WO2011048443A1 - Neuronal exocytosis inhibiting peptides derived from c subunit of v-atpase and cosmetic and pharmaceutical compositions containing said peptides - Google Patents

Abstract

The present invention refers to peptides derived from the carboxy end of protein C subunit of V-ATPase, useful as inhibitors of neuronal exocytosis, and to their use in cosmetic and/or therapeutic applications

Description

NEURONAL EXOCYTOSIS INHIBITING PEPTIDES DERIVED FROM C SUBUNIT OF V-ATPASE AND COSMETIC AND PHARMACEUTICAL

COMPOSITIONS CONTAINING SAID PEPTIDES

FIELD OF THE INVENTION

This invention refers to novel peptides derived from the carboxy end of the C subunit of theV-ATPase, useful as inhibitors of neuronal exocytosis, and to their use in therapeutic and/or cosmetic applications.

BACKGROUND OF THE INVENTION

The basis or mechanism for the formation of facial wrinkles is the tensing of the muscles of the epidermis that drag the skin inwards. This muscular tension is the result of hyperactivity of the nerves innervating the facial muscles. Nerve hyperactivity is characterized by the uncontrolled and excessive release of neurotransmitters that excite muscle fibers. Because of this, the molecules that control neuronal exocytosis contribute to relaxing muscular tension, and consequently, to eliminating wrinkles.

Botulinum toxins are a family of bacterial neurotoxins produced by Clostridium Botulinum . 7 different serotypes are known (serotypes A, B, C, D, E, F and G) with an average molecular weight of 150 kDa. These toxins inhibit acetylcholine exocytosis in the neuromuscular junction (nerve-muscle synapse) of the skeletal muscle (Schiavo, G., et al).

At a molecular level, botulinum toxins are proteases that degrade neuronal proteins involved in the exocytosis mechanism activated by the calcium ion . For example, botulinum toxin A, the one most commonly used clinically and cosmetically [because of its applications in eliminating facial wrinkles and asymmetry, and to mitigate the symptomatology of spastic diseases], cleaves the neuronal protein SNAP-25. This protein (SNAP-25) plays a key role in neurosecretion, as it is involved in the formation of a protein complex (known as SNARE complex or fusion complex), which directs and controls the release of acetylcholine accumulated in vesicles. The nucleus of said fusion complex is made up of proteins SNAP-25 and syntaxin, located in the presynaptic plasma membrane, and protein synaptobrevin (or VAMP), located in the vesicular plasma membrane . The main function of the fusion complex is to bring the vesicle loaded with neurotransmitter (i.e acetylcholine, glutamate, ...) nearer to the presynaptic plasma membrane and put it in contact with same. In this way, in response to an elevated concentration of calcium, the fusion of both plasma membranes is encouraged, thus producing the release of the neurotransmitter. Therefore, said vesicle docking and fusion protein complex (SNARE) is a key target in controlling neurosecretion. Cleaving any of the proteins that make up the fusion complex prevents its assembly, and therefore inhibits vesicle release and neuronal exocytosis.

The power of botulinum toxins and, in particular, serotype A (BOTOX TM ) to inhibit neurosecretion, as well as their neuronal selectivity (they only act on neurons) is being widely used therapeutically to correct spastic ailments such as dystonias, strabismus, tics, blepharospasm, facial scoliosis, etc... Botulinum toxin A (botulinum A) is, moreover, an effective agent for eliminating facial wrinkles and asymmetry. In fact, the administration of BOTOX TM is the first effective non-surgical therapy to eliminate the signs of aging.

Therapeutic and cosmetic treatment with BOTOX TM consists of a localized injection of diluted pharmaceutical preparations (botulinum A-hemagglutinin complex, 500 kDa) in the areas where muscular tension is localized. The paralytic effects of the toxin are reversible with an average duration of 6 months. The treatment, therefore, requires repeated injections of BOTOX TM . The main problem with this treatment is the chance that it may trigger an immune reaction against the pharmaceutical preparation due to the fact that, because of its molecular size, it may be recognized by the patient's immune system. The appearance of antibodies against botulinum A is a serious problem, as it contributes to a clear decrease in the treatment's effectiveness. This loss of effectiveness in treatment with BOTOX TM means the need to increase the preparation's concentration level in later treatments, which in turn produces a potentiation of the immune response. As an alternative, the use of different botulinum toxin (BoTox) serotypes has been discussed, such as BoTox B, BoTox F and BoTox E. Nevertheless, the application of pharmaceutical preparations with different serotypes cannot be considered a solution to the problem, as sooner or later the immune reaction may once again occur. In addition, treatment with botulinum toxins is expensive, mainly because of the lability and instability of the pharmaceutical preparations containing them.

There is, therefore, a pressing need to develop molecules that mimic the paralytic effects of the botulinum toxins, but with simpler and more stable molecular structures, which do not cause adverse immune reactions, and whose manufacturing costs are low. Small size molecules, i.e.oligopeptidic-type molecules meet these requirements. Amino acid sequences that inhibit neuronal exocytosis have been described. Specifically, it has been proven that peptides with more than 20 amino acids, deriving from the C-terminal sequence of SNAP-25, block the release of catecholamines from permeabilized chromaffin cells (Gutierrez, L.M et al. 1995). Likewise, peptides deriving from the amino acid sequences of proteins syntaxin and VAMP have been described that can also affect the exocytotic process (Augine, G.J. et al 1996). Although these peptides show biological activity, their later development as cosmetic and/or therapeutic agents has not occurred, most likely due to their size, as this complicates their development as useful therapeutic agents and makes it more expensive. Therefore, there is a need to find molecules of a smaller size that can be applied in cosmetics and medicine.

While working on regulation of the molecular mechanism of neurotransmitter release, inventors of the instant invention have discovered a direct molecular interaction occurring between the c-subunit of the membrane component of V-ATPase (called V0) and SNAREs. Using the yeast 2 hybrid system and pull-down experiments with bacterially-expressed fusion proteins, they have demonstrated a direct interaction between

V0 c-subunit and the v-SNARE VAMP2 (also called synaptobrevin, also target of several botulinum neurotoxins). The different domain on the c-subunit likely to bind VAMP2 were mapped ,and, in order to explore the functional relevance of this interaction, various derived peptide isolated from c-subunit domain were tested.

Thus, the invention provides a solution to existing need , namely the provision of a soluble peptide with a sequence of twelve amino acids (POF16) as well as derived peptides corresponding to portion of the V0 c-subunit sequence (loop 3-4 domain) that VAMP2 binds to and which have the property to interfere with the neurotransmitter release.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a polypeptide comprising at least 6 consecutive amino acid selected in the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l (Vo ATPase subunit C) or a function-conservative variant which is able to at least partially inhibit neuronal exocytosis.

More particularly, the polypeptide, has a length of 6 to 20 amino acids and contains an sequence made up of 6 to 12 adjacent amino acids contained in the carboxy end of the c-subunit of the membrane component of V-ATPase (called V0), which inhibits neuronal exocytosis. An additional object of the invention is a nucleic acid that essentially codes for the polypeptides provided by this invention. The plasmids and vectors that contain said nucleic acid (also identified as constructions), as well as the cells transformed with said plasmids or vectors that express a peptide of the invention, also constitute additional objects of this invention.

Another additional object of this invention is a cosmetic composition that includes at least one peptide provided by the invention. The use of the polypeptides of the invention in the preparation of said cosmetic composition, as well as methods of cosmetic treatment that includes the application of said cosmetic composition top the epiderm, constitute additional objects of this invention.

Another additional object of this invention is a pharmaceutical composition that includes at least one peptide provided by this invention, or alternatively, a vector containing a nucleic acid that codes for one of the peptides of the invention. The use of the peptides and vectors (constructions) of the invention in the preparation of said pharmaceutical compositions, as well as the method of treating humans or animals encompassed by the application of said cosmetic composition, constitute additional objects of this invention.

Another additional object of the invention is a combination of drugs that includes at least one of the peptides provided by the invention, along with, at least, one drug intended as a second therapeutic target which may be the same as or different from the therapeutic target at which the peptide provided by this invention is aimed.

DETAILED DESCRIPTION OF THE INVENTION

While working on different constructions of the c-subunit of V-ATPase (called VO), the inventors showed that small peptides derived from region implicated in the interaction with VAMP2 could play a role in the process of neurotransmitter release.

More particularly, they observed that the twelve amino acids of the carboxy- terminal end of this polypeptide play a major role in the interaction between the c-subunit of V-ATPase and VAMP2 and derived polypeptides have the property to inhibit the neurotransmitter release.

Definitions

"Function-conservative variants" are peptides derived from the peptide of the invention in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, most preferably at least 85%, and even more preferably at least 90 %, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80 %, preferably greater than 85 %, preferably greater than 90 % of the amino acids are identical, or greater than about 90 %, preferably grater than 95 %, are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA, etc.

The term "V-ATPase" or Vacular proton ATPase, or "VATPase Vo" or "Vo VATPase" or "Vo" refers to large multi-molecular enzymatic complex expressed in all eukaryotic cells, with the primary function of proton pumping on cellular compartiment. It is omnipresent in intracellular membrane compartments, including synaptic vesicles (Stevens and Forgac, 1997) where it generates vesicular proton gradients and membrane potential that underlie GABA/monoamine and glutamate uptake (Moriyama et al., 1992). V-ATPase is composed of two reversibly-associated sectors, a peripheral multi-subunit

VI sector that hydro lyses ATP and a membrane V0 sector that translocates protons. In mammals, V0 is composed of a rotor of six subunits (5 c-subunits and 1 c"-subunit) and single copies of a, d and e-subunits. The V-ATPase c-subunit can be from any source, but typically is a mammalian (e.g., human and non- human primate, rodent,...) V-ATPase, and more particularly a rat V-ATPase. Typically, the amino acid sequence of the rat V-

ATPase c-subunit is provided by SEQ ID NO: l and the nucleic sequence of the ADNc is NCBI referenced as NM_130823.

As shown in Figure 1A, the sequence of V-ATPase c-subunit is divided in different regions. In its broadest meaning, the term "preventing" or "prevention" refers to preventing the disease or condition from occurring in a patient which has not yet been diagnosed as having it.

In its broadest meaning, the terms "treating" or "treatment" refer to reversing, alleviating, inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

The term "patient" refers to any subject (preferably human) afflicted with or susceptible to be afflicted with i.e. a neuronal exocytosis-mediated pathological diseases and/or disorders or for the treatment of facial wrinkles and/or asymmetry.

Within the scope of this invention are included cosmetically and/or pharmaceutically acceptable salts of the peptide of the invention. The term "cosmetically and/or pharmaceutically acceptable salts" includes salts customarily used to form metal salts or salts formed by adding free acids or bases. The nature of the salt is not critical, as long as it is cosmetically and/or pharmaceutically acceptable. Cosmetically and/or pharmaceutically acceptable salts of the peptide of the invention may be obtained from acids or bases, organic or inorganic, by conventional methods which are well known to technicians in these matters, by making the appropriate acid or base react with the peptide of the invention.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other non desired reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically or cosmetically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

Polypeptides derived from the activation peptide of V-ATPase

A first object of the present invention relates to a polypeptide comprising at least 6 consecutive amino acid selected in the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l or a function-conservative variant which is able to at least partially inhibit neuronal exocytosis.

In one embodiment SEQ ID N° 1 is not a polypeptide of the invention.

This invention provides a peptide deriving from the carboxy end of the protein V- ATPase c-subunit. More specifically, the invention provides a polypeptide, herein designated as the polypeptide of the invention, which has a sequence of 6 to 12 adjacent amino acids contained in SEQ. ID. No. 1 [see the section regarding the SEQUENCE LIST].

The invention also includes peptides which are substantially homologous to the polypeptide of the invention.

In one particular embodiment, the peptide of the invention has a length of 6 to 20 amino acids, and preferably from 6 to 12 amino acids.

In one embodiment, said polypeptide comprises the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l (SEQ ID N°2) .

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 119 to 128 of SEQ ID NO 1 : (SEQ ID N°3)

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 121 to 128 of SEQ ID NO:l (SEQ ID N°4)

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 123 to 128 of SEQ ID NO:l . (SEQ ID N°5)

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 117 to 126 of SEQ ID NO:l (SEQ ID N°6).

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 117 to 124 of SEQ ID NO:l (SEQ ID N°7)..

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 117 to 122 of SEQ ID NO:l (SEQ ID N°8).

In a particular embodiment, said polypeptide comprises the amino acid sequence ranging from positions 119 to 126 of SEQ ID NO:l (SEQ ID N°9).

In a particular embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO:2.

The amino acids that make up the structural units of the peptide of the invention may have D- or L-configuration.

The amino acid from the amino end may have an acetylated terminal amino group, and the amino acid from the carboxyl end may have an amidated terminal carboxyl group.

Therefore, this invention also includes derivatives of the peptide of the invention in which the amino -terminal end is acetylated and/or in those where the carboxy-terminal end is amidated.

Particular examples of polypeptides of the invention are those polypeptides that have sequences of amino acids shown in SEQ. ID No. 2 to SEQ. ID No. 9. In addition, the polypeptide of the invention may undergo reversible chemical modifications in order to increase its bioavailability (including stability and fat solubility) and its ease in passing through the blood-brain barrier and epithelial tissue. Examples of such reversible chemical modifications include the esterification of the carboxylate groups of glutamic and aspartic amino acids with an acetyl-methyl group, by which the negative charge of the amino acid is eliminated and its hydrophobicity is increased. This esterification is reversible, as the ester link formed is recognized by intracellular esterases which hydro lyze it, giving back the charge to the aspartic and glutamic residues. The net effect is an accumulation of intracellular polypeptide, as the internalized, de-esterified polypeptide cannot cross the cell membrane.

Another example of such reversible chemical modifications include the addition of a further peptidic sequence, which allows the increase of the membrane permability, such as a TAT peptide or Penetratin peptide (see - Charge-Dependent Translocation of the Trojan .A Molecular View on the Interaction of the Trojan Peptide Penetratin with the Polar Interface of Lipid Bilayers. Biophysical Journal, Volume 87, Issue 1, 1 July 2004,

Pages 332-343)

The polypeptide of the invention may be obtained through conventional methods of solid-phase chemical polypeptide synthesis, following Fmoc and/or Boc-based methodology (see Pennington, M.W. and Dunn, B.N. (1994). Peptide synthesis protocols.

Humana Press, Totowa.).

Alternatively, the polypeptide of the invention may be obtained through conventional methods based on recombinant DNA technology, e.g., through a method that, in brief, includes inserting the nucleic acid sequence coding for the polypeptide of the invention into an appropriate plasmid or vector, transforming competent cells for said plasmid or vector, and growing said cells under conditions that allow the expression of the polypeptide of the invention and, if desired, isolating and (optionally) purifying the polypeptide of the invention through conventional means known to experts in these matters. The nucleic acid sequence that codes for the polypeptide of the invention may be easily deduced from the correspondence that exists between the amino acids and the nucleotide codons that code for such amino acids. In this case, an additional object of the invention is an isolated nucleic acid sequence that codes for the polypeptide of the invention. In one particular embodiment, said nucleic acid is selected from single-strand DNA, double-stranded DNA, and RNA. Additional objects of this invention are plasmids and expression vectors that contain said nucleic acid sequence that codes for the polypeptide of the invention, as well as prokaryotic or eukaryotic cells that express the polypeptide of the invention. A review of the principles of recombinant DNA technology may be found, for example, in the text book entitled "Principles of Gene Manipulation: An Introduction to Genetic Engineering," R.W. Old & S.B. Primrose, published by Blackwell

Scientific Publications, 4th Edition (1989).

The polypeptide of the invention is able to at least partially inhibit neuronal exocytosis, probably through a mechanism that involves interfering with the assembly of the fusion protein complex (SNARE) and/or its thermal destabilization and/or its zipping completion.

As described the invention also includes peptides which are functionally equivalent to the polypeptide of the invention or " function-conservative variant". In the sense used in this description, the expression "functionally equivalent" means that the peptide in question has at least one of the biological activities of the peptide of the invention, such as, for example, the ability to at least partially inhibit neuronal exocytosis

The neuronal-exocytosis (neurosecretion) inhibiting capabilities of the polypeptides of the invention will become evident to the skilled person by implementing a simple test to evaluate the kinetic of said polypeptidepeptides in inhibiting the release of neurotransmitters (as in the exemple of this application in Nicotinic SCG neurons) This kinetic of the inhibition is measured as a reduction in the Excitatory Post Synaptic

Potential, EPSP (see Ma H, Mochida S. ".et al . 2007). Thus following peptide injection, the kinetics of inhibition of neurotransmission gives an indication as to which step in ACh release is perturbed (ie. the quicker the effect, the nearer to the fusion step the interaction occurs). Similar to inhibition kinetics observed with BoNT and tetanus (TeNT) neurotoxins, the injection of polypeptide SEQ ID N°2 triggered a rapid inhibition of neurotransmission in contrast to the use of a scrambled control peptide sequence which had no effect (see Exemple below).

Another test could be to assess the strength of said polypeptides in inhibiting the release of neurotransmitters induced by calcium in chromaffin cells permeabilized with a detergent (see Quetglas S, et al 2002). Briefly, calcium induced release of either tritiated norepinephrin or transfected growth hormon could be measured after cell membrane permeabilization..

The docapeptide of the invention [SEQ. ID. No. 2], at a concentration of 1.5 to 2 mM, blocked approximately 20% of the acetylcholine release from Nicotinic SCG neurons and up to 70% of the glutamate release in glutamatergic cortical pyramidal neurons.

Taken all together, these results indicate that peptides derived from the carboxyl end of the c-subunit V-ATPase interfering with the interaction between VAMP2 and c- subunit V- ATPase, could inhibit neurotransmitters exocytosis,.

In one particular embodiment, said polypeptides may have a cyclic structure by adding a neutral polypeptide linker that should help reaching a sufficient length to allow a constrained presentation of the polypeptide of the invention. A review of the cyclic peptide technology may be found, for example, in "Conformation and Biological Activity of Cyclic Peptides" Prof. Dr. Horst Kessler * Angewandte Chemie International Edition in

English Volume 21 Issue 7, Pages 512 - 523.

The mechanism of action of the peptides of the is prima facie similar to that of botulinum toxins, thus affecting the formation and/or stability of the fusion protein complex; so that the polypeptides of the invention can be considered to have cosmetic/therapeutic applications identical or similar to those described for botulinum toxin. Therefore, the polypeptides of the invention may be regarded as efficacious, stable, safe and economical substitutes for botulinum toxins, both for the treatment of facial wrinkles and/or asymmetry and in the treatment of the symptomatology of spastic diseases, allowing to consider their use as neuroprotectors in the treatment of neurological disorders and neurodegenerative diseases.

Taken together, the results obtained with the polypeptides of the invention, along with their stability and structural simplicity and the chemical diversity that can be obtained therefore, keeping in mind the composition of the carboxyl ends of c-subunit V- ATPase,, provide the peptides of the invention with a large cosmetic and/or therapeutic potential.

The polypeptides of the invention may thus be used for pathological neuronal exocyto sis-mediated cosmetic and/or therapeutic purposes.

Nucleic acids, vectors and recombinant host cells

A further object of the present invention relates to a nucleic acid molecule encoding polypeptides according to the invention.

A "coding sequence" or a sequence "encoding" an expression product, such as a R A, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. These nucleic acid molecules may be obtained by conventional methods well known to those skilled in the art, in particular by site-directed mutagenesis of the gene encoding the native protein.

Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) may be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

Any expression vector for animal cell may be used, as long as a gene encoding a polypeptide or chimeric derivative of the invention can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4) and the like.

Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.

Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells,

GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like.

The invention also includes gene delivery systems comprising a nucleic acid molecule of the invention, which can be used in gene therapy in vivo or ex vivo. This includes for instance viral transfer vectors such as those derived from retrovirus, adenovirus, adeno associated virus, lentivirus, which are conventionally used in gene therapy. This also includes gene delivery systems comprising a nucleic acid molecule of the invention and a non-viral gene delivery vehicle. Examples of non viral gene delivery vehicles include liposomes and polymers such as polyethylenimines, cyclodextrins, histidine/lysine (HK) polymers, etc.

A subject of the present invention is also a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule according to the invention.

The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or R A sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

Preferably, for expressing and producing the polypeptides and, and in particular the protein V-ATPase c-subunit derivatives in accordance with the invention, eukaryotic cells, in particular mammalian cells, and more particularly human cells, will be chosen.

Typically, cell lines such as CHO, BHK-21, COS-7, CI 27, PER.C6 or HEK293 could be used, for their ability to process to the right post-translational modifications of the derivatives.

The construction of expression vectors in accordance with the invention, the transformation of the host cells can be carried out using conventional molecular biology techniques. The V-ATPase c-subunit derivatives of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the derivative expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractionated precipitation, in particular ammonium sulphate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.

Cosmetic methods uses and compositions

The invention provides a cosmetic composition that includes a cosmetically effective amount of at least one peptide of the invention, along with at least one cosmetically acceptable adjuvant.

For cosmetic applications, the peptides of the invention may be applied by means any medium that produces contact between the peptide and the location where it is to act in a mammal's body, preferably in humans.

The cosmetically effective amount of peptide that are applied, as well as the dosage for the treatment of facial wrinkles and/or asymmetry with the peptides and/or cosmetic compositions of the invention, will depend on numerous factors, including the age and condition of the person desiring treatment, the severity of the wrinkles and/or facial asymmetry, the method and frequency of application and the particular peptide to be used.

The presentation of the cosmetic compositions containing the polypeptides of the invention may be in any form that is suitable for application, e.g., solid, liquid or semisolid, such as creams, ointments, gels or solutions, and the application of these compositions may be by any suitable means, preferably topically, so they will include the cosmetically acceptable adjuvants necessary to make up the desired form of administration. In a preferred and particular embodiment, the peptides of the invention are encapsulated in liposomes, along with (optionally) another or other (COOH) peptide(s), which are added to the other components of the cosmetic preparation. A review of the different cosmetic forms for applying active compounds and of the adjuvants necessary for obtaining same may be found, for example, in the text book "Cosmetolog a de Harry" (Harry's Cosmetology), Wilkinson & Moore, Ed. D az de Santos (1990).

Therefore, an additional object of this invention is the use of the peptides of the invention in the preparation of cosmetic compositions for the treatment of facial wrinkles and/or asymmetry.

The invention also provides a method for the cosmetic treatment of facial wrinkles and/or asymmetry in mammals, preferably humans, which consists of applying a cosmetically effective amount of at least one peptide of the invention to the mammal that has facial wrinkles and/or asymmetry, along with (optionally) one or more (COOH) peptides, preferably in the form of a cosmetic composition containing it.

Therapeutic methods, uses and Pharmaceutical compositions

A further object of the present invention relates to the use of polypeptides of the invention for the treatment of spastic diseases, for example, dystonias, strabismus, blepharospasm, facial scoliosis, tics, etc.; and/or as neuroprotectors in the treatment of neurological disorders and/or neurodegenerative diseases.

Among said neurological disorders are acute neurological diseases, for example, those that take place in the first stages of cerebral ischemia. It is a known fact that during an ischemic process an uncontrolled release of the neurotransmitter glutamate takes place in the affected area. This neurotransmitter interacts with specific neuronal membrane receptors causing a massive influx of calcium ions inside the neuron. The intracellular calcium causes the release of more glutamate, thus triggering a chain reaction. Moreover, the massive, prolonged influx of calcium inside the neurons causes their death, which translates into the formation of necrotic tissue in the ischemic zone. Clearly, the progress of the ischemic damage can be stopped, at least partially, if the uncontrolled glutamate exocytosis is controlled. Therefore, the polypeptides of the invention, because of their ability to inhibit exocytosis, may be suitable for preventing and/or slowing down the neuronal death that is characteristic of an ischemic process, and so would be useful in the treatment of neuropathologies that occur because of excessive glutamate exocytosis, such as, for example, senile dementia, Alzheimer's-related dementia, AIDS-related dementia, epilepsy, amiotrophic sclerosis, multiple/lateral sclerosis, etc. In this case, application in the treatment of neurological diseases would be similar to the one described for botulinum toxin A (see Clarke, C.E. (1992). Therapeutic potential of botulinum toxin in neurological disorders. Quart. J. Med. 299, 197-205. 18).

The polypeptides of the invention could therefore form part of a combined therapy (aimed at several therapeutic targets) with the objective of more effectively stopping neurodegeneration.

An additional object of this invention is a pharmaceutical composition which includes a therapeutically effective amount of at least one polypeptide of the invention, along with at least one pharmaceutically acceptable excipient. In one particular embodiment, said pharmaceutical composition also contains one or more (COOH) peptides. Alternatively, the pharmaceutical composition of the invention may contain a therapeutically effective amount of a vector that contains at least one nucleic acid sequence that codes for a polypeptide of the invention, along with at least one adjuvant and/or a pharmaceutically acceptable excipient. Said vector may be used in gene therapy.

By a "therapeutically effective amount" is meant a sufficient amount of the chimeric derivative of the invention to treat pathological neuronal exocytosis-mediated pathological diseases and/or disorders, at a reasonable benefit/risk ratio applicable to any medical treatment.

It will be understood that the total daily dosage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The active products of the invention (polypeptides or vectors) may be administered for the treatment of pathological neuronal exocytosis, manifested, for example, by spastic diseases, neurological disorders or neurodegenerative diseases, through any medium that produces contact between the polypeptide and the place where it is to act in a mammal's body, preferably in humans. The therapeutically effective amount of the active product of the invention [peptides or vectors (constructions)] that should be administered, as well as the dosage for the treatment of a pathological condition with the peptides and/or pharmaceutical compositions of the invention, will depend on numerous factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration and the particular peptide to be used.

The presentation of the pharmaceutical compositions that contain the peptides or vectors (constructions) of the invention may be in any form that is suitable for administration, e.g., solid, liquid or semi-solid, such as creams, ointments, gels or solutions, and these compositions may be administered by any suitable means, for example, orally, parenterally or topically, so they will include the pharmaceutically acceptable excipients necessary to make up the desired form of administration. A review of the different pharmaceutical forms for administering medicines and of the excipients necessary for obtaining same may be found, for example, in the "Tratado de Farmacia Gal nica" (Treatise on Galenic Pharmacy), C. Faul i Trillo, 1993, Luz n 5, S.A. Ediciones,

Madrid.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral- route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The polypetides of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The polypeptide of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

As was previously mentioned, the peptides of the invention could form part of a combined therapy for the purpose of more effectively stopping neurodegeneration. In this case, the invention provides a pharmaceutical composition that includes at least one peptide of the invention; along with (optionally) another or other neuronal-exocytosis inhibiting compound(s), and along with at least one drug intended for another therapeutic target, selected from the group formed by a neuronal glutamate receptor blocker, a calcium chelator, an anti-oxidant, a free-radical destroyer and their combinations. In one particular embodiment, said composition that is useful in combined therapy may contain at least one peptide of the invention, along with (optionally) another or other neuronal exocytosis inhibiting compound(s) and a neuronal glutamate receptor blocker. In another embodiment of this invention, said composition could contain at least one peptide of the invention, along with (optionally) another or other neuronal exocytosis inhibiting compound(s), a neuronal glutamate receptor blocker, a calcium chelator, an anti-oxidant and/or a free-radical destroyer.

An additional object of this invention is the use of the peptides of the invention or of vectors that contain at least one sequence that codes for a polypeptide of the invention, in the preparation of a medicine for the treatment of pathological neuronal exocytosis- mediated pathological diseases and/or disorders, such as, for example, spastic diseases, neurological disorders and/or neurodegenerative diseases.

In addition, the invention provides a method for the treatment in mammals of pathological neuronal exocytosis-mediated pathological diseases and disorders such as, for example, spastic diseases, neurological disorders and/or neurodegenerative diseases, which consists of administering to said mammal suffering from said pathological disease or disorder a therapeutically effective amount of at least one peptide of the invention, or of a vector containing at least one DNA sequence that codes for a peptide of the invention, preferably in the form of a pharmaceutical composition that contains it. In one particular embodiment of this invention, said pharmaceutical composition contains, in addition to the peptide or peptides of the invention, one or more (COOH) peptides.

