Involvement of IL-6 and IL-1 receptor antagonist on intellectual disability.

Imbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with several neurodevelopmental disorders and their role in neuronal development is being investigated. To assess the possible influence of cytokines on the onset of intellectual disability (ID), we studied the polymorphisms of thirteen proinflammatory cytokine genes in 81 patients and 61 healthy controls. We demonstrated a significant association of interleukin-6 (IL-6) single-nucleotide polymorphism (SNP) (-174 G/C and nt565 G/A), and interleukin-1 receptor antagonist (IL-1RA) (Mspa-I 11100) SNP with ID. Moreover, the IL-6 SNPs is an unfavorable genetic predisposition for females. The evaluation of circulating levels of IL-6 and IL-1RA showed that the serum concentrations of IL-6 were significantly higher in ID patients than in controls. These data suggest that functional cytokine gene polymorphisms may influence the development of ID.

Signal transduction pathways involved in PPARß/δ-induced neuronal differentiation.

Neuroblastomas are pediatric tumors originating from neuroblasts in the developing peripheral nervous system. The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of survival and differentiation of specific neuronal populations in the central and peripheral nervous system. Patients whose neuroblastoma tumors express high levels of BDNF and TrkB have an unfavorable prognosis. We have previously reported on the neuronal differentiating activity of peroxisome proliferator-activated receptors (PPAR)ß/δ natural and synthetic ligands by modulating BDNF/TrkB pathway, suggesting their potential use as new therapeutic strategies for neuroblastoma. The validation of new therapeutic agents implies the understanding of their mechanisms of action. Herein, we report the effects of activated-PPARß/δ on signal transduction pathways known to be involved in neuronal differentiation, such as ERK1,2 and BDNF pathways. The results obtained, using also PPARß/δ silencing, indicating a neuronal differentiating effect PPARß/δ-dependent through BDNF-P75-ERK1,2 pathways, further support a role for PPARß/δ in neuronal differentiation and pointing towards PPARß/δ as a modulator of pathways crucial for neuronal differentiation. These findings open new perspectives in the formulation of potential therapeutic approaches to be used as adjuvant treatment with the standard therapies.

Attention-deficit hyperactivity disorder and intellectual disability: a study of association with brain-derived neurotrophic factor gene polymorphisms.

Symptoms of attention-deficit hyperactivity disorder (ADHD) have been found in several studies of children with intellectual disabilities (ID) but the two diseases are not always associated. Several lines of evidence implicate the involvement of brain-derived neurotrophic factor (BDNF) in ADHD, and it may also be relevant in ID due to its known involvement in the development of the central nervous system (CNS) and in learning/memory functions. We genotyped paediatric patients with ADHD and ID for the Val66Met and 270 C/T polymorphisms in BDNF. Diagnosis of ADHD and ID was confirmed by the clinicians in accordance with DSM-IV criteria. The G/A genotype of the Val66Met SNP was associated with both ADHD and ID, and the G allele was significantly associated with ADHD. The C/C genotype of the C270T SNP was significantly overrepresented in both ADHD and ID groups compared with the controls. Data suggest that both BDNF polymorphisms could play a role in the etiology of ADHD. In addition, we present the first results suggesting that these BDNF SNPs are significantly associated with ID.

Neuronal response of peroxisomal and peroxisome-related proteins to chronic and acute Abeta injury.

The central role of peroxisomes in ROS and lipid metabolism and their importance in brain functioning are well established. The aim of this work was to study the modulation of peroxisomal and peroxisome-related proteins in cortical neurons in vitro challenged with chronic or acute Abeta treatment, in order to investigate whether peroxisomes represent one of the cellular target of Abeta in these cells. The expression of peroxisomal (PMP70, catalase, acyl-CoA oxidase and thiolase), peroxisome-related (PPARalpha, insulin-degrading enzyme) and anti-oxidant (SOD1, SOD2, GSTP1) proteins was studied. The results obtained, demonstrating an early upregulation of the peroxisomal proteins during the chronic challenge, followed by their dramatic impairment after acute challenge, suggest that peroxisomes represent one of the first line of defence against Abeta-mediated oxidative injury. Our results support the notion that substances able to activate PPARalpha and/or to induce peroxisomal proliferation may constitute a novel preventive and/or therapeutic tool against neurodegenerative diseases.

Investigation on the possible relationship existing between the HLA-DR gene and attention deficit hyperactivity disorder and/or mental retardation.

This study examines the possible relationship existing between the HLA-DR gene and attention deficit hyperactivity disorder (ADHD) and/or mental retardation (MR). The diagnosis of ADHD and mental retardation were established through clinical interviews with the parents, children and teachers, according to the criteria in DSM-IV. HLA-DRB1 genotyping was performed both by polymerase chain reaction-sequence specific primers (PCR-SSP) and by sequence based typing (SBT) in a cohort of 81 affected children and a sample of 100 healthy controls. Here, we report a positive association of HLA-DR4 with ADHD but not with MR. The study adds confirmation to the role of the HLA-DRB1 in the etiology of some types of childhood neuropsychiatric illnesses.

PPARbeta agonists trigger neuronal differentiation in the human neuroblastoma cell line SH-SY5Y.

Neuroblastomas are pediatric tumors originating from immature neuroblasts in the developing peripheral nervous system. Differentiation therapies could help lowering the high mortality due to rapid tumor progression to advanced stages. Oleic acid has been demonstrated to promote neuronal differentiation in neuronal cultures. Herein we report on the effects of oleic acid and of a specific synthetic PPARbeta agonist on cell growth, expression of differentiation markers and on parameters responsible for the malignancy such as adhesion, migration, invasiveness, BDNF, and TrkB expression of SH-SY5Y neuroblastoma cells. The results obtained demonstrate that many, but not all, oleic acid effects are mediated by PPARbeta and support a role for PPARbeta in neuronal differentiation strongly pointing towards PPAR ligands as new therapeutic strategies against progression and recurrences of neuroblastoma.

Expression of peroxisome proliferator-activated receptors (PPARs) and retinoic acid receptors (RXRs) in rat cortical neurons.

Neuronal differentiation is a complex process involving the sequential expression of several factors. The important role of lipid molecules in brain development is well known. Many fatty acid cell signaling activities are mediated by peroxisome proliferator-activated receptors (PPARs). PPARs are ligand-activated transcription factors belonging to the steroid, thyroid and retinoid nuclear receptor superfamily. They are activated by fatty acids and their derivatives. Different isotypes of PPARs (alpha, beta/delta and gamma) have distinct physiological functions depending on their different ligand activation profiles and tissue distribution. PPARs have been involved in neural cell differentiation and death as well as in inflammation and neurodegeneration. Although PPARs have been described in neurons by in situ studies, the presence and possible modulation of these receptors during neuronal differentiation has not been explored yet. In this study we analyzed the expression of PPARs and of their heterodimeric partners, RXRs, in embryonic rat cortical neurons during their in vitro maturation. Our results demonstrate the presence of PPARs alpha, beta/delta and gamma and of RXRs beta and gamma. PPARalpha, beta/delta and gamma are differentially modulated during culture time suggesting that they may be involved in neuronal maturation. In particular, we point toward the PPARbeta/delta isotype as a key factor in neuronal differentiation.

Interleukin 1-beta modulates the effects of hypoxia in neuronal culture.

In order to study the role of interleukin-1beta (IL-1beta) in homeostasis, hypoxia and recovery of neuronal cells, we studied the expression and release of tumor necrosis factor-alpha (TNF-alpha) and nerve growth factor (NGF), in relation to the presence or absence of this cytokine in culture medium. Moreover, we evaluated cell mortality in the same conditions. For this aim, we used untreated and IL-1beta pre-immunoneutralized hippocampal neuronal cultures exposed to mild hypoxic stress and left to reoxygenate. Semiquantitative reverse-transciptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) determined gene expression and protein levels. Mild hypoxic stress provokes a decrease in both the expression and release of TNF-alpha and NGF. IL-1beta neutralization results in an inversion of this pattern since treated hypoxic cultures exhibited an increase of both expression and release of NGF. In pretreated hypoxic cells the increased expression of TNF-alpha was not followed by a rise in release. Reoxygenation reversed the observed effects in both cultures and the levels of cytokine expression and release were approaching control values. Our data show that in physiological conditions IL-1beta may have a neuroprotective action through positive modulation of NGF. Contrary to that, in presence of insult, IL-1beta may have an opposite role, since neutralization provoked an increase of expression and release of NGF. In addition, we demonstrated that neuronal cells are biochemically capable, not only of maintaining and recovering the homeostasis, but also of activating the appropriate response to insult. IL-1beta may have a pivotal role in this mechanism through the modulation of NGF and to a lesser degree of TNF-alpha.

RT-PCR analysis of immune-modulating factors in PBMCs from patients with cancer: reduced IL-2 and increased IL-2-receptor (p55) expression characterize gastroenteric neoplasms.

BACKGROUND: To determine the potential contribution of cytokines associated with lymphocyte activation to the pathogenesis of immune impairment in gastroenteric cancer, we examined the expression of cytokine mRNA in peripheral blood mononuclear cells (PBMCs) and tumor-draining lymph nodes (LNs). MATERIAL AND METHODS: Using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique, mRNA transcripts for interleukin (IL)-4, IL-2, IL-5, IL-6, IL-1 beta, IFN gamma, IL-10 and IL-2-receptor(IL-2R)(p55) were detected in PBMCs from 16 patients with gastroenteric cancer, undergoing surgical resection, and from 13 healthy donors. RESULTS: Patients expressed a different pattern of cytokines. Significantly increased IL-2R(p55) and reduced IL-2 mRNA expression were found in patients (p = 0.034, p = 0.043). IL-10 expression was significantly correlated to IL-2R(p55) expression in both groups (p = 0.011, p = 0.009). In LNs, the results reflected those from PBMCs of the same patient. CONCLUSIONS: Gastroenteric cancer showed a suppressive pattern of cytokine expression, suggesting an impairment of T cell activation that can be evidenced from the specific pattern of PBMC cytokine expression.

Transplantation of mesencephalic cell suspension in dopamine-denervated striatum of the rat. II. Effects on corticostriatal transmission.

The present study has been designed to investigate whether intrastriatal implantation of mesencephalic dopamine (DA)-synthetizing neurons into the striatum (ST) of rats whose substantia nigra (SN) was previously destroyed by 6-hydroxydopamine (6-OHDA) restores the pattern of corticostriatal transmission from the medial prelimbic and sensorimotor cortices. In 6-month-old normal animals electrical stimulation of these two functionally unrelated cortices evoked a short latency and brief excitation in 81.6% of neurons recorded in the dorsolateral ST. This percentage decreased significantly (70.6%) in age-matched animals whose dopaminergic nigrostriatal pathway was unilaterally destroyed by 6-OHDA 3 months before recording. However a significant increase in neurons (36.9%) which could be simultaneously activated from the two cortices in comparison to intact rats was noted. In addition the lesion caused a significant decrease in the threshold current required to evoke activation of striatal neurons from the sensorimotor cortex. The increase in the number of striatal neurons responding simultaneously to cortical stimulations demonstrates that destruction of the dopaminergic nigrostriatal pathway causes a loss of the focusing action of DA on corticostriatal transmission. Transplantation of embryonic mesencephalic neurons appears to reestablish this action since the number of convergent responses was significantly decreased in grafted animals (23.5%) in comparison to denervated (36.9%) and sham-grafted (35.1%) animals. Furthermore, the grafts showed a trend to increase current intensities required to evoke activation of striatal cells from both cortices. The action of grafted mesencephalic neurons over prelimbic and sensorimotor cortical inputs to the dorsal ST could be involved in recovery of grafted animals in the correct execution of complex sensorimotor tasks requiring integration of different cortical signals within the ST.

Blockade of N-methyl-D-aspartate receptor prevents hypoxic neuronal death and cytokine release.

Neuronal mortality, interleukin-1 beta (IL1 beta) and tumor necrosis factor-alpha (TNF alpha) release were measured in hypoxic hippocampal neuronal cultures. Release of IL 1 beta and TNF alpha was already observed in normoxic cultures, but after hypoxia it was increased approximately 2-fold. Pretreatment with 2-amino-5-phosphonovaleric acid (APV), the N-methyl-D-aspartate (NMDA) receptor antagonist, not only decreased neuronal mortality as expected, but also dramatically lowered cytokine release. However, there was no relationship between the neuronal mortality and the release of each cytokine both in untreated hypoxic cultures and in APV-pretreated ones. We conclude that IL 1 beta and TNF alpha release in hypoxia are dependent on the activation of the NMDA receptor, but that this is not the main mechanism of hypoxia damage in in vitro neuronal cultures.

