Surfing on Membrane Waves: Microvilli, Curved Membranes, and Immune Signaling.

Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.

Molecular basis of immunogenicity to botulinum neurotoxins and uses of the defined antigenic regions.

Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design.

Reduction of established antibody responses against botulinum neurotoxin A by synthetic monomethoxypolyethylene glycol peptide conjugates.

In cervical dystonia, injection of botulinum neurotoxin (BoNT) A or B into affected neck muscle reduces symptoms but may elicit anti-toxin antibodies (Abs) that block responsiveness to treatment. Previously, we localized the BoNT/A and BoNT/B sites that bind mouse or human blocking Abs. We also reported that site-specific auto-Abs can be suppressed by a monomethoxypolyethylene glycol (mPEG)-epitope conjugate. So we elicited here anti-toxin Abs in outbred mice by immunization with sublethal-suboptimal doses of active BoNT/A and determined the efficacy of selected mPEG-epitopes in reducing established anti-BoNT/A Abs. We tested in outbred mice four synthetic mPEG-N(α)-epitopes [N8 (residues 547-565), N25 (785-803), C15 (1051-1069), C31 (1275-1296)] of BoNT/A in tolerance against ongoing anti-toxin Abs. After short immunizations, tolerization with an mPEG-peptide reduced Abs to correlate peptide and caused varying Ab reductions to the other 3 peptides. Anti-N8 Abs were unaffected by mPEG-N25 tolerization, but mPEG-N8 and mPEG-N25 caused drop in anti-BoNT/A Abs. After long immunization with BoNT/A, tolerization with mPEG-N8 lessened anti-N8 Abs. Anti-C15 Abs decreased by tolerization with mPEG-C15 or any other mPEG-peptide. Anti-N25 Abs were not altered by mPEG-N25, but decreased after tolerization with mPEG-C15. Anti-C31 Abs disappeared on day 474 by tolerization with mPEG-C31 or mPEG-N8, mPEG-N25 or mPEG-C15. When an Ab response returns, a decrease can be re-established by re-administering the correlate mPEG-peptide. The method may be beneficial for extending BoNT treatment in immunoresistant patients.

Single and binge methamphetamine administrations have different effects on the levels of dopamine D2 autoreceptor and dopamine transporter in rat striatum.

Methamphetamine (METH) is a central nervous system psychostimulant with a high potential for abuse. At high doses, METH causes a selective degeneration of dopaminergic terminals in the striatum. Dopamine D2 receptor antagonists and dopamine transporter (DAT) inhibitors protect against neurotoxicity of the drug by decreasing intracellular dopamine content and, consequently, dopamine autoxidation and production of reactive oxygen species. In vitro, amphetamines regulate D2 receptor and DAT functions via regulation of their intracellular trafficking. No data exists on axonal transport of both proteins and there is limited data on their interactions in vivo. The aim of the present investigation was to examine synaptosomal levels of presynaptic D2 autoreceptor and DAT after two different regimens of METH and to determine whether METH affects the D2 autoreceptor-DAT interaction in the rat striatum. We found that, as compared to saline controls, administration of single high-dose METH decreased D2 autoreceptor immunoreactivity and increased DAT immunoreactivity in rat striatal synaptosomes whereas binge high-dose METH increased immunoreactivity of D2 autoreceptor and had no effect on DAT immunoreactivity. Single METH had no effect on D2 autoreceptor-DAT interaction whereas binge METH increased the interaction between the two proteins in the striatum. Our results suggest that METH can affect axonal transport of both the D2 autoreceptor and DAT in an interaction-dependent and -independent manner.

Inhibitory role of diazepam on autoimmune inflammation in rats with experimental autoimmune encephalomyelitis.

