Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

PDMP blocks brefeldin a-induced retrograde membrane transport from Golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not ¿rescue¿ the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Calcium homeostasis in the central nervous system: adaptation to neurodegeneration.

Here we review the results of our recent studies on neurodegeneration together with data on cerebral calcium precipitation in animal models and humans. A model that integrates the diversity of mechanisms involved in neurodegeneration is presented and discussed based on the functional relevance of calcium precipitation.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Synaptic depression and slow oscillatory activity in a biophysical network model of the cerebral cortex

Short-term synaptic depression (STD) is a form of synaptic plasticity that has a large impact on network computations. Experimental results suggest that STD is modulated by cortical activity, decreasing with activity in the network and increasing during silent states. Here, we explored different activity-modulation protocols in a biophysical network model for which the model displayed less STD when the network was active than when it was silent, in agreement with experimental results. Furthermore, we studied how trains of synaptic potentials had lesser decay during periods of activity (UP states) than during silent periods (DOWN states), providing new experimental predictions. We next tackled the inverse question of what is the impact of modifying STD parameters on the emergent activity of the network, a question difficult to answer experimentally. We found that synaptic depression of cortical connections had a critical role to determine the regime of rhythmic cortical activity. While low STD resulted in an emergent rhythmic activity with short UP states and long DOWN states, increasing STD resulted in longer and more frequent UP states interleaved with short silent periods. A still higher synaptic depression set the network into a non-oscillatory firing regime where DOWN states no longer occurred. The speed of propagation of UP states along the network was not found to be modulated by STD during the oscillatory regime; it remained relatively stable over a range of values of STD. Overall, we found that the mutual interactions between synaptic depression and ongoing network activity are critical to determine the mechanisms that modulate cortical emergent patterns.

Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Role of Myotonic Dystrophy Protein Kinase [DMPK] in Glucose Homeostasis and Muscle Insulin Action

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3′-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk−/−) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk−/− mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk−/− mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

Role of Myotonic Dystrophy Protein Kinase [DMPK] in Glucose Homeostasis and Muscle Insulin Action

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3′-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk−/−) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk−/− mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk−/− mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

Effects of neurotrophins on synaptic protein expression in the visual cortex of dark reared rats.

Total lack of visual experience [dark rearing (DR)] is known to prolong the critical period and delay development of sensory functions in mammalian visual cortex. Recent results show that neurotrophins (NTs) counteract the effects of DR on functional properties of visual cortical cells and exert a strong control on critical period duration. NTs are known to modulate the development and synaptic efficacy of neurotransmitter systems that are affected by DR. However, it is still unknown whether the actions of NTs in dark-reared animals involve interaction with neurotransmitter systems. We have studied the effects of DR on the expression of key molecules in the glutamatergic and GABAergic systems in control and NT-treated animals. We have found that DR reduced the expression of the NMDA receptor 2A subunit and its associated protein PSD-95 (postsynaptic density-95), of GRIP (AMPA glutamate receptor interacting protein), and of the biosynthetic enzyme GAD (glutamic acid decarboxylase). Returning dark-reared animals to light for 2 hr restored normal expression of the above-mentioned proteins almost completely. NT treatment specifically counteracts DR effects; NGF acts primarily on the NMDA system, whereas BDNF acts primarily on the GABAergic system. Finally, the action of NT4 seems to involve both excitatory and inhibitory systems. These data demonstrate that different NTs counteract DR effects by modulating the expression of key molecules of the excitatory and inhibitory neurotransmitter systems

Bienestar del lechón en la fase de lactación, destete y transición

Tanto el momento del nacimiento como del destete implican para el lechón un conjunto de cambios importantes a los cuales debe responder el lechón para restablecer su equilibrio fisiológico interno (homeostasis). La capacidad de adaptación del lechón a estos cambios va a influir no sólo su bienestar en estas fases, sino otros parámetros productivos durante el engorde. A continuación se describen los fenómenos más destacables en relación al bienestar del lechón durante estas fases productivas: la mortalidad neonatal, el estrés del destete y de la formación de grupos durante la transición.

