Inger Lauritzen

Polyunsaturated fatty acids are potent neuroprotectors

Results reported in this work suggest a potential therapeutic value of polyunsaturated fatty acids for cerebral pathologies as previously proposed by others for cardiac diseases. We show that the polyunsaturated fatty acid linolenic acid prevents neuronal death in an animal model of transient global ischemia even when administered after the insult. Linolenic acid also protects animals treated with kainate against seizures and hippocampal lesions. The same effects have been observed in an in vitro model of seizure‐like activity using glutamatergic neurons and they have been shown to be associated with blockade of glutamatergic transmission by low concentrations of distinct polyunsaturated fatty acids. Our data suggest that the opening of background K+ channels, like TREK‐1 and TRAAK, which are activated by arachidonic acid and other polyunsaturated fatty acids such as docosahexaenoic acid and linolenic acid, is a significant factor in this neuroprotective effect. These channels are abundant in the brain where they are located both pre‐ and post‐synaptically, and are insensitive to saturated fatty acids, which offer no neuroprotection.

TREK‐1 is a heat‐activated background K+ channel

Peripheral and central thermoreceptors are involved in sensing ambient and body temperature, respectively. Specialized cold and warm receptors are present in dorsal root ganglion sensory fibres as well as in the anterior/preoptic hypothalamus. The two‐pore domain mechano‐gated K+ channel TREK‐1 is highly expressed within these areas. Moreover, TREK‐1 is opened gradually and reversibly by heat. A 10°C rise enhances TREK‐1 current amplitude by ∼7‐fold. Prostaglandin E2 and cAMP, which are strong sensitizers of peripheral and central thermoreceptors, reverse the thermal opening of TREK‐1 via protein kinase A‐mediated phosphorylation of Ser333. Expression of TREK‐1 in peripheral sensory neurons as well as in central hypothalamic neurons makes this K+ channel an ideal candidate as a physiological thermoreceptor.

Polycystin-1 and -2 Dosage Regulates Pressure Sensing

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.

A phospholipid sensor controls mechanogating of the K + channel TREK‐1

TREK‐1 (KCNK2 or K2P2.1) is a mechanosensitive K2P channel that is opened by membrane stretch as well as cell swelling. Here, we demonstrate that membrane phospholipids, including PIP2, control channel gating and transform TREK‐1 into a leak K+ conductance. A carboxy‐terminal positively charged cluster is the phospholipid‐sensing domain that interacts with the plasma membrane. This region also encompasses the proton sensor E306 that is required for activation of TREK‐1 by cytosolic acidosis. Protonation of E306 drastically tightens channel–phospholipid interaction and leads to TREK‐1 opening at atmospheric pressure. The TREK‐1–phospholipid interaction is critical for channel mechano‐, pHi‐ and voltage‐dependent gating.

The KCNQ2 potassium channel: splice variants, functional and developmental expression. Brain localization and comparison with KCNQ3

Benign familial neonatal convulsions, an autosomal dominant epilepsy of newborns, are linked to mutations affecting two six‐transmembrane potassium channels, KCNQ2 and KCNQ3. We isolated four splice variants of KCNQ2 in human brain. Two forms generate, after transient expression in COS cells, a potassium‐selective current similar to the KCNQ1 current. L‐735,821, a benzodiazepine molecule which inhibits the KCNQ1 channel activity (EC50=0.08 μM), also blocks KCNQ2 currents (EC50=1.5 μM). Using in situ hybridization, KCNQ2 and KCNQ3 have been localized within the central nervous system, in which they are expressed in the same areas, mainly in the hippocampus, the neocortex and the cerebellar cortex. During brain development, KCNQ3 is expressed later than KCNQ2.

The structure, function and distribution of the mouse TWIK-1 K+ channel

The two P domain K+ channel mTWIK‐1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK‐1 currents are K+‐selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK‐1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK‐1 subunits dimerize via a disulfide bridge. TWIK‐1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK‐1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.

Cross-talk between the mechano-gated K2P channel TREK-1 and the actin cytoskeleton.

TREK‐1 (KCNK2) is a K2P channel that is highly expressed in fetal neurons. This K+ channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK‐1 markedly alters the cytoskeletal network and induces the formation of actin‐ and ezrin‐rich membrane protrusions. The genetic inactivation of TREK‐1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK‐1 mechano‐sensitivity. Thus, the dialogue between TREK‐1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.

Expression of group II phospholipase A2 in rat brain after severe forebrain ischemia and in endotoxic shock

Secretory phospholipase A2 type II is known to be involved in various inflammatory processes. This paper describes the changes in secretory phospholipase A2 (PLA2) gene expression induced by ischemia and endotoxic shock. Type I PLA2 (pancreatic type) is not expressed in ischemic and endotoxic-shock brains but both ischemia and endotoxin injection induce type II PLA2 expression. The first phase of PLA2 II gene expression following the ischemic insult occurs 1-6 h after ischemia. During that period, PLA2 II gene expression is slightly enhanced and it returns to control levels after 1 day. A second phase corresponding to higher levels of induction of mRNA for PLA2 appears at a later period after ischemia between 7 and 18 days. In situ hybridization shows that PLA2 gene expression in the ischemic brain is localized in regions known to be vulnerable to ischemia (hippocampus and neocortex). Endotoxic shock which leads to a major inflammatory state induces an abundant expression of the PLA2 II mRNA in the brain and this high level of expression appears in a large number of brain structures. The results suggest that the early phase of ischemia-induced PLA2 gene expression could be an additional element in mechanisms leading to neuronal death. The later phase of increased PLA2 mRNA levels is more probably related to the inflammatory response associated to neuronal degeneration.

Ryanodine Receptor Blockade Reduces Amyloid-β Load and Memory Impairments in Tg2576 Mouse Model of Alzheimer Disease

In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP695), or APP harboring the Swedish double mutation (APPswe) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.

An immunocytochemical study on the distribution of two G-protein-gated inward rectifier potassium channels (GIRK2 and GIRK4) in the adult rat brain.

G-protein-gated inward rectifier potassium channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain, and were recently shown to be candidates for genetic mutations leading to neuronal cell death. This report describes the localization of G-protein-gated inward rectifier potassium channel-2 and G-protein-gated inward rectifier potassium channel-4 proteins in the rat brain, as assessed by immunocytochemistry. G-protein-gated inward rectifier potassium channel-2 immunoreactivity was widely distributed throughout the brain, with the strongest staining seen in the hippocampus, septum, granule cell layer of the cerebellum, amygdala and substantia nigra pars compacta. In contrast, G-protein-gated inward rectifier potassium channel-4 immunoreactivity was restricted to some neuronal populations, such as Purkinje cells and neurons of the globus pallidus and the ventral pallidum. The presence of G-protein-gated inward rectifier potassium channel-2 immunoreactivity in substantia nigra pars compacta dopaminergic neurons was confirmed by showing its co-localization with tyrosine hydroxylase by double immunocytochemistry, and also by selectively lesioning dopaminergic neurons with the neurotoxin 6-hydroxydopamine. At the cellular level both proteins were localized in neuronal cell bodies and dendrites, but clear differences were seen in the degree of dendritic staining among neuronal groups. For some neuronal groups the staining of distal dendrites (notably dendritic spines) was strong, while for others the cell body and proximal dendrites were preferentially labelled. In addition, some of the results suggest that G-protein-gated inward rectifier potassium channel-2 protein could be localized in distal axonal terminal fields. A knowledge of the distribution of G-protein-gated inward rectifier potassium channel proteins in the brain could help to elucidate their physiological roles and to evaluate their potential involvement in neurodegenerative processes in animal models and human diseases.