Glutamate and GABA are the main excitatory and inhibitory neurotransmitters in the CNS, and both may be involved in the neuronal dysfunction in neurodegenerative conditions. We have recently found that glutamate release was decreased in isolated synaptosomes from the rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. In contrast to control animals where GABA induced a decrease in the evoked glutamate release, which was abolished by picrotoxin (a GABA(A) antagonist), synaptosomes from EAE rats showed a loss in the inhibition of the glutamate release mediated by GABA with a concomitant diminution of the flunitrazepam-sensitive GABA(A) receptor density. We have presently further evaluated the relevance of the GABAergic system in EAE by treating rats challenged for the disease with the GABA agonist diazepam. Administration of diazepam during 6 days starting at day 6 or 11 after EAE active induction led to a marked decrease of the disease incidence and histological signs associated with the disease. Cellular reactivity and antibody responses against the encephalitogenic myelin basic protein were also diminished. Beyond the effects of diazepam on the autoimmune, inflammatory response, we report also a positive effect on neurotransmission. Treatment with diazepam inhibited the previously described reduction in glutamate release in the frontal cortex synaptosomes from EAE animals. These data suggest that an endogenous inhibitory GABAergic system within the immune system is involved in the diazepam effect on EAE and indicate that increasing GABAergic activity potently ameliorates EAE.

Immune recognition of BoNTs A and B: how anti-toxin antibodies that bind to the heavy chain obstruct toxin action.

We localized the BoNT regions that bind blocking Abs from 28 BoNT/A- and 30 BoNT/B-treated dystonia patients who became unresponsive to, and whose sera protected mice against LD100 of, the correlate BoNT. We analyzed Ab binding to BoNT/A- and BoNT/B-peptide panels, each of which consisted of 60, 19-residue peptides that overlapped consecutively by 5 residues and covered the entire H chain of the correlate toxin. Abs bound to a limited set of peptides but levels varied with patient, consistent with responses to each epitope being under separate MHC control. BoNT/B-treated patients had higher anti-toxin Ab levels and bound more H regions (at least 11) than BoNT/A-treated patients (5 regions). The epitopes were on surface areas that did not correlate with surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. Some epitopes within the two toxins display substantial homology and occupy equivalent 3-D locations, occasionally showing a small shift relative to one another, consistent with recognition adjustments accommodating structural differences between the two BoNTs. Blocking Abs bound to BoNT/A at sites that coincided or overlapped with those involved in synaptosome-binding, thus preventing its binding and blocking its entry into the neuron. On BoNT/B, Ab-binding regions overlapped with the sites that bind to mouse and rat synaptotagmin II or to ganglioside, thereby explaining Ab blocking of BoNT/B action.

Inhibition by human sera of botulinum neurotoxin-A binding to synaptosomes: a new assay for blocking and non-blocking antibodies.

The mouse protection assay (MPA), which is an in vivo assay, is currently the most widely used method for monitoring blocking antibodies (Abs) in botulinum neurotoxin (BoNT)-treated patients. In recent studies we found that a number of the regions on the heavy (H) subunit of BoNT/A that bind blocking mouse Abs coincided, or overlapped, with the regions that bind to mouse synaptosomes (snps). This suggested that blocking anti-BoNT/A Abs would be expected to inhibit BoNT/A binding to snps. In the present work, we analyzed sera from 58 cervical dystonia (CD) patients who had been treated with BOTOX (a preparation of BoNT/A serotype) for blocking Abs by MPA and by their abilities to inhibit in vitro the binding of 125I-labeled active BoNT/A or inactive toxin (toxoid) to mouse brain snps. With active 125I-labeled BoNT/A-snps binding, the MPA-positive sera (n = 30) displayed inhibition levels that were distinctly higher (mean = 21.1 +/- 5.8) than those obtained with MPA-negative sera (n = 28) (mean = -1.3 +/- 3.9; p < 0.0001) or control sera (n = 19) (mean = -3.4 +/- 2.8; p < 0.0001). Similarly, inhibition levels by MPA-positive sera of 125I-labeled toxoid snp-binding (mean = 48.6 +/- 8.7) were distinctly higher than inhibition by MPA-negative sera (mean=10.0+/-7.6; p < 0.0001) or control sera (mean = 1.8 +/- 6.9; p < 0.0001). Thus, using labeled active toxin or toxoid, the inhibition assay correlated very well with the MPA. The inhibitory activity of the non-protective sera generally correlated with the duration of survival after toxin challenge (correlation coefficients of inhibition: active toxin = 0.445; p = 0.0167; inactive toxoid = 0.774; p < 0.0001). It is concluded that the snp-inhibition assay reported here is reliable, reproducible and correlates very well with the MPA. It requires much less serum (0.75% of the amount needed for the MPA) and is considerably less costly than the MPA. With either 125I-labeled active toxin or toxoid, it is possible to distinguish CD sera that have blocking Abs from those that lack such Abs. Since the results with the toxoid were as discriminating as those of the active toxin, it would not even be necessary to use active toxin in these assays.

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis.

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.

Neuronal antigens modulate the immune response associated with experimental autoimmune encephalomyelitis.

Rats primed with bovine myelin (BM) in complete Freunds adjuvant, develop acute experimental autoimmune encephalomyelitis (EAE). We have previously described that intraperitoneal administration prior to the active induction of the disease of a bovine synaptosomal fraction (BSF) and BM were effective ways of suppressing EAE. We found that both treatments diminish the incidence of the disease and reduced biochemical and histological alterations of the central nervous system (CNS). To characterize this suppression process, in this study we examined the antigen-specific immune response in animals protected from EAE. Lymph node mononuclear cells derived from sick EAE rats, as well as from those protected by BM and BSF, showed strong myelin basic protein (MBP) proliferation. Analysis of the humoral response against MBP showed a significant diminution of IgG2b anti-MBP titres in protected BM and BSF rats in contrast to sick EAE rats whose condition could be related to a diminished anti-MBP Th1 response. Finally, cells from rats protected by BSF and BM reduced the incidence of EAE when they were adoptively transferred into animals prior to active induction of the disease. These results suggest that a mechanism based on the generation of regulatory cells and immune deviation could account for the EAE suppression mediated by myelin as well as synaptosomal antigens.

Anti-basal ganglia antibody abnormalities in Sydenham chorea.

Anti-basal ganglia antibodies (ABGA) were measured in nine children with Sydenham chorea (SC) and compared to nine controls. Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) methods were used to detect ABGA against supernatant (S1), pellet, and synaptosomal preparations from adult and pediatric postmortem caudate, putamen, and globus pallidus. ELISA optical density (OD) values were higher in SC patients than controls across all preparations, but did not reach a level of significance. Although WB identified multiple bands in all subjects, discriminant analysis showed that the mean binding patterns of SC patients were significantly different from control, most notably in the caudate S1 fraction (Wilks' lambda=0.011, p<0.0001). Numerous antigens contributed to differences between groups; the two most defining molecular masses were at 126 and 113 kDa. In contrast to WB with discriminant analysis, ELISA measurements did not significantly differentiate between the SC group and controls.

Interleukin-1beta-dependent changes in the hippocampus following parenteral immunization with a whole cell pertussis vaccine.

Neurological side effects are a major cause of concern following immunization with a number of vaccines, especially the whole cell pertussis vaccine (Pw). In this study we report that IL-1beta concentrations were significantly increased in the hippocampus following subcutaneous (s.c.) injection of Pw, and that this was accompanied by increased activity of the stress-activated kinase, c-Jun-N-terminal kinase (JNK) and a decrease in glutamate release. These effects were mimicked by s.c injection of active pertussis toxin (PT) or lipopolysaccharide (LPS). Incubation of hippocampal synaptosomes in the presence of Pw, PT or LPS also resulted in increased JNK activation and decreased glutamate release, effects which were mimicked by IL-1beta and blocked by the IL-1 receptor antagonist (IL-ra). Our observations are consistent with the hypothesis that IL-1beta induced by active bacterial toxins present in vaccine preparations, mediate the neurochemical and perhaps the neurological effects of Pw.

Mice overexpressing Bcl-2 in their neurons are resistant to myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE).

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by destruction of myelin. Recent studies have indicated that axonal damage is involved in the pathogenesis of the progressive disability of this disease. To study the role of axonal damage in the pathogenesis of MS-like disease induced by myelin oligodendrocyte glycoprotein (MOG), we compared experimental autoimmune encephalomyelitis (EAE) in wild-type (WT) and transgenic mice expressing the human bcl-2 gene exclusively in neurons under the control of the neuron-specific enolase (NSE) promoter. Our study shows that, following EAE induction with pMOG 35-55, the WT mice developed significant clinical manifestations with complete hind-limb paralysis. In contrast, most of the NSE-bcl-2 mice (16/27) were completely resistant, whereas the others showed only mild clinical signs. Histological examination of CNS tissue sections showed multifocal areas of perivascular lymphohistiocytic inflammation with loss of myelin and axons in the WT mice, whereas only focal inflammation and minimal axonal damage were demonstrated in NSE-bcl-2 mice. No difference could be detected in the immune potency as indicated by delayed-type hypersensitivity (DTH) and T-cell proliferative responses to MOG. We also demonstrated that purified synaptosomes from the NSE-bcl-2 mice produce significantly lower level of reactive oxygen species (ROS) following exposure to H2O2 and nitric oxide (NO) than WT mice. In conclusion, we demonstrated that the expression of the antiapoptotic gene, bcl-2, reduces axonal damage and attenuates the severity of MOG-induced EAE. Our results emphasize the importance of developing neuroprotective therapies, in addition to immune-specific approaches, for treatment of MS.

Glycerol-induced seizure: involvement of IL-1beta and glutamate.

It is widely accepted that interleukin-1beta (IL-1beta), a cytokine produced not only by cells of the immune system but also by cells of the central nervous system, modulates hippocampal function. Here we investigate the effect of a seizure-induced increase in hippocampal IL-1beta concentration on neurotransmitter release. We report that oral administration of glycerol evoked seizure activity in BALB/c mice. Associated with these convulsions was an induction of IL-1beta gene expression and a significant increase in cytokine protein in the hippocampus. Release of glutamate in synaptosomes prepared from hippocampi of these animals was reduced compared to control. These results are consistent with previous data suggesting a modulatory effect of IL-1beta on glutamate release in hippocampus.

Antiphospholipid antibodies permeabilize and depolarize brain synaptoneurosomes.

Antiphospholipid antibodies (aPL) are associated with neurological diseases such as stroke, migraine, epilepsy and dementia and are thus associated with both vascular and non-vascular neurological disease. We have therefore examined the possibility that these antibodies interact directly with neuronal tissue by studying the electrophysiological effects of aPL on a brain synaptosoneurosome preparation. IgG from patients with high levels of aPL and neurological involvement was purified by protein-G affinity chromatography as was control IgG pooled from ten sera with low levels of aPL. Synaptoneurosomes were purified from perfused rat brain stem. IgG from the patient with the highest level of aPL at a concentration equivalent to 1:5 serum dilution caused significant depolarization of the synaptoneurosomes as determined by accumulation of the lipophylic cation [3H]-tetraphenylphosphonium. IgG from this patient as well as IgG from two elderly patients with high levels of aPL were subsequently shown to permeabilize the synaptosomes to labeled nicotinamide adenine dinucleotide (NAD) and pertussis toxin-ADP-ribose transferase (PTX-A protein) as assayed by labeled ADP-ribosylation of G-proteins in the membranes. No such effects were seen with the control IgG. aPL may thus have the potential to disrupt neuronal function by direct action on nerve terminals. These results may explain some of the non-thromboembolic CNS manifestations of the antiphospholipid syndrome.

Suppression of acute experimental allergic encephalomyelitis by intraperitoneal administration of synaptosomal antigens.

The effect of a synaptosomal fraction isolated from bovine brain was examined on acute experimental allergic encephalomyelitis (EAE) in Wistar rats. Intraperitoneal administration of the animals with low doses of saline-soluble synaptosomal antigens 10 and 3 days previous to the active induction of the disease was an effective way of suppressing EAE. This treatment diminished the incidence and severity of EAE, reverted the appearance of central nervous system histological and biochemical alterations, and produced changes in the autoimmune humoral response against the encephalitogenic myelin basic protein. The phenomenon observed by treatment with synaptosomal fraction is similar to the previously described suppression mediated by myelin antigens. Taking into account that affinity-purified antibodies and T lymphocytes specific for myelin basic protein can also recognize several neuronal proteins, among them the specific synaptosomal protein synapsin I, can be suggested that antigen-driven bystander suppression could be a mechanism by which synaptosomal proteins suppress the response against myelin antigens.

Monoclonal antibodies neutralizing the toxin II from Androctonus australis hector scorpion venom: usefulness of a synthetic, non-toxic analog.

Scorpion venom contains toxins that act on ion channels. Some are responsible for the noxious effects observed when people are stung by scorpions. The study of the neutralization of these molecules and the production of monoclonal antibodies (mAbs) should prove valuable. Toxin II from Androctonus australis hector scorpion (AahII) is one of the most potent toxins and has been well-characterized and studied. Producing mAbs against such molecules is often difficult due to their toxicity. We used a synthetic, non-toxic analog, (Abu)8-AahII, to obtain mAbs which recognize and neutralize the native toxin AahII. Sets of peptides spanning the entire sequence of AahII were assayed to identify the binding sites of the mAbs. The various mAbs recognized only the largest peptides (12-17 residues). They recognized peptides corresponding to different parts of the AahII sequence, suggesting that several regions of the (Abu)8-AahII sequence mimic AahII epitopes and then elicit mAbs directed against toxin.

[Anti-idiotypic monoclonal antibodies against latrotoxin interact with rat brain synaptosomes and modify latrotoxin-induced calcium entry into synaptosomes]. / Antiidiotipicheskie monokonal'nye antitela protiv latrotoksina vzaimdeistvuiut s sinaptosomami iz mozga krysy i modifitsiruiut latrotoksinindutsirovannyi vkhod kal'tsiia v sinaptosomy.

Hybridoma lines producing anti-idiotypic monoclonal antibodies (AImAbs) were prepared by fusing splenocytes of mice immunized with alpha-latrotoxin (LT) and P3-X63Ag8.653 myeloma line cells. AImAbs (1) bind to the rat brain synaptosomes, (2) do not affect the LT binding to the high-affinity receptor, and (3) do not react with LT in solution. The effect of AImAbs on the LT-induced 45Ca2+ influx into rat brain synaptosomes was studied. Some antibodies (6.6D11 and 11.7B7) were found to strongly inhibit this process. The result obtained indicate that the presynaptic membrane contains unidentified components interacting with LT. The distortion of the interaction of LT with these unknown components affects the LT-induced calcium influx into synaptosomes.

Release of somatostatin-like immunoreactivity from enriched enteric nerve varicosities of rat ileum.

Synaptosomes were isolated from rat ileum by various steps of differential centrifugation. The peptide content for somatostatin-like immunoreactivity was used as marker for neuronal membranes. The enriched synaptosomal fraction (P2) showed a good enrichment of somatostatin content (4-fold) in comparison to the post-nuclear supernatant. The basal release of somatostatin-like immunoreactivity was 26 +/- 3 pg/mg tissue protein. KCl-evoked depolarization (65 mM) caused a significant increase of somatostatin-like immunoreactivity release (72 +/- 11 pg/mg, n = 12, P < 0.001) compared to basal release. In Ca(2+)-free medium the evoked release of somatostatin-like immunoreactivity was abolished. A substantial increase of somatostatin-like immunoreactivity release (52 +/- 7 pg/mg, n = 12, P < 0.05) was also observed in the presence of the Ca2+ ionophore A-23187. The cholinergic agonist carbachol elicited a dose-dependent release of somatostatin-like immunoreactivity (10(-7) M: 54 +/- 8 pg/mg, 10(-6) M: 63 +/- 6 pg/mg, 10(-5) M: 53 +/- 5 pg/mg, n = 12, P < 0.001), which was blocked by atropine (10(-6) M: 35 +/- 6 pg/mg, n = 12, P < 0.001), but not by hexamethonium. Other presynaptic modulating substances such as serotonin, the selective neurokinin-B agonist [beta Asp4,MePhe7]neurokinin B-(4-10), neurotensin, cholecystokinin-8, caerulein and pentagastrin had no stimulatory effect on release of somatostatin-like immunoreactivity. In summary, somatostatin-like immunoreactivity can be released from enteric synaptosomes by both depolarization with KCl and cholinergic stimulation via a muscarinic mechanism. The synaptosomes of intrinsic nerves offer an approach to study release of neuronal somatostatin on the subcellular level.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.