WO2003095483A1

WO2003095483A1 - Dna encoding for a gtpase activating protein

Info

Publication number: WO2003095483A1
Application number: PCT/EP2003/004886
Authority: WO
Inventors: Volker Endris; Gudrun Rappold
Original assignee: Universitaetsklinikum Heidelberg
Current assignee: Universitaetsklinikum Heidelberg
Priority date: 2002-05-10
Filing date: 2003-05-10
Publication date: 2003-11-20
Anticipated expiration: 2004-11-10
Also published as: AU2003240617A1

Abstract

The present invention relates to a novel human gene involved in regulation of signal transduction pathways, and the proteins and polypeptides encoded thereby. More specifically, the invention relates to MEGAP and its isoforms that have a role in signal transduction regulating activity or functions of neuronal cells, a disruption thereof leading to abnormal development or function of neuronal structures that are important for normal cognitive or physical function, e.g. migration, activation, and/or branching.

Description

DNA ENCODING FOR A GTPASE ACTIVATING PROTEIN

Remarkable progress in understanding the genetic traits of idiopathic mental retardation (MR) has occurred in recent years. Several genes could be identified on the human X chromosome using positional cloning strategies and their contribution to the mental handicap was accomplished by mutation detection (1,2). These analyses revealed for most of these genes an apparent common molecular mechanism in contributing to the same intracellular pathways involving the Rho-class of small GTP-coupled proteins. This family of ras-homologous proteins is comprised of several members, with RhoA, Racl and Cdc42 as best studied examples. Their functions are diverse, but have ascribed roles in outgrowth of axons and dendrites in vivo, activation of the MAP kinase cascades and cytoskeleton organisation (3-5). The correct temporal and spatial activity is accomplished by the tight regulation of the active GTP-bound and the inactive GDP state. This situation is achieved by the mutual function of GEF (guanine exchange factors) and GAP (GTPase activating proteins) proteins. It has become evident, that mental retardation seems to be a consequence of the inproper regulation of the small GTPases or their downstream targets. For example, ARHGEF6 represents a guanine exchange factor for Racl and Cdc42 and was identified by the molecular analysis of a X;21 reciprocal translocation (6). In a similar way, Oligophreninl, a GAP for RhoA, was found to be interrupted in a female patient carrying a balanced X;12 translocation associated with MR (7,8).

In the study underlying the present invention, a patient with a balanced de novo translocation t(X;3)(pl l.2;p25) with one of its breakpoints mapping within the 3p^" syndrome deleted region (9-12) was analyzed. This young woman shows hypotonia and severe mental retardation, features characteristic for 3p^" patients, but not microcephaly, growth failure, heart and renal defects and the 3p^" typical facial abnormalities (13,14). The translocation breakpoint on chromosome X is located outside of any coding region. However, the breakpoint on chromosome 3 interrupts a previously unknown gene, which we teraied MEGAP (Mental disorder associated GAP protein). MEGAP shares structural and functional similarities with other members of the RhoGAP protein family and is highly expressed in fetal and adult brain tissue. The C-terminal part of MEGAP has recently been shown to play a critical role in the Slit-Robo signal transduction pathway (15). The inventors concluded that the phenotype observed in our patient is due to a misregulation of a neuronal signal transduction machinery controlling the correct migration of neurons and their axonal connectivity.

A gene with similar sequence to the one described herein is the subject of a patent application by Glenn et al. (WO 00/75320), published December 14, 2000. However, Glenn et al. fail to describe or intimate any role of the gene product in the growth, migration or any other behaviour of neuronal cells. Neither do Glenn et al. describe a neuronally specific expression of said gene.

Therefore, it is the object of the present invention to provide a novel gene product, which is involved in the growth, migration or any other behaviour of neuronal cells and which is, therefore, capable of being used as a therapeutical agent in the treatment of disorders associated with an impaired neuronal function.

This object is solved by the subject-matter of the independent claims. Preferred embodiments are set forth in the dependent claims.

As it has already been shown, MEGAP is involved in signal transduction pathways, which are triggered by binding of the extracellular ligand Slit to the transmembrane receptor Robol. MEGAP is responsible for the signal transduction via Robol to Racl. Racl is inactivated by the enzymatic function of MEGAP and thus, Racl induced alterations of the actine cytoskeleton are eliminated. An important question, which is to be posed in this context, is the activation of MEGAP by transmembrane receptor Robol. Phosphorylations of amino acids serine, threonine or tyrosine play an important role in the regulation of the signal transduction pathways. By adding one or more phosphate groups by protein kinases at specific sites within a molecul, the activity of the protein can be altered. The present inventors could show that MEGAP is specifically phosphorylated in the C-terminal region at Tyrosine.

The invention corresponds to a DNA coding for human MEGAPa, or its splice-variants MEGAPb and MEGAPc (Fig. 8), which code for an amino acid sequence from Met at the 1^st site to Met at the 1099^st site, or an DNA in which at least one nucleotide is inserted, is deleted or is replaced with an other nucleotide, relative to the DNA coding for MEGAP, or its splice-variants MEGAPb and MEGAPc, and which code for a protein having the capacity of being an GTPase activating protein.

Thus, the invention is directed to a nucleotide molecule comprising a nucleotide sequence coding for MEGAPa (SEQ ID NO: 1), MEGAPb (SEQ ID NO: 2) or MEGAPc (SEQ ID NO: 3) having activity as GTPase activating protein, or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID NO: 1 , 2 or 3, provided that:

a) said variants hybridize under moderately stringent conditions to a nucleic acid which codes for a protein of SEQ ID NO: 1, 2 or 3, and b) said variants code for a protein with activity as GTPase activating protein.

According to a preferred embodiment, the above mentioned nucleotide molecule comprises a nucleotide sequence coding for amino acids 426-939 of MEGAPa (SEQ ID NO: 1), amino acids 426-915 of MEGAPb (SEQ ID NO: 2) or amino acids 1-488 of MEGAPc (SEQ ID NO: 3) or variants thereof.

The above nucleotide sequences encode for a protein product that is preferably capable of enhancing the GTPase activity of Racl or Cdc42Hs, as it is outlined in Example 7 of this application.

As defined above, "variants" are according to the invention especially such nucleic acids or nucleotide molecule, which contain one or more substitutions, insertions and/or deletions when compared to the nucleic acids of SEQ ID No. 1, 2 or 3. These lack preferably one, but also 2, 3, 4, or more nucleotides 5' or 3' or within the nucleic acid sequence, or these nucleotides are replaced by others.

The nucleic acid sequences of the present invention also comprise such nucleic acids which contain sequences in essence equivalent to the nucleic acids coding for a protein as described in SEQ ID No. 1, 2 or 3. According to the invention nucleic acids can show for example at least about 60%, preferably at least about 75%, more typically at least about 85% or more than about 90% sequence identity to the nucleic acids coding for a protein as described in SEQ ID No. 1, 2 or 3.

The nucleic acid variants according to the invention also comprise nucleic acid fragments which contain more than 10, preferably more than 15, more than 20, more than 25 or more than 30 and up to 50 nucleotides. The term oligonucleotide includes fragments containing 10 to 50 nucleotides and parts thereof. These sequences can be in any order as long as at least 10 successive nucleotides are according to the invention. These oligonucleotides can be preferably used as primer, for example for RT-PCR or as a probe for in situ hybridization, or as a capture molecule in a solid phase assay including hybridization- based assays, such as GeneChip type assay systems (GeneChip is a trademark of Affymetrix Inc).

According to the state of the art an expert can test which possible variations derived from these revealed nucleic acid sequences according to the invention are partially or are not appropriate for specific applications like hybridization and PCR assays. The nucleic acids and oligonucleotides of the inventions can also be part of longer DNA or RNA sequences, e.g. flanked by restriction enzyme sites.

Amplification and detection methods are according to the state of the art. The methods are described in detail in protocol books which are known to the expert. Such books are for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and all subsequent editions. PCR-methods are described for example in Newton, PCR, BIOS Scientific Publishers Limited, 1994 and all subsequent editions. In particular, nucleotid variants are contemplated, which encode those variants of the proteins of the invention, which comprise for example deletions, insertions and/or substitutions as compared to SEQ ED NO: 1, 2 or 3, which cause for so-called "silent" changes. Those variants are considered to be part of the invention.

For example, such changes in the nucleic acid sequence are considered to cause a substitution with an equivalent amino acid. Preferably are such amino acid substitutions the result of substitutions which substitute one amino acid with a similar amino acid with similar structural and/or chemical properties, i.e. conservative amino acid substitutions.

Amino acid substitutions can be performed on the basis of similarity in polarity, charges, solubility, hydrophobic, hydrophilic, and/or amphipathic (amphiphil) nature of the involved residues. Examples for hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar, neutral amino acids include glycine, serine, threonine, cysteine, thyrosine, asparagine and glutamine. Positively (basic) charged amino acids include arginine, lysine and histidine. And negatively charged amino acids include aspartic acid and glutamic acid.

"Insertions" or "deletions" usually range from one to five amino acids. The allowed degree of variation can be experimentally determined via methodically applied insertions, deletions or substitutions of amino acids in a polypeptide molecule using recombinant DNA methods. The resulting variants can be tested for their biological activity.

Nucleotide changes, which affect the N-terminal and C-terminal part of the protein or polypeptide, often do not change the protein activity, because these parts are often not involved in the biological activity. It can be desired to eliminate one or more of the cysteins of the sequence, since cysteines can cause the unwanted formation of multimers when the protein is produced recombinant. Multimers may complicate purification procedures. Each of the suggested modifications is in range of the current state of the art, and under the retention of the biological activity of the encoded products.

According to a further embodiment, the nucleic acids of the present invention comprise transcriptional products of one of the above nucleic acids, e.g. mRNA, as well as nucleic acids, which selectively hybridize to said transcriptional products of the nucleic acids under moderate stringent conditions.

The terms "nucleotide molecule" or "nucleic acid sequence" refer to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms "nucleic acid", "nucleotide molecule" and "polynucleotide" are used interchangeably herein to refer to a heteropolymer of nucleotides.

The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes to the complement of the disclosed nucleotide sequences under moderately stringent or stringent hybridization conditions; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the herein disclosed proteins; or a polynucleotide that encodes a polypeptide comprising an additional specific domain or truncation of the disclosed proteins.

Stringency of hybridization, as used herein, refers to conditions under which polynucleotide duplexes are stable. As known to those of skill in the art, the stability of duplex is a function of sodium ion concentration and temperature (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2^nd Ed. (Cold Spring Harbor Laboratory, (1989)). Stringency levels used to hybridize can be readily varied by those of skill in the art.

The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5 x Denhart's solution, 6 x SSPE, 0.2% SDS at 42°C, followed by washing in 1 x SSPE, 0.2% SDS, at 50°C. Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers.

As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the DNA; with greater than about 90% identity to said DNA being especially prefeired. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50%) formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2 x SSPE, 0.2% SDS, at 65°C.

The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable duplex in 0.018M NaCl at 65°C. (i.e., if a duplex is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1 x SSPE, and 0.1% SDS at 65°C.

Further, nucleic acid hybridization techniques can be used to identify and obtain a nucleic acid within the scope of the invention. Briefly, any nucleic acid having some homology to a sequence set forth in this invention, or fragment thereof, can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Such similar nucleic acid then can be isolated, sequenced, and analyzed to determine whether they are within the scope of the invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a radioisotope such as ³²P, an enzyme, digoxygenin, or by biotinylation.

The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, 2^nd edition, Cold Spring Harbor Laboratory, Plainview, NY.

Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in this invention can be used to identify a nucleic acid identical to or similar to a nucleic acid sequence set forth in the group of nucleic acids of the present invention. In addition, probes longer or shorter than 20 nucleotides can be used. Any cell containing a nucleic acid within the scope of the invention is itself within the scope of the invention. This includes, without limitation, prokaryotic and eukaryotic cells. It is noted that cells containing a nucleic acid of the invention are not required to express the nucleic acid. In addition, the nucleic acid can be integrated into the genome of the cell or maintained in an- episomal state. In other words, cells can be stably or transiently transfected with a nucleic acid of the invention.

Generally, the term "nucleic acid" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e. g., chemically synthesized) DNA.

The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, the nucleic acid can be circular or linear.

In a further embodiment, the present invention includes the invention of a vector (construct) comprising a nucleic acid (nucleotide molecule) according to the invention. This vector is preferably an expression vector which contains a nucleic acid according to the invention and one or more regulatory nucleic acid sequences.

Numerous vectors are known to be appropriate for the transformation of bacterial cells, for example plasmids and bacteriophages, like the phage λ, and are frequently used as vectors for bacterial hosts. Viral vectors can be used in mammalian and insect cells to express exogenous DNA fragments, e.g. SN 40 and poly oma virus.

The transformation of the host cell can be done alternatively directly using "naked DΝA" without the use of a vector.

According to a preferred embodiment, the expression vector comprises a nucleic acid sequence as defined herein and, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers.

Preferably, the vector is a plasmid or an adenovirus vector. The proteins according to the invention can be produced either in eukaryotic or prokaryotic host cells. Examples for eukaryotic cells include mammalian, plant, insect and fungus (e.g. yeast) cells. Appropriate prokaryotic cells include bacterial cells such as Escherichia coli and Bacillus subtilis. E. coli derived BL21, XL-1 Blue, Tg-1 cells are preferred.

Preferred mammalian host cells are CHO, COS, HeLa, 293T, HEH or BHK cells.

Alternatively, the polypeptides according to the invention can be produced in transgenic plants (e.g. potatoes, tobacco) or in transgenic animals, for example in transgenic goats or sheep.

According to a preferred embodiment, said cell is an insect cell and said vector is a baculovirus-derived vector, the insect cell more preferably being SF9, or Ffl5.

According to a further aspect, the present invention is directed to a protein product which is encoded by a nucleotide as defined hereinabove.

According to a still further aspect, the invention includes a process for the production of a protein molecule as defined herein, comprising the steps of

(i) inserting a nucleotide molecule as defined herein into an expression vector comprising, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers,

(ii) introducing said vector into a host cell as disclosed above,

(iii) culturing said cell under conditions allowing for expression of said peptide or enzyme to occur within said cell, and

(iv) isolating said protein molecule from said cell, from the supernatant, or from a cell compartment, within or without the plasma membrane thereof.

According to a preferred embodiment, the vector comprises a second coding region for a second protein molecule which is fused to the coding region of the protein molecule of the invention, i.e. a MEGAP protein or a variant thereof. Said second coding region preferably codes for a signal peptide, which signal peptide preferably causes the protein or peptide which it is fused to to be transported into the periplasmatic space of the host cell.

According to a preferred embodiment, said second coding region codes for a peptide for which a specific binding partner exists, wherein said specific binding partner preferably is an antibody.

In the above process, preferably said vector is pGEX, wherein said second coding regions code for Glutathion-S-transf erase, and the process of isolating the peptide comprises the use of GST-affinity chromatography and release of the GTPase activity containing protein molecule by thrombin cleavage.

Alternatively, in the above process, said vector preferably is pGEX, wherein said second coding regions codes for a Histidine-tag, and the process of isolating the peptide comprises the use of Histidine affinity chromatography.

In a further embodiment, the present invention includes a molecule capable of binding specifically to an epitope of a MEGAP protein or variant thereof as defined hereinbefore. According to a preferred embodiment, this molecule is an antibody or antigen-binding part thereof, or an aptamer.

The above MEGAP -binding compounds can further be small molecules, recombinant phages, or peptides. Suitable molecules are e.g., anticalins, described in EP1017814. Said European patent also describes the process of preparing such anticalins with the ability to bind a specific target. Further suitable molecules are Trinectins (Phylos Inc., Lexington, Massachusetts, USA, and Xu et al., Chem. Biol. 9:933, 2002). Another kind of suitable molecule are affybodies (see Hansson et al., Immunotechnology 4(3-4):237-52, 1999, and Henning et al., Hum Gene Ther. 13(12): 1427-39, 2002, and references therein).

Also included in the scope of the invention are antisense nucleic acids, aptamers, siRNA molecules, RNAi molecules, and the like molecules known to the person of skill in the art. These molecules may be used to diminish or abolish the transcription of the MEGAP gene and/or the expression of MEGAP. Such diminishing or abolishing may be useful in situations where increased MEGAP activity is not desired, or where it is desired to abolish MEGAP activity entirely, for example for experimental purposes.

Alternatively, the present invention provides aptamers, which are directed against MEGAP of the present invention. Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms.

The antibody is preferably selected from a group, which consists of polyclonal antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies and synthetic antibodies. The antibody according to the invention can be additionally linked to a toxic and/or a detectable agent.

The term "antibody", is used herein for intact antibodies as well as antibody fragments, which have a certain ability to selectively bind to an epitop (antigen-binding part). Such fragments include, without limitations, Fab, F(ab')₂ und Fv antibody fragment. The term "epitop" means any antigen determinant of an antigen, to which the paratop of an antibody can bind. Epitop determinants usually consist of chemically active surface groups of molecules (e.g. amino acid or sugar residues) and usually display a three-dimensional structure as well as specific physical properties.

The antibodies according to the invention can be produced according to any known procedure. For example the pure complete protein according to the invention or a part of it can be produced and used as immunogen, to immunize an animal and to produce specific antibodies.

The production of polyclonal antibodies is commonly known. Detailed protocols can be found for example in Green et al, Production of Polyclonal Antisera, in Immimochemical Protocols (Manson, editor), pages 1 - 5 (Humana Press 1992) und Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols In Immunology, section 2.4.1 (1992). In addition, the expert is familiar with several techniques regarding the purification and concentration of polyclonal antibodies, as well as of monoclonal antibodies (Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

The production of monoclonal antibodies is as well commonly known. Examples include the hybridoma method (Kohler and Milstein, 1975, Nature, 256:495-497, Coligan et al, section 2.5.1 - 2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988).), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBN-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

In brief, monoclonal antibodies can be attained by injecting a mixture which contains the protein according to the invention into mice. The antibody production in the mice is checked via a serum probe. In the case of a sufficient antibody titer, the mouse is sacrificed and the spleen is removed to isolate B-cells. The B cells are fused with myeloma cells resulting in hybridomas. The hybridomas are cloned and the clones are analyzed. Positive clones which contain a monoclonal antibody against the protein are selected and the antibodies are isolated from the hybridoma cultures. There are many well established techniques to isolate and purify monoclonal .antibodies. Such techniques include affinity chromatography with protein A sepharose, size-exclusion chromatography and ion exchange chromatography. Also see for example, Coligan et al., section 2.7.1 - 2.7.12 and section „Immunglobulin G (IgG)", in Methods In Molecular Biology, volume 10, pages 79 - 104 (Humana Press 1992).

According to a still further embodiment, the invention as hereinabove described provides a hybridoma cell line which produces a monoclonal antibody which specifically binds to MEGAP protein according to the invention.

According to a further aspect, the present invention is directed to a nucleotide molecule suitable as a primer in a primer extension reaction comprising from about 11 to about 100 nucleotides of a nucleotide molecule as defined herein for use in the detection of the presence in a sample of mRΝA coding for a MEGAP protein as defined herein, or for use in the detection of the presence in a sample of the MEGAP gene, or for use in the detection in a sample of the disruption of the MEGAP gene. According to preferred embodiment, the nucleotide primer is suitable for the detection of a 3p-breakpoint.

According to a further aspect, the present invention provides an immunogenic peptide comprising at least 9 contiguous amino acids of the sequence of the protein molecule of the invention for use in the detection of the MEGAP protein molecule as defined herein.

A diagnostic kit according to the invention comprises the antibody as defined herein or the immunogenic peptide (see above), or the above disclosed nucleotide primer for the detection of MEGAPa, MEGAPb or MEGAPc in a sample of a subject.

This diagnostic kit is preferably suitable for the detection of impaired neuronal function in an individual, preferably a human being. The impaired neuronal function preferably is the detection of impaired neuronal migration, impaired ability to form and/or elongate axons and/or neurons, in a directed or non-directed manner, impaired ability to form contacts and/or synapses with other neurons.

Alternatively, the diagnostic kit may find application for use in the detection of involvement of MEGAPa, MEGPb or MEGAPc in spinal cord injury, stroke, Alzheimer's, and 3p- syndrome.

According to a further aspect, a pharmaceutical composition is provided, which contains a MEGAP protein or variant thereof, a nucleotide molecule or a vector, as defined herein, and a pharmaceutically acceptable carrier and/or diluent.

The pharmaceutical composition may be used in the treatment of a disorder or condition associated with impaired neuronal function, for example impaired migration, impaired ability to form and/or elongate axons and/or neurons, in a directed or non-directed manner.

The impaired function may be an impaired ability to form contacts and/or synapses with other neurons. The disorder or condition treated further may be selected from spinal cord injury, stroke, Alzheimer's, and 3p- syndrome. The active components of the present invention, i.e. proteins, nucleic acids etc. are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

The term "pharmaceutical acceptable" is defined as non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve a synergistic effects or to minimize adverse or unwanted effects.

Techniques for the formulation or preparation and application/medication of compounds of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. A therapeutically effective dose relates to the amount of a compound which is sufficient to improve the symptoms, for example a treatment, healing, prevention or improvement of such conditions. An appropriate application can include for example oral, dermal, rectal, transmucosal or intestinal application and parenteral application, including intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal injections. The intravenous injection is the preferred treatment of a patient.

A typical composition for an intravenous infusion can be produced such that it contains 250 ml sterile Ringer solution and for example 10 mg MEGAP protein. See also Remington's Pharmaceutical Science (15. edition, Mack Publishing Company, Easton, Ps., 1980).

An amount which is adequate to reach the aforesaid effect is defined as "therapeutically effective dose". Amounts, which are effective for these applications, depend on the severity of the condition and the general condition of the patient and his immune system. However, the dose range is usually between 0.01 and 100 mg protein per dose with a dose of 0.1 to 50 mg and from 1 to 10 mg per patient. Single or multiple applications after a daily, weekly or monthly treatment regimen can be performed with application rate and samples chosen by the physician in charge.

A method for the treatment of impaired neuronal migration, impaired ability to form and/or elongate axons and/or neurons, in a directed or non-directed manner, impaired ability to form contacts and/or synapses with other neurons, spinal cord injury, stroke, Alzheimer's, and 3p- syndrome, according to the invention comprises administering to a subject in need thereof a therapeutically effective amount of nucleic acid, a vector, a protein molecule or a pharmaceutical composition as defined herein.

Preferably, the vector is an adenovirus vector, which more preferably is administered together with agents that facilitate DNA uptake by cells.

According to a further aspect, the invention provides a transgenic animal where the gene for MEGAP has been modified so as to ablate the expression of functional MEGAP gene products therefrom.

Further, the invention is directed to a nucleotide molecule comprising from about nucleotide number 134881 to about nucleotide number 134981 of the nucleotide sequence of SEQ ID NO:4, or a sequence wherein one or more nucleotides have been exchanged, deleted or inserted.

Preferred nucleotide sequences hereof are:

from about nucleotide number 134781 to about 134981 of SEQ ID NO:4.

from about nucleotide number 134681 to about 134981 of SEQ ID NO:4.

from about nucleotide number 134581 to about 134981 of SEQ ID NO:4.

from about nucleotide number 134481 to about 134981 of SEQ ID NO:4. from about nucleotide number 134381 to about 134981 of SEQ ID NO:4.

from about nucleotide number 134281 to about 134981 of SEQ IDNO:4.

from about nucleotide number 134181 to about 134981 of SEQ ID NO:4.

from about nucleotide number 133681 to about 134981 of SEQ ID NO:4.

from about nucleotide number 133181 to about 134981 of SEQ ID NO:4.

from about nucleotide number 132681 to about 134981 of SEQ _DNO:4.

from about nucleotide number 132181 to about 134981 of SEQ ID NO:4.

from about nucleotide number 131681 to about 134981 of SEQ ID NO:4.

from about nucleotide number 131181 to about 134981 of SEQ ID NO:4.

from about nucleotide number 130681 to about 134981 of SEQ ID NO:4.

from about nucleotide number 130021 to about 134981 of SEQ _D NO:4.

According to a further aspect, the invention provides a vector comprising one of the nucleotide sequences disclosed above.

The invention is further directed to a promoter capable of driving the expression of a gene specifically in brain and kidney, comprising a nucleotide sequence as defined hereinabove.

The invention further provides a vector capable of providing expression of a nucleotide sequence inserted therein, comprising 5' to said nucleotide sequence the nucleotide sequence of said promoter and a composition comprising said vector. The composition may further comprise agents that facilitate the uptake of DNA by cells. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The invention is now further illustrated by the accompanying drawings:

Fig. 1 shows the genomic organisation of the MEGAP gene on chromosome 3p25 and localisation of the translocation breakpoints in the patient.

Fig. 1 a, Physical mapping of MEGAP.

Fig. 1 b, Schematic drawing of the breaking event.

Fig. 2 shows the peptide sequence of MEGAP and multiple alignment of the rhoGAP domain.

Fig. 2 a, Amino acid sequence of the isoforai MEGAPa containing the GAP domain, the SH3 and the FEPJCIP4 (FCH) domain.

Fig. 2 b, Multiple alignment of GAP domains in MEGAP and other human rhoGAP containing proteins.

Fig. 3 shows expression analysis of MEGAP.

Fig. 3 a, Multiple tissue northern blots from adult and fetal tissues (Clontech).

Fig. 3 b, Schematic drawing of exons and motifs of MEGAP. The position of the start (ATG) and stop (TGA) codon are indicated.

Fig. 3 c, Expression of MEGAP in different brain regions.

Fig. 4 shows alternative splicing of exon 12.

Fig. 4 a, Alternative splicing forms in brain and kidney of fetal and adult origin.

Fig. 4 b, Predicted peptide sequences of exons 11 to 13 of isoforms MEGAPa, b and c.

Fig. 5: shows distribution of Megap mRNA in adult mouse brain Fig. 6 shows Filter binding assay showing GAP activity of MEGAP towards Racl and Cdc42Hs, but no activity towards RhoA.

Fig. 7. The sequence shows the promotor seqence of MEGAP S'upstream of exon 1.

Fig. 8. cDNA sequences of the isoforms of human MEGAPa,b,c genes.

Fig. 9.. Fluorescence in situ hybridisation of BAC RP11-203C04 and BAC RP11-334L22 on metaphase spreads from the patient carrying the balanced translocation (upper panel) (case report) and patient 150500 with 3p^" syndrome (lower panel). The BAC clones were labeled with Biotin using the standard nick-translation procedure. Biotin was detected using FITC conjugated Streptavidin. Chromosomes were counterstained with DAPI. Upper panel: BAC RPl 1-203 C04 shows signals on the normal chromosome 3 and both derivative chromosomes, indicating its localisation at the translocation breakpoint. Lower panel: BAC RPl 1-334L22 shows only one signal on the normal chromosome 3 on metaphase spreads of patient 150500, indicating loss of one MEGAP copy.

Fig.10 shows the sequence of both derivative chromosomes in the vicinity of the breakpoint and the expression of MEGAP in different regions of human brain.

Fig. 10 a. Sequence of both derivative chromosomes in the vicinity of the breakpoint. Bold: chromosome 3 specific sequence. Normal style: chromosome X specific sequence. Underlined: LTR sequence. Italic type: CAA repeats. Numbers at the beginning and the end correspond to the base positions in the reference sequence from clones RPl 1- 203C04 (AC066583) and RP3-339A18 (Z97054), respectively.

Fig. 10 b. Expression of MEGAP in different brain regions. Northerns were hybridised with two different probes from the 3'-UTR (upper panel: bp 5396-5596, lower panel: bp 7414-7715). Notice the lack oftranscri.pt II and HI in the lower panel and the loss of transcript III in the upper panel. Examples:

Materials and Methods:

Case Report

The proband is a 16 year old female, the second child of healthy non-consanguineous parents (father, 47 years; mother, 45 years). Her brother of 26 years is not affected; there are no further affected individuals in this family. Pregnancy was reported to be uneventful. Delivery occurred at 40 weeks of gestation by breech presentation. Birth weight was 3720 g, length 55 cm and head cfrcumference 35 cm. Her height is 163 cm (25.pc) and head circumference 55 cm (75.pc). Mental and motor developmental delay was evident in the early stages of life. She could stand freely at the age of 4 years and only walk at the age of 5.5 years. Clinical findings at the age of 16 years include an extremely psychomotoric retardation with an atactic gait, jerky arm movements, very low mental performance, periodical auto-aggressive behaviour and absence of speech. She shows a triangular face with a high nasal bridge, micrognathia, short philtrum, high and narrow palate, slender hands, muscle hypotonia and scoliosis. In the last three years three epileptic seizures occurred. The patient presents severe attention deficits; IQ testing was therefore not possible. Magnetic resonance imagery scan or further imaging diagnostics have been rejected by the parents.

Case 150500 is a 3 year old male, diagnosed with psychomotor retardation and epicanthus. Cytogenetically, a terminal deletion of chromosome 3p was diagnosed, with the breakpoint residing in band 3p25. Cases PI - P10 have been previously described (10, 12). Case Pl l is an adult without intellectual impairment, who has a cytogenetically visible deletion of chromosome 3p and a normal phenotype.

Genomic clones and deletion mapping

YAC clones from Xpll.2 and 3p25 were chosen from the Whitehead radiation hybrid maps WCX.l l and WC3.1 and purchased from the german Resource Center (RZPD). PAC and BAC clones were obtained from the Resource Center, Oakland, (RPCI 1, 3, 11) or from Research Genetics, Huntsville (CIT-B, -C). Cosmids were purchased from the RZPD after screening the LL03NC01 'AC chromosome 3 and LL0XNC01 'U' chromosome X specific human cosmid libraries (Lawrence Livermore National Laboratory, Livermore, CA). Overlapping clones were confirmed by PCR and hybridisation analysis. Fluorescence in situ hybridisation (FISH) was performed as described elsewhere. LOH analysis was performed as described by Green et al. (12).

RACE analysis

Human total RNA of adult brain (Invitrogen) was reverse transcribed using the gene specific primer Ex5.1 (5'-GAGCAGGTTCATGCTGAGGT-3') (SEQ ID NO: 5) and Superscript II reverse transcriptase (Gibco BRL). After second strand synthesis, the cDNA was ligated with the 2R adapter (Gibco BRL) and subsequently amplified with adapter primer 2R (Gibco BRL) and nested gene specific primer Ex5nl (5'- TCATGCTGAGGTCTCCTGACT-3') (SEQ ID NO: 6). Specific amplification was achieved using a second round of nested PCR using the nested 2R primer (Gibco BRL) and primer Ex4n2 (5'-CTCATTGGTCACCTTCAGGA-3') (SEQ ID NO:7). The PCR product was cloned into pCR2.1 vector using the TOPO TA cloning kit (Invitrogen) and sequenced. Putative exons were aligned with the genomic sequence and confirmed by RT- PCR analysis (Fig. 7)

Expression studies

Human multiple tissue northern blots were purchased from Clontech Laboratories (USA). RT-PCR generated fragments covering exons 1-4 (bp 27-569) or a β-Actin cDNA probe were labeled with α-[³²P]-dCTP (Amersham Pharmacia) using a random primed labeling protocol. The probes were hybridised to the northerns over night at 65°C as recommended by the manufacturer, washed with 2x SSC and 0.2x SSC at 65°C and exposed for 24 hours at -80°C (Actin: 2-3 hours at room temperature).

In situ hybridisation

Three months old male B6 mice were perfused with 4% paraformaldehyde (PFA) in PBS. After dissection the brains were postfixed over night in 4% PFA and sectioned in 50 micron slices with the vibratome (Leica NT 1000S). Sense and antisense riboprobes for Megap prepared by in vitro transcription of cDΝA encoding for exons 1-19 of the murine homolog were labeled with DIG-11-UTP by T3 or T7 polymerases (Roche). Floating sections were hybridised over night at 65°C with the DIG-labeled riboprobes and visualized by alkaline phosphatase conjugated anti-DIG antibody. Alternative splicing analysis

Total human RNA from different tissues was reverse transcribed with an oligo-dT primer using Superscript II RT (Gibco BRL). PCR amplification of exons 9 to 14 was carried out in an Eppendorff Gradient Cycler with primer Ex9for (5'- GCCACCATGCAGACATTACA-3') (SEQ ID NO: 8) and Exl4rev (5'- ACTCTGAAGATCCCCTGCTG-3') (SEQ ID NO: 9) with an initial denaturation step of 94°C for 2 minutes, 33 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final 3 minutes extension step at 72°C. PCR products were analysed on an agarose gel, reeluted from the gel and cloned into pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen). Clones were sequenced and compared with the published genomic sequence.

GAP assay

MEGAPb-GST fusion protein was generated by subcloning a Sail fragment of KIAA0411 (residues 1-1468, corresponding to bp 1377-2849 of MEGAPb), containing the GAP and SH3 domain, into the pGEX4T3 vector (Amersham Pharmacia). Isoform a was generated by replacement of the Psyl / Eco81I fragment (bases 1323-1813). After transfection into E.coli BL21, protein expression was induced with 1 mM IPTG. Recombinant proteins were purified using Glutathione beads in NETN-buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 1 mM DTT) and washed with 20 mM Tris-HCl, 0.1 mM DTT. Vectors containing the full-length coding sequence for the three small GTPases RhoA, Racl and Cdc42Hs were obtained from the Guthrie cDNA resource center and subcloned into pQE30 vectors (Qiagen). Proteins were prepared under native conditions as recommended, but including 5 mM MgCl₂ to each of the buffers (35). Purified proteins were dialysed over night against 10 mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 0.1 mM DTT. Protein concentrations were determined by SDS-Page and BCA-assay (Pierce).

GAP assays were performed as described (36). Briefly, 0.3 μM of purified and dialysed GTPases were incubated for 10 min at 30°C with 10 μCi γ-[³ P] GTP (Amersham Pharmacia) in 20 μl of 20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5 mM EDTA and 0.1 mM DTT. The reaction was terminated by adding 5 μl MgCl₂ (0.1 M) on ice. Three microliters of this reaction were incubated at 30°C with GST-tagged MEGAP or ARHGAP1-GST protein. Five microliters were removed after 0, 3, 6, 9, 12 and 15 min and mixed with 500 μl of icecold Stop-buffer. The samples were filtered through BA85 nitrocellulose membrane (Schleicher&Schuell) and washed with 10 ml icecold Stop-buffer (50 mM Tris- HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl₂). The amount of radioactivity bound to each of the GTPases was analysed in a szintillation counter.

Example 1: Physical mapping and characterisation of the translocation breakpoints

Cytogenetic analysis of a female patient with severe mental retardation revealed a de novo balanced translocation t(X;3)(pl l.2;p25). To characterise the breakpoints on both derivative chromosomes, we performed fluorescence in situ hybridisation (FISH) (Fig. 9) on the patient's metaphase chromosomes. Using YAC, PAC/BAC and cosmid clones we could narrow down the breakpoint region to an interval of approximately 40 kb (Fig. l ).

It shows the physical mapping of MEGAP. The precise order of markers for the chromosomal regions 3p25 and Xpl 1.2 is shown at the top and the bottom, as well as the orientation of telomere and centromere. BAC, PAC and cosmid clones used for FISH mapping of the respective breakpoints are shown. Both BAC RP11-203C04 and RP5- 1036D20 showed signals on the normal and the derivative chromosome, thus containing the translocation breakpoint. The localisation of the 22 exons of MEGAP is indicated as vertical red lines. The entire gene encompasses approximately 250 kb of genomic sequence, with exon 1 separated from exon 2 by about 100 kb. The breakpoint on the derivative chromosome 3 is located between exons 3 and 4 of MEGAP, as shown in the middle part of the scheme. Exons are indicated here as red boxes. Markers D3S2405 and SHGC-57635 (white boxes) are . located within intronic sequences of MEGAP. Marker IB772 (white box) resides on the X chromosome.

Subsequent Southern and PCR analysis showed aberrant products in the patients DNA samples but not in controls (data not shown). Sequence comparison of the cloned junction fragments confirmed that the translocation is balanced.

The X chromosomal breakpoint is situated within a truncated HERN element, about 12kb from the next LTR element (Fig. lb).

The breakpoint on the X chromosome is located in a remnant human endogenous retroviral element (HERV), next to the flanking long terminal repeat (LTR) sequence. The breakpoint region on chromosome 3 shows several CAA trinucleotid repeats on both sides of the translocation. The position of primers used for amplification of the junction fragments are indicated as arrows.

We analysed several hundred kilobases around the breakpoint and excluded this region as causative for the observed phenotype. Analysis of chromosome 3 specific sequences at the breakpoint identified two partially overlapping cDNA clones, KIAA0411 and KIAA1156, derived from adult human brain (Kazusa Institute, Japan). Alignment of these cDNAs to the genomic sequence revealed that both clones reside distal to the breakpoint.

Example 2: Isolation of a novel gene on chromosome 3p25

RT-PCR with primers corresponding to the KIAA clones indicated that both cDNAs, overlapping by 162 bp, are derived from a single transcriptional unit. To identify the 5' end and the start codon of the transcript, we performed 5'-RACE. The resulting PCR product was sequenced and compared to the genomic sequence. We identified three additional putative exons, all located proximal to the breakpoint (Fig. la). Thus, this transcript bridges the breakpoint on chromosome 3 of the patient. Alignment of the cDNA clone KIAA04il to the genomic sequence established that it contains exons 9 to 22, whereas KIAA1156 contains exons 4 to 11. Just recently, database searches revealed another unnamed sequence (AX056907) that corresponds to one of the isoforms (see below) that we isolated, however lacked the 5' and 3' untranslated regions of the gene.

The longest (8327 bp) mRNA encoded by this gene has a putative start codon in the first exon 103 bp downstream of the consensus cDNA sequence. A stop codon in exon 22 defines an open reading frame of 3299 bp which encodes a putative protein of 1099 aa (Fig. 2a).

The blue shaded region shows the GAP domain. The green shaded region shows the SH3 domain and the yellow shaded region shows the FER/CIP4 (FCH) domain.

Thus, the mRNA contains a long 3'-UTR of about 5 kb, encoded by a large part of the last exon.

Example 3: Screening of 3p^" patients

Recent studies (10, 12, 14) indicated that patients carrying terminal deletions of chromosome 3p are associated with mental retardation and further multiple recurring symptoms depending on the size of their deletion. The deletion size is variable but generally encompass several Megabases of DNA, as shown by micros atellite analyses. To find out if MEGAP is deleted in these patients, we carried out a combined FISH / LOH study in 11 patients diagnosed with terminal deletions of chromosome 3p (Fig. 9; Patient 150500). Four cases (PI, P2, P5 and P7) were found deleted for D3S1597, a marker approximately 50 kilobases proximal to MEGAP (Fig. lα and Tab. 1). Cases P4 and P6 were not informative for D3S1597, but showed deletions in the more proximal marker D3S1038. In patients P3 (GM10922), P8 (GM10985) and P150500, we carried out fluorescence in situ hybridisations with BAC clones RP11-203C04 and RP11-334L22, which contain MEGAP. We could show, that in all three patients P3, P8 and 150500 both BACs presented one signal on the normal chromosome 3, but were deleted on the derivative chromosome 3 (Tab. 1). Most interestingly, we also examined a patient with a terminal deletion of chromosome 3p, but without diagnosed intellectual impairment. We could show that marker D3S1597 is still present on both alleles. Together, these data indicate that the loss of one MEGAP copy is associated with the mental handicap in 3p^" patients.

Example 4: MEGAP shares homologies with other RhoGAP proteins

Comparison of the predicted amino acid sequence with the SWISSPROT database revealed that the protein shows significant sequence similarity to srGAPl and 2, as well as ARHGAP4 (15,16). Compared to ARHGAP4, both proteins show 50% amino acid identity and 69% similarity. Strikingly, all sizes of the individual exons 1 to 19 are conserved. ARHGAP4 is an X-linked gene and has been shown to contain three functional domains. All three domains were also identified in the predicted peptide sequence of the novel gene and of srGAPl and srGAP2. A rhoGAP motif of 149 amino acids was identified between aa 520 and 670, sharing 30-40% identity with other known members of the family of Rho- GTPase activating proteins (RhoGAP) such as Oligophreninl, BCRl and n-Chimaerin (Fig. 2b).

Boxes of higher sequence conservation are indicated as described (37). Blue shaded boxes show amino acids conserved in all five proteins. Red shaded boxes show identical amino acids in four of the proteins. Green shaded boxes schow amino acids conserved in three of five proteins. The arrow shows the conserved arginine residue in box 1 (16). The rhoGAP motif contains three boxes of higher sequence conservation. A conserved arginine residue in box 1 (position 542) has been shown to be particularly important for efficient domain function (17). Besides the rhoGAP domain, an SH3 and a FER-CIP4 (FCH) domain were identified at the C- and N-terminal end, respectively (Fig. 3b).

The schematic drawing of exons and motifs of MEGAP shows the position of the start (ATG) and stop (TGA) codon. Introns are depicted as black lines and are not drawn to scale. Grey boxes show 5' and 3'-UTR. Yellow boxes show the FEPJCIP4 (FCH) domain. Blue boxes show the rhoGAP domain. The green box shows the SH3 domain. Black bars indicate the localisation of probes used for hybridisation of the northerns. Orange shaded bars show the localisation of putative polyA tails in the 3'-UTR.

Because of its relation to other members of the rhoGAP protein family we termed the gene MEGAP.

Example 5: Expression analysis

Since MEGAP was identified in a patient with mental and motor deficits, we investigated the MEGAP mRNA expression in whole brain and in specific brain regions, as well as in a series of other adult tissues. Figure 3α shows that MEGAP is predominantly and highly expressed in adult and fetal brain, with low expression detectable in kidney tissue and very low expression in other tissues.

Multiple tissue northern blots from adult and fetal tissues (Clontech) containing 1-2 μg polyA⁺ RNA per lane, were hybridised with α-[³²P]-dCTP labeled probes containing exons 1 to 4 of MEGAP (bp 27-569). The filters were hybridised over night at 65°C, stringently washed at 65°C and exposed to X-ray films for 24 hours at -80°C. A control hybridisation with β-actin shows signals at 2.0 kb in all lanes, with additional bands at 1.6-1.8 kb in heart and skeletal muscle as described (38).

In the adult brain, the MEGAP gene is expressed in all tested regions including cerebellum, cortex, occipital pole, frontal and temporal lobe, amygdala, hippocampus, substantia nigra and thalamus. Besides the predicted 8.3 kb full length transcript, two additional isoforms II and UI of about 7 and 4.5 kb were observed. The 4.5 kb fragment was not detected using a probe derived from position 5396-5596 of the gene. Using a probe from position 7414- 7715, the 4.5 and the 7 kb transcript were not detected (Fig. 3c).

Hybridisation of exons 1-4 results in three signals (I, π, III). The longest band (I) corresponds to the predicted 8.3 kb full-length transcript. Signals at 7 kb (II) and 4.5 kb (UI) represent transcripts with differing 3'-UTRs.

These results strongly suggest that the three different transcript sizes I, II and HI are due to different polyadenylation sites in the 3 -UTR.

To further investigate the temporal and spatial expression pattern of MEGAP, we isolated its mouse ortholog. We used RT-PCR with mouse brain cDNA to clone and sequence the entire open reading frame of the murine homolog. The mouse Megap open reading frame shows 92% nucleotide homology with the human cDNA; the predicted proteins share 97% identity (data not shown). These data were confirmed by a partial database entry (AF338472), which corresponds to the human isoform a but lacks the 5 '-part of the sequence. We then performed a series of RNA in situ hybridisations on sections of the adult mouse brain. Megap shows a distinct expression pattern with the highest level of expression seen throughout the neurons of the hippocampus and specific parts of the cortex and olfactory bulb (Fig. 5a, c).

In situ hybridisation with digoxigenin labelled Megap antisense riboprobe on coronar (a) and sagital sections (c) showing strong expression in the olfactory bulb, amygdala, piriform cortex, posterior parietal associative area, the granular layer of the cerebellum and particularly strong expression in the hippocampal formation. Hybridisation with Megap sense DIG-RNA (Fig. 5b) gave no signal. (Abbrevations: A, amygdala; Cx, Cortex; DG, dentate gyrus; Gl, granular layer; Hi, hippocampus; OB, olfactory bulb; Pir, piriform cortex; PPta, posterior parietal associative area.

The high expression in these brain structures strongly suggests that MEGAP may play a role in processes underlying synaptic plasticity, higher cognitive function and learning and memory. Example 6: Multiple MEGAP isoforms are generated by alternative splicing

Besides the different transcript sizes detected by northern analysis, RT-PCR analysis indicates that MEGAP mRNA contains three alternatively spliced forms (Fig. 4α).

Alternative splicing forms were found in brain and kidney of fetal and adult origin. 33 cycles of RT-PCR were performed with oligo-dT transcribed cDNAs using primers derived from exons 9 and 14. PCR products at 482, 412 and 383 bp correspond to isoforms a, b and c, respectively. Isoforms a and b of MEGAP differ in the use of an alternative exon 12. The presence of exon 12b leads to a transcript 72 bp shorter than isoform MEGAPa, leading to an open reading frame of 3227 bp and coding for a predicted protein of 1075 amino acids. Isoform MEGAPc lacks exons 12, leading to a frame shift and a premature stop as indicated.

These isoforms differ in the use of exon 12. The largest and most abundant transcript MEGAPa comprises exon 12α (Fig. 2). The smaller exon 12b is present in both KIAA cDNA clones and in AX056907. In isoform MEGAPc, exon 12 is skipped completely, leading to a frame shift and a premature stop nine amino acids after the splice acceptor site, just prior to the rhoGAP domain (Fig. 4b).

Predicted peptide sequences of exons 11 to 13 of isoforms MEGAPa, b and c is shown. The asteriks indicate a stop codon due to the frame shift in exon 13.

Interestingly, this truncated form can be detected in all analysed tissues by RT-PCR (e.g. adult kidney), but is absent in whole brain and in all analysed brain subregions (data not shown). Among the investigated tissues, the MEGAPa isoform seems to be the most prominent transcript as determined by RT-PCR (data not shown). Taken into account the different isoforms I - IE and a - c, nine different transcripts and three protein isofoims of MEGAP may exist.

Example 7: MEGAP enhances the GTPase-activity of Racl and Cdc42Hs, but not of RhoA substrates.

Prior analysis of proteins containing a rhoGAP domain has revealed that some are substrate specific while others offer a broad spectrum of reactivity. To test the ability of MEGAP to enhance the GTPase activity of RhoA, Racl and Cdc42Hs in vitro, we performed a GAP assay using recombinant proteins expressed in Escherichia coli. GST- bound MEGAP isoform a (amino acid residues 426-939) and isoform b (amino acid residues 426-915) were incubated with γ-[³²P]-GTP preloaded GTPases at 30°C and the amount of GTP hydrolysis was measured in a filter binding assay. As control, we used GST-tagged ARHGAPl (residues 198-439 aa), which was previously shown to enhance the hydrolysis rate of Cdc42Hs, RhoA and Racl (18).

As shown in Figure 6, MEGAP strongly activates Racl GTPase function and to a lower extent Cdc42Hs. In contrast, MEGAP shows no activity towards RhoA (Fig. 6c), while ARHGAPl is able to efficiently enhance its intrinsic GTPase activity. No difference in activity was detectable between the two isoforms α and b, suggesting that the alternative use of exon 12α or b does not influence its activity in the in vitro assay.

The Filter binding assay shows GAP activity of MEGAP towards Racl and Cdc42Hs. As described, ARHGAPl shows strong activity towards RhoA, Racl and Cdc42Hs. MEGAP however shows almost no activity towards RhoA, a strong activity towards Racl and a lower, but still significant activity towards Cdc42Hs. 0.3 μM of γ-[³²P]-GTP labeled ρ21 were incubated at 30°C in the absence or presence of equimolar concentrations of the GAP domains of MEGAP (amino acid residues 426-939) or ARHGAPl (amino acid residues 198-438). Aliquots of the reaction were taken eveiy three minutes and immediately passed through a 0.45 μm nitrocellulose membrane. The amount of radioactivity bound to the filter was measured in a szintillation counter. Activity at t=0 minutes was set to 100%>.

Example 8: MEGAP-inducible Neuroblastoma cell lines

For a further characterization of MEGAP, in the inventors 's laboratory, inducible cell lines have been developed based on the Tet-on system, in which the expression of MEGAPa and b constructs can be induced by addition of doxycycline. As cell lines, human neuroblastoma line SHSY-5Y and murine neuroblastoma cell line Neuro2a have been used. The cell lines were transfacted with MEGAP constructs within vector pcDNA4/TO/mycHis (Invitrogen) and with a vector coding for a tetracycline repressor and were selectioned by means of blasticidin/zeocine. After several weeks of selection single clones where picked up and were tested for an inducible expression of MEGAP. Microarray Hybridisation

Starting from the stable MEGAP-inducible neuroblastoma cell line SHSY-5Y studies were performed to show, how the expression pattern of a cell was altered by expression of MEGAP. RNA of MEGAP induced and not-induced SHSY-5Y cells were hybridisized on a microarray with about 30.000 transcripts and quantitative differences in the expression pattern between both cell lines were measured.

Example 9: Preparation of a polyclonal antibody against MEGAP

Polyclonal antibodies were developed against three different epitopes of MEGAP. The epitopes are located in the N-terminal region of MEGAP (antibody MEGAP5: epitope: N- RHEDRPQRRSSVK-C; (SEQ ID NO: 8)) and in the C-terminus (MEGAP3: epitope: N- MRSTCGSTRHSSLG -C (SEQ ID NO:9) und MEGAP19: N- HELRELERQNTVKQ -C (SEQ ID NO: 10)).

Three rabbits were infected against these immunogenes each and antibodies were obtained following 180 days. The antibodies were subjected to affinity purification and tested. Antibody MEGAP5 and MEGAP 19 recognize proteins having a size of about 160kDa in non-transfected HEK293 or Neuro2a cells. Additionally, antibody MEGAP5 recognizes a protein having a size of about lOOkDa. The size of about 160 kDa, which is about 30 kDa more than the calculated size of MEGAP indicates that MEGAP was post-translationally modified. The protein having about lOOkDa, which is only recognized by the N-terminal antibody MEGAP5 seems to be a truncated protein without C-terminus.

Interaction MEGAP - WANE1

Recently, it could be shown that MEGAP interacts with a further protein, WANE1, via its SH3 domain (Soderling et al, Nat Cell Biol. 2002 Dec;4(12):970-5). WAVE1 in turn binds to an Arp2/3 complex, which is responsible for the nucleation of actine monomers to the actine cytoskeleton. The activation of these complexes is dependent on Racl activity, which in turn is regulated by MEGAP. Moreover, it was shown that mice having a knockout of the WAVE1 gene show symptoms which are similar to those of the patient having the MEGAP involving X;3 translocation and of patients having 3p-syndrome (Soderling et al. Proc Natl Acad Sci USA. 2003 Feb 18;100(4):1723-8). These include an impaired locomotion, reduced learning ability, a reduced memory ability as well as dwarfism. This underlines the importance of MEGAP/WAVEl complexes in connection with cognitive and mental handicaps.

The invention is preferably directed to the following embodiments:

1. A nucleotide molecule comprising a nucleotide sequence essentially coding for amino acids 426-939 of MEGAPa or for the amino acid sequence 426-939 of MEGAPa wherein one or more amino acids have been exchanged, inserted or deleted.

2. A nucleotide sequence of emb. 1 which encodes for a protein product that is capable of enhancing the GTPase activity of Racl.

3. A nucleotide sequence of emb. 1 which encodes for a protein product that is capable of enhancing the GTPase activity of Cdc42Hs.

4. A nucleotide sequence of any one of emb.s 2-3 which encodes a protein product that essentially fails to enhance the GTPase-activity of a RhoA substrate.

5. A nucleotide sequence of any one of emb.s 1-4, selected from the group of molecules comprising the sequence of MEGAPa, sequences essentially identical to MEGAPa, and/or MEGAPa sequences wherein one or more nucleotides are exchanged, deleted or inserted.

6. A nucleotide molecule according to any one of the preceding emb.s which is DNA.

7. A nucleotide molecule according to any one of the preceding emb.s which is RNA.

8. A vector comprising the nucleotide molecule of one of the preceding emb.s .

9. A protein product encoded by the MEGAPa part of the nucleotide of any one of the preceding emb.s.

10. A nucleotide molecule comprising a nucleotide sequence essentially coding for amino acids 1-939 of MEGAPa, or for amino acids 1-915 of MEGAPb, or for one of the said amino acid sequences wherein one or more amino acids have been exchanged, inserted or deleted, or a nucleotide molecule of any one of emb.s 1-8, or a protein product of emb. 9, or a protein molecule consisting essentially of amino acids 1-939 of MEGAPa, or essentially of amino acids 1-915 of MEGAPb, or of amino acids 1-939 of MEGAPa or of amino acids 1-915 of MEGAPb, wherein one or more amino acids have been exchanged, inserted or deleted, for use in the treatment of a disorder or condition associated with impaired neuronal function.

11. The nucleotide or protein molecule of emb. 10 wherein the protein product encoded by the nucleotide molecule or the protein molecule is capable of binding to other proteins via its SH3 domain. 12. The nucleotide or protein molecule of emb.s 10 or 11 wherein the protein product encoded by the nucleotide molecule or the protein molecule comprises a GTPase activity.

13. The nucleotide or protein molecule of emb.s 10 11, or 12 wherein the protein product encoded by the nucleotide molecule or the protein molecule is capable of enhancing the GTPase activity of Racl.

14. The nucleotide or protein molecule of emb.s 10, 11, 12 or 13 wherein the protein product encoded by the nucleotide molecule or the protein molecule is capable of enhancing the GTPase activity of Cdc42Hs.

15. The nucleotide or protein molecule of emb.s 10, 11, 12 13 or 14 wherein the protein product encoded by the nucleotide molecule or the protein molecule essentially fails to enhance the GTPase-activity of a RhoA substrate.

16. The nucleotide or protein molecule of any one of emb.s 10-15 wherein the impaired function is impaired migration.

17. The nucleotide or protein molecule any one of emb.s 10-16 wherein the impaired function is impaired ability to form and/or elongate axons and/or neurons, in a directed or non-directed manner.

18. The nucleotide or protein molecule any one of emb.s 10-17 wherein the impaired function is impaired ability to form contacts and or synapses with other neurons.

19. The nucleotide or protein molecule of any one of emb.s 10 to 18 wherein the disorder or condition is selected from spinal cord injury, stroke, Alzheimer's, and 3p- syndrome.

20. A process for the production of a protein molecule according to emb. 9 or of a protein molecule consisting essentially of amino acids 1-939 of MEGAPa, or of amino acids 1- 939 of MEGAPa wherein one or more amino acids have been exchanged, inserted or deleted, comprising the steps of

(i) inserting a nucleotide molecule according to any one of emb.s 1-18 or 10-19 or a nucleotide molecule coding for the protein molecule of any one of emb.s 9-19, into an expression vector comprising, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers,

(ii) introducing said vector into a cell,

(iii) culturing said cell under conditions allowing for expression of said peptide or enzyme to occur within said cell, (iv) isolating said protein molecule from said cell, from the supernatant, or from a cell compartment, within or without the plasma membrane thereof.

21. The process of emb. 20, wherein the cell is derived from a bacteria, insect, fungus, animal, or plant.

22. The process of emb. 21 wherein said cell is derived from yeast.

23. The process of emb. 21 wherein said cell is an insect cell and said vector is a baculovirus-derived vector.

24. The process of emb. 23 wherein said cell is SF9, or HI5.

25. The process of emb. 21 wherein said cell is derived from bacteria.

26. The process of emb. 25 wherein said cell is derived from E.coli.

27. The process of emb. 26 wherein the cell is a BL21 cell.

28. The process of any one of emb.s 20 to 27 wherein said vector comprises a second coding region for a second protein molecule which is fused to the coding region of the protein molecule of emb.s 20 to 27.

29. The process of emb. 28, wherein said second coding region codes for a signal peptide.

30. The process of emb. 29, wherein said signal peptide causes the protein or peptide which it is fused to to be transported into the periplasmatic space of the host cell.

31. The process of any one of emb.s 20 to 30, wherein said second coding region codes for a peptide for which a specific binding partner exists.

32. The process of emb. 31, wherein said specific binding partner is an antibody.

33. The process of emb. 31, wherein said vector is pGEX, wherein said second coding regions codes for Glutathion-S-transf erase, and the process of isolating the peptide comprises the use of GST-affinity chromatography and release of the GTPase activity containing protein molecule by thrombin cleavage.

34. The process of emb. 31 wherein said vector is pGEX, wherein said second coding regions codes for a Histidine-tag, and the process of isolating the peptide comprises the use of Histidine affinity chromatography.

35. An antibody against the protein molecule of any one of emb.s 9-19, for use in the detection of the protein molecule of any one of emb.s 9-19. 36. An immunogenic peptide comprising at least 9 contiguous amino acids of the sequence of the protein molecule of any one of emb. s 9- 19, for use in the detection of the protein molecule of any one of emb.s 9-19.

37. A nucleotide molecule suitable as a primer in a primer extension reaction comprising from about 11 to about 100 nucleotides of a nucleotide molecule of any one of emb.s 1- 8 or 10-19, for use in the detection of the presence in a sample of mRNA coding for a protein of any one of emb.s 1-19, or for use in the detection of the presence in a sample of the MEGAP gene, or for use in the detection in a sample of the disruption of the MEGAP gene.

38. The nucleotide primer of emb. 37, suitable for the detection of a 3p- Breakpoint.

39. A diagnostic kit comprising the antibody of emb. 35 or the immunogenic peptide of emb. 36, or the nucleotide primer of emb. 37, for the detection of MEGAPa or MEGAPb in a sample of a subject.

40. A diagnostic kit for the detection of impaired neuronal function, comprising the antibody of emb. 35, or the immunogenic peptide of emb. 36, or the nucleotide primer of emb. 37.

41. The diagnostic kit of emb. 40, for use on the detection of impaired neuronal migration, impaired ability to form and/or elongate axons and or neurons, in a directed or non- directed manner, impaired ability to form contacts and/or synapses with other neurons.

42. The diagnostic kit of emb. 40, for use on the detection of involvement of MEGAPa or MEGPb in spinal cord injury, stroke, Alzheimer's, and 3p- syndrome.

43. A vector capable of directing in a host cell the production of a protein molecule according to emb. 9 or of a protein molecule consisting essentially of amino acids 1- 939 of MEGAPa, or of amino acids 1-939 of MEGAPa wherein one or more amino acids have been exchanged, inserted or deleted, comprising a nucleotide molecule according to any one of emb.s 1-18 or 10-19 or a nucleotide molecule coding for the protein molecule of any one of emb.s 9-19, within an expression vector comprising, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers.

44. A method for the treatment of impaired neuronal migration, impaired ability to form and/or elongate axons and or neurons, in a directed or non-directed manner, impaired ability to form contacts and or synapses with other neurons, spinal cord injury, stroke, Alzheimer's, and 3p- syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of nucleic acid coding for a protein molecule of any one of emb.s 9-19.

45. The method of emb. 44 wherein the nucleic acid molecule is comprised within the vector of emb. 43.

46. The method of emb. 45 wherein the vector is an adenovirus vector.

47. The method of emb. 45 wherein the vector is administered together with agents that facilitate DNA uptake by cells.

48. A transgenic animal where the gene for MEGAP has been modified so as to ablate the expression of functional MEGAP gene products therefrom.

49. A host cell containing anucleotide molecule of any one of emb.s 1-8 or 10-19.

50. The host cell of emb. 49 containing a vector of emb. 43.

51. The host cell of emb. 50 which is a bacterial cell.

52. The host cell of emb. 51 which is derived fromE. coli.

53. The host cell of emb. 52 which is BL21, XL-1 Blue, Tg-1.

54. A pharmaceutical composition comprising as active ingredient a vector of emb. 43, and or a protein molecule of any one of emb.s 9-19, for use in the treatment of a disorder or condition associated with impaired neuronal function.

55. The pharmaceutical composition according to emb. 54, wherein the impaired function is impaired migration.

56. The pharmaceutical composition according to emb. 54, wherein the impaired function is impaired ability to form and or elongate axons and/or neurons, in a directed or non- directed manner.

57. The pharmaceutical composition according to emb. 54, wherein the impaired function is impaired ability to form contacts and/or synapses with other neurons.

58. The pharmaceutical composition according to emb. 54, wherein the disorder or condition is selected from spinal cord injury, stroke, Alzheimer's, and 3ρ- syndrome.

59. A nucleotide molecule comprising from about nucleotide number 134881 to about nucleotide number 134981 of the nucleotide sequence shown in Fig. 7, or a sequence wherein one or more nucleotides have been exchanged, deleted or inserted.

60. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134781 to about 134981 of the sequence shown in Fig. 7.

61. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134681 to about 134981 of the sequence shown in Fig. 7. 62. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134581 to about 134981 of the sequence shown in Fig. 7.

63. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134481 to about 134981 of the sequence shown in Fig. 7.

64. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134381 to about 134981 of the sequence shown in Fig. 7.

65. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134281 to about 134981 of the sequence shown in Fig. 7.

66. The nucleotide sequence of emb. 59, comprising from about nucleotide number 134181 to about 134981 of the sequence shown in Fig. 7.

67. The nucleotide sequence of emb. 59, comprising from about nucleotide number 133681 to about 134981 of the sequence shown in Fig. 7.

68. The nucleotide sequence of emb. 59, comprising from about nucleotide number 133181 to about 134981 of the sequence shown in Fig. 7.

69. The nucleotide sequence of emb. 59, comprising from about nucleotide number 132681 to about 134981 of the sequence shown in Fig. 7.

70. The nucleotide sequence of emb. 59, comprising from about nucleotide number 132181 to about 134981 of the sequence shown in Fig. 7.

71. The nucleotide sequence of emb. 59, comprising from about nucleotide number 131681 to about 134981 of the sequence shown in Fig. 7.

72. The nucleotide sequence of emb. 59, comprising from about nucleotide number 131181 to about 134981 of the sequence shown in Fig. 7.

73. The nucleotide sequence of emb. 59, comprising from about nucleotide number 130681 to about 134981 of the sequence shown in Fig. 7.

74. The nucleotide sequence of emb. 59, comprising from about nucleotide number 130021 to about 134981 of the sequence shown in Fig. 7.

75. A vector comprising the nucleotide sequence of any one of emb.s 59-74.

76. A promoter capable of driving the expression of a gene specifically in brain and kidney, comprising a nucleotide sequence of any one of emb.s 59-74.

77. A vector capable of providing expression of a nucleotide sequence inserted therein, comprising 5' to said nucleotide sequence the nucleotide sequence of the promoter of emb. 76.

78. A composition comprising the vector of emb. 79. A composition of emb. 78, comprising agents that facilitate the uptake of DNA by cells.

References

1. Chelly, J. (1999) Hum. Mol. Genet. 8, 1833-1838.

2. Chelly, J. (2000) Am. J. Med. Genet. 94, 364-366.

3. Threadgill, R., Bobb, K. and Ghosh, A. (1997) Neuron 19, 625-634.

4. Luo, L. (2000) Nat. Rev. Neurosci. 1, 173-180.

5. Ramakers, G.J.A. (2000) Am. J. Med. Genet. 94, 367-371.

6. Kutsche, K., Yntema, H., Brandt, A., Jantke, I, Nothwang, H.G., Orth, U, Boavida, M.G., David, D., Chelly, J., Fr ns, IP. et al. (2000) Nat. Genet. 26, 247-250.

7. Bienvenu, T., Der-Sarkissian, H., Billuart, P., Tissot, M., Des Portes, N, Brills, T., Chabrolle, J.P., Chauveau, P., Cherry, M., Kahn, A. et al. (1997) Eur. J. Hum. Genet. 5, 105-109.

8. Billuart, P., Bienvenu, T., Ronce, Ν, des Portes, N., Ninet, M.C., Zemni, R., Crollius, H.R., Carrie, A., Fauchereau, F., Cherry, M. et al. (1998) Nature 392, 823-826.

9. Νarahara, K., Kikkawa, K., Murakami, M., Hiramoto, K., Νamba, H., Tsuji, K., Yokoyama, Y. and Kimoto, H. (1990) Am. J. Med. Genet. 35, 269-273.

10. Phipps, M.E. Latif, F., Prowse, A., Payne, S.J., Dietz-Band, J., Leversha, M., Affara, Ν.A., Moore, A.T., Tolmie, J., Schinzel, A. et al. (1994) Hum. Mol. Genet. 3, 903-908.

11. Drumheller, T., MacGillivray, B.C., Behmer, D., MacLeod, P. McFadden, D.E., Roberson, J., Venditti, C, Chorney, K., Chorney, M. and Smith, D.I. (1996) J. Med. Genet. 33, 842-847.

12. Green, E.K., Priestley, M.D., Waters, J., Maliszewska, C, Latif, F. and Maher, E.R. (2000) J. Med. Genet. 37, 581-587.

13. Nerjaal, M. and De Νef, I (1978) Am. J. Dis. Child. 132, 43-45.

14. Mowrey, P.Ν., Chorney, M.J., Venditti, C.P., Latif, F., Modi, W.S., Lerman, M.I., Zbar, B., Robins, D.B., Rogan, P.K. and Ladda, R.L. (1993) Am. J. Med. Genet. 46, 623-629.

15. Wong, K., Ren, X.R., Huang, Y.Z., Xie, Y., Liu, G., Saito, H., Tang, H, Wen, L., Brady-Kalnay, S.M., Mei, L., Wu, J.Y., Xiong, W.C., Rao, Y. (2001) Cell 107, 209- 221.

16. Tribioli, C. Droetto, S., Bione, S., Cesareni, G., Torrisi, M.R., Lotti, L.N., Lanfrancone, L., Toniolo, D. and Pelicci, P. (1996) Proc. Natl. Acad. Sci. USA 93, 695-699.

17. Graham, D.L., Eccleston, J.F. and Lowe, P.Ν. (1999) Biochemistry 38, 985-991.

18. Lamarche-Nane, Ν. and Hall, A. (1998) J. Biol. Chem. 273, 29172-29177. 19. Couvert, P., Bienvenu, T., Aquaviva, C, Poirier, K., Moraine, C, Gendrot, C, Nerloes, A., Andres, C, Le Fevre, A.C., Souville, I. et al. (2001) Hum. Mol. Genet. 10, 941-946.

20. Merienne, K., Jacquot, S., Pannetier, S., Zeniou, M., Bankier, A., Gesz, J., Mandel, J.L., Mulley, J., Sassone-Corsi, P. and Hanauer, A. (1999) Nat. Genet. 22, 13-14.

21. Gecz, J., Barnett, S., Liu, J., Hollway, G., Donnelly, A., Eyre, H, Eshkevari, H.S., Baltazar, R., Grunn, A., Νagaraja, R et al. (1999) Genomics 62, 356-368.

22. Chelly, J. and Mandel, J.L. (2001) N t. Rev. Genet., 2, 669-680.

23. Gέcz, J. and Mulley, J. (2001) GenomeRes. 10, 157-163.

24. Sotgia, F., Minetti, C. andLisanti, M.P. (1999) FEBSLett. 3, 177-180.

25. Angeloni, D., Lindor, Ν.M., Pack, S., Latif, F., Wie, M.H. and Lerman, M.I. (1999) Am. J. Med. Genet. 86, 482-485.

26. Higgins, J.J., Rosen, D.R., Loveless, J.M., Clyman, J.C. and Grau, M.J. (2000) Neurology 55, 335-340.

27. Decker, C.J. and Parker, R. (1995) Curr. Opin. CellBiol. 7, 386-392.

28. Billuart, P., Chelly, J., Carrie, A., Ninet, M.C., Couvert, P., McDonell, Ν., Zemni, R., Kahn, A., Moraine, C, Beldjord, C. et al. (2000) Ann. Genet. 43, 5-9.

29. D'Adamo, P., Menegon, A., Lo Νigro, C, Grasso, M., Gulisano, M., Tamanini, F., Bienvenu, T., Gedeon, A.K., Oostra, B., Wu, S.K. et al. (1998) Nat. Genet. 19, 134- 139.

30. Carrie, A., Jun, L., Bienvenu, T., Ninet, M.C., McDonell, Ν., Couvert, P., Zemni, R, Cardona, A., Nan Buggenhout, G., Frints, S. et al. (1999) Nat. Genet. 23, 25-31.

31. Antonarakis, S. E. and Nan Aelst, L. (1998) Nat. Genet. 19, 106-108.

32. Brose, K., Bland, K.S., Wang, K.H., A nott, D., Henzel, W., Goodman, C.S., Tessier- Lavigne, M., Kidd, T. (1999) Cell 96, 795-806.

33. Wu, W., Wong, K., Chen, J.H., Jiang, Z.H., Dupuis, S., Wu, J.Y., Rao, Y. (1999) Nature 400, 331-336.

34. Zemni, R Bienvenu, T., Ninet, M.C., Sefiani, A., Carrie, A., Billuart, P., McDonell, Ν., Couvert, P., Francis, F., Chafey, P. et al. (2000) Nat. Genet. 24, 167-170.

35. Self, A.J. and Hall, A. (1995) Methods Enzymol. 256, 3-10.

36. Richnau, Ν. and Aspensrrom, P. (2001) J. Biol Chem. 276, 35060-35070.

37. Lancaster, C.A., Taylor-Harris, P.M., Self, A.J., Brill, S., van Erp, HE. and Hall, A. (1994) J. Biol. Chem. 269, 1137-1142.

38. Pari, G., Jardine, K. andMcBumey, M.W. (1991) Mol. Cell. Biol. 11, 4796-4803.

Claims

1. A nucleotide molecule comprising a nucleotide sequence coding for MEGAPa (SEQ ID NO: 1), MEGAPb (SEQ ID NO: 2) or MEGAPc (SEQ ID NO: 3) having activity as GTPase activating protein, or variants thereof, wherein the variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the nucleotide molecule coding for the protein of SEQ ID NO: 1, 2 or 3, provided that:

2. The nucleotide molecule of claim 1, comprising a nucleotide sequence coding for amino acids 426-939 of MEGAPa (SEQ ID NO: 1), amino acids 426-915 of MEGAPb (SEQ ID NO:2) or amino acids 1-488 of MEGAPc (SEQ ID NO:3) or variants thereof.

3. The nucleotide sequence of claim 1 or 2, which codes for a protein product that is capable of enhancing the GTPase activity of Racl.

4. The nucleotide sequence of claim 1 or 2, which codes for a protein product that is capable of enhancing the GTPase activity of Cdc42Hs.

5. The nucleotide molecule according to any one of the preceding claims which is DNA.

6. The nucleotide molecule according to any one of the preceding claims which is RNA.

7. A vector comprising the nucleotide molecule of one of the preceding claims.

8. An expression vector, which comprises the nucleic acid sequence of any of claims 1-6 and one or more regulatory sequences.

9. The expression vector, which comprises the nucleic acid sequence of any of claims 1-6 and, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers.

10. The vector of claim 7 - 9, which is a plasmid or an adenovirus vector.

11. A host, which has been transformed with the vector of any of claims 7-10.

12. The host cell of claim 11, wherein the cell is derived from a bacteria, insect, fungus, animal, or plant.

13. The host cell of claim 12, wherein said cell is derived from yeast.

14. The host cell of claim 12, wherein said cell is an insect cell and said vector is a baculovirus-derived vector.

15. The host cell of claim 14, wherein said cell is SF9, or HI5.

16. The host cell of claim 12 which is derived from E. coli.

17. The host cell of claim 16 which is BL21, XL- 1 Blue, Tg-l.

18. A protein product which is encoded by a nucleotide molecule of any one of claims 1-6.

19. A process for the production of a protein molecule according to claim 18, comprising the steps of

(i) inserting a nucleotide molecule according to any one of claims 1-6 into an expression vector comprising, in operable sequence, a promoter, optionally one or more enhancers, polyadenylation signals, terminators, origins of replication, and selectable markers,

(ii) introducing said vector into a host cell of one of claims 11-17,

(iii) culturing said cell under conditions allowing for expression of said peptide or enzyme to occur within said cell, and (iv) isolating said protein molecule from said cell, from the supernatant, or from a cell compartment, within or without the plasma membrane thereof.

20. The process of claim 19, wherein said vector comprises a second coding region for a second protein molecule which is fused to the coding region of the protein molecule of claim 18.

21. The process of claim 20, wherein said second coding region codes for a signal peptide.

22. The process of claim 21, wherein said signal peptide causes the protein or peptide which it is fused to to be transported into the periplasmatic space of the host cell.

23. The process of any one of claims 20 to 22, wherein said second coding region codes for a peptide for which a specific binding partner exists.

24. The process of claim 23, wherein said specific binding partner is an antibody.

25. The process of claim 23, wherein said vector is pGEX, wherein said second coding region codes for Glutathion-S-transferase, and the process of isolating the peptide comprises the use of GST-affinity chromatography and release of the GTPase activity containing protein molecule by thrombin cleavage.

26. The process of claim 23, wherein said vector is pGEX, wherein said second coding region codes for a Histidine-tag, and the process of isolating the peptide comprises the use of Histidine affinity chromatography.

27. A molecule capable of binding specifically to an epitope of a protein as defined in claim 18.

28. The molecule of claim 27 which is an antibody or antigen-binding part thereof, anticalin, trinectin, aptamer, or an affybody.

29. The antibody of claim 28, wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, and a synthetic antibody.

30. A hybridoma which produces a monoclonal antibody of claim 29.

31. A nucleotide molecule suitable as a primer in a primer extension reaction comprising from about 11 to about 100 nucleotides of a nucleotide molecule of any one of claims 1-6 for use in the detection of the presence in a sample of mRNA coding for a protein of claim, or for use in the detection of the presence in a sample of the MEGAP gene, or for use in the detection in a sample of the disruption of the MEGAP gene.

32. The nucleotide primer of claim 31 , suitable for the detection of a 3p- Breakpoint.

33. An immunogenic peptide comprising at least 9 contiguous amino acids of the sequence of the protein molecule of claim 18, for use in the detection of the protein molecule of claim 18.

34. A diagnostic kit comprising the antibody of claim 29 or the immunogenic peptide of claim 33, or the nucleotide primer of claim 31 or 32, for the detection of MEGAPa, MEGAPb or MEGAPc in a sample of a subject.

35. A diagnostic kit for the detection of impaired neuronal function, comprising the antibody of claim 29, or the immunogenic peptide of claim 33, or the nucleotide primer of claims 31 or 32.

36. The diagnostic kit of claim 35, for use in the detection of impaired neuronal migration, impaired ability to form and or elongate axons and/or neurons, in a directed or non- directed manner, impaired ability to form contacts and or synapses with other neurons.

37. The diagnostic kit of claim 35, for use in the detection of involvement of MEGAPa, MEGAPb or MEGAPc in spinal cord injury, stroke, Alzheimer's, and 3p- syndrome.

38. A pharmaceutical composition containing a protein of claim 18, a nucleotide of one of claims 1-6 or a vector of one of claims 7-10, and a pharmaceutically acceptable carrier and/or diluent.

39. The pharmaceutical composition of claim 38 for use in the treatment of a disorder or condition associated with impaired neuronal function.

40. The pharmaceutical composition of claim 39, wherein the impaired function is impaired migration.

41. The pharmaceutical composition according to claim 39, wherein the impaired function is impaired ability to form and/or elongate axons and/or neurons, in a directed or non- directed manner.

42. The pharmaceutical composition according to claim 39, wherein the impaired function is impaired ability to form contacts and/or synapses with other neurons.

43. The pharmaceutical composition according to claim 39, wherein the disorder or condition is selected from spinal cord injury, stroke, Alzheimer's, and 3p- syndrome.

44. A method for the treatment of impaired neuronal migration, impaired ability to form and/or elongate axons and/or neurons, in a directed or non-directed manner, impaired ability to form contacts and/or synapses with other neurons, spinal cord injury, stroke, Alzheimer's, and 3p- syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of nucleic acid of one of claims 1-6, a vector of one of claims 7-10, a protein molecule of claim 18 or a pharmaceutical composition of claim 38-43.

45. The method of claim 44 wherein the vector is an adenovirus vector.

46. The method of claim 44 wherein the vector is administered together with agents that facilitate DNA uptake by cells.

47. A transgenic animal where the gene for MEGAP as defined in one of claims 1-6 has been modified so as to ablate the expression of functional MEGAP gene products therefrom.

48. A nucleotide molecule comprising from about nucleotide number 134881 to about nucleotide number 134981 of the nucleotide sequence of SEQ ID NO:4, or a sequence wherein one or more nucleotides have been exchanged, deleted or inserted.

49. The nucleotide sequence of claim 48, comprising from about nucleotide number 134781 to about 134981 of SEQ IDNO:4.

50. The nucleotide sequence of claim 48, comprising from about nucleotide number 134681 to about 134981 of SEQ IDNO:4.

51. The nucleotide sequence of claim 48, comprising from about nucleotide number 134581 to about 134981 of SEQ IDNO:4.

52. The nucleotide sequence of claim 48, comprising from about nucleotide number 134481 to about 134981 of SEQ IDNO:4.

53. The nucleotide sequence of claim 48, comprising from about nucleotide number 134381 to about 134981 of SEQ IDNO:4.

54. The nucleotide sequence of claim 48, comprising from about nucleotide number 134281 to about 134981 of SEQ IDNO:4.

55. The nucleotide sequence of claim 48, comprising from about nucleotide number 134181 to about 134981 of SEQ IDNO:4.

56. The nucleotide sequence of claim 48, comprising from about nucleotide number 133681 to about 134981 of SEQ ID NO:4.

57. The nucleotide sequence of claim 48, comprising from about nucleotide number 133181 to about 134981 of SEQ ID O:4.

58. The nucleotide sequence of claim 48, comprising from about nucleotide number 132681 to about 134981 of SEQ IDNO:4.

59. The nucleotide sequence of claim 48, comprising from about nucleotide number 132181 to about 134981 of SEQ IDNO:4.

60. The nucleotide sequence of claim 48, comprising from about nucleotide number 131681 to about 134981 of SEQ IDNO:4.

61. The nucleotide sequence of claim 48, comprising from about nucleotide number 131181 to about 134981 ofSEQ IDNO:4.

62. The nucleotide sequence of claim 48, comprising from about nucleotide number 130681 to about 134981 of SEQ IDNO:4.

63. The nucleotide sequence of claim 48, comprising from about nucleotide number 130021 to about 134981 of SEQ IDNO:4.

64. A vector comprising the nucleotide sequence of any one of claims 48-63.

65. A promoter capable of driving the expression of a gene specifically in brain and kidney, comprising a nucleotide sequence of any one of claims 48-63.

66. A vector capable of providing expression of a nucleotide sequence inserted therein, comprising 5' to said nucleotide sequence the nucleotide sequence of the promoter of claim 65.

67. A composition comprising the vector of claim 66.

68. A composition of claim 69, comprising agents that facilitate the uptake of DNA by cells.

69. The monoclonal or polyclonal antibody of claim 29, which is directed against one of the epitopes of SEQ ID NO: 8, 9 or 10.