum of parkin(-/-) mice.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disease, is primarily characterized by neurodegeneration in the substantia nigra pars compacta, resulting in motor impairment. Loss-of-function mutations in parkin are the major cause of the early onset familial form of the disease. Although rodents deficient in parkin (parkin(-/-) ) have some dopaminergic system dysfunction associated with central oxidative stress and energy metabolism deficiencies, these animals only display nigrostriatal pathway degeneration under inflammatory conditions. This study investigated the impact of the inflammatory stimulus induced by lypopolisaccharide (LPS) on tetrahydrobiopterin (BH4) synthesizing enzymes (de novo and salvage pathways), since this cofactor is essential for dopamine synthesis. The mitochondrial content and architecture was investigated in the striatum of LPS-exposed parkin(-/-) mice. As expected, the LPS (0.33 mg/kg; i.p.) challenge compromised spontaneous locomotion and social interaction with juvenile parkin(-/-) and WT mice. Moreover, the genotype impacted the kinetics of the investigation of the juvenile. The inflammatory scenario did not induce apparent changes in mitochondrial ultrastructure; however, it increased the quantity of mitochondria, which were of smaller size, and provoked the perinuclear distribution of the organelle. Furthermore, the BH4 de novo biosynthetic pathway failed to be up-regulated in the LPS challenge, a well-known stimulus for its activation. The LPS treatment increased sepiapterin reductase (SPR) expression, suggesting compensation by the salvage pathway. This might indicate that dopamine synthesis is compromised in parkin(-/-) mice under inflammatory conditions. Finally, this scenario impaired the striatal expression of the transcription factor BDNF, possibly favoring cell death.

Diphenyl diselenide administration enhances cortical mitochondrial number and activity by increasing hemeoxygenase type 1 content in a methylmercury-induced neurotoxicity mouse model.

Interest in biochemistry of organoselenium compound has increased in the last decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of diphenyl diselenide (PhSe)2 (5 µmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Swiss male mice were divided into four experimental groups: control, (PhSe)2 (5 µmol/kg, subcutaneous administration), MeHg (40 mg/L, in tap water), and MeHg + (PhSe)2. After the treatment (21 days), the animals were killed and the cerebral cortex was analyzed. Electron microscopy indicated an enlarged and fused mitochondria leading to a reduced number of organelles, in the MeHg-exposed mice. Furthermore, cortical creatine kinase activity, a sensitive mitochondrial oxidative stress sensor, was almost abolished by MeHg. Subcutaneous (PhSe)2 co-treatment rescued from MeHg-induced mitochondrial alterations. (PhSe)2 also behaved as an enhancer of mitochondrial biogenesis, by increasing cortical mitochondria content in mouse-receiving (PhSe)2 alone. Mechanistically, (PhSe)2 (1 µM; 24 h) would trigger the cytoprotective Nrf-2 pathway for activating target genes, since astroglial cells exposed to the chalcogen showed increased content of hemeoxygenase type 1, a sensitive marker of the activation of this via. Thus, it is proposed that the (PhSe)2-neuroprotective effect might be linked to its mitoprotective activity.

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia.

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17ß-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17ß-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.

Testosterone abrogates TLR4 activation in prostate smooth muscle cells contributing to the preservation of a differentiated phenotype.

Prostate smooth muscle cells (pSMCs) are capable of responding to inflammatory stimuli by secreting proinflammatory products, which causes pSMCs to undergo dedifferentiation. Although it has been proposed that androgens decrease proinflammatory molecules in many cells and under various conditions, the role of testosterone in the prostate inflammatory microenvironment is still unclear. Therefore, our aim was to evaluate if testosterone was able to modulate the pSMCs response to bacterial LPS by stimulating primary pSMC cultures, containing testosterone or vehicle, with LPS (1 or 10 µg/ml) for 24-48 h. The LPS challenge induced pSMCs dedifferentiation as evidenced by a decrease of calponin and alpha smooth muscle actin along with an increase of vimentin in a dose-dependent manner, whereas testosterone abrogated these alterations. Additionally, an ultrastructural analysis showed that pSMCs acquired a secretory profile after LPS and developed proteinopoietic organelles, while pSMCs preincubated with testosterone maintained a more differentiated phenotype. Testosterone downregulated the expression of surface TLR4 in control cells and inhibited any increase after LPS treatment. Moreover, testosterone prevented IκB-α degradation and the LPS-induced NF-κB nuclear translocation. Testosterone also decreased TNF-α and IL6 production by pSMCs after LPS as quantified by ELISA. Finally, we observed that testosterone inhibited the induction of pSMCs proliferation incited by LPS. Taken together, these results indicate that testosterone reduced the proinflammatory pSMCs response to LPS, with these cells being less reactive in the presence of androgens. In this context, testosterone might have a homeostatic role by contributing to preserve a contractile phenotype on pSMCs under inflammatory conditions.

