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

Nitrergic modulation of ion channel function in regulating neuronal excitability

Nitric oxide (NO) signaling in the brain provides a wide range of functional properties in response to neuronal activity. NO exerts its effects through different signaling pathways, namely, through the canonical soluble guanylyl cyclase-mediated cGMP production route and via post-translational protein modifications. The latter pathways comprise cysteine S-nitrosylation and 3-nitrotyrosination of distinct tyrosine residues. Many ion channels are targeted by one or more of these signaling routes, which leads to their functional regulation under physiological conditions or facilities their dysfunction leading to channelopathies in many pathologies. The resulting alterations in ion channel function changes neuronal excitability, synaptic transmission, and action potential propagation. Transient and activity-dependent NO production mediates reversible ion channel modifications via cGMP and S-nitrosylation signaling, whereas more pronounced and longer-term NO production during conditions of elevated oxidative stress leads to increasingly cumulative and irreversible protein 3-nitrotyrosination. The complexity of this regulation and vast variety of target ion channels and their associated functional alterations presents a challenging task in assessing and understanding the role of NO signaling in physiology and disease

App-Based Evaluation of Older People’s Fall Risk Using the mHealth App Lindera Mobility Analysis: Exploratory Study

Background: Falls and the risk of falling in older people pose a high risk for losing independence. As the risk of falling progresses over time, it is often not adequately diagnosed due to the long intervals between contacts with health care professionals. This leads to the risk of falling being not properly detected until the first fall. App-based software able to screen fall risks of older adults and to monitor the progress and presence of fall risk factors could detect a developing fall risk at an early stage prior to the first fall. As smartphones become more common in the elderly population, this approach is easily available and feasible. Objective: The aim of the study is to evaluate the app Lindera Mobility Analysis (LIN). The reference standards determined the risk of falling and validated functional assessments of mobility. Methods: The LIN app was utilized in home- and community-dwelling older adults aged 65 years or more. The Berg Balance Scale (BBS), the Tinetti Test (TIN), and the Timed Up & Go Test (TUG) were used as reference standards. In addition to descriptive statistics, data correlation and the comparison of the mean difference of analog measures (reference standards) and digital measures were tested. Spearman rank correlation analysis was performed and Bland-Altman (B-A) plots drawn. Results: Data of 42 participants could be obtained (n=25, 59.5%, women). There was a significant correlation between the LIN app and the BBS (r=-0.587, P<.001), TUG (r=0.474, P=.002), and TIN (r=-0.464, P=.002). B-A plots showed only few data points outside the predefined limits of agreement (LOA) when combining functional tests and results of LIN. Conclusions: The digital app LIN has the potential to detect the risk of falling in older people. Further steps in establishing the validity of the LIN app should include its clinical applicability

Rab11 rescues synaptic dysfunction and behavioural deficits in a Drosophila model of Huntington's disease

Synapse abnormalities in Huntington's disease (HD) patients can precede clinical diagnosis and neuron loss by decades. The polyglutamine expansion in the huntingtin (htt) protein that underlies this disorder leads to perturbations in many cellular pathways, including the disruption of Rab11-dependent endosomal recycling. Impairment of the small GTPase Rab11 leads to the defective formation of vesicles in HD models and may thus contribute to the early stages of the synaptic dysfunction in this disorder. Here, we employ transgenic Drosophila melanogaster models of HD to investigate anomalies at the synapse and the role of Rab11 in this pathology. We find that the expression of mutant htt in the larval neuromuscular junction decreases the presynaptic vesicle size, reduces quantal amplitudes and evoked synaptic transmission and alters larval crawling behaviour. Furthermore, these indicators of early synaptic dysfunction are reversed by the overexpression of Rab11. This work highlights a potential novel HD therapeutic strategy for early intervention, prior to neuronal loss and clinical manifestation of disease

Plum modulates Myoglianin and regulates synaptic function in D. melanogaster

Alterations in the neuromuscular system underlie several neuromuscular diseases and play critical roles in the development of sarcopenia, the age-related loss of muscle mass and function. Mammalian Myostatin (MST) and GDF11, members of the TGF-β superfamily of growth factors, are powerful regulators of muscle size in both model organisms and humans. Myoglianin (MYO), the Drosophila homologue of MST and GDF11, is a strong inhibitor of synaptic function and structure at the neuromuscular junction in flies. Here, we identified Plum, a transmembrane cell surface protein, as a modulator of MYO function in the larval neuromuscular system. Reduction of Plum in the larval body-wall muscles abolishes the previously demonstrated positive effect of attenuated MYO signalling on both muscle size and neuromuscular junction structure and function. In addition, downregulation of Plum on its own results in decreased synaptic strength and body weight, classifying Plum as a (novel) regulator of neuromuscular function and body (muscle) size. These findings offer new insights into possible regulatory mechanisms behind ageing- and disease-related neuromuscular dysfunctions in humans and identify potential targets for therapeutic interventions

The C terminus of p73 is essential for hippocampal development

The p53 family member p73 has a complex gene structure, including alternative promoters and alternative splicing of the 3′ UTR. This results in a complex range of isoforms whose biological relevance largely remains to be determined. By deleting exon 13 (which encodes a sterile α motif) from the Trp73 gene, we selectively engineered mice to replace the most abundantly expressed C-terminal isoform, p73α, with a shorter product of alternative splicing, p73β. These mice (Trp73Δ13/Δ13) display severe neurodevelopmental defects with significant functional and morphological abnormalities. Replacement of p73α with p73β results in the depletion of Cajal–Retzius (CR) cells in embryonic stages, thus depriving the developing hippocampus of the pool of neurons necessary for correct hippocampal architecture. Consequently, Trp73Δ13/Δ13 mice display severe hippocampal dysgenesis, reduced synaptic functionality and impaired learning and memory capabilities. Our data shed light on the relevance of p73 alternative splicing and show that the full-length C terminus of p73 is essential for hippocampal development

Medium-Chain Fatty Acids Rescue Motor Function and Neuromuscular Junction Degeneration in a Drosophila Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterised by progressive degeneration of the motor neurones. An expanded GGGGCC (G4C2) hexanucleotide repeat in C9orf72 is the most common genetic cause of ALS and frontotemporal dementia (FTD); therefore, the resulting disease is known as C9ALS/FTD. Here, we employ a Drosophila melanogaster model of C9ALS/FTD (C9 model) to investigate a role for specific medium-chain fatty acids (MCFAs) in reversing pathogenic outcomes. Drosophila larvae overexpressing the ALS-associated dipeptide repeats (DPRs) in the nervous system exhibit reduced motor function and neuromuscular junction (NMJ) defects. We show that two MCFAs, nonanoic acid (NA) and 4-methyloctanoic acid (4-MOA), can ameliorate impaired motor function in C9 larvae and improve NMJ degeneration, although their mechanisms of action are not identical. NA modified postsynaptic glutamate receptor density, whereas 4-MOA restored defects in the presynaptic vesicular release. We also demonstrate the effects of NA and 4-MOA on metabolism in C9 larvae and implicate various metabolic pathways as dysregulated in our ALS model. Our findings pave the way to identifying novel therapeutic targets and potential treatments for ALS

Nitric Oxide Signalling Augments Neuronal Voltage-Gated L-Type (CaV1) and P/Q-Type (CaV2.1) Channels in the Mouse Medial Nucleus of the Trapezoid Body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (CaV) within this nucleus. Whole-cell patch recordings were made from brain slices from P13–15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 µM) and pharmacological blockers used to isolate Ca2+ current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 µM) or Diethyl-ammonium-nonoate (DEA, 100 µM) were made to elucidate NO-dependent modulation of the expressed CaV subtypes. A differential effect of NO on the calcium channel subtypes was observed: CaV1 and CaV2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (CaV2.2) and R-type (CaV2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36±0.04 nA to 0.64±0.11 nA and P/Q+R-type from 0.55±0.09 nA to 0.94±0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways

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