A new mouse model of ATR-X syndrome carrying a common patient mutation exhibits neurological and morphological defects.

ATRX is a chromatin remodelling ATPase that is involved in transcriptional regulation, DNA damage repair and heterochromatin maintenance. It has been widely studied for its role in ALT-positive cancers, but its role in neurological function remains elusive. Hypomorphic mutations in the X-linked ATRX gene cause a rare form of intellectual disability combined with alpha-thalassemia called ATR-X syndrome in hemizygous males. Clinical features also include facial dysmorphism, microcephaly, short stature, musculoskeletal defects and genital abnormalities. As complete deletion of ATRX in mice results in early embryonic lethality, the field has largely relied on conditional knockout models to assess the role of ATRX in multiple tissues. Given that null alleles are not found in patients, a more patient-relevant model was needed. Here, we have produced and characterized the first patient mutation knock-in model of ATR-X syndrome, carrying the most common causative mutation, R246C. This is one of a cluster of missense mutations located in the chromatin-binding domain and disrupts its function. The knock-in mice recapitulate several aspects of the patient disorder, including craniofacial defects, microcephaly, reduced body size and impaired neurological function. They provide a powerful model for understanding the molecular mechanisms underlying ATR-X syndrome and testing potential therapeutic strategies.

RyR-mediated Ca2+ release elicited by neuronal activity induces nuclear Ca2+ signals, CREB phosphorylation, and Npas4/RyR2 expression.

The expression of several hippocampal genes implicated in learning and memory processes requires that Ca2+ signals generated in dendritic spines, dendrites, or the soma in response to neuronal stimulation reach the nucleus. The diffusion of Ca2+ in the cytoplasm is highly restricted, so neurons must use other mechanisms to propagate Ca2+ signals to the nucleus. Here, we present evidence showing that Ca2+ release mediated by the ryanodine receptor (RyR) channel type-2 isoform (RyR2) contributes to the generation of nuclear Ca2+ signals induced by gabazine (GBZ) addition, glutamate uncaging in the dendrites, or high-frequency field stimulation of primary hippocampal neurons. Additionally, GBZ treatment significantly increased cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation-a key event in synaptic plasticity and hippocampal memory-and enhanced the expression of Neuronal Per Arnt Sim domain protein 4 (Npas4) and RyR2, two central regulators of these processes. Suppression of RyR-mediated Ca2+ release with ryanodine significantly reduced the increase in CREB phosphorylation and the enhanced Npas4 and RyR2 expression induced by GBZ. We propose that RyR-mediated Ca2+ release induced by neuronal activity, through its contribution to the sequential generation of nuclear Ca2+ signals, CREB phosphorylation, Npas4, and RyR2 up-regulation, plays a central role in hippocampal synaptic plasticity and memory processes.

Ryanodine receptor-mediated Ca2+ release and atlastin-2 GTPase activity contribute to IP3-induced dendritic Ca2+ signals in primary hippocampal neurons.

Neuronal Ca2+ signals are fundamental for synaptic transmission and activity-dependent changes in gene expression. Voltage-gated Ca2+ channels and N-methyl-d-aspartate receptors play major roles in mediating external Ca2+ entry during action potential firing and glutamatergic activity. Additionally, the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels expressed in the endoplasmic reticulum (ER) also contribute to the generation of Ca2+ signals in response to neuronal activity. The ER forms a network that pervades the entire neuronal volume, allowing intracellular Ca2+ release in dendrites, soma and presynaptic boutons. Despite its unique morphological features, the contributions of ER structure and of ER-shaping proteins such as atlastin - an ER enriched GTPase that mediates homotypic ER tubule fusion - to the generation of Ca2+ signals in dendrites remains unreported. Here, we investigated the contribution of RyR-mediated Ca2+ release to IP3-generated Ca2+ signals in dendrites of cultured hippocampal neurons. We also employed GTPase activity-deficient atlastin-2 (ATL2) mutants to evaluate the potential role of atlastin on Ca2+ signaling and ER-resident Ca2+ channel distribution. We found that pharmacological suppression of RyR channel activity increased the rising time and reduced the magnitude and propagation of IP3-induced Ca2+ signals. Additionally, ATL2 mutants induced specific ER morphological alterations, delayed the onset and increased the rising time of IP3-evoked Ca2+ signals, and caused RyR2 and IP3R1 aggregation and RyR2 redistribution. These results indicate that both RyR and ATL2 activity regulate IP3-induced Ca2+ signal dynamics through RyR-mediated Ca2+-induced Ca2+ release, ER shaping and RyR2 distribution.

Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) Channels.

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments we study the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. Our results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. Our results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo.

Astaxanthin Protects Primary Hippocampal Neurons against Noxious Effects of Aß-Oligomers.

Increased reactive oxygen species (ROS) generation and the ensuing oxidative stress contribute to Alzheimer's disease pathology. We reported previously that amyloid-ß peptide oligomers (AßOs) produce aberrant Ca(2+) signals at sublethal concentrations and decrease the expression of type-2 ryanodine receptors (RyR2), which are crucial for hippocampal synaptic plasticity and memory. Here, we investigated whether the antioxidant agent astaxanthin (ATX) protects neurons from AßOs-induced excessive mitochondrial ROS generation, NFATc4 activation, and RyR2 mRNA downregulation. To determine mitochondrial H2O2 production or NFATc4 nuclear translocation, neurons were transfected with plasmids coding for HyperMito or NFATc4-eGFP, respectively. Primary hippocampal cultures were incubated with 0.1 µM ATX for 1.5 h prior to AßOs addition (500 nM). We found that incubation with ATX (≤10 µM) for ≤24 h was nontoxic to neurons, evaluated by the live/dead assay. Preincubation with 0.1 µM ATX also prevented the neuronal mitochondrial H2O2 generation induced within minutes of AßOs addition. Longer exposures to AßOs (6 h) promoted NFATc4-eGFP nuclear translocation and decreased RyR2 mRNA levels, evaluated by detection of the eGFP-tagged fluorescent plasmid and qPCR, respectively. Preincubation with 0.1 µM ATX prevented both effects. These results indicate that ATX protects neurons from the noxious effects of AßOs on mitochondrial ROS production, NFATc4 activation, and RyR2 gene expression downregulation.

Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5' AMP-activated protein kinase (AMPK).

This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 µM compound C AMPK inhibitor or 2 mM AMP+40 µM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

Vitamin C and oxidative stress in the seminiferous epithelium.

In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These findings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis.

Vitamin C and oxidative stress in the seminiferous epithelium

In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These findings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis.

High resolution measurement of the glycolytic rate.

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K(+), supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.

Algunas consideraciones sobre la apoptosis

Desde las primeras descripciones clásicas de Virchow se reconocía un tipo de muerte celular: la necrosis. Sin embargo, a finales del siglo pasado, Walter Flemming describió un tipo de muerte celular con características morfológicas diferentes, al que denominó cromatólisis. Tuvo que transcurrir casi un siglo hasta que este tipo de muerte celular fuese adecuadamente caracterizado por Kerr, quien en 1972 propuso el término apoptosis para denominarlo(AU)

Pseudoquistes pancreáticos. Morbilidad y mortalidad en el Hospital General Docente en el período 1984 - 2004

Se realiza un estudio en 16 pacientes egresados del servicio de cirugía general del hospital general docente de Guantánamo "Dr. Agostinho Neto", con el diagnóstico de Pseudoquiste Pancreático en el período comprendido entre los años 1984 y 2004, en el que se pudieron obtener las tasas de 3,4 por cada 100 mil ingresos hospitalarios y de 4,3 por cada 10 mil ingresos en el servicio de cirugía general. El alcoholismo fue la primera causa de los Pseudoquistes, con el 62,5 por ciento. La edad promedio fue de 44 años, con una DS de 13,1 y una oscilación de 23 a 65 años. Los pacientes masculinos predominaron, para un 92,8 por ciento . El tipo de operación que se realizó con mayor frecuencia fue la cistoyeyunostomía (37.5 por ciento), seguida por la cistoduodenostomía (31,2 por ciento). No hubo mortalidad quirúrgica. La mortalidad global fue de 6.2por ciento, y el promedio de supervivencia de 7 años con una DS de 5 años(AU)