No abstract

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

SummaryFrontotemporal dementia (FTD) is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs) from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.

The time course of inactivation of voltage-activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvb subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce 'open-channel block'. Open-channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid-induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (Ih). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and Ih properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that Ih was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1–4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of Ih found in WAG/Rij rats compensate each other in a way that leaves Ih availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of Ih on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in Ih in older animals.

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.

Key pointsr During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively.r The modulation of specific K + channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity.r In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. r These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. r The present report introduces PIP 2 and DAG as new elements of signal transduction in the thalamus. AbstractThe activity of two-pore domain potassium channels (K 2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K + channel (TWIK)-related acid-sensitive K + (TASK) channels and TWIK-related K + (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCβ), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and diacylglycerol (DAG) acting downstream of PLCβ was probed. The standing outward current (I SO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP 2 level in TC neurons, we here show that the scavenging of PIP 2 (by neomycin) results in an increased muscarinic effect on I SO whereas increased availability of PIP 2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP 2 but no other S. G. Meuth and T. Budde contributed equally to this work. phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP 2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCβ as well as the application of OAG resulted in membrane depolarization, while PIP 2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP 2 and DAG in K 2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP 2 depletion) and TASK (via DAG) channels following MAChR stimulation.

The membrane potential is mainly maintained by the K(+) concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high-molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration-dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high-molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high-molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high-molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism.