Pierre-Alexandre Favier
email: favier@enib.fr

Pierre De Loor
email: deloor@enib.fr

From decision to action : intentionality, a guide for the specification of intelligent agents' behaviour

Keywords: multi-agent systems, intentional agent, in-line prototyping, behavioural specification, cross-paradigm programming

This article introduces a reflexion about behavioural specification for interactive and participative agent-based simulation in virtual reality. Within this context, it is necessary to reach a high level of expressivness in order to enforce interactions between the designer and the behavioural model during the in-line prototyping. This requires to consider the need of semantic very early in the design process. The Intentional agent model is here exposed as a possible answer. It relies on a mixed imperative and declarative approach which focuses on the link between decision and action. The design of a tool able to simulate virtual environment implying agents based on this model is discussed.

Introduction

This article presents a work in progress about behavioural specification for multiagent systems and the study of a tool designed to interact with such systems. This is a part of the research area of the LI2 a which is concerned by the study of methodologies and tools for agents' behaviour specification and execution, within the context of interactive and participative simulation. Our aim is to enable in-line prototyping of such agents : we want the end-user to be able to test and modify his model (agent's behaviour) at simulation time. This work introduces a reflexion around our agent-oriented language oRis. [START_REF] Harrouet | oRis : un environnement de simulation interactive multi-agents[END_REF] This is an interpreted language wich is able to schedule the different activities of many agents and to dynamically modify the code of each instance through a run-time code parsing mechanism. oRis, combined with a dedicated rendering library, was succesfully used for really heterogenous applications dealing with domains as different as biology, 2,3 dynamic problem solving systems, [START_REF] De Loor | Solving distributed an dynamic constraints using an emotional metaphor: Application to the timetabling problem[END_REF] interactive simulator [START_REF] Bodic | Multimodal interfaces evaluation with virtual reality simulation[END_REF] or virtual environment for training. [START_REF] Querrec | Multiagents systems for virtual environment for training. application to fire-fighting[END_REF][START_REF] Querrec | Les systèmes multi-agents pour les Environnements Virtuels de Formation[END_REF] This last a Laboratoire d'Ingénierie Informatique (Computer science Engineering Laboratory) application field, virtual environment for training, is typically the kind of application we are targeting at : they imply need of a very high level specification since they need design of avatar with complex behaviours. If the end user is a teacher of a really specific domain, we can't expect him to be a multi-agent systems' expert at the same time. The design tool should provide an intuitive interface, allowing the designer to remain as close as possible to his domain ontology during the whole process.

This implies that the model should accept domain specific keywords as symbols for the behaviour specification. The user should therefore be able to specify and modify his model, manipulating his everyday vocabulary. This is not only a matter of interface, it implies the usage a behavioural model which can manipulate domain dependent symbols during the specification process on one hand, and on the other hand which can produce a "feedback" to the user with those symbols too : if the end user isn't a computer science expert, the system have to be able to produce on the fly some explanations using his vocabulary. This need of a semantic regards both the design process of the behaviour and its execution. This consideration must be kept in mind in order to propose a kind of behavioural model which favors high level interactions with the end-user and the associated execution tool.

oRis was a partial answer to this problem : it allows on-line prototyping, but it requires the end user to be familiar with its syntax, wich is very close to C++. The reflexion exposed here deals with a new behavioural specification model proposition, and with the prerequities of the tool capable to execute it : we rely on the experience aquired through the building and the usage of oRis to create a new framework for the design and the execution of multi-agent systems closer to the end user.

As it was previously said, the building of such a tool can't be separated from the elaboration of the behavioural model : these two aspects of this approach emulate each other.

Next sections of this article details our approach as a compromise : our desired mixing of reactive and symbolic approaches of behaviour specification induces a cross-paradigm (imperative and declarative) implementation of the execution model. Intentionality is introduced as a link between those different aspects of the behaviour specification and the intentional agent model is described. Finally, the first implementation of the framework is explained and some of the potential evolutions of this proposition are discussed as a conclusion.

Need of a Specification Framework

From conception to execution

Among the numerous methodologies that implement an agent's behaviour in a virtual reality simulation 8 , it's usually very hard to chose the most appropriated one. Actually, this choice is rather a matter of habits of the designer or of industrial constraint. But the designer is not always the end-user : the implementation of a behaviour is a "translation" from the targeted domain to a definition in a com-puter language. Sometimes the computer science expert is able to learn enougth about the application domain to process coherently, but most of the time he has to interpret the model expressed by an expert of the application domain. Another approach is to ask this expert to learn computer science and to perform to the implementation. Those ways are not suitable for complex systems : they imply very specific knowledge from both the programmer and the designer. Though, a tool built for the design of such systems has to provide an interface between those two aspects of the implementation process usable by all the involved person. This link between the software implementation and the conceptual design of the behaviour should be used on one hand at design time, in order to help the communication within the developpment team, and on the other hand at run-time if we want to enable on-line prototyping : if we expect the designer (the end user) to interactively modify his model, we should provide a framework which can support the execution of agents specified and manipulated through domain dependent concepts; we can't ask an ethologist who is tuning a simulation involving fishes to dynamically modify the java class implementing their behaviour. This in-line prototyping objective implies a coherence from specification to execution : the model must be defined and manipulated (executed) the same way; definition and execution are not different steps anymore. So, our aim is to reach this complete coherence in the whole specification process in our framework and, ideally, to build a complete methodology of behaviour specification based on this framework. A framework capable of such an abstract control level should rely on very generic mechanisms. The definition of the necessary set of those mechanisms is the main obstacle, there is no unique correct choice. Since the priority is given to the possibility of a manipulation through the domain dependent concepts and vocabulary, the class of expressible behaviour has to be restricted in order to define a minimal and consistent set of basics behavioural mechanisms to be implemented in the framework. The following describes the kind of behavioural model we expect to specify and execute.

Meaning of action

Our approach of behaviour specification is a compromise between reactive approaches [START_REF] Maes | The dynamics of action selection[END_REF][START_REF] Brooks | A robust layered control system for a mobile robot[END_REF][START_REF] Arbib | Schema theory[END_REF][START_REF] Arkin | Behavior-Based Robotics[END_REF][START_REF] Arnaud | Des moutons et des robots[END_REF] and symbolical approaches [START_REF] Cohen | Intention is choice with commitment[END_REF][START_REF] Shapiro | Specifying communicative multi-agent systems[END_REF]16,[START_REF] Bordini | Agentspeak(xl): Efficient intention selection in bdi agents via decision-theoretic task scheduling[END_REF] . The former are usually simple and easy to process automatically, and they are often a efficient for real-time rendering which is an important aspect in interactive simulation of virtual environment. Nevertheless they usually are not suitable for in-line prototyping because of lack of semantic in the model. The later, symbolical approaches, bring expressiveness and a good abstraction level; they are more confortable for non computer-science expert though they suffer from completeness and synchronization problem within the context of dynamic and open systems such as virtual environment : execution time for complex symbolic resolution could lead to coherence problem.

To get rid of as many drawbacks of those existing approaches as possible, the one considered here is a mix of reactive and symbolic approaches : it's an action selection method which adds some semantical information about meaning of those actions. We don't want to provide a logic about action as in [START_REF] Pratt | Action logic and pure induction[END_REF][START_REF] Lamport | The temporal logic of actions[END_REF][START_REF] Baral | Reasoning about effects of concurrent actions[END_REF] but we constraint the designer to add to every implemented method the needed symbols for reasoning. Each action (including perceptions which are here considered as particular actions) is in charge of the provision of some information about its side effect on the agentsome of its properties -in a qualitative way. This enables a symbolical reasoning about effects of actions which are defined following a reactive approach.

This first simple definition of our approach might look ambiguous, but it is a sufficient basis to discuss some important aspects of its implementation before its refinment, which is the next section purpose.

A cross-paradigm implementation

If we consider the behaviour specification as a translation from the application domain into a language computable by the machine, as previously suggested, we have to choose very carefully the final implementation language in order to keep a strong coherence within those different aspects.

Some computer languages are very close to the machine model. Those are imperative languages which need the user to be familiar with very technical concepts such as memory allocation, assignement,. . . They are difficult to use correctly but allow very efficient processing because they are really close to the hardware structure of the computer. They might be a good choice for 3D rendering and immersion in a virtual environment, but they are not suitable for an easy specification of complex behaviour : the building of an application enabling of in-line prototyping from scratch is a matter of specialist, 21 not of the domain expert. It is really hard to design an agent capable of dynamic modification of its own code. Some other languages were designed to be close to the end-user and to favour a high level abstraction, trying to reach the natural language as interface between machine and human beeing. Those are for example declarative or functional languages, such as Prolog. They are closer to our natural reasoning than C++ or Java. They bring the expressiveness we expect for the end-user. They are not nevertheless suitable for the definition of side effects of the agent's action on itself and its environment in a dynamic context because of their performances. But the specification of a behaviour as an enumeration of behavioural rules seems really interesting : with a language such as Prolog the knowledge base is totally independent from the inference mechanism. One can deeply modify a logic program adding or deleting some clauses, even at run time. This is typically the kind of flexibility which can enable a high level in-line prototyping.

We have chosen to use both imperative and declaratice programming : imperative programming for the definition of side effects and perception (methods' implementation) together with declarative logic programming for the reasoning (action selection).

In order to keep the usage of such a tool simple, we have to provide a link between these two parts of the behaviour specification. For the framework's coherence, and to maximize expressiveness in the model, this link must be an automatic and implicit mechanism providing an interface between imperative and declarative parts. Moreover it must also be a therorical link between those two aspects of the specification : it must link decision part with action and perception part from the designer point of view, and, relying on the same concept, it must link the imperative and the declarative parts from the implementation point of view. The figure 1 illustrates the role of this link, which is the main point of our approach. The next section introduces the concept of intentionality as a possible symbolical link between the different parts of this framework.

Intentionality as a Semantical Link

A mixed imperative / declarative programming approach

The choices exposed in the previous sections are the fundation of the intentional model. In one sentence we could say that it's an action selection based on the qualitative explicitation in each action of its effects on the agent itself and its environment. [START_REF] Favier | Cross-paradigm Programming for Behavioural Specification in Multiagent Systems, Intentionality : From Decision to Action[END_REF] Actions and perceptions are coded following an imperative approach (methods) whereas decision (action selection) is made by an inference engine which exploits a knowledge base filled in with behavioural rules. To keep coherence of the agent's state, the decision part must not modify the agent or its environment : only actions are able of side effect. This way, the logic part implied in the decision process is kept as "pure" as possible (no extra-logic predicates needed for some side effects), and a separation is deeply layed down between the action specification, which is the computer science expert job, and the behaviour specification, which is the role of the designer (our end-user). The decision process only needs to consult the value of the agent's properties and must select an action, or a set of actions, which best fit the situation. This is the unique interface between the two parts which has to be defined using a symbolic aproach. This favours the setting of a very meaningfull semantic link between those two parts from specification to execution : the needed interface between imperative and declarative part is entirely defined and implemented in the framework; the designer is constrained to follow our approach if he wants to use this tool. This is the first step through the building of a behaviour specification methodology.

Figure 2 illustrates the behavioural model architecture as it has been described up to this point of this paper. We now have to define the implicit link between the two parts of the behaviour specification.

The implicit link represented in this figure must not be specified by the user. It has to be entirely supported by the plateform. This ensures greater independence between the two aspects of the behaviour specification, which is required by the need of expressiveness for in-line prototyping. Of course, the decision process can't be separated from the agent's state and environment or it wouldn't be accurate. Therefore, although the decision process can't modify the agent's properties, it must still be able to consult them at any time : one of the main feature of the framework should be the ability to consult on the fly the value of a property from the reasoning process. Initializing a logic inference with properties' values and wait for the result is not a good idea : as it was previously stated, logic inference process could be very slow regarding to the global simulation. Thereby, the decision part of the behaviour should be processed in parallel to agent's methods (actions and perceptions), and should be able to consult the value of a property at the exact time it's needed : during the whole process, which may implies many backtracks, the value of a property could become obsolete. Perceptive actions are in charge of updating of those properties' values. This is the place where to put some domain dependent semantic in the model in order to increase expressiveness for the end-user. As every perception method is specific to an agent, its implementation can support the symbolic conversion of a property's value accordingly to domain vocabulary, e.g. : whatever is the unit system used in the simulation plateform, the symbol nearPredator can be updated in the method lookAround() of the agent, if this symbol is meaningfull for this kind of agent. This requires from the user the enumeration of all the needed symbols for the decision process at design time. As those symbols depend on agent's state, they can be exhaustively enumerated : whatever your agent can see in a dynamic environment, the corresponding symbols (prey, predator, obstacle,. . . ) are statically designed. This avoids the completeness problem of pure symbolical approaches in dynamic environment, and brings some more semantic in pure reactive approaches.

Decision

Of course, the inference capabilities is limited by this static enumeration

To enhance coherence of the whole specification process, this link must not only be a link between decision part and action part of the model, but, what's more, a semantical link between specification and execution of the behaviour. So, if a symbolic exchange between decision and action is the technical selected choice, we now have to chose the semantical meaning of such a link. This choice will define the range of model which can be expressed and execute by this framework. Next section introduces the notion of intentionality as a first proposition for this semantical choice.

Intentionality

It has been previously explained that, in this approach, methods are in charge of the production of symbols needed by the decision process. In order to obtain a systematic execution process of behaviour defined this way, and to progress in the building of a specification methodology laying on this framework, the semantic of those symbols has to be exactly defined. The chosen class of expressible behaviour could be qualified as "rational behaviour" : we won't focus on pure reflex behaviour, neither on behaviour based on complex reflexion process. In our cross topic of virtual environments and agentbased simulations, we want to obtain adaptable and credible behaviour. This means that an agent is expected to select the "good" method, accordingly to its dynamic context. But this selection of the appropriated method can't be processed without explicitly expressing the meaning of every method in the behavioural model itself. A behaviour definition is frequently very alike an enumeration of use case, selecting directly a method from some attributes using a if. . . then-style structure. This implies that the meaning of each method is only known at design time and totally disapears at simulation time : action selection is hard coded. This prevents the usage of this meaning within the agent, which yet would be part of in-line prototyping, or to generate some explaination automatically. . . . Our approach is to introduce a symbolic expression of the meaning of every action as an indirection between decision and action : the designer must provide all the information needed by a generic decision process to select an action. This obligation is a first step for the building of a design methodology. The set of behaviour expressible in this framework is limited by the generic aspect of this decision process.

The selection process must be template and does not make use of any logic of action : selection is only based on the context and known meaning of actions. But the context is perceived through perception methods, so it is totally defined by the way of agent's properties (internal represention of the environment). As well, the role of the decision process of the agent is to select an action (or many of them) accordingly to its properties and to its intention : an agent can "decide" to climb because he has got the intention to go higher. The notion of intention is here considered as a willing over a property, a complete analysis of this notion, as described in [START_REF] Searle | Intentionality, an essay in the philosophy of mind[END_REF] ,has to be considered as a guide to enforce coherence during the implementation of the final framework. This led us to the definition of the kind of symbol methods have to produce : every method should export its influence over agent's properties. So, intentionality in the intentional agent's model is seen as the willing to modify some of its properties accordingly to specific tendencies.

Four tendencies have been chosen : increase,reduce,maintain and independent.

For example the method eat could be declared : eat() increase energy. This information about the meaning of the method eat can be used by the decision process to select the correct method. There's no need to define at design time which use case could lead the agent to will to increase its energy : this is the role of the designer to declare, statically or dynamically, the behavioral rules producing this intention. A simple symbolical and qualitative solver based on those four operators provides a generic decision process : whatever properties and action are, they all are treated with this system, for every agent. This allows the selection of one or many action which best respond to agent's intention. There's no guarantee this selection will satisfy every expectation, but such a selection can be done even if it doesn't fit exactly the considered intention. This permits an action selection within an overconstrained context : we don't expect an agent to find an exact matching between its intention and its methods. As the environment is highly dynamic, the agent must be able to select a method even if there's no exact solution at this time. We don't want to create an animation from an exact preprocessed solution : the simulation is created by the dynamic solving by the agent. Some solution can occur or disappear dymically with the modification of the environment. In this context, trying to always find an exact and complete solution in advance trough a planification process is nonsense. [START_REF] Favier | Programming agent with purposes: Application to autonomous shooting in virtual environment[END_REF] Figure 3 below shows the architecture of the intentional agent model : actions are defined by imperative methods, decision is designed as a set of behavioural rules expressed as logic rules and a link between those two parts is ensured by a qualitative and symbolic solver. This is a first attempt to implement a model based on the framework exposed in section 2 : the lacking implicit link of fig. 2 is this solver; its semantic towards the model is brought by the notion of intentionality.

Let's take the example of a very simple cat's behaviour. Let the method lookAround provides the symbol danger. This method is in charge of the treatment of any state of the cat's environment to provide this symbol if applicable. A dog, a car or anything else dangerous should be correctly perceived as a danger. The symbolical adaptations from the environment to the properties of the agent are processed in the methods. Let some behavioural rules be, asking the cat to eat in order to increase its fitness, and refraining it from eating when it's in danger. This will result in the intention to reduce the danger, if it exists. This intention is not sufficient to select the method run() if there's no further information on its effect on agent's properties, danger in particular. If an information is added to provide the link between action and decision, the method can be selected. So the designer has to define : run() reduce danger. This indirection may seem useless, but it adds some semantical information about the meaning of the action. This is the major benefit of the model : this information is specified with a high level of abstraction, using a domain dependent vocabulary.

In the same example, we can imagine our cat knows how to mew, but it "thinks" it only increases its sex appeal. If at run time, the designer, or any learning process, adds the following information : mew() reduce danger, the corresponding method is now selectable, and an alternative behaviour has been defined. The in-line prototyping through high level and domain dependent concept manipulation has been reached.

The information about the symbol provided by the perceptive methods can be used to trigger perception on demand, in order to optimize the whole execution of the behaviour : some perception methods are expensive to process but not necessary all the time.

Figure 4 below sums up the cat's behaviour specification using the intentional model. Keywords ensure and provide are used to specify links between respectively perception and decision in the first place, and decision and action in the second place.

Implementation

The first step of the application of such a proposition is the building of a tool able to execute it. Primary tests have been led using oRis and Prolog. oRis is an imperative and interpreted language. It provides many agent oriented functions : scheduler for parallel activities, message exchange protocol between entities, dynamic code modification,. . . This brings most of the needed functions of our model. We wrapped GNU Prolog's engine in oRis to add the required first logic order capabilities. The wrapper offers the ability to create many prolog context : every agent owns a separate set of stacks to handle all fact and predicate it needs. When a query is performed, the appropriate context is restored within the prolog engine. This is totally transparent to the user which can consider that every agent owns a separated prolog engine. The main asset of this implementation is that there's only one instance of prolog, statically linked with the application : performance remains correct when many agents are implied in the simulation. As this is an add-on to oRis, it doesn't provide all expressiveness we expect : those two languages weren't designed with the idea of such an interface. Another problem is that GNU Prolog is monolithic : when starting a query, you have to wait for the first result; it's hard to schedule many agents as their behaviour become more complex. This first implementation was only a test for a preliminary try in order to validate the basis of our approach.

We are now working with an implementation based on our new C++ library, ARéVi, which offers scheduling and rendering features together with SWI Prolog which is capable of multi-threading resolution. This allows us to explore deeper the proposition of the intentional model. But it is still a test plateform : this implementation only provide a simple bridge between C++ and Prolog, e.g. : the predicate get-Property(+PropertyName, ?Value) has been created to get at any time the value of a C++ property from Prolog part. Some other simple mechanisms allow to give tendencies back to the C++ simulation engine from the Prolog decision process. If a first implementation of intentional agent model is possible with the help of this tool, there is a lack of expressivness of the so created framework because of the deep difference between the two involved language. The binding between C++ attribute and Prolog variable is still partially handled by the designer.

In parallel, we now are building a new language from scratch based on oRis and ARéVI including in its virtual machine the Warren's abstract machine instruction set. [START_REF] Warren | An abstract prolog instruction set[END_REF] This will allow a native support of the needed link between the two aspects of the behavioural specification, with a deep control of the scheduling of mixed imperative / declarative code. This tool will be able to execute any kind of model based on our mixed approach of behaviour specification.

Conclusion

We have presented our reflexion around the need of a greater coherence in the behaviour specification process. This increases the coherence of the whole framework and enables a high level in-line prototyping with a reduced set of concepts : the user interaction with the behavioural model is the same at design time and at run-time. This is one more step toward the interactive prototyping of an agent's behaviour by a non computer science expert. This need should be considered from the early begining of the building of an execution plateform in order to provide an easy interface to the designer. This approach led us to the proposition of a new tool and to the specification of the model we could simulate within this framework : intentional agent model. The building of a new tool from scratch ensures the usage of the same concepts from conception to execution. We consider it is the only way to reach the expressivness we are targeting at.

Another direction of our further work is the behavioural inheritance : as the behaviour is totally defined by a set of logic rules, it's easier to modify or enrich. Therefore, we now want to define some inheritance mechanisms to allow an agent to inherit -statically or dynamicly -and to share its knowledge. For a prey / predator example, it would permit the creation of a new kind of predator at run-time of the behaviour of a standard predator, and the addition or deletion of some clauses of the purchase rule.

The last direction we'd like to explore is a direct benefit of the semantic added in the model. As the meaning of methods is partially expressed within the model, we could expect a greater easiness in the application of symbolical learning and explanation generation process with intentional agent.

Fig. 1 .

 1 Fig. 1. Suggested model architecture

Fig. 2 .

 2 Fig. 2. Suggested model architecture

Fig. 3 .

 3 Fig. 3. Intentional agent model architecture


María María Teresa Moral-Naranjo

Fernanda Montenegro

Encarnación Muñoz-Delgado

Francisco J Campoy

Dr Cecilio J Vidal
email: cevidal@um.es

The levels of

Keywords: caveolae, cholinesterases, ecto-5'-nucleotidase (CD73), muscular dystrophy

Introduction

Nearly half of patients (30-40%) suffering from congenital muscular dystrophy (CMD) shows a deficiency of the laminin-2 chain (called merosin). Thus, the term 'merosin-deficient CMD' (MCMD) applies to the disease arising from anomalies in the LAMA2 gene that encodes merosin [START_REF] Jiménez-Mallebrera | Congenital muscular dystrophy: molecular and cellular aspects[END_REF][START_REF] Lisi | Congenital muscular dystrophies: new aspects of an expanding group of disorders[END_REF]. LAMA2 mutations affect laminin-2 ( 2 1 1) and laminin-4 ( 2 2 1). At the extracellular matrix laminin-2 links the sarcolemma through integrin 7 1D and -dystroglycan, a protein of the dystrophin-glycoprotein complex that spanning the sarcolemma joins the extracellular matrix with the cytoskeleton of the myofibre.

Laminin-2 is needed to seal the sarcolemma with the basement membrane. Its absence renders the sarcolemma fragile and permeable to calcium, and its increase leads to myofibre damage and muscle wasting [START_REF] Jiménez-Mallebrera | Congenital muscular dystrophy: molecular and cellular aspects[END_REF][START_REF] Lisi | Congenital muscular dystrophies: new aspects of an expanding group of disorders[END_REF]. Despite the above, the molecular etiopathogenesis of MCMD remains unsolved due to the variety of functions played by laminin-2. In humans and mice, MCMD leads to muscle degeneration and nerve dysmyelination [START_REF] Moral-Naranjo | Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice[END_REF], and, although several mouse strains (Lama2dy, dy 2j , dy w , and dy 3k ) are used for unveiling laminin-2 functions, the deficiency of merosin makes it the Lama2dy mouse a suitable model for MCMD studies [START_REF] Jiménez-Mallebrera | Congenital muscular dystrophy: molecular and cellular aspects[END_REF].

Lipid rafts are membrane domains rich in cholesterol, sphingolipids and specific proteins, which have been shown to be involved in signal transduction [START_REF] Lajoie | Lattices, rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane[END_REF]. The rafts are surrounded by less organized lipid/protein mixtures referred to as non-raft membranes [START_REF] Garner | Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers[END_REF][START_REF] Hanzal-Bayer | Lipid rafts and membrane traffic[END_REF].

Caveolae, the omega-shaped invaginations of the plasma membrane, are particular lipid rafts that contain the scaffolding protein caveolin (Cav) [START_REF] Hnasko | The biology of caveolae: lessons from caveolin knockout mice and implications for human disease[END_REF]; non-caveolar rafts are devoid of caveolin.

Acetylcholinesterase (AChE; EC 3.1.1.7) regulates synaptic and neurohumoral cholinergic activity by hydrolysing acetylcholine. Both 5' and 3'-alternatively spliced AChE mRNA variants have been identified [START_REF] Meshorer | Virtues and woes of AChE alternative splicing in stressrelated neuropathologies[END_REF][START_REF] Fernández-Gómez | Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8[END_REF]. 3' spliced mRNAs generate the three classical AChE catalytic subunits, T, H and R [START_REF] Meshorer | Virtues and woes of AChE alternative splicing in stressrelated neuropathologies[END_REF][START_REF] Massoulié | Old and new questions about cholinesterases[END_REF]. AChE-T is by far the major and the most physiologically relevant subunit in mammalian brain and muscle. In muscle, AChE-T polymerizes into globular (G 1 , G 2 and G 4 ) and asymmetric (A 4 , A 8 and A 12 ) forms, which, after appropriate processing, reach specific cell stores [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF][START_REF] Guerra | Acetylcholinesterase and molecular interactions at the neuromuscular junction[END_REF]. The asymmetric species are the principal if not the only AChE forms in the neuromuscular junction (NMJ) of fast-twitch muscle [START_REF] Rotundo | Targeting acetylcholinesterase to the neuromuscular synapse[END_REF]. The AChE-R subunit can replace the AChE-T protein in the oligomers of brain [START_REF] Meshorer | Virtues and woes of AChE alternative splicing in stressrelated neuropathologies[END_REF] and the AChE-H subunit incorporates glycosylphosphatidylinositol (GPI) and generates GPI-anchored G 1 and G 2 species [START_REF] Nieto-Cerón | Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice[END_REF]. Though functionally unrelated, AChE and ecto-5'nucleotidase (eNT; CD73) share some properties. Both AChE-H forms and eNT lie in the cell membrane as GPI-linked dimers, where they hydrolyse neurotransmitters (acetylcholine and AMP) [START_REF] García-Ayllón | Identification of inactive ecto-5'-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2 dy mice[END_REF].

The importance of lipid rafts for membrane traffic [START_REF] Hanzal-Bayer | Lipid rafts and membrane traffic[END_REF] besides the increased number of caveolae in muscle of dystrophin-deficient mice [START_REF] Vaghy | Increased caveolin-3 levels in mdx mouse muscles[END_REF] and of patients with Duchenne muscular dystrophy [START_REF] Repetto | Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy[END_REF] prompted us to study firstly whether the merosin deficiency affected or not the population of lipid rafts in muscle. Moreover, bearing in mind: 1) the incorporation of GPIlinked proteins in lipid rafts [START_REF] Garner | Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers[END_REF]; 2) the presence in tissues of GPI-linked AChE-H protein;

and 3) the role of rafts in targeting nicotinic receptors (nAChR) to the NMJ [START_REF] Zhu | Lipid rafts serve as a signaling platform for nicotinic acetylcholine receptor clustering[END_REF], our second goal was to look at the possible sorting of GPI-anchored AChE to lipid rafts as a means to justify the presence of AChE-H mRNA and protein in muscle and other tissues. Besides, given the abnormal pattern of AChE forms in merosin-deficient muscle [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF] and the capacity of AChE to bind laminin, caveolin and other protein partners [START_REF] Johnson | Non-enzymatic developmental functions of acetylcholinesterase -the question of redundancy[END_REF][START_REF] Paraoanu | Acetylcholinesterase in cell adhesion, neurite growth and network formation[END_REF][START_REF] Park | Interactions of acetylcholinesterase with caveolin-1 and subsequently with cytochrome c are required for apoptosome formation[END_REF] it might be that the merosin deficiency disrupted AChE transport toward the surface membrane of muscle.

Since both caveolar and non-caveolar rafts exist, our third objective was to explore whether the content of non-caveolar rafts increased in merosin-deficient muscle. For this, advantage of the widely accepted use of catalytically active eNT as a marker of non-caveolar

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rafts was taken [START_REF] Matschke | Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma[END_REF][START_REF] Ostapkowicz | Lipid rafts remodelling in estrogen receptor-negative breat cancer is reversed by histone deacetylase inhibitor[END_REF]. Some of the results reported here have been published in a preliminary form [START_REF] Moral-Naranjo | Targeting of acetylcholinesterase to lipid rafts of muscle[END_REF].

Methods

Materials

Acetylthiocholine and butyrylthiocholine iodide, 5,5'-dithio-bis-2-nitrobenzoic acid, tetraisopropyl pyrophosphoramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), AMP, , -methylene-ADP, Fiske-Subbarow reagent, Triton X-100 and a cocktail of antiproteinases made of bacitracin (1 mg/ml), benzamidine (2 mM), pepstatin A (10 μg/ml), leupeptin (20 μg/ml), aprotinin (20 U/ml) and soybean trypsin inhibitor (0.1 mg/ml). The muscle was ground in Ultra-Turrax at low speed, five times for 10 s with 40 s intervals, and then in a glass-glass Potter (10 strokes). After centrifugation at 4ºC (5 min at 1,000 x g) the supernatant was mixed with its volume of 80% sucrose in MBS.

The mixture (4 ml) was placed on centrifuge tubes and, after adding a 30-5% continuous sucrose gradient over the mixture, the tubes were centrifuged at 165,000 x g, for 16-20 h at 4ºC, in a SW41 Beckman rotor. Twelve fractions of 1 ml were collected from the top of the gradient and the Triton-resistant membrane fractions, containing the lipid rafts, were identified by their content in the protein markers of caveolar or non-caveolar rafts, i.e., Cav3 and eNT activity. More than 95% of the protein remained within the 40% sucrose layer, which contained the Triton X-100-soluble membranes. Fourteen raft preparations of NM and twelve of DM were analyzed.

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Immunolabelling of Cav3 in muscle sections

Frozen muscle sections (0.4-0.5 mm thick) were fixed in buffered 4% paraformaldehyde, washed three times with a saline phosphate buffer, incubated with 0.1% Triton X-100 in the above buffer for 1 h, and washed again. After blocking with 3% bovine serum albumin in phosphate buffer, muscle sections were soaked overnight in anti-Cav3

antibodies (1:300) washed and incubated with Alexa Fluor 488 goat anti-mouse IgG Fluor 488. For image analysis, the mean integrated optical density of 10 microscopic fields of NM and DM sections was recorded using a Microm image processing MIP 4.5 software.

Statistical differences between normal and dystrophic muscle sections in Cav3 labelling were examined by the Mann-Whitney rank sum test.

Enzymatic assays

eNT activity was assayed with 1 mM AMP and the released phosphate was determined with a mixture of ammonium molibdate and Fiske-Subbarow reagent [START_REF] Martínez-Martínez | The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds[END_REF]. One unit of eNT activity (U) is the amount of enzyme that hydrolyzes one nmol of AMP per min at 37°C.

Cholinesterase (ChE) activity in the gradient fractions was assayed by the Ellman method [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF]; AChE with 1 mM acetylthiocholine plus 50 µM Iso-OMPA and BuChE with 1 mM butyrylthiocholine plus 10 µM BW284c51. Inhibition of BuChE activity by Triton X-100 was relieved by adding 0.5% Brij 96 to the assay mixture. ChE activity is given in arbitrary units, one unit referring to an increase of 0.001 absorbance units per microliter of sample, and per minute. In sedimentation analysis, ChE activity was normalized for the volume of sample loaded onto the gradient. Activity values for NM and DM were compared by the Mann-Whitney rank sum test. resistant and Triton-soluble membranes were resolved by sedimentation analysis. For this, samples and sedimentation markers were loaded on 5-20% sucrose gradients containing 0.5% Brij 96. After centrifugation at 165,000 x g, 20 h at 4ºC in a SW41 Beckman rotor, fractions were collected from the tube bottom, assayed for AChE and BuChE activities, and sedimentation coefficients of AChE forms were calculated [START_REF] Moral-Naranjo | Molecular forms of acetyl-and butyrylcholinesterase in normal and dystrophic mouse brain[END_REF]. The presence of GPI in the raft-bound AChE forms was tested by incubating samples with PIPLC (3 U/ml), 2 h at 37ºC.

Identification of AChE forms located at lipid rafts

Removal of the hydrophobic domain in GPI-linked AChE was assessed by sedimentation analysis.

Lectin interaction assays

Lectin binding assays were used for testing possible differences between oligoglycans linked to homologous AChE forms in erythrocyte and lipid rafts of muscle. For this, lipid raft extracts (0.5 ml) were mixed with the same volume of Sepharose 4B (control) and of agaroselinked Lens culinaris agglutinin, wheat germ agglutinin and Ricinus communis agglutinin.

After overnight incubation, agarose-bound AChE was removed and unbound AChE activity was quantified by sedimentation analysis [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF].

Real time PCR for measuring the relative content of ChE mRNAs

Possible changes in the content of ChE mRNAs as the result of muscular dystrophy were examined by RT-PCR, using five NM and five DM samples. Details of RNA extraction from muscle and of assessment of its higher amount in DM than NM have been reported

elsewhere [START_REF] Morote-García | The increased ecto-5'-nucleotidase activity in muscle, heart and liver of laminin 2-deficient mice is not caused by an elevation in the mRNA content[END_REF]. For reverse transcription, 5 µg of total RNA (pre-treated with DNase I) was heated 10 min at 70ºC and cooled. A mixture of buffer, dNTPs, dithiothreitol, random primers and ribonuclease inhibitor was added, and all heated 2 min at 42ºC. Then, 200 units

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RESULTS

The content of caveolin-3 increases in merosin-deficient mouse muscle

Lipid rafts (Triton-resistant membranes) of muscle were isolated taking advantage of their insolubility in Triton X-100 and buoyancy in sucrose gradients. The rafts of NM concentrated in a sharp band (N1) in fractions 6-8 of the gradient (out of 12; supplementary that in NM slices (Fig. 2D) lent strong support to the proposal.

Catalytically active eNT prefers raft membranes and inactive eNT non-raft membranes

Since the floating membranes likely consist of Cav3-free and Cav3-bearing rafts (noncaveolar and caveolar rafts), we next investigated whether the production of the former was boosted by the deficiency of merosin. For this, we exploited the consideration of eNT activity as a marker of non-caveolar rafts [START_REF] Matschke | Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma[END_REF][START_REF] Ostapkowicz | Lipid rafts remodelling in estrogen receptor-negative breat cancer is reversed by histone deacetylase inhibitor[END_REF]. The fact that both eNT activity and Cav3 concentrated in the same membrane fractions of NM (Fig. 3) supported co-migration of caveolar and non-caveolar rafts, an idea corroborated by the increased levels of both Cav3 and eNT activity (2.3 ± 0.6 times, n = 5, p = 0.038) in the raft-rich fractions of DM (Fig. 3).

Although eNT activity was confined to rafts, Western blots detected eNT protein in fractions with and without rafts (Fig. 3B). According to our previous results [START_REF] Morote-García | The increased ecto-5'-nucleotidase activity in muscle, heart and liver of laminin 2-deficient mice is not caused by an elevation in the mRNA content[END_REF], the bands at 68-72 kDa in the gel lanes with rafts of NM (rich in eNT activity) should correspond to active eNT and the triplet at 72-78 kDa in non-raft fractions (lacking activity) to inactive eNT. Whilst non-catalytic eNT protein was similarly labelled in non-rafts of NM and DM (Fig. 3) both eNT activity and eNT protein (blot signal) rose in rafts of DM (2.3 ± 0.6 and 3.4 ± 1.3 times (n = 4, p = 0.017), respectively), and this confirmed previous observations concerning the relationship between muscular dystrophy and the increase of eNT activity in muscle [START_REF] Morote-García | The increased ecto-5'-nucleotidase activity in muscle, heart and liver of laminin 2-deficient mice is not caused by an elevation in the mRNA content[END_REF].

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Moral-Naranjo et al. AChE in lipid rafts 12

Raft microdomains of muscle contain AChE activity

The reports showing interaction of AChE with Cav1 [START_REF] Park | Interactions of acetylcholinesterase with caveolin-1 and subsequently with cytochrome c are required for apoptosome formation[END_REF] and the need of caveolae for directing rapsyn/nAChR to the sarcolemma [START_REF] Zhu | Lipid rafts serve as a signaling platform for nicotinic acetylcholine receptor clustering[END_REF] prompted us to explore the presence of ChEs in rafts of muscle. Although AChE and BuChE activities were mostly measured in non-raft membranes (Fig. 4A), on the basis of the protein content in the collected fractions (ChE activity/mg of protein) a significant part of AChE activity (but no BuChE activity at all) was found associated with rafts (Fig. 4A). The nearly five-fold (4.6 ± 2.1, n = 5, p = 0.024) higher AChE activity/mg protein in rafts of DM than of NM (Fig. 4A) revealed a link between the density of rafts and the level of raft-bound AChE activity, while the unequal distribution of AChE activity and Cav3 in the raft-containing fractions (Fig. 4B) pointed to a preference of AChE for non-caveolar rafts, an idea strongly supported by its enrichment in rafts of Caco-2 cells (authors' observations) that do not express Cav1 [START_REF] Patlolla | Overexpression of caveolin-1 in experimental colon adenocarcinomas and human cancer cell lines[END_REF].

AChE located at lipid rafts of muscle consists of GPI-anchored dimers

The targeting of GPI-anchored proteins to rafts [START_REF] Garner | Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers[END_REF], the recent observation of GPIanchored AChE in rafts of cultured neuroblastoma/glioma hybrid cells transfected with Torpedo AChE-H [START_REF] Liu | Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositolanchored protein microdomains[END_REF], and the identification of AChE protein (Fig. 4) and enzyme activity (Fig. 5) in rafts of muscle prompted us to examine whether the raft-bound AChE consisted of GPI-linked species. Sedimentation analysis revealed that raft membranes contained only amphiphilic AChE dimers (G 2 A forms), whereas non-raft membranes exhibited G 1 A , G 2 A and occasionally hydrophilic tetramers (G 4 H forms) (Fig. 5). The almost complete conversion of the raft-associated G 2 A AChE species into G 2 H forms with PIPLC (supplementary Fig. S3A) allowed us to identify for the first time GPI-anchored AChE dimers in lipid rafts of muscle.

As regards their adsorption to lectins, the raft-residing dimers were fully bound by Lens

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Moral-Naranjo et al. AChE in lipid rafts

13 culinaris agglutinin and wheat germ agglutinin, and almost fully bound by Ricinus communis agglutinin (supplementary Fig. S3B). This lectin interaction pattern differed from the one displayed by AChE dimers of sarcoplasmic reticulum [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF] and blood cells [START_REF] Nieto-Cerón | Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice[END_REF].

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Discussion

The content of caveolin-3 increases in merosin-deficient mouse muscle

The insolubility of lipid rafts in Triton X-100 and their flotation in sucrose gradients allowed us to isolate the lipid raft populations. They appeared as a single band (N1) in preparations of normal muscle and two bands (D1 and D2) in those of merosin-deficient dystrophic muscle (supplementary Fig. S1). The almost 5-fold higher Western blot signal for Cav3 in D1+D2 than N1 (Fig. 1C) and stronger Cav3 labelling in Lama 2dy mouse muscle sections (Fig. 1D) supported a relationship between the deficiency of merosin and, therefore, of laminin-2 in muscle and the up-regulation of Cav3. In addition, the increase of Cav3 in merosin-deficient muscle (this report) and dystrophin-deficient muscle [START_REF] Repetto | Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy[END_REF][START_REF] Merrick | Muscular dystrophy begins early in embryonic development deriving from stem cells loss and disrupted muscle formation[END_REF] links muscular dystrophy with caveolae expansion and agrees with the dystrophic phenotype of the Cav3 over-expressing mouse [START_REF] Galbiati | Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype[END_REF].

The high level of cholesterol in dystrophic muscle [START_REF] Fischbeck | Freeze-fracture analysis of plasma membrane cholesterol in Duchenne muscle[END_REF], its influence on the size and shape of rafts [START_REF] Repetto | Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy[END_REF] and its activating effect on Cav expression [START_REF] Pol | Cholesterol and fatty acids regulate dynamic caveolin trafficking through the Golgi complex and between the cell surface and lipid bodies[END_REF] might explain the recovery of two raft populations (D1 and D2) in merosin-deficient muscle and its increased content of Cav3. The role that Cav3 fulfils in myoblast fusion, the lower density of rafts as myofibres mature [START_REF] Draeger | Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells[END_REF], the muscular damage produced by statins, that lower cholesterol level and, therefore, the caveolae population [START_REF] Draeger | Statin therapy induces ultrastructural changes in skeletal muscle in patients without myalgia[END_REF], and our data pointing to an expansion of caveolar and non-caveolar (see below) rafts in DM all agree with the postulated impairment of myofiber differentiation as a leading factor for muscular dystrophy [START_REF] Oexle | Cause of progression in Duchenne muscular dystrophy: impaired differentiation more probable than replicative aging[END_REF]. In addition, the high level of calcium in dystrophic muscle [START_REF] Whitehead | Muscle damage in mdx (dystrophic) mice: role of calcium and reactive oxygen species[END_REF] and its activating effect on caveolin synthesis [START_REF] Fawzi-Grancher | Role of Ca 2+ in the effects of shear stress and TNF-on caveolin-1 expression[END_REF] make the deregulation of calcium homeostasis a probable cause for the excess of Cav3 in dystrophic muscle.

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Moral-Naranjo et al. AChE in lipid rafts 15

The increase of raft-associated eNT activity supports expansion of non-caveolar rafts in merosin-deficient muscle

The resistance of eNT activity to detergent extraction [START_REF] Martínez-Martínez | The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds[END_REF], its absence from caveolae [START_REF] Liu | Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositolanchored protein microdomains[END_REF], and the higher eNT activity/mg of protein in rafts (Fig. 3A) than whole muscle [START_REF] García-Ayllón | Identification of inactive ecto-5'-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2 dy mice[END_REF] all agree with the consideration of eNT activity as a marker of non-caveolar rafts [START_REF] Matschke | Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma[END_REF]. The rise of Cav3 and eNT activity in the Triton-resistant membranes of DM supports expansion of caveolar and non-caveolar rafts, and the raft localization of active eNT (that converts AMP into adenosine) as well as adenosine receptors and adenosine deaminase [START_REF] Ginés | Involvement of caveolin in ligand-induced recruitment and internalization of A1 adenosine receptor and adenosine deaminase in an epithelial cell line[END_REF] gives functional significance to the increase of eNT activity in dystrophic muscle [START_REF] García-Ayllón | Identification of inactive ecto-5'-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2 dy mice[END_REF].

Lipid rafts of muscle contains GPI-anchored dimers

AChE of muscle has been thoroughly studied because this enzyme is required for the correct coupling of excitation with contraction. The overwhelming amount and functional relevance in muscle of the AChE-T subunit (compared with AChE-H and AChE-R subunits)

have boosted research onto regulatory aspects of the AChE-T mRNA expression, translation, oligomerisation of AChE-T subunits, assembly with non-catalytic (structural) subunits, and transport of oligomers to specific stores of muscle [START_REF] Massoulié | Old and new questions about cholinesterases[END_REF]. In fast muscle, AChE occurs as A 12 forms made of T subunits at the NMJ and as PRiMA-bearing tetramers (G 4 A species) at the sarcolemma, whereas in slow fibres appears as G 1 , G 4 A , A 4 and A 8 forms at synaptic and nonsynaptic areas [START_REF] Rotundo | Targeting acetylcholinesterase to the neuromuscular synapse[END_REF][START_REF] Xie | Regulation of a transcript encoding the proline-rich membrane anchor of globular muscle acetylcholinesterase. The suppressive roles of myogenesis and innervating nerves[END_REF].

As regards the translation of the muscle AChE-H mRNA, despite the long time elapsed since its observation [START_REF] Legay | Developmental regulation of acetylcholinesterase transcripts in the mouse diaphragm: alternative splicing and focalization[END_REF], the lack of information on AChE-H protein challenged the capacity of muscle for translating the AChE-H mRNA. The identification of G 2 A AChE species in rafts of muscle and their conversion into G 2 H forms with PIPLC (supplementary Fig. S2A)

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Moral-Naranjo et al. AChE in lipid rafts 16
demonstrate for the first time that muscle can translate the AChE-H mRNA and the existence of an as yet undetected pool of raft-residing AChE-H protein in the form of GPI-anchored dimers. This pool of dimers had probably remained unnoticed in whole muscle studies owing to the greater quantity of AChE-T than of AChE-H tailored molecules. The concentration in rafts of the latter permitted their identification.

The raft-residing AChE-H dimers were fully bound by Lens culinaris agglutinin and wheat germ agglutinin, and almost totally by Ricinus communis agglutinin (suppl. Fig. S2B), this lectin binding pattern differing substantially from that displayed by AChE dimers of sarcoplasmic reticulum [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF] and blood cells [START_REF] Nieto-Cerón | Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice[END_REF]. Thus, along with the report showing AChE in caveolin-free membranes of erythrocytes [START_REF] Salzer | Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts[END_REF], the differences between the AChE dimers in rafts of muscle and in erythrocytes in PIPLC sensitivity and extent of lectin binding (suppl.

Fig. S2) [START_REF] Nieto-Cerón | Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice[END_REF] ruled out a possible erythrocytic origin of the raft-bound AChE. Finally, the lower binding extent of Ricinus communis agglutinin with the AChE dimers of nerves [START_REF] Moral-Naranjo | Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice[END_REF] than of rafts (suppl. Fig. S3B) discarded their possible neural origin.

The abundance in whole muscle and internal stores of asymmetric and tetrameric AChE-T [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF], which are lacking in rafts (Fig. 5), and the rise of AChE activity (as dimers) in rafts of DM suggested package of particular subsets of AChE forms in distinct transport vesicles for their delivery to particular subcellular compartments of the myofibre.

5'-Alternative AChE mRNAs

Our RT-PCR results demonstrate that mouse muscle expresses all five 5' alternative AChE transcripts described to date and the three 3' alternative AChE-T, AChE-R and AChE-H mRNAs (Fig. 6). The mRNAs with exons E1c and E1e prevailed over those with exons E1d, E1b and E1a in mouse muscle (this work) and brain [START_REF] Fernández-Gómez | Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8[END_REF], and this prevalence allowed us to report for the first time the capacity of muscle and brain to express AChE

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transcripts containing the E1e exon which encodes the N-terminal extension. Our mRNAs data agree with those of the Soreq's group, who also observed abundant E1c-containing transcripts in most mouse tissues, including muscle, and undetectable or very low levels of mRNAs with E1d, E1b and E1a in muscle [START_REF] Meshorer | Combinatorial complexity of 5' alternative acetylcholinesterase transcripts and protein products[END_REF]. The expression level of E1e mRNAs in mouse organs was not analyzed in that work. Nevertheless, the substantial amount of E1e mRNAs in mouse muscle with respect to the total AChE transcripts that we observe agrees with the presence in human brain of the homologous 5'-spliced mRNA variant [START_REF] Toiber | N-Acetylcholinesterase-induced apoptosis in Alzheimer's disease[END_REF]. Finally, the increasing abundance of AChE-H, AChE-R and AChE-T mRNAs in mouse muscle (Fig. 6) also coincides with previous observations [START_REF] Meshorer | Combinatorial complexity of 5' alternative acetylcholinesterase transcripts and protein products[END_REF].

The identification in mouse muscle (this work) and brain [START_REF] Fernández-Gómez | Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8[END_REF] of E1e-containing AChE mRNAs that translate into N-terminally extended AChE subunits and whose extension potentially allows membrane anchorage [START_REF] Meshorer | Virtues and woes of AChE alternative splicing in stressrelated neuropathologies[END_REF] points to the production of N-extended AChE variants in muscle and brain. With respect to muscle, the strong labelling in lipid rafts of the ~66-72 kDa double protein band (Fig. 4B) that has been assigned to human N-extended AChE protein [START_REF] Meshorer | Combinatorial complexity of 5' alternative acetylcholinesterase transcripts and protein products[END_REF] supports its production in mouse muscle. With respect to brain, it is worth mentioning the presence in human cortical neurons of both E1e-containing AChE transcripts and N-AChE-T protein and their increased levels in neurons of patients suffering from Alzheimer disease [START_REF] Toiber | N-Acetylcholinesterase-induced apoptosis in Alzheimer's disease[END_REF]. This report prompts for an investigation focused on the identification in tissues of the various N-extended AChE proteins and their functional roles. We shall undertake this study in the near future using selected cell lines and antibodies against the Nterminal domain of N-extended AChE.

The prevalence of AChE-T mRNA over AChE-R and AChE-H mRNAs in muscle (Fig. 6) confirms the need of AChE-T protein for appropriate functioning, and the lower quantity of AChE-T and PRiMA mRNAs in mouse muscle (~9,500 and 3,500 copies/million copies of -actin mRNA) (Fig. 6) than brain (35,000 and 8,000 copies) [START_REF] Fernández-Gómez | Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8[END_REF] agrees with the functional forms that probably support neuromuscular transmission in AChE-null mice, which lack the homologous AChE forms [START_REF] Li | Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse[END_REF].

The PRiMA subunit is down-regulated in merosin deficient muscle

The comparable amounts of AChE and BuChE mRNAs in healthy and merosindeficient muscle contrasted with the sixteen-fold lower level of the PRiMA mRNA in merosin-deficient than in normal muscle (Fig. 6). This down-regulation of PRiMA would explain the reported decrease of G 4 A AChE and BuChE in dystrophin-deficient and merosindeficient dystrophic muscle [START_REF] Cabezas-Herrera | Glycosylation of acetylcholinesterase forms in microsomal membranes from normal and dystrophic Lama2dy mouse muscle[END_REF][START_REF] Gisiger | Localization of the pool of G 4 acetylcholinesterase characterizing fast muscles and its alteration in murine muscular dystrophy[END_REF]. The role of PRiMA in converting G 4 AChE into G 4 A forms, and, hence, in attenuating the synthesis of asymmetric AChE [START_REF] Xie | Regulation of a transcript encoding the proline-rich membrane anchor of globular muscle acetylcholinesterase. The suppressive roles of myogenesis and innervating nerves[END_REF], might account for the similar levels of G 4 A AChE in dystrophic fast-twitch and normal slow-twitch muscles, that are lower than in healthy fast-twitch muscle [START_REF] Bacou | Acetylcholinesterase forms in fast and slow rabbit muscle[END_REF]. Down-regulation of PRiMA may be inherent to dystrophy; it may arise from early myogenin synthesis [START_REF] Yablonka-Reuveni | Satellite cells from dystrophic (mdx) mice display accelerated differentiation in primary cultures and isolated myofibers[END_REF] and its suppressive action on PRiMA [START_REF] Xie | Regulation of a transcript encoding the proline-rich membrane anchor of globular muscle acetylcholinesterase. The suppressive roles of myogenesis and innervating nerves[END_REF]. The need of PRiMA for targeting G 4 A AChE to the cell membrane in differentiating neurons [START_REF] Xie | Transcriptional regulation of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in neuron: an inductive effect of neuron differentiation[END_REF] could mean that the down-regulation of PRiMA is responsible for the loss of G 4 A AChE and BuChE in merosin-deficient nerves [START_REF] Moral-Naranjo | Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice[END_REF], but further work is needed to confirm this.

Possible significance of AChE located in muscle lipid rafts

The role of lipid rafts in targeting nAChR [START_REF] Stetzkowski-Marden | Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes[END_REF] AChE-H mRNA (Fig. 6). This increased level of raft-bound AChE activity may arise from:

1) release of mRNA-blocking micro-RNA; 2) faster translation; 3) improved posttranslational maturation, including folding of AChE-H polypeptides, efficiency of GPI addition, conversion of catalytically incompetent into catalytic molecules, evasion from proteolysis, dimerization and exit to the Golgi [START_REF] Massoulié | Old and new questions about cholinesterases[END_REF]; and 4) favourable incorporation therein of the glypiated AChE in rafts and further targeting to the sarcolemma. Elucidation of these possible reasons deserves further investigation.

Concerning the functional significance of raft-bound AChE, the presence of muscarinic receptors (mAChR) in lipid rafts of muscle [START_REF] Furlan | Developing skeletal muscle cells express functional muscarinic acetylcholine receptors coupled to different intracellular signaling system[END_REF][START_REF] Allen | Lipid rafts microdomains and neurotransmitter signalling[END_REF] supports a role for raft-bound AChE in cholinergic responses. The increased level of AChE activity in rafts of dystrophic muscle may contribute to the pathology by lowering the availability of acetylcholine. In addition, given 1) the non-enzymatic actions of AChE [START_REF] Massoulié | Old and new questions about cholinesterases[END_REF]; 2) its capacity to interact with laminin [START_REF] Johnson | Non-enzymatic developmental functions of acetylcholinesterase -the question of redundancy[END_REF][START_REF] Paraoanu | Acetylcholinesterase in cell adhesion, neurite growth and network formation[END_REF][START_REF] Johnson | Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain[END_REF]; and 3) the similar structural motifs in AChE, syndecans, perlecans and other heparin sulfate proteoglycans [START_REF] Johnson | Non-enzymatic developmental functions of acetylcholinesterase -the question of redundancy[END_REF], it is conceivable that the raft-bound AChE can contribute to the stabilization of the basal lamina of muscle.

Conclusions

The results reported herein demonstrate for the first time that the deficiency of merosin produces an increase in the content of Cav3 and of raft-bound eNT activity in muscle. These observations, besides the increased density of caveolae in dystrophin-deficient muscle [START_REF] Vaghy | Increased caveolin-3 levels in mdx mouse muscles[END_REF][START_REF] Repetto | Increased number of caveolae and caveolin-3 overexpression in Duchenne muscular dystrophy[END_REF], support the notion that regardless of the primary defect the dystrophic phenotype causes an increase in the population of caveolar and non-caveolar lipid rafts. The results are compatible with the notion of overproduction of lipid rafts in the damaged myofibre for

( 1 :

 1 1,000). The slides were observed under a Leica TCS SP2 AOBS confocal module equipped with an HCX PLAN APO-CS 40.0 x 1.25 oil immersion lens. Confocal images A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Moral-Naranjo et al. AChE in lipid rafts 8 were obtained by excitation with an Ar laser at 488 nm and an emission of 519 nm for Alexa

Fractions with lipid rafts

  were pooled and centrifuged. The pellet was extracted with a Tris-Triton X-100 buffer (150 mM NaCl, 5 mM EDTA, 60 mM octyl-glucoside, 1% Triton X-100 in 10 mM Tris-HCl, pH 8.0). Fractions with Triton-soluble membranes were also pooled and dialyzed in MBS for sucrose removal. AChE and BuChE forms in the Triton-A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Moral-Naranjo et al. AChE in lipid rafts 9

Fig. S1A )

 S1A Fig.S1A) and the rafts of DM distributed between a strong band (D1) in fractions 6-8 and a

3. 5 .

 5 The levels of AChE and BuChE mRNAs are unaffected and that of PRiMA mRNA decreases in merosin-deficient muscle A comparison of the content of AChE and BuChE mRNAs in NM and DM could improve current understanding on the capacity of muscle for expressing the range of AChE mRNAs that differ in the choice of transcription initiation sites and alternative splicing (Figs.1 and 6). In addition, the study might unveil possible differences between normal and dystrophic muscle in the splicing pattern of the ACHE gene. Figure 6 shows that the AChE mRNAs with E1c and E1e exons prevail over those with E1d, E1b or E1a exons in mouse muscle, and the AChE-T mRNA over the AChE-R and AChE-H mRNAs. It is worth noting that the E1e-containing mRNA encodes for an AChE subunit with an N-terminal extension which potentially allows membrane anchorage. The levels of BuChE and PRiMA mRNAs exceeded those of AChE-R and AChE-H mRNAs. The lack of differences in the content of AChE and BuChE mRNAs between NM and DM contrasted with the sixteen-fold lower level of the PRiMA mRNA in merosin-deficient than in normal muscle (210 vs. 3500 copies; n = 5, p <0.001; Fig. 6), which demonstrated for the first time down-regulation of PRiMA in dystrophic muscle.

  the PRiMA-linked tetramers) for brain. The identification of BuChE and PRiMA mRNAs in muscle confirms its capacity for making the G 4A BuChE

  and our data revealing raft-bound AChE highlight the importance of rafts in targeting cholinergic proteins. The higher content of raftconfined AChE activity (in the form of AChE-H dimers) in Lama2dy muscle compared to A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Moral-Naranjo et al. AChE in lipid rafts 19 wild type muscle (Figs. 4, 5, and supp. Fig. S3A) contrasts with their similar levels of

Fig. 1 .

 1 Fig. 1. Primers used in the PCR assays to quantify AChE, BuChE and PRiMA mRNAs. A)

Fig. 2 .

 2 Fig. 2. Triton X-100-resistant lipid rafts were isolated from normal muscle (NM) and

Fig. 3 .Fig. 4 .

 34 Fig. 3. Distribution of eNT activity and eNT protein in raft and non-raft membranes of NM

Fig. 5 .

 5 Fig. 5. Sedimentation profiles with cholinesterase forms in raft and non-raft fractions of NM

Fig. 6 .

 6 Fig. 6. Expression of AChE, BuChE and PRiMA mRNAs. (A) Agarose gels showing the RT-

ACCEPTED MANUSCRIPTMoral-Naranjo et al. AChE in lipid rafts

Acknowledgments

This research was supported by the Ministerio de Ciencia y Tecnología of Spain (SAF2001/0279) and Fundación Séneca de la Comunidad Autónoma de la Región de Murcia (PI 83/00840/FS01). M.T.M.-N. and M.F.M. hold scholarships from CajaMurcia and the Fundación Séneca, respectively.