The following examples serve to illustrate the nature of this invention and should not be considered in a restricting sense as regards said invention.

FIGURES

Figure 1: VAMP2 and c-subunit interaction determinants

(A) Alignments of c-subunit recombinant constructions used in Y2H and their interaction with full length VAMP2.

(B) Binary interaction results using controls, c-subunit truncation constructs and full length VAMP2, VAMP2-ATM as well as VAMP2-stxTM constructs.

All experiments were reproduced at least 4 times Figure 2: c-subunit loop 3.4 triggers binding to VAMP2

(A) Pull down experiments of GST-VAMP2-2-92 (GVA) and GV-W₈₉,9oP with immobilised PH-L3.4 or PH-L3.4s. One and 10 μΜ L3.4 displaced respectively 16 and 64% of GVA binding to PH-L3.4.

(B) Effect in ELISA of L3.4, L3.4s, PH-L3.4 and PH-L3.4s on GVA binding to immobilised full length c2-155. In contrast to L3.4s and PH-L3.4s, L3.4 as well as PH- L3.4 efficiently displaced the binding.

(C) Effect in pull down of L3.4 and L3.4s on GVA binding to immobilised c-2- 155. Over 60 % of the binding is inhibited in the presence of L3.4 peptide.

Figure 3: V0 c-subunit loop 3.4 peptide inhibits neurotransmission at L5-L5 excitatory synapses

_.(A) Effect of the active peptide (L3.4). Top, representative examples of synaptic currents recorded 2 minutes (left) and 45 minutes (right in grey) after whole-cell access. Bottom, time course of the inhibition of the normalized synaptic current produced by the peptide. Dotted line represents the baseline level.

(B) Effect of the inactive peptide (L3.4s). Note the stability of synaptic transmission.

(C) Inhibition observed with bath application of bafilomycin.

Figure 4: Effect of L3.4 on cholinergic neurotransmission and SNARE- liposome fusion

(Al) Excitatory postsynaptic potentials (EPSPs) from one representative experiment recorded 1 min before, 5 and 20 min after the start of 3 min presynaptic injection of L3.4. (A2) The first order derivative of EPSPs before and 20 min after L3.4 injection shown in Al . Dotted line indicates the peak of EPSPs. (A3) The peak amplitude of EPSPs shown in Al was normalized to that before the injection.

(B) Normalized EPSP amplitudes (circles) and smoothed values (lines). EPSP was recorded every 10 sec in the presence of L3.4 or L3.4s. (C) Decrease in amplitudes of EPSP elicited by 0.1, 0.25 and 0.5 Hz (n=5) action potentials at 20 min after peptides injection. Mean ± SEM **, p<0.01; *, p<0.05, Unpaired Student t-test.

(D, E) Interaction of L3.4 peptide with the membrane-proximal domain of VAMP2 inhibits SNARE-mediated liposome fusion. Membrane fusion was measured upon mixing t-SNARE and v-SNARE liposomes in the absence or presence of 50 μΜ L3.4, means + SEM, n=4. Inhibition by L3.4 was observed with v-SNARE liposomes containing wild type VAMP2 but not VAMP2-W₈9,9oA.

EXAMPLE Material & Methods Reagents

All oligonucleotides were purchased from MWG Biotech (Germany). Glutathione Sepharose was from GE Healthcare and Ni-NTA agarose from QIAGEN. Peptides (L3.4: GVRGTAQQPRLF; L3.4s: GQATVQPLGRRF) were synthesized by Activotec (Southampton, UK). All proteins purifications were performed in the presence of protease inhibitors (Complete, Roche).

Expression constructs and cloning procedures

All sequences are from rat. For Y2H studies, VAMP2 constructs were either full length or contained syntaxin transmembrane domain VAMP2-STXTM. Except for two hybrid studies where full length VAMP2B was used, all VAMP2B constructs lacked a transmembrane domain (TM) (amino acids 2-92). All constructs were generated by standard polymerase chain reaction (PCR) using a commercial Y2H adult rat brain cDNA library (Origine). VAMP2 construct encompassing residues 2-92 (forward 5'- GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'- GCGTCGACTTAGTTTTTCCACCAGTATTTGCG-3') was amplified and inserted between EcoRl and Sail sites of pGEX4T-l (GVA). VAMP2 2-31 (Forward 5'- GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'-

GCGTCGACTTACAGTCTCCTGTTACTGGTAAG-3', VAMP2 28-76 (Forward 5'- CGAATTC AGT AAC AGGAGACTGC AGC-3 ' + Reverse 5'-GCGTCGACTTACTGG GAGGCCCCTGCCTG-3') and VAMP2 28-92 (Forward 5'-

CGAATTC AGT AAC AGGAGACTGC AGC-3 ' + Reverse 5'-

GCGTCGACTTAGTTTTTCCACCAGTATTTGCG-3') were amplified and inserted between EcoR I and Sal I restriction sites into pGEX-5X-l (respectively GV-2-31, GV- 28-76 and GV-28-92). His tagged VAMP2-2-92 (His-VAMP2) was also obtained by inserting VAMP2 in pET28a using EcoRl and Sail sites. Site-directed mutagenesis (Stratagene) was used to introduce point mutations and generate His tagged (His-VAMP2- 2-92-W89,90A) and GST tagged VAMP2-2-92-W89,90A (GV- W89,90A; Forward 5'- GCGAATTCTCGGCTACCGCTGCCAC-3'+ Reverse 5'- GCGTCGACTTAGTTTTTcgccgcGTATTTGCG-3'), GST-VAMP2-2-92-W90A (GV-

W90A; Forward 5 '-CTCAAGCGCAAATACTGGccgAAAAACTAAGTCGAG-3 ' + Reverse 5'-CTCGACTTAGTTTTTcggCCAGTATTTGCGCTTGAG-3')) as well as GST-VAMP2-2-92-W89,90P (GV- W89,90P; Forward 5'-

CTCAAGCGC AAATACccgccgAAAAACTAAGTCGAG-3 ' + Reverse 5'- CTCGACTTAGTTTTTcggcggGTATTTGCGCTTGAG-3'). The following primers were used for VAMP2-STXTM (Forward 5'-GCGAATTCTCGGCTACCGCTGCCAC-3' + Reverse 5'- GCGTCGACCTATCCAAAGATGCCCCCGATGGTGGAGGCGATGATGATGCCCA GAATCACACAGCAAATGATGATCATGATGTTTTTCCACCAGTATTTGCG-3'). The reverse primer covers the TM of syntaxin and residues 86 to 92 of VAMP2. VAMP2-

2-92 was used as a matrix for this PCR. HSV tag (QPELAPEDPED) was introduced as follow at the 5' of pET28a (Novagen) "multiple cloning site" to generate pET28-HSV- Nter: 5' phosphorylated primers encoding HSV tag sequence ( Forward 5'- GATCCCAGCCTGAACTCGCTCCAGAGGATCCGGAAGATG-3' Reverse 5'- AATTCATCTTCCGGATCCTCTGGAGCGAGTTCAGGCTGG-3') were annealed and ligated to BamHl, EcoRl open pET28a. Full length V0 c-subunit (residues 2-155) was amplified and inserted between Sacl and Sail of pEGFPC3 and pET28a-HSV-Nter to generate pEGFPc3-c-2-155 and pET28-HSV-c-2-155 respectively. All truncated c-subunit constructs were amplified by PCR and cloned between EcoRl and Sail sites in pEGFPc2, pET28a-HSV-Nter, or EcoRl and Xhol for the yeast two hybrid vectors (Origine): pGilda

(for c-subunit) and pJG-4-5 (for VAMP2). The following c-subunit truncation constructs were thus prepared: pEGFPc-2-c2-155 (E-c-2-155); pGilda-c2-155 (BD-c2-155); pGilda- c-2-76 (BD-c-2-76); pGilda-c-2-36 (BD-c-2-36); pGilda-c-37-155 (BD-c37-155); pGilda- c-77-155 (BD-c-77-155); pGilda-c-117-155 (BD-c-117-155). BD-c-2-155 (full length), BD-c-2-76 (from the N-terminus until the end of TM2), BD-c-2-36 (from the N-terminus until the end of TM1), c-37-155 (from the beginning of loop 1.2 until the C-terminus) , c77-155 (from the beginning of loop 2.3 until the C-terminus) , c- 117-155 (from the beginning of loop 3.4 until the C-terminus). c- 117-155 with scrambled loop 3.4 sequence (GQATVQPLGR F/ ggtcaggccactgtccagcctctgggccggcgattc) pET28a-HSV-c-l 17-155-

L3.4s (c-117-155-L3.4s) was generated by PCR suing the following primers (Forward 5'- gcgaattcggtcaggccactgtccagcctctgggccgg-3' + Reverse 5'- gcgtcgacCTACTTTGTGGAGAGGATTAG-3') on a matrix of a full length c-subunit construct with scrambled loop 3.4 sequence pET28a-HSV-c-2-155-L3.4s (c-2-155-L3.4s). This later construct was made as follows using three consecutive PCRs: Using Platinum

Taq DNA polymerase (Pfx) (Invitrogen) and as amplification matrix pET28a-HSV-c-l- 155, overlapping PCR fragments were generated using a) 1- EcoRl flanked c-subunit forward primer (5'-GCGAATTCGCTGACATCAAGAACAACCC) and 2- reverse encoding the scrambled L3.4 sequence (5'- gaatcgccggcccagaggctggacagtggcctgaccAGCATCTCCGACAATGCC-3') b) 3- Forward primer encoding the scrambled L3.4 sequence (5'- ggtcaggccactgtccagcctctgggccggcgattcGTGGGCATGATCCTGATCC-3') and 4- a Sail flanked reverse T7 terminator primer (5 '-GCTAGTTATTGCTCAGCGG) that primes 3' to the vector multiple cloning site. One tenth of both purified newly generated fragments were mixed in the presence of Pfx and in the absence of additional primers or matrix and subjected to 10 cycles to generate the new mutated matrix. Primers 1 +4 were then added to the tube and cycling continued to amplify the full length mutated construct. The consequent PCR product was digested with EcoRl + Sail and ligated to pET28a-HSV- Nter.

Syntaxin la / SNAP25 heterodimer (co-expressed from pGEX-KG and pET28a respectively) were provided by G. Schiavo (London Research Institute, London). PH-L3.4 and PH-L3.4s were constructed as follow: Using Bbsl and Sail restriction sites, L3.4 and scrambled L3.4s encoding phosphorylated complementary oligonucleotides were introduced between -strands 6 and 6' of the PH domain of human cytohesinl in pET21d [Bedet, 2006 #46]. PH-L3.4: Forward primer (5'- aggccGGTGTCCGGGGCACTGCCCAGCAGCCTCGACTGTTCg-3'); Reverse primer (5'- tcgacGAACAGTCGAGGCTGCTGGGCAGTGCCCCGGACACCg-3'). PH-L3.4s: Forward primer (5'- aggccGGTCAGGCCACTGTCCAGCCTCTGGGCCGACGGTTCg- 3'); Reverse primer

tcgacGAACCGTCGGCCCAGAGGCTGGACAGTGGCCTGACCg-3').

Bacterial protein expression

VAMP2 and c-subunit were expressed in Rosetta2 bacteria (Novagen). VAMP2 constructs were expressed using classical protocols. Crude supernatants of ultracentrifuged "French press" bacterial lysates were stored at -20°C. Bacterial strains expressing c-subunit constructs were cultured in pH 5.7 phosphate-buffered LB medium and protein expression was induced with 1 mM IPTG for 4-5 hours. Bacterial pellets were stored at -20°C until protein purification.

Purification of His-HSV tagged c-subunit constructs

Ten grams of dry bacterial pellet were resuspended in 100 ml of buffer A (10 mM Tris-Cl, 100 mM NaH2P04 pH 8.0, 500 mM NaCl, 6M GuCl, 1% CHAPS (w/v), 20% glycerol, 20 mM imidazol, 10 mM β-mercaptoethanol). The extract was sonicated to achieve complete homogenization and incubated overnight in the presence of DNAse I (20 Mg/ml) at 4°C with 2-3 ml of Ni-NTA beads (Qiagen). prewashed with buffer A. Beads were then loaded in a column and subjected at gravity flow to successive washes with buffer A containing decreasing amounts of GuCl (4M, 2M 0.5M, 0M). A last wash with buffer B (20 mM Hepes pH 7.5, 150 mM NaCl, 0.5% CHAPS) was performed before collecting purified and renatured proteins by elution at 4°C in buffer B containing 250 mM imidazole. Protein concentration was determined using the micro BCA assay (Pierce) and eluted proteins were stored at -20°C in single use aliquots. Purification of GST and His tagged SNAREs

Bacterial pellets of His tagged proteins were French pressed in 20 mM Tris pH 8.0, 500 mM NaCl, 10%> glycerol buffer and purified on Ni-NTA beads in the presence of 0.1 %> Triton X-100. A last wash was made by adding to the French press buffer 50 mM imidazole and 0.5 mM DTT. Proteins were eluted in French press buffer containing 500 mM imidazole and 0.5 mM DTT and dialyzed overnight against 25 mM Hepes pH 7.4, 140 mM KC1 and 0.5 mM DTT. Protein aliquots were stored at -80°C. t-SNAREs were co-expressed and purified using standard methods and were either eluted or cleaved from their tags by thrombin. Bacterial pellets of GST tagged VAMP2-2-92 (GV ) and VAMP2-2-92-W89, 90A (GV- W89, 90A) were French pressed in 25 mM Tris pH 8.0, 150 mM NaCl buffer and purified on glutathione Sepharose beads in the presence of 0.1% Triton X-100. Proteins were eluted in Tris 50 mM pH 8.0, 10 mM reduced glutathione and frozen at -20°C until used.

Pull down experiments

Using standard procedures, VAMP2 constructs were freshly purified on glutathione beads from frozen cleared bacterial extracts. Per tube, a 1 ml mixture of Ι μΜ GST and GST- VAMP2 constructs (GVA, GV-2-31, GV-28-76, GV-28-92, GV-W89,90A, GV-W89,90P or GV-W90A) was immobilized. For E-c-2-155, pre-transfected HEK 293 cells from a 10 cm culture dish were solubilized in 1 ml of 25 mM Tris pH 7.5, 150 mM NaCl, 1% CHAPS supplemented with protease inhibitors (Complete, Roche). One fifth of this extract was incubated with the corresponding pre-loaded glutathione beads pellet. For HSV-tagged c-2- 155, 2-10 iiM was added to the corresponding glutathione beads in 1 ml binding buffer: 25 mM Tris pH 7.5, 150 mM NaCl, 0.5% CHAPS. His tagged HSV-c-2-155, PH-L3.4 or PH- L3.4s were freshly purified on Nickel beads. Per tube, 100 iiM of purified GVA or GVA W89,90A was added in the presence of increasing two concentrations of L3.4 or L3.4s concentrations (1 and, 10 μΜ).

In all conditions, incubation was overnight at 4°C. Beads were washed four times with binding buffer and bound proteins were eluted in SDS sample buffer. Proteins were separated by SDS PAGE, and c-subunit binding to VAMP2 was probed by Western blotting using either monoclonal anti-GFP (Roche) or polyclonal anti-HSV antibodies (AbCaM) (1 :2500) and Immobilon western (Millipore) or super signal West pico (Pierce) HRP chemiluminescence substrates. GVA binding to immobilized c-2-155 or PH constructs was probed using goat polyclonal anti GST antibodies (GE healthcare). ELISA

Nunc Maxisorp 96 wells plates were coated over night with purified His-HSV-c2-155 (2 g/ml) in NaHC03 100 mM pH 8.9 buffer. After a 1 h blocking step using Tris 25 mM pH 7.5, NaCl 150 mM, Casein 1%, 0.05% Tween 20, 300 iiM of GVA or GV-W89,90P in Tris 25 mM pH 7.5, NaCl 150 mM, BSA 0.1%, 0.05% Tween 20 were added in the presence or absence of increasing concentrations of L3.4 or L3.4s peptides. The chromogenic HRP substrate TMB (3,3 ^',5,5^'-tetramethylbenzidine) was then used to detect VAMP2 binding using anti GST (GE healthcare) and an HRP coupled secondary antidody (Jackson ImmunoResearch) . V-ATPase mediated proton uptake

The following protocol was inspired from (Goncalves et al, 2000) with modifications: four rat brains were homogeneized in 0.32 M sucrose, 10 mM HEPES-KOH, 0.2 mM EGTA in the presence of protease inhibitor cocktail (Sigma), then centrifuged for 10 min at 900 x g. Three ml of supernatant were layered onto a 4 ml 0.8 M sucrose cushion and centrifuged 20 min at 257.000 x g. Synaptosome pellets were then resuspended in hypotonic buffer (10 mM Tris-HCl pH 8.5, 1 mM PMSF) and centrifuged 20 min at 40.000 x g. The synaptic vesicle enriched supernatant was then adjusted to 10 mM Tris-HCl pH 8.5, 60 mM sucrose, 140 mM KC1, 2 mM MgC12, 50 μΜ EGTA (assay buffer). ATP-dependent proton transport was monitored by the quenching of acridine orange (AO) fluorescence. When the synaptic vesicle lumen is acidified, membrane permeable AO becomes protonated, and trapped within the synaptic vesicle. The high AO concentration achieved results in a red-shift and self-quenching of fluorescence. In 100 μΐ final reaction buffer, a 5-fold dilution of enriched synaptic vesicle preparation was incubated at 30°C in the presence of 2 μΜ AO (Sigma). Proton uptake was triggered by adding 500 μΜ Mg-ATP. Experiments were carried out in Nunclon fluorescence plates using a Biotek Sirius HT injector plate reader and KC4 software. Fluorescence at 525 nm was monitored with data acquisition every 15 seconds.

Preparation of liposomes

Lipids were from Avanti Polar Lipids. 850 nmoles of dried lipids (85% (mol/mol) 1- palmitoyl, 2-oleoyl phosphatidylcholine (PC) + 15% 1,2-dioleoylphosphatidylserine (PS)) were resuspended in 25 mM HEPES-KOH pH 7.4, 140 mM KC1, 0.25 mM DTT,1.5% (w/v) sodium cholate (Sigma). Liposomes were obtained by rapid dilution and extensive dialysis in the resuspension buffer.

Preparation of proteoliposomes

Reconstitution of v-SNAREs and t-SNAREs liposomes was carried out essentially as previously described (Weber et al, 1998). Lipids were from Avanti Polar Lipids. Dried lipids were resuspended in VAMP2 or syntaxin 1 / SNAP25 heterodimer solutions in presence of 1.5% (w/v) sodium cholate (Sigma), at a 1/100 (mol/mol) protein-to-lipid ratio. Lipid compositions were 85% (mol/mol) 1 -palmitoyl, 2-oleoyl phosphatidylcholine (PC), 15% 1,2- dioleoylphosphatidylserine (PS) for the acceptor v-SNARE vesicles and 83% PC, 15% PS, 1.5% (mol/mol) N-(7-nitro-2- 1 ,3-benzoxadiazol-4-yl)- 1 ,2-dipalmitoyl phosphatidylethanolamine (NBD-PE), and 1.5% (mol/mol) N-(lissamine rhodamine B sulfonyl)-l,2-dipalmitoyl phosphatidylethanolamine (Rhodamine-PE) for the donor t-SNARE liposomes. Liposomes were obtained by rapid dilution and extensive dialysis in 25 mM HEPES-KOH pH 7.4, 140 mM KC1, 1 mM DTT in the presence of Bio Beads (Biorad), unincorporated proteins and aggregates were removed by a 4 hour centrifugation (250000xg) on a discontinuous Optiprep (Abcys) gradient.

In vitro fusion assay and data analysis

In each fusion reaction, 2 μΐ donor liposomes were mixed on ice with 10 μΐ acceptor liposomes in 25 mM HEPES-KOH pH 7.4, 140 mM KC1, 1 mM DTT, 3 mM EGTA, then NBD dequenching was monitored at 37°C with a Biotek Sirius HT injector plate reader and data acquisition every 2 min. L3.4 (50 μΜ) was preincubated 30 min on ice with donor liposomes prior to v- and t-SNARE mixing (Controls for SNARE-dependency of fusion were performed by preincubating 10 μΜ His-VAMP21-92 using the W89,90A mutant, to preclude unspecific inhibition of fusion due to interaction of VAMP2-1-92 with anionic phospholipids (Quetglas et al, 2002) (Quetglas et al, 2000), for 2 hours with donor liposomes). Each assay condition was carried out in triplicate. For data analysis, the lowest values obtained in controls, after the initial drop in fluorescence due to fluorophore warming (Tucker et al., 2004), were set to t=0 and all the conditions were analyzed from the same time point. Fluorescence values obtained by maximal dequenching with excess sodium cholate at the end of each reaction were set to 100%. Mean traces from each experiment were normalized to the control fluorescence level at 60 min, and presented ± SEM (n=4).

Binding analysis by SPR

SPR experiments were conducted using a Biacore 3000 (GE Healthcare). All experiments were performed at 25°C with HBS EP (lOmM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing Triton X-100 0.005% as running buffer. Purified His- VAMP 1-92 {2000- 3000 resonance units (RU)} was covalently coupled in 10 mM sodium acetate pH 5 to a CM5 chip via amine group linkage using standard coupling procedures (Quetglas et al, 2002). c-117-155 or c-117-155-L3.4s were injected at 30 μΐ / min using Kinject mode. All flow cells were regenerated for 40 sec between samples with 50 mM octylglucoside diluted in running buffer. Data obtained over a non-functionalized flow cell were subtracted from all runs to account for non-specific binding (less than 5% of total binding). Interactions were fitted globally across all concentrations to a 1 : 1 Langmuir binding model and rate constants were derived using BIA Evaluation 4.1 software. The affinity constant, KD, was calculated from the ratio of the rate constants kd (dissociation rate) versus ka (association rate).

Preparation, recording and data analysis from cortical slices

Cortical slices and dual whole-cell recordings were obtained as detailed previously

(Boudkkazi et al, 2007). Loop 3.4 (L3.4) or scrambled (L3.4s) synthetic c-subunit peptides were injected (2 mM) into the presynaptic neurons. Bafilomycin Al was purchased from Euromedex (France) and was bath applied (final concentration, 5 μΜ). All paired-pulse protocols were performed at a frequency of 20 Hz. Synaptic responses were averaged following alignment of the presynaptic action potentials using automatic peak detection (Detectivent 4.0, N. Ankri INSERM). Data are means ± SEM.

Synaptic transmission from SCG neurons

6-8 weeks cultures, EPSP recording and injection of peptides (1.5 mM; 5 mM showed no further effect) were performed as described previously (Mochida et al, 1994). EPSPs were recorded at 0.1, 0.25 Hz or 0.5 Hz. The peak amplitudes were normalized to the values before injection. The averaged and smoothed values (Origin 7.5) obtained from an eight-point moving average algorithm were plotted against recording time with t=0 corresponding to the start of 3 min presynaptic injection of L3.4, L3.4s (a scrambled control peptide). Data are mean ± SEM. A two-tailed Student t-test was applied as indicated.

Results

VAMP2 interacts with VP c-subunit

Yeast two hybrid (Y2H) methods have not been widely used to probe interactions between proteins with transmembrane (TM) domains. However detection of V0 c-subunit as a partner for βΐ integrin (Skinner and Wildeman, 1999) and identification of a full length (FL) syntaxin 1 clone in a screen using β SNAP as bait (O El Far - unpublished results) encouraged us to pursue Y2H analysis of in vivo interactions between SNAREs and V0 subunits. Therefore, we used the LexA Y2H system to probe for interactions between c-subunit and VAMP2.

Our results showed that c-subunit binds to VAMP2 via an interaction involving the C- terminal third of c-subunit, indicating that the cytoplasmically-oriented loop 3.4 is very likely to constitute the VAMP2-binding domain (Figure 1 A). Control experiments confirmed robust binding of FL VAMP2 to syntaxinl and homodimerization of FL VAMP2 (Figure IB). Interestingly, FL VAMP2 displayed an interaction with c-subunit (Figure 1) while a VAMP2 construct lacking the TM (VAMP2-ATM) did not. Two explanations can be envisaged: i, VAMP2 TM carries the c-subunit binding site ii, VAMP2 TM is required to anchor VAMP2 in the inner nuclear membrane nearby its tetraspan binding partner. To discriminate between these possibilities, we generated a VAMP2 construct in which the TM was replaced by that of the t-SNARE syntaxinl (VAMP2-stxTM). VAMP2-stxTM interacted with c-subunit, while FL syntaxinl did not (Figure IB). These results indicate that VAMP2 harbours a c-subunit binding site located outside of its TM. VAMP2 TM is thus not necessary for binding to c- subunit but confers transport of VAMP2 to the inner nuclear membrane where interaction takes place.

V0 c-subunit is an extremely hydrophobic protein with four TM domains connected by very short loops and discrete N and C-termini. Analysis of native proteins was unsuccessful since both commercially available and specifically designed anti-peptide antibodies against exposed conserved regions of V0 c-subunit, failed to recognise rat proteins (data not shown). We therefore expressed GFP-tagged full length c-subunit (E-c-2-155) in HEK 293 cells. Pulldown assays detected the specific binding of multimeric forms of E-c-2-155 to bacterially- expressed GST-VAMP2-2-92 (GVA), but not to GST, immobilised on glutathione beads (). These data indicate that c-subunit can associate with VAMP2 and that the TM region of VAMP is not necessary for interaction, consistent with Y2H data.

Due to limited expression yields in HEK 293 and in order to address direct binding interactions, we produced bacterially-expressed tagged recombinant c-subunit. Major toxicity was encountered when overexpressing c-subunit in E. coli but was overcome by acidifying the culture medium. A specific protocol to express and purify HSV-tagged c-subunit was developed which allowed the recovery of milligram amounts of otherwise insoluble fusion proteins. The profile of c-2-155 expression in western blots showed, in addition to monomers and dimers, higher molecular weight c-subunit multimers that resisted dissociation by SDS. Pull down experiments using pure recombinant c-subunit and immobilised GVA established a direct interaction. Mapping of c-subunit interaction sites on VAMP2 indicated that only VAMP constructs containing the calmodulin (CaM) binding motif at amino acids 76-92 (Quetglas et al, 2000) bound to c-2-155 . Furthermore mutation of juxtamembrane tryptophans (W89W90) known to be involved in CaM binding (Quetglas et al, 2002) completely abolished c-subunit binding. To consolidate binding data, we used soluble His- tagged VAMP2-ATM (His-VAMP2) to displace c-subunit binding to immobilized GVA. A, a tenfold molar excess of His-VAMP2 reduced c-subunit binding to background levels while His-VAMP2-W89,₉oA had no effect. Surface plasmon resonance (SPR) measurements confirmed pull-down assays (data not shown) and ELISA assays using biotynilated, wild type or mutated (Ws₉,₉oA) VAMP2 peptides (amino acids 77-94) corroborated the binding domain delimitation on VAMP2 as well as the importance of Ws₉ W90 in c-subunit binding (data not shown).

Furthermore a recombinant C-terminal region of the c-subunit containing loop 3.4 (c- 117-155) displayed by SPR high affinity binding to VAMP2 with (Kd = 1.9 + 0.7 nM,) and the interaction was completely abolished when loop 3.4 sequence was scrambled in the c-117- 155-L3.4s construct. SPR data thus substantiated Y2H assays, underlining the importance of c-subunit loop 3.4 in binding to VAMP2. In order to verify whether c-subunit loop 3.4 (L3.4) was sufficient to trigger binding to VAMP2, and to mimic the TM surrounding constrain of this loop, we cloned it as well as its corresponding scrambled peptide (identical to the scrambled insert in c-117-115 L3.4s) between two β-strands of the PH (Plextrin Homology) domain of Cytohesin 6 ([Bedet, 2006 #46]). In contrast to PH-L3.4s, pull down assays (Figure 2 A) using immobilised PH-L3.4 specifically bound GVA in a Ws9,9o dependant manner and 10 μΜ of free L3.4 peptide inhibited this binding by over 60 %.To summarize, results in vivo and in vitro indicate specific molecular interactions between the membrane- proximal domain of VAMP2 and loop 3.4 of the tetraspan V-ATPase c-subunit, both of which are cytoplasmically-oriented motifs.

10 μΜ of L3.4 inhibited GVA interaction with immobilised c-2-155 by 36% in ELISA assay (Figure 2B) and over 60%> by pull down (Figure 2C). In contrast, L3.4s did not inhibit binding in ELISA and reached 10.3% in pull down assays (Figure 2C). One micromolar of PH-L3.4 was sufficient to specifically inhibit over 63% GVA binding to immobilised c-2-155 in ELISA (Figure 2B).

L3.4 peptide decreases release probability in connected L5-L5 pyramidal pairs

To address the physiological importance of the interaction, we examined the ability of

L3,4 to interfere with neurotransmitter release in acute rat cortical slices. Dual recordings in acute rat cortical slices from connected glutamatergic L5 pyramidal neurons (Debanne et al., 2008) were performed while injecting a synthetic peptide corresponding to loop 3.4 (L3.4) or its scrambled sequence (L3.4s) into the presynaptic cell. Ten minutes after whole-cell access to the presynaptic neuron, EPSC amplitude slowly but robustly decreased and stabilised to 32.5 ± 4.8% (n = 3, Mann- Whitney test p<0.01) of the control amplitude after 40 minutes (Figure 3A). In contrast, no reduction in amplitude occurred when the presynaptic pipette contained the scrambled L3.4s peptide (100.6 ± 1.7%, n = 3, Mann- Whitney test p>0.10; Figure 3B). While reduced transmitter release could result from interference with the interaction between VAMP2 and c-subunit, an alternative possibility must be addressed: L3.4 peptides might inhibit V-ATPase proton pump activity and induce a default in vesicle loading with transmitter, leading to "firing blanks" (ie. fusion of synaptic vesicles that are empty or have reduced contents). We therefore studied the effects on synaptic response amplitudes of the specific V-ATPase inhibitor bafilomycin Al, which induced a comparable reduction in EPSC (61.3 ± 6.5%; n = 3, Mann- Whitney test p<0.01; Figure 3C).

In order to rule out the possibility that the L3.4 peptide reduces release by inhibiting the proton pump, we compared its effect on release probability with that of bafilomycin Al . While no change was observed in paired pulse ratio (PPR) in neurons injected with L3.4s peptide (100.3 ± 1%>; n = 3, Mann- Whitney test p<0.01, not shown), a decrease in release probability, translated by an increase in the paired pulse ratio (PPR), was observed in L3.4- injected neurons (162.7 ± 11.7% of the control PPR, n = 3, Mann- Whitney test p<0.01;). Importantly, the opposite effect was observed in bafilomycin-treated neurons in which, in accordance with a previous report (Harrison and Jahr, 2003), a decrease in PPR (77.3 ± 2.6%> of the control, n = 3, Mann- Whitney test p<0.01;) was measured. This clearly demonstrates that the mechanisms underlying inhibition of neurotransmitter release by L3.4 and bafilomycin are distinct, suggesting that inhibition by L3.4 is likely to translate interference with the VAMP2 / c-subunit interaction rather then proton pump inhibition. To directly address effects on proton pump activity we performed in vitro assays with synaptic vesicle- enriched membrane fractions from rat brain. Intra-luminal acidification by V-ATPase was triggered by addition of Mg2+/ATP and monitored fluorimetrically by acridine orange quenching. Results demonstrated that synaptic vesicle acidification was not affected at a concentration as high as 1 mM L3.4 peptide, while it was robustly inhibited by bafilomycin or EDTA. These data are consistent with the conclusion that inhibition of neurotransmitter release by L3.4 peptide entails decreased release probability and is not a consequence of suppressing proton transport.

L3.4 peptide inhibits neurotransmitter release in cholinergic rat superior cervical ganglion (SCG) neurons In order to ascertain whether v-SNARE interactions with V0 might constitute a general presynaptic mechanism we also examined the action of L3.4 peptide at peripheral cholinergic synapses. SCG neurons provide a well-established culture system in which many agents that perturb neurotransmitter release mechanisms have already been studied. Furthermore unlike cortical neurons, SCG neurons have extremely short axons, thus somatically- injected peptides reach nerve terminals very rapidly. Hence, this experimental system allows direct evaluation of the kinetics of effects, providing an indication as to which step in exocytosis is perturbed when a protein-protein interaction is disrupted (Ma and Mochida, 2007).

When L3.4 was injected into SCG neurons, inhibition started after around 1 min (1.1 ±

0.2 min, n=6) (Figure 4A and 4B) reaching a plateau within 10 min (11.6 ± 2.4 min, n=6), whereas the scrambled peptide L3.4s had no effect (Figure 4B). This rapid inhibition is comparable to that observed with agents that perturb SNARE assembly (Mochida, 1998) which suggests that L3.4 peptide affects a late step in exocytosis. The time to peak of the decreased EPSP wave form was not affected (Figure 4A1), however the speed of rise to the peak (Figure 4A2) and the decay from the peak were slightly slower (Figure 4A3) suggesting that the kinetics of synaptic vesicle fusion might be modified in the presence of the peptide. In agreement with experiments in cortical pairs, an effect of the peptide on proton pumping and thus on acetylcholine transport during vesicle recycling, appears unlikely since the EPSP decrease was independent of stimulation frequency (synaptic activity) (Figure 4C) (Ma and Mochida, 2007; Su et al, 2001). Furthermore in these strong peripheral synapses, inhibition by microinjected bafilomycin Al displayed onset at around 40 min at 0.2Hz (data not shown) which was very slow compared to that of L3.4 peptide at an even lower stimulation frequency (Figure 4B).

In order to confirm that L3.4 did not interfere with the classical recycling pathway, reserve vesicles and the rapidly releasable vesicle pool were depleted by a 3 min train of presynaptic action potentials at 5 Hz (Lu et al, 2009; Ma and Mochida, 2007) and the recovery of EPSP amplitude was measured every 1 sec. At the end of the train, the EPSP amplitude was within baseline noise levels 4, and subsequently recovered at two distinct fast and slow rates. L3.4 did not affect the recovery rates from synaptic vesicle depletion by either a 5 Hz train 4 or a 10 Hz train (data not shown). Together, these results consolidate data from L5-L5 synapses. Furthermore they indicate that L3.4 peptide does not affect vesicle recycling and thus are consistent with the view that it perturbs the interaction of VAMP2 and c-subunit of V-ATPase V0 at a late step of exocytosis, close to fusion. L3.4 peptide inhibits SNARE dependent in vitro membrane fusion

Action of V0 at a late step is consistent with recent reports indicating a role downstream of SNARES. We hypothesized that binding to the membrane-proximal domain of VAMP serves to position c-subunit hexamers at the interface between vesicle and target membranes as trans SNARE complexes assemble. A dominant negative effect of L3.4 peptide implies that the peptide can bind directly to full length membrane-anchored VAMP2, like a c-subunit surrogate and thus might itself modulate SNARE assembly. In order to directly assess the effect of L3.4 peptide on trans SNARE-assembly, we measured SNARE liposome fusion in vitro using a standard fluorescence dequenching assay. Mixing acceptor v- SNARE (VAMP2) with donor t-SNARE (syntaxin 1 / SNAP-25 dimer) liposomes, generates an increase in NBD fluorescence following membrane fusion and lipid mixing (Weber et al., 1998). The SNARE-dependency of fusion was confirmed by adding soluble VAMP2 cytosolic domain (VAMP2 2-92) 4. As shown in Figure 4D, L3.4 inhibited fusion up to 30.9 + 1.3% (n=4). Interestingly, in assays using v-SNARE liposomes containing mutant VAMP2- Ws , oA that is unable to bind to c-subunit, fusion was not inhibited by L3.4 (Figure 4E). In addition, SPR analysis showed that up to 5 μΜ L3.4 peptide were unable to bind protein free PC/PS liposomes. At the same concentration, a VAMP2 peptide encompassing residues 77-94 robustly bound these liposomes in a comparable way to GVA . These results demonstrate that L3.4 peptide binds directly to the membrane-proximal domain of VAMP2 implicated in interaction with the c-subunit and partially restricts SNARE assembly.

Discussion

Analysis of links between SNAREs and V-ATPase subunits by Y2H, GST pull- down from lysates of transfected HEK293 cells and binding studies with bacterially- expressed proteins, has uncovered a direct molecular interaction between the v-SNARE VAMP2 (synaptobrevin) and V0 c-subunits? The c-subunit binding domain on VAMP2 was mapped to the juxtamembrane region, which also constitutes a calmodulin-binding motif (Quetglas et al., 2000). We delimited the VAMP2 binding site on c-subunit to the C- terminal quarter (amino acids 117-155) corresponding to the loop 3.4-TM4 plus three C- terminal amino acids. Thus the association occurs via two cytoplasmically-oriented and membrane-proximal sequences which is topologically consistent with a cis interaction between intrinsic vesicle membrane proteins. In order to assess the functional relevance of this interaction, we chose acute peptide interference in synaptically-coupled pairs of neurons. We measured neurotransmitter release by monitoring postsynaptic responses after injection of the loop 3.4 peptide (L3.4) sequence in two different neuronal systems. This approach allowed us to analyse the kinetics of the onset of inhibition in a given pair and to determine whether effects occur during vesicle-recycling or at later steps, close to fusion. Glutamatergic neurotransmission between two connected pairs of pyramidal neurones in acute cortical slices was robustly inhibited by loop 3.4 peptide injection while the scrambled peptide had no effect. Inhibition was not exclusive to glutamatergic synapses since cholinergic neurotransmission in SCG neuronal cultures was also inhibited, though to a lesser extent

(22%) than in cortical neurons. This may be due to the different mode of peptide injection. Peptide was transiently microinjected (3 min) into SCG neurons, while it diffused constantly into cortical neurons from a recording patch electrode. Alternatively L3.4 peptide might inhibit a specific neurotransmitter release mode that is less prevalent in SCG neurons.

