Functional interplay between plasma membrane Ca2+-ATPase, amyloid β-peptide and tau

Accepted Manuscript Title: Functional interplay between Plasma Membrane Ca2+ -ATPase, amyloid ␤-peptide and tau Author: Ana M. Mata PII: DOI: Reference:

S0304-3940(17)30641-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.08.004 NSL 33004

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

25-5-2017 12-7-2017 1-8-2017

Please cite this article as: Ana M.Mata, Functional interplay between Plasma Membrane Ca2+-ATPase, amyloid ␤-peptide and tau, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Functional interplay between Plasma Membrane Ca2+-ATPase, amyloid β-peptide and tau

Author names and affiliations: Ana M. Mata Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06006 Badajoz, Spain. [email protected]

Corresponding autor: Ana M. Mata Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06006 Badajoz, Spain. [email protected]

Highlights:   

The plasma membrane Ca2+-ATPase (PMCA) activity is inhibited by Aβ and tau. Calmodulin prevents and reverses inhibitory effects of Aβ and tau on PMCA activity. The phospholipids charge modulates the inhibition of PMCA activity by Aβ and tau.

Abstract: It is well known that dysregulation of Ca2+ homeostasis is involved in Alzheimer´s disease (AD), a neurodegenerative disorder characterized by the presence of toxic aggregates of amyloid β-peptide (Aβ) and neurofibrillary tangles of tau. Alteration of calcium signaling has been linked to Aβ and tau pathologies, although the understanding of underlying molecular and cellular mechanisms is far from clear. This review summarizes the functional inhibition of plasma membrane Ca2+-ATPase (PMCA) by Aβ and tau, and its modulation by calmodulin and the ionic nature of phospholipids. The data obtained until now in our laboratory suggest that PMCA injury linked to Aβ and tau can be significantly involved in the cascade of events leading to intracellular calcium overload associated to AD.

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Abbreviations: AD, Alzheimer's disease; Aβ, amyloid β-peptide; CaM, calmodulin; PMCA, plasma membrane Ca2+-ATPase; PC, phosphatidylcholine; PS, phosphatidylserine

Keywords: PMCA, calcium, Alzheimer’s disease, Aβ, tau, calmodulin Contents 1. Introduction 2. Differential sensitivity of calcium pumps to Aβ and tau 3. The inhibition of PMCA by Aβ and tau is modulated by calmodulin and phosphatidylserine. 3.1. Calmodulin blocks the inhibition of PMCA by Aβ and tau 3.2. Phosphatidylserine mediates the inhibition of PMCA by tau, but not by Aβ 4. Concluding remarks Acknowledgements References 1. Introduction Calcium ion (Ca2+) is an intracellular messenger involved in many cellular functions. In the nervous system, cytosolic free Ca2+ regulates significant mechanisms such as neurotransmitter release, neuronal excitability, synaptic plasticity, gene transcription, neural development or neuronal death, which are activated by a temporary increase in cytosolic Ca2+. However, in the resting neuron, the intracellular Ca2+ concentration is kept much lower (around 100 nM) that in the extracellular space (about 1.2 mM). And failure of these cells to maintain this 10,000-fold gradient seems to be a common pathway leading to neuronal disorders and cell death. Among all systems involved in Ca2+ homeostasis neural cells contains very high-affinity Ca2+ pumps in the plasma membrane (PMCA), sarco(endo)plasmic reticulum (SERCA) and secretory pathway (SPCA), that play an essential role in the maintenance of low cytosolic Ca2+ concentrations. In 1984, Khachaturian proposed the calcium hypothesis linking alteration of intracellular Ca2+ homeostasis with the aging process and dementia [1], and very recently a new update of this hypothesis [2] has been published due to the increasing number of evidences linking dysregulation of Ca2+ homeostasis with agingrelated diseases such as Alzheimer’s disease (AD). AD is the most prevalent form of dementia in old age population. Although a huge effort is underway to elucidate the disease in order to develop efficient treatments, up to now the mechanisms are not fully understood at molecular and cellular levels. Two key characteristic hallmarks of AD are the aggregation of amyloid-β peptide (Aβ) and tau protein to form extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), respectively. In early-mild AD, it has been noticed a progressive loss of functional synapses in hippocampal and related cortical brain regions [3], and it has been shown that Aβ impairs synaptic performance acting both at pre- and post-synaptic sites [4]. Moreover, many studies have pointed out that nonfibrillar Aβ peptides are strongly neurotoxic and can access the cytosolic space of neurons, see e.g. [5]. The role of different Aβ aggregation states in the early stages of

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AD is still a matter of debate, reviewed in [6]. Concerning tau protein, it is well stablished that its aggregation into NFTs and spreading is associated with the progression of AD. In fact, NFTs have a stereotypical spatiotemporal progression that correlates with the severity of the cognitive decline and defines six Braak stages, clinically used for the pathological diagnosis of AD. More recently, however, it has been reported that cellular uptake of extracellular monomeric tau may lead to cells infection, promoting co-aggregation with endogenous tau and further release out of the cells, thus spreading the infection [7].On the other hand, dysregulation of intracellular calcium homeostasis has been shown both in sporadic and familial forms of AD (see reviews [8-10]), and this can trigger alterations of synaptic morphology and neuronal apoptosis which will eventually lead to cognitive impairment [11]. However, the molecular basis for the sustained alteration of calcium homeostasis in early-mild AD are not well understood and are likely to be different in sporadic and in familial AD. But a sustained alteration of cytosolic calcium concentration can by itself potentiate Aβ peptides formation and tau hyperphosphorylation [12]. An increasing body of evidences suggests that Aβ induces oxidative stress which plays a key role in initial events and progression of AD pathology [13-15]. A link between tau and oxidative stress has also been reported [16-18], see also reviews [19, 20]. However, it is not clear whether oxidative stress is induced by tau phosphorylation or is an early factor that can contribute to tauopathy. Besides, lipid peroxidation and protein oxidation were shown to be increased in most regions of brain affected by AD, but not in cerebellum [21], and several studies have shown dysregulation of lipids in AD-affected brains compared to age-matched controls [22, 23]. This is particularly relevant for dysregulation of intracellular calcium homeostasis, as it has been shown that lipid peroxidation and the lipid peroxidation product 4-hydroxynonenal inhibits PMCA activity [24]. Moreover, calmodulin (CaM) has been found to become more oxidized in aged animals and it has been proved that CaM oxidation leads to inhibition of target proteins by non-productive association and stabilization of their inactive state [25]. This has been experimentally demonstrated for the PMCA [26]. In addition, PMCA is associated with lipid rafts microdomains of the neuronal plasma membrane [27, 28], and cholesterol has been shown to be dysregulated in ADaffected brains [29]. Furthermore, increasing evidences indicate that both Aβ [30, 31] and tau [32, 33] interact with membrane phospholipids, and acidic phospholipids also activate the PMCA to levels close to those attained by CaM stimulation [34]. Bearing in mind the vital role that Ca2+-ATPases play in the control of Ca2+ homeostasis, we have been developing studies to experimentally assess if these pumps are functionally affected by Aβ and tau, the main components of the two major pathological hallmarks of AD. In this review we analyze the following features: i) the differential sensitivity of calcium pumps to Aβ and tau, and ii) the modulation of the inhibitory effects of Aβ and tau on PMCA. 2. Differential sensitivity of calcium pumps to Aβ and tau. Calcium pumps play a central role in the control of intracellular Ca2+ homeostasis in all healthy eukaryotic cells and particularly in neuronal cells (reviewed in [35-37]). Therefore, dysregulation of Ca2+ pumps can a priori account, at least in part, for the alteration of intracellular calcium levels which have been reported to be very important factors of the progression of AD [12]. In addition, the involvement of specific isoforms in particular diseases suggests a functional role of specific Ca2+-ATPases isoforms in the control of different Ca2+-mediated cellular processes (see review [38]).

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Early functional studies performed in our laboratory with membranes prepared from postmortem human brain (cerebrum) revealed a Ca2+ dependence dysregulation of PMCA activity in AD membranes compared to the typical bell-shaped Ca2+ dependence found in age-matched controls and in cerebellum from both, AD and control samples [39]. Interestingly, the addition of Aβ inhibited ATPase activity in membranes from human control samples, at the optimum pCa, with an IC50 of 0.75 µM, but did not have any effect on ATPase activities in membranes from AD-brain and from AD or control cerebellum tissues. In contrast, the Aβ did not affect intracellular SERCA and SPCA activities. Similar effects were observed in synaptosomes and in purified synaptosomal PMCA from pig cerebrum [39, 40]. Therefore, the Ca2+-dependence of PMCA activity found in AD-affected membranes seems to be linked to its inhibition by Aβ. In addition, this study revealed that Aβ-mediated PMCA inhibition was only observed in cerebrum but not in cerebellum, an area which is not significantly affected in AD patients. Since these membranes contain complex mixtures of PMCA isoforms and different lipids and the purified PMCA preparation is also a mixture of isoforms [41] we analyzed Aβ effects on the activity of PMCA isoforms overexpressed in COS cells [39]. This study revealed that Aβ inhibited mainly isoforms hPMCA4b and rPMCA3b, and did not have any effect on hPMCA2b, an isoform that is highly expressed in cerebellum. In addition, removal of the CaM binding domain (CaMBD) on both, the purified PMCA (by proteolysis) and the hPMCA4b (using truncated forms) resulted in a loss of Aβ effects on Ca2+- ATPase activity, suggesting that this domain is involved in PMCA-Aβ binding [42]. Interestingly, the Ca2+-ATPase activity is also sensitive to inhibition by tau protein [41, 42]. The inhibitory effect is specific for PMCA and is produced in the nM range of tau (IC50 ~ 1.5-3 nM) in membranes from brain mouse, human medium frontal gyrus, and cell cultures [41, 42]. However, the following significant differences were found with respect to Aβ effect on PMCA: a) tau inhibits PMCA activity in human brain membranes from both, AD and control samples, b) tau does not affect the Ca2+ dependence of PMCA activity, c) tau also inhibits hPMCA2b, besides isoforms 3 and 4, d) the inhibitory effect of tau is dependent on the ionic nature of phospholipids used to activate the delipidated PMCA, and e) tau does inhibit the truncated hPMCA4b-L1086* lacking the CaMBD (not inhibited by Aβ) [40], but it does not inhibit the shortest form of hPMCA4b-R1052* without the whole cytosolic C-tail [41]. Although several factors may be involved in the functional effects of Aβ and tau on PMCA, these observations pointed out the involvement of residues along the C-terminal tail (which is lacking in the intracellular SERCA and SPCA pumps) in the modulatory actions of Aβ and tau. Furthermore, our results suggested that the CaMBD may be a target for Aβ binding [40], while tau may bind the C-terminal tail on a site close to the transmembrane domain 10 [41]. Another important component that is likely to link Aβ and tau with PMCA dysfunction is the possible oxidation of PMCA caused by Aβ and tau, which can lead to a decrease of ATPase activity [40]. Oxidative stress has been found to affect PMCA pump function, decreasing its activity either as a result of PMCA exposure to oxidants in synaptic membranes [43], human blood purified preparations [44] and neuronal cultures [45], or by an increase in ROS production associated with aging and neurodegeneration [46]. Several studies have shown that neuronal cell lines can internalize both Aβ [47, 48] and tau protein [49-51]. We have reported that treatment of SHSY5Y cells with tau caused a decrease of cell viability in parallel with an increase in ROS production, and an inhibition of the PMCA Ca2+-ATPase activity similar to that described by our laboratory in membranes prepared from animal species and human

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samples [41]. Analogous response was observed with primary cultures of rat hippocampal neurons treated with Aβ (unpublished results). Comparable effects were reported when additions were done to non-treated cells, indicating that their role as modulators of Ca2+-ATPase activity are not due to their effects on cell survival. However, a direct demonstration and identification of the oxidative modification(s) of PMCA that may be induced by Aβ and/or tau is still lacking. 3. The inhibition of PMCA by Aβ and tau is modulated by calmodulin and phosphatidylserine The functional inhibition of PMCA by Aβ and tau is likely a factor contributing to the observed dysregulation of cytosolic calcium in the brain of AD patients. Therefore, agents that could interfere with the inhibitory effects of these two molecules on PMCA activity should be expected to provide protection against brain neurodegeneration in AD. A vast number of functional studies performed with purified proteins and overexpressed native and truncated isoforms have revealed that PMCA is highly regulated, and is primarily activated by CaM and acidic phospholipids [34], reviewed in [35, 38, 52, 53]. This pump contains most of its regulatory sites in the cytosolic Cterminal tail. Among them, a key site is the CaMBD which turns to be an autoinhibitory domain that interacts with two sites of the protein located in their largest cytosolic loops, close to the active site [54, 55]. The previously reported lack of Aβ or tau effects on C-terminal constructs of hPMCA4b, lacking the CaMBD or whole C-terminal tail, respectively, let us to rise the hypothesis that some residues located along the cytosolic C-terminal tail can play a major role in the interaction of PMCA with the Aβ peptide and with tau. Consequently, we first focused our attention on CaM as a potential modulator of PMCA inhibition by Aβ and tau. 3.1. Calmodulin can block the inhibition of PMCA by Aβ and tau The Ca2+-CaM complex prevents and reverses the inhibitory effect of Aβ on the activities of purified PMCA and hPMCA4b [40]. Then, it is possible that CaM also binds to Aβ, avoiding the toxic effect of the peptide on the pump. Recently, we have applied fluorescence quenching assays to gain further insight on Aβ/CaM interaction. Taking advantage of the sensitivity of fluorescent Badan (6-bromoacetyl-2dimethylaminonaphthalene) derivatives of CaM and Aβ(1-42) HiLyte™-Fluor555 we have just shown [56] that Aβ binds with very high affinity to CaM (Kd=0.98±0.11 nM) and that this interaction involves the neurotoxic Aβ(25-35) fragment. These findings indicate that CaM and CaM-derived peptides may represent a potential therapeutic venue to target the neurotoxic action of Aβ in AD. As indicated above, PMCA oxidation may be one of the mechanisms involved in its inhibition by Aβ. In a recent study we have suggested that CaM could prevent this oxidative effect by its interaction with the pump [40], based on the protection produced by CaM against the oxidative inactivation of PMCA by H2O2 [44] More recently we have shown that CaM also decreases and even prevents the inhibition of PMCA activity by tau [41]. Noteworthy, exogenous CaM was also able to prevent the effects of Aβ (unpublished results) and tau [41] on ROS production, cell death and PMCA activity in neuroblastoma SHSY5Y cell cultures. However, in aged AD brains CaM protection against PMCA inhibition by Aβ and tau should be impaired because of the decrease of CaM levels [57] and also by CaM oxidation [25]. 3.2. Phosphatidylserine mediates the inhibition of PMCA by tau, but not by Aβ.

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Phosphatidylserine (PS) is a major activator of neuronal PMCA [34]. Recently, we have described that tau inhibits the PMCA activity only when is reconstituted in acidic phospholipids like PS but not in the presence of pure uncharged phospholipids, such as phosphatydilcholine (PC) [41]. Moreover, tau was shown to be a potent PMCA inhibitor, with a Ki value of 1.5±0.2 nM. In contrast, PMCA inhibition by Aβ is independent of the ionic nature of the phospholipids [39]. Although several authors have reported that tau binds to acidic phospholipids with higher affinity than to zwitterionic lipids [32, 33, 58], we concluded that PS sequestration by tau can, at most, have a modest contribution to the inhibition of PMCA activity because: (1) the dissociation constant reported for tau/PS is >0.2 μM [33], e.g. more than 100-fold higher than the Ki obtained for tau, and (2) under our assay conditions the total PS concentration was 17.3 μM, i.e. more than 104-fold higher than the Ki obtained for tau. The use of native and truncated variants of hPMCA4b expressed in COS cells allowed us to increase our knowledge about the relationship between tau inhibitory effects and PMCA structure [41]. A priori, hPMCA4b is an interesting target for tau in AD because: (i) it is also inhibited by Aβ, and (ii) b-splice variants of PMCA contain a larger C-terminal tail than a-splice variants with a PDZ domain-binding sequence that interacts with synaptic proteins [59]. Noteworthy, we found that tau inhibits the truncated isoform lacking the CaMBD, but not the isoform lacking the full C-terminal tail. Therefore, these results strongly support a direct binding of tau to a site located between the CaMBD and the transmembrane domain 10. In addition, an increase in ionic strength has been shown to decrease the inhibitory effect of tau on PMCA activity [42], pointing out that electrostatic interactions play a major role in tau/PMCA interaction. In fact, tau has a C-terminal domain rich in positively charged residues, that binds to the acidic surface of microtubules [60], and an acidic domain that interacts with neuronal plasma membrane components [61]. Therefore, tau could grasp negatively charged PS with PMCA domains enriched in Lys and/or Arg. Basic amino acids-rich sequences are present in the cytosolic loop between transmembrane helices 2 and 3 of the PMCA, which contains one of the two identified PS binding sites of PMCA [62], and is close to the catalytic domain which is altered upon binding of CaM to PMCA [37]. Since CaM also gives protection against the inhibition of PMCA by tau, the possibility that tau also interacts with this PS-binding site of PMCA cannot be excluded, as briefly discussed in [41]. Thus, to gain a deeper understanding regarding the inhibition of PMCA by tau is necessary to map more precisely tau binding site(s) in the three-dimensional structure of hPMCA4b, a task currently underway in our laboratory. 4. Concluding remarks: Experimental data point out that PMCA is functionally inhibited not only by Aβ but also by tau. The PMCA inhibition by these two important molecules in the onset and progression of AD can be a significant feature in the cascade of events leading to disruption of Ca2+ homeostasis associated with AD. Then, PMCA can be considered as a target for therapeutic agents that can prevent or block the neurotoxic actions of Aβ and tau.

Acknowledgements: I want to thank all past and present members of my laboratory and colleagues who have carry out or contributed to the studies described in this review and are mentioned in our

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references. Among them, I am deeply grateful to Prof. Carlos Gutierrez-Merino for his continuous support, constructive comments and help in the writing of the manuscript, and also to Dr. Isaac Corbacho, for his careful reading and assistance with the proofreading of the manuscript. This work was supported by Grants from Ministerio de Economía y Competitividad (MINECO, BFU2014-53641-P) and Junta de Extremadura (GR15139) to the Research Group BBB008, both co-financed by FEDER funds.

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Figure captions Figure 1: Functional effects of Aβ and tau on PMCA activity, based on studies carried out with purified pig brain PMCA reconstituted in PC or in PS, and in hPMCA4b membranes (Ext: extracellular space; Cyt: cytosol; Mbs: membranes).