Membrane targeting, shedding and protein ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2011 | 116 | 742–746

doi: 10.1111/j.1471-4159.2010.07032.x

,

*Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK àI.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, St. Petersburg, Russia

Abstract The early stages of Alzheimer’s disease are characterized by cholinergic deficits and the preservation of cholinergic function through the use of acetylcholinesterase inhibitors is the basis for current treatments of the disease. Understanding the causes for the loss of basal forebrain cholinergic neurons in neurodegeneration is therefore a key to developing new therapeutics. In this study, we review novel aspects of cholinesterase membrane localization in brain and propose mechanisms for its lipid domain targeting, secretion and protein-protein interactions. In erythrocytes, acetylcholinesterase (AChE) is localized to lipid rafts through a GPI anchor. However, the main splice form of AChE in brain lacks a transmembrane peptide anchor region and is bound to the ‘prolinerich membrane anchor’, PRiMA, in lipid rafts. Furthermore, AChE is secreted (‘shed’) from membranes and this shedding is stimulated by cholinergic agonists. Immunocytochemical

studies on rat brain have shown that membrane-associated PRiMA immunofluorescence is located selectively at cholinergic neurons of the basal forebrain and striatum. A strong association of AChE with the membrane via PRiMA seems therefore to be a specific requirement of forebrain cholinergic neurons. a7 nicotinic acetylcholine receptors are also associated with lipid rafts where they undergo rapid internalisation on stimulation. We are currently probing the mechanism(s) of AChE shedding, and whether this process and its apparent association with a7 nicotinic acetylcholine receptors and metabolism of the Alzheimer’s amyloid precursor protein is determined by its association with lipid raft domains either in normal or pathological situations. Keywords: acetylcholinesterase, Alzheimer disease, amyloid, lipid rafts, PRiMA, shedding, secretase. J. Neurochem. (2011) 116, 742–746.

Alzheimer’s disease (AD) is uniquely characterized by the selective deposition in the brain of aggregates of the amyloid b-peptide (Ab) derived from aberrant metabolism of the amyloid precursor protein (APP) particularly in cortical and hippocampal areas. In AD, there is also an inexplicable failure of the cholinergic innervation of the cerebral cortex that arises from basal forebrain cholinergic neurons (Schliebs and Arendt 2006). Current treatments for AD rely largely on inhibitors of acetylcholinesterase (AChE) to provide temporary symptomatic relief but they do not halt the progress of the disease. A major question therefore is what causes the basal forebrain cholinergic neurons to be selectively vulnerable. In this mini-review, we highlight several features of

cholinergic biology, particularly in relation to AChE and its membrane localisation and protein interactions, which may facilitate the amyloidogenic deposition pathway. In this

742

Received September 7, 2010; revised manuscript received September 22, 2010; accepted September 22, 2010. Address correspondence and reprint requests to Prof. Anthony J. Turner, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail: [email protected] Abbreviations used: Ab, amyloid b-peptide; AChE, acetylcholinesterase; AD, Alzheimer’s disease; APP, amyloid precursor protein; GPI, glycosyl-phosphatidylinositol; nAChR, nicotinic acetylcholine receptor; PRiMA, proline-rich membrane anchor.

Ó 2011 The Authors Journal of Neurochemistry Ó 2011 International Society for Neurochemistry, J. Neurochem. (2011) 116, 742–746

Brain acetylcholinesterase | 743

context the partial localisation of AChE to lipid raft domains provides a new perspective to cholinergic biology and to amyloidogenesis.

of 40 amino acid residues, called the T peptide. Disulphide bonding between AChET subunits via the T peptide gives rise to amphipathic homodimers and homotetramers of the enzyme. The T peptide, which is organised as an a-helix, also allows the AChE complexes to bind to hydrophobic, proline-rich domains of specific membrane-anchoring proteins such as the collagen-like Q subunit at the neuromuscular junction, and the proline-rich membrane anchor (PRiMA) in the brain (Perrier et al. 2002). Most of the AChE in the CNS is thus in the form of tetrameric AChET (designated G4) bound to PRiMA (Navaratnam et al. 2000; Perrier et al. 2002). It has been proposed that the interaction of AChE with PRiMA forms the basis of a larger multiprotein complex (Navaratnam et al. 2000). If so, two key questions emerge: (i) how do these interactions direct the trafficking and sub-synaptic localisation of AChE, and (ii) does dysregulation of these interactions and subcellular localization play any role in CNS pathology and neurodegeneration given the sensitivity of the cholinergic system to loss in AD? As the primary role of AChE is to hydrolyse ACh, nicotinic receptors (nAChRs), and especially the desensitising a7* nAChR, may be particularly reliant on the action of AChE to prevent their desensitisation by ACh. Consistent with this hypothesis is our previous demonstration of high levels of AChE and a7* nAChR expression on the membranes of basal forebrain cholinergic neurons (Henderson 1981, 1989; Henderson et al. 2005). Both AChE and a7* nAChR have been proposed to interact with the Ab, a toxic metabolite of APP when in oligomeric form and the main component of the senile plaques in AD (Inestrosa et al. 1996; Wang et al. 2000; Small et al. 2007). Both reversible and irreversible cholinesterase inhibitors also cause changes

Acetylcholinesterase localisation and membrane anchorage Although the principal function of AChE is the hydrolysis of the neurotransmitter acetylcholine, it also displays a number of non-catalytic, non-classical functions. For example, exogenous AChE has been shown to increase neurite outgrowth, an effect that is not blocked by typical cholinesterase inhibitors (Grisaru et al. 1999; Greenfield et al. 2008). The cholinergic system, in general, plays a role in the proliferation and differentiation of embryonic stem cells consistent with high levels of both AChE and butyrylcholinesterase in the embryonic nervous system (Landgraf et al. 2010). The cholinergic system also has roles in cell migration and proliferation and differentiation of various haematopoietic cells (Small et al. 1996). These various functional properties of AChE depend to a great extent on the existence of multiple molecular forms of this enzyme deriving from various transcripts of the AChE gene and representing different products of post-translational modification of encoded polypeptides. Thus, AChE exists in several different molecular forms which are dependent on alternative splicing of the AChE gene (Fig. 1). These range from the soluble ‘read-through’ form (AChER) and those bound to the membrane by association with different types of membrane anchors. The most common transcript of AChE in the mammalian nervous system and in muscle is AChET. This encodes an AChE isoform characterized by the presence of a C-terminal peptide

(a) AChE-T

Fig. 1 Current view of alternative protein forms of AChE and their anchoring to the membrane. (a) Soluble forms of AChE are represented by the globular mono-, di- and tetrameric forms of AChE-T (amphiphilic G1, G2, and non-amphiphilic G4), and the monomeric read-through form (AChE-R). (b) Membrane-bound forms of AChE are represented by the non-amphiphilic globular G4 AChE-T form anchored in the neuronal cell membrane via the proline-rich protein PRiMA (1), or at the neuromuscular junction via the collagen-tailed protein, collagen-like Q subunit (ColQ) (2). In erythrocytes and in Torpedo brain, a dimeric G2 hydrophobic form of AChE (AChE-H) is linked to the cell membrane via a GPI anchor (3). AChE-H is also up-regulated in dystrophic muscle.

G1a

(b) (1)

AChE-R

G2a

G4na

AChE-T muscle:

(3)

(2)

AChE-T brain:

A4

A8

AChE-H

A12

erythrocytes:

G4 GPI-G2 ColQ

ColQ

ColQ P

PRiMA


744 | D. Hicks et al.

in the processing of APP (Pakaski et al. 2001). AChE is detectable in amyloid plaques and its sequestration therein is attributed to the affinity between AChE and Ab. The association between AChE and Ab alters the enzymic properties of AChE, that is, its pH optimum and inhibitor sensitivity (Geula and Mesulam 1989), and renders the Ab more neurotoxic (Inestrosa et al. 1996; Alvarez et al. 1998). This accelerated assembly of Ab into fibrils appears to involve the peripheral anionic binding site on AChE (Inestrosa et al. 1996). Such studies lead to the conclusion that the presence of high levels of AChE and a7* nAChR, most likely in complex with other proteins in basal forebrain neurons, may render these neurons more susceptible to injury by Ab. In reciprocal fashion, the degeneration of Ab-associated cholinergic structures is seen in transgenic Tg2576 mice that express the Swedish double mutation of human APP (Lu¨th et al. 2003).

of AChE described above and resembles in some respects the physiological roles of secreted (‘shed’) forms of APP (Ring et al. 2007) possibly implying the existence of a cellular ‘receptor’ for shed AChE. AChE is also released from membranes and/or internalised under pathological circumstances such as excitotoxic stimulation, hypoxia or exposure to Ab (Malatova´ and Marsala 1993; Rodrı´guez-Ithurralde et al. 1998). Whether this is a protective or pathogenic mechanism is, as yet, unknown. The knowledge that AChE secretion is dependent upon calcium ions has led to the suggestion that AChE secretion is physiological and dependent upon exocytosis (Appleyard and Smith 1987). Subsequently, carbachol-induced apparent exocytosis of AChE from cell lines was demonstrated using reversible and non-reversible, and penetrative and nonpenetrative AChE inhibitors (Schweitzer 1993). We have previously demonstrated that the carbachol-stimulated AChE release has characteristics in common with the shedding of other membrane proteins, such as the a-secretase, nonamyloidogenic shedding of APP (Nalivaeva and Turner 1999). This shedding was inhibited by typical a-secretase inhibitors such as batimastat. Thus, a-secretase-like activity may not only inhibit the production of Ab but may also inhibit toxic AChE–Ab complexes from accumulating at the cell membranes of basal forebrain cholinergic neurons. To date, however, the mechanisms of AChE membrane release have received little attention although, in common with APP, it is likely that one or more of the ADAMs (A Disintegrin And Metalloproteinase) family of metalloproteinases are involved (Fig. 2) (van Goor et al. 2009).

The origin of soluble forms of AChE In addition to the membrane-associated form of AChE in the brain, soluble forms of AChE exist and have functional roles. The soluble read-through form of AChE is a rare transcript variant whose expression is up-regulated in response to stress (Salas et al. 2008). In addition, membrane-bound AChE in the CNS, both in vitro and in vivo, is released as a hydrophilic, soluble form upon electrical, pharmacological or behavioural stimulation (Appleyard 1992). The mechanism and function of this ‘secretion’ is unclear but it has been linked with the proposed non-cholinergic, non-catalytic roles

Lipid ra

APP

s A P P β

and

Non-lipid ra

domains

AChE β-secretase α-secretase?

α-secretase

γ-secretase

AICD Fig. 2 Schematic representation of the proteolytic processing of the Alzheimer’s amyloid precursor protein (APP) by secretases and possible involvement of a-secretase in release of a soluble form of AChE-T anchored in the lipid rafts via PRiMA. Cleavage of APP by b- and c-secretases results in formation of an extracellular APP fragment sAPPb, amyloid b-peptide (Ab, shown in black) and the APP

intracellular domain (AICD), which acts as a transcriptional regulator (Belyaev et al. 2009). a-Secretase cleavage prevents formation of Ab and generates sAPPa, p3 (not shown) and AICD. There is evidence that a-secretase-like metallopeptidase can also cleave AChE possibly at the PRiMA site (Nalivaeva and Turner 1999). This process, like APP cleavage by b-secretase, probably takes place in the lipid rafts.


Brain acetylcholinesterase | 745

Lipid-raft association of AChE and its functional consequences Lipid rafts are membrane compartments rich in cholesterol and sphingolipids and they play a key role in cell signalling and in Ab formation in AD (Cordy et al. 2006). For example, exclusively targeting the APP b-secretase to lipid rafts by attachment of a glycosyl-phosphatidylinositol (GPI)anchor causes a substantial increase in Ab production (Cordy et al. 2003) and key proteins of the amyloidogenic pathway have post-translational modifications facilitating their co-location in rafts (Vetrivel and Thinakaran 2010). Whether or not the processing of APP follows an amyloidogenic or non-amyloidogenic route is partly determined by factors that interfere with the integrity of lipid rafts, for example, fluctuations in cholesterol levels, hypoxia and oxidative stress (Cordy et al. 2003, 2006; Ehehalt et al. 2003). One of the splice forms of AChE found in mammalian erythrocytes (and paradoxically in Torpedo brain) is hydrophobic in nature (referred to as AChE-H) (Fig. 1) and possesses a GPI-anchor thus localizing this form exclusively to lipid rafts (Massoulie´ et al. 2005). This suggests that at least a proportion of mammalian brain AChE may also require raft localization for some of its neuronal functions, necessitating an alternative anchoring strategy, because AChET does not possess any membrane anchoring domain and is membrane-localized solely via PRiMA. A recent study (Xie et al. 2010) endorses this hypothesis and shows a significant proportion of neuronal AChE is raft-localized and that this process is determined through its interaction with PRiMA. PRiMA is, like APP, a type I transmembrane protein and it exists as two alternatively spliced forms both of which can act as membrane anchors for AChET. The preferential raft localisation of PRiMA appears to be a consequence of a 13-residue cholesterol-binding motif bordering its transmembrane and cytoplasmic domains. Both cholesterol depletion and mutation of the cholesterol-binding motif reduced the raft association of PRiMA. Additionally, palmitoylation of a cytoplasmic cysteine residue may contribute to the raft association (Xie et al. 2010). Whether the shedding of AChE is confined to raft-localised enzyme, or like APP, to non-raft domains, remains to be elucidated. In a separate study (Henderson et al. 2010) we have shown, by using an immunocytochemical approach, a strong plasma membrane co-localization of AChE and PRiMA associated with the somata, dendrites and axons of cholinergic neurons, particularly those that innervate forebrain and brainstem structures. In contrast, PRiMA immunoreactivity was not detectable on neighbouring GABAergic neurons or on dopaminergic neurons. PRiMA expression increases during brain development, in parallel with AChE, but changes in PRiMA with aging and neurodegeneration have not been documented to date. The mitogen-activated protein kinase signalling pathway appears to have a key role in regulating

both PRiMA and AChE co-expression (Xie et al. 2009) and is promoted by a variety of growth factors, including nerve growth factor and brain-derived neurotrophic factor, as well as the neural cell-adhesion molecule. Another site where the GPI-anchored form of AChE is highly expressed is the sarcolemma from dystrophic muscle where there is a corresponding loss of PRiMA expression and of PRiMAcontaining AChE tetramers (Moral-Naranjo et al. 2010). This up-regulation of AChE-H in dystrophic muscle correlates with a striking increase in the density of lipid rafts (5fold higher levels of caveolin-3) in the tissue compared to wild-type. These changes may reflect attempts by the damaged muscle fibre to accelerate protein transport, including AChE, to the sarcolemma for repair (MoralNaranjo et al. 2010).

Conclusions The selective loss of cholinergic neurons in AD remains an enigma yet perhaps provides an opportunity for novel therapeutic intervention in addition to the use of AChE inhibitors. In this context, AChE clearly plays a central role and its subcellular localization, shedding and protein-protein interactions with Ab itself, with a7* nAChRs and with other as yet unidentified proteins are key components of the amyloidogenic process. The localization of AChE to membrane lipid rafts although its association with PRiMA, a site where amyloidogenic processing predominates and where a7* nAChRs congregate, should allow the unravelling of the links between lipid rafts, amyloidogenic processing and its facilitation in cholinergic neurons.

