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.

Effect of long-term antihypertensive treatment on cerebrovascular structure and function in hypertensive rats.

Midlife hypertension is an important risk factor for cognitive impairment and dementia, including Alzheimer's disease. We investigated the effects of long-term treatment with two classes of antihypertensive drugs to determine whether diverging mechanisms of blood pressure lowering impact the brain differently. Spontaneously hypertensive rats (SHR) were either left untreated or treated with a calcium channel blocker (amlodipine) or beta blocker (atenolol) until one year of age. The normotensive Wistar Kyoto rat (WKY) was used as a reference group. Both drugs lowered blood pressure equally, while only atenolol decreased heart rate. Cerebrovascular resistance was increased in SHR, which was prevented by amlodipine but not atenolol. SHR showed a larger carotid artery diameter with impaired pulsatility, which was prevented by atenolol. Cerebral arteries demonstrated inward remodelling, stiffening and endothelial dysfunction in SHR. Both treatments similarly improved these parameters. MRI revealed that SHR have smaller brains with enlarged ventricles. In addition, neurofilament light levels were increased in cerebrospinal fluid of SHR. However, neither treatment affected these parameters. In conclusion, amlodipine and atenolol both lower blood pressure, but elicit a different hemodynamic profile. Both medications improve cerebral artery structure and function, but neither drug prevented indices of brain damage in this model of hypertension.

Cerebellar presence of immune cells in patients with neuro-coeliac disease.

Although various neurodegenerative disorders have been associated with coeliac disease (CD), the underlying neuropathological link between these brain and gut diseases remains unclear. We postulated that the neuronal damage sporadically observed in CD patients is immune-mediated. Our aim was to determine if the loss of neurons, especially Purkinje cells, coincides with microglia activation and T- and B-cell infiltration in the cerebellum of patients with CD and a concomitant idiopathic neurological disease affecting the cerebellum (NeuroCD). Post-mortem cerebellar tissue was collected of validated NeuroCD cases. Gender- and age-matched genetic spinocerebellar ataxia (SCA) controls and non-neurological controls (NNC) were selected based on clinical reports and pathological findings. Cerebellar tissue of seventeen patients was included (6 NeuroCD, 5 SCA, 6 NNC). In SCA cases we found that the Purkinje cell layer was 58.6% reduced in comparison with NNC. In NeuroCD cases this reduction was even more prominent with a median reduction of 81.3% compared to NNC. Marked increased numbers of both CD3+ and CD8+ cells were observed in the NeuroCD but not in SCA patients. This coincided with significantly more microglial reactivity in NeuroCD patients. These findings demonstrate that the massive loss of Purkinje cells in the cerebellum of neuro CD patients is accompanied by local innate and T-cell mediated immune responses.

Absence of tissue transglutaminase reduces amyloid-beta pathology in APP23 mice.

AIMS: Alzheimer's disease (AD) is characterised by amyloid-beta (Aß) aggregates in the brain. Targeting Aß aggregates is a major approach for AD therapies, although attempts have had little to no success so far. A novel treatment option is to focus on blocking the actual formation of Aß multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post-translational modifications in Aß resulting in covalently cross-linked, stable and neurotoxic Aß oligomers. In vivo absence of TG2 in the APP23 mouse model may provide evidence that TG2 plays a key role in development and/or progression of Aß-related pathology. METHODS: Here, we compared the effects on Aß pathology in the presence or absence of TG2 using 12-month-old wild type, APP23 and a crossbreed of the TG2-/- mouse model and APP23 mice (APP23/TG2-/-). RESULTS: Using immunohistochemistry, we found that the number of Aß deposits was significantly reduced in the absence of TG2 compared with age-matched APP23 mice. To pinpoint possible TG2-associated mechanisms involved in this observation, we analysed soluble brain Aß1-40 , Aß1-42 and/or Aß40/42 ratio, and mRNA levels of human APP and TG2 family members present in brain of the various mouse models. In addition, using immunohistochemistry, both beta-pleated sheet formation in Aß deposits and the presence of reactive astrocytes associated with Aß deposits were analysed. CONCLUSIONS: We found that absence of TG2 reduces the formation of Aß pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aß deposition in AD.

The Transglutaminase-2 Interactome in the APP23 Mouse Model of Alzheimer's Disease.

Amyloid-beta (Aß) deposition in the brain is closely linked with the development of Alzheimer's disease (AD). Unfortunately, therapies specifically targeting Aß deposition have failed to reach their primary clinical endpoints, emphasizing the need to broaden the search strategy for alternative targets/mechanisms. Transglutaminase-2 (TG2) catalyzes post-translational modifications, is present in AD lesions and interacts with AD-associated proteins. However, an unbiased overview of TG2 interactors is lacking in both control and AD brain. Here we aimed to identify these interactors using a crossbreed of the AD-mimicking APP23 mouse model with wild type and TG2 knock-out (TG2-/-) mice. We found that absence of TG2 had no (statistically) significant effect on Aß pathology, soluble brain levels of Aß1-40 and Aß1-42, and mRNA levels of TG family members compared to APP23 mice at 18 months of age. Quantitative proteomics and network analysis revealed a large cluster of TG2 interactors involved in synaptic transmission/assembly and cell adhesion in the APP23 brain typical of AD. Comparative proteomics of wild type and TG2-/- brains revealed a TG2-linked pathological proteome consistent with alterations in both pathways. Our data show that TG2 deletion leads to considerable network alterations consistent with a TG2 role in (dys)regulation of synaptic transmission and cell adhesion in APP23 brains.

The thermodynamic theory of action potential propagation: a sound basis for unification of the physics of nerve impulses.

The thermodynamic theory of action potential propagation challenges the conventional understanding of the nerve signal as an exclusively electrical phenomenon. Often misunderstood as to its basic tenets and predictions, the thermodynamic theory is virtually ignored in mainstream neuroscience. Addressing a broad audience of neuroscientists, we here attempt to stimulate interest in the theory. We do this by providing a concise overview of its background, discussion of its intimate connection to Albert Einstein's treatment of the thermodynamics of interfaces and outlining its potential contribution to the building of a physical brain theory firmly grounded in first principles and the biophysical reality of individual nerve cells. As such, the paper does not attempt to advocate the superiority of the thermodynamic theory over any other approach to model the nerve impulse, but is meant as an open invitation to the neuroscience community to experimentally test the assumptions and predictions of the theory on their validity.

Tissue Transglutaminase Expression Associates With Progression of Multiple Sclerosis.

OBJECTIVE: The clinical course of multiple sclerosis (MS) is variable and largely unpredictable pointing to an urgent need for markers to monitor disease activity and progression. Recent evidence revealed that tissue transglutaminase (TG2) is altered in patient-derived monocytes. We hypothesize that blood cell-derived TG2 messenger RNA (mRNA) can potentially be used as biomarker in patients with MS. METHODS: In peripheral blood mononuclear cells (PBMCs) from 151 healthy controls and 161 patients with MS, TG2 mRNA was measured and correlated with clinical and MRI parameters of disease activity (annualized relapse rate, gadolinium-enhanced lesions, and T2 lesion volume) and disease progression (Expanded Disability Status Scale [EDSS], normalized brain volume, and hypointense T1 lesion volume). RESULTS: PBMC-derived TG2 mRNA levels were significantly associated with disease progression, i.e., worsening of the EDSS over 2 years of follow-up, normalized brain volume, and normalized gray and white matter volume in the total MS patient group at baseline. Of these, in patients with relapsing-remitting MS, TG2 expression was significantly associated with worsening of the EDSS scores over 2 years of follow-up. In the patients with primary progressive (PP) MS, TG2 mRNA levels were significantly associated with EDSS, normalized brain volume, and normalized gray and white matter volume at baseline. In addition, TG2 mRNA associated with T1 hypointense lesion volume in the patients with PP MS at baseline. CONCLUSION: PBMC-derived TG2 mRNA levels hold promise as biomarker for disease progression in patients with MS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with MS, PBMC-derived TG2 mRNA levels are associated with disease progression.

Lower scores in pharmacokinetics than in pharmacodynamics assessment among students in a health and life sciences curriculum.

In pharmacology teaching, pharmacokinetics (PK) and pharmacodynamics (PD) may be defined as part of the 'general pharmacology' domain, whereas effects of drugs on the autonomic nervous system and clinical trial design might be defined as part of the 'medical' and 'clinical' pharmacology domain, respectively. We recently designed a pharmacology course covering these domains for second year Health and Life Sciences students at the Vrije Universiteit Amsterdam (VU). We used a combination of lectures, problem-based learning and practicals to transfer knowledge to students in order for them to acquire sufficient knowledge and insight to solve real-world pharmacological problems. To evaluate whether we 1) successfully aligned our course objectives with both our teaching strategy and assessment, and 2) to identify topics in our course that would benefit from improvement in teaching strategy and/or effort, we determined success rate of the exam questions in above-defined pharmacology domains. We analyzed 3 consecutive second year cohorts (n = 377) of students enrolled in our course, and found a statistically significant reduction in success rate in exam questions of the general pharmacology domain (especially in PK), compared to domains covering 'medical' and 'clinical' pharmacology. In addition, we found lower success rates for 'knows how' questions compared to 'knows' questions in the combined PK/PD domain. Our data show that we overall succeeded in aligning our course objectives with both our teaching strategy and assessment, but that outcomes on the PK domain might benefit from additional attention.

Tissue Transglutaminase contributes to myelin phagocytosis in interleukin-4-treated human monocyte-derived macrophages.

Macrophages exert either a detrimental or beneficial role in Multiple Sclerosis (MS) pathology, depending on their inflammatory environment. Tissue Transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been described as a novel marker for anti-inflammatory, interleukin-4 (IL-4) polarized macrophages (M(IL-4)), which represent a subpopulation of macrophages with phagocytic abilities. Since TG2 is expressed in macrophages in active human MS lesions, we questioned whether TG2 drives the differentiation of M(IL-4) into an anti-inflammatory phenotype and whether it plays a role in the phagocytosis of myelin by these cells. In macrophage-differentiated THP-1 monocytes, TG2 was increased upon IL-4 treatment. Reducing TG2 expression impairs the differentiation of M(IL-4) macrophages into an anti-inflammatory phenotype and drives them into a pro-inflammatory state. In addition, reduced TG2 expression resulted in increased presence of myelin basic protein in macrophages upon myelin exposure of M(IL-4) macrophages. Moreover, the elevated presence of an early endosome marker and equal expression of a lysosome marker compared to control macrophages, suggest that TG2 plays a role in phagosome maturation in M(IL-4) macrophages These data suggest that tuning macrophages into TG2 producing anti-inflammatory cells by IL-4 treatment may benefit effective myelin phagocytosis in e.g. demyelinating MS lesions and open avenues for successful regeneration.

Interaction between tissue transglutaminase and amyloid-beta: Protein-protein binding versus enzymatic crosslinking.

Self-interaction, chaperone binding and posttranslational modification of amyloid-beta (Aß) is essential in the initiation and propagation of Aß aggregation. Aggregation results in insoluble Aß deposits characteristic of Alzheimer's disease (AD) brain lesions, i.e. senile plaques and cerebral amyloid angiopathy. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes posttranslational modifications including the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds (molecular crosslinks), and colocalizes with Aß deposits in AD. Two independent groups recently found that apart from the induction of Aß oligomerization, the blood-derived transglutaminase member FXIIIa forms stable protein-protein complexes with Aß independent of the transamidation reaction. Here, we investigated whether also tTG forms rigid protein complexes with Aß in the absence of catalytic activation. We found that both Aß1-40 and Aß1-42 are substrates for tTG-catalyzed crosslinking. In addition, in the absence of calcium or the presence of a peptidergic inhibitor of tTG, stable tTG-Aß1-40 complexes were found. Interestingly, the stable complexes between tTG and Aß1-40, were only found at 'physiological' concentrations of Aß1-40. Together, our data suggest that depending on the Aß species at hand, and on the concentration of Aß, rigid protein-complexes are formed between tTG and Aß1-40 without the involvement of the crosslinking reaction.

Tissue Transglutaminase Appears in Monocytes and Macrophages but Not in Lymphocytes in White Matter Multiple Sclerosis Lesions.

Leukocyte infiltration is an important pathological hallmark of multiple sclerosis (MS) and is therefore targeted by current MS therapies. The enzyme tissue transglutaminase (TG2) contributes to monocyte/macrophage migration and is present in MS lesions and could be a potential therapeutic target. We examined the cellular identity of TG2-expressing cells by immunohistochemistry in white matter lesions of 13 MS patients; 9 active and chronic active lesions from 4 patients were analyzed in detail. In these active MS lesions, TG2 is predominantly expressed in leukocytes (CD45+) but not in cells of the lymphocyte lineage, that is, T cells (CD3+) and B cells (CD20+). In general, cells of the monocyte/macrophage lineage (CD11b+ or CD68+) are TG2+ but no further distinction could be made regarding pro- or anti-inflammatory macrophage subtypes. In conclusion, TG2 is abundantly present in cells of the monocyte/macrophage lineage in active white matter MS lesions. We consider that TG2 can play a role in MS as it is associated with macrophage infiltration into the CNS. As such, TG2 potentially presents a novel target for therapeutic intervention that can support available MS therapies targeting lymphocyte infiltration.

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

[This corrects the article DOI: 10.1371/journal.pone.0196433.].

Differential Expression of Tissue Transglutaminase Splice Variants in Peripheral Blood Mononuclear Cells of Primary Progressive Multiple Sclerosis Patients.

Multiple Sclerosis (MS) is an inflammatory and neurodegenerative disorder of the central nervous system (CNS) characterized by inflammation and immune cell infiltration in the brain parenchyma. Tissue transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been shown to be present in infiltrating MHC-II positive cells in lesions of patients suffering from MS. Moreover, TG2 mRNA levels in peripheral blood mononuclear cells (PBMC)-derived from primary progressive (PP)-MS patients correlated with clinical parameters, thus highlighting the importance of TG2 in MS pathology. In the present study, we further characterized TG2 expression by measuring the mRNA levels of full-length TG2 and four TG2 alternative splice variants in PBMCs derived from PP-MS patients and healthy control (HC) subjects. In PP-MS-derived PBMCs, TG2 variant V4b was significantly higher expressed, and both V4a and V4b variants were relatively more expressed in relation to full-length TG2. These observations open new avenues to unravel the importance of TG2 alternative splicing in the pathophysiology of PP-MS.

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.

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.

Monocyte-derived tissue transglutaminase in multiple sclerosis patients: reflecting an anti-inflammatory status and function of the cells?

BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.

Enhanced interstitial fluid drainage in the hippocampus of spontaneously hypertensive rats.

Hypertension is associated with cognitive decline and various forms of dementia, including Alzheimer's disease. In animal models of hypertension, many of Alzheimer's disease characteristics are recapitulated, including brain atrophy, cognitive decline, amyloid ß accumulation and blood brain barrier dysfunction. Removal of amyloid ß and other waste products depends in part on clearance via the brain interstitial fluid (ISF). Here we studied the impact of hypertension on ISF drainage, using spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). At 8 months, high (500 kD) and low (3 kD) fluorescent molecular weight tracers released passively into the hippocampus showed a drastically enhanced spreading in SHR. Tracer spreading was inhomogeneous, with accumulation at ISF-CSF borders, around arteries, and towards the stratum lacunosum moleculare. These locations stained positively for the astrocyte marker GFAP, and aquaporin 4. Despite enhanced dispersion, clearance of tracers was not affected in SHR. In conclusion, these data indicate enhanced bulk flow of ISF in the hippocampus of hypertensive rats. ISF drains along astrocytes towards the cerebrospinal fluid compartment, which leads to sieving of high molecular weight solutes. Sieving may lead to a local increase in the concentration of waste products and potentially promotes the aggregation of amyloid ß.

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.

Development of fluorine-18 labeled peptidic PET tracers for imaging active tissue transglutaminase.

INTRODUCTION: The protein-protein crosslinking activity of the enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including celiac disease, lung-, liver- and kidney fibrosis, cancer and neurodegenerative diseases. This study aims at developing a TG2 PET tracer based on the peptidic irreversible TG2 inhibitor Z006. METHODS: Initially, the carbon-11 labeling of Z006 at the diazoketone position was explored. Subsequently, a set of analogues that allow for fluorine-18 labeling was synthesized. Two potent analogues, 6f and 6g, were radiolabeled with fluorine-18 and biodistribution and metabolite analysis in Wistar rats was performed. The identity of the main metabolite of [18F]6g was elucidated using LC-MS/MS. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 breast cancer tissue using [18F]6g was performed. RESULTS: [18F]6f and [18F]6g were obtained in 20 and 9% yields, respectively. Following administration to healthy Wistar rats, rapid metabolism of both tracers was observed. Remarkably, full conversion to just one single metabolite was observed for one of the tracers, [18F]6g. By LC-MS/MS analysis this metabolite was identified as C-terminally saponified [18F]6g. This metabolite was also found to be a potent TG2 inhibitor in vitro. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 tumor sections using [18F]6g demonstrated high specific and selective binding of [18F]6g to active TG2. CONCLUSIONS: Whereas based on the intensive metabolism [18F]6f seems unsuitable as a TG2 PET tracer, the results warrant further evaluation of [18F]6gin vivo.

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.