The role of fixed delays in neuronal rate models

Fixed delays in neuronal interactions arise through synaptic and dendritic processing. Previous work has shown that such delays, which play an important role in shaping the dynamics of networks of large numbers of spiking neurons with continuous synaptic kinetics, can be taken into account with a rate model through the addition of an explicit, fixed delay. Here we extend this work to account for arbitrary symmetric patterns of synaptic connectivity and generic nonlinear transfer functions. Specifically, we conduct a weakly nonlinear analysis of the dynamical states arising via primary instabilities of the stationary uniform state. In this way we determine analytically how the nature and stability of these states depend on the choice of transfer function and connectivity. While this dependence is, in general, nontrivial, we make use of the smallness of the ratio in the delay in neuronal interactions to the effective time constant of integration to arrive at two general observations of physiological relevance. These are: 1 - fast oscillations are always supercritical for realistic transfer functions. 2 - Traveling waves are preferred over standing waves given plausible patterns of local connectivity.

A numerical solver for a nonlinear Fokker-Planck equation representation of neuronal network dynamics

To describe the collective behavior of large ensembles of neurons in neuronal network, a kinetic theory description was developed in [13, 12], where a macroscopic representation of the network dynamics was directly derived from the microscopic dynamics of individual neurons, which are modeled by conductance-based, linear, integrate-and-fire point neurons. A diffusion approximation then led to a nonlinear Fokker-Planck equation for the probability density function of neuronal membrane potentials and synaptic conductances. In this work, we propose a deterministic numerical scheme for a Fokker-Planck model of an excitatory-only network. Our numerical solver allows us to obtain the time evolution of probability distribution functions, and thus, the evolution of all possible macroscopic quantities that are given by suitable moments of the probability density function. We show that this deterministic scheme is capable of capturing the bistability of stationary states observed in Monte Carlo simulations. Moreover, the transient behavior of the firing rates computed from the Fokker-Planck equation is analyzed in this bistable situation, where a bifurcation scenario, of asynchronous convergence towards stationary states, periodic synchronous solutions or damped oscillatory convergence towards stationary states, can be uncovered by increasing the strength of the excitatory coupling. Finally, the computation of moments of the probability distribution allows us to validate the applicability of a moment closure assumption used in [13] to further simplify the kinetic theory.

Estudio del bypass gástrico en Y de Roux y de la gastrectomía tubular sobre el metabolismo de la glucosa y la secreción de hormonas intestinales. Estudio prospectivo y randomizado

Introducció: El bypass gàstric en Y de Roux laparoscòpic (BGYRL) és la tècnica quirúrgica d’elecció per al tractament de l’obesitat mòrbida. La gastrectomia tubular laparoscòpica (GTL) és una tècnica bariàtrica restrictiva amb resultats molt prometedors en relació a la pèrdua de pes, però se’n desconeixen els efectes metabòlics i endocrins. L’objectiu d’aquest estudi és l’avaluació de les diferències en la resposta del metabolisme de la glucosa i la secreció d’hormones intestinals entre ambdós procediments. Material i mètodes: Es va dissenyar un estudi prospectiu i aleatoritzat per la realització del BGYRL i la GTL, ambdós realitzats via laparoscòpica. Totes les pacients es varen avaluar abans, i als 3 i 12 mesos de la intervenció. Se’ls va prendre una mostra de sang venosa després d’un dejuni de 12 hores i als 10 i 60 minuts després de la ingesta d’Ensure® 420 kcal per realitzar les determinacions dels nivells plasmàtics de glucosa, insulina, grelina, leptina, pèptid relacionat amb glucagó (GLP-1), pèptid YY (PYY) i polipèptid pancreàtic (PP). Resultats: Es varen incloure 15 pacients (totes de gènere femení, edat mitja de 48±9 anys, IMC de 44±2.7 kg/m2 amlitat preoperatòria) dels quals 7 van ser aleatoritzats a BGYRL i 8 a GTL. No hi van haver diferències entre ambdós grups en edat, IMC preoperatori, classificació ASA i determinacions hormonals preoperatòries. Després de la cirurgia, s’observa un descens de la glicèmia i insulinèmia, amb una reducció de l´índex HOMA-IR en ambdós grups. Postoperatòriament, es detecta una disminución de les concentracions de la leptina en dejú i després de la ingesta, significativament menor en el grup de BGYRL. Mentre que els nivells de grelina en dejú només descendeixen de forma significativa en el grup de la GTL. Després de la ingesta es produeix una augment dels nivells de GLP-1, significativament major en el grup de BGYRL. Conclusions: Tan el BGYRL como la GTL s’associen a una significativa pèrdua de pes, encara que aquesta fou significativament superior en el BGYRL. Ambdós procediments han millorat notablement l’homeostasi de la glucosa. Només la GTL va rehuir els nivells de grelina tan en dejú com després de la ingesta, mentre que els nivells de GLP-1 i PYY s’elevaren després de la cirurgia, sense diferències estadísticament significatives entre ambdues tècniques.