Sellar xanthogranuloma as a diagnostic challenge: a report on five cases.

Xanthogranulomas are considered rare tumors, with their sellar and non-sellar frequency ranging from 1.6 to 7% among intracranial lesions, and described as a separate entity by the World Health Organization in 2000. The diagnosis of sellar xanthogranulomas is challenging, given their uncertain origin and clinical course. In addition, the limited reporting of sellar xanthogranuloma cases and the absence of characteristic images make these entities difficult to distinguish from other cystic lesions of the sellar region, such as adamantinomatous craniopharyngiomas, Rathke's cleft cysts, pituitary tumors, arachnoid cysts, epidermoid cysts, and dermoid cysts. Here, we describe the clinical presentation, radiological findings, immunohistochemical/histopathological analysis, and the ultrastructural examination by transmission electron microscopy of five sellar xanthogranulomas cases reported in two care centers in Cordoba, Argentina. Two males and three females between 37 and 73 years of age (average 51.8 years) presented with persistent headaches, generalized endocrine defects, and visual problems. MRI revealed cystic formations in the sellar region, which usually projected into adjacent tissues such as the suprasellar region or cavernous sinuses, and compressed other structures such as the optic chiasm, pituitary gland, and cranial nerves. All patients underwent surgical intervention to remove the tumor tissue. The histopathological analysis of the samples showed cellular tissue with a xanthogranulomatous appearance, inflammatory cellular infiltrate (mainly lymphocytes and macrophages), fibroblasts, abundant collagen fibers, and hemorrhages. An ultrastructural analysis helped to identify cellular infiltrates and granules resulting from tumor cell activity. The data support the hypothesis that sellar xanthogranulomas could occur as an inflammatory reaction secondary to the rupture and hemorrhage of a previous cystic process, thereby generating an expansion of the tumor body toward adjacent tissues. The information obtained from these cases contributes to the current knowledge about this disease's origin and clinical and histological evolution. However, the scarcity of patients and the observed phenotypic heterogeneity make its diagnosis still challenging. Undoubtedly, more investigations are needed to provide additional information in order to be able to achieve a more accurate diagnosis and effective treatment of this rare disease.

Architects of Pituitary Tumour Growth.

The pituitary is a master gland responsible for the modulation of critical endocrine functions. Pituitary neuroendocrine tumours (PitNETs) display a considerable prevalence of 1/1106, frequently observed as benign solid tumours. PitNETs still represent a cause of important morbidity, due to hormonal systemic deregulation, with surgical, radiological or chronic treatment required for illness management. The apparent scarceness, uncommon behaviour and molecular features of PitNETs have resulted in a relatively slow progress in depicting their pathogenesis. An appropriate interpretation of different phenotypes or cellular outcomes during tumour growth is desirable, since histopathological characterization still remains the main option for prognosis elucidation. Improved knowledge obtained in recent decades about pituitary tumorigenesis has revealed that this process involves several cellular routes in addition to proliferation and death, with its modulation depending on many signalling pathways rather than being the result of abnormalities of a unique proliferation pathway, as sometimes presented. PitNETs can display intrinsic heterogeneity and cell subpopulations with diverse biological, genetic and epigenetic particularities, including tumorigenic potential. Hence, to obtain a better understanding of PitNET growth new approaches are required and the systematization of the available data, with the role of cell death programs, autophagy, stem cells, cellular senescence, mitochondrial function, metabolic reprogramming still being emerging fields in pituitary research. We envisage that through the combination of molecular, genetic and epigenetic data, together with the improved morphological, biochemical, physiological and metabolically knowledge on pituitary neoplastic potential accumulated in recent decades, tumour classification schemes will become more accurate regarding tumour origin, behaviour and plausible clinical results.

Neuronal ceroid lipofuscinosis in the South American-Caribbean region: An epidemiological overview.

Neuronal ceroid lipofuscinoses (NCLs) comprise 13 hereditary neurodegenerative pathologies of very low frequency that affect individuals of all ages around the world. All NCLs share a set of symptoms that are similar to other diseases. The exhaustive collection of data from diverse sources (clinical, genetic, neurology, ophthalmology, etc.) would allow being able in the future to define this group with greater precision for a more efficient diagnostic and therapeutic approach. Despite the large amount of information worldwide, a detailed study of the characteristics of the NCLs in South America and the Caribbean region (SA&C) has not yet been done. Here, we aim to present and analyse the multidisciplinary evidence from all the SA&C with qualitative weighting and biostatistical evaluation of the casuistry. Seventy-one publications from seven countries were reviewed, and data from 261 individuals (including 44 individuals from the Cordoba cohort) were collected. Each NCL disease, as well as phenotypical and genetic data were described and discussed in the whole group. The CLN2, CLN6, and CLN3 disorders are the most frequent in the region. Eighty-seven percent of the individuals were 10 years old or less at the onset of symptoms. Seizures were the most common symptom, both at onset (51%) and throughout the disease course, followed by language (16%), motor (15%), and visual impairments (11%). Although symptoms were similar in all NCLs, some chronological differences could be observed. Sixty DNA variants were described, ranging from single nucleotide variants to large chromosomal deletions. The diagnostic odyssey was probably substantially decreased after medical education activities promoted by the pharmaceutical industry and parent organizations in some SA&C countries. There is a statistical deviation in the data probably due to the approval of the enzyme replacement therapy for CLN2 disease, which has led to a greater interest among the medical community for the early description of this pathology. As a general conclusion, it became clear in this work that the combined bibliographical/retrospective evaluation approach allowed a general overview of the multidisciplinary components and the epidemiological tendencies of NCLs in the SA&C region.

Epidermal growth factor induces a sexually dimorphic proliferative response of lactotroph cells through protein kinase C-ERK1/2-Pit-1 in vitro.

Lactotroph cells display morphological and functional heterogeneity, a feature which is closely related to the oestrogenic environment. In this study, we focused on sex-related differences linked to the proliferative and secretory responses of lactotrophs exposed to EGF in vitro. Furthermore, we addressed the involvement of the PKCε/ERK1/2 signalling pathway and the contribution of Pit-1 in the EGF actions in primary pituitary cultures from male and female rats. EGF promoted a differential proliferative activity on PRL cells, which was closely associated to the sex, as revealed by the uptake of bromodeoxyuridine (BrdU). In females, the mitogenic activity was up to nine times greater, whereas in males, the number of BrdU-labelled PRL cells was only doubled compared to control. However, in both models, EGF had a similar effectiveness in promoting PRL secretion. EGF also induced a significant increase in the PKCε, P -ERK 1/2, and Pit-1 protein levels, which were higher in females than in males. Pre-incubation with BIM blocked EGF-induced ERK 1/2 activation and Pit-1 expression. These results suggest a sexually dimorphic response of lactotroph cells to the proliferative effects of EGF, with the PKCε/ERK1/2 Pit-1 pathway being involved in this action.

ConBr lectin modulates MAPKs and Akt pathways and triggers autophagic glioma cell death by a mechanism dependent upon caspase-8 activation.

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.

GH3B6 pituitary tumor cell proliferation is mediated by PKCalpha and PKCepsilon via ERK 1/2-dependent pathway.

BACKGROUND: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. METHODS: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. RESULTS: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. CONCLUSION: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.

Pivotal role of NF-κB in cellular senescence of experimental pituitary tumours.

The molecular mechanisms underlying the capability of pituitary tumours to avoid unregulated cell proliferation are still not well understood. However, the NF-κB transcription factor, which is able to modulate not only cellular senescence but also tumour progression, has emerged as a targeted candidate. This work was focused on the NF-κB role in cellular senescence during the progression of experimental pituitary tumours. Also, the contribution of the signalling pathways in senescence-associated NF-κB activation and the senescence-associated secretory phenotype (SASP) and pro-survival-NF-κB target genes transcription were analysed. A robust NF-κB activation was seen at E20-E40 of tumour development accompanied by a marked SA-ß-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-ß as specific SASP-related NF-κB target genes as well as Bcl-2 and Bcl-xl pro-survival genes was shown to be accompanied by increases in the p-p38 MAPK protein levels, starting at the E20 stage and strengthening from 40 to 60 days of tumour growth. It is noteworthy that p-JNK displayed a similar pattern of activation during pituitary tumour development, while p-AKT and p-ERK1/2 were downregulated. By employing a pharmacological strategy to abrogate NF-κB activity, we demonstrated a marked reduction in SA-ß-Gal activity and a slight decrease in Ki67 immunopositive cells after NF-κB blockade. These results suggest a central role for NF-κB in the regulation of the cellular senescence programme, leading to the strikingly benign intrinsic nature of pituitary adenomas.

Thyroid tumor cells-fibroblasts crosstalk: role of extracellular vesicles.

Tumor-stroma crosstalk leads to a tumor-promoting microenvironment. In this milieu, extracellular vesicles (EVs) are protagonists in cell-cell communication. Despite thyroid cancer being the most common endocrine malignancy, the contribution of the tumor microenvironment to thyroid cancer progression is still largely underexplored. We focused on the role of thyroid tumor cell-fibroblast interaction and EVs as mediators of tumor-stroma interplay, in the promotion of thyroid tumor aggressiveness. Thyroid tumor (TPC-1, 8505c) or non-tumor thyroid cells (NThyOri) were co-cultured with human fibroblasts (Fb). Thyroid cell migration was investigated by the wound-healing assay and actin-network staining. Cell-CD147 expression was characterized by flow cytometry. EVs, obtained by ultracentrifugation of conditioned media (CMs), were characterized by transmission electron-microscopy and CD81 and CD147 expression. Metalloproteinases (MMPs) were evaluated by zymography in CMs. A migratory phenotype was triggered in thyroid tumor cells treated with CMs from Fb or from Fb-thyroid tumor cell co-cultures. Fb-thyroid cell co-cultures induced the secretion of proMMP9 and proMMP2 and led to a significant MMP2 activation in CMs. Fb, thyroid cells and Fb-thyroid cell co-cultures released EVs, and remarkably, EVs released by Fb-thyroid tumor cell co-cultures induced the secretion of proMMP2 and the expression of MMP2 from normal Fb. A significant CD147 expression was demonstrated in Fb-thyroid tumor cell-derived EVs. These findings reveal the role of Fb and thyroid tumor cell-Fb interaction in the promotion of a microenvironment suitable for thyroid tumor progression. Moreover, they highlight, for the first time, the role of thyroid tumor cell-Fb interaction in the production of specialized EVs.

[Surgical treatment in acromegaly: experience in Córdoba]. / Tratamiento quirúrgico en acromegalia: experiencia en Córdoba

