María Teresa Espinosa-García

Diagnostic accuracy, effectiveness and cost for cognitive impairment and dementia screening of three short cognitive tests applicable to illiterates.

Background Illiteracy, a universal problem, limits the utilization of the most widely used short cognitive tests. Our objective was to assess and compare the effectiveness and cost for cognitive impairment (CI) and dementia (DEM) screening of three short cognitive tests applicable to illiterates. Methods Phase III diagnostic test evaluation study was performed during one year in four Primary Care centers, prospectively including individuals with suspicion of CI or DEM. All underwent the Eurotest, Memory Alteration Test (M@T), and Phototest, applied in a balanced manner. Clinical, functional, and cognitive studies were independently performed in a blinded fashion in a Cognitive Behavioral Neurology Unit, and the gold standard diagnosis was established by consensus of expert neurologists on the basis of these results. Effectiveness of tests was assessed as the proportion of correct diagnoses (diagnostic accuracy [DA]) and the kappa index of concordance (k) with respect to gold standard diagnoses. Costs were based on public prices at the time and hospital accounts. Results The study included 139 individuals: 47 with DEM, 36 with CI, and 56 without CI. No significant differences in effectiveness were found among the tests. For DEM screening: Eurotest (k = 0.71 [0.59–0.83], DA = 0.87 [0.80–0.92]), M@T (k = 0.72 [0.60–0.84], DA = 0.87 [0.80–0.92]), Phototest (k = 0.70 [0.57–0.82], DA = 0.86 [0.79–0.91]). For CI screening: Eurotest (k = 0.67 [0.55–0.79]; DA = 0.83 [0.76–0.89]), M@T (k = 0.52 [0.37–0.67]; DA = 0.80 [0.72–0.86]), Phototest (k = 0.59 [0.46–0.72]; DA = 0.79 [0.71–0.86]). There were no differences in the cost of DEM screening, but the cost of CI screening was significantly higher with M@T (330.7±177.1€, mean±sd) than with Eurotest (294.1±195.0€) or Phototest (296.0±196.5€). Application time was shorter with Phototest (2.8±0.8 min) than with Eurotest (7.1±1.8 min) or M@T (6.8±2.2 min). Conclusions Eurotest, M@T, and Phototest are equally effective. Eurotest and Phototest are both less expensive options but Phototest is the most efficient, requiring the shortest application time.

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast

BACKGROUND STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

The Role of Mitochondria in Syncytiotrophoblast Cells: Bioenergetics and Steroidogenesis

Human placenta maintains pregnancy. The mitochondria of this tissue synthesize pregnenolone (P5) from cholesterol through a transport chain formed by adrenodoxin, adrenodoxin reductase and cytochrome P450scc (CYP11A1; EC 1.14.15.6) , which breaks up the lateral chain of cholesterol. P5 is transformed into progesterone (P4) within mitochondria by the 3-OH-steroid-dehydrogenase-5-6isomerase (3HSD). The particular hormone(s) or substance(s) that modulate P4 synthesis during pregnancy is currently unknown (Strauss et al., 1996; Martinez & Strauss, 1997); nevertheless, the presence of cAMP analogues stimulated P4 synthesis in trophoblastic cells, suggesting that a hormonal signal or another kind of signal may modulate the concentration of this second messenger into the cells (Ringler et al., 1989; Strauss et al., 1992). Although P4 synthesis was suggested to be the main function of the placenta, the analysis of P450scc cytochrome concentration shows that placental mitochondria have a lower content of P450scc than respiratory chain cytochromes (Table 1), even when it is compared to adrenal gland mitochondria, suggesting that placental mitochondria participate in other functions different to that from steroidogenesis.

PKA tightly bound to human placental mitochondria participates in steroidogenesis and is not modified by cAMP

INTRODUCTION Protein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work. METHODS Placental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of (32)P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography. RESULTS The PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas βII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme. DISCUSSION The lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated. CONCLUSIONS The phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases.

Even a Chronic Mild Hyperglycemia Affects Membrane Fluidity and Lipoperoxidation in Placental Mitochondria in Wistar Rats

It is known the deleterious effects of diabetes on embryos, but the effects of diabetes on placenta and its mitochondria are still not well known. In this work we generated a mild hyperglycemia model in female wistar rats by intraperitoneal injection of streptozotocin in 48 hours-old rats. The sexual maturity onset of the female rats was delayed around 6–7 weeks and at 16 weeks-old they were mated, and sacrificed at day 19th of pregnancy. In placental total tissue and isolated mitochondria, the fatty acids composition was analyzed by gas chromatography, and lipoperoxidation was measured by thiobarbituric acid reactive substances. Membrane fluidity in mitochondria was measured with the excimer forming probe dipyrenylpropane and mitochondrial function was measured with a Clark-type electrode. The results show that even a chronic mild hyperglycemia increases lipoperoxidation and decreases mitochondrial function in placenta. Simultaneously, placental fatty acids metabolism in total tissue is modified but in a different way than in placental mitochondria. Whereas the chronic mild hyperglycemia induced a decrease in unsaturated to saturated fatty acids ratio (U/S) in placental total tissue, the ratio increased in placental mitochondria. The measurements of membrane fluidity showed that fluidity of placenta mitochondrial membranes increased with hyperglycemia, showing consistency with the fatty acids composition through the U/S index. The thermotropic characteristics of mitochondrial membranes were changed, showing lower transition temperature and activation energies. All of these data together demonstrate that even a chronic mild hyperglycemia during pregnancy of early reproductive Wistar rats, generates an increment of lipoperoxidation, an increase of placental mitochondrial membrane fluidity apparently derived from changes in fatty acids composition and consequently, mitochondrial malfunction.

Contribution of Potassium in Human Placental Steroidogenesis

The role of K(+) on steroidogenesis in isolated mitochondria from the human placenta was explored. Cholesterol uptake and progesterone synthesis were stimulated by K(+), and by the further addition of ATP. In the presence of glibenclamide or quinine (inhibitors of the K(+) channel mito-K(ATP)), the synthesis of progesterone was improved, indicating that K(+) acts outside the mitochondria. Valinomycin, a K(+)-ionophore, inhibited mitochondrial steroidogenesis only in the absence of K(+). The mitochondrial K(+) channel in human placental mitochondria is formed by the subunit Kir 6.1 which was detected by Western blot with polyclonal antibodies. These results suggest that K(+) contributes placental mitochondrial steroidogenesis facilitating cholesterol uptake and intermembrane translocation through a mechanism non-dependent of the transport of K(+) inside the mitochondria.

Effect of calcium on mitochondria from human term placenta

We describe here the effects of free Ca2+ on several functions of mitochondria from human term placenta. In the presence of 0.1 microM free Ca2+, an inhibitory effect on both ADP-induced respiration and succinate-DCPIP reductase activity was observed. At the same Ca2+ concentration, ATPase activity as well as various segments of the respiratory chain were stimulated. However, a higher free Ca2+ concentration (0.3 microM) was needed to stimulate progesterone synthesis. Our results suggest that Ca2+ plays an important role in the metabolic functions of mitochondria from human term placenta.

The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells

Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy.