Maria V. Tejada-Simon

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.

Cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis.

Viral infections are though to play an important role in the pathogenesis of multiple sclerosis (MS) potentially through molecular mimicry. An identical sequence was found in both myelin basic protein (MBP, residues 96–102), a candidate autoantigen for MS, and human herpesvirus‐6 (HHV‐6 U24, residues 4–10) that is a suspected viral agent associated with MS. In this study, we showed that greater than 50% of T cells recognizing MBP93‐105 cross‐reacted with and could be activated by a synthetic peptide corresponding to residues 1 to 13 of HHV‐6 U24 in MS patients. The estimated precursor frequency of these cross‐reactive T cells recognizing both peptides, MBP93‐105 and HHV‐6 (U24)1‐13, was significantly elevated in MS patients compared with that in healthy controls. These cross‐reactive CD4+ T cells represented the same Th1 phenotype as that of monospecific T cells recognizing MBP93‐105. There were increased antibody titers for both peptide HHV‐6 (U24)1‐13 and peptide MBP93‐105 in the same patients with MS compared with those in healthy controls, suggesting B‐cell sensitization to the antigens in MS patients. The study provides important evidence in the understanding of the potential role of HHV‐6 infection/reactivation in the activation of autoimmune reactivity to MBP and its implication in the pathogenesis of MS. Ann Neurol 2003

Removal of FKBP12 Enhances mTOR-Raptor Interactions, LTP, Memory, and Perseverative/Repetitive Behavior

FK506-binding protein 12 (FKBP12) binds the immunosuppressant drugs FK506 and rapamycin and regulates several signaling pathways, including mammalian target of rapamycin (mTOR) signaling. We determined whether the brain-specific disruption of the FKBP12 gene in mice altered mTOR signaling, synaptic plasticity, and memory. Biochemically, the FKBP12-deficient mice displayed increases in basal mTOR phosphorylation, mTOR-Raptor interactions, and p70 S6 kinase (S6K) phosphorylation. Electrophysiological experiments revealed that FKBP12 deficiency was associated with an enhancement in long-lasting hippocampal long-term potentiation (LTP). The LTP enhancement was resistant to rapamycin, but not anisomycin, suggesting that altered translation control is involved in the enhanced synaptic plasticity. Behaviorally, FKBP12 conditional knockout (cKO) mice displayed enhanced contextual fear memory and autistic/obsessive-compulsive-like perseveration in several assays including the water maze, Y-maze reversal task, and the novel object recognition test. Our results indicate that FKBP12 plays a critical role in the regulation of mTOR-Raptor interactions, LTP, memory, and perseverative behaviors.

Moderate treadmill exercise prevents oxidative stress-induced anxiety-like behavior in rats.

Recent work has suggested correlation of oxidative stress with anxiety-like behavior. There also is evidence for anxiolytic effects of physical exercise. However, a direct role of oxidative stress in anxiety is not clear and a protective role of physical exercise in oxidative stress-mediated anxiety has never been addressed. In this study, we have utilized rats to test direct involvement of oxidative stress with anxiety-like behavior and have identified oxidative stress mechanisms likely involved in anxiolytic effects of physical exercise. Intraperitoneal injections at non-toxic dose of l-buthionine-(S,R)-sulfoximine (BSO), an agent that increases oxidative stress markers, increased anxiety-like behavior of rats compared to vehicle-treated control rats. Prior 2 weeks treatment with the antioxidant, tempol attenuated BSO-induced anxiety-like behavior of rats suggesting a role of oxidative stress in this phenomenon. Moreover, moderate treadmill exercise prevented BSO-induced anxiety-like behavior of rats and also prevented BSO-mediated increase in oxidative stress markers in serum, urine and brain tissue homogenates from hippocampus, amygdala and locus coeruleus. Thus increasing oxidative stress increases anxiety-like behavior of rats. Moreover, antioxidant or treadmill exercise training both reduce oxidative stress in the rat brain regions implicated in anxiety response and prevent anxiety-like behavior of rats.

