Fatma Simsek-Duran

Adapter protein 14-3-3 is required for a presynaptic form of LTP in the cerebellum

Long-term potentiation (LTP) of granule cell–Purkinje cell synapses in the mouse cerebellum requires phosphorylation by protein kinase A of the active-zone protein RIM1α at Ser413. Here, we show that the adapter protein 14-3-3 readily binds phosphorylated Ser413 in RIM1α, and that presynaptic transfection with a dominant-negative 14-3-3η mutant, or a RIM1α mutant with enhanced 14-3-3 binding, inhibits LTP. Thus, RIM1α phosphorylation triggers presynaptic LTP in part through recruitment of 14-3-3 to phospho-Ser413–RIM1α.

Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

Background In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus ) and mouse (Mus musculus) ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. Methodology/Principal Findings Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM) analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. Conclusions/Significance In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

The association of reproductive senescence with mitochondrial quantity, function, and DNA integrity in human oocytes at different stages of maturation.

OBJECTIVE To determine the impact of reproductive aging on oocyte mitochondrial quantity, function, and DNA (mtDNA) integrity. DESIGN Prospective observational study. SETTING IVF clinic in a tertiary academic care center. PATIENT(S) One hundred two oocytes from 32 women undergoing IVF. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Adenosine triphosphate (ATP) levels, mtDNA number, and mtDNA deletion occurrence in individual oocytes. RESULT(S) Oocyte ATP content increases with maturation (786 ± 87 fmol, 1,037 ± 57 fmol, and 1,201 ± 59 fmol for prophase 1 [P1], metaphase 1 [M1], and metaphase 2 [M2] oocytes, respectively), whereas mtDNA copy numbers do not change (64,500 ± 20,440, 180,000 ± 44,040, and 143,000 ± 31,210 for P1, M1, and M2 oocytes, respectively). Stepwise multiple regression analysis identified developmental stage as a determinant of oocyte ATP, whereas number of oocytes retrieved and cycle day 3 FSH level were determinants of mtDNA copy number. Of the 15 oocytes found to possess the 5-kb mtDNA deletion, 10 were arrested or degenerated oocytes. CONCLUSION(S) Although no direct association was found between female age and oocyte mitochondrial quantity and function, the number of mitochondria was predicted by ovarian reserve indicators. As the oocyte matures, ATP content increases.

Synapsin II and calcium regulate vesicle docking and the cross-talk between vesicle pools at the mouse motor terminals

The synapsins, an abundant and highly conserved family of proteins that associate with synaptic vesicles, have been implicated in regulating the synaptic vesicle cycle. However, it has not been determined whether synapsin directly regulates the number of docked vesicles. Here we document that reducing Ca2+ concentration [Ca2+]o in the extracellular medium from 2 to 0.5 mm led to an approximately 40% decrease in both docked and undocked synaptic vesicles in wild‐type nerve terminals of the mouse diaphragm. The same treatment reduced the number of undocked vesicles in nerve terminals derived from synapsin II gene deleted animals, but surprisingly it did not decrease vesicle docking, indicating that synapsin II inhibits docking of synaptic vesicles at reduced [Ca2+]o. In accordance with the morphological findings, at reduced [Ca2+]o synapsin II (−) terminals had a higher rate of quantal neurotransmitter release. Microinjection of a recombinant synapsin II protein into synapsin II (−) terminals reduced vesicular docking and inhibited quantal release, indicating a direct and selective synapsin II effect for regulating vesicle docking and, in turn, quantal release. To understand why [Ca2+]o has a prominent effect on synapsin function, we investigated the effect of [Ca2+]o on the distribution of synaptic vesicles and on the concentration of intraterminal Ca2+. We found that reduced [Ca2+]o conditions produce a decrease in intracellular Ca2+ and overall vesicle depletion. To explore why at these conditions the role of synapsin II in vesicle docking becomes more prominent, we developed a quantitative model of the vesicle cycle, with a two step synapsin action in stabilizing the vesicle store and regulating vesicle docking. The results of the modelling were in a good agreement with the observed dependence of vesicle distribution on synapsin II and calcium deficiency.

The role of RIM1α in BDNF-enhanced glutamate release

Brain-derived neurotrophic factor (BDNF) is known to activate proline-directed Ser/Thr protein kinases and to enhance glutamatergic transmission via a Rab3a-dependent molecular pathway. The identity of molecular targets in BDNFs action on Rab3a pathway, a synaptic vesicle protein involved in vesicle trafficking and synaptic plasticity, is not fully known. Here we demonstrate that BDNF enhances depolarization-evoked efflux of [(3)H]-glutamate from nerve terminals isolated from the CA1 region of the hippocampus. BDNF also potentiated hyperosmotic shock-evoked [(3)H]-glutamate efflux, indicating an effect on the size of the readily releasable pool. This effect of BDNF was completely abolished in nerve terminals derived from Rim1alphaKO (Rab3 interacting molecule 1alpha null mutant) mice. Using in vitro phosphorylation assays we identified two novel phosphorylation sites, Ser447 and Ser745 that were substrates for ERK2, a proline-directed kinase known to be activated by BDNF. The pSer447 site was phosphorylated under resting conditions in hippocampal CA1 nerve terminals and its phosphorylation was enhanced by BDNF treatment, as indicated by the use of a pSer447-RIM1alpha antibody we have developed. Together these findings identify RIM1alpha, a component of the Rab3a molecular pathway in mediating presynaptic plasticity, as a necessary factor in BDNFs enhancement of [(3)H]-glutamate efflux from hippocampal CA1 nerve terminals and indicate a possible role for RIM1alpha phosphorylation in BDNF-dependent presynaptic plasticity.

The role of active zone protein Rab3 interacting molecule 1 alpha in the regulation of norepinephrine release, response to novelty, and sleep

Sleep mechanisms and synaptic plasticity are thought to interact to regulate homeostasis and memory formation. However, the influences of molecules that mediate synaptic plasticity on sleep are not well understood. In this study we demonstrate that mice lacking Rab3 interacting molecule 1 alpha (RIM1 alpha) (Rim1 alpha KO), a protein of the synaptic active zone required for certain types of synaptic plasticity and learning, had 53+/-5% less baseline rapid eye movement (REM) sleep compared with their wild type littermates. Also, compared with wild type littermates, exposure of the mice to an open field or to a novel object induced more robust and longer lasting locomotion suggesting altered habituation. This difference in exploratory behavior correlated with genotype specific changes in REM and deregulated release of norepinephrine in the cortex and basal amygdala of the Rim1 alpha KO mice. Also, moderate sleep deprivation (4 h), a test of the homeostatic sleep response, induced REM sleep rebound with different time course in Rim1 alpha KO and their wild type littermates. As norepinephrine plays an important role in regulating arousal and REM sleep, our data suggest that noradrenergic deficiency in Rim1 alpha KO animals impacts exploratory behavior and sleep regulation and contributes to impairments in learning.

Different natriuretic responses in obese and lean rats in response to nitric oxide reduction.

BACKGROUND Nitric oxide (NO) is an important regulator of renal sodium transport and participates in the control of natriuresis and diuresis. In obesity, the nitric oxide bioavailability was reportedly reduced, which may contribute to the maintenance of hypertension. The aim of this study was to determine the effect of NO depletion on renal sodium handling in a model of diet-induced obesity hypertension. METHODS Obese hypertensive (obesity-prone (OP)) and lean normotensive (obesity-resistant (OR)) Sprague-Dawley rats were treated with 1.2 mg/kg/day N(G)-nitro-L-arginine-methyl ester (L-NAME) for 4 weeks to inhibit NO synthesis. Acute pressure natriuresis and diuresis were measured in response to an increase in perfusion pressure. NHE3 and Na(+), K(+)-ATPase protein expression were measured by Western blot and NHE3 activity was determined as the rate of pH change in brush border membrane vesicles. NHE3 membrane localization was determined by confocal microscopy. RESULTS L-NAME did not significantly attenuate the natriuretic and diuretic responses to increases in renal perfusion pressure (RPP) in OP rats while inducing a significant reduction in OR rats. Following chronic NO inhibition, NHE3 protein expression and activity and Na(+), K(+)-ATPase protein expression were significantly increased in the OR but not in the OP group. Immunofluorescence studies indicated that the increase in NHE3 activity could be, at least in part, due to NHE3 membrane trafficking. CONCLUSIONS Obese hypertensive rats have a weaker natriuretic response in response to NO inhibition compared to lean rats and the mechanism involves different regulation of the apical sodium exchanger NHE3 expression, activity, and trafficking.

The effects of pentoxifylline on skeletal muscle contractility and neuromuscular transmission during hypoxia

Objectives: The objective of this study was to investigate the effects of pentoxifylline (PTX), a drug that is mainly used for indications related to tissue hypoxia, on hypoxia-induced inhibition of skeletal muscle contractility and neuromuscular transmission in mice. We hypothesized that chronic PTX treatment alters skeletal muscle contractility and hypoxia-induced dysfunction. Materials and Methods: Mice were treated with 50 mg/kg PTX or saline intraperitoneally for a week. Following ether anesthesia, diaphragm muscles were removed; isometric muscle contractions and action potentials were recorded. Time to reach neuromuscular blockade and the rate of recovery of muscle contractility were assessed during hypoxia and re-oxygenation. Results: The PTX group displayed 90% greater twitch amplitudes (P < 0.01). Hypoxia depressed twitch contractions and caused neuromuscular blockade in both groups. However, neuromuscular blockade occurred earlier in PTX-treated animals (P < 0.05). Muscle contractures developed during hypoxia were more pronounced in the PTX group (P < 0.05). Re-oxygenation reduced contracture and indirect muscle contractions resumed. The rate of recovery of contractions was faster (P < 0.05) and the amplitude of contractions was greater (P < 0.01) in the PTX group. PTX treatment increased amplitude (P < 0.05) and shortened action potential (P < 0.05) without altering resting membrane potential, excitation threshold, and neurotransmitter release. Conclusion: Chronic PTX treatment increases diaphragm contractility, but amplifies hypoxia-induced contractile dysfunction in mice. These results may implicate important clinical consequences for clinical usage of PTX in hypoxia-related conditions.