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The processing of the amyloid precursor protein (APP) is one of the key events contributing to Alzheimer’s disease (AD) etiology. Canonical cleavages by β- and γ-secretases lead to Aβ production which accumulate in amyloid plaques. Recently, the matrix metalloprotease MT5-MMP, referred to as η-secretase, has been identified as a novel APP cleaving enzyme producing a transmembrane fragment, ηCTF that undergoes subsequent cleavages by α- and β-secretases yielding the Aηα and Aηβ peptides, respectively. The functions and contributions of ηCTF and its related fragments to AD pathology are poorly understood. In this study, we designed a novel immunological probe referred to as ηCTF-NTer antibody that specifically interacts with the N-terminal part of ηCTF targeting ηCTF, Aηα, Aηβ but not C99, C83 and Aβ. We examined the fate and localization of ηCTF fragment in various cell models and in mice. We found that overexpressed ηCTF undergoes degradation in the proteasomal and autophagic pathways and accumulates mainly in the Golgi and in endosomes. Moreover, we observed the presence of ηCTF in small extracellular vesicles (sEVs) purified from neuroblastoma cells or from mouse brains expressing ηCTF. Importantly, the expression of ηCTF in fibroblasts devoid on APP leads to Aβ production demonstrating its contribution to the amyloidogenic pathway. Finally, we observed an ηCTF-like immunoreactivity around amyloid plaques and an age-dependent accumulation of ηCTF in the triple-transgenic mouse AD model. Thus, our study suggests that the ηCTF fragment likely contributes to AD pathology by its exosomal spreading and involvement in Aβ production

© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way., Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer’s disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the β-secretase-derived APP-CTF fragment (C99) combined with β- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aβ triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aβ to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD. Electronic supplementary material The online version of this article (10.1007/s00401-020-02234-7) contains supplementary material, which is available to authorized users.

Background: Alzheimer’s disease (AD) is associated with extracellular accumulation and aggregation of amyloid β (Aβ) peptides ultimately seeding in senile plaques. Recent data show that their direct precursor C99 (βCTF) also accumulates in AD-affected brain as well as in AD-like mouse models. C99 is consistently detected much earlier than Aβ, suggesting that this metabolite could be an early contributor to AD pathology. C99 accumulates principally within endolysosomal and autophagic structures and its accumulation was described as both a consequence and one of the causes of endolysosomalautophagic pathology, the occurrence of which has been documented as an early defect in AD. C99 was also accompanied by C99-derived C83 (αCTF) accumulation occurring within the same intracellular organelles. Both these CTFs were found to dimerize leading to the generation of higher molecular weight CTFs, which were immunohistochemically characterized in situ by means of aggregate-specific antibodies. Discussion: Here, we discuss studies demonstrating a direct link between the accumulation of C99 and C99-derived APP-CTFs and early neurotoxicity. We discuss the role of C99 in endosomal-lysosomalautophagic dysfunction, neuroinflammation, early brain network alterations and synaptic dysfunction as well as in memory-related behavioral alterations, in triple transgenic mice as well as in newly developed AD animal models. Conclusion: This review summarizes current evidence suggesting a potential role of the β -secretasederived APP C-terminal fragment C99 in Alzheimer’s disease etiology

Genetic, biochemical, and anatomical grounds led to the proposal of the amyloid cascade hypothesis centered on the accumulation of amyloid beta peptides (Aβ) to explain Alzheimer's disease (AD) etiology. In this context, a bulk of efforts have aimed at developing therapeutic strategies seeking to reduce Aβ levels, either by blocking its production (γ- and β-secretase inhibitors) or by neutralizing it once formed (Aβ-directed immunotherapies). However, so far the vast majority of, if not all, clinical trials based on these strategies have failed, since they have not been able to restore cognitive function in AD patients, and even in many cases, they have worsened the clinical picture. We here propose that AD could be more complex than a simple Aβ-linked pathology and discuss the possibility that a way to reconcile undoubted genetic evidences linking processing of APP to AD and a consistent failure of Aβ-based clinical trials could be to envision the pathological contribution of the direct precursor of Aβ, the β-secretase-derived C-terminal fragment of APP, βCTF, also referred to as C99. In this review, we summarize scientific evidences pointing to C99 as an early contributor to AD and postulate that γ-secretase should be considered as not only an Aβ-generating protease, but also a beneficial C99-inactivating enzyme. In that sense, we discuss the limitations of molecules targeting γ-secretase and propose alternative strategies seeking to reduce C99 levels by other means and notably by enhancing its lysosomal degradation.

Brains that are affected by Alzheimer&rsquo, s disease (AD) are characterized by the overload of extracellular amyloid &beta, (A&beta, ) peptides, but recent data from cellular and animal models propose that A&beta, deposition is preceded by intraneuronal accumulation of the direct precursor of A&beta, C99. These studies indicate that C99 accumulation firstly occurs within endosomal and lysosomal compartments and that it contributes to early-stage AD-related endosomal-lysosomal-autophagic defects. Our previous work also suggests that C99 accumulation itself could be a consequence of defective lysosomal-autophagic degradation. Thus, in the present study, we analyzed the influence of the overexpression of the transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, on C99 accumulation occurring in both AD cellular models and in the triple-transgenic mouse model (3xTgAD). In the in vivo experiments, TFEB overexpression was induced via adeno-associated viruses (AAVs), which were injected either into the cerebral ventricles of newborn mice or administrated at later stages (3 months of age) by stereotaxic injection into the subiculum. In both cells and the 3xTgAD mouse model, exogenous TFEB strongly reduced C99 load and concomitantly increased the levels of many lysosomal and autophagic proteins, including cathepsins, key proteases involved in C99 degradation. Our data indicate that TFEB activation is a relevant strategy to prevent the accumulation of this early neurotoxic catabolite.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/− /PS1 +/−). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

BackgroundWe recently demonstrated an endolysosomal accumulation of the β-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Moreover, we showed that the treatment with the γ-secretase inhibitor (D6) led to further increased endolysosomal APP-CTF levels, but also revealed extracellular APP-CTF-associated immunostaining. We here hypothesized that this latter staining could reflect extracellular vesicle (EV)-associated APP-CTFs and aimed to characterize these γ-secretase inhibitor-induced APP-CTFs.MethodsEVs were purified from cell media or mouse brains from vehicle- or D6-treated C99 or APPswedishexpressing cells/mice and analyzed for APP-CTFs by immunoblot. Combined pharmacological, immunological and genetic approaches (presenilin invalidation and C99 dimerization mutants (GXXXG)) were used to characterize vesicle-containing APP-CTFs. Subcellular APP-CTF localization was determined by immunocytochemistry.ResultsPurified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains. Surprisingly, EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight (HMW) APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts. Accordingly, the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869. By combined pharmacological, immunological and genetic approaches, we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83. Immunocytochemical analysis showed that monomers were localized mainly to thetrans-Golgi network, whereas oligomers were confined to endosomes and lysosomes, thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs. The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due to γ-secretase blockade, since it similarly occurred in presenilin-deficient fibroblasts. Further, our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis, γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN.ConclusionsThis is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models, the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified by γ-secretase inhibition. Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset, progression and spreading.

International audience; Recent studies have suggested deep brain stimulation (DBS) as a promising therapy in patients with Alzheimer's disease (AD). Particularly, the stimulation of the forniceal area was found to slow down the cognitive decline of some AD patients, but the biochemical and anatomical modifications underlying these effects remain poorly understood. We evaluated the effects of chronic forniceal stimulation on amyloid burden, inflammation, and neuronal loss in a transgenic Alzheimer rat model TgF344-AD, as well as in age-matched control rats. 18-month-old rats were surgically implanted with electrodes in stereotactic conditions and connected to a portable microstimulator for chronic DBS in freely moving rats. The stimulation was continuous during 5 weeks and animals were immediately sacrificed for immunohistochemical analysis of pathological markers. Implanted, but non-stimulated rats were used as controls. We found that chronic forniceal DBS in the Tg-AD rat significantly reduces amyloid deposition in the hippocampus and cortex, decreases astrogliosis and microglial activation and lowers neuronal loss, as determined by NeuN staining. In control animals, the stimulation neither affects neuroinflammation nor neuronal count. In the Tg-F344-AD rat model, 5 weeks of forniceal DBS decreased amyloidosis, inflammatory responses, and neuronal loss in both cortex and hippocampus. These findings strongly suggest a neuroprotective effect of DBS and support the beneficial effects of targeting the fornix in Alzheimer's disease patients.

Neuronal network dysfunction and cognitive decline constitute the most prominent features of Alzheimer’s disease (AD), although mechanisms causing such impairments are yet to be determined. Here we report that virus-mediated delivery of the active spliced transcription factor X-Box binding protein 1s (XBP1s) in the hippocampus rescued spine density, synaptic plasticity and memory function in a mouse model of AD. XBP1s transcriptionally activated Kalirin-7 (Kal7), a protein that controls synaptic plasticity. In addition, we found reduced levels of Kal7 in primary neurons exposed to Aβ oligomers, transgenic mouse models and human AD brains. Short hairpin RNA-mediated knockdown of Kal7 altered synaptic plasticity and memory formation in naive mice. Further, reduction of endogenous Kal7 compromised the beneficial effects of XBP1s in Alzheimer’s model. Hence, our findings reveal that XBP1s is neuroprotective through a mechanism that engages Kal7 pathway with therapeutic implications in AD pathology.

International audience; Keywords: C99 Ab 2ÂTgAD 3ÂTgAD Alzheimer's disease Behavior Actimetry Morris water maze a b s t r a c t The triple transgenic mouse model (3ÂTgAD: APPswe, Tau P301L , PS1 M146V) recapitulates both amyloid b (Ab)-and tau-related lesions as well as synaptic and memory deficits. In these mice, we reported an early apathy-like behavior and alterations in synaptic plasticity appearing concomitantly with intraneuronal accumulation of C99 in the subiculum. To delineate the genuine contribution of C99 on the above phenotypes, we generated double transgenic mice (2ÂTgAD: APPswe, Tau P301L) that accumulate C99 without Ab deposition or hyperphosphorylation of tau and compared them to 3ÂTgAD mice. Here, we show that both TgAD mice display similar decreases in long-term potentiation and in spontaneous lo-comotor activity measured by actimetry suggesting that the synaptic alterations and the apathy-like behavior were likely linked to C99 rather than Ab. However, spatial learning alterations, assessed by the Morris water maze task, are more pronounced in 3ÂTgAD than in 2ÂTgAD, suggesting that both Ab and C99 contribute to defects in the acquisition of spatial information. Finally, even if similar results are observed in males, cognitive and non-cognitive deficits are more severe in females.

The β-amyloid precursor protein undergoes cleavages by β- and γ-secretasses yielding amyloid-β peptides (Aβ) that accumulate in Alzheimer's disease. Subsequently, Aβ peptides are targets of additional truncations or endoproteolytic cleavages explaining the diversity of Aβ-related fragments recovered in cell media or pathologic human fluids. Here, we focused on Aβ1-34 (Aβ34) that has been detected both in vitro and in vivo and that derives from the hydrolysis of Aβ by β-secretase. We have obtained and fully characterized by immunologic and biochemical approaches, a polyclonal antibody that specifically recognizes the C-terminus of Aβx-34. We present immunohistochemical evidence for the presence of Aβx-34 in the brain of 3xTg mice and Alzheimer's disease-affected human brains. Finally, we demonstrate a neprilysin-mediated degradation process of Aβ34 and the ability of synthetic Aβ34 to protect HEK cells overexpressing either wild type or Swedish-mutated β-amyloid precursor protein from apoptosis.

Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers’s disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the β-secretase-derived βAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aβ independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer’s disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1577-6) contains supplementary material, which is available to authorized users.

International audience; Apathy is an early and common neuropsychiatric syndrome in Alzheimer's disease (AD) patients. In clinical trials, apathy is associated with decreased motor activity that can be monitored by actigraphy. The triple transgenic mouse AD model (3xTgAD) has been shown to recapitulate the biochemical lesions as well as many of the synaptic and cognitive alterations associated with AD. In the present work we found that these mice also develop an early and consistent apathy-like behavior as evidenced by a drastic decrease in spontaneous activity measured by actimetry. We recently established that these mice also display an intraneuronal accumulation of the β-secretase-derived βAPP fragment (C99) appearing early, in absence of Aβ. Interestingly, we found that the apathy-like behavior observed in 3xTgAD mice was temporally associated with C99 accumulation and synaptic alterations. Since it is well known that the genetic background can strongly influence behavior and can induce transcriptional variability in animal models, we decided to determine the influence of genetic background on the above-described alterations. We backcrossed 3xTgAD mice to C57BL/6 and found that the genetic background had no influence on either C99 accumulation or synaptic plasticity alterations, but strongly affected the apathy-like behavior.

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.

International audience; One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-β (Aβ) peptides. Aβ peptides are derived from combined proteolytic cleavages undergone on the amyloid-β protein precursor (AβPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aβ peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AβPP first undergoes β- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aβ peptides but similarly gives rise to an AβPP IntraCellular Domain (AICD fragment) that could modulate the transcription of several genes linked to AD pathology. It is therefore striking that AICD theoretically derives from both amyloidogenic and non-amyloidogenic AβPP processing pathways. Here we show that AICD predominantly derives from C99 by means of recombinant substrates and transiently transfected cells expressing C99. Our data suggest a preferred pathogenic pathway for AICD production and suggests that this fragment, in addition to C99 and Aβ peptides, could contribute to AD pathology.

Autosomal dominant polycystic kidney disease (ADPKD) is a multisystem disorder characterized by renal, hepatic and pancreatic cyst formation and cardiovascular complications. The condition is caused by mutations in the PKD1 or PKD2 gene. In mice with reduced expression of Pkd1, dissecting aneurysms with prominent media thickening have been seen. To study the effect of selective disruption of Pkd1 in vascular smooth muscle cells (SMCs), we have generated mice in which a floxed part of the Pkd1 gene was deleted by Cre under the control of the SM22 promotor (SM22-Pkd1(del/del) mice). Cre activity was confirmed by X-gal staining using lacZ expressing Cre reporter mice (R26R), and quantitative PCR indicated that in the aorta Pkd1 gene expression was strongly reduced, whereas Pkd2 levels remained unaltered. Histopathological analysis revealed cyst formation in pancreas, liver and kidneys as the result of extravascular Cre activity in pancreatic ducts, bile ducts and in the glomerular Bowman's capsule. Remarkably, we did not find any spontaneous gross structural blood vessel abnormalities in mice with somatic Pkd1 gene disruption in SMCs or simultaneous disruption of Pkd1 in SMCs and endothelial cells (ECs). Extensive isometric myographic analysis of the aorta did not reveal differences in response to KCl, acetylcholine, phenylephrin or serotonin, except for a significant increase in contractility induced by phenylephrin on arteries from 40 weeks old Pkd1(del/+) germ-line mice. However, SM22-Pkd1(del/del) mice showed significantly reduced decrease in heart rate on angiotensin II-induced hypertension. The present findings further demonstrate in vivo, that adaptation to hypertension is altered in SM22-Pkd1(del/del) mice. Laboratory Investigation (2011) 91, 24-32; doi:10.1038/labinvest.2010.159; published online 20 September 2010

Specialized chemo- and nutrient-sensing cells share a common electrophysiological mechanism by transducing low O(2), high CO(2) and low glucose stimuli into a compensatory cellular response: the closing of background K(+) channels encoded by the K(2P) subunits. Inhibition of the TASK K(2P) channels by extracellular acidosis leads to an increased excitability of brainstem respiratory neurons. Moreover, hypoxic down-modulation of TASK channels is implicated in the activation of glomus cells in the carotid body. Stimulation of both types of cell leads to an enhanced ventilation and to cardiocirculatory adjustments. Differential modulation of TASK channels by acidosis and high glucose alters excitability of the hypothalamic orexin neurons, which influence arousal, food seeking and breathing. These recent results shed light on the role of TASK channels in sensing physiological stimuli.

The 2P domain K(+) channel TASK-3 is highly expressed in cerebellar granule neurons where it has been proposed to underlie the K(+) leak conductance, IKso. In a previous work we showed that expression of TASK-3 increases in cerebellar granule neurons as they mature in culture. Here we show that within the cerebellum, levels of TASK-3 mRNA increase as granule neurons migrate to their adult positions and receive excitatory mossy fiber input. To understand the mechanism of this increase in TASK-3 expression we used an in vitro model culturing the neurons in either depolarizing conditions mimicking neuronal activity (25K, 25 mm KCl) or in conditions which approach deafferentation (5K, 5 mm KCl). An important increase in TASK-3 mRNA is uniquely observed in 25K and is specific since other background K(+) channel levels remain unchanged or decrease. The rise in TASK-3 mRNA leads to an increase in TASK-3 protein and the IKso conductance resulting in hyperpolarization. Blocking L-type calcium channels or their downstream effector calcineurin, abrogates TASK-3 expression and IKso, leading to hyperexcitability. This is the first study demonstrating that depolarization-induced Ca(2+) entry can directly regulate cellular excitability by dynamically regulating the transcription of a resting K(+) conductance. The appearance of this conductance may play an important role in the transition of depolarized immature neurons to their mature hyperpolarized state during neuronal development.

TREK-1 (KCNK2) is a K(2P) 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.

Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.

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 degrees C rise enhances TREK-1 current amplitude by approximately 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.

International audience; 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 polyunsatur-ated 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 poly-unsaturated 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 docosahexa-enoic acid and linolenic acid, is a signi®cant 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.

TRAAK is the sole member of the emerging class of 2P domain K+ channels to be exclusively expressed in neuronal cells. TRAAK produces baseline K+ currents which are strongly stimulated by arachidonic acid and by mechanical stretch, and which are insensitive to the classical K+ channel blockers tetraethylammonium, Ba2+, and Cs+. This report describes the immunolocalization of TRAAK in brain, spinal cord, and retina of the adult mouse. The most striking finding is the widespread distribution of the TRAAK immunoreactivity, with a prominent staining of the cerebellar cortex, neocortex, hippocampus, dentate gyrus, subiculum, the dorsal hippocampal commissure, thalamus, caudate-putamen, olfactory bulb, and several nuclei in the brainstem. Virtually all neurons express TRAAK, and the highest immunoreactivity was seen in soma, and to a lesser degree in axons and/or dendrites in most areas in brain and spinal cord. In the retina, the TRAAK protein is concentrated to the soma of ganglion cells and to the dendrites of all other neurons. Taken together, these results show a wide distribution of TRAAK, a mechanosensitive and arachidonic acid-stimulated neuron-specific baseline K+ channel, in brain, spinal cord and retina.

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 microM), also blocks KCNQ2 currents (EC50 = 1.5 microM). 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.

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.

The main target for degeneration associated with the weaver mutation is the cerebellum. Expression of the GIRK2 mRNA and protein was studied in cerebellum of 12- and 22-day-old normal and weaver mice. In 12-day-old mice, GIRK2 is expressed at highest levels in the external granule layer (EGL) and in lower levels in the newly forming internal granule layer (IGL). In the weaver cerebellum, a high hybridization signal and dark immunostaining was observed in the EGL due to the higher density of non-migrated cells. In 22-day-old weaver cerebella, there are only few remaining granule cells existing as scattered cells within the IGL and molecular layer. GIRK2 is expressed in these neurons but the majority of cells expressing GIRK2 in these cerebella are Purkinje cells that are also affected by the weaver mutation (position, shape) but have not died. Normal cerebellar granule neurons but not homozygous mutant neurons in primary cultures and cerebellar slices of 8-day-old mice displayed inward rectifier K + currents. Taken together, these findings suggest that cell loss in the weaver cerebellum is not directly related to a differential content of GIRK2 in the affected neurons during development. The lethal effect of the weaver mutation in specific neurons is probably due to a combination of the abnormal function of the inward rectifier K + channels and other factors specific to the vulnerable neurons. © 1997 Elsevier Science B.V. All rights reserved.

IT has been suggested that a mutation in a G-protein-gated inward rectifier K + channel (GIRK2) is responsible for inducing cell death in the cerebellum of homozygous weaver (wv/wv) mutant mice. These mice also display a progressive, massive loss of mesencephalic dopaminergic neurones. Using an immunocytochemical method, we detected GIRK2-positive cell bodies and fibres in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) of control (+/+) mice. Cell counts of both GIRK2- and tyrosine hydroxylase (TH)-positive neurones demonstrated a marked loss of SNC cell bodies, especially in 12-month-old (12M) wv/wv mice. A considerable proportion of GIRK2-positive cell bodies were preserved, however. In addition, no loss of GIRK2-positive neurones was observed in the VTA of 12M wv/wv mice, despite of a significant reduction in TH-positive cell bodies. These results suggest that expression of the mutated channel is not a sufficient condition to induce cell death in the ventral mesencephalon of the wv/wv mice.

Background In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins (PS) [1], the catalytic core in gamma-secretase complexes cleaving the amyloid precursor protein (APP) thereby generating amyloid beta peptides. Here we investigate whether APP perturbs ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence amyloid beta production.

Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated β-amyloiḍprecursoṛprotein (βAPPswe), P310L-Tau (TauP301L) and physiological levels of M146V-presenilin-1 (PS1M146V) display extracellular amyloid-β peptides (Aβ) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal Aβ that has been linked to synaptic dysfunction and cognitive deficits. Here, we provide immunohistological, genetic and pharmacological evidences for early, age-dependent and hippocampus-specific accumulation of the β-secretase-derived βAPP fragment C99 that is observed in 3 month-old mice and enhanced by pharmacological blockade of γ-secretase. Notably, intracellular Aβ is only detectable several months later and appears, as is the case for C99, in enlarged cathepsin B-positive structures, while extracellular Aβ deposits are detected around 12 months of age and beyond. Early C99 production occurs mainly in the CA1/subicular interchange area of the hippocampus corresponding to the first region exhibiting plaques and tangles in old mice. The examination of two other mice models harboring mutated βAPP but endogenous wild type PS1 and Tau protein (TgCRND8 or Tg2576) indicate that C99 levels are largely higher in all animal models than in their respective control mice. Furthermore, the comparison of 3xTgAD mice with double transgenic mice bearing the βAPPswe and TauP301L mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation could not be accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by γ-secretase. Altogether, our work identifies C99 as the earliest βAPP catabolite and main contributor to the intracellular βAPP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mice model.

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.

SummaryAutosomal-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.

The K + channel TASK-3 is highly expressed in cerebellar granule neurons where it encodes the K + current IKso. Besides the role of TASK-3 in controlling cellular excitability and shaping neuronal responses, it has recently been proposed to contribute to the development and maturation of neurons in the cerebellum. K + dependent apoptosis and tumorigenesis have also been attributed to TASK-3 over-expression. Transcription of TASK-3 is strongly dependent on depolarization-induced Ca 2+ -entry. To understand the mechanisms involved in TASK-3 regulation, we have characterized a minimal promoter which specifically expresses in cellular backgrounds expressing endogenous TASK-3. Moreover, we have cloned and characterized the 5′ and 3′ untranslated regions of TASK-3. Both regions contribute to inhibit expression of a reporter gene. Given the direct consequence of membrane potential on TASK-3 expression, this is an important first step towards the understanding of the complex regulation of this gene.

Rat mature cerebellar granule, unlike hippocampal neurons, die by apoptosis when cultured in a medium containing a physiological concentration of K+ but survive under high external K+ concentrations. Cell death in physiological K+ parallels the developmental expression of the TASK-1 and TASK-3 subunits that encode the pH-sensitive standing outward K+ current IKso. Genetic transfer of the TASK subunits in hippocampal neurons, lacking IKso, induces cell death, while their genetic inactivation protects cerebellar granule neurons. Neuronal death of cultured rat granule neurons is also prevented by conditions that specifically reduce K+ efflux through the TASK-3 channels such as extracellular acidosis and ruthenium red. TASK leak K+ channels thus play an important role in K+-dependent apoptosis of cerebellar granule neurons in culture.

Many members of the two-pore-domain potassium (K + ) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I KAA , a background K + current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I KAA , like TREK-1, was outwardly rectifying in physiological K + conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I KAA was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I KAA was inhibited by chlorophenylthio-cAMP (500 μmol/L) and also by stimulation of β-adrenergic receptors with isoproterenol (1 μmol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by β-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.

Hypoxia and inhibitors of mitochondrial respiration impair the regulatory volume decrease (RVD) of cerebellar granule neurons after hypotonic swelling. RVD is linked to the opening of volume-regulated anion channels (VRACs). VRACs are outwardly rectifying, inactivate slowly during maintained depolarization, and are permeable to the cellular organic osmolyte taurine. Channel activation requires nonhydrolytic ATP binding and is not modulated by intracellular ADP. VRAC opening is reversibly depressed by hypoxia and by mitochondrial inhibitors such as oligomycin, rotenone, and antimycin A. These results demonstrate that neuronal VRAC activation and swelling are both tightly linked to cellular energy. Moreover, the findings reported in this work may have a particular significance for inherited mitochondrial human diseases, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which cause brain swelling and edema.

(-)-Cromakalim, a typical K+-channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 microM) extracellular glutamate in primary cultures of hippocampus. (-)-Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c-fos mRNA transcription. (-)-Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (-)-cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (-)-cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (-)-cromakalim return to their original size in a few hours, whereas non-(-)-cromakalim-treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate-induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (-)-cromakalim. Glibenclamide, a potent blocker of the ATP-sensitive K+ channel, abolishes all the beneficial effects of (-)-cromakalim. These findings strongly suggest that (-)-cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.

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.

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.

Severe forebrain ischemia induces a large increase in expression of NMDA receptor subunits in rat brain. One week after ischemia, levels of NMDA-R1 mRNAs in the CA1 pyramidal cells of hippocampus are 7 times higher than those observed in control rats. At 7 days postischemia, an enhanced immunostaining of the NMDA-R1 subunit was observed in all hippocampal structures indicating that changes in mRNA levels are accompanied by changes in receptor protein level. Riluzole, a potent inhibitor of glutamate release and CNQX, a selective AMPA/kainate antagonist, drastically reduced the ischemia-induced expression of mRNAs for three NMDA receptor subunits while d -AP5, a selective NMDA antagonist, had essentially no effect. Therefore ischemia-induced expression of NMDA receptor subunits is associated with glutamate release and proceeds via an AMPA/kainate pathway. These results together with those of other groups concerning ischemia effects on AMPA and GABA A receptor levels, suggest an important role of the induced expression of NMDA receptor subunits in the deleterious effects of ischemia.

Lysophospholipids (LPLs) are important intermediates in the synthesis and degradation of membrane phospholipids. Here we show that certain LPLs, particularly lysophosphatidylcholine and lysophosphatidylinositol, prevent neuronal death both in an in vivo model of transient global ischemia and in an in vitro model of excitotoxicity using primary cultures of cerebellar granule cells exposed to high extracellular concentrations of glutamate (20–40 μmol/L). The intravenous injection of lysophosphatidylcholine or lysophosphatidylinositol at a concentration of 200 nmol/kg induced a survival of CA1 pyramidal neurons as high as approximately 95%, even when the treatment was started 30 minutes after 15-minute global ischemia. In contrast, lysophosphatidic acid induced no protection. This work also provides evidence that a pretreatment with lysophosphatidylcholine or lysophosphatidylinositol (200 nmol/kg) injected as long as 3 days before a severe 6-minute ischemia provided a potent tolerance against neurodegeneration. Neuroprotection was also observed in in vitro experiments with LPLs. Taken together, in vivo and in vitro data suggest a potential therapeutic use of LPLs as antiischemic compounds. The potential role of 2P-domain K+channels as targets of LPLs in this potent neuroprotective effect is discussed.

Mutations of the degenerins (deg-1, mec-4, mec-10) are the major known causes of hereditary neurodegeneration in the nematode Caenorhabditis elegans. We cloned a neuronal degenerin (MDEG) from human and rat brain. MDEG is an amiloride-sensitive cation channel permeable for Na+, K+, and Li+. This channel is activated by the same mutations which cause neurodegeneration in C. elegans. Like the hyperactive C. elegans degenerin mutants, constitutively active mutants of MDEG cause cell death, suggesting that gain of function of this novel neuronal ion channel might be involved in human forms of neurodegeneration.