Rocío Ruiz

Role of follicle-stimulating hormone receptor Ser680Asn polymorphism in the efficacy of follicle-stimulating hormone

OBJECTIVE To evaluate the association between FSH efficacy and FSHR alleles. DESIGN Retrospective study. SETTING University-based fertility unit and a private center for biomedical research. PATIENT(S) One hundred two women with ovarian function who were undergoing controlled ovarian stimulation (COS). Women were categorized as poor responders (< or =3 ovarian follicles at the end of the cycle) or normal responders (>3 follicles). INTERVENTION(S) Daily administration of exogenous FSH. MAIN OUTCOME MEASURE(S) Number of good or poor responders. RESULT(S) The allele frequency and genotype distribution of the Ser680Asn marker differed significantly between groups. Cycle cancellations were increased (21%) among women who were homozygous for Ser680 compared with Ser/Asn and Asn/Asn patients, and 36% of poor-responders were homozygous for Ser680. CONCLUSION(S) The results support a role for FSHR gene in COS outcome. However, the weight of this factor is probably low. The Ser680 allele may act in concert with other environmental and genetic factors that contribute to FSH efficacy.

Altered Intracellular Ca2+ Homeostasis in Nerve Terminals of Severe Spinal Muscular Atrophy Mice

Low levels of survival motor neuron (SMN) protein result in spinal muscular atrophy (SMA), a severe genetic disease characterized by motor impairment and premature lethality. Although SMN is a ubiquitous protein, motor neurons are much more vulnerable to low levels of SMN than other cells. To gain insight into the pathogenesis of SMA, we have compared synaptic function of motor terminals in wild-type and severe SMA mice at different ages and in two proximal muscles. Our results show that mutant muscle fibers fire normal action potentials and that multi-innervated terminals are functional. By studying the characteristics of the three main components of synaptic transmission in nerve terminals (spontaneous, evoked, and asynchronous release), we found that the kinetics of the postsynaptic potentials are slowed and evoked neurotransmitter release is decreased by ∼55%. In addition, asynchronous release is increased ∼300%, indicating an anomalous augmentation of intraterminal bulk Ca2+ during repetitive stimulation. Together, these results show that the reduction of SMN affects synaptic maturation, evoked release, and regulation of intraterminal Ca2+ levels.

Active Zones and the Readily Releasable Pool of Synaptic Vesicles at the Neuromuscular Junction of the Mouse

Synchronous neurotransmitter release is a highly regulated process that takes place at specializations at the presynaptic membrane called active zones (AZs). The relationships between AZs, quantal release, and vesicle replenishment are not well understood in a mature synapse. We have measured the number, distribution, and other properties of AZs in mouse motor nerve terminals and combined these observations with electrophysiological estimates of the size of the readily releasable pool (RRP) of synaptic vesicles. On average, we counted 850 AZs per terminal. Assuming two primary docked vesicles per AZ, we predict a total of ∼1700 vesicles optimally positioned for exocytosis. Electrophysiological estimates of the size of the RRP, using a simple kinetic model that assumes exponential depletion of the initial pool and refilling by recruitment, gave an average value of 1730 quanta during 100 Hz stimulation, in satisfying agreement with the morphology. At lower stimulus frequencies, however, the model revealed that the estimated RRP size is smaller, suggesting that not all AZs participate in release at low stimulation frequencies.

SMN Requirement for Synaptic Vesicle, Active Zone and Microtubule Postnatal Organization in Motor Nerve Terminals

Low levels of the Survival Motor Neuron (SMN) protein produce Spinal Muscular Atrophy (SMA), a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs) and Active Zones (AZs), reduction in the size of the readily releasable pool (RRP), and the recycling pool (RP) of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.

Ciliary neurotrophic factor-induced sprouting preserves motor function in a mouse model of mild spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5. Depending on the levels of SMN protein produced from a second SMN gene (SMN2), different forms of the disease are distinguished. In patients with milder forms of the disease, type III or type IV SMA that normally reach adulthood, enlargement of motor units is regularly observed. However, the underlying mechanisms are not understood. Smn(+/-) mice, a mouse model of type III/IV SMA, reveal progressive loss of motor neurons and denervation of motor endplates starting at 4 weeks of age. Loss of spinal motor neurons between 1 month and 12 months reaches 40%, whereas muscle strength is not reduced. In these animals, amplitude of single motor unit action potentials in the gastrocnemic muscle is increased more than 2-fold. Confocal analysis reveals pronounced sprouting of innervating motor axons. As ciliary neurotrophic factor (CNTF) is highly expressed in Schwann cells, we investigated its role for a compensatory sprouting response and maintenance of muscle strength in this mouse model. Genetic ablation of CNTF results in reduced sprouting and decline of muscle strength in Smn(+/-) mice. These findings indicate that CNTF is necessary for a sprouting response and thus enhances the size of motor units in skeletal muscles of Smn(+/-) mice. This compensatory mechanism could guide the way to new therapies for this motor neuron disease.

Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptophluorin

We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wild-type controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free (“empty”) vesicles or other organelles during intense stimulation.

Association of NOS3 gene with metabolic syndrome in hypertensive patients

Recent data from animal models indicate that the eNOS null mice present a phenotype that resemble the human metabolic syndrome (hypertension, insulin resistance and hypertriglyceridemia). In this work, we have studied whether NOS3 gene, previously related to endothelial dysfunction, might have a role in metabolic syndrome susceptibility in hypertensive patients. To carry out the study, we genotyped 105 hypertensive patients < or = 60 years old with two polymorphisms of NOS3 gene: 1132 T>C and 7164 G>T (GeneBank:AF519768.1). To check the allelic frequency of these polymorphisms in our geographical area, we also genotyped 94 unselected healthy controls (control group). To perform sample genotyping, we designed a novel FRET system coupled to real time PCR. There were no differences in genotypic distribution or allelic frequency between hypertensive patients and the control group. However, we observed that 786CC genotype was significantly more frequent in hypertensive patients with metabolic syndrome than in those without the syndrome (p=0.0022). When both polymorphisms were analyzed, we identified the 786C894G as the risk haplotype for metabolic syndrome susceptibility (p=0.011). These data suggest a role of the NOS3 gene in the pathogenesis of metabolic syndrome in hypertensive patients.

Cysteine string protein‐α is essential for the high calcium sensitivity of exocytosis in a vertebrate synapse

Cysteine string protein (CSPα) is a synaptic vesicle protein present in most central and peripheral nervous system synapses. Previous studies demonstrated that the deletion of CSPα results in postnatal sensorial and motor impairment and premature lethality. To understand the participation of CSPα in neural function in vertebrates, we have studied the properties of synaptic transmission of motor terminals in wild‐type and CSPα knockout mice. Our results demonstrate that, in the absence of CSPα, fast Ca2+‐triggered release was not affected at postnatal day (P)14 but was dramatically reduced at P18 and P30 without a change in release kinetics. Although mutant terminals also exhibited a reduction in functional vesicle pool size by P30, further analysis showed that neurotransmission could be ‘rescued’ by high extracellular [Ca2+] or by the presence of a phorbol ester, suggesting that an impairment in the fusion machinery, or in vesicle recycling, was not the primary cause of the dysfunction of this synapse. The specific shift to the right of the Ca2+ dependence of synchronous release, and the lineal dependence of secretion on extracellular [Ca2+] in mutant terminals after P18, suggests that CSPα is indispensable for a normal Ca2+ sensitivity of exocytosis in vertebrate mature synapses.

Morphological and functional remodelling of the neuromuscular junction by skeletal muscle PGC-1α

The neuromuscular junction (NMJ) exhibits high morphological and functional plasticity. In the mature muscle, the relative levels of physical activity are major determinants of NMJ function. Classically, motor neuron-mediated activation patterns of skeletal muscle have been thought of as the major drivers of NMJ plasticity and the ensuing fiber-type determination in muscle. Here we use muscle-specific transgenic animals for the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) as a genetic model for trained mice to elucidate the contribution of skeletal muscle to activity-induced adaptation of the NMJ. We find that muscle-specific expression of PGC-1α promotes a remodeling of the NMJ, even in the absence of increased physical activity. Importantly, these plastic changes are not restricted to post-synaptic structures, but extended to modulation of pre-synaptic cell morphology and function. Therefore, our data indicate that skeletal muscle significantly contributes to the adaptation of the NMJ subsequent to physical activity.

Relationship between oxidative and occupational stress and aging in nurses of an intensive care unit

Stressful conditions lead to formation of excessive reactive oxygen species (ROS) and cause oxidative stress and aging. The aim of this study was to determine superoxide dismutase (SOD) and catalase (CAT) activity, and malondialdehyde (MDA) levels in nurses of a hospital intensive care unit according to demographic and occupational parameters, and to analyse the relationship with aging. Thirty-two nurses working in an intensive care unit and 35 aged-matched healthy individuals of both sexes as a control group were surveyed. No significant variations with respect to sex were detected in SOD, CAT, MDA and burnout levels. MDA levels increased with age in both the control group and the nurses, and we observed significant differences in MDA levels between the control group and nurses for all age groups. Significant variations in MDA levels were detected between single (286.12 ± 8.41) and married (318.82 ± 6.02), people, between those who frequently practice some kind of sport (281.41 ± 7.32) and those who never participate in sport (298.24 ± 8.11) ,and between those who frequently eat fruit and greens (289.75 ± 8.41) and those who never eat them (315.12 ± 7.21). Significant differences were detected between smokers and nonsmokers in SOD, CAT and MDA, but not for alcohol, coffee, tea or cola consumption. Higher SOD activity and MDA levels were detected in nurses on evening and night shifts (P < 0.01); these nurses also scored significantly higher on burnout subscales. These results suggest that: (1) occupational stress increases oxidative stress levels as a response to elevated ROS generation; (2) occupational stress increases MDA levels as a response to an elevation in free radical generation and can lead to aging; (3) working evening and night shifts increases oxidative and burnout levels. It is evident that preventive changes in job conditions and lifestyle are necessary to improve the quality of life of nurses who work in intensive care units.