Motoneuronal inflammasome activation triggers excessive neuroinflammation and impedes regeneration after sciatic nerve injury.

BACKGROUND: Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1ß. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury. METHODS: After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing. RESULTS: In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4-L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1ß. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels. CONCLUSIONS: Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Stress Resilience is Associated with Hippocampal Synaptoprotection in the Female Rat Learned Helplessness Paradigm.

The synaptogenic hypothesis of major depressive disorder implies that preventing the onset of depressive-like behavior also prevents the loss of hippocampal spine synapses. By applying the psychoactive drugs, diazepam and fluoxetine, we investigated whether blocking the development of helpless behavior by promoting stress resilience in the rat learned helplessness paradigm is associated with a synaptoprotective action in the hippocampus. Adult ovariectomized and intact female Sprague-Dawley rats (n = 297) were treated with either diazepam, fluoxetine, or vehicle, exposed to inescapable footshocks or sham stress, and tested in an active escape task to assess helpless behavior. Escape-evoked corticosterone secretion, as well as remodeling of hippocampal spine synapses at a timepoint representing the onset of escape testing were also analyzed. In ovariectomized females, treatment with diazepam prior to stress exposure prevented helpless behavior, blocked the loss of hippocampal spine synapses, and muted the corticosterone surge evoked by escape testing. Although fluoxetine stimulated escape performance and hippocampal synaptogenesis under non-stressed conditions, almost all responses to fluoxetine were abolished following exposure to inescapable stress. Only a much higher dose of fluoxetine was capable of partly reproducing the strong protective actions of diazepam. Importantly, these protective actions were retained in the presence of ovarian hormones. Our findings indicate that stress resilience is associated with the preservation of spine synapses in the hippocampus, raising the possibility that, besides synaptogenesis, hippocampal synaptoprotection is also implicated in antidepressant therapy.

Upregulation of Nucleotide-Binding Oligomerization Domain-, LRR- and Pyrin Domain-Containing Protein 3 in Motoneurons Following Peripheral Nerve Injury in Mice.

Neuronal injuries are accompanied by release and accumulation of damage-associated molecules, which in turn may contribute to activation of the immune system. Since a wide range of danger signals (including endogenous ones) are detected by the nucleotide-binding oligomerization domain-, LRR- and pyrin domain-containing protein 3 (NLRP3) pattern recognition receptor, we hypothesized that NLRP3 may become activated in response to motor neuron injury. Here we show that peripheral injury of the oculomotor and the hypoglossal nerves results in upregulation of NLRP3 in corresponding motor nuclei in the brainstem of mice. Although basal expression of NLRP3 was observed in microglia, astroglia and neurons as well, its upregulation and co-localization with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, suggesting inflammasome activation, was only detected in neurons. Consequently, increased production of active pro-inflammatory cytokines interleukin-1ß and interleukin-18 were detected after hypoglossal nerve axotomy. Injury-sensitive hypoglossal neurons responded with a more pronounced NLRP3 upregulation than injury-resistant motor neurons of the oculomotor nucleus. We further demonstrated that the mitochondrial protector diazoxide was able to reduce NLRP3 upregulation in a post-operative treatment paradigm. Our results indicate that NLRP3 is activated in motoneurons following acute nerve injury. Blockade of NLRP3 activation might contribute to the previously observed anti-inflammatory and neuroprotective effects of diazoxide.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Diazoxide blocks or reduces microgliosis when applied prior or subsequent to motor neuron injury in mice.

Diazoxide (DZX), an anti-hypertonic and anti-hypoglycemic drug, was shown to have anti-inflammatory effects in several injured cell types outside the central nervous system. In the brain, the neuroprotective potential of DZX is well described, however, its anticipated anti-inflammatory effect after acute injury has not been systematically analyzed. To disclose the anti-inflammatory effect of DZX in the central nervous system, an injury was induced in the hypoglossal and facial nuclei and in the oculomotor nucleus by unilateral axonal transection and unilateral target deprivation (enucleation), respectively. On the fourth day after surgery, microglial analysis was performed on tissue in which microglia were DAB-labeled and motoneurons were labeled with immunofluorescence. DZX treatment was given either prophylactically, starting 7 days prior to the injury and continuing until the animals were sacrificed, or postoperatively only, with daily intraperitoneal injections (1.25 mg/kg; in 10 mg/ml dimethyl sulfoxide in distilled water). Prophylactically + postoperatively applied DZX completely eliminated the microglial reaction in each motor nuclei. If DZX was applied only postoperatively, some microglial activation could be detected, but its magnitude was still significantly smaller than the non-DZX-treated controls. The effect of DZX could also be demonstrated through an extended period, as tested in the hypoglossal nucleus on day 7 after the operation. Neuronal counts, determined at day 4 after the operation in the hypoglossal nucleus, demonstrated no loss of motor neurons, however, an increased Feret's diameter of mitochondria could be measured, suggesting increased oxidative stress in the injured cells. The increase of mitochondrial Feret's diameter could also be prevented with DZX treatment.

ApoE-Targeting Increases the Transfer of Solid Lipid Nanoparticles with Donepezil Cargo across a Culture Model of the Blood-Brain Barrier.

Pharmacological treatment of central nervous system (CNS) disorders is difficult, because the blood-brain barrier (BBB) restricts the penetration of many drugs into the brain. To solve this unmet therapeutic need, nanosized drug carriers are the focus of research efforts to develop drug delivery systems for the CNS. For the successful delivery of nanoparticles (NPs) to the brain, targeting ligands on their surface is necessary. Our research aim was to design a nanoscale drug delivery system for a more efficient transfer of donepezil, an anticholinergic drug in the therapy of Alzheimer's disease across the BBB. Rhodamine B-labeled solid lipid nanoparticles with donepezil cargo were prepared and targeted with apolipoprotein E (ApoE), a ligand of BBB receptors. Nanoparticles were characterized by measurement of size, polydispersity index, zeta potential, thermal analysis, Fourier-transform infrared spectroscopy, in vitro release, and stability. Cytotoxicity of nanoparticles were investigated by metabolic assay and impedance-based cell analysis. ApoE-targeting increased the uptake of lipid nanoparticles in cultured brain endothelial cells and neurons. Furthermore, the permeability of ApoE-targeted nanoparticles across a co-culture model of the BBB was also elevated. Our data indicate that ApoE, which binds BBB receptors, can potentially be exploited for successful CNS targeting of solid lipid nanoparticles.

Experimental Motor Neuron Disease Induced in Mice with Long-Term Repeated Intraperitoneal Injections of Serum from ALS Patients.

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice.

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier.

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.

Examination of the Role of Mitochondrial Morphology and Function in the Cardioprotective Effect of Sodium Nitrite Administered 24 h Before Ischemia/Reperfusion Injury.

Background: We have previous evidence that in anesthetized dogs the inorganic sodium nitrite protects against the severe ventricular arrhythmias, resulting from coronary artery occlusion and reperfusion, when administered 24 h before. The present study aimed to examine, whether in this effect changes in mitochondrial morphology and function would play a role. Methods: Thirty dogs were infused intravenously either with saline (n = 15) or sodium nitrite (0.2 µmol/kg/min; n = 15) for 20 min, and 24 h later, 10 dogs from each group were subjected to a 25 min period of occlusion and then reperfusion of the left anterior descending coronary artery. The severity of ischaemia and ventricular arrhythmias were examined in situ. Left ventricular tissue samples were collected either before the occlusion (5 saline and 5 nitrite treated dogs) or, in dogs subjected to occlusion, 2 min after reperfusion. Changes in mitochondrial morphology, in complex I and complex II-dependent oxidative phosphorylation (OXPHOS), in ATP, superoxide, and peroxynitrite productions were determined. Results: The administration of sodium nitrite 24 h before ischemia/reperfusion significantly attenuated the severity of ischaemia, and markedly reduced the number and incidence of ventricular arrhythmias. Nitrite also attenuated the ischaemia and reperfusion (I/R)-induced structural alterations, such as reductions in mitochondrial area, perimeter, and Feret diameter, as well as the increase in mitochondrial roundness. The administration of nitrite, however, enhanced the I/R-induced reduction in the mitochondrial respiratory parameters; compared to the controls, 24 h after the infusion of nitrite, there were further significant decreases, e.g., in the complex I-dependent OXPHOS (by -20 vs. -53%), respiratory control ratio (by -14 vs. -61%) and in the P/E control coupling ratio (by 2 vs. -36%). Nitrite also significantly reduced the I/R-induced generation of superoxide, without substantially influencing the ATP production. Conclusions: The results suggest that sodium nitrite may have an effect on the mitochondria; it preserves the mitochondrial structure and modifies the mitochondrial function, when administered 24 h prior to I/R. We propose that nitrite affects primary the phosphorylation system (indicated by the decreased P/E ratio), and the reduction in superoxide production would result from the subsequent suppression of the ROS producing complexes; an effect which may certainly contribute to the antiarrhythmic effect of nitrite.

Presymptomatically applied AMPA receptor antagonist prevents calcium increase in vulnerable type of motor axon terminals of mice modeling amyotrophic lateral sclerosis.

Increased intracellular calcium (Ca), which might be the consequence of an excess influx through Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, plays a crucial role in degeneration of motor neurons. Previously we demonstrated that the presymptomatic application of AMPA receptor antagonist, talampanel, could reduce Ca elevation in spinal motor neurons of mice carrying the G93A mutation of superoxide dismutase 1 (SOD1), modeling amyotrophic lateral sclerosis (ALS). It remained to be examined whether the remote, functionally semi-autonomous motor axon terminals could be rescued from the Ca overload, or if the terminals, where the degeneration possibly starts, already experience intractable changes at early time points. Thus using electron microscopic techniques, we measured the Ca level of motor axon terminals in the interosseus muscle of the SOD1 mutant animals, which are prototypes of vulnerable nerve endings in ALS. In line with the results obtained in the perikarya, talampanel treatment could reduce Ca increase evoked by the presence of mutant SOD1 in the axon terminals if the treatment was started presymptomatically but not at an early symptomatic stage. We also tested the Ca level in the cell bodies and axon terminals of the oculomotor neurons, which are resistant to the disease. Neither Ca increase, nor talampanel effect could be demonstrated at either time point. This is consistent with the observations that oculomotor neurons contain increased level of Ca buffer, which could reduce excess Ca load, and they also express glutamate receptor subunit type 2, which renders AMPA receptors impermeable to Ca.

Calcium in the pathomechanism of amyotrophic lateral sclerosis - Taking center stage?

Amyotrophic lateral sclerosis is an incurable, relentlessly progressive disease primarily affecting motor neurons. The cause of the disease, except for the mutations identified in a small fraction of patients, is unknown. The major mechanisms contributing to the degeneration of motor neurons have already been disclosed and characterized, including excitotoxicity, oxidative stress, mitochondrial dysfunction, and immune/inflammatory processes. During the progression of the disease these toxic processes are not discrete, but each facilitates the deleterious effect of the other. However, due to their common reciprocal calcium dependence, calcium ions may act as a common denominator and through a positive feedback loop may combine the individual pathological processes into a unified escalating mechanism of neuronal destruction. This mini-review provides an overview of the mutual calcium dependence of the major toxic mechanisms associated with amyotrophic lateral sclerosis.

[Calcium ion is a common denominator in the pathophysiological processes of amyotrophic lateral sclerosis]. / Az amyotrophiás lateralsclerosis patofiziológiai tényezoinek központi kapcsolóeleme, a kalcium.

Amyotrophic lateral sclerosis (ALS), the most frequent motor neuron disease is characterized by progressive muscle weakness caused by the degeneration of the motor neurons in the spinal cord and motor cortex. However, according to the recent observations, ALS is a rather complex syndrome which frequently involves symptoms of cognitive impairment. Therefore, ALS cases can be interpreted in a clinico-pathological spectrum spanning from the classical ALS involving only the motor system to the fronto-temporal dementia. The progression of the disease, however, manifested in the degeneration of the upper and lower motor neurons, is based on the same complex pathobiology. The main elements of the pathomechanism, such as oxidative stress, excitotoxicity, immune/inflammatory processes and mitochondrial dysfunction are well described already, which operate in orchestrated way and amplify the deleterious effect of each other. It is assumed that calcium ions act as a catalyst in this interaction, hence each of the individual mechanisms has strong, positive and reciprocal calcium dependence thus may combine the individual pathological processes into a unified escalating mechanism of neuronal destruction. This review provides an overview of the role of calcium in connecting and amplifying the major mechanisms which lead to degeneration of the motor neurons in ALS.

Stress induces equivalent remodeling of hippocampal spine synapses in a simulated postpartum environment and in a female rat model of major depression.

Stress and withdrawal of female reproductive hormones are known risk factors of postpartum depression. Although both of these factors are capable of powerfully modulating neuronal plasticity, there is no direct electron microscopic evidence of hippocampal spine synapse remodeling in postpartum depression. To address this issue, hormonal conditions of pregnancy and postpartum period were simulated in ovariectomized adult female Sprague-Dawley rats (n=76). The number of hippocampal spine synapses and the depressive behavior of rats in an active escape task were investigated in untreated control, hormone-withdrawn 'postpartum', simulated proestrus, and hormone-treated 'postpartum' animals. After 'postpartum' withdrawal of gonadal steroids, inescapable stress caused a loss of hippocampal spine synapses, which was related to poor escape performance in hormone-withdrawn 'postpartum' females. These responses were equivalent with the changes observed in untreated controls that is an established animal model of major depression. Maintaining proestrus levels of ovarian hormones during 'postpartum' stress exposure did not affect synaptic and behavioral responses to inescapable stress in simulated proestrus animals. By contrast, maintaining pregnancy levels of estradiol and progesterone during 'postpartum' stress exposure completely prevented the stress-induced loss of hippocampal spine synapses, which was associated with improved escape performance in hormone-treated 'postpartum' females. This protective effect appears to be mediated by a muted stress response as measured by serum corticosterone concentrations. In line with our emerging 'synaptogenic hypothesis' of depression, the loss of hippocampal spine synapses may be a novel perspective both in the pathomechanism and in the clinical management of postpartum affective illness.

Axotomy Leads to Reduced Calcium Increase and Earlier Termination of CCL2 Release in Spinal Motoneurons with Upregulated Parvalbumin Followed by Decreased Neighboring Microglial Activation.

BACKGROUND: Motoneurons with naturally elevated calcium binding protein content, such as parvalbumin, are more resistant against injury. Furthermore, increase of intracellular calcium, which plays a pivotal role in injury of neurons, could be moderated by elevating their calcium binding proteins. OBJECTIVE: To test whether by elevating parvalbumin content of motoneurons, activation of neighboring microglial cells, a robust component of the inflammatory reaction after injury, could be influenced. METHODS: Mice overexpressing neuronal parvalbumin were derived and the spinal motoneurons were challenged by cutting the sciatic nerve. At postoperative days 1, 4, 7, 14 and 21 the change of the chemokine ligand 2 immunostaining in the motoneurons and the activation of microglial cells, measured as alterations in CD11b immunostaining were determined. Calcium level of motoneurons was tested electron microscopically at postoperative day 7. RESULTS: After axotomy, increased level of chemokine ligand 2 was detected in the lumbar motoneurons. The staining intensity reached its maximum at day 7 and decayed faster in transgenic mice compared to controls. Microglial activation around motoneurons attenuated faster in parvalbumin overexpressing mice, too, but the decrease of microglial activation was delayed compared to the decline of the chemokine ligand 2 signal. At the time when the microglial reaction peaked, no intracellular calcium increase was detected in the motoneurons of transgenic mice, in contrast to the twofold increase in wild type animals. CONCLUSION: Increased calcium buffering capacity, which augments resistance of motoneurons against calcium-mediated injury, leads to earlier termination of motoneuronal emission of CCL2 followed by a reduction of neighboring microglial activation after axotomy.

Intraperitoneally administered IgG from patients with amyotrophic lateral sclerosis or from an immune-mediated goat model increase the levels of TNF-α, IL-6, and IL-10 in the spinal cord and serum of mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the selective loss of the upper and lower motor neurons (MNs). Neuroinflammation has been implicated in the pathogenesis of the sporadic form of the disease. We earlier developed immune-mediated animal models of ALS and demonstrated humoral and cellular immune reactions in the nervous system and in the sera of patients and animals. The accumulation of immunoglobulin G (IgG), an elevated intracellular level of calcium, ultrastructural alterations in the MNs, and activation of the microglia were noted in the spinal cord of ALS patients. Similar alterations developed in mice inoculated intraperitoneally with IgG from ALS patients or from an immune-mediated goat model. METHODS: We have now examined whether the intraperitoneal injection of mice with IgG from sporadic ALS patients or from immunized goats with the homogenate of the anterior horn of the bovine spinal cord is associated with changes in the pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines in the spinal cord and serum of the mice. The levels of cytokines were measured by ELISA. RESULTS: Intraperitoneally administered IgG from the ALS patients induced subclinical signs of MN disease, while the injection of IgG from immunized goats resulted in a severe respiratory dysfunction and limb paralysis 24 h after the injections. Significantly increased levels of TNF-α and IL-10 were detected in the spinal cord of the mice injected with the human ALS IgG. The level of IL-6 increased primarily in the serum. The IgG from the immunized goats induced highly significant increases in the levels of all three cytokines in the serum and the spinal cord of mice. CONCLUSIONS: Our earlier experiments had proved that when ALS IgG or IgG from immune-mediated animal models was inoculated into mice, it was taken up in the MNs and had the ability to initiate damage in them. The pathological process was paralleled by microglia recruitment and activation in the spinal cord. The present experiment revealed that these forms of IgG cause significant increases in certain cytokine levels locally in the spinal cord and in the serum of the inoculated mice. These results suggest that IgG directed to the MNs may be an initial element in the damage to the MNs both in human ALS and in its immune-mediated animal models.

Aromatase and estrogen receptor beta expression in the rat olfactory bulb: Neuroestrogen action in the first relay station of the olfactory pathway?

The expression pattern of aromatase (ARO), the enzyme converting androgens to estrogens, was analyzed in the olfactory bulb of adult male rats and was compared with the distribution of estrogen receptor beta (ERbeta), the main estrogen receptor isoform expressed in this brain region. A strong ARO immunolabeling obtained with a specificity tested antibody was observed in juxtaglomerular neurons of the glomerular layer and a weaker immunoreaction was detected in the mitral cell layer of the main olfactory bulb, while the granule cell layer of the main olfactory bulb as well as all layers in the accessory olfactory bulb showed faint immunolabeling. Fluorescence double labeling experiments revealed that ARO detected in juxtaglomerular neurons of the main olfactory bulb colocalized with tyrosine hydroxylase (TH) and glutamic acid decarboxylase 67 (GAD67), while no colocalization between ARO and the calcium binding proteins calretinin (CR) and calbindin (CB) was observed. Furthermore, the TH immunoreactive neurons expressed metabotropic glutamate receptor 1 (mGluR1) too. ERß immunoreactivity, in contrast to ARO, was detected in all layers of both the main and accessory olfactory bulb. In the glomerular layer of the main olfactory bulb it was expressed in TH and GAD67 containing juxtaglomerular neurons, and it colocalized with CR, CB and even with glial fibrillary acidic protein too. Our morphological findings suggest that ARO expression is a novel feature of dopaminergic/GABAergic juxtaglomerular neurons in the adult rat main olfactory bulb, and raise the possibility that ARO activity may change in function of olfactory input via mGluR1. In situ estrogen production in the olfactory bulb in turn may modulate interglomerular circuits through ERbeta.

Highly immunoreactive IgG antibodies directed against a set of twenty human proteins in the sera of patients with amyotrophic lateral sclerosis identified by protein array.

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS.

Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

An MND/ALS phenotype associated with C9orf72 repeat expansion: abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline.

The transactive response DNA binding protein (TDP-43) proteinopathies describe a clinico-pathological spectrum of multi-system neurodegeneration that spans motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and frontotemporal lobar degeneration (FTLD). We have identified four male patients who presented with the clinical features of a pure MND/ALS phenotype (without dementia) but who had distinctive cortical and cerebellar pathology that was different from other TDP-43 proteinopathies. All patients initially presented with weakness of limbs and respiratory muscles and had a family history of MND/ALS. None had clinically identified cognitive decline or dementia during life and they died between 11 and 32 months after symptom onset. Neuropathological investigation revealed lower motor neuron involvement with TDP-43-positive inclusions typical of MND/ALS. In contrast, the cerebral pathology was atypical, with abundant star-shaped p62-immunoreactive neuronal cytoplasmic inclusions in the cerebral cortex, basal ganglia and hippocampus, while TDP-43-positive inclusions were sparse. This pattern was also seen in the cerebellum where p62-positive, TDP-43-negative inclusions were frequent in granular cells. Western blots of cortical lysates, in contrast to those of sporadic MND/ALS and FTLD-TDP, showed high p62 levels and low TDP-43 levels with no high molecular weight smearing. MND/ALS-associated SOD1, FUS and TARDBP gene mutations were excluded; however, further investigations revealed that all four of the cases did show a repeat expansion of C9orf72, the recently reported cause of chromosome 9-linked MND/ALS and FTLD. We conclude that these chromosome 9-linked MND/ALS cases represent a pathological sub-group with abundant p62 pathology in the cerebral cortex, hippocampus and cerebellum but with no significant associated cognitive decline.