Diadenosine pentaphosphate regulates dendrite growth and number in cultured hippocampal neurons.

During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (Ap5A), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that Ap5A negatively modulates the dendrite's growth and number by inducing transient intracellular calcium increases in the dendrites' growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of Ap5A on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by Ap5A. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by Ap5A, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that Ap5A exerts a negative influence on dendritic growth.

Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-based Metabolomics and Analysis of Sample Degradation.

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

Tissue-nonspecific alkaline phosphatase promotes axonal growth of hippocampal neurons.

Axonal growth is essential for establishing neuronal circuits during brain development and for regenerative processes in the adult brain. Unfortunately, the extracellular signals controlling axonal growth are poorly understood. Here we report that a reduction in extracellular ATP levels by tissue-nonspecific alkaline phosphatase (TNAP) is essential for the development of neuritic processes by cultured hippocampal neurons. Selective blockade of TNAP activity with levamisole or specific TNAP knockdown with short hairpin RNA interference inhibited the growth and branching of principal axons, whereas addition of alkaline phosphatase (ALP) promoted axonal growth. Neither activation nor inhibition of adenosine receptors affected the axonal growth, excluding the contribution of extracellular adenosine as a potential hydrolysis product of extracellular ATP to the TNAP-mediated effects. TNAP was colocalized at axonal growth cones with ionotropic ATP receptors (P2X7 receptor), whose activation inhibited axonal growth. Additional analyses suggested a close functional interrelation of TNAP and P2X7 receptors whereby TNAP prevents P2X7 receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore inhibition of P2X7 receptor reduced TNAP expression, whereas addition of ALP enhanced P2X7 receptor expression. Our results demonstrate that TNAP, regulating both ligand availability and protein expression of P2X7 receptor, is essential for axonal development.

Interaction between ghrelin and the ghrelin receptor (GHS-R1a), a NMR study using living cells.

The study of the interaction of ghrelin (1), the endogenous ligand for the GH secretagogues receptor (GHS-R1a), and des-acyl ghrelin (2) with the GHS-R1a by NMR using living cells is presented, using GHS-R1a stably transfected cell lines (CHO and HEK 293) and wild type cells. Therefore, the interaction of 1 and 2 with the GHS-R1a receptor has been performed using quasi-physiological conditions. Ghrelin (1), showed a higher number of residues affected by chemical shift perturbation (CSP) or chemical shift exchange (CSE) effects: Ser3, Phe4, Leu5, Val12, Gln13/Gln14, Lys16/Lys19, Glu17 and Lys24 were much more affected in 1 than in des-acyl ghrelin (2). The chemical shift index CSI values indicated the presence of a possible alpha-helical region between Glu8 and Lys20 for ghrelin (1). After analysing the NMR data, two possible structures have arisen, which present different proline rotamers: the EEZE and the EZEZ conformers, at positions Pro7, Pro21, Pro22 and Pro27, respectively, keeping a left-handed alpha-helix from Glu8 to Lys20. These experimental evidences might imply that the GHS-R1a receptor is acting as a prolyl-cis/trans isomerase.

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum.

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells.

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

Is the ubiquitin-proteasome system impaired in Huntington's disease?

Ubiquitylated inclusion bodies (IBs) found in Huntington's disease (HD) postulate an impaired ubiquitin-proteasome system. However, this hypothesis remains controversial. In vitro-generated polyglutamine aggregates failed to inhibit purified proteasomes, while filamentous huntingtin aggregates isolated from mice resulted in inhibition. However, similarly isolated IBs did not, thus suggesting that IB formation is protective by sequestering smaller inhibitory aggregates. Accordingly, proteasome-activity assays in IB-containing mouse brain homogenates did not show decreased activity. On the contrary, some of the endoproteolytic proteasome activities increased, probably due to altered subunit composition. However, activity was found decreased in postmortem human HD tissue. Finally, evidence supporting the hypothesis was found in HD cell models expressing fluorescent ubiquitin-proteasome system reporters but not in retina of SCA-7 mice with similar reporters. In summary, it seems that mutant huntingtin, probably in intermediate aggregate forms, has the potential to inhibit proteasome activity, but the global status of the system in HD brain tissue is not yet fully elucidated.

Role of CaCMKII in the cross talk between ionotropic nucleotide and nicotinic receptors in individual cholinergic terminals.

Ionotropic P2X receptors for ATP are formed, to date, by seven different subunits named P2X (Torres et al., 1999; Cunha and Ribeiro, 2000; North and Surprenant, 2000; Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003; Illes and Ribeiro, 2004), which are cloned from various mammalian species (Illes and Ribeiro, 2004). These subunits can occur as homo- or hetero-oligomeric assemblies of more than one subunit (North and Surprenant, 2000), except P2X (Miras-Portugal et al., 2003) receptor, which has been described not to coassemble with other subunits (Torres et al., 1999). They are abundantly expressed in the peripheral and central nervous systems and exhibit high permeability to Ca2+ ions (Cunha and Ribeiro, 2000). The existence of presynaptic ionotropic receptors for nucleotides, either for ATP or dinucleotides, has been reported in isolated synaptic terminals from mammalian brain, and both exhibit good permeability to Ca2+ ions (Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003). Studies on isolated single terminals have confirmed the existence of independent and specific responses to ATP and dinucleotides on the same or different terminals (Miras-Portugal et al., 1999; Díaz-Hernández et al., 2002; Hervás et al., 2005; Sánchez-Nogueiro et al., 2005). The activation of presynaptic ionotropic nucleotide receptors can induce the release of other neurotransmitters such as acetylcholine, glutamate, or GABA. In these specific terminals, ionotropic nucleotide receptors can be modulated by interaction with metabotropic receptors, such as GABAB and adenosine receptors (Khakh and Henderson, 1998; Gómez-Villafuertes et al., 2001), and ionotropic, such as nicotinic cholinergic receptors (Díaz-Hernández et al., 2004; Sánchez-Nogueiro et al., 2005). Here, we discuss a relevant finding on the interaction between ionotropic nucleotide and nicotinic receptors in cholinergic synaptic terminals and the role of CaCMKII in this interaction.

The ubiquitin-proteasome system in Huntington's disease.

The main histopathological feature of Huntington's disease (HD) is the presence of protein aggregates that are gathered into inclusion bodies. So far the mechanisms that lead to inclusion formation as well as their role in the pathogenesis of HD are not totally understood. However, it is well established that inclusion bodies contain components of the ubiquitin-proteasome system. Accordingly, it has been postulated that impairment of this machinery can be one of the causes of this disorder. In this review, the authors summarize the state of current knowledge about this hypothesis.

Incidence and severity of infections according to the development of neutropenia during combined therapy with pegylated interferon-alpha2a plus ribavirin in chronic hepatitis C infection.

The evolution of treatment of chronic hepatitis C virus infection has led to improved therapeutic efficacy. However, a major problem is the presence of side effects that require modification or withdrawal of drug therapy in 15-20% of cases. This could potentially influence the lack of sustained viral response in 50% of the cases. Side effects are common, even with pegylated interferon. This study aimed to assess the incidence and severity of infections based on the development of neutropenia associated with combined therapy with pegylated interferon-alpha2a plus ribavirin in 209 patients with chronic hepatitis C infection. All patients were administered pegylated interferon-alpha2a (180 microg/week) plus ribavirin (800 mg/day for 24 weeks in cases of nongenotype 1, or 1000-1200 mg/day for 48 weeks for genotype 1, according to whether patients weighed more or less than 75 kg). Patients with preexisting neutropenia of any cause or cirrhosis were excluded. Neutropenia was defined as a neutrophil count (NC) of <1500 cells/microl. Neutropenia was classified into three levels during treatment: 750

Dependencia en el anciano artrósico e intervención enfermera / Dependency in the arthrosic elderly and nursing intervention

La artrosis como enfermedad degenerativa osteoarticular incide de forma directa en la capacidad de independencia del individuo, sobre todo en el anciano. En este último se complejiza enormemente la respuesta ante la necesidad de desplazarse o de moverse condicionada, no ya solamente por factores orgánicos y fisiológicos, sino que, además, se intercalan aspectos de carácter psicológico, económico, social, cultural, etc. que convierten, no ya la necesidad sino incluso el placer del movimiento, en una verdadera tortura para el anciano amén de restar, considerablemente, su capacidad de independencia. Realizamos en el presente trabajo una valoración de dichos condicionantes en pacientes ancianos tributarios de tratamiento protésico en las articulaciones de la cadera y la rodilla, al tiempo que comprobamos la necesidad de implementar actuaciones enfermeras, en este tipo de pacientes, que rentabilicen mejor los recursos terapéuticos a través de una adecuada educación para la salud. (AU)

Independent receptors for diadenosine pentaphosphate and ATP in rat midbrain single synaptic terminals.

Diadenosine pentaphosphate (Ap5A) and adenosine 5'-triphosphate (ATP) stimulate a intrasynaptosomal calcium concentration [Ca(2+)](i) increase via specific purinergic receptors in rat midbrain synaptosomes, although nothing is known about their distribution in presynaptic terminals. A microfluorimetric technique to measure [Ca(2+)](i) increase using the dye FURA-2AM, has permitted study of the presence of dinucleotide and P2X receptors in independent isolated synaptic terminals. Our results demonstrate the existence of three populations of synaptosomes: one with dinucleotide receptors (12%), another with P2X receptors (20%) and a third with both (14%). It has been possible to demonstrate that the activation of these receptors occurs only in the presence of extracellular Ca(2+) and that it is also coupled with voltage-dependent Ca(2+) channels. Finally 54% of the synaptosomes that responded to K(+) did not present any calcium increase mediated by the nucleotides used. In summary, ATP and dinucleotides exhibit specific ionotropic receptors that can coexist or not on the same synaptic terminal.

Adenosine triphosphate and diadenosine pentaphosphate induce [Ca(2+)](i) increase in rat basal ganglia aminergic terminals.

Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap(5)A, which stimulate a [Ca(2+)](i) increase in the presynaptic terminals via specific ionotropic receptors, termed P2X, and diadenosine polyphosphate receptors. Aminergic terminals from rat brain basal ganglia were characterized by immunocolocalization of synaptophysin and the vesicular monoamine transporter VMAT2 and represent 29% of the total. These aminergic terminals respond to ATP and/or Ap(5)A with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique. This technique, which allows single synaptic terminals to be studied, showed that roughly 8.2% +/- 1.6% of the aminergic terminals respond to ATP, 16.9% +/- 1.3% respond to Ap(5)A, 32.6% +/- 0.8% to both, and 42.3% +/- 1.5% of them have no response. Immunological studies performed with antibodies against ionotropic ATP receptor subunits showed positive labelling with anti-P2X(3) antibodies in 39% of the terminals. However, colocalization studies of VMAT and P2X(3) receptor subunit indicate that only 25% of the aminergic terminals also contain this receptor subtype. These results demonstrate that the aminergic terminals from the rat brain basal ganglia are to a large extent under the modulation of presynaptic nucleotide and dinucleotide receptors.

Presence of different ATP receptors on rat midbrain single synaptic terminals. Involvement of the P2X(3) subunits.

Adenosine 5'-triphosphate (ATP) stimulates a [Ca(2+)](i) increase via specific ionotropic receptors, termed P2X receptors, in rat midbrain presynaptic terminals. A microfluorimetric technique enabled study of the [Ca(2+)](i) increase in isolated single synaptic terminals, showing that 33.4+/-2.5% of them responded to ATP. Immunological studies carried out, after functional studies, with specific anti-P2X receptor subunit antibodies showed only positive labelling with anti-P2X(3) antibodies in 23.5+/-1.7% of the terminals. All positively P2X(3) labelled synaptic terminals responded to ATP. Nevertheless, not all of them responded to alpha,beta-meATP, these representing 6.7+/-1.5% of the total. In addition, 9.8+/-2.3% of the terminals responded to ATP but exhibit negative P2X(3)-labelling. These results demonstrate the existence of a heterogeneous population of ionotropic ATP receptors at the presynaptic level.

Diadenosine polyphosphate receptors. from rat and guinea-pig brain to human nervous system.

Diadenosine polyphosphates are a family of naturally occurring nucleotidic compounds present in secretory vesicles together with other chemical messengers. The exocytotic release of these compounds permits them to stimulate receptors termed "purinoceptors" or "ATP receptors." Purinoceptors for nucleotides are named P2 in contrast with those sensitive to nucleosides (P1). P2 receptors are further subdivided into metabotropic P2Y receptors, further divided into 5 subtypes, and ionotropic P2X receptors, with 7 different subtypes. Diadenosine polyphosphates can activate recombinant P2Y(1), P2Y(2), and P2Y(4) and recombinant homomeric P2X(1), P2X(2), P2X(3), P2X(4), and P2X(6). Heteromeric P2X receptors change their sensitivity to diadenosine polyphosphates when co-assembly between different subunits occurs. Diadenosine polyphosphates can activate specific receptors termed dinucleotide receptors or P4 receptors, which are insensitive to other nucleosides or nucleotides. The P4 receptor is a receptor-operated Ca(2)+ channel present in rat brain synaptic terminals, stimulated by diadenosine pentaphosphate and diadenosine tetraphosphate. This receptor is strongly modulated by protein kinases A and C and protein phosphatases. The dinucleotide receptor is present in different brain areas, such as midbrain (in rat and guinea-pig), cerebellum (in guinea-pig), and cortex (in human).

Presynaptic signalling mediated by mono- and dinucleotides in the central nervous system.

Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap(5)A, which elicit a Ca(2+) entrance to the presynaptic terminals. Studies of isolated single terminals showed that not all the terminals contain ionotropic receptors for nucleotides, in fact only 46% of them do. Of these, 12% responded only to the dinucleotide Ap(5)A, and 20% to the mononucleotide ATP. At the presynaptic level, diinosine pentaphosphate, Ip(5)I, is a good tool to specifically block dinucleotide responses, which are inhibited at low nM concentration, versus the high microM concentrations required to block ATP responses. There is evidence for a presynaptic control of mononucleotide and dinucleotide responses, mediated by metabotropic and ionotropic receptors. Stimulation of adenosine A1 receptors increases the affinity of dinucleotide receptors by five orders of magnitude, from 30 microM to 680 pM for control and in the presence of A1 agonist, respectively.

Modulation of the dinucleotide receptor present in rat midbrain synaptosomes by adenosine and ATP.

Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap(5)A), elicits Ca(2+) transients at concentrations ranging from 10(-7) to 10(-3) M with a single-phase curve and an EC(50) value of 56.21+/-1.82 microM. Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose-response control curve into a biphasic one presenting two EC(50) values of 6.47+/-1.25 nM and 11.16+/-0.83 microM respectively. The adenosine A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) reversed the biphasic concentration-response for Ap(5)A curve in the presence of AP, to a monophasic one with an EC(50) value of 76.05+/-7.51 microM. The application of adenosine deaminase produced the same effect as DPCPX, the EC(50) value for Ap(5)A, in the presence of AP being 18.62+/-4.03 microM. Activation of the adenosine A(1) receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap(5)A to the left, resulting in a monophasic curve with an EC(50) of 5. 01+/-0.02 pM. The destruction of extrasynaptosomal nucleotides by AP or the addition of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a broad P2 antagonist compound, enhance maximal effect of the Ap(5)A up to 55.6% on the dose response curve, thus suggesting a negative modulation by P2 receptors. In a summary, ATP and adenosine present at the extra-synaptosomal space, are relevant natural modulators of the dinucleotide receptor, via P2 and adenosine A(1) receptors respectively.

Presence of dinucleotide and ATP receptors in human cerebrocortical synaptic terminals.

Human cerebrocortical synaptic terminals elicited concentration-dependent Ca2+ transients after Ap5A (diadenosine pentaphosphate) and ATP stimulation, with EC50 values of 23.44 +/- 3.70 microM and 11.48 +/- 2.12 microM, respectively. The lack of cross-desensitisation and the selective antagonism by Ip5I (diinosine pentaphosphate), suggests the activation of a dinucleotide receptor by Ap5A, and a P2X receptor by ATP. Ap5A Ca2+ transients were partially abolished by omega-conotoxin GVI-A (53%), suggesting the participation of a N-type Ca2+ channel in the dinucleotide response. ATP effect on Ca2+ entry was abolished by nicardipine (44%) and by omega-conotoxin GVI-A (52%), suggesting the participation of L- and N-type Ca2+ channels. These data suggest that Ap5A and ATP activate dinucleotide and P2X receptors, respectively, in human brain synaptic terminals.