Modulation of actin dynamics by Rac1 to target cognitive function.

The small GTPase Rac1 is well known for regulating actin cytoskeleton reorganization in cells. Formation of extensions at the surface of the cell is required for migration and even for cell invasion and metastases. Because an elevated level and hyperactivation of this protein has been associated with metastasis in cancer, direct regulators of Rac1 are currently envisioned as a potential strategy to treat certain cancers. Less research, however, has been done regarding the role of this small GTP-binding protein in brain development, where it has an important role in dendritic spine morphogenesis through the regulation of actin. Alteration of dendritic development and spinogenesis has been often associated with mental disorders. Rac1 is associated with and required for learning and the formation of memories in the brain. Rac1 appears to be dysregulated in certain neurodevelopmental disorders that present all these three alterations: mental retardation, atypical synaptic plasticity and aberrant spine morphology. Thus, to develop novel therapies for rescuing cognitive impairment, a reasonable approach might be to target this protein, Rac1, which plays a pivotal role in directing signals that regulate actin dynamics, which in turn might have an effect in spine cytoarchitecture and synaptic function. It is possible that novel drugs that regulate Rac1 activation and function could modulate actin cytoskeleton and spine dynamics, representing potential candidates to repair intellectual disability in disorders associated with spine abnormalities. Herein, we present a list of the current Rac1 inhibitors that might fulfill this role together with a summary of the latest findings concerning their function as they relate to neuronal studies. While the small GTPase Rac1 is well known for regulating actin cytoskeleton reorganization in different type of cells, it appears to be also required for learning and the formation of memories in the brain. Abnormal regulation of this protein has been associated with cognitive disabilities, atypical synaptic plasticity and abnormal morphology of dendritic spines in certain neurodevelopmental disorders. Thus, modulation of Rac1 activity using novel inhibitors might be a strategy to reestablish cognitive function.

Modulation of dendritic spines and synaptic function by Rac1: a possible link to Fragile X syndrome pathology.

Rac1, a protein of the Rho GTPase subfamily, has been implicated in neuronal and spine development as well as the formation of synapses with appropriate partners. Dendrite and spine abnormalities have been implicated in several psychiatric disorders such as Fragile X syndrome, where neurons show a high density of long, thin, and immature dendritic spines. Although abnormalities in dendrites and spines have been correlated with impaired cognitive abilities in mental retardation, the causes of these malformations are not yet well understood. Fragile X syndrome is the most common type of inherited mental retardation caused by the absence of FMRP protein, a RNA-binding protein implicated in the regulation of mRNA translation and transport, leading to protein synthesis. We suggest that FMRP might act as a negative regulator on the synthesis of Rac1. Maintaining an optimal level of Rac1 and facilitating the reorganization of the cytoskeleton likely leads to normal neuronal morphology during activity-dependent plasticity. In our study, we first demonstrated that Rac1 is not only associated but necessary for normal spine development and long-term synaptic plasticity. We further showed that, in Fmr1 knockout mice, lack of FMRP induces an overactivation of Rac1 in the mouse brain and other organs that have been shown to be altered in Fragile X syndrome. In those animals, pharmacological manipulation of Rac1 partially reverses their altered long-term plasticity. Thus, regulation of Rac1 may provide a functional link among deficient neuronal morphology, aberrant synaptic plasticity and cognition impairment in Fragile X syndrome.

Pharmacological inactivation of the small GTPase Rac1 impairs long-term plasticity in the mouse hippocampus.

Neuronal development involves several discrete morphological steps requiring migration of newborn neurons to characteristic locations, extension of axons and dendrites into proper target regions, and formation of synapses with appropriate partners. Small GTPases such as Rac1, are believed to be critical regulators of these processes. We have previously reported that Rac1 is highly expressed in mouse hippocampus, where NMDA receptor activation causes Rac1 to translocate to the membrane in a manner similar to that observed in other non-neuronal cells. Additionally Rac1 has been seen to play a role in activation of signal transduction pathways associated with hippocampal learning and memory. Because of the established role of LTP and LTD in learning and memory processes, in this study we investigate whether Rac1 plays also an active and critical role in these types of long-term synaptic plasticity. We found that activation of Rac1 is associated with long-term plasticity, both LTP and LTD. Rac1 appears to have a transient role during the induction of NMDA receptor-dependent LTP, but does not have an effect on LTP maintenance and expression. Similar results were found for NMDA receptor-dependent induction of LTD, while mGluR-dependent LTD was shown to be significantly altered but not abolished. The results of these experiments provide essential knowledge regarding the signaling mechanisms that underlie synaptic plasticity, as well as learning and memory processes, which in turn offers insights into the basis of diseases involving memory impairment, such as Fragile X syndrome, Alzheimer's disease, William's syndrome, Angelman syndrome (AS), and schizophrenia.

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.

Translocation and activation of Rac in the hippocampus during associative contextual fear learning.

Small G proteins including Rac are mediators of changes in neuronal morphology associated with synaptic plasticity. Previous studies in our laboratory showed that Rac is highly expressed in the adult mouse hippocampus, a brain area that exhibits robust synaptic plasticity and is crucial for the acquisition of memories. In this study, we investigated whether Rac was involved in NMDA receptor-dependent associative fear learning in the area CA1 of adult mouse hippocampus. We found that Rac translocation and activation was increased in the hippocampus following associative fear conditioning in mice, and that these increases are blocked by intraperitoneal injection of the NMDA receptor channel blocker MK801 at the acquisition stage. Our data indicate that NMDA receptor-dependent associative fear learning alters Rac localization and function in the mouse hippocampus.

NMDA receptor activation induces translocation and activation of Rac in mouse hippocampal area CA1.

Neuronal development requires several discrete morphological steps that are believed to involve the small GTPase Rac. For example, neural activity, through NMDA receptors and/or AMPA receptors, activates Rac leading to elaboration of dendritic arbors. In the current study, we have conducted studies which indicate that Rac might be an important molecule involved in morphological plasticity in the adult mouse. We demonstrate that Rac is expressed at synapses in the adult mouse hippocampus. We also demonstrate that treatment of hippocampal slices with NMDA induces membrane translocation and activation of Rac in area CA1. Interestingly, we also find that there is an increase in Rac that is associated with NMDA receptor complexes following NMDA receptor activation. Taken together, our data are consistent with the idea that Rac could be participating in NMDA receptor-dependent changes in morphology that occur during synaptic plasticity and memory formation in the adult mouse hippocampus.

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 MBP(93-105) 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, MBP(93-105) 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 MBP(93-105). There were increased antibody titers for both peptide HHV-6 (U24)(1-13) and peptide MBP(93-105) 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.

Skewed autoantibody reactivity to the extracellular domain of myelin oligodendrocyte glycoprotein in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is found to induce both autoreactive T-cell and antibody responses associated with demyelinating pathology and is implicated in the pathogenesis of multiple sclerosis (MS). In this study, we addressed the potential association of anti-MOG immune responses with MS by examining, comparatively, both the T-cell and antibody responses to recombinant MOG fragments in MS patients and healthy subjects. T cells recognizing MOG were detected in MS patients as well as in healthy subjects, and their precursor frequency in the blood was not increased in patients with MS. MOG-reactive T cells isolated from both MS patients and healthy subjects exhibited a similar cytokine profile, producing interleukin (IL)-4, IL-10 and tumour necrosis factor (TNF), but not interferon-gamma (IFN-gamma), and recognized predominantly the extracellular (residues 1-60) and the transmembrane/cytoplasmic (residues 154-218) domains of MOG. In contrast, anti-MOG antibodies derived from MS patients displayed a skewed reactivity pattern, even though the occurrence and titres of serum anti-MOG antibodies were only slightly elevated in MS patients. MS-derived autoantibodies were predominantly directed at the 1-60 region of MOG, while naturally occurring anti-MOG antibodies derived from healthy individuals reacted selectively to the 154-218 domain. These differences were statistically significant. The findings of this study are consistent with the presence of anti-MOG antibodies within demyelinating lesions of MS and their role in the induction of demyelinating pathology in animal models. The study has important implications in the understanding of the autoimmune processes in MS.

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

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.

Detection of viral DNA and immune responses to the human herpesvirus 6 101-kilodalton virion protein in patients with multiple sclerosis and in controls.

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.

Effects of Lactobacillus spp. on Cytokine Production by RAW 264.7 Macrophage and EL-4 Thymoma Cell Lines.

We have hypothesized that lactobacilli used in fermented dairy products can stimulate immune function via enhancing cytokine secretion by leukocytes. To test the effects of lactobacilli on cytokine production, RAW 264.7 cells (macrophage model) and EL4.IL-2 thymoma cells (T helper cell model) were cultured in the presence of 16 representative strains of heat-killed Lactobacillus spp. cells. In unstimulated RAW 264.7 cells, most lactobacilli, when present at concentrations from 106 to 108 bacterial cells per ml, induced marked tumor necrosis factor alpha (TNF-α) production (up to 411-fold) compared to the negligible TNF-α in controls. A strain-dependent increase in interleukin 6 (IL-6) production (up to 88-fold) was also observed without stimulation at concentrations of 108 bacteria per ml. Upon concurrent stimulation of RAW 264.7 cells with lipopolysaccharide, both IL-6 and TNF-α production were enhanced between 4.2- and 10.6-fold and 1.8- and 8.7-fold, respectively, when cultured with 108 lactobacilli per ml. In unstimulated EL4.IL-2 cells, lactobacilli had no effect on interleukin 2 (IL-2) or interleukin 5 (IL-5) production. Upon stimulation of EL4.IL-2 cells with phorbol 12-myristate-13-acetate, IL-2 secretion increased up to 1.9-fold in the presence of 106 L. bulgaricus Lr 79 cells per ml whereas this cytokine decreased in the presence of 107 or 108 lactobacilli per ml. In contrast, IL-5 secretion increased in the presence of increasing concentrations of lactobacilli (up to 3.1-fold with 108 L. bulgaricus NCK 231 cells per ml). The results indicated that direct interaction of most lactobacilli with macrophages resulted in a concentration-dependent enhancement of cytokine production, whereas the effects on cytokine production by the T-cell model were smaller and strain dependent. The in vitro approaches employed here should be useful in further characterization of the effects of lactobacilli on the gut and systemic immune systems.