Naturally Processed HLA Class II Peptides Reveal Highly Conserved Immunogenic Flanking Region Sequence Preferences That Reflect Antigen Processing Rather Than Peptide-MHC Interactions

MHC class II heterodimers bind peptides 12–20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRs. We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus

Previous postsynaptic density (PSD) isolation methodologies have utilized either whole brain or discrete brain regions of relatively large mammals such as dogs and rats. The present report details a simple and highly effective procedure for the rapid isolation of PSDs from small amounts of adult mouse hippocampus that has several advantages. First, by substituting synaptoneurosomes for synaptosomes as starting material, we have decreased the steps, time, and amount of tissue required to isolate PSDs. Second, by modifying critical steps in the synaptic isolation protocols we were able to isolate PSDs from less than 200 mg of mouse hippocampi in 3 h. Electron micrographs of isolated synaptoneurosomes showed presynaptic vesicles and densely stained membranes representing PSDs. Morphological examination of these PSDs by electron microscopy revealed a preparation that seems to be quite pure, with little or no membrane contamination. A comparison by Western blot analysis of synaptoneurosome and PSD fractions suggests that this technique yields a purified sample. Moreover, two different protocols using swing and fixed bucket rotors were used for this small-scale PSD isolation and both resulted in a very pure partition, supporting the idea that this procedure is reliable and consistent.

T cell vaccination in multiple sclerosis: results of a preliminary study.

Abstract Myelin basic protein (MBP)-reactive T cells are potentially involved in the pathogenesis of multiple sclerosis (MS), and can be depleted by subcutaneous inoculations with irradiated autologous MBP-reactive T cells (T cell vaccination). This preliminary open label study was undertaken to evaluate whether depletion of MBP-reactive T cells would be clinically beneficial to patients with MS. Fifty-four patients with relapsing-remitting (RR) MS (n=28) or secondary progressive (SP) MS (n=26) were immunized with irradiated autologous MBP-reactive T cells and monitored for changes in rate of relapse, expanded disability scale score (EDSS) and MRI lesion activity over a period of 24 months. Depletion of MBP-reactive T cells correlated with a reduction (40 %) in rate of relapse in RR-MS patients as compared with the pre-treatment rate in the same cohort. However, the reduction in EDSS was minimal in RR-MS patients while the EDSS was slightly increased in SP-MS patients over a period of 24 months. Serial semi-quantitative MRI examinations suggest stabilization in lesion activity as compared with baseline MRI. The findings suggest some potential clinical benefit of T cell vaccination in MS and encourage further investigations to evaluate the treatment efficacy of T cell vaccination in controlled trials.

Reactivity pattern and cytokine profile of T cells primed by myelin peptides in multiple sclerosis and healthy individuals.

Autoreactive T cells specific for candidate myelin antigens, including myelin basic protein (MBP) and proteolipid protein (PLP), are thought to play an important role in the pathogenesis of multiple sclerosis (MS). Myelin‐reactive T cells primed in vivo by myelin breakdown products or microbial cross‐reactive antigens during the disease processes may exhibit a reactivity pattern and cytokine profile different from those in the normal T cell repertoire. In this study, we examined the precursor frequency, the reactivity pattern and cytokine profile of myelin‐reactive T cells that were primed in vitro with overlapping peptides of MBP and PLP in patients with MS and healthy individuals. The results revealed that T cells specific for peptides of MBP and PLP occurred at a relatively higher precursor frequency in patients with MS than that in healthy individuals. We identified a number of dominant T cell epitopes within MBP and PLP, some of which were not previously detected using whole myelin antigens as the primary stimuli. Some residues represented common immunodominant regions that were detected in both MS patients and healthy controls while others were associated only with MS. MBP‐reactive T cell lines generally exhibited a Th0‐like cytokine profile. There was significantly increased Th1 cytokine production (i. e. TNF and IFN‐γ) among MS‐derived T cell lines. PLP‐reactive T cell lines had a distinct cytokine profile, producing predominantly TNF‐α and little or not IFN‐γ and IL‐4. The findings have important implications in the understanding of the role of myelin‐reactive T cells in MS.

Th2 immune regulation induced by T cell vaccination in patients with multiple sclerosis.

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In this study, we demonstrated that subcutaneous inoculations with irradiated autologous MBP‐reactive T cell clones (T cell vaccination) elicited CD8+ antiidiotypic T cell responses and CD4+ Th2 cell responses in patients with MS. Both regulatory cell types induced by T cell vaccination contributed to the inhibition of MBP‐reactive T cells while they differed in the recognition pattern and functional properties. We describe for the first time that the Th2 regulatory cells reacted with activated but not resting T cells in the context of MHC class II molecules and inhibited the proliferation of MBP‐reactive T cells through the secretion of IL‐4 and IL‐10. The T‐T cell interaction mediated by Th2 regulatory cells was independent of the antigen specificity of activated T cells. The findings have important implications for our understanding of the regulatory mechanism induced by T cell vaccination.

Detection of Viral DNA and Immune Responses to the Human Herpesvirus 6 101-Kilodalton Virion Protein in Patients with Multiple Sclerosis and in Controls

ABSTRACT Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.