Maria Teresa Ciotti

Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells

Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of -60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10-1000 microM; n = 16). Dose-response analysis of this effect revealed an IC50 of 48.9 microM. Topiramate also made the steady-state inactivation curve of this current shift toward more negative values (midpoint of the inactivation curve -46.9 mV under control conditions and -56.5 mV during topiramate application; n = 5). We propose that these effects may contribute to control the sustained depolarizations with repetitive firing of action potentials that occur within neuronal networks during seizure activity. Therefore they may represent a mechanism of action for this novel anticonvulsant drug.

CXC chemokines interleukin-8 (IL-8) and growth-related gene product α (GROα) modulate Purkinje neuron activity in mouse cerebellum

Abstract We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROα by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROα may play a neuromodulatory role on mouse cerebellum.

Abnormal Striatal GABA Transmission in the Mouse Model for the Fragile X Syndrome

BACKGROUND Structural and functional neuroimaging studies suggest abnormal activity in the striatum of patients with the fragile X syndrome (FXS), the most common form of inherited mental retardation. METHODS Neurophysiological and immunofluorescence experiments in striatal brain slices. We studied the synaptic transmission in a mouse model for FXS, as well as the subcellular localization of fragile X mental retardation protein (FMRP) and brain cytoplasmic (BC1) RNA in striatal axons. RESULTS Our results show that absence of FMRP is associated with apparently normal striatal glutamate-mediated transmission, but abnormal gamma-aminobutyric acid (GABA) transmission. This effect is likely secondary to increased transmitter release from GABAergic nerve terminals. We detected the presence of FMRP in axons of striatal neurons and observed a selective increase in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs, mIPSCs) in fmr1-knockout mice. We also observed reduced paired-pulse ratio of evoked IPSCs, a finding that is consistent with the idea that transmitter release probability from striatal GABAergic nerve terminals is higher than normal in these mutants. Finally, we have identified the small noncoding BC1 RNA as a critical coplayer of FMRP in the regulation of striatal synaptic transmission. CONCLUSIONS Understanding the physiologic action of FMRP and the synaptic defects associated with GABA transmission might be useful to design appropriate pharmacologic interventions for FXS.

Role of the autophagic-lysosomal system on low potassium- induced apoptosis in cultured cerebellar granule cells

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal–lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF‐1 and forskolin, two well‐known inhibitors of CGC apoptosis, as well as by adenovirus‐mediated overexpression of Bcl‐2. 3‐Methyladenine (3‐MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3‐MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase‐3 activation which, in turn, appears to be mediated by cathepsin B, as CA074‐Me, a selective inhibitor of this enzyme, fully blocks the processing of pro‐caspase‐3. Immunofluorescence analysis demonstratesd that cathepsin B, normally confined inside the lysosomal‐endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

Chemokine CX3CL1 protects rat hippocampal neurons against glutamate-mediated excitotoxicity.

Excitotoxicity is a cell death caused by excessive exposure to glutamate (Glu), contributing to neuronal degeneration in many acute and chronic CNS diseases. We explored the role of fractalkine/CX3CL1 on survival of hippocampal neurons exposed to excitotoxic doses of Glu. We found that: CX3CL1 reduces excitotoxicity when co-applied with Glu, through the activation of the ERK1/2 and PI3K/Akt pathways, or administered up to 8 h after Glu insult; CX3CL1 reduces the Glu-activated whole-cell current through mechanisms dependent on intracellular Ca2+; CX3CL1 is released from hippocampal cells after excitotoxic insult, likely providing an endogenous protective mechanism against excitotoxic cell death.

MicroRNA-101 Regulates Amyloid Precursor Protein Expression in Hippocampal Neurons

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Aβ) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3′-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Aβ load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1β (IL-lβ). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Aβ, suggesting a possible role for miR-101 in neuropathological conditions.

SDF‐1α‐mediated modulation of synaptic transmission in rat cerebellum

The functional expression of the seven‐transmembrane domain G protein‐coupled chemokine receptor CXCR‐4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti‐CXCR‐4 Ab, and by evaluating the calcium responses to the physiological agonist stromal‐derived cell factor‐1α (SDF‐1α) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR‐4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF‐1α, both in cell bodies and in neuronal processes. Whole‐cell patch‐clamped PNs in cerebellar slices responded to SDF‐1α application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF‐1α‐induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR‐4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

NGF and BDNF signaling control amyloidogenic route and Aβ production in hippocampal neurons

Here, we report that interruption of NGF or BDNF signaling in hippocampal neurons rapidly activates the amyloidogenic pathway and causes neuronal apoptotic death. These events are associated with an early intracellular accumulation of PS1 N-terminal catalytic subunits and of APP C-terminal fragments and a progressive accumulation of intra- and extracellular Aβ aggregates partly released into the culture medium. The released pool of Aβ induces an increase of APP and PS1 holoprotein levels, creating a feed-forward toxic loop that might also cause the death of healthy neurons. These events are mimicked by exogenously added Aβ and are prevented by exposure to β- and γ-secretase inhibitors and by antibodies directed against Aβ peptides. The same cultured neurons deprived of serum die, but APP and PS1 overexpression does not occur, Aβ production is undetectable, and cell death is not inhibited by anti-Aβ antibodies, suggesting that hippocampal amyloidogenesis is not a simple consequence of an apoptotic trigger but is due to interruption of neurotrophic signaling.

Ligand-independent CXCR2 Dimerization

Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product β) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.

Oxidative Stress Induces p53-Mediated Apoptosis in Glia: p53 Transcription-Independent Way to Die

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress‐induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H2O2‐induced oxidative stress protocol followed by a recovery period. We evaluated the effects of pifithrin‐α (PF‐α), which has been reported to protect neurons from ischemic insult by specifically inhibiting p53 DNA‐binding activity. Strikingly, PF‐α was unable to prevent oxidative stress‐induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z‐VAD‐fmk‐sensitive apoptotic response requires a caspase‐dependent MDM‐2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.