Sialylated intravenous immunoglobulin suppress anti-ganglioside antibody mediated nerve injury.

The precise mechanisms underlying the efficacy of intravenous immunoglobulin (IVIg) in autoimmune neurological disorders including Guillain-Barré syndrome (GBS) are not known. Anti-ganglioside antibodies have been reported to be pathogenic in some variants of GBS, and we have developed passive transfer animal models to study anti-ganglioside antibody mediated-endoneurial inflammation and associated neuropathological effects and to evaluate the efficacy of new therapeutic approaches. Some studies indicate that IVIg's anti-inflammatory activity resides in a minor sialylated IVIg (sIVIg) fractions and is dependent on an innate Th2 response via binding to a specific ICAM3-grabbing nonintegrin related 1 receptor (SIGN-R1). Therefore the efficacy of IVIg, IVIg fractions with various IgG Fc sialylation status, and the involvement of Th2 pathway were examined in one of our animal model of antibody-mediated inhibition of axonal regeneration. We demonstrate that both IVIg and sIVIg ameliorated anti-glycan antibody mediated-pathological effect, whereas, the unsialylated fractions of IVIg were not beneficial in our model. Tenfold lower doses of sIVIg compared to whole IVIg provided equivalent efficacy in our studies. Moreover, we found that whole IVIg and sIVIg significantly upregulates the gene expression of IL-33, which itself can provide protection from antibody-mediated nerve injury in our model. Our results support that the SIGN-R1-Th2 pathway is involved in the anti-inflammatory effects of IVIg on endoneurium in our model and elements of this pathway including IL-33 can provide novel therapeutics in inflammatory neuropathies.

Fluorescently-tagged anti-ganglioside antibody selectively identifies peripheral nerve in living animals.

Selective in vivo delivery of cargo to peripheral nervous system (PNS) has broad clinical and preclinical applications. An important applicability of this approach is systemic delivery of fluorescently conjugated ligands that selectively label PNS, which could allow visualization of peripheral nerves during any surgery. We examine the use of an anti-ganglioside monoclonal antibody (mAb) as selective neuronal delivery vector for surgical imaging of peripheral nerves. Systemic delivery of an anti-ganglioside mAb was used for selective intraneuronal/axonal delivery of fluorescent agents to visualize nerves by surgical imaging in living mice. In this study, we show that intact motor, sensory, and autonomic nerve fibers/paths are distinctly labeled following a single nanomolar systemic injection of fluorescently labeled anti-ganglioside mAb. Tissue biodistribution studies with radiolabeled mAb were used to validate neuronal uptake of fluorescently labeled mAb. Implications of this proof of concept study are that fluorescent conjugates of anti-ganglioside mAbs are valuable delivery vectors to visualize nerves during surgery to avoid nerve injury and monitor nerve degeneration and regeneration after injury. These findings support that antibodies, and their derivatives/fragments, can be used as selective neuronal delivery vector for transport of various cargos to PNS in preclinical and clinical settings.

Amyloid ß-induced impairments in hippocampal synaptic plasticity are rescued by decreasing mitochondrial superoxide.

Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer's disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid ß peptide (Aß1-42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by diphenyliodonium (DPI) or by genetically deleting gp91(phox), the key enzymatic component of NADPH oxidase, had no effect on Aß-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aß-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aß-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aß-induced impairments in hippocampal synaptic plasticity.

Manganese-enhanced magnetic resonance imaging (MEMRI).

The use of manganese ions (Mn(2+)) as an MRI contrast agent was introduced over 20 years ago in studies of Mn(2+) toxicity in anesthetized rats (1). Manganese-enhanced MRI (MEMRI) evolved in the late nineties when Koretsky and associates pioneered the use of MEMRI for brain activity measurements (2) as well as neuronal tract tracing (3). Currently, MEMRI has three primary applications in biological systems: (1) contrast enhancement for anatomical detail, (2) activity-dependent assessment and (3) tracing of neuronal connections or tract tracing. MEMRI relies upon the following three main properties of Mn(2+): (1) it is a paramagnetic ion that shortens the spin lattice relaxation time constant (T(1)) of tissues, where it accumulates and hence functions as an excellent T(1) contrast agent; (2) it is a calcium (Ca(2+)) analog that can enter excitable cells, such as neurons and cardiac cells via voltage-gated Ca(2+) channels; and (3) once in the cells Mn(2+) can be transported along axons by microtubule-dependent axonal transport and can also cross synapses trans-synaptically to neighboring neurons. This chapter will emphasize the methodological approaches towards the use of MEMRI in biological systems.

Reactive oxygen species in the regulation of synaptic plasticity and memory.

