Alexander M. Herrmann

The TASK1 channel inhibitor A293 shows efficacy in a mouse model of multiple sclerosis

The two-pore domain potassium channel TASK1 (KCNK3) has recently emerged as an important modulator in autoimmune CNS inflammation. Previously, it was shown that T lymphocytes obtained from TASK1(-/-) mice display impaired T cell effector functions and that TASK1(-/-) mice show a significantly reduced disease severity in myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We here evaluate a potent and specific TASK1 channel inhibitor, A293, which caused a dose-dependent reduction of T cell effector functions (cytokine production and proliferation). This effect was abolished in CD4(+) T cells from TASK1(-/-) mice but not in cells from TASK3(-/-) mice. In electrophysiological measurements, A293 application induced a significant reduction of the outward current of wildtype T lymphocytes, while there was no effect in TASK1(-/-) cells. Preventive and therapeutic application of A293 significantly ameliorated the EAE disease course in wildtype mice while it had no significant effect in TASK1(-/-) mice and was still partly effective in TASK3(-/-) mice. In summary, our findings support the concept of TASK1 as an attractive drug target for autoimmune disorders.

Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes

Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K(V)1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K(2P)) channels in the RVD of human CD4(+) T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K(2P)5.1 and K(2P)18.1 as the most important K(2P) channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K(2P)5.1 and K(2P)18.1 on the RVD was comparable to that of K(V)1.3. K(2P)9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K(2P) channels.

Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

IntroductionCD4+ T cells express K2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K2P5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K2P5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients.MethodsExpression levels of K2P5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K2P5.1.ResultsK2P5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K2P5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K2P5.1 expression to disease activity parameters during a longitudinal follow-up for six months.ConclusionsDisease activity in RA patients correlates strongly with K2P5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K2P5.1 as a potential biomarker for disease activity and differential diagnosis.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

BackgroundNeural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes.MethodsWild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations.ResultsFollowing T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter.ConclusionGlutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

4-Aminopyridine ameliorates mobility but not disease course in an animal model of multiple sclerosis ☆

Neuropathological changes following demyelination in multiple sclerosis (MS) lead to a reorganization of axolemmal channels that causes conduction changes including conduction failure. Pharmacological modulation of voltage-sensitive potassium channels (K(V)) has been found to improve conduction in experimentally induced demyelination and produces symptomatic improvement in MS patients. Here we used an animal model of autoimmune inflammatory neurodegeneration, namely experimental autoimmune encephalomyelitis (EAE), to test the influence of the K(V)-inhibitor 4-aminopyridine (4-AP) on various disease and immune parameters as well as mobility in MOG₃₅₋₅₅ immunized C57Bl/6 mice. We challenged the hypothesis that 4-AP exerts relevant immunomodulatory or neuroprotective properties. Neither prophylactic nor therapeutic treatment with 4-AP altered disease incidence or disease course of EAE. Histopathological signs of demyelination and neuronal damage as well as MRI imaging of brain volume changes were unaltered. While application of 4-AP significantly reduced the standing outward current of stimulated CD4(+) T cells compared to controls, it failed to impact intracellular calcium concentrations in these cells. Compatibly, KV channel inhibition neither influenced CD4(+) T cell effector functions (proliferation, IL17 or IFNγ production). Importantly however, despite equal disease severity scores 4-AP treated animals showed improved mobility as assessed by 2 independent methods, 1) foot print and 2) rotarod analysis (0.332 ± 0.03, n=7 versus 0.399 ± 0.08, n=14, p<0.001, respectively). Our data suggest that 4-AP while having no apparent immunomodulatory or direct neuroprotective effects, significantly ameliorates conduction abnormalities thereby improving gait and coordination. Improvement of mobility in this experimental model supports trial data and clinical experience with 4-AP in the symptomatic treatment of MS.

Glatiramer Acetate Attenuates Pro-Inflammatory T Cell Responses but Does Not Directly Protect Neurons from Inflammatory Cell Death

Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.

Melanocortin-1 receptor activation is neuroprotective in mouse models of neuroinflammatory disease

The approved drug NDP-MSH ameliorates neurodegeneration in mouse models of neuroinflammatory disease. A repurposed drug for neuroinflammatory disease Treatment of inflammation-associated progressive neurodegenerative disorders, such as relapsing remitting multiple sclerosis, is challenging. In a new study, Mykicki et al. demonstrate that the drug NDP-MSH, currently approved for treating porphyria, showed potent anti-inflammatory and neuroprotective effects in two mouse models of neuroinflammatory disease. The drug, acting via the melanocortin-1 and orphan nuclear 4 receptors, prevented neuronal cell death by inducing regulatory T cells and preventing the infiltration of pathogenic T cells into the central nervous system. In inflammation-associated progressive neuroinflammatory disorders, such as multiple sclerosis (MS), inflammatory infiltrates containing T helper 1 (TH1) and TH17 cells cause demyelination and neuronal degeneration. Regulatory T cells (Treg) control the activation and infiltration of autoreactive T cells into the central nervous system (CNS). In MS and experimental autoimmune encephalomyelitis (EAE) in mice, Treg function is impaired. We show that a recently approved drug, Nle4-d-Phe7–α-melanocyte–stimulating hormone (NDP-MSH), induced functional Treg, resulting in amelioration of EAE progression in mice. NDP-MSH also prevented immune cell infiltration into the CNS by restoring the integrity of the blood-brain barrier. NDP-MSH exerted long-lasting neuroprotective effects in mice with EAE and prevented excitotoxic death and reestablished action potential firing in mouse and human neurons in vitro. Neuroprotection by NDP-MSH was mediated via signaling through the melanocortin-1 and orphan nuclear 4 receptors in mouse and human neurons. NDP-MSH may be of benefit in treating neuroinflammatory diseases such as relapsing-remitting MS and related disorders.

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis.

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.

Metabolic and Homeostatic Changes in Seizures and Acquired Epilepsy—Mitochondria, Calcium Dynamics and Reactive Oxygen Species

Acquired epilepsies can arise as a consequence of brain injury and result in unprovoked seizures that emerge after a latent period of epileptogenesis. These epilepsies pose a major challenge to clinicians as they are present in the majority of patients seen in a common outpatient epilepsy clinic and are prone to pharmacoresistance, highlighting an unmet need for new treatment strategies. Metabolic and homeostatic changes are closely linked to seizures and epilepsy, although, surprisingly, no potential treatment targets to date have been translated into clinical practice. We summarize here the current knowledge about metabolic and homeostatic changes in seizures and acquired epilepsy, maintaining a particular focus on mitochondria, calcium dynamics, reactive oxygen species and key regulators of cellular metabolism such as the Nrf2 pathway. Finally, we highlight research gaps that will need to be addressed in the future which may help to translate these findings into clinical practice.

The quality of cortical network function recovery depends on localization and degree of axonal demyelination.

Myelin loss is a severe pathological hallmark common to a number of neurodegenerative diseases, including multiple sclerosis (MS). Demyelination in the central nervous system appears in the form of lesions affecting both white and gray matter structures. The functional consequences of demyelination on neuronal network and brain function are not well understood. Current therapeutic strategies for ameliorating the course of such diseases usually focus on promoting remyelination, but the effectiveness of these approaches strongly depends on the timing in relation to the disease state. In this study, we sought to characterize the time course of sensory and behavioral alterations induced by de- and remyelination to establish a rational for the use of remyelination strategies. By taking advantage of animal models of general and focal demyelination, we tested the consequences of myelin loss on the functionality of the auditory thalamocortical system: a well-studied neuronal network consisting of both white and gray matter regions. We found that general demyelination was associated with a permanent loss of the tonotopic cortical organization in vivo, and the inability to induce tone-frequency-dependent conditioned behaviors, a status persisting after remyelination. Targeted, focal lysolecithin-induced lesions in the white matter fiber tract, but not in the gray matter regions of cortex, were fully reversible at the morphological, functional and behavioral level. These findings indicate that remyelination of white and gray matter lesions have a different functional regeneration potential, with the white matter being able to regain full functionality while cortical gray matter lesions suffer from permanently altered network function. Therefore therapeutic interventions aiming for remyelination have to consider both region- and time-dependent strategies.