Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes

Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (eLTP, <1 hour) had initially been explained either by presynaptic increases in glutamate release or by direct modification of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional post-synaptic AMPARs, sourced either from an intracellular reserve pool by exocytosis or from nearby extra synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during eLTP is still unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion dramatically impaired synaptic potentiation of Schaffer collateral/commissural inputs to cornu ammonis area 1 (CA1) in cultured slices, acute slices and in vivo. Our data also identifies distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus (DH) before fear conditioning, indicated that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein-protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning

Beta-Amyloid Peptides Enhance the Proliferative Response of Activated CD4+CD28+ Lymphocytes from Alzheimer Disease Patients and from Healthy Elderly

Alzheimer's disease (AD) is the most frequent form of dementia among elderly. Despite the vast amount of literature on non-specific immune mechanisms in AD there is still little information about the potential antigen-specific immune response in this pathology. It is known that early stages of AD include β-amyloid (Aβ)- reactive antibodies production and inflammatory response. Despite some evidence gathered proving cellular immune response background in AD pathology, the specific reactions of CD4+ and CD8+ cells remain unknown as the previous investigations yielded conflicting results. Here we investigated the CD4+CD28+ population of human peripheral blood T cells and showed that soluble β-amyloids alone were unable to stimulate these cells to proliferate significantly, resulting only in minor, probably antigen-specific, proliferative response. On the other hand, the exposure of in vitro pre-stimulated lymphocytes to soluble Aβ peptides significantly enhanced the proliferative response of these cells which had also lead to increased levels of TNF, IL-10 and IL-6. We also proved that Aβ peptide-enhanced proliferative response of CD4+CD28+ cells is autonomous and independent from disease status while being associated with the initial, ex vivo activation status of the CD4+ cells. In conclusion, we suggest that the effect of Aβ peptides on the immune system of AD patients does not depend on the specific reactivity to Aβ epitope(s), but is rather a consequence of an unspecific modulation of the cell cycle dynamics and cytokine production by T cells, occurring simultaneously in a huge proportion of Aβ peptide-exposed T lymphocytes and affecting the immune system performance

Blocking Mineralocorticoid Receptors prior to Retrieval Reduces Contextual Fear Memory in Mice

BACKGROUND: Corticosteroid hormones regulate appraisal and consolidation of information via mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respectively. How activation of these receptors modulates retrieval of fearful information and the subsequent expression of fear is largely unknown. We tested here whether blockade of MRs or GRs during retrieval also affects subsequent expression of fear memory. METHODOLOGY/PRINCIPAL FINDINGS: Mice were trained in contextual or tone cue fear conditioning paradigms, by pairing mild foot shocks with a particular context or tone respectively. Twenty-four hours after training, context-conditioned animals were re-exposed to the context for 3 or 30 minutes (day 2); tone-conditioned animals were placed in a different context and re-exposed to one or six tones. Twenty-four hours (day 3) and one month later, freezing behavior to the aversive context/tone was scored again. MR or GR blockade was achieved by giving spironolactone or RU486 subcutaneously one hour before retrieval on day 2. Spironolactone administered prior to brief context re-exposure reduced freezing behavior during retrieval and 24 hours later, but not one month later. Administration of spironolactone without retrieval of the context or immediately after retrieval on day 2 did not reduce freezing on day 3. Re-exposure to the context for 30 minutes on day 2 significantly reduced freezing on day 3 and one month later, but freezing was not further reduced by spironolactone. Administration of spironolactone prior to tone-cue re-exposure on day 2 did not affect freezing behavior. Treatment with RU486 prior to re-exposure did not affect context or tone-cue fear memories at any time point. CONCLUSIONS/SIGNIFICANCE: We conclude that MR blockade prior to retrieval strongly reduces the expression of contextual fear, implying that MRs, rather than GRs, play an important role in retrieval of emotional information and subsequent fear expression

An Efficient Strategy to Induce and Maintain In Vitro Human T Cells Specific for Autologous Non-Small Cell Lung Carcinoma

BACKGROUND: The efficient expansion in vitro of cytolytic CD8+ T cells (CTLs) specific for autologous tumors is crucial both for basic and translational aspects of tumor immunology. We investigated strategies to generate CTLs specific for autologous Non-Small Cell Lung Carcinoma (NSCLC), the most frequent tumor in mankind, using circulating lymphocytes. PRINCIPAL FINDINGS: Classic Mixed Lymphocyte Tumor Cultures with NSCLC cells consistently failed to induce tumor-specific CTLs. Cross-presentation in vitro of irradiated NSCLC cells by autologous dendritic cells, by contrast, induced specific CTL lines from which we obtained a high number of tumor-specific T cell clones (TCCs). The TCCs displayed a limited TCR diversity, suggesting an origin from few tumor-specific T cell precursors, while their TCR molecular fingerprints were detected in the patient's tumor infiltrating lymphocytes, implying a role in the spontaneous anti-tumor response. Grafting NSCLC-specific TCR into primary allogeneic T cells by lentiviral vectors expressing human V-mouse C chimeric TCRalpha/beta chains overcame the growth limits of these TCCs. The resulting, rapidly expanding CD4+ and CD8+ T cell lines stably expressed the grafted chimeric TCR and specifically recognized the original NSCLC. CONCLUSIONS: This study defines a strategy to efficiently induce and propagate in vitro T cells specific for NSCLC starting from autologous peripheral blood lymphocytes

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands

Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

The SIV/rhesus macaque model for HIV/AIDS is a powerful system for examining the contribution of T cells in the control of AIDS viruses. To better our understanding of CD8(+) T-cell control of SIV replication in CD4(+) T cells, we asked whether TCRs isolated from rhesus macaque CD8(+) T-cell clones that exhibited varying abilities to suppress SIV replication could convey their suppressive properties to CD8(+) T cells obtained from an uninfected/unvaccinated animal.We transferred SIV-specific TCR genes isolated from rhesus macaque CD8(+) T-cell clones with varying abilities to suppress SIV replication in vitro into CD8(+) T cells obtained from an uninfected animal by retroviral transduction. After sorting and expansion, transduced CD8(+) T-cell lines were obtained that specifically bound their cognate SIV tetramer. These cell lines displayed appropriate effector function and specificity, expressing intracellular IFNγ upon peptide stimulation. Importantly, the SIV suppression properties of the transduced cell lines mirrored those of the original TCR donor clones: cell lines expressing TCRs transferred from highly suppressive clones effectively reduced wild-type SIV replication, while expression of a non-suppressing TCR failed to reduce the spread of virus. However, all TCRs were able to suppress the replication of an SIV mutant that did not downregulate MHC-I, recapitulating the properties of their donor clones.Our results show that antigen-specific SIV suppression can be transferred between allogenic T cells simply by TCR gene transfer. This advance provides a platform for examining the contributions of TCRs versus the intrinsic effector characteristics of T-cell clones in virus suppression. Additionally, this approach can be applied to develop non-human primate models to evaluate adoptive T-cell transfer therapy for AIDS and other diseases

Microtubules as Platforms for Assaying Actin Polymerization In Vivo

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process

Inhibition of Soluble Tumor Necrosis Factor Ameliorates Synaptic Alterations and Ca2+ Dysregulation in Aged Rats

The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4–6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca2+ channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling