Irene Carunchio

Increased persistent sodium current determines cortical hyperexcitability in a genetic model of amyotrophic lateral sclerosis

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly(93)-->Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.

Modulation of AMPA receptors in cultured cortical neurons induced by the antiepileptic drug levetiracetam

Summary:u2002 Purpose: The present study explores the hypothesis that the antiepileptic mechanism of action of levetiracetam (LEV) is related to effects on α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor channels in mouse cortical neurons in culture.

Brivaracetam (ucb 34714) inhibits Na + current in rat cortical neurons in culture

Brivaracetam (ucb 34714; BRV), a new antiepileptic drug (AED) candidate, is a pyrrolidone derivative displaying a markedly higher affinity than levetiracetam (LEV; Keppra) to the synaptic vesicle protein SV2A, shown to be the brain-specific binding site of LEV. The higher affinity for SV2A correlates significant antiepileptic activity in animal epilepsy models in vitro and in vivo. Since many AEDs act upon inhibiting neuronal Na(+) currents, this study explored putative activity of BRV on the properties of these currents. Voltage-activated Na(+) currents were recorded by whole-cell patch-clamp on neuronal somas of rat neocortical neurons, grown in dissociated cell culture for up to 12 days. BRV, dissolved at the desired final concentration (between 0.2microM and 1mM) was applied by a multi-barrel pipette system near the soma of the recorded neuron. BRV produced a concentration-dependent inhibition of voltage-dependent Na(+) currents with IC(50) values of 41microM at the holding potential of -100mV, and of 6.5microM at the holding potential of -60mV. The voltage-dependence of activation and the kinetics of fast inactivation were not modified in the presence of BRV (30microM). Conversely, the recovery from fast inactivation was significantly slower and the voltage of half-maximal inactivation was shifted toward hyperpolarized value after BRV perfusion in a concentration-dependent manner. Furthermore, BRV (30microM) induced a significant use-dependent block at 50Hz stimulation frequency. These results indicate that BRV is able to modulate the voltage-activated Na(+) inflow in cortical neurons, which conceivably might contribute to the antiepileptic activity of this drug.

Altered calcium homeostasis in motor neurons following AMPA receptor but not voltage-dependent calcium channels' activation in a genetic model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by a substantial loss of motor neurons in the spinal cord, brain stem and motor cortex. By combining electrophysiological recordings with imaging techniques, clearance/buffering capacity of cultured spinal cord motor neurons after a calcium accumulation has been analyzed in response to AMPA receptors (AMPARs) activation and to depolarizing stimuli in a genetic mouse model of ALS (G93A). Our studies demonstrate that the amplitude of the calcium signal in response to AMPARs or voltage-dependent calcium channels activation is not significantly different in controls and G93A motor neurons. On the contrary, in G93A motor neurons, the [Ca(2+)](i) recovery to basal level is significantly slower compared to control neurons following AMPARs but not voltage-dependent calcium channels activation. This difference was not observed in G93A cultured cortical neurons. This observation is the first to indicate a specific alteration of the calcium clearance linked to AMPA receptors activation in G93A motor neurons and the involvement of AMPA receptor regulatory proteins controlling both AMPA receptor functionality and the sequence of events connected to them.

Increased levels of p70S6 phosphorylation in the G93A mouse model of Amyotrophic Lateral Sclerosis and in valine-exposed cortical neurons in culture

The higher risk factor for Amyotrophic Lateral Sclerosis (ALS) among Italian soccer players is a question that is still debated. One of the hypotheses that have been formulated to explain a possible link between ALS and soccer players is related to the abuse of dietary supplements and drugs for enhancing sporting performance. In particular, it has been reported that branched-chain amino acids (BCAAs) are widely used among athletes as nutritional supplements. To observe the possible effect of BCAAs on neuronal electrical properties, we performed electrophysiological experiments on Control cultured cortical neurons and on neurons after BCAA treatment. BCAA-treated neurons showed hyperexcitability and rapamycin was able to suppress it and significantly reduce the level of mTOR, Akt and p70S6 phosphorylation. Interestingly, the hyperexcitability previously reported in cortical neurons from a genetic mouse model of ALS (G93A) was also reversed by rapamycin treatment. Moreover, both G93A and valine-treated neurons presented significantly higher levels of Pp70S6 when compared to control neurons, strongly indicating the involvement of this substrate in ALS pathology. Finally, we performed electrophysiological experiments on motor cortex slices from Control and G93A mice and those fed with a BCAA-enriched diet. We observed that neuron excitability was comparable between G93A and BCAA-enriched diet mice, but was significantly higher than in Control mice. These findings, besides strongly indicating that BCAAs specifically induce hyperexcitability, seem to suggest the involvement of p70S6 substrate in ALS pathology.

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: Evidence for the involvement of the delayed rectifier potassium current

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

SP protects cerebellar granule cells against β-amyloid-induced apoptosis by down-regulation and reduced activity of Kv4 potassium channels

The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimers disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (A beta) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both A beta(25-35)- and A beta(1-42)-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism. Since in CGCs the fast inactivating or A-type K(+) current (I(KA)) was potentiated by A beta treatment through up-regulation of Kv4 subunits, we investigated whether I(KA) and the related potassium channel subunits could be involved in the SP anti-apoptotic activity. Patch-clamp experiments showed that the A beta-induced increase of I(KA) current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression. These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in A beta-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.

GABAA receptors present higher affinity and modified subunit composition in spinal motor neurons from a genetic model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the selective degeneration of motor neurons in the spinal cord, brainstem and cerebral cortex. In this study we have analysed the electrophysiological properties of GABAA receptors and GABAA alpha1 and alpha2 subunits expression in spinal motor neurons in culture obtained from a genetic model of ALS (G93A) and compared with transgenic wild type SOD1 (SOD1) and their corresponding non transgenic litter mates (Control). Although excitotoxic motor neuron death has been extensively studied in relation to Ca2+‐dependent processes, strong evidence indicates that excitotoxic cell death is also remarkably dependent on Cl− ions and on GABAA receptor activation. In this study we have analysed the electrophysiological properties of GABAA receptors and the expression of GABAAα1 and α2 subunits in cultured motor neurons obtained from a genetic model of amyotrophic lateral sclerosis (G93A) and compared them with transgenic wild‐type Cu,Zn superoxide dismutase and their corresponding non‐transgenic littermates (Control). In all tested motor neurons, the application of γ‐aminobutyric acid (GABA) (0.5–100u2003μm) evoked an inward current that was reversibly blocked by bicuculline (100u2003μm), thus indicating that it was mediated by the activation of GABAA receptors. Our results indicate that the current density at high GABA concentrations is similar in control, Cu,Zn superoxide dismutase and G93A motor neurons. However, the dose‐response curve significantly shifted toward lower concentration values in G93A motor neurons and the extent of desensitization also increased in these neurons. Finally, multiplex single‐cell real‐time polymerase chain reaction and immunofluorescence revealed that the amount of GABAAα1 subunit was significantly increased in G93A motor neurons, whereas the levels of α2 subunit were unchanged. These data show that the functionality and expression of GABAA receptors are altered in G93A motor neurons inducing a higher Cl− influx into the cell with a possible consequent neuronal excitotoxicity acceleration.

Persistent elevation of D-Aspartate enhances NMDA receptor-mediated responses in mouse substantia nigra pars compacta dopamine neurons

Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive functions. NMDA receptors play a crucial role in modulating the activity of these cells. Considering that the amino-acid D-Aspartate has been recently shown to be an endogenous NMDA receptor agonist, the aim of the present study was to examine the effects of D-Aspartate on the functional properties of nigral dopamine neurons. We compared the electrophysiological actions of D-Aspartate in control and D-aspartate oxidase gene (Ddo(-/-)) knock-out mice that show a concomitant increase in brain D-Aspartate levels, improved synaptic plasticity and cognition. Finally, we analyzed the effects of L-Aspartate, a known dopamine neuron endogenous agonist in control and Ddo(-/-) mice. We show that D- and L-Aspartate excite dopamine neurons by activating NMDA, AMPA and metabotropic glutamate receptors. Ddo deletion did not alter the intrinsic properties or dopamine sensitivity of dopamine neurons. However, NMDA-induced currents were enhanced and membrane levels of the NMDA receptor GluN1 and GluN2A subunits were increased. Inhibition of excitatory amino-acid transporters caused a marked potentiation of D-Aspartate, but not L-Aspartate currents, in Ddo(-/-) neurons. This is the first study to show the actions of D-Aspartate on midbrain dopamine neurons, activating not only NMDA but also non-NMDA receptors. Our data suggest that dopamine neurons, under conditions of high D-Aspartate levels, build a protective uptake mechanism to compensate for increased NMDA receptor numbers and cell hyper-excitation, which could prevent the consequent hyper-dopaminergia in target zones that can lead to neuronal degeneration, motor and cognitive alterations.

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.