Signaling pathways mediating a selective induction of nitric oxide synthase II by tumor necrosis factor alpha in nerve growth factor-responsive cells

BACKGROUND: Inflammation and oxidative stress play a critical role in neurodegeneration associated with acute and chronic insults of the nervous system. Notably, affected neurons are often responsive to and dependent on trophic factors such as nerve growth factor (NGF). We previously showed in NGF-responsive PC12 cells that tumor necrosis factor alpha (TNFα) and NGF synergistically induce the expression of the free-radical producing enzyme inducible nitric oxide synthase (iNOS). We proposed that NGF-responsive neurons might be selectively exposed to iNOS-mediated oxidative damage as a consequence of elevated TNFα levels. With the aim of identifying possible therapeutic targets, in the present study we investigated the signaling pathways involved in NGF/TNFα-promoted iNOS induction. METHODS: Western blotting, RT-PCR, transcription factor-specific reporter gene systems, mutant cells lacking the low affinity p75NTR NGF receptor and transfections of TNFα/NGF chimeric receptors were used to investigate signalling events associated with NGF/TNFα-promoted iNOS induction in PC12 cells. RESULTS: Our results show that iNOS expression resulting from NGF/TNFα combined treatment can be elicited in PC12 cells. Mutant PC12 cells lacking p75NTR did not respond, suggesting that p75NTR is required to mediate iNOS expression. Furthermore, cells transfected with chimeric TNFα/NGF receptors demonstrated that the simultaneous presence of both p75NTR and TrkA signaling is necessary to synergize with TNFα to mediate iNOS expression. Lastly, our data show that NGF/TNFα-promoted iNOS induction requires activation of the transcription factor nuclear factor kappa B (NF-κB). CONCLUSION: Collectively, our in vitro model suggests that cells bearing both the high and low affinity NGF receptors may display increased sensitivity to TNFα in terms of iNOS expression and therefore be selectively at risk during acute (e.g. neurotrauma) or chronic (e.g. neurodegenerative diseases) conditions where high levels of pro-inflammatory cytokines in the nervous system occur pathologically. Our results also suggest that modulation of NFκB-promoted transcription of selective genes could serve as a potential therapeutic target to prevent neuroinflammation-induced neuronal damage

Pharmacologically Distinct Nicotinic Acetylcholine Receptors Drive Efferent-Mediated Excitation in Calyx-Bearing Vestibular Afferents

Electrical stimulation of vestibular efferent neurons rapidly excites the resting discharge of calyx/dimorphic (CD) afferents. In turtle, this excitation arises when acetylcholine (ACh), released from efferent terminals, directly depolarizes calyceal endings by activating nicotinic ACh receptors (nAChRs). Although molecular biological data from the peripheral vestibular system implicate most of the known nAChR subunits, specific information about those contributing to efferent-mediated excitation of CD afferents is lacking. We sought to identify the nAChR subunits that underlie the rapid excitation of CD afferents and whether they differ from α9α10 nAChRs on type II hair cells that drive efferent-mediated inhibition in adjacent bouton afferents. We recorded from CD and bouton afferents innervating the turtle posterior crista during electrical stimulation of vestibular efferents while applying several subtype-selective nAChR agonists and antagonists. The α9α10 nAChR antagonists, α-bungarotoxin and α-conotoxin RgIA, blocked efferent-mediated inhibition in bouton afferents while leaving efferent-mediated excitation in CD units largely intact. Conversely, 5-iodo-A-85380, sazetidine-A, varenicline, α-conotoxin MII, and bPiDDB (N,N-dodecane-1,12-diyl-bis-3-picolinium dibromide) blocked efferent-mediated excitation in CD afferents without affecting efferent-mediated inhibition in bouton afferents. This pharmacological profile suggested that calyceal nAChRs contain α6 and β2, but not α9, nAChR subunits. Selective blockade of efferent-mediated excitation in CD afferents distinguished dimorphic from calyx afferents by revealing type II hair cell input. Dimorphic afferents differed in having higher mean discharge rates and a mean efferent-mediated excitation that was smaller in amplitude yet longer in duration. Molecular biological data demonstrated the expression of α9 in turtle hair cells and α4 and β2 in associated vestibular ganglia

Loss of α-Calcitonin Gene-Related Peptide (αCGRP) Reduces Otolith Activation Timing Dynamics and Impairs Balance

Calcitonin gene-related peptide (CGRP) is a neuroactive peptide that is thought to play a role at efferent synapses in hair cell organs including the cochlea, lateral line, and semicircular canal. The deletion of CGRP in transgenic mice is associated with a significant reduction in suprathreshold cochlear nerve activity and vestibulo–ocular reflex (VOR) gain efficacy when compared to littermate controls. Here we asked whether the loss of CGRP also influences otolithic end organ function and contributes to balance impairments. Immunostaining for CGRP was absent in the otolithic end organs of αCGRP null (-/-) mice while choline acetyltransferase (ChAT) immunolabeling appeared unchanged suggesting the overall gross development of efferent innervation in otolithic organs was unaltered. Otolithic function was assessed by quantifying the thresholds, suprathreshold amplitudes, and latencies of vestibular sensory-evoked potentials (VsEPs) while general balance function was assessed using a modified rotarod assay. The loss of αCGRP in null (-/-) mice was associated with: (1) shorter VsEP latencies without a concomitant change in amplitude or thresholds, and (2) deficits in the rotarod balance assay. Our findings show that CGRP loss results in faster otolith afferent activation timing, suggesting that the CGRP component of the efferent vestibular system (EVS) also plays a role in otolithic organ dynamics, which when coupled with reduced VOR gain efficacy, impairs balance

2016 Classification Criteria for Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis: A European League Against Rheumatism/American College of Rheumatology/Paediatric Rheumatology International Trials Organisation Collaborative Initiative

Objective To develop criteria for the classification of macrophage activation syndrome (MAS) in patients with systemic juvenile idiopathic arthritis (JIA). Methods A multistep process, based on a combination of expert consensus and analysis of real patient data, was conducted. A panel of 28 experts was first asked to classify 428 patient profiles as having or not having MAS, based on clinical and laboratory features at the time of disease onset. The 428 profiles comprised 161 patients with systemic JIA-associated MAS and 267 patients with a condition that could potentially be confused with MAS (active systemic JIA without evidence of MAS, or systemic infection). Next, the ability of candidate criteria to classify individual patients as having MAS or not having MAS was assessed by evaluating the agreement between the classification yielded using the criteria and the consensus classification of the experts. The final criteria were selected in a consensus conference. Results Experts achieved consensus on the classification of 391 of the 428 patient profiles (91.4%). A total of 982 candidate criteria were tested statistically. The 37 best-performing criteria and 8 criteria obtained from the literature were evaluated at the consensus conference. During the conference, 82% consensus among experts was reached on the final MAS classification criteria. In validation analyses, these criteria had a sensitivity of 0.73 and a specificity of 0.99. Agreement between the classification (MAS or not MAS) obtained using the criteria and the original diagnosis made by the treating physician was high (κ = 0.76). Conclusion We have developed a set of classification criteria for MAS complicating systemic JIA and provided preliminary evidence of its validity. Use of these criteria will potentially improve understanding of MAS in systemic JIA and enhance efforts to discover effective therapies, by ensuring appropriate patient enrollment in studies

Expert consensus on dynamics of laboratory tests for diagnosis of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis

Objective: To identify which laboratory tests that change over time are most valuable for the timely diagnosis of macrophage activation syndrome (MAS) complicating systemic juvenile idiopathic arthritis (sJIA). Methods: A multistep process, based on a combination of expert consensus and analysis of real patient data, was conducted. A panel of experts was first asked to evaluate 115 profiles of patients with MAS, which included the values of laboratory tests at the pre-MAS visit and at MAS onset, and the change in values between the two time points. The experts were asked to choose the 5 laboratory tests in which change was most important for the diagnosis of MAS and to rank the 5 selected tests in order of importance. The relevance of change in laboratory parameters was further discussed and ranked by the same experts at a consensus conference. Results: Platelet count was the most frequently selected test, followed by ferritin level, aspartate aminotransferase (AST), white cell count, neutrophil count, and fibrinogen and erythrocyte sedimentation rate. Ferritin was most frequently assigned the highest score. At the end of the process, platelet count, ferritin level and AST were the laboratory tests in which the experts found change over time to be most important. Conclusions: We identified the laboratory tests in which change over time is most valuable for the early diagnosis of MAS in sJIA. The dynamics of laboratory values during the course of MAS should be further scrutinised in a prospective study in order to establish the optimal cut-off values for their variation

Loss of α-Calcitonin Gene-Related Peptide (αCGRP) Reduces Otolith Activation Timing Dynamics and Impairs Balance

Calcitonin gene-related peptide (CGRP) is a neuroactive peptide that is thought to play a role at efferent synapses in hair cell organs including the cochlea, lateral line, and semicircular canal. The deletion of CGRP in transgenic mice is associated with a significant reduction in suprathreshold cochlear nerve activity and vestibulo– ocular reflex (VOR) gain efficacy when compared to littermate controls. Here we asked whether the loss of CGRP also influences otolithic end organ function and contributes to balance impairments. Immunostaining for CGRP was absent in the otolithic end organs of aCGRP null (-/-) mice while choline acetyltransferase (ChAT) immunolabeling appeared unchanged suggesting the overall gross development of efferent innervation in otolithic organs was unaltered. Otolithic function was assessed by quantifying the thresholds, suprathreshold amplitudes, and latencies of vestibular sensory-evoked potentials (VsEPs) while general balance function was assessed using a modified rotarod assay. The loss of aCGRP in null (-/-) mice was associated with: (1) shorter VsEP latencies without a concomitant change in amplitude or thresholds, and (2) deficits in the rotarod balance assay. Our findings show that CGRP loss results in faster otolith afferent activation timing, suggesting that the CGRP component of the efferent vestibular system (EVS) also plays a role in otolithic organ dynamics, which when coupled with reduced VOR gain efficacy, impairs balance

The mammalian efferent vestibular system utilizes cholinergic mechanisms to excite primary vestibular afferents

Abstract Electrical stimulation of the mammalian efferent vestibular system (EVS) predominantly excites primary vestibular afferents along two distinct time scales. Although roles for acetylcholine (ACh) have been demonstrated in other vertebrates, synaptic mechanisms underlying mammalian EVS actions are not well-characterized. To determine if activation of ACh receptors account for efferent-mediated afferent excitation in mammals, we recorded afferent activity from the superior vestibular nerve of anesthetized C57BL/6 mice while stimulating EVS neurons in the brainstem, before and after administration of cholinergic antagonists. Using a normalized coefficient of variation (CV*), we broadly classified vestibular afferents as regularly- (CV*  0.1) and characterized their responses to midline or ipsilateral EVS stimulation. Afferent responses to efferent stimulation were predominantly excitatory, grew in amplitude with increasing CV*, and consisted of fast and slow components that could be identified by differences in rise time and post-stimulus duration. Both efferent-mediated excitatory components were larger in irregular afferents with ipsilateral EVS stimulation. Our pharmacological data show, for the first time in mammals, that muscarinic AChR antagonists block efferent-mediated slow excitation whereas the nicotinic AChR antagonist DHβE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic