Thinking about the action potential: the nerve signal as a window to the physical principles guiding neuronal excitability

Ever since the work of Edgar Adrian, the neuronal action potential has been considered as an electric signal, modeled and interpreted using concepts and theories lent from electronic engineering. Accordingly, the electric action potential, as the prime manifestation of neuronal excitability, serving processing and reliable “long distance” communication of the information contained in the signal, was defined as a non-linear, self-propagating, regenerative, wave of electrical activity that travels along the surface of nerve cells. Thus, in the ground-breaking theory and mathematical model of Hodgkin and Huxley (HH), linking Nernst’s treatment of the electrochemistry of semi-permeable membranes to the physical laws of electricity and Kelvin’s cable theory, the electrical characteristics of the action potential are presented as the result of the depolarization-induced, voltage- and time-dependent opening and closure of ion channels in the membrane allowing the passive flow of charge, particularly in the form of Na+ and K+ -ions, into and out of the neuronal cytoplasm along the respective electrochemical ion gradient. In the model, which treats the membrane as a capacitor and ion channels as resistors, these changes in ionic conductance across the membrane cause a sudden and transient alteration of the transmembrane potential, i.e., the action potential, which is then carried forward and spreads over long(er) distances by means of both active and passive conduction dependent on local current flow by diffusion of Na+ ion in the neuronal cytoplasm. However, although highly successful in predicting and explaining many of the electric characteristics of the action potential, the HH model, nevertheless cannot accommodate the various non-electrical physical manifestations (mechanical, thermal and optical changes) that accompany action potential propagation, and for which there is ample experimental evidence. As such, the electrical conception of neuronal excitability appears to be incomplete and alternatives, aiming to improve, extend or even replace it, have been sought for. Commonly misunderstood as to their basic premises and the physical principles they are built on, and mistakenly perceived as a threat to the generally acknowledged explanatory power of the “classical” HH framework, these attempts to present a more complete picture of neuronal physiology, have met with fierce opposition from mainstream neuroscience and, as a consequence, currently remain underdeveloped and insufficiently tested. Here we present our perspective that this may be an unfortunate state of affairs as these different biophysics-informed approaches to incorporate also non-electrical signs of the action potential into the modeling and explanation of the nerve signal, in our view, are well suited to foster a new, more complete and better integrated understanding of the (multi)physical nature of neuronal excitability and signal transport and, hence, of neuronal function. In doing so, we will emphasize attempts to derive the different physical manifestations of the action potential from one common, macroscopic thermodynamics-based, framework treating the multiphysics of the nerve signal as the inevitable result of the collective material, i.e., physico-chemical, properties of the lipid bilayer neuronal membrane (in particular, the axolemma) and/or the so-called ectoplasm or membrane skeleton consisting of cytoskeletal protein polymers, in particular, actin fibrils. Potential consequences for our view of action potential physiology and role in neuronal function are identified and discussed

Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology

Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer’s Disease

The pathologic lesions of Alzheimer’s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-β (Aβ) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. “Professional chaperones”, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. “Amateur” chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of Aβ, and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood–brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention

Tissue transglutaminase in Alzheimer's disease: involvement in pathogenesis and its potential as a therapeutic target

Protein misfolding and the formation of stable insoluble protein complexes by self-interacting proteins, in particular amyloid-β and tau protein, play a central role in the pathogenesis of Alzheimer's disease (AD). Unfortunately, the underlying mechanisms that trigger the misfolding of self-interacting proteins that eventually results in formation of neurotoxic dimers, oligomers, and aggregates remain unclear. Elucidation of the driving forces of protein complex formation in AD is of crucial importance for the development of disease-modifying therapies. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that induces the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds, which results in both intra- and intermolecular protein cross-links. These tTG-catalyzed intermolecular cross-links induce stable, rigid, and insoluble protein complexes, whereas intramolecular cross-links change the conformation of proteins. Inhibition of tTG-catalyzed cross-linking counteracts the formation of protein aggregates, as observed in disease-models of other protein misfolding diseases, in particular Parkinson's and Huntington's diseases. Although data of tTG activity in AD models is limited, there is compelling evidence from both in vitro and postmortem human brain tissue of AD patients that point toward a crucial role for tTG in the pathogenesis of AD. Here, we review these data on the role of tTG in the initiation and development of protein aggregates in AD, and discuss the possibility to use inhibitors of the cross-linking activity of tTG as a new therapeutic approach for A

Catalytically active tissue transglutaminase colocalises with Aβ pathology in Alzheimer's disease mouse models

Alzheimer's disease (AD) is characterised by amyloid-beta (Aβ) protein deposition in the brain. Posttranslational modifications in Aβ play an important role in Aβ deposition. Tissue transglutaminase (tTG) is an enzyme involved in posttranslational cross-linking of proteins. tTG levels and activity are increased in AD brains, and tTG is associated with Aβ deposits and lesion-associated astrocytes in AD cases. Furthermore, Aβ is a substrate of tTG-catalysed cross-linking. To study the role of tTG in Aβ pathology, we compared tTG distribution and activity in both the APPSWE/PS1ΔE9 and APP23 mice models with human AD. Using immunohistochemistry, we found association of both tTG and in situ active tTG with Aβ plaques and vascular Aβ, in early and late stages of Aβ deposition. In addition, tTG staining colocalised with Aβ-associated reactive astrocytes. Thus, alike human AD cases, tTG was associated with Aβ depositions in these AD models. Although, distribution pattern and spatial overlay of both tTG and its activity with Aβ pathology was substantially different from human AD cases, our findings provide evidence for an early role of tTG in Aβ pathology. Yet, species differences should be taken into account when using these models to study the role of tTG in Aβ pathology

Is monocyte- and macrophage-derived tissue transglutaminase involved in inflammatory processes?

Monocytes and macrophages are key players in inflammatory processes following an infection or tissue damage. Monocytes adhere and extravasate into the inflamed tissue, differentiate into macrophages, and produce inflammatory mediators to combat the pathogens. In addition, they take up dead cells and debris and, therefore, take part in the resolution of inflammation. The multifunctional enzyme tissue Transglutaminase (TG2, tTG) is known to participate in most of those monocyte- and macrophage-mediated processes. Moreover, TG2 expression and activity can be regulated by inflammatory mediators. In the present review, we selectively elaborate on the expression, regulation, and contribution of TG2 derived from monocytes and macrophages to inflammatory processes mediated by those cells. In addition, we discuss the role of TG2 in certain pathological conditions, in which inflammation and monocytes and/or macrophages are prominently present, including atherosclerosis, sepsis, and multiple sclerosis. Based on the studies and considerations reported in this review, we conclude that monocyte- and macrophage-derived TG2 is clearly involved in various processes contributing to inflammation. However, TG2’s potential as a therapeutic target to counteract the possible detrimental effects or stimulate the potential beneficial effects on monocyte and macrophage responses during inflammation should be carefully considered. Alternatively, as TG2-related parameters can be used as a marker of disease, e.g., in celiac disease, or of disease-stage, e.g., in cancer, we put forward that this could be subject of research for monocyte- or macrophage-derived TG2 in inflammatory diseases

Characterization of Transglutaminase 2 activity inhibitors in monocytes in vitro and their effect in a mouse model for multiple sclerosis.

The neurodegenerative disease multiple sclerosis (MS) is pathologically characterized by the massive influx of immune cells into the central nervous system. This contributes to demyelination and axonal damage which causes symptoms such as motor and cognitive dysfunctions. The migration of leukocytes from the blood vessel is orchestrated by a multitude of factors whose determination is essential in reducing cellular influx in MS patients and the experimental autoimmune encephalomyelitis (EAE) animal model. The here studied enzyme tissue Transglutaminase (TG2) is present intracellularly, on the cell surface and extracellularly. There it contributes to cellular adhesion and migration via its transamidation activity and possibly by facilitating cellular interaction with the extracellular matrix. Previous data from our group showed reduced motor symptoms and cellular infiltration after using a pharmacological TG2 transamidation activity inhibitor in a rat EAE model. However, it remained elusive if the cross-linking activity of the enzyme resulted in the observed effects. To follow-up, we now characterized two new small molecule TG2 activity inhibitors, BJJF078 and ERW1041E. Both compounds are potent inhibitor of recombinant human and mouse Transglutaminase enzyme activity, mainly TG2 and the close related enzyme TG1. In addition they did not affect the binding of TG2 to the extracellular matrix substrate fibronectin, a process via which TG2 promotes cellular adhesion and migration. We found, that ERW1041E but not BJJF078 resulted in reduced EAE disease motor-symptoms while neither caused apparent changes in pathology (cellular influx), Transglutaminase activity or expression of inflammation related markers in the spinal cord, compared to vehicle treated controls. Although we cannot exclude issues on bioavailability and in vivo efficacy of the used compounds, we hypothesize that extracellular TG1/TG2 activity is of greater importance than (intra-)cellular activity in mouse EAE pathology

Effects of a single, oral 60 mg caffeine dose on attention in healthy adult subjects

Caffeine induces positive effects on sustained attention, although studies assessing the acute effects of low caffeine dose (<75 mg) on sustained attention are limited and use short-term tests. Therefore, we investigated the acute effects of a 60 mg dose of caffeine on sustained attention in tests lasting up to 45 minutes using 82 low or non-caffeine-consuming healthy male (n=41) and female (n=41) adults aged between 40 and 60 years. Vigilance was measured using Mackworth Clock test, Rapid Visual Information Processing Test, adaptive tracking test, saccadic eye movement and attention switch test. Effects on mood and fatigue were analysed using Bond and Lader and Caffeine Research visual analogue scales, and Samn-Perelli questionnaire. Saliva sampling was performed for both compliance and caffeine pharmacokinetic analysis. Administration of a 60 mg caffeine dose resulted in a significant improvement in sustained attention compared with the placebo. Also a significantly improved peak saccadic velocity and reaction time performance was found, and decreased error rate. Significantly increased feelings of alertness, contentment and overall mood after caffeine treatment compared with placebo were observed. This study demonstrated that in healthy adult subjects oral administration of a single 60 mg caffeine dose elicited a clear enhancement of sustained attention and alertness, measured both in multiple objective performances and in subjective scales

Development of carbon-11 labeled acryl amides for selective PET imaging of active tissue transglutaminase

Introduction Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of forming metabolically and mechanically stable crosslinks between the γ-carboxamide of a glutamine acyl-acceptor substrate and the ε-amino functionality of a lysine acyl-donor substrate resulting in protein oligomers. High TG2 crosslinking activity has been implicated in the pathogenesis of various diseases including celiac disease, cancer and fibrotic and neurodegenerative diseases. Development of a PET tracer specific for active TG2 provides a novel tool to further investigate TG2 biology in vivo in disease states. Recently, potent irreversible active site TG2 inhibitors carrying an acrylamide warhead were synthesized and pharmacologically characterized. Methods Three of these inhibitors, compound 1, 2 and 3, were successfully radiolabeled with carbon-11 on the acrylamide carbonyl position using a palladium mediated [11C]CO aminocarbonylation reaction. Ex vivo biodistribution and plasma stability were evaluated in healthy Wistar rats. Autoradiography was performed on MDA-MB-231 tumor sections. Results [11C]1, -2 and -3 were obtained in decay corrected radiochemical yields of 38–55%. Biodistribution showed low uptake in peripheral tissues, with the exception of liver and kidney. Low brain uptake of < 0.05% ID/g was observed. Blood plasma analysis demonstrated that [11C]1 and [11C]2 were rapidly metabolized, whereas [11C]3 was metabolized at a more moderate rate (63.2 ± 6.8 and 28.7 ± 10.8% intact tracer after 15 and 45 min, respectively). Autoradiography with [11C]3 on MDA-MB-231 tumor sections showed selective and specific binding of the radiotracer to the active state of TG2. Conclusions Taken together, these results identify [11C]3 as the most promising of the three compounds tested for development as PET radiotracer for the in vivo investigation of TG2 activity

In vivo evaluation of two tissue transglutaminase PET tracers in an orthotopic tumour xenograft model

Background: The protein cross-linking enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including cancer. Recently, the synthesis and initial evaluation of two high-potential radiolabelled irreversible TG2 inhibitors were reported by us. In the present study, these two compounds were evaluated further in a breast cancer (MDA-MB-231) tumour xenograft model for imaging active tissue transglutaminase in vivo. Results: The metabolic stability of [11C]1 and [18F]2 in SCID mice was comparable to the previously reported stability in Wistar rats. Quantitative real-time polymerase chain reaction analysis on MDA-MB-231 cells and isolated tumours showed a high level of TG2 expression with very low expression of other transglutaminases. PET imaging showed low tumour uptake of [11C]1 (approx. 0.5 percentage of the injected dose per gram (%ID/g) at 40–60 min p.i.) and with relatively fast washout. Tumour uptake for [18F]2 was steadily increasing over time (approx. 1.7 %ID/g at 40–60 min p.i.). Pretreatment of the animals with the TG2 inhibitor ERW1041E resulted in lower tumour activity concentrations, and this inhibitory effect was enhanced using unlabelled 2. Conclusions: Whereas the TG2 targeting potential of [11C]1 in this model seems inadequate, targeting of TG2 using [18F]2 was achieved. As such, [18F]2 could be used in future studies to clarify the role of active tissue transglutaminase in disease