Introduction: Acromegaly prevalence is 35-70 / million. Transsphenoidal surgery is the first-line treatment, with a remission rate of 80% for microadenomas and 50% for macroadenomas. Our aim was to evaluate the surgical results in Córdoba and determine predictive remission factors due to the lack of records. Methods: Retrospective-descriptive study of patients with surgery as the first therapeutic line. Remission criteria: IGF1 normalization for age/sex, with GH ≤1.0 g/L. Test X2 and Fisher's exact test with p<0.05. Results: 38 patients were included: 61% women and 39% men; Average age 45 years. Most frequent chief complaint: headache and acral growth (26%), visual disturbances (20%). Macroadenomas were the 84% of the tumors. Of 37 patients, 54% underwent microscopic surgery, 38% endoscopic and 8% transcranial. The 29% of patients showed post-operative complications and diabetes insipidus was the most frequent (10%). The percentage of them was: 33% transcranial surgery, 29% endoscopic and 25% microscopic (p = 0.557). The biochemical remission at 6 months was 34% and at 12 months 55% (p= 0.0001). No significant differences between the endoscopic and microscopic approach (p = 0.071). Of 36 patients, 31% showed complete tumor resection. The subjective clinical improvement was 88%. There weren´t predictive remission factors with significant differences. Conclusion: The surgical biochemical remission was similar to the bibliography. We didn´t find predictive remission factors but a larger number of patients could modify these results.

Introducción: La acromegalia tiene una prevalencia de 35-70/millón. La cirugía transesfenoidal es el tratamiento de elección, siendo la tasa de remisión del 80% en microadenomas y 50% en macroadenomas. Debido a la falta de registros, nos propusimos evaluar los resultados quirúrgicos en Córdoba y determinar factores predictivos de remisión. Métodos: Estudio retrospectivo-descriptivo de pacientes con cirugía como primera línea terapéutica. Criterios de remisión: normalización de IGF1 para edad/sexo, con GH ≤1,0 g/L.Test X2 y test exacto de Fisher y p<0,05. Resultados: Se incluyeron 38 pacientes: 61% mujeres y 39% hombres; edad promedio 45 años. Motivos de consulta más frecuentes: cefalea y crecimiento acral (26%), alteraciones visuales (20%). El 84% de los tumores fueron macroadenomas. De 37 pacientes, 54% se sometieron a cirugía microscópica, 38% endoscópica y 8% transcraneal. El 29% evidenció complicaciones postquirúrgicas, siendo la diabetes insípida la más frecuente (10%). El porcentaje de las mismas fue: cirugía transcraneal el 33%, endoscópica 29% y microscópica 25% (p= 0,557). La remisión bioquímica a los 6 meses fue de 34% y a los 12 meses 55% (p=0,0001). Sin diferencias significativas entre la vía endoscópica y microscópica (p=0,071). De 36 pacientes el 31% evidenció resección tumoral completa. La mejoría clínica subjetiva fue del 88%. No hubo factores predictivos de remisión bioquímica estadísticamente significativos. Conclusión: La remisión bioquímica con la cirugía fue similar a la bibliografía. No encontramos factores predictivos de remisión pero un número mayor de casos podría modificar estos resultados.

Bromocriptine induces parapoptosis as the main type of cell death responsible for experimental pituitary tumor shrinkage.

Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.

Chronic Metabolic Derangement-Induced Cognitive Deficits and Neurotoxicity Are Associated with REST Inactivation.

Chronic metabolic alterations may represent a risk factor for the development of cognitive impairment, dementia, or neurodegenerative diseases. Hyperglycemia and obesity are known to imprint epigenetic markers that compromise the proper expression of cell survival genes. Here, we showed that chronic hyperglycemia (60 days) induced by a single intraperitoneal injection of streptozotocin compromised cognition by reducing hippocampal ERK signaling and by inducing neurotoxicity in rats. The mechanisms appear to be linked to reduced active DNA demethylation and diminished expression of the neuroprotective transcription factor REST. The impact of the relationship between adiposity and DNA hypermethylation on REST expression was also demonstrated in peripheral blood mononuclear cells in obese children with reduced levels of blood ascorbate. The reversible nature of epigenetic modifications and the cognitive impairment reported in obese children, adolescents, and adults suggest that the correction of the anthropometry and the peripheral metabolic alterations would protect brain homeostasis and reduce the risk of developing neurodegenerative diseases.

Activation of PKC epsilon induces lactotroph proliferation through ERK1/2 in response to phorbol ester.

The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.

Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.

Anti-glioma properties of DVL, a lectin purified from Dioclea violacea.

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity.

Trastuzumab inhibits pituitary tumor cell growth modulating the TGFB/SMAD2/3 pathway.

In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.