Interferon-ß1a modulates glutamate neurotransmission in the CNS through CaMKII and GluN2A-containing NMDA receptors.

Interferons (IFNs) are widely expressed cytokines with antiviral and immune-modulating effects and have been utilised for the treatment of several human pathological conditions. In particular, the immune-modulatory drug IFN-ß is utilized in the treatment of multiple sclerosis (MS), a chronic autoimmune and neurodegenerative disorder of the central nervous system (CNS). Although the effects of IFN-ß on immune cells functions have been widely investigated, information about the ability of the drug to modulate neuronal transmission in the CNS is still largely lacking. The aim of this study was to investigate the ability of IFN-ß1a to modulate excitatory synaptic transmission in the CNS. Whole cell patch-clamp electrophysiological recordings were performed in the nucleus striatum, one of the CNS grey matter structures that is prone to degenerate during the course of MS. We demonstrate that the drug IFN-ß1a, independently from its known peripheral immune-modulating action, is able to directly modulate synaptic transmission. In particular, we demonstrated that IFN-ß1a reduces the amplitude of striatal excitatory post-synaptic currents, indicating an inhibitory effect on glutamate neurotransmission, and in particular on its NMDA component. The inhibitory effect of IFN-ß1a on striatal glutamate neurotransmission was found to be mediated by a novel post-synaptic mechanism requiring Ca(2+), CaMKII and the GluN2A subunit of the NMDA receptor, without the involvement of the classic STAT1 pathway. The evidence of a novel neuro-modulating effect of IFN-ß shed light on the mechanisms of action of the drug and on the complex bidirectional interaction occurring between the immune and the nervous system. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Interferon-ß1a protects neurons against mitochondrial toxicity via modulation of STAT1 signaling: electrophysiological evidence.

Multiple sclerosis, one of the main causes of non-traumatic neurological disability in young adults, is an inflammatory and neurodegenerative disorder of the central nervous system. Although the pathogenesis of neuroaxonal damage occurring during the course of the disease is still largely unknown, there is accumulating evidence highlighting the potential role of mitochondria in multiple sclerosis-associated neuronal degeneration. The aim of the present study was to investigate, by utilizing electrophysiological techniques in brain striatal slices, the potential protective effects of interferon-ß1a, one of the most widely used medication for multiple sclerosis, against acute neuronal dysfunction induced by mitochondrial toxins. Interferon-ß1a was found to exert a dose-dependent protective effect against the progressive loss of striatal field potential amplitude induced by the mitochondrial complex I inhibitor rotenone. Interferon-ß1a also reduced the generation of the rotenone-induced inward current in striatal spiny neurons. Conversely, interferon-ß1a did not influence the electrophysiological effects of the mitochondrial complex II inhibitor 3-nitropropionic acid. The protective effect of interferon-ß1a against mitochondrial complex I inhibition was found to be dependent on the activation of STAT1 signaling. Conversely, endogenous dopamine depletion and the modulation of the p38 MAPK and mTOR pathways did not influence the effects of interferon-ß1a. During experimental autoimmune encephalomyelitis (EAE) striatal rotenone toxicity was enhanced but the protective effect of interferon-ß1a was still evident. These results support future studies investigating the role played by specific intracellular signaling pathways in mediating the potential link among inflammation, mitochondrial impairment and neuroaxonal degeneration in multiple sclerosis.

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca(2+) increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca(2+) increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

Ischemic-LTP in striatal spiny neurons of both direct and indirect pathway requires the activation of D1-like receptors and NO/soluble guanylate cyclase/cGMP transmission.

Striatal medium-sized spiny neurons (MSNs) are highly vulnerable to ischemia. A brief ischemic insult, produced by oxygen and glucose deprivation (OGD), can induce ischemic long-term potentiation (i-LTP) of corticostriatal excitatory postsynaptic response. Since nitric oxide (NO) is involved in the pathophysiology of brain ischemia and the dopamine D1/D5-receptors (D1-like-R) are expressed in striatal NOS-positive interneurons, we hypothesized a relation between NOS-positive interneurons and striatal i-LTP, involving D1R activation and NO production. We investigated the mechanisms involved in i-LTP induced by OGD in corticostriatal slices and found that the D1-like-R antagonist SCH-23390 prevented i-LTP in all recorded MSNs. Immunofluorescence analysis confirmed the induction of i-LTP in both substance P-positive, (putative D1R-expressing) and adenosine A2A-receptor-positive (putative D2R-expressing) MSNs. Furthermore, i-LTP was dependent on a NOS/cGMP pathway since pharmacological blockade of NOS, guanylate-cyclase, or PKG prevented i-LTP. However, these compounds failed to prevent i-LTP in the presence of a NO donor or cGMP analog, respectively. Interestingly, the D1-like-R antagonism failed to prevent i-LTP when intracellular cGMP was pharmacologically increased. We propose that NO, produced by striatal NOS-positive interneurons via the stimulation of D1-like-R located on these cells, is critical for i-LTP induction in the entire population of MSNs involving a cGMP-dependent pathway.

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

Clinical pharmacogenetics of methotrexate.

It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity.

Altered regulation of glutamate release and decreased functional activity and expression of GLT1 and GLAST glutamate transporters in the hippocampus of adolescent rats perinatally exposed to Delta(9)-THC.

The long-term effects of perinatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) exposure - from gestational day (GD) 15 to postnatal day (PND) 9 - on hippocampal glutamatergic neurotransmission were studied in slices from the 40-day-old offspring of Delta(9)-THC exposed (Delta(9)-THC-rats) and vehicle-exposed (control) dams. Basal and in K+-evoked endogenous hippocampal glutamate outflow were both significantly decreased in Delta(9)-THC-rats. The effect of short Delta(9)-THC exposure (0.1microM) on K(+)-evoked glutamate release disclosed a loss of the stimulatory effect of Delta(9)-THC on hippocampal glutamate release in Delta(9)-THC-rats, but not in controls. In addition, l-[(3)H]-glutamate uptake was significantly lower in hippocampal slices from Delta(9)-THC-rats, where a significant decrease in glutamate transporter 1 (GLT1) and glutamate/aspartate transporter (GLAST) protein was also detected. Collectively, these data demonstrate that perinatal exposure to cannabinoids induces long-term impairment in hippocampal glutamatergic neurotransmission that persist into adolescence.