Development of vulnerability to hypoxic damage in in vitro hippocampal neurons.

We investigated the relationship between sensitivity to hypoxia and culture age in in vitro hippocampal neurons. Hypoxia was induced by 24 hr incubation in an oxygen-free environment. Up to 6 days in vitro (DIV) mortality was very low or negligible, with few exceptions. Starting at 7 DIV, significant mortality began to be observed; in the age range 7 10 DIV, mortality of 50% or more was observed in five out of 11 experiments (45%) and average mortality was 51 +/- 15% (mean +/- standard deviation, N = 11). In older (12 18 DIV) cultures, mortality of 50% or more was the rule (13 out of 13 experiments) and average mortality was 83 +/- 16% (mean +/- standard deviation, N = 13). The data could be fitted by a sigmoid line (r = 0.87, P < 10(-6) in which 50% mortality corresponds to 8.6 DIV. The N-methyl-D-aspartate antagonist amino-phosphono-valerate and the nitric oxide synthase inhibitor nitroarginine both provided protection. Degree of protection was comparable for the two compounds, but was not observed in cultures younger than approximately 7 DIV. By contrast exogenous creatine was not protective, at variance with findings from other models. The data represent the first description of how sensitivity to hypoxic damage varies during the lifetime of an in vitro neuronal hippocampal culture. Moreover, they suggest the hypothesis that some maturational changes occurring at 79 days in vitro may make previously resistant in vitro neurons significantly sensitive to hypoxic damage, and that at least some of these changes may reflect the development of N-methyl-D-aspartate-mediated glutamatergic transmission.

Cytokine contribution to the repair processes and homeostasis recovery following anoxic insult: a possible IFN-gamma-regulating role in IL-1beta neurotoxic action in physiological or damaged CNS.

The cytokine contribution to the central nervous system (CNS) is currently being investigated, but conflicting results have frequently been found. Since, as stressed in the immune system, the cytokine action can be profoundly influenced by the synergistic and antagonistic interactions of the various cytokines present in the medium, a productive effect of the cytokine network in the repair processes and homeostasis recovery may also be dependent on their balanced response in the CNS. We, therefore, hypothesize that it is necessary to study the cytokine contribution to CNS events, evaluating cytokines both in their network and individually. Recent studies focus on interleukin 1 (IL-1) as a cytokine of primary importance for the outcome of diseases, and it seems to exert this role by regulating the synthesis of new proteins such as gamma interferon (IFN-gamma) cytokine which has been reported as having a very important early role in the balance of interactions in the cytokine network. On this basis, to verify the above hypothesis, we determined the release level of IL-1beta and IFN-gamma cytokines in media of cultured rat hippocampal neurons under physiological and anoxic conditions with and without 2-amino-5-phosphonopentanoate (AP5; an N-methyl-D-aspartate receptor antagonist). Our results show that these cytokines are released in these media, and the anoxic insult seems to determine an increase in IL-1beta and a decrease in IFN-gamma release levels as compared to those under the physiological condition. Moreover, while the IL-1beta and IFN-gamma releases covary positively in the physiological media (because an increase in the IL1-beta level is paralleled by an increase in IFN-gamma), the anoxic insult renders this interaction negative (as the IL-1beta level increase corresponds to a decrease in the IFN-gamma level). These data led us to suggest that IFN-gamma may have a physiological regulating role in the IL-1 neurotoxic action and homeostasis recovery following an insult. In fact, the addition of AP5 before anoxic exposure resulted in a significant increase of cellular survival, but also in a decrease of IL-1beta release and a re-establishment of IL-1beta and IFN-gamma release relations to the initial condition without stress. Our overall results, therefore, sustain our hypothesis on the importance of studying the role of cytokines both in their network and individually.

Transplantation of mesencephalic cell suspension in dopamine-denervated striatum of the rat.I. Effects on spontaneous activity of striatal neurons.

These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway. Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine. Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side. Sham-grafted rats received the suspension medium only. The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine. Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months. The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates. The signs of increased neuronal activity were also present in sham-grafted animals. The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate. The effects of the intrastriatal graft appeared to be present within a radius of 1.5-2 mm from the core of the grafted area. The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording. The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains.

Transplantation of Mesencephalic Cell Suspension in Dopamine-Denervated Striatum of the Rat

These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway. Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine. Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side. Sham-grafted rats received the suspension medium only. The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine. Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months. The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates. The signs of increased neuronal activity were also present in sham-grafted animals. The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate. The effects of the intrastriatal graft appeared to be present within a radius of 1.5-2 mm from the core of the grafted area. The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording. The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains.

Short-latency excitation of hindlimb motoneurons induced by electrical stimulation of the pontomesencephalic tegmentum in the rat.

The monosynaptic reflex response evoked by stimulating the dorsal root L6 was greatly facilitated when a low intensity conditioning stimulus was applied to the pontomesencephalic tegmentum (PT) 1-2 ms in advance. When increasing the stimulus strength or the number of stimuli, motor discharges were recorded in the ventral roots and in nerves innervating hindlimb muscles. The lowest threshold site for reflex facilitation was found in a region just ventral to the superior colliculus. A descending volley was recorded from the medulla midline, in the region of the medial longitudinal fascicle (MLF) and from the spinal cord surface at thoracic and lumbar level. The latency of the descending volley and of the motor responses indicates that excitation of hindlimb motoneurons was due to activation of a disynaptic pathway having a relay in the lower brainstem. All spinal and peripheral responses evoked by PT stimulation disappeared when a small electrolytic lesion was placed in the MLF 1-2 mm rostral to the obex. The results show that in the rat the PT region may exert a powerful facilitatory action on hindlimb motoneurons.

Influence of prelimbic and sensorimotor cortices on striatal neurons in the rat: electrophysiological evidence for converging inputs and the effects of 6-OHDA-induced degeneration of the substantia nigra.

These studies were designed to investigate whether there are convergent prelimbic and sensorimotor cortical inputs onto striatal neurons in the rat and whether dopaminergic (DA) nigrostriatal fibers regulate these inputs. The influence of the nigrostriatal DA system was assessed in rats with either small or large 6-hydroxydopamine-induced lesions of the substantia nigra. In normal rats 39 out of 74 neurons (52.7%) were excited by stimulation of both the prelimbic and the sensorimotor cortex. No marked change in corticostriatal transmission was evident in rats with small 6-OHDA-induced lesions (defined as 10-35% decrease in [3H]DA uptake in striatal synaptosomes). In rats with large lesions (75-85% decrease in striatal [3H]DA uptake), however, a complete rearrangement of the corticostriatal transmission occurred. This was evident in a decrease of thresholds to obtain cortical responses, by modifications of the discharge properties of striatal neurons receiving input from cortices and by an increase in the number of neurons responding to cortical stimulation. In addition, a significantly higher percentage of striatal neurons responded to stimulation of both prelimbic and sensorimotor cortices in rats with large lesions than in rats with small lesions or in control rats. This data suggests that: (1) no functional separation of prelimbic and sensorimotor cortical inputs occurs in the rat striatum, (2) the nigrostriatal DA system exerts a focusing effect on these inputs, (3) the striatum is actively involved in the integrative processing of descending cortical information.

Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons.

Extracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-alpha-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-D-glutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.

[Dopamine-glutamate interaction and cholinesterase activity in the corpus striatum of the adult rat]. / Interazione dopamina-glutamato ed attività colinesterasica nello striato del ratto adulto.

Electrophysiological, microiontophoretic and neuroanatomical techniques have been employed to investigate the relationships between intrastriatal sites of dopamine/glutamate (DA/GLU) interaction and inhomogeneities for acetylcholinesterase. The sites where iontophoretically applied DA antagonized the excitatory effects of iontophoretic GLU or cortical stimulation showed no topographic arrangement in the dorsolateral parts of the striatum. The data suggest that DA/GLU interaction in the striatum of the adult rat may occur independently from distribution of acetylcholinesterase.