Acknowledgements We thank the U.K. Medical Research Council and the Russian Academy of Science Programme ‘Fundamental Sciences to Medicine’ and Russian Foundation for Basic Research (10-04-01156) for their financial assistance, and the Biotechnology and Biological Sciences Research Council for their PhD studentship support for DH and DJ.

References Alvarez A., Alarcoń R., Opazo C. et al. (1998) Stable complexes involving acetylcholinesterase and amyloid-b peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer’s fibrils. J. Neurosci. 18, 3213–3223. Appleyard M. E. (1992) Secreted acetylcholinesterase: non-classical aspects of a classical enzyme. Trends Neurosci. 15, 485–490. Appleyard M. E. and Smith A. D. (1987) Spontaneous and carbacholevoked in vivo secretion of acetylcholinesterase from the hippocampus of the rat. Neurochem. Int. 11, 397–406. Belyaev N. D., Nalivaeva N. N., Makova N. Z. and Turner A. J. (2009) Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease. EMBO Rep. 10, 94–100.


746 | D. Hicks et al.

Cordy J. M., Hussain I., Dingwall C., Hooper N. M. and Turner A. J. (2003) Exclusively targeting b-secretase to lipid rafts by GPI-anchor addition up-regulates b-site processing of the amyloid precursor protein. Proc. Natl Acad. Sci. USA 100, 11735–11740. Cordy J. M., Hooper N. M. and Turner A. J. (2006) The involvement of lipid rafts in Alzheimer’s disease. Mol. Membr. Biol. 23, 111–122. Ehehalt R., Keller P., Haass C., Thiele C. and Simons K. (2003) Amyloidogenic processing of the Alzheimer b-amyloid precursor protein depends on lipid rafts. J. Cell Biol. 160, 113–123. Geula C. and Mesulam M. (1989) Special properties of cholinesterases in the cerebral cortex of Alzheimer’s disease. Brain Res. 498, 185– 189. van Goor H., Melenhorst W. B., Turner A. J. and Holgate S. T. (2009) Adamalysins in biology and disease. J. Pathol. 219, 277–286. Greenfield S. A., Zimmermann M. and Bond C. E. (2008) Non-hydrolytic functions of acetylcholinesterase. The significance of C-terminal peptides. FEBS J. 275, 604–611. Grisaru D., Sternfeld M., Eldor A., Glick D. and Soreq H. (1999) Structural roles of acetylcholinesterase variants in biology and pathology. Eur. J. Biochem. 264, 672–686. Henderson Z. (1981) A projection from acetylcholinesterase-containing neurones in the diagonal band to the occipital cortex of the rat. Neuroscience 6, 1081–1088. Henderson Z. (1989) Acetylcholinesterase on the dendrites of central cholinergic neurons: an electron microscopical study in the ferret. Neuroscience 28, 95–108. Henderson Z., Boros A., Janzso G., Westwood A. J., Monyer H. and Halasy K. (2005) Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent. J. Physiol. 562, 165–182. Henderson Z., Matto N., John D., Nalivaeva N. N. and Turner A. J. (2010) Co-localization of PRiMA with acetylcholinesterase in cholinergic neurons of rat brain: an immunocytochemical study. Brain Res. 1344, 34–42. Inestrosa N. C., Alvarez A., Pe´rez C. A., Moreno R. D., Vicente M., Linker C., Casanueva O. I., Soto C. and Garrido J. (1996) Acetylcholinesterase accelerates assembly of amyloid-b-peptides into Alzheimer’s fibrils: possible role of the peripheral site of the enzyme. Neuron 16, 881–891. Landgraf D., Barth M., Layer P. G. and Sperling L. E. (2010) Acetylcholine as a possible signaling molecule in embryonic stem cells: studies on survival, proliferation and death. Chem. Biol. Interact. 187, 115–119. Lu¨th H. J., Apelt J., Ihunwo A. O., Arendt T. and Schliebs R. (2003) Degeneration of b-amyloid-associated cholinergic structures in transgenic APPSW mice. Brain Res. 977, 16–22. Malatova´ Z. and Marsala J. (1993) Cholinergic enzymes in spinal cord infarction. Biochemical and histochemical changes. Mol. Chem. Neuropathol. 19, 283–296. Massoulie´ J., Bon S., Perrier N. and Falasca C. (2005) The C-terminal peptides of acetylcholinesterase: cellular trafficking, oligomerization and functional anchoring. Chem. Biol. Interact. 157–158, 3–14. Moral-Naranjo M. T., Montenegro M. F., Munõz-Delgado E., Campoy F. J. and Vidal C. J. (2010) The levels of both lipid rafts and raftlocated acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency. Biochim. Biophys. Acta 1802, 754–764.

Nalivaeva N. N. and Turner A. J. (1999) Does acetylcholinesterase secretion involve an ADAMs-like metallosecretase? Lett. Peptide Sci. 6, 343–348. Navaratnam D. S., Fernando F. S., Priddle J. D., Giles K., Clegg S. M., Pappin D. J., Craig I. and Smith A. D. (2000) Hydrophobic protein that copurifies with human brain acetylcholinesterase: amino acid sequence, genomic organization, and chromosomal localization. J. Neurochem. 74, 2146–2153. Pakaski M., Rakonczay Z. and Kasa P. (2001) Reversible and irreversible acetylcholinesterase inhibitors cause changes in neuronal amyloid precursor protein processing and protein kinase C level in vitro. Neurochem. Int. 38, 219–226. Perrier A. L., Massoulie´ J. and Krejci E. (2002) PRiMA: the membrane anchor of acetylcholinesterase in the brain. Neuron 33, 275–285. Ring S., Weyer S. W., Kilian S. B. et al. (2007) The secreted b-amyloid precursor protein ectodomain APPsa is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J. Neurosci. 27, 7817–7826. Rodrı´guez-Ithurralde D., Maruri A. and Rodrı´guez X. (1998) Motor neurone acetylcholinesterase release precedes neurotoxicity caused by systemic administration of excitatory amino acids and strychnine. J. Neurol. Sci. 160(Suppl. 1), S80–S86. Salas R., Main A., Gangitano D. A., Zimmerman G., Ben-Ari S., Soreq H. and De Biasi M. (2008) Nicotine relieves anxiogenic-like behavior in mice that overexpress the read-through variant of acetylcholinesterase but not in wild-type mice. Mol. Pharmacol. 74, 1641–1648. Schliebs R. and Arendt T. (2006) The significance of the cholinergic system in the brain during aging and in Alzheimer’s disease. J. Neural Transm. 113, 1625–1644. Schweitzer E. S. (1993) Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells. J. Cell Sci. 106, 731–740. Small D. H., Michaelson S. and Sberna G. (1996) Non-classical actions of cholinesterases: role in cellular differentiation, tumorigenesis and Alzheimer’s disease. Neurochem. Int. 28, 453–483. Small D. H., Maksel D., Kerr M. L. et al. (2007) The b-amyloid protein of Alzheimer’s disease binds to membrane lipids but does not bind to the a7 nicotinic acetylcholine receptor. J. Neurochem. 101, 1527–1538. Vetrivel K. S. and Thinakaran G. (2010) Membrane rafts in Alzheimer’s disease b-amyloid production. Biochim. Biophys. Acta 1801, 860– 867. Wang H. Y., Lee D. H., Davis C. B. and Shank R. P. (2000) Amyloid peptide Ab(1–42) binds selectively and with picomolar affinity to a7 nicotinic acetylcholine receptors. J. Neurochem. 75, 1155– 1161. Xie H. Q., Choi R. C., Leung K. W., Chen V. P., Chu G. K. and Tsim K. W. (2009) Transcriptional regulation of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in neuron: an inductive effect of neuron differentiation. Brain Res. 1265, 13–23. Xie H. Q., Liang D., Leung K. W., Chen V. P., Zhu K. Y., Chan W. K., Choi R. C., Massoulie´ J. and Tsim K. W. (2010) Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons. J. Biol. Chem. 285, 11537–11546.