The brain is a metabolically active organ exhibiting high oxygen consumption and robust production of reactive oxygen species (ROS). The large amounts of ROS are kept in check by an elaborate network of antioxidants, which sometimes fail and lead to neuronal oxidative stress. Thus, ROS are typically categorized as neurotoxic molecules and typically exert their detrimental effects via oxidation of essential macromolecules such as enzymes and cytoskeletal proteins. Most importantly, excessive ROS are associated with decreased performance in cognitive function. However, at physiological concentrations, ROS are involved in functional changes necessary for synaptic plasticity and hence, for normal cognitive function. The fine line of role reversal of ROS from good molecules to bad molecules is far from being fully understood. This review focuses on identifying the multiple sources of ROS in the mammalian nervous system and on presenting evidence for the critical and essential role of ROS in synaptic plasticity and memory. The review also shows that the inability to restrain either age- or pathology-related increases in ROS levels leads to opposite, detrimental effects that are involved in impairments in synaptic plasticity and memory function.

Neuronal and vascular oxidative stress in Alzheimer's disease.

The brain is a highly metabolically active organ producing large amounts of reactive oxygen species (ROS). These ROS are kept in check by an elaborate network of antioxidants. Although ROS are necessary for signaling and synaptic plasticity, their uncontrolled levels cause oxidation of essential macromolecules such as membrane lipids, nucleic acids, enzymes and cytoskeletal proteins. Indeed, overproduction of ROS and/or failure of the antioxidant network lead to neuronal oxidative stress, a condition associated with not only aging but also Alzheimer's disease (AD). However, the specific source of excessive ROS production has not yet been identified. On one hand, amyloid beta (Aß) has been extensively shown to act as an oxidant molecule. On the other hand, oxidative stress has been shown to precede and exacerbate Aß pathology. This review will address the involvement of oxidative stress in the context of neuronal as well as vascular dysfunction associated with AD.

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice.

BACKGROUND: While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits. RESEARCH DESIGN AND METHODS: T(1)-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively. RESULTS: STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged. CONCLUSION: Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.

Mitochondrial superoxide contributes to blood flow and axonal transport deficits in the Tg2576 mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by the progressive decline in cognitive functions and the deposition of aggregated amyloid beta (Abeta) into senile plaques and the protein tau into tangles. In addition, a general state of oxidation has long been known to be a major hallmark of the disease. What is not known however, are the mechanisms by which oxidative stress contributes to the pathology of AD. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, we used a mouse model of AD and genetically boosted its ability to quench free radicals of specific mitochondrial origin. We found that such manipulation conferred to the AD mice protection against vascular as well as neuronal deficits that typically affect them. We also found that the vascular deficits are improved via antioxidant modulation of the endothelial nitric oxide synthase, an enzyme primarily responsible for the production of nitric oxide, while neuronal deficits are improved via modulation of the phosphorylation status of the protein tau, which is a neuronal cytoskeletal stabilizer. CONCLUSIONS/SIGNIFICANCE: These findings directly link free radicals of specific mitochondrial origin to AD-associated vascular and neuronal pathology.

In vitro and in vivo magnetic resonance imaging (MRI) detection of GFP through magnetization transfer contrast (MTC).

Green fluorescent protein (GFP) is a widely utilized molecular marker of gene expression. However, its use in in vivo imaging has been restricted to transparent tissue mainly due to the tissue penetrance limitation of optical imaging. Here, we report a novel approach to detect GFP with Magnetization transfer contrast (MTC) magnetic resonance imaging (MRI). MTC is an MRI methodology currently utilized to detect macromolecule changes such as decrease in myelin and increase in collagen content. We employed MTC MRI imaging to detect GFP both in vitro and in in vivo mouse models. We demonstrated that our approach produces values that are protein specific, and concentration dependent. This approach provides a flexible, non-invasive in vivo molecular MRI imaging strategy that is dependent upon the presence and concentration of the GFP reporter.

Overexpression of SOD-2 reduces hippocampal superoxide and prevents memory deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by impaired cognitive function and the deposition of extracellular amyloid plaques and intracellular tangles. Although the proximal cause of AD is not well understood, it is clear that amyloid-beta (Abeta) plays a critical role in AD pathology. Recent studies also implicate mitochondrial abnormalities in AD. We investigated this idea by crossing mice that overexpress mitochondrial superoxide dismutase (SOD-2) with the Tg2576 mouse model of AD that overexpresses the human amyloid precursor protein carrying the Swedish mutation (K670N:M671L). We found that overexpression of SOD-2 decreased hippocampal superoxide, prevented AD-related learning and memory deficits, and reduced Abeta plaques. Interestingly, SOD-2 overexpression did not affect the absolute levels of Abeta(1-40) and Abeta(1-42), but did significantly reduce the Abeta(1-42) to Abeta(1-40) ratio, thereby shifting the balance toward a less amyloidogenic Abeta composition. These findings directly link mitochondrial superoxide to AD pathology and demonstrate the beneficial effects of a mitochondrial anti-oxidant enzyme, hence offering significant therapeutic implications for AD.

Inhibition of transcription factor activity by nuclear compartment-associated Bcl-2.

Using a reporter gene assay in PC12, HEK293, HeLa, and NIH-3T3 cells, we show that the anti-apoptotic protein Bcl-2 significantly inhibits transcriptional activation of various transcription factors, including NF kappa B, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (Delta BH4) also inhibited transcription, whereas a Bcl-2 mutant lacking its transmembrane domain (Delta TM) was ineffective. Furthermore, Bcl-2 chimeric proteins containing transmembrane domains from the mitochondrial protein monoamine oxidase B (MaoB) or the endoplasmic reticulum protein cytochrome b(5) showed no effect on transcription factor activity. Subcellular localization studies showed that under conditions of transient transfection, the active Bcl-2 forms (wild type and Delta BH4) were predominantly found in the nuclear fraction, whereas the non-active forms (Delta TM, MaoB, and cytochrome b(5)) were in the non-nuclear fraction. Additionally, stably expressed Bcl-2 loses its ability to inhibit transcriptional activation and localizes predominantly to the non-nuclear fraction. Expression of FKBP38 (a chaperone that shuttles Bcl-2 to the mitochondria) removes co-expressed Bcl-2 from the nuclear fraction and reverses its effect on transcription factor activity. Finally, using an inducible gene expression system, we show that nuclear compartment-associated Bcl-2 prevents entry of NF kappa B subunits to the nucleus without affecting NF kappa B release from its cytosolic inhibitory sub-unit I kappa B alpha. These results suggest that (a) Bcl-2 suppresses transcriptional activity of multiple transcription factors; (b) Bcl-2 does not interfere with NF kappa B activation but prevents entrance of its active subunits to the nucleus; (c) membrane anchoring is required for this function of Bcl-2; and (d) association of Bcl-2 with the nuclear compartment is also necessary. We speculate that nuclear compartment-associated Bcl-2 may affect nuclear trafficking of multiple factors necessary for transcriptional activity.

Bcl-2 phosphorylation is not required for its effects on NFkappaB activity.

We have previously shown that in the aged CNS there is a free radical-dependent upregulation of Bcl-2 associated with perturbations of NFkappaB function. Both Bcl-2 and NFkappaB are key players in the cellular machinery devoted to cope with stress and regulate neuronal apoptosis. To study whether effects on NFkappaB are part of the Bcl-2 anti-apoptotic mechanism, we examined the effect of Bcl-2 on NFkappaB transcriptional activity in PC12 cells and determined the role thereby of Bcl-2 phosphorylation (required for the anti-apoptotic function of Bcl-2). Our results indicate that over-expression of Bcl-2 significantly decreases NFkappaB-promoted transcription and that this effect is independent of Bcl-2 phosphorylation. We conclude that such Bcl-2 effect on NFkappaB activity is distinct from its anti-apoptotic action.

Nociceptive behaviour induced by dental application of irritants to rat incisors: a new model for tooth inflammatory pain.

Animal models simulating acute human pulpitis are still lacking. The rat incisors present a particular situation where most of their innervation is considered to be unmyelinated and concentrated mainly in the tooth pulp. This study reports on a new model for dental pain induced by inflammatory agents applied to the tooth pulps of incisors. In different groups of rats, artificial crowns were fixed on the lower incisors, after cutting 1-2mm of their distal extremities. A volume of 7-10 microl of solutions of saline, capsaicin (1-10mg/ml) or formalin (2.5% or 5%) was injected in the crown cavity, and the nociceptive behaviour was quantitated following a devised scoring method of four scales. Intradental application of capsaicin produced nociceptive scores in the form of one plateau for 1-2h depending on the concentration used. Similar results were obtained with intradental application of formalin 2.5%. The one plateau of nociceptive scores obtained with formalin contrasts with the biphasic aspect of nociceptive behaviour described with the intradermal formalin test. This discrepancy could be attributed to a difference in the types of afferent fibres involved in each situation. Pretreatment with morphine (2 mg/kg) attenuated, in a naloxone-reversible manner, the nociceptive behaviour observed following intradental application of capsaicin. Pretreatment with meloxicam (a cyclo-oxygenase-2 inhibitor) exerted a less pronounced attenuation of the nociceptive scores when compared with morphine. These results provide evidence for the validity of the described model for the simulation of tooth pulp inflammatory pain in awake animals.