Nitrergic modulation of neuronal excitability in the mouse hippocampus is mediated via regulation of Kv2 and voltage‐gated sodium channels

Regulation of neuronal activity is a necessity for communication and information transmission. Many regulatory processes which have been studied provide a complex picture of how neurons can respond to permanently changing functional requirements. One such activity-dependent mechanism involves signaling mediated by nitric oxide (NO). Within the brain, NO is generated in response to neuronal NO synthase (nNOS) activation but NO-dependent pathways regulating neuronal excitability in the hippocampus remain to be fully elucidated. This study was set out to systematically assess the effects of NO on ion channel activities and intrinsic excitabilities of pyramidal neurons within the CA1 region of the mouse hippocampus. We characterized whole-cell potassium and sodium currents, both involved in action potential (AP) shaping and propagation and determined NO-mediated changes in excitabilities and AP waveforms. Our data describe a novel signaling by which NO, in a cGMP-independent manner, suppresses voltage-gated Kv2 potassium and voltage-gated sodium channel activities, thereby widening AP waveforms and reducing depolarization-induced AP firing rates. Our data show that glutathione, which possesses denitrosylating activity, is sufficient to prevent the observed nitrergic effects on potassium and sodium channels, whereas inhibition of cGMP signaling is also sufficient to abolish NO modulation of sodium currents. We propose that NO suppresses both ion channel activities via redox signaling and that an additional cGMP-mediated component is required to exert effects on sodium currents. Both mechanisms result in a dampened excitability and firing ability providing new data on nitrergic activities in the context of activity-dependent regulation of neuronal function following nNOS activation

Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background: Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology: We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions: This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.DFG/REBIRT

Neuropeptides in the Antennal Lobe of the Yellow Fever Mosquito, Aedes aegypti.

For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592-608, 2014. (c) 2013 Wiley Periodicals, Inc

beta-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia

Alzheimer\u27s disease is the world\u27s most common neurodegenerative disorder. It is associated with neuroinflammation involving activation of microglia by beta-amyloid (Abeta) deposits. Based on previous studies showing apoptosis-associated speck-like protein containing a CARD (ASC) binding and cross-seeding extracellular Abeta, we investigate the propagation of ASC between primary microglia and the effects of ASC-Abeta composites on microglial inflammasomes and function. Indeed, ASC released by a pyroptotic cell can be functionally built into the neighboring microglia NOD-like receptor protein (NLRP3) inflammasome. Compared with protein-only application, exposure to ASC-Abeta composites amplifies the proinflammatory response, resulting in pyroptotic cell death, setting free functional ASC and inducing a feedforward stimulating vicious cycle. Clustering around ASC fibrils also compromises clearance of Abeta by microglia. Together, these data enable a closer look at the turning point from acute to chronic Abeta-related neuroinflammation through formation of ASC-Abeta composites

Characterizing microglial senescence: Tau as a key player.

peer reviewedThe highest risk factor for the development of neurodegenerative diseases like tauopathies is aging. Many physiological decrements underlying aging are linked to cellular senescence. Senescent cells are characterized by an irreversible growth arrest and formation of a senescence-associated secretory phenotype (SASP), a proinflammatory secretome that modifies the cellular microenvironment and contributes to tissue deterioration. Microglia, the innate immune cells in the brain, can enter a senescent state during aging. In addition, senescent microglia have been identified in the brains of tau-transgenic mice and patients suffering from tauopathies. While the contribution of senescent microglia to the development of tauopathies and other neurodegenerative diseases is a growing area of research, the effect of tau on microglial senescence remains elusive. Here, we exposed primary microglia to 5 and 15 nanomolar (nM) of monomeric tau for 18 h, followed by a recovery period of 48 h. Using multiple senescence markers, we found that exposure to 15 nM, but not 5 nM of tau increased levels of cell cycle arrest and a DNA damage marker, induced loss of the nuclear envelope protein lamin B1 and the histone marker H3K9me3, impaired tau clearance and migration, altered the cell morphology and resulted in formation of a SASP. Taken together, we show that exposure to tau can lead to microglial senescence. As senescent cells were shown to negatively impact tau pathologies, this suggests the presence of a vicious circle, which should be further investigated in the future

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with alpha-synuclein (alpha-syn) fibrils and their clearance. We found that microglia exposed to alpha-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer alpha-syn from overloaded microglia to neighboring naive microglia where the alpha-syn cargo got rapidly and effectively degraded. Lowering the alpha-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of alpha-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an on-demand functional network in order to improve pathogenic alpha-syn clearance

Inhibition of neuroinflammatory nitric oxide signalling supresses protein glycation and recovers neuronal dysfunction in prion disease

Background: Several neurodegenerative diseases associated with protein misfolding (Alzheimer’s, Parkinson’s disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated post-translational modifications impact upon protein functions that can exacerbate pathology. Non-enzymatic and irreversible glycation signalling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood.Methods: Here we investigated the potential therapeutic effects of supressing neurotoxic NO signalling during early progression of prion disease. Tg37 mice aged 3-4 weeks were inoculated by intracerebral injection with either 1% brain homogenate of Rocky Mountain Laboratory (RML) scrapie prion protein or control normal brain homogenate (NBH). Hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analysed and electrophysiological characterisations of pyramidal neurons were performed in 6-10 weeks old RML and NBH mice. Mice were injected with a NO synthase (NOS) inhibitor and the time course of disease markers was compared to controls. Electrophysiology, immunoblotting and immunocytochemistry studies were performed to identify the effects of NOS inhibition on neurophysiology, glycation, prion protein misfolding and cell death. Statistical analyses employed two-tailed unpaired Student’s t-test, one-way or two-way ANOVA as required and data were considered significant with P<0.05.Results: Increased neuroinflammatory signalling was observed in mice between 6 and 10 weeks post inoculation (w.p.i.) with scrapie prion protein which was characterised by enhanced nitrergic stress and associated with a decline in hippocampal neuronal function by 9 w.p.i.. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg abolished the functional degeneration of hippocampal neurons in prion mice. We further found that this intervention in diseased mice ameliorated 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation signalling. Furthermore, L-NAME application reduced the expression of the receptor for advanced glycation end products and the accumulation of hippocampal prion misfolding.Conclusions: Our data suggest that alleviating nitrergic stress during early phases of neurodegeneration reduces neurotoxic post-translational NO signalling and glycation-assisted prion misfolding in the hippocampus, a mechanism which might be applicable to other protein misfolding neurodegenerative conditions

Differential interaction with TREM2 modulates microglial uptake of modified Aβ species.

Funder: Canadian Institutes of Health Research; Id: http://dx.doi.org/10.13039/501100000024Funder: Alzheimer's Association (Zenith Award)Funder: UK Alzheimer Society and ARUKFunder: Wellcome Trust Collaborative Award in ScienceRare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid β peptides (Aβ). Aβ peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post-translational modifications result in the production of several distinct Aβ variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post-translational modifications of Aβ affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aβ, and that phosphorylation of Aβ increases this interaction. Phosphorylation of Aβ also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aβ variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aβ species in preclinical models of Alzheimer's disease

Neuroimmune Connections in Aging and Neurodegenerative Diseases

In recent years, the inter-relationship between the innate immune system and the central nervous system (CNS) has moved to the forefront of biomedical research, with the discovery that these two physiological systems modulate each other by a steady mutual interaction. During normal brain aging, but also under certain pathological conditions, this crosstalk can go beyond physiological control, resulting in an unresolved inflammatory response of the CNS-resident immune cells that might initiate and propagate the progression of severe tissue damage and neurodegeneration. In this review, we focus on the impact of CNS-resident cells of the innate immune system for the development of neurodegenerative diseases, review immune pathway genes that have been identified, and discuss the vicious cycle between inflammation and neurodegeneration

Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored