A novel spirocyclic tropanyl-Δ²-isoxazoline derivative enhances citalopram and paroxetine binding to serotonin transporters as well as serotonin uptake.

A group of spirocyclic tropanyl-Δ(2)-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC(50) >> 10 µM), while some had an IC(50) value in the range 5-10 µM (8a-c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3'-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5'(4'H)-isoxazole 7a, was able to enhance at a concentration of 10 µM both [(3)H]citalopram and [(3)H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of B(max)) and [(3)H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.

Neural precursors (NPCs) from adult L967Q mice display early commitment to "in vitro" neuronal differentiation and hyperexcitability.

The pathogenic factors leading to selective degeneration of motoneurons in ALS are not yet understood. However, altered functionality of voltage-dependent Na(+) channels may play a role since cortical hyperexcitability was described in ALS patients and riluzole, the only drug approved to treat ALS, seems to decrease glutamate release via blockade or inactivation of voltage-dependent Na(+) channels. The wobbler mouse, a murine model of motoneuron degeneration, shares some of the clinical features of human ALS. At early stages of the wobbler disease, increased cortical hyperexcitability was observed. Moreover, riluzole reduced motoneuron loss and muscular atrophy in treated wobbler mice. Here, we focussed our attention on specific electrophysiological properties, like voltage-activated Na(+) currents and underlying regenerative electrical activity, as read-outs of the neuronal maturation process of neural stem/progenitor cells (NPCs) isolated from the subventricular zone (SVZ) of adult early symptomatic wobbler mice. In self-renewal conditions, the rate of wobbler NPC proliferation "in vitro" was 30% lower than that of healthy mice. Conversely, the number of wobbler NPCs displaying early neuronal commitment and action potentials was significantly higher. Upon switching from proliferative to differentiative conditions, NPCs underwent significant changes in the key properties of voltage gated Na(+) currents. The most notable finding, in cells with neuronal morphology, was an increase in Na(+) current density that strictly correlated with an increased probability to generate action potentials. This feature was remarkably more pronounced in neurons differentiated from wobbler NPCs that upon sustained stimulation, displayed short trains of pathological facilitation. In agreement with this result, an increase in the number of c-Fos positive cells, a surrogate marker of neuronal network activation, was observed in the mesial cortex of the wobbler mice "in situ". Thus these findings, all together, suggest that a state of early neuronal hyperexcitability may be a major contributor of motoneuron vulnerability.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease.

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Increased [³H]D-aspartate release and changes in glutamate receptor expression in the hippocampus of the mnd mouse.

Neuronal ceroid lipofuscinoses (NCLs) are a group of hereditary childhood diseases characterized mainly by lipopigment accumulation and a multisystemic pattern of symptoms including mental retardation, seizures, motor impairment, and blindness. The mnd mouse, carrying a mutation in the Cln8 gene, has been proposed as a model of epilepsy with mental retardation (EPMR, ornorthern epilepsy). We recently showed neuronal hyperexcitability and seizure hypersusceptibility in mnd mice. To elucidate the cellular mechanisms related to hippocampal hyperexcitability, the glutamatergic transmission and the expression of postsynaptic glutamate receptors were investigated in hippocampus. A significant increase in either spontaneous or KCl-stimulated overflow of [³H]D-aspartate was found in mnd mice compared with controls. This increase was maintained after DL-threo-ß-benzyloxyaspartic acid (TBOA) treatment, suggesting a nonrelevant role for transporter-mediated release and supporting the involvement of exocytotic [³H]D-aspartate release. Accordingly, Ca²âº-dependent overflow induced by ionomycin was also increased in mnd mice. Levels of glutamate 1-3 AMPA receptor subunits were increased, and levels of the NR2A NMDA receptor subunit were decreased in the hippocampus of mnd mice, suggesting an adaptive response to glutamate overstimulation.

Human skeletal muscle stem cell antiinflammatory activity ameliorates clinical outcome in amyotrophic lateral sclerosis models.

Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle-derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm-SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.

Synthesis and molecular modeling of 1H-pyrrolopyrimidine-2,4-dione derivatives as ligands for the α1-adrenoceptors.

Three different series of 1H-pyrrolopyrimidine-2,4-dione derivatives were designed and synthesized as ligands for the α(1)-adrenergic receptors (α(1)-ARs). A microwave-assisted protocol was developed in order to improve purity and yields of some final products. The majority of the synthesized compounds, tested in binding assays, displayed α(1)-AR affinities in the nanomolar range. Highest affinity values were found in derivatives 10b and 10c (K(i)=1.4 nM for both) whereas compound 10e was endowed with the best profile in term of α(1)-AR affinity (K(i)=2.71 nM) coupled with high selectivity towards 5-HT(1A) receptors (K(i) >10,000). Molecular docking studies were performed on human α(1)-ARs and human 5-HT(1A) receptors in order to rationalize the observed experimental affinity and selectivity; these computational studies helped to clarify molecular requirements for the design of high-selective α(1)-adrenergic ligands.

Cerebral cortex demyelination and oligodendrocyte precursor response to experimental autoimmune encephalomyelitis.

Experimentally induced autoimmune encephalomyelitis (EAE) in mice provides an animal model that shares many features with human demyelinating diseases such as multiple sclerosis (MS). To what extent the cerebral cortex is affected by the process of demyelination and how the corollary response of the oligodendrocyte lineage is explicated are still not completely known aspects of EAE. By performing a detailed in situ analysis of expression of myelin and oligodendrocyte markers we have identified areas of subpial demyelination in the cerebral cortex of animals with conventionally induced EAE conditions. On EAE-affected cerebral cortices, the distribution and relative abundance of cells of the oligodendrocyte lineage were assessed and compared with control mouse brains. The analysis demonstrated that A2B5(+) glial restricted progenitors (GRPs) and NG2(+)/PDGFR-α(+) oligodendrocyte precursor cells (OPCs) were increased in number during "early" disease, 20 days post MOG immunization, whereas in the "late" disease, 39 days post-immunization, they were strongly diminished, and there was an accompanying reduction in NG2(+)/O4(+) pre-oligodendrocytes and GST-π mature oligodendrocytes. These results, together with the observed steady-state amount of NG2(-)/O4(+) pre-myelinating oligodendrocytes, suggested that oligodendroglial precursors attempted to compensate for the progressive loss of myelin, although these cells appeared to fail to complete the last step of their differentiation program. Our findings confirm that this chronic model of EAE reproduces the features of neocortex pathology in progressive MS and suggest that, despite the proliferative response of the oligodendroglial precursors, the failure to accomplish final differentiation may be a key contributing factor to the impaired remyelination that characterizes these demyelinating conditions.

Different early ER-stress responses in the CLN8(mnd) mouse model of neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by epilepsy, progressive motor and cognitive decline, blindness, and by the accumulation of autofluorescent lipopigment. Late-infantile onset forms (LINCL) include those linked to mutations in CLN8 gene, encoding a transmembrane protein at the endoplasmic reticulum (ER). In the motor neuron degeneration (mnd) mouse model of the CLN8-LINCL (CLN8(mnd)), we carried out an analysis of ER stress-related molecules in CNS structures that exhibit a variable rate of disease progression (early retinal degeneration and delayed brain and motoneuron dysfunction). At the presymptomatic state of 1-month-old CLN8(mnd) mice, we found an upregulation of GRP78 and activation of the transcription factor-6 (ATF6) in all structures examined, an activation of a CHOP-dependent pathway in the cerebellum, hippocampus and retina, a caspase-12-dependent pathway in the retina and no activation of these two pathways in the cerebral cortex and spinal cord. An increased CHOP expression was detected in the cortex and spinal cord at the early symptomatic state (4 months). Caspase-3 cleavage occurred presymptomatically in the cerebellum, hippocampus and retina, and symptomatically in the cerebral cortex and spinal cord. We also monitored activation of NF-κB, which is engaged in the alarming phase of ER stress, together with increased levels of TRAF2, TNF-α and TNFR1, and no activation of ASK-1/JNK signalling pathway, all over mnd structures. The results suggest that early ER-stress responses distinctly combined and ER-stress pathways integrated with inflammatory responses may contribute to the progression of the CLN8(mnd) disease in CNS structures.

Neuronal hyperexcitability and seizures are associated with changes in glial-neuronal interactions in the hippocampus of a mouse model of epilepsy with mental retardation.

Hippocampal excitability and the metabolic glial-neuronal interactions were investigated in 22-week-old mice with motor neuron degeneration (mnd), a model of progressive epilepsy with mental retardation. Mnd mice developed spontaneous spikes in the hippocampus and were more susceptible to kainate-induced seizures compared with control mice. Neuronal hyperexcitability in their hippocampus was confirmed by the selective increase of c-Fos positive nuclei. Glial activation and pro-inflammatory cytokines over-expression were observed in the hippocampus of mnd mice, even in the absence of marked hippocampal neurodegeneration, as suggested by unchanged amounts of neuroactive amino acids and N-acetyl aspartate. Concentration of other amino acids, including GABA and glutamate, was not changed as well. However, ex vivo(13) C magnetic resonance spectroscopy, after simultaneous injection of [1-(13) C]glucose and [1,2-(13) C]acetate, followed by decapitation, showed decreased [1,2-(13) C]GABA formation from hippocampal astrocytic precursors and a marked reduction in [4,5-(13) C]glutamate derived from glutamine. We suggest that astrocyte dysfunction plays a primary role in the pathology and that mnd mice are of value to investigate early pathogenetic mechanism of progressive epilepsy with mental retardation.

Are descending control pathways of the lower urinary tract and pain overlapping systems?

The functions of the lower urinary tract (LUT) are dependent upon neural circuits located in the brain, spinal cord and peripheral ganglia, organized as on-off switching circuits to regulate storage and periodic elimination of urine. Damage or disease in any of the nervous pathways controlling the lower urinary tract can cause impairment of normal bladder function. Nociceptive information from different organs are delivered to the dorsal horn of the spinal cord where a network of descending pathways projecting from cerebral structures either suppress or potentiate the passage of the nociceptive messages to the brain. Some of the central structures involved in the micturition reflexes and pain modulation are common, e.g. nucleus raphe magnum, nucleus locus coeruleus alpha, periacqueductal grey, etc. Functionally, however, their effects may be similar or contrasting. The central micturition reflexes and descending control pathways of pain also utilize common transmitters and transmitter systems with similar or different effects on micturition and pain, suggesting a certain degree of overlapping between these systems. All these findings have provided a rich palette of novel mechanisms potentially available for the improved control of LUT and pain. The proliferation of potential analgesic drug targets for the therapeutic manipulation of descending control of pain is testimony of a more (in comparison with LUT) intensive research programme in this field. Nevertheless, with the exception of parenteral administration of micro-opioids and spinal application of alpha(2)-AR agonists, no other approach has been extensively validated in the clinic. Great effort should be invested in the characterization of central mechanisms controlling the micturition reflexes, although the possibility to find novel drugs for micturition disorders with central effect appears to be problematic.

Neural precursor-derived astrocytes of wobbler mice induce apoptotic death of motor neurons through reduced glutamate uptake.

In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway. NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.

Neuropathologic and biochemical changes during disease progression in liver X receptor beta-/- mice, a model of adult neuron disease.

In amyotrophic lateral sclerosis (ALS), there is selective degeneration of motor neurons that leads to paralysis and death. Although the etiology of ALS is unclear, its heterogeneity suggests that a combination of factors (endogenous and/or environmental) may induce progressive motor neuron stress that results in the activation of different cell death pathways. Alterations of brain cholesterol homeostasis have recently been considered as possible cofactors in many neurodegenerative disorders, including ALS. The liver X receptor beta (LXRbeta) receptor is involved in lipogenesis and cholesterol metabolism, and we previously found that adult-onset motor neuron pathology occurs in LXRbeta mice. Here, we investigated neuromuscular alterations of LXRbeta mice from ages 3 to 24 months. Increased cholesterol levels, gliosis, and inflammation preceded motor neuron loss and clinical disease onset; the mice showed progressivemotor neuron deficits starting from age 7 months. The numbers ofmotor neurons and neuromuscular junctions were decreased in 24-month-old mice, but neither paralysis nor reduced life span was observed. Moreover, other spinal neurons were also lost in these mice. These results suggest that LXRbeta may inhibit neuroinflammation and maintain cholesterol homeostasis, and that LXRbeta mice represent a potential model for investigating the role of cholesterol in ALS and other neurodegenerative disorders.

Novel, potent, and selective quinoxaline-based 5-HT(3) receptor ligands. 1. Further structure-activity relationships and pharmacological characterization.

We investigated the pharmacological profile of a novel series of quinoxaline-based 5-HT(3) receptor ligands bearing an extra basic moiety on the piperazine N-4. High affinity and selectivity were dependent on the electronic properties of the substituents, and at cardiac level 3a and 3c modulated chronotropy but not inotropy. In von Bezold-Jarisch reflex test 3a-c were partial agonists while 3i was a full agonist. Preliminary pharmacokinetic studies indicated that 3a is a brain penetrating agent.

Proteomic profiling of cervical and lumbar spinal cord reveals potential protective mechanisms in the wobbler mouse, a model of motor neuron degeneration.

The wobbler mouse is a model of selective motor neuron degeneration in the cervical spinal cord. Comparing cervical and lumbar tracts of control and diseased mice at the early stage of pathology by proteomic analysis, we identified 31 proteins by peptide mass fingerprint after tryptic digestion and MALDI-TOF analysis, that were differently represented among the four experimental groups. In healthy mice, patterns of protein expression differed between cervical and lumbar tract: proteins of cellular energetic metabolism pathway showed lower expression in the cervical tract, while cellular trafficking proteins were overrepresented. In wobbler mice, these differences disappeared and the expression pattern was similar between cervical and lumbar spinal cord. We found that most of the proteins differentially regulated in wobbler with respect to control cervical tract were related to astrogliosis or involved in glutamate-glutamine cycle, energy transduction and redox functions. Proteins overrepresented in the wobbler lumbar spinal cord were cytoskeleton proteins and cellular transport proteins, in particular the vesicle fusing ATPase and the isoform 2 of syntaxin-binding protein 1, involved in vesicle trafficking. We suggest that overexpression of proteins involved in vesicle trafficking, together with proteins counteracting mitochondrial dysfunction can have neuroprotective effects, preserving lumbar spinal cord motor neurons in wobbler mice.

Release of [3H]D-aspartate induced by K+-stimulation is increased in the cervical spinal cord of the wobbler mouse: a model of motor neuron disease.

The Ca2+-dependent exocytotic release of [3H]D-aspartate and [3H]GABA evoked by 15 mM KCl depolarization was studied in wobbler mice, an animal model of selective motor neuron degeneration in the cervical tract of the spinal cord. Neurotransmitters release was studied in superfusion using synaptosomes purified from the cervical or lumbar tract of the spinal cord. The early symptomatic stage (4 weeks) and the late symptomatic stage (12 weeks) of the disease were considered. Results showed that the KCl-induced release of [3H]D-aspartate was significantly increased in synaptosomes of the cervical region in wobbler mice with respect to healthy control mice, while the basal outflow was unchanged; this alteration was present both at 4 and 12 weeks. On the contrary, the KCl-induced release of [3H]D-aspartate from the lumbar spinal cord did not differ in wobbler and control mice. The KCl-induced release of [3H]GABA from cervical and lumbar spinal cord synaptosomes was unmodified at 4 weeks of age while it was moderately but significantly reduced in wobbler mice at 12 weeks, selectively in the cervical spinal cord. No changes in K(m) and V(max) for [3H]L-glutamate uptake were found in spinal cord synaptosomes from wobbler mice, compared to controls, both at 4 and 12 weeks of age. Taken together our data indicate the presence of an increased glutamate release in the affected region of spinal cord in wobbler mice, suggesting a possible involvement of altered glutamate homeostasis already in early stages of the wobbler disease, in the absence of appreciable changes in the uptake process.

Specific targeting of peripheral serotonin 5-HT(3) receptors. Synthesis, biological investigation, and structure-activity relationships.

The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT(3) receptor (5-HT(3)R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated cardiac parameters by direct interaction with myocardial 5-HT(3)Rs. The potent 5-HT(3)R ligands 4h and 4n modulate chronotropy (right atrium) but not inotropy (left atrium) at the cardiac level, being antagonist and partial agonist, respectively. Preliminary pharmacokinetic studies indicate that (S)-4n and 4a, representatives of the novel 5-HT(3)R ligands, possess poor blood-brain barrier permeability, being the prototypes of peripherally acting 5-HT(3)R modulators endowed with a clear-cut pharmacological activity at the cardiac level. The unique properties of 4h and 4n, compared to their previously described centrally active N-methyl analogue 5a, are mainly due to the hydrophilic groups at the distal piperazine nitrogen. These analogues represent novel pharmacological tools for investigating the role of peripheral 5-HT(3)R in the modulation of cardiac parameters.

Synthesis, chiral resolution and pharmacological evaluation of a 2,3-benzodiazepine-derived noncompetitive AMPA receptor antagonist.

The resolution of 1-(4-aminophenyl)-3,5-dihydro-3-N-ethylcarbamoyl-5-methyl-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (R,S)-(+/-)-5 by chiral HPLC and assignment of the absolute configuration of the two enantiomers was carried out. Compound (R,S)-(+/-)-5 and its enantiomers were tested in a binding assay to evaluate their affinity for AMPA receptors. Enantiomer (S)-(-)-5 appears to be more potent than its optical antipode (R)-(+)-5. In a primary culture of rat cerebellar granule cells, which express AMPA receptors, (R,S)-(+/-)-5 and (S)-(-)-5 inhibited kainate- induced [Ca(2+)](i) increase, thus confirming the antagonism at the AMPA receptor.

Discovery of a new class of potential multifunctional atypical antipsychotic agents targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and effects on behavior.

Dopamine D(3) antagonism combined with serotonin 5-HT(1A) and 5-HT(2A) receptor occupancy may represent a novel paradigm for developing innovative antipsychotics. The unique pharmacological features of 5i are a high affinity for dopamine D(3), serotonin 5-HT(1A) and 5-HT(2A) receptors, together with a low affinity for dopamine D(2) receptors (to minimize extrapyramidal side effects), serotonin 5-HT(2C) receptors (to reduce the risk of obesity under chronic treatment), and for hERG channels (to reduce incidence of torsade des pointes). Pharmacological and biochemical data, including specific c-fos expression in mesocorticolimbic areas, confirmed an atypical antipsychotic profile of 5i in vivo, characterized by the absence of catalepsy at antipsychotic dose.

Endogenous erythropoietin as part of the cytokine network in the pathogenesis of experimental autoimmune encephalomyelitis.

Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS.