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Synaptic plasticity in hepatic encephalopathy – A molecular perspective
Archives of Biochemistry and Biophysics 536 (2013) 183–188
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Review
Synaptic plasticity in hepatic encephalopathy – A molecular perspective Shuping Wen, Annett Schroeter, Nikolaj Klöcker ⇑ Institute of Neural and Sensory Physiology, Medical Faculty, University of Düsseldorf D-40225 Düsseldorf, Germany
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Article history: Available online 25 April 2013 Keywords: Hepatic encephalopathy NMDA receptor AMPA receptor Synaptic plasticity Membrane protein complex Functional proteomics
a b s t r a c t Hepatic encephalopathy (HE)1 is a common neuropsychiatric complication of both acute and chronic liver disease. Clinical symptoms may include motor disturbances and cognitive dysfunction. Available animal models of HE mimic the deficits in cognitive performance including the impaired ability to learn and memorize information. This review explores the question how HE might affect cognitive functions at molecular levels. Both acute and chronic models of HE constrain the plasticity of glutamatergic neurotransmission. Thus, long-lasting activity-dependent changes in synaptic efficiency, known as long-term potentiation (LTP) and long-term depression (LTD) are significantly impeded. We discuss molecules and signal transduction pathways of LTP and LTD that are targeted by experimental HE, with a focus on ionotropic glutamate receptors of the AMPA-subtype. Finally, a novel strategy of functional proteomic analysis is presented, which, if applied differentially, may provide molecular insight into disease-related dysfunction of membrane protein complexes, i.e. disturbed ionotropic glutamate receptor signaling in HE. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Hepatic encephalopathy (HE) is a common neuropsychiatric complication of both acute liver failure and chronic liver disease. The spectrum of clinical manifestations is remarkably broad and comprises characteristic deficits in psychomotor and cognitive performance even before overt neurological signs. The pathophysiological basis of HE is just as complex involving association of several factors [1]. There is, however, a general consensus that ammonia plays a crucial role among the pathogenetic factors, hence being the main target of therapeutic treatment strategies [2–6]. Available animal models of HE reproduce many of the clinical symptoms [7], including dysfunctions in learning and memory. Rat models of portocaval shunting and toxic cirrhosis show alterations in associative learning, spatial memory, and cognitive flexibility [8–11], and chronic dietary hyperammonemia can slow learning of avoidance and conditional discrimination behavior [12].
⇑ Corresponding author. Address: Institute of Neural and Sensory Physiology, Medical Faculty, University of Düsseldorf, Universitätsstr 1, D-40225 Düsseldorf, Germany. Fax: +49 0211 81 14231. E-mail address: [email protected] (N. Klöcker). 1 Abbreviations used: Hepatic encephalopathy, HE; long-term potentiation, LTP; long-term depression, LTD; ionotropic glutamate receptors, iGluRs; AMPA receptor, AMPAR; NMDA receptor, NMDAR; Transmembrane AMPAR Regulatory Proteins, TARPs; soluble guanlyate cyclase, sGC; neuronal nitric oxide synthase, nNOS; nitric oxide, NO; reactive oxygen species, ROS; Transmembrane AMPAR Regulatory Proteins, TARPs; cornichon homologues 2 and 3, CNIH-2, CNIH-3; cystein-knot AMPAR modulating protein 44, CKAMP44; germline-specific gene 1-like protein, GSG1-like; endocytic recycling compartment, ERC. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.04.008
This article reviews the available experimental data on how HE might impair cognitive performance at molecular levels. Being aware that there are multiple means, by which activity generated by experience modifies CNS circuitry [13–18], we will restrict our focus to the phenomenon of long-lasting, activity-dependent changes in synaptic efficiency known as long-term potentiation (LTP) and long-term depression (LTD). After summarizing the key features of this leading cellular model for learning and memory, we will discuss its pathophysiological alterations in experimental HE. Finally, a recently developed molecular approach will be introduced, which, in the future, may provide unbiased information on how HE and other diseases constrain synaptic plasticity at the molecular level. Physiology of NMDAR-dependent LTP and LTD in CA1 Long-lasting, activity-dependent changes in the efficacy of synaptic communication are the leading experimental model for the capability of the brain to learn and store information. In the early 1970s, Bliss and Lomo first described that repetitive activation of glutamatergic synapses in the hippocampus, a brain region central for learning and memory, would lead to an increase in synaptic strength lasting for hours or even days [19]. Since then, it has become clear that there exist multiple forms of synaptic plasticity in brain characterized by the two electrophysiological phenomena of long-term potentiation (LTP) and long-term depression (LTD), which may employ distinct molecular mechanisms with respect to brain region and the type of synapse involved [20,21]. Here, we will focus on the key molecules required for the induction of associative LTP and LTD in the hippocampal CA1 region [22,23].
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During basal synaptic transmission, presynaptically released glutamate binds predominantly to two different subtypes of ionotropic glutamate receptors (iGluRs), the AMPA receptor (AMPAR) and the NMDA receptor (NMDAR). AMPARs are ligand-gated ion channels permeable to monovalent cations (Na+ and K+) providing most of the inward current of the postsynaptic glutamatergic response when the neuron is at resting potential. NMDARs are also ligand-gated cation channels, but they show a strong voltagedependence due to a voltage-dependent block by extracellular Mg2+. Under low-frequency transmission, they contribute only little to the postsynaptic response. However, if the postsynaptic neuron is depolarized, for instance when LTP is induced, the Mg2+ block is released allowing Na+ and Ca2+ to enter into the postsynaptic compartment. Depending on its temporal and spatial profile, the rise in intracellular Ca2+ will then trigger LTP or LTD [21,24– 26]. The voltage-dependent Mg2+ block of the NMDAR hence accounts for the associative or Hebbian nature of these forms of synaptic plasticity. The literature describes a perplexing multitude of signal transduction pathways that are activated by the NMDAR-dependent Ca2+ influx when LTP or LTD is induced [25]. Most of these pathways, however, do not seem to be required for LTP or LTD, but are rather modulating in function. The key players of downstream signaling are a set of protein kinases and phosphatases indicating the involvement of phosphoproteins in the induction of synaptic plasticity [27,28]. One of the major phosphoproteins implicated in both LTP and LTD at different types of synapses is the AMPAR protein complex, which can be phosphorylated by CaMKII, PKC, and PKA, and dephosphorylated by calcineurin [29]. Another class of signaling components in LTP induction includes molecules that might function as retrograde messengers. Released from the postsynapse, they are hypothesized to modify presynaptic function and might thereby contribute to changes in synaptic strength. Among such molecules, nitric oxide, endocannabinoids, carbon monoxide, and platelet-activating factor have been studied in greater detail [30]. Again, it has not been shown yet that any of those factors is mandatory for LTP induction, although undoubtedly they may modulate synaptic strength. With respect to the postsynaptic events leading to increases or decreases in synaptic efficacy, it is generally accepted that the regulation of AMPARs plays the key role in glutamatergic synapses of both hippocampus and neocortex [20]. Thus, changes in the number of synaptic AMPARs and in their biophysical properties translate into changes in charge transfer during synaptic transmission. Using electrophysiological and optical methods, AMPARs have been shown to vary in their density at postsynaptic sites depending on synaptic activity [26,31–33]. During induction of LTP, exocytosis of AMPARs into extrasynaptic domains creates a reserve pool, from which they are increasingly recruited into the postsynaptic density [34–37]. On the opposite, induction of LTD decreases the postsynaptic number of AMPARs by promoting their endocytosis [38]. Experiments have shown that endocytosis occurs at specialized perisynaptic zones, whose availability is controlled by synaptic activity [39]. Those endocytic zones also provide AMPARs for the endocytic recycling compartment at the base of synaptic spines for re-insertion [40,41]. A great number of studies have indicated that both exocytosis and endocytosis of AMPARs is regulated by phosphorylation and dephosphorylation of specific serine residues of their subunits. Thus, PKA phosphorylation of S845 in the AMPAR subunit GluA1 facilitates plasma membrane insertion of the receptors, whereas phosphorylation of S818 by PKC is thought to be critical for actual targeting of the receptors into the postsynaptic density [42–44]. A third phospho-site in GluA1, S831 and a target of CaMKII, is thought to contribute to LTP by increasing single channel conductance of the receptors [45,46]. Whereas CaMKII is, however, required for LTP
induction, phosphorylation of S831 is not, indicating that there are most likely more important substrates for CaMKII activity [47–49]. For LTD induction, studies have involved dephosphorylation of GluA1 at S845 and phosphorylation of S880 in GluA2 [50– 54]. But as with other signal transduction components of LTP or LTD, phosphorylation of AMPARs might vary in site and requirement depending on the type of synapse looked at [20]. Indeed, for LTP in hippocampal CA1, the relevance of the GluA1 C-terminal tail has been fundamentally questioned in a very recent elegant study using a single-cell molecular replacement strategy replacing all endogenous GluAs with defined subunits [34]. Besides endo- and exocytosis of AMPARs, also their recruitment to the postsynaptic density might be modulated by phosphorylation and dephosphorylation events. As detailed below, the Transmembrane AMPAR Regulatory Proteins (TARPs) are important constituents of native AMPARs and are able to recruit GluA subunits to the postsynaptic density via C-terminal interaction with the postsynaptic scaffold protein PSD-95 [55–57]. It is hypothesized that CaMKII-mediated phosphorylation of the C-terminal tail of TARP c-2 increases its avidity for binding to PSD-95, as the electrostatic interaction between clusters of positively charged residues in the TARP C-terminus with negatively charged head groups of plasma membrane phospholipids is weakened by newly added, negatively charged C-terminal phosphate modifications [36,58,59]. Thus, CaMKII phosphorylation of TARPs would facilitate diffusional trapping of AMPARs and thereby increase their density in the postsynapse. However, this view has been questioned recently by demonstrating that PDZ binding of the predominant TARP isoform in hippocampus, c-8, controls in fact synaptic transmission, but seems not to be required for induction of LTP [60]. As summarized in Fig. 1, induction of associative NMDAR-dependent LTP and LTD in hippocampal CA1 involves the following key steps: activation of NMDARs, intracellular rise of Ca2+, activation of signal transduction pathways of protein kinases (CaMKII and others) and phosphatases, and finally the regulation of the number and biophysical properties of postsynaptic AMPARs. For LTP and LTD to be long lasting, it is widely accepted that both new protein synthesis and regulated protein degradation are required [61–67]. However, much less is known about the identity of the newly synthesized proteins and their mechanistic contributions [68].
Pathophysiology of LTP and LTD in models of HE As the induction of LTP and LTD are considered to be the leading electrophysiological models for learning and memory function of the brain, which is impaired in HE, it stands to reason that they were investigated in more detail in acute and chronic models of HE. For mimicking acute conditions of HE, brain slices were acutely preincubated with ammonium chloride, whereas for mimicking chronic HE, brain slices were prepared from animal models of chronic HE such as portocaval anastomosis and high-ammonia diet [7,69]. Both acute or chronic hyperammonemic conditions diminished NMDAR-dependent LTP in hippocampus and neostriatum [70–75]. Moreover, it has recently been shown that also NMDARindependent LTD in corticostriatal synapses is impaired by acute exposure of brain slices to ammonia [76]. Both the induction and maintenance phases of LTP and LTD were affected by ammonia. Interestingly, electrophysiogical studies in brain slices from rats suffering from portocaval shunting showed a stronger reduction of LTP compared to rats fed on a high-ammonia diet indicating that additional factors besides ammonia contribute to reduced synaptic plasticity [71]. How do ammonia and/or other mediators of HE constrain synaptic plasticity? As HE leads to an imbalance between inhibitory and excitatory neurotransmission [77], a number of studies have
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Fig. 1. Model of NMDAR-dependent AMPAR regulation. If presynaptically released glutamate activates NMDARs, the postsynaptic Ca2+ influx induces a signal transduction pathway involving activation and autophosphorylation of CaMKII. Activated CaMKII promotes exocytosis of AMPARs from the endocytic recycling compartment (ERC) into extrasynaptic plasma membrane domains, from which they are recruited into the postsynaptic density. Moreover, phosphorylation of AMPARs can increase their single channel conductance. Synaptic AMPARs will be endocytosed from perisynaptic sites destined for re-supplying the ERC or for degradation.
addressed the question whether HE altered the expression of neurotransmitter receptors. To this end, investigators quantified receptor densities by receptor subtype-specific radioactive ligand binding in brain slices or synaptic membrane preparations from animal models of chronic HE. The results revealed highly discrepant changes in the densities of NMDARs, AMPARs, and kainate receptors: they could be downregulated, upregulated, or not changed at all in various brain regions tested [70][78–84]. A more recent study has comprehensively analyzed a broad spectrum of neurotransmitter receptor densities by autoradiography in postmortem human brain tissue of patients having suffered from liver cirrhosis and HE [85]. This study has also systematically controlled for changes in ligand affinities that would confound the quantification of receptor densities. Nevertheless, the investigators observed a remarkable inter-individual variability reflecting the controversial results published and concluded from their data that alterations in neurotransmitter receptor densities do not play the key role in causing the neuropsychiatric and motor disturbances in HE [85]. With respect to impaired synaptic plasticity in HE, it is rather difficult to draw any mechanistic conclusions from those studies. The employed methodology does neither distinguish reliably between synaptic and extrasynaptic surface populations of receptors nor does it address the modulation of their trafficking
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to the postsynapse, which is one of the three key steps in NMDAR-dependent synaptic plasticity. Also, ligand dissociation constants were determined for crude membrane preparations averaging over various subcellular receptor populations, which might show differential composition and hence differential ligand binding affinities (see below). Other studies focused on the effects of ammonia on putative signal transduction pathways induced by NMDAR activity [86– 89]. Chronic exposure of neurons to ammonia has been reported to alter the phosphorylation state of a number of known PKC substrates [90]. In cerebellar granule cells, long-term exposure to low dose ammonia differentially changes the expression level and subcellular distribution of PKC depending on its isoform and mechanism of activation [91]. Chronic ammonia treatment also alters the basal and NMDA-induced phosporylation of the essential NMDAR subunit GluN1, which is at least in part mediated by PKC [92]. As surface trafficking and specific plasma membrane localization of NMDARs are regulated by phosphorylation [93–95], this observation might explain the decrease in receptor surface trafficking in cerebellar granule cells exposed to ammonia [92,96]. Another enzyme activated downstream of NMDAR signaling is soluble guanlyate cyclase (sGC): NMDAR-mediated Ca2+ influx activates neuronal nitric oxide synthase (nNOS) producing nitric oxide (NO), which in turn elevates the intracellular cGMP concentration by activating sGC. Pharmacological modulation of this pathway indicates some involvement in LTP induction and in learning and memory function of the brain [97–104]. Chronic exposure to ammonia has been reported to impair the glutamate-mediated activation of the signal transduction pathway when using NOS activity and cGMP levels as a readout [105–107]. Restoring normal cGMP concentrations by intracerebral administration of cGMP or by pharmacological inhibition of its enzymatic degradation also restored impaired learning abilities in in vivo models of HE, which was taken as a proof of concept that ammonia exerts its effects on synaptic plasticity by inhibiting NO signal transduction [108– 111]. It has to be noted, however, that the NO/cGMP pathway is not sufficient to generate LTP in hippocampal CA1. In a seminal study, three independent laboratories have convincingly shown that an increase in cGMP level either alone or paired with repetitive synaptic stimulation cannot potentiate synaptic transmission in CA1 nor overcome the block of LTP by NMDAR inhibition [112]. How does HE or rather its mediators act at the molecular level to impair signal transduction pathways and receptor trafficking? For ammonia, the best-characterized mediator of HE, traditional hypotheses included changes in pH, membrane potential, and energy metabolism [87]. Recent experimental evidence assigns oxidative and nitrosative stress a central role in the molecular mechanism of action of ammonia and inflammatory mediators of HE [3,113,114]. Among the functional consequences of the enhanced release of reactive oxygen species (ROS) in models of acute and chronic HE is selective RNA oxidation [115,116], which might target protein synthesis necessary for LTP (and LTD) maintenance. This may occur irrespective of whether ROS are released from neurons, adjacent astrocytes or activated microglia [115], [117–119]. However, antioxidants scavenging ROS and inhibiting neuronal NOS could only partially alleviate the impaired induction of hippocampal LTP [74], indicating further molecular actions, by which HE mediators affect the key molecules of synaptic plasticity. In line with this hypothesis, a very recent study in post mortem brain samples from patients with HE has revealed differential regulation not only of genes associated with oxidative stress and microglial activation, but also with receptor signaling, inflammatory pathways, cell proliferation, and apoptotic cell death [120]. In summary, there have been many phenomenological studies on how HE affects well-known modulators of synaptic plasticity. The results might be sufficient to explain much of the consequent
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cognitive impairment [121]. However, a recent study indicated that also some of the key molecules required for induction of LTP and LTD may be affected in HE [122]. Thus, in hippocampal synaptosomes and brain slices of rats suffering from portocaval shunting, a significant decrease in both basal and stimulus-induced phosphorylation of CaMKII-T286 was reported, which is a prerequisite for hippocampal NMDAR-dependent LTP [48]. In good agreement, also the phosphorylation of a known substrate of CaMKII, that is GluA1-S831, was significantly reduced. Finally, both the basal synaptic expression and the stimulus-induced synaptic recruitment of the AMPAR subunit GluA1 was abolished, which explains the reported basal decrease in AMPAR-mediated postsynaptic responses and the lack of synaptic potentiation in CA1 of rats with portocaval shunting [71]. However, the molecular mechanism for such impaired synaptic trafficking of GluA1 under conditions of chronic HE remains elusive, as the discussed phosphorylation of GluA1S831 is indeed permissive but not required for GluA1 trafficking or LTP induction [34,47,122]. We will therefore take a closer look at the composition of native AMPARs and some of the important determinants of their trafficking next.
The molecular diversity of native AMPARs AMPARs mediate most of the fast excitatory neurotransmission in the CNS. Conducting depolarizing non-selective cation currents under conditions of basal neuronal activity, these ligand-gated ion channels set the strength of glutamatergic neurotransmission. The core of AMPARs forms as hetero-tetrameric combinations of the four pore-lining a-subunits GluA1, GluA2, GluA3, and GluA4. Functional diversity is enhanced by alternative splicing and RNA editing of the GluA subunits [123–126]. As known for voltagegated ion channels for many years [127–131], it has now become clear that also ligand-gated ion channels including the AMPARs are protein complexes containing auxiliary b-subunits [132–137]. Like in voltage-gated ion channels, such b-subunits modulate not only the biophysical properties of the receptor a-subunits under native conditions, but also determine their subcellular targeting and surface expression. Groundbreaking work revealed that in cerebellar granule neurons, stargazin, a small tetraspanning membrane protein with a homology to the voltage-gated calcium channel subunit c-1, and hence called c-2, is a prerequisite for functional surface expression of AMPARs [57]. Stargazin turned out to be the founding member of the family of the Transmembrane AMPAR Regulatory Proteins (TARPs) that modulate both the subcellular distribution and the biophysical properties of native AMPAR complexes [134,138,139]. As the prototypic TARP, stargazin enhances surface expression of AMPARs and synaptic targeting and recycling of the receptors by mediating an interaction with the postsynaptic scaffolding protein PSD-95 [57,140,141]. In addition, stargazin increases charge transfer through individual AMPARs: it slows channel deactivation and desensitization, changes their ligand binding affinity, and reduces current rectification by polyamines [142,143]. We and others have employed proteomic tools to identify further constituents of native AMPAR complexes including the cornichon homologues 2 and 3 (CNIH-2, CNIH-3), the cystein-knot AMPAR modulating protein 44 (CKAMP44), and the claudin homolog germline-specific gene 1-like protein (GSG1-like) [144–147]. For a more comprehensive molecular analysis of native AMPARs, we have recently developed a sophisticated experimental approach combining multi-epitope and target knockout-controlled affinity purifications with high-resolution mass spectrometric quantification of protein complexes separated on native gels [148]. The methodology of such functional proteomic analysis is detailed in Schulte et al. [149]. Briefly, membrane protein
assemblies are solubilized from brain plasma membrane enriched fractions by the use of detergents. As the latter have inevitable effects on protein–protein interactions, which can range from breaking to stabilizing or even supporting otherwise unlikely interactions, it is necessary to control for solubilization artifacts. Whereas conventional denaturing gel electrophoresis and Western blotting may assess solubilization efficiency, the integrity of the solubilized protein complexes can be monitored by native gel electrophoresis as it allows for quantifying the assembly of respective target proteins into higher molecular weight complexes. Successfully solubilized ion channel complexes can then be affinity-purified by the use of antibodies [147][150–153]. Concerns with respect to antibody specificity and to the selection of subpopulations of protein complexes by sterical interference of protein interactions with antibody epitopes can be adequately addressed by using a multi-epitope affinity purification approach, which includes several antibodies directed against different target epitopes, and by using antibody target knockout tissue as a control [148]. Liquid nano-HPLC coupled high-resolution mass spectrometry is finally the method of choice for unbiased identification of the proteins co-purified with the respective target protein [154–156]. It also yields the quantitative information, which is required for the evaluation of collected data sets along defined criteria such as protein abundance, specificity, and consistency [149]. The described experimental approach has unraveled an unanticipated molecular complexity of native AMPARs in brain [148]. Besides the known AMPAR constituents, we have identified 21 novel constituents that are mostly transmembrane or secreted proteins of low molecular mass. Subsequent biochemical, cell biological, and electrophysiological experiments indicate that the properties of native AMPARs, including their trafficking and their gating, strongly depend on their particular subunit composition. Together with TARP c-8, the two cornichon homologs CNIH-2 and CNIH-3 associate with the majority of native AMPARs [147,148,157]. Cornichon had originally been described as a cargo exporter of soluble growth factors of the TGF-a family from the endoplasmic reticulum [158–160]. AMPARs exploit this property of the two cornichon homologs, which promote receptor surface expression by preferential ER export [161]. Moreover, AMPARs break with the phylogenetically conserved role of cornichon as a simple cargo exporter and recruit CNIH-2 and CNIH-3 to the cell surface membrane, where they serve as AMPAR auxiliary subunits. Besides increasing the surface number of channels, CNIH-2 and CNIH-3 slow deactivation and desensitization of AMPARs and thereby increase charge transfer [147,157,161,162]. Besides TARPs and CNIHs, the AMPAR constituents CKAMP44 and GSG1-l have been characterized in more detail. Both CKAMP44 and GSG1-l significantly delay the recovery of receptors from the desensitized state [144,146,148]. For CKAMP44, which features a PDZ type II ligand binding motif at its C-terminal end serving as an anchor at the postsynaptic density, over-expression and genetic deletion experiments suggest its involvement in short-term plasticity of glutamatergic synapses in the hippocampus [146]. Though most native AMPAR constituents are still awaiting a detailed functional characterization, it is quite obvious that the neurobiology of these receptors and hence also their involvement in synaptic plasticity is much more complex than previously assumed. Needless to state that also their mechanistic involvement in the pathophysiology of HE needs to be revisited, as the disease might target each or combinations of AMPAR complex constituents.
Perspective Given the experimental evidence that HE affects not only the modulators of synaptic plasticity, but also the required key mole-
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cules for LTP/LTD such as iGluRs, we think that their systematic molecular analysis would not only afford a better understanding of the pathophysiological basis of HE, but also identify novel strategies for its treatment. Most if not all voltage-gated ion channels contain auxiliary bsubunits, which not only influence but indeed determine nearly every aspect of channel biology [127–131]. They may control anterograde and/or retrograde subcellular traffic, direct the channel into specific plasma membrane domains, determine their surface half-life, and shape their gating characteristics. The significance of viewing voltage-gated ion channels as complexes of more than just pore-lining subunits is emphasized by the numerous channelopathies caused by dysfunctional b-subunits [163–166]. In recent years, it has become clear that also ligandgated ion channels are formed as protein complexes of highly diverse constituents [132–134][136,148]. A comprehensive functional proteomic analysis as described above provides an unbiased experimental approach to identify and further investigate the molecular composition of such ion channel protein complexes [149]. It is highly advantageous over conventional assays including yeast-two-hybrid, split-ubiquitin, and genetic screens, as it allows comprehensive and direct insight into native protein assemblies. Moreover, if performed differentially, comparing physiological versus pathophysiological conditions, it might yield quantitative information on disease-related changes in ion channel composition and their post-transcriptional and post-translational modification, including alternative splicing, editing, phosphorylation, etc. As these parameters determine both the subcellular distribution and the biophysical properties of ion channels in general, differential functional proteomics may provide the key to understand disease-related dysfunction of a given ion channel at the molecular level, i.e. the functional impairment of iGluR signaling in HE. Acknowledgments Work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to N. K. (SFB974 TP B05). References [1] D. Häussinger, F. Schliess, Gut 57 (2008) 1156. [2] T. Zhan, W. Stremmel, Deutsches Ärzteblatt International 109 (2012) 180. [3] J. Albrecht, M. Zielin´ska, M.D. Norenberg, Biochemical Pharmacology 80 (2010) 1303. [4] M. Bismuth, N. Funakoshi, J.-F. Cadranel, P. Blanc, European Journal of Gastroenterology & Hepatology 23 (2011) 8. [5] P. Desjardins, T. Du, W. Jiang, L. Peng, R.F. Butterworth, Neurochemistry International 60 (2012) 690. [6] C.F. Rose, Clinical Pharmacology and Therapeutics 92 (2012) 321. [7] R.F. Butterworth, M.D. Norenberg, V. Felipo, P. Ferenci, J. Albrecht, A.T. Blei, Liver 29 (2009) 783. [8] M. Méndez, M. Méndez-López, L. López, M.Á. Aller, J. Árias, J.M. Cimadevilla, J.L. Árias, Behavioural Brain Research 188 (2008) 32. [9] M. Méndez, M. Méndez-López, L. López, M.Á. Aller, J. Arias, J.L. Arias, Behavioural Brain Research 198 (2009) 346. [10] M. Méndez, M. Méndez-López, L. López, M.A. Aller, J. Arias, J.L. Arias, Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia 18 (2011) 690. [11] M. Wesierska, H.D. Klinowska, I. Adamska, I. Fresko, J. Sadowska, J. Albrecht, Behavioural Brain Research 171 (2006) 70. [12] M.A. Aguilar, J. Miñarro, V. Felipo, Experimental Neurology 161 (2000) 704. [13] G. Buzsáki, Rhythms of the Brain, Oxford University Press, New York, 2006. [14] W.C. Abraham, M.F. Bear, Trends in Neurosciences 19 (1996) 126. [15] H.W. Kessels, R. Malinow, Neuron 61 (2009) 340. [16] D.E. Feldman, Annual Review of Neuroscience 32 (2009) 33. [17] W. Zhang, D.J. Linden, Nature Reviews Neuroscience 4 (2003) 885. [18] G.G. Turrigiano, S.B. Nelson, Nature Reviews Neuroscience 5 (2004) 97. [19] T. Bliss, T. Lomo, Journal of Physiology 232 (1973) 331. [20] H.-K. Lee, A. Kirkwood, Seminars in Cell & Developmental Biology 22 (2011) 514. [21] R.C. Malenka, M.F. Bear, Neuron 44 (2004) 5. [22] R.C. Malenka, R.A. Nicoll, Trends in Neurosciences 16 (1993) 521. [23] T. Bliss, G.L. Collingridge, Nature 361 (1993) 31–39.
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Review
Synaptic plasticity in hepatic encephalopathy – A molecular perspective Shuping Wen, Annett Schroeter, Nikolaj Klöcker ⇑ Institute of Neural and Sensory Physiology, Medical Faculty, University of Düsseldorf D-40225 Düsseldorf, Germany
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Article history: Available online 25 April 2013 Keywords: Hepatic encephalopathy NMDA receptor AMPA receptor Synaptic plasticity Membrane protein complex Functional proteomics
a b s t r a c t Hepatic encephalopathy (HE)1 is a common neuropsychiatric complication of both acute and chronic liver disease. Clinical symptoms may include motor disturbances and cognitive dysfunction. Available animal models of HE mimic the deficits in cognitive performance including the impaired ability to learn and memorize information. This review explores the question how HE might affect cognitive functions at molecular levels. Both acute and chronic models of HE constrain the plasticity of glutamatergic neurotransmission. Thus, long-lasting activity-dependent changes in synaptic efficiency, known as long-term potentiation (LTP) and long-term depression (LTD) are significantly impeded. We discuss molecules and signal transduction pathways of LTP and LTD that are targeted by experimental HE, with a focus on ionotropic glutamate receptors of the AMPA-subtype. Finally, a novel strategy of functional proteomic analysis is presented, which, if applied differentially, may provide molecular insight into disease-related dysfunction of membrane protein complexes, i.e. disturbed ionotropic glutamate receptor signaling in HE. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Hepatic encephalopathy (HE) is a common neuropsychiatric complication of both acute liver failure and chronic liver disease. The spectrum of clinical manifestations is remarkably broad and comprises characteristic deficits in psychomotor and cognitive performance even before overt neurological signs. The pathophysiological basis of HE is just as complex involving association of several factors [1]. There is, however, a general consensus that ammonia plays a crucial role among the pathogenetic factors, hence being the main target of therapeutic treatment strategies [2–6]. Available animal models of HE reproduce many of the clinical symptoms [7], including dysfunctions in learning and memory. Rat models of portocaval shunting and toxic cirrhosis show alterations in associative learning, spatial memory, and cognitive flexibility [8–11], and chronic dietary hyperammonemia can slow learning of avoidance and conditional discrimination behavior [12].
⇑ Corresponding author. Address: Institute of Neural and Sensory Physiology, Medical Faculty, University of Düsseldorf, Universitätsstr 1, D-40225 Düsseldorf, Germany. Fax: +49 0211 81 14231. E-mail address: [email protected] (N. Klöcker). 1 Abbreviations used: Hepatic encephalopathy, HE; long-term potentiation, LTP; long-term depression, LTD; ionotropic glutamate receptors, iGluRs; AMPA receptor, AMPAR; NMDA receptor, NMDAR; Transmembrane AMPAR Regulatory Proteins, TARPs; soluble guanlyate cyclase, sGC; neuronal nitric oxide synthase, nNOS; nitric oxide, NO; reactive oxygen species, ROS; Transmembrane AMPAR Regulatory Proteins, TARPs; cornichon homologues 2 and 3, CNIH-2, CNIH-3; cystein-knot AMPAR modulating protein 44, CKAMP44; germline-specific gene 1-like protein, GSG1-like; endocytic recycling compartment, ERC. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.04.008
This article reviews the available experimental data on how HE might impair cognitive performance at molecular levels. Being aware that there are multiple means, by which activity generated by experience modifies CNS circuitry [13–18], we will restrict our focus to the phenomenon of long-lasting, activity-dependent changes in synaptic efficiency known as long-term potentiation (LTP) and long-term depression (LTD). After summarizing the key features of this leading cellular model for learning and memory, we will discuss its pathophysiological alterations in experimental HE. Finally, a recently developed molecular approach will be introduced, which, in the future, may provide unbiased information on how HE and other diseases constrain synaptic plasticity at the molecular level. Physiology of NMDAR-dependent LTP and LTD in CA1 Long-lasting, activity-dependent changes in the efficacy of synaptic communication are the leading experimental model for the capability of the brain to learn and store information. In the early 1970s, Bliss and Lomo first described that repetitive activation of glutamatergic synapses in the hippocampus, a brain region central for learning and memory, would lead to an increase in synaptic strength lasting for hours or even days [19]. Since then, it has become clear that there exist multiple forms of synaptic plasticity in brain characterized by the two electrophysiological phenomena of long-term potentiation (LTP) and long-term depression (LTD), which may employ distinct molecular mechanisms with respect to brain region and the type of synapse involved [20,21]. Here, we will focus on the key molecules required for the induction of associative LTP and LTD in the hippocampal CA1 region [22,23].
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During basal synaptic transmission, presynaptically released glutamate binds predominantly to two different subtypes of ionotropic glutamate receptors (iGluRs), the AMPA receptor (AMPAR) and the NMDA receptor (NMDAR). AMPARs are ligand-gated ion channels permeable to monovalent cations (Na+ and K+) providing most of the inward current of the postsynaptic glutamatergic response when the neuron is at resting potential. NMDARs are also ligand-gated cation channels, but they show a strong voltagedependence due to a voltage-dependent block by extracellular Mg2+. Under low-frequency transmission, they contribute only little to the postsynaptic response. However, if the postsynaptic neuron is depolarized, for instance when LTP is induced, the Mg2+ block is released allowing Na+ and Ca2+ to enter into the postsynaptic compartment. Depending on its temporal and spatial profile, the rise in intracellular Ca2+ will then trigger LTP or LTD [21,24– 26]. The voltage-dependent Mg2+ block of the NMDAR hence accounts for the associative or Hebbian nature of these forms of synaptic plasticity. The literature describes a perplexing multitude of signal transduction pathways that are activated by the NMDAR-dependent Ca2+ influx when LTP or LTD is induced [25]. Most of these pathways, however, do not seem to be required for LTP or LTD, but are rather modulating in function. The key players of downstream signaling are a set of protein kinases and phosphatases indicating the involvement of phosphoproteins in the induction of synaptic plasticity [27,28]. One of the major phosphoproteins implicated in both LTP and LTD at different types of synapses is the AMPAR protein complex, which can be phosphorylated by CaMKII, PKC, and PKA, and dephosphorylated by calcineurin [29]. Another class of signaling components in LTP induction includes molecules that might function as retrograde messengers. Released from the postsynapse, they are hypothesized to modify presynaptic function and might thereby contribute to changes in synaptic strength. Among such molecules, nitric oxide, endocannabinoids, carbon monoxide, and platelet-activating factor have been studied in greater detail [30]. Again, it has not been shown yet that any of those factors is mandatory for LTP induction, although undoubtedly they may modulate synaptic strength. With respect to the postsynaptic events leading to increases or decreases in synaptic efficacy, it is generally accepted that the regulation of AMPARs plays the key role in glutamatergic synapses of both hippocampus and neocortex [20]. Thus, changes in the number of synaptic AMPARs and in their biophysical properties translate into changes in charge transfer during synaptic transmission. Using electrophysiological and optical methods, AMPARs have been shown to vary in their density at postsynaptic sites depending on synaptic activity [26,31–33]. During induction of LTP, exocytosis of AMPARs into extrasynaptic domains creates a reserve pool, from which they are increasingly recruited into the postsynaptic density [34–37]. On the opposite, induction of LTD decreases the postsynaptic number of AMPARs by promoting their endocytosis [38]. Experiments have shown that endocytosis occurs at specialized perisynaptic zones, whose availability is controlled by synaptic activity [39]. Those endocytic zones also provide AMPARs for the endocytic recycling compartment at the base of synaptic spines for re-insertion [40,41]. A great number of studies have indicated that both exocytosis and endocytosis of AMPARs is regulated by phosphorylation and dephosphorylation of specific serine residues of their subunits. Thus, PKA phosphorylation of S845 in the AMPAR subunit GluA1 facilitates plasma membrane insertion of the receptors, whereas phosphorylation of S818 by PKC is thought to be critical for actual targeting of the receptors into the postsynaptic density [42–44]. A third phospho-site in GluA1, S831 and a target of CaMKII, is thought to contribute to LTP by increasing single channel conductance of the receptors [45,46]. Whereas CaMKII is, however, required for LTP
induction, phosphorylation of S831 is not, indicating that there are most likely more important substrates for CaMKII activity [47–49]. For LTD induction, studies have involved dephosphorylation of GluA1 at S845 and phosphorylation of S880 in GluA2 [50– 54]. But as with other signal transduction components of LTP or LTD, phosphorylation of AMPARs might vary in site and requirement depending on the type of synapse looked at [20]. Indeed, for LTP in hippocampal CA1, the relevance of the GluA1 C-terminal tail has been fundamentally questioned in a very recent elegant study using a single-cell molecular replacement strategy replacing all endogenous GluAs with defined subunits [34]. Besides endo- and exocytosis of AMPARs, also their recruitment to the postsynaptic density might be modulated by phosphorylation and dephosphorylation events. As detailed below, the Transmembrane AMPAR Regulatory Proteins (TARPs) are important constituents of native AMPARs and are able to recruit GluA subunits to the postsynaptic density via C-terminal interaction with the postsynaptic scaffold protein PSD-95 [55–57]. It is hypothesized that CaMKII-mediated phosphorylation of the C-terminal tail of TARP c-2 increases its avidity for binding to PSD-95, as the electrostatic interaction between clusters of positively charged residues in the TARP C-terminus with negatively charged head groups of plasma membrane phospholipids is weakened by newly added, negatively charged C-terminal phosphate modifications [36,58,59]. Thus, CaMKII phosphorylation of TARPs would facilitate diffusional trapping of AMPARs and thereby increase their density in the postsynapse. However, this view has been questioned recently by demonstrating that PDZ binding of the predominant TARP isoform in hippocampus, c-8, controls in fact synaptic transmission, but seems not to be required for induction of LTP [60]. As summarized in Fig. 1, induction of associative NMDAR-dependent LTP and LTD in hippocampal CA1 involves the following key steps: activation of NMDARs, intracellular rise of Ca2+, activation of signal transduction pathways of protein kinases (CaMKII and others) and phosphatases, and finally the regulation of the number and biophysical properties of postsynaptic AMPARs. For LTP and LTD to be long lasting, it is widely accepted that both new protein synthesis and regulated protein degradation are required [61–67]. However, much less is known about the identity of the newly synthesized proteins and their mechanistic contributions [68].
Pathophysiology of LTP and LTD in models of HE As the induction of LTP and LTD are considered to be the leading electrophysiological models for learning and memory function of the brain, which is impaired in HE, it stands to reason that they were investigated in more detail in acute and chronic models of HE. For mimicking acute conditions of HE, brain slices were acutely preincubated with ammonium chloride, whereas for mimicking chronic HE, brain slices were prepared from animal models of chronic HE such as portocaval anastomosis and high-ammonia diet [7,69]. Both acute or chronic hyperammonemic conditions diminished NMDAR-dependent LTP in hippocampus and neostriatum [70–75]. Moreover, it has recently been shown that also NMDARindependent LTD in corticostriatal synapses is impaired by acute exposure of brain slices to ammonia [76]. Both the induction and maintenance phases of LTP and LTD were affected by ammonia. Interestingly, electrophysiogical studies in brain slices from rats suffering from portocaval shunting showed a stronger reduction of LTP compared to rats fed on a high-ammonia diet indicating that additional factors besides ammonia contribute to reduced synaptic plasticity [71]. How do ammonia and/or other mediators of HE constrain synaptic plasticity? As HE leads to an imbalance between inhibitory and excitatory neurotransmission [77], a number of studies have
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Fig. 1. Model of NMDAR-dependent AMPAR regulation. If presynaptically released glutamate activates NMDARs, the postsynaptic Ca2+ influx induces a signal transduction pathway involving activation and autophosphorylation of CaMKII. Activated CaMKII promotes exocytosis of AMPARs from the endocytic recycling compartment (ERC) into extrasynaptic plasma membrane domains, from which they are recruited into the postsynaptic density. Moreover, phosphorylation of AMPARs can increase their single channel conductance. Synaptic AMPARs will be endocytosed from perisynaptic sites destined for re-supplying the ERC or for degradation.
addressed the question whether HE altered the expression of neurotransmitter receptors. To this end, investigators quantified receptor densities by receptor subtype-specific radioactive ligand binding in brain slices or synaptic membrane preparations from animal models of chronic HE. The results revealed highly discrepant changes in the densities of NMDARs, AMPARs, and kainate receptors: they could be downregulated, upregulated, or not changed at all in various brain regions tested [70][78–84]. A more recent study has comprehensively analyzed a broad spectrum of neurotransmitter receptor densities by autoradiography in postmortem human brain tissue of patients having suffered from liver cirrhosis and HE [85]. This study has also systematically controlled for changes in ligand affinities that would confound the quantification of receptor densities. Nevertheless, the investigators observed a remarkable inter-individual variability reflecting the controversial results published and concluded from their data that alterations in neurotransmitter receptor densities do not play the key role in causing the neuropsychiatric and motor disturbances in HE [85]. With respect to impaired synaptic plasticity in HE, it is rather difficult to draw any mechanistic conclusions from those studies. The employed methodology does neither distinguish reliably between synaptic and extrasynaptic surface populations of receptors nor does it address the modulation of their trafficking
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to the postsynapse, which is one of the three key steps in NMDAR-dependent synaptic plasticity. Also, ligand dissociation constants were determined for crude membrane preparations averaging over various subcellular receptor populations, which might show differential composition and hence differential ligand binding affinities (see below). Other studies focused on the effects of ammonia on putative signal transduction pathways induced by NMDAR activity [86– 89]. Chronic exposure of neurons to ammonia has been reported to alter the phosphorylation state of a number of known PKC substrates [90]. In cerebellar granule cells, long-term exposure to low dose ammonia differentially changes the expression level and subcellular distribution of PKC depending on its isoform and mechanism of activation [91]. Chronic ammonia treatment also alters the basal and NMDA-induced phosporylation of the essential NMDAR subunit GluN1, which is at least in part mediated by PKC [92]. As surface trafficking and specific plasma membrane localization of NMDARs are regulated by phosphorylation [93–95], this observation might explain the decrease in receptor surface trafficking in cerebellar granule cells exposed to ammonia [92,96]. Another enzyme activated downstream of NMDAR signaling is soluble guanlyate cyclase (sGC): NMDAR-mediated Ca2+ influx activates neuronal nitric oxide synthase (nNOS) producing nitric oxide (NO), which in turn elevates the intracellular cGMP concentration by activating sGC. Pharmacological modulation of this pathway indicates some involvement in LTP induction and in learning and memory function of the brain [97–104]. Chronic exposure to ammonia has been reported to impair the glutamate-mediated activation of the signal transduction pathway when using NOS activity and cGMP levels as a readout [105–107]. Restoring normal cGMP concentrations by intracerebral administration of cGMP or by pharmacological inhibition of its enzymatic degradation also restored impaired learning abilities in in vivo models of HE, which was taken as a proof of concept that ammonia exerts its effects on synaptic plasticity by inhibiting NO signal transduction [108– 111]. It has to be noted, however, that the NO/cGMP pathway is not sufficient to generate LTP in hippocampal CA1. In a seminal study, three independent laboratories have convincingly shown that an increase in cGMP level either alone or paired with repetitive synaptic stimulation cannot potentiate synaptic transmission in CA1 nor overcome the block of LTP by NMDAR inhibition [112]. How does HE or rather its mediators act at the molecular level to impair signal transduction pathways and receptor trafficking? For ammonia, the best-characterized mediator of HE, traditional hypotheses included changes in pH, membrane potential, and energy metabolism [87]. Recent experimental evidence assigns oxidative and nitrosative stress a central role in the molecular mechanism of action of ammonia and inflammatory mediators of HE [3,113,114]. Among the functional consequences of the enhanced release of reactive oxygen species (ROS) in models of acute and chronic HE is selective RNA oxidation [115,116], which might target protein synthesis necessary for LTP (and LTD) maintenance. This may occur irrespective of whether ROS are released from neurons, adjacent astrocytes or activated microglia [115], [117–119]. However, antioxidants scavenging ROS and inhibiting neuronal NOS could only partially alleviate the impaired induction of hippocampal LTP [74], indicating further molecular actions, by which HE mediators affect the key molecules of synaptic plasticity. In line with this hypothesis, a very recent study in post mortem brain samples from patients with HE has revealed differential regulation not only of genes associated with oxidative stress and microglial activation, but also with receptor signaling, inflammatory pathways, cell proliferation, and apoptotic cell death [120]. In summary, there have been many phenomenological studies on how HE affects well-known modulators of synaptic plasticity. The results might be sufficient to explain much of the consequent
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cognitive impairment [121]. However, a recent study indicated that also some of the key molecules required for induction of LTP and LTD may be affected in HE [122]. Thus, in hippocampal synaptosomes and brain slices of rats suffering from portocaval shunting, a significant decrease in both basal and stimulus-induced phosphorylation of CaMKII-T286 was reported, which is a prerequisite for hippocampal NMDAR-dependent LTP [48]. In good agreement, also the phosphorylation of a known substrate of CaMKII, that is GluA1-S831, was significantly reduced. Finally, both the basal synaptic expression and the stimulus-induced synaptic recruitment of the AMPAR subunit GluA1 was abolished, which explains the reported basal decrease in AMPAR-mediated postsynaptic responses and the lack of synaptic potentiation in CA1 of rats with portocaval shunting [71]. However, the molecular mechanism for such impaired synaptic trafficking of GluA1 under conditions of chronic HE remains elusive, as the discussed phosphorylation of GluA1S831 is indeed permissive but not required for GluA1 trafficking or LTP induction [34,47,122]. We will therefore take a closer look at the composition of native AMPARs and some of the important determinants of their trafficking next.
The molecular diversity of native AMPARs AMPARs mediate most of the fast excitatory neurotransmission in the CNS. Conducting depolarizing non-selective cation currents under conditions of basal neuronal activity, these ligand-gated ion channels set the strength of glutamatergic neurotransmission. The core of AMPARs forms as hetero-tetrameric combinations of the four pore-lining a-subunits GluA1, GluA2, GluA3, and GluA4. Functional diversity is enhanced by alternative splicing and RNA editing of the GluA subunits [123–126]. As known for voltagegated ion channels for many years [127–131], it has now become clear that also ligand-gated ion channels including the AMPARs are protein complexes containing auxiliary b-subunits [132–137]. Like in voltage-gated ion channels, such b-subunits modulate not only the biophysical properties of the receptor a-subunits under native conditions, but also determine their subcellular targeting and surface expression. Groundbreaking work revealed that in cerebellar granule neurons, stargazin, a small tetraspanning membrane protein with a homology to the voltage-gated calcium channel subunit c-1, and hence called c-2, is a prerequisite for functional surface expression of AMPARs [57]. Stargazin turned out to be the founding member of the family of the Transmembrane AMPAR Regulatory Proteins (TARPs) that modulate both the subcellular distribution and the biophysical properties of native AMPAR complexes [134,138,139]. As the prototypic TARP, stargazin enhances surface expression of AMPARs and synaptic targeting and recycling of the receptors by mediating an interaction with the postsynaptic scaffolding protein PSD-95 [57,140,141]. In addition, stargazin increases charge transfer through individual AMPARs: it slows channel deactivation and desensitization, changes their ligand binding affinity, and reduces current rectification by polyamines [142,143]. We and others have employed proteomic tools to identify further constituents of native AMPAR complexes including the cornichon homologues 2 and 3 (CNIH-2, CNIH-3), the cystein-knot AMPAR modulating protein 44 (CKAMP44), and the claudin homolog germline-specific gene 1-like protein (GSG1-like) [144–147]. For a more comprehensive molecular analysis of native AMPARs, we have recently developed a sophisticated experimental approach combining multi-epitope and target knockout-controlled affinity purifications with high-resolution mass spectrometric quantification of protein complexes separated on native gels [148]. The methodology of such functional proteomic analysis is detailed in Schulte et al. [149]. Briefly, membrane protein
assemblies are solubilized from brain plasma membrane enriched fractions by the use of detergents. As the latter have inevitable effects on protein–protein interactions, which can range from breaking to stabilizing or even supporting otherwise unlikely interactions, it is necessary to control for solubilization artifacts. Whereas conventional denaturing gel electrophoresis and Western blotting may assess solubilization efficiency, the integrity of the solubilized protein complexes can be monitored by native gel electrophoresis as it allows for quantifying the assembly of respective target proteins into higher molecular weight complexes. Successfully solubilized ion channel complexes can then be affinity-purified by the use of antibodies [147][150–153]. Concerns with respect to antibody specificity and to the selection of subpopulations of protein complexes by sterical interference of protein interactions with antibody epitopes can be adequately addressed by using a multi-epitope affinity purification approach, which includes several antibodies directed against different target epitopes, and by using antibody target knockout tissue as a control [148]. Liquid nano-HPLC coupled high-resolution mass spectrometry is finally the method of choice for unbiased identification of the proteins co-purified with the respective target protein [154–156]. It also yields the quantitative information, which is required for the evaluation of collected data sets along defined criteria such as protein abundance, specificity, and consistency [149]. The described experimental approach has unraveled an unanticipated molecular complexity of native AMPARs in brain [148]. Besides the known AMPAR constituents, we have identified 21 novel constituents that are mostly transmembrane or secreted proteins of low molecular mass. Subsequent biochemical, cell biological, and electrophysiological experiments indicate that the properties of native AMPARs, including their trafficking and their gating, strongly depend on their particular subunit composition. Together with TARP c-8, the two cornichon homologs CNIH-2 and CNIH-3 associate with the majority of native AMPARs [147,148,157]. Cornichon had originally been described as a cargo exporter of soluble growth factors of the TGF-a family from the endoplasmic reticulum [158–160]. AMPARs exploit this property of the two cornichon homologs, which promote receptor surface expression by preferential ER export [161]. Moreover, AMPARs break with the phylogenetically conserved role of cornichon as a simple cargo exporter and recruit CNIH-2 and CNIH-3 to the cell surface membrane, where they serve as AMPAR auxiliary subunits. Besides increasing the surface number of channels, CNIH-2 and CNIH-3 slow deactivation and desensitization of AMPARs and thereby increase charge transfer [147,157,161,162]. Besides TARPs and CNIHs, the AMPAR constituents CKAMP44 and GSG1-l have been characterized in more detail. Both CKAMP44 and GSG1-l significantly delay the recovery of receptors from the desensitized state [144,146,148]. For CKAMP44, which features a PDZ type II ligand binding motif at its C-terminal end serving as an anchor at the postsynaptic density, over-expression and genetic deletion experiments suggest its involvement in short-term plasticity of glutamatergic synapses in the hippocampus [146]. Though most native AMPAR constituents are still awaiting a detailed functional characterization, it is quite obvious that the neurobiology of these receptors and hence also their involvement in synaptic plasticity is much more complex than previously assumed. Needless to state that also their mechanistic involvement in the pathophysiology of HE needs to be revisited, as the disease might target each or combinations of AMPAR complex constituents.
Perspective Given the experimental evidence that HE affects not only the modulators of synaptic plasticity, but also the required key mole-
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cules for LTP/LTD such as iGluRs, we think that their systematic molecular analysis would not only afford a better understanding of the pathophysiological basis of HE, but also identify novel strategies for its treatment. Most if not all voltage-gated ion channels contain auxiliary bsubunits, which not only influence but indeed determine nearly every aspect of channel biology [127–131]. They may control anterograde and/or retrograde subcellular traffic, direct the channel into specific plasma membrane domains, determine their surface half-life, and shape their gating characteristics. The significance of viewing voltage-gated ion channels as complexes of more than just pore-lining subunits is emphasized by the numerous channelopathies caused by dysfunctional b-subunits [163–166]. In recent years, it has become clear that also ligandgated ion channels are formed as protein complexes of highly diverse constituents [132–134][136,148]. A comprehensive functional proteomic analysis as described above provides an unbiased experimental approach to identify and further investigate the molecular composition of such ion channel protein complexes [149]. It is highly advantageous over conventional assays including yeast-two-hybrid, split-ubiquitin, and genetic screens, as it allows comprehensive and direct insight into native protein assemblies. Moreover, if performed differentially, comparing physiological versus pathophysiological conditions, it might yield quantitative information on disease-related changes in ion channel composition and their post-transcriptional and post-translational modification, including alternative splicing, editing, phosphorylation, etc. As these parameters determine both the subcellular distribution and the biophysical properties of ion channels in general, differential functional proteomics may provide the key to understand disease-related dysfunction of a given ion channel at the molecular level, i.e. the functional impairment of iGluR signaling in HE. Acknowledgments Work was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to N. K. (SFB974 TP B05). References [1] D. Häussinger, F. Schliess, Gut 57 (2008) 1156. [2] T. Zhan, W. Stremmel, Deutsches Ärzteblatt International 109 (2012) 180. [3] J. Albrecht, M. Zielin´ska, M.D. Norenberg, Biochemical Pharmacology 80 (2010) 1303. [4] M. Bismuth, N. Funakoshi, J.-F. Cadranel, P. Blanc, European Journal of Gastroenterology & Hepatology 23 (2011) 8. [5] P. Desjardins, T. Du, W. Jiang, L. Peng, R.F. Butterworth, Neurochemistry International 60 (2012) 690. [6] C.F. Rose, Clinical Pharmacology and Therapeutics 92 (2012) 321. [7] R.F. Butterworth, M.D. Norenberg, V. Felipo, P. Ferenci, J. Albrecht, A.T. Blei, Liver 29 (2009) 783. [8] M. Méndez, M. Méndez-López, L. López, M.Á. Aller, J. Árias, J.M. Cimadevilla, J.L. Árias, Behavioural Brain Research 188 (2008) 32. [9] M. Méndez, M. Méndez-López, L. López, M.Á. Aller, J. Arias, J.L. Arias, Behavioural Brain Research 198 (2009) 346. [10] M. Méndez, M. Méndez-López, L. López, M.A. Aller, J. Arias, J.L. Arias, Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia 18 (2011) 690. [11] M. Wesierska, H.D. Klinowska, I. Adamska, I. Fresko, J. Sadowska, J. Albrecht, Behavioural Brain Research 171 (2006) 70. [12] M.A. Aguilar, J. Miñarro, V. Felipo, Experimental Neurology 161 (2000) 704. [13] G. Buzsáki, Rhythms of the Brain, Oxford University Press, New York, 2006. [14] W.C. Abraham, M.F. Bear, Trends in Neurosciences 19 (1996) 126. [15] H.W. Kessels, R. Malinow, Neuron 61 (2009) 340. [16] D.E. Feldman, Annual Review of Neuroscience 32 (2009) 33. [17] W. Zhang, D.J. Linden, Nature Reviews Neuroscience 4 (2003) 885. [18] G.G. Turrigiano, S.B. Nelson, Nature Reviews Neuroscience 5 (2004) 97. [19] T. Bliss, T. Lomo, Journal of Physiology 232 (1973) 331. [20] H.-K. Lee, A. Kirkwood, Seminars in Cell & Developmental Biology 22 (2011) 514. [21] R.C. Malenka, M.F. Bear, Neuron 44 (2004) 5. [22] R.C. Malenka, R.A. Nicoll, Trends in Neurosciences 16 (1993) 521. [23] T. Bliss, G.L. Collingridge, Nature 361 (1993) 31–39.
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