As L3.4 peptide mimics a c-subunit cytoplasmic linker sequence, we were concerned that it might modify intramolecular interactions between V-ATPase subunits and inhibit proton pumping, which would then compromise neurotransmitter loading into synaptic vesicles. In this case empty vesicles might still fuse without generating an EPSP, thus a reduced EPSP could result from blockade of the proton pump. Several observations allow us to rule out this possibility. Firstly we compared the effects on neurotransmission of L3.4 peptide with those of bafilomycin Al, a specific inhibitor of the V-ATPase that blocks proton transport. In the strong synapses of peripheral SCG neurons, bafilomycin Al only modified EPSPs after prolonged stimulation, while L3.4 peptide reduced EPSPs much more rapidly, even with lower stimulation frequencies. This observation is consistent with the idea that bafilomycin affects a step in vesicle recycling (eg. reduced loading with transmitter due to a defective proton gradient) while L3.4 peptide inhibits a downstream step closer to vesicle fusion. As expected in the weaker synapses of central glutamatergic neurons the onset of inhibition by bafilomycin Al was faster and induced gradual inhibition of EPSCs similar to that observed with L3.4. However, the effects of bafilomycin Al and L3.4 on short term plasticity were completely different. L3.4 increased the paired pulse ratio (PPR) by over 62% while bafilomycin Al produced the opposite effect with a PPR decrease of about 23%. Finally high concentrations of L3.4 peptide had no effect on bafilomycin-sensitive proton transport by V-ATPase in vitro, in a membrane fraction enriched in synaptic vesicles. All these results indicate that the inhibition of neurotransmitter release by L3.4 peptide is not a consequence of perturbing V-ATPase proton pump activity.

In SCG neurons microinjected peptides reach nerve terminals very rapidly. The L3.4 peptide induced very rapid inhibition (onset at around 1 min) which is typical of agents that perturb neurotransmission at late steps in exocytosis (eg. SNARE interacting Synprint peptide from voltage-gated N-type calcium channel (Mochida et al., 1996), P/Q type calcium channel blockers (Mochida et al., 2003). Consistent with this interpretation the L3.4 peptide induced a slightly slower speed of rise and decay of the EPSP, suggesting that the kinetics of synaptic vesicle fusion might be modified in the presence of the peptide. Furthermore experiments involving high frequency stimulation and recovery to examine whether L3.4 peptide perturbed the classical recycling pathway indicated that it did not modify the re-filling rate of the readily-releasable or reserve vesicle pools. Hence, taken together these results suggest that inhibition by L3.4 takes place at a step close to Ca2+-triggering and fusion pore opening.

Thus data obtained using the c-subunit domain that binds v-SNAREs supports the hypothesis that V0 sector is involved in vesicle fusion. What is the evidence that the partner sequence in VAMP is functionally important? The c-subunit interaction site on VAMP2 is located in the C-terminal domain and binding was completely abolished by mutations in membrane-proximal tryptophan residues (W89W90). Interestingly the same tryptophan residues are known to be involved in Ca2+-dependent calmodulin binding or acidic phospholipid binding to VAMP2 and in (Quetglas et al, 2002). Thus the consequences of mimetic peptide injection and mutagenesis in this region have already been investigated, establishing the importance of VAMP2 W89W90 in Ca2+-dependent exocytosis from chromaffin and PC12 cells (Quetglas et al, 2002). The tryptophan motif has been implicated in SNARE complex dimers (Fdez et al, 2008). Furthermore expression of the VAMP W89A,W90A mutant in VAMP KO neurons induced a 2-fold decrease in evoked release compared to wild-type rescue but does not affect SNARE assembly nor binding of complexin or synaptotagmin to the SNARE complex (Maximov et al, 2009). Hence several reports support the functional importance of VAMP2 juxtamembrane region, but owing to the multiplicity of potential binding partners in this domain, further investigation will be necessary to determine unequivocally whether c- subunit is involved. Nevertheless the fact that c-subunit and Ca2+-dependent calmodulin binding domains on the v-SNARE overlap suggests that interactions may be regulated. How might V0 c-subunit co-operate with SNARE proteins in neurotransmitter release? That SNARE protein zipping pulls trans membranes towards each other to favour fusion is a widely accepted concept. However the precise role of the hexameric V0 c- subunit is still an open question. Intriguingly c-subunits purified from Torpedo (reviewed by (Morel et al, 2001) or yeast (Peters et al, 2001) can form a regulated pore in vitro. Evidence from yeast suggests that SNARE pairing precedes the assembly of trans V0-V0 complexes which delineate a channel-like structure at the fusion site (Peters et al., 2001).

Our data indicates that L3.4 peptide binding to membrane-inserted VAMP2 reduced SNARE-dependent liposome fusion in vitro, while this peptide failed to inhibit fusion when v-SNARE liposomes contained mutant VAMP2-W89,90A. These results are consistent with a hypothesis in which the c-subunit rotor of V0 associates with VAMP2 to promote a distinct mode of fusion, possibly by forming a transmembrane pore. In this mode of fusion we speculate that only loose SNARE assembly is required, thus c-subunit itself (or a peptide that mimics this domain) might prevent complete SNARE complex zipping. Our findings thus establish a direct molecular link between SNARE scaffolding and pore-forming V-ATPase subunits, and we anticipate that regulation of these interactions might control transitions between distinct neurotransmitter release modes.

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Claims

1. A polypeptide comprising at least 6 consecutive amino acid selected in the amino acid sequence ranging from positions 117 to 128 of SEQ ID NO: l or a function- conservative variant which is able to at least partially inhibit neuronal exocytosis.

2. A polypeptide according to claim 1 wherein the consecutive amino acid sequence has a length of to 6 amino acids to 12 amino acids.

3. A polypeptide according to claim 1 to 2, characterized by the fact that it has a length of 6 to 20 amino acids.

4. A polypeptide according to claim 1, characterized by the fact that it is substantially homologous to a peptide that has a sequence of 6 to 12 consecutive amino sequence ranging from positions 117 to 128 of SEQ ID NO: l and which is able to at least partially inhibit neuronal exocytosis.

5. A polypeptide according to claim 1 to 4, characterized by the fact that the amino acid from the amino end has an acetylated terminal amino group and/or from the carboxyl end has an amidated terminal carboxyl group.

6. A polypeptide according to claim 1 to 5, characterized by the fact that it has an amino acid sequence selected from the amino acid sequences shown in SEQ. ID. No. 2 , SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 8, SEQ. ID. No. 9.

7. A polypeptide according to claim 1 to 6, characterized by the fact that it also contains a reversible modification in order to increase its bioavailability and its ease in passing through the blood-brain barrier and epithelial tissue.

8. An isolated nucleic acid sequence, characterized by the fact that it codes for a polypeptide according to any of the claims from 1 to 6.

9. A nucleic acid sequence according to claim 8, characterized by the fact that said nucleic acid is selected among single-strand DNA, double-stranded DNA, and RNA.

10. A plasmid, characterized by the fact that it has a nucleic acid sequence according to claim 8 to 9.

11. An expression vector, characterized by the fact that it contains a nucleic acid sequence according to claim 8.

12. A prokaryotic or eukaryotic cell, characterized by the fact that it expresses a polypeptide according to any of the claims from 1 to 7.

13. A cosmetic composition that includes a cosmetically effective amount of at least one polypeptide according to any of the claims from 1 to 7, along with at least one cosmetically acceptable adjuvant.

14. Use of a polypeptide according to any of the claims from 1 to 7 in the preparation of a cosmetic composition for the treatment of facial wrinkles and/or asymmetry.

15. A method for the cosmetic treatment in humans of facial wrinkles and/or asymmetry that includes applying a cosmetically effective amount of at least one polypeptide according to any of the claims from 1 to 7 to said human who has facial wrinkles and/or asymmetry.

16. A pharmaceutical composition that includes a therapeutically effective amount of at least one polypeptide according to any of the claims from 1 to 7, along with at least one pharmaceutically acceptable excipient.

17. A pharmaceutical composition according to claim 16 that includes, also optionally, a drug selected from the group formed by a neuronal glutamate receptor blocker, a calcium chelator, an anti-oxidant, a free-radical destroyer and their combinations, and optionally, another or other neuronal-exocytosis inhibiting compound(s).

18. A pharmaceutical composition that includes a therapeutically effective amount of a vector that contains at least one nucleic acid sequence according to claim 8, that codes for a polypeptide according to any of the claims from 1 to 7, along with at least one pharmaceutically acceptable adjuvant and/or excipient.

19. Use of a polypeptide according to any of the claims from 1 to 7 in the preparation of a medicine for the treatment of pathological neuronal exocytosis-mediated pathological diseases and/or disorders.

20. Use of a vector that contains at least one nucleic acid sequence according to claim 8, that codes for a polypeptide according to any of the claims from 1 to 7, in the preparation of a medicine for the treatment of pathological neuronal exocytosis-mediated pathological diseases and/or disorders.