WO2007101991A1

WO2007101991A1 - Target for the enhancement qf cognitive function

Info

Publication number: WO2007101991A1
Application number: PCT/GB2007/000777
Authority: WO
Inventors: David Martin Hunt; Anthony Moore; Sanjay Mull Sisodiya; Pamela Thompson
Original assignee: Ucl Business Plc
Priority date: 2006-03-07
Filing date: 2007-03-07
Publication date: 2007-09-13
Also published as: GB0604611D0

Abstract

A method for identifying an agent suitable for use in the enhancement of cognition, which method comprises determining whether or not a test substance binds and/or modulates the Rab3A-interacting molecule (RJMSl) gene, thereby to determine whether or not the test substance is suitable for use in the enhancement of cognition.

Description

TARGET FOR THE ENHANCEMENT QF COGNITIVE FUNCTION

Field of the invention The invention relates to methods for the identification of agents useful in the enhancement of cognitive function. The invention also relates to the enhancement of cognitive function.

Background to the invention The existence of a genetic foundation for human intelligence and its variation between individuals is well-established, but the identities and roles of its constitutive genes remain elusive. The therapeutic possibilities deriving from discovery of any such genetic bases are potentially important: learning disabilities and cognitive decline are associated with reduced quality of life, increased morbidity and a shorter life-span. Disabling cognitive decline typifies neurodegenerative conditions that afflict increasingly ageing populations in developed countries: for example, the prevalence of Alzheimer's disease is expected to quadruple in the United States by 2047. Whilst knowledge of the pathophysiology and genetics of learning disability and cognitive decline grows, the genetic evolution and generation of human intelligence remains comparatively unknown and subject to intense research.

Recently, accelerated evolution of genes subserving neurodevelopment has been implicated as part of the molecular explanation of the advance of the human nervous system: many of the identified candidate genes regulate brain size and behaviour, and some encode proteins critical for synaptic function. At the whole organism level, numerous studies have linked inter-individual differences in intelligence with brain structure on imaging.

Summary of the invention

Genetic influences on human cognitive abilities are poorly understood: few mutations are known to directly impair cognition. Studying the brain structural and functional consequences of a mutation in RIMSl known to cause a retinal dystrophy surprisingly revealed significantly enhanced cognitive abilities segregating with the mutation and eye phenotype. Though no evidence was found for an effect of common variation in RIMSl on cognitive variation in a population cohort, or for RIMSl accelerated evolution in the primate lineage, this first human mutation shown directly to increase intelligence emphasises the role of the synapse in human cognitive abilities.

In accordance with the present invention, there is thus provided a method for identifying an agent suitable for use in the enhancement of cognition, which method comprises determining whether or not a test substance binds and/or modulates the Rab3 A-interacting molecule (RIMSl) gene, thereby to determine whether or not the test substance is suitable for use in the enhancement of cognition.

The invention also provides: a method for the preparation of a pharmaceutical composition, which method comprises:

(a) identifying an agent suitable for use in the enhancement of cognition by a method as set out above; and

(b) formulating the agent identified in (a) with a pharmaceutically acceptable carrier or diluent; a method for the enhancement of cognition in a host, which method comprises: (a) identifying an agent suitable for use in the enhancement of cognition by a method as set out above;

(b) formulating the inhibitor identified in (a) with a pharmaceutically acceptable carrier or diluent; and

(c) administering to the host a therapeutically effective amount of the pharmaceutical composition formulated in (b); use of an agent which is capable of binding to or modulating the activity of the RIMSl gene in the manufacture of a medicament for use in the enhancement of cognition; an agent which is capable of binding to or modulating the activity of the RIMSl gene for use in the enhancement of cognition; a pharmaceutical composition comprising an agent which is capable of binding to or modulating the activity of the RIMSl gene and a pharmaceutically acceptable carrier or diluent; a method for the enhancement of cognition in a host, which method comprises modulating the RIMSl gene in the host; an agent identified by a method of the invention; an agent identified by a method of the invention for use in the enhancement of cognition; use of an agent identified by a method of the invention in the manufacture of a medicament for use in the enhancement of cognition; a pharmaceutical composition comprising an agent identified by a method of the invention and a pharmaceutically acceptable carrier or diluent; a method for the enhancement of cognition in a host, which method comprises administering to the host a therapeutically effective amount of an agent identified by a method of the invention; and a method for predicting the level of cognitive function in a subject, which method comprises typing the RIMSl gene of the subject.

Brief description of the drawings Figure 1 shows the kindred studied. Filled symbols represent affected individuals, unfilled symbols unaffected individuals. Note that the kindred is an extension of that presented by Johnson et al. (2003) and Kelsell et al. (1998), reordered by age of generation II.

Figure 2 shows the distribution of VIQ measures in affected (top row of symbols in key) and unaffected (bottom row of symbols in key) members of the kindred.

Figure 3 shows serial reformatted Tl -weighted magnetic resonance images from subject 11:2, shown in (a) coronal and (b) axial planes. Note the widened CSF spaces around an area of cortical malformation, shown encircled in selected images. Detailed inspection in three-dimensions suggested the malformation was most likely an area of polymicrogyria. Figure 4 shows in situ hybridisation for Riml (a) and Pax6 (b) in adult mouse olfactory bulb, showing expression of both genes.

Brief description of the sequence listing SEQ ID NO: 1 sets out the nucleic acid sequence of the coding region of the human RIMSl gene.

SEQ ID NO: 2 sets out the amino acid sequence of the human RIMSl polypeptide.

Detailed description of the invention

Throughout this specification, the word "comprise", or variations such as "comprised" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the general knowledge in Australia or elsewhere.

In an effort to identify candidate genes influencing human brain development and function, the inventors have espoused a paradigm that evaluates brain structure and function in individuals known to have mutations in genes expressed in the eye and the brain, who have been ascertained by their (often dramatic) ocular phenotype, but in whom a neurological phenotype was not fully appreciated. In this way, roles have been demonstrated for the homeobox genes PAX6, PITX2, SOX2 and OTX2 in human brain development and function, including cognition and memory. RIMSl encodes a synaptic active zone protein (Rimlo;) necessary both for preserving normal probability of synaptic neurotransmitter release and for long-term presynaptic potentiation at brain synapses. Mice lacking Riml a protein show severely impaired learning and memory. The inventors examined a kindred with a dominantly-inherited cone-rod dystrophy due to a missense mutation (Arg844His) in RIMSl, leading to visual loss from the third decade onwards. In situ hybridisation confirmed RIMSl expression in foetal and adult human brain. Magnetic resonance imaging revealed bilateral posterior polymicrogyria in two affected individuals (see Figure 3).

Unexpectedly and remarkably, affected members in the kindred were shown to have enhanced cognitive function in contrast to their unaffected kin. In the context of the search for genetic factors underlying human behavioural genetic findings and contributing to cognitive differences between humans, the inventors also examined the potential role of RIMSl variation in modulating cognitive function in normal individuals, by seeking association between common RIMSl variation and cognitive variation in a large cohort of well-phenotyped normal individuals. The inventors also examined the evolution of RIMSl and its expression in human brain development.

Verbal intelligence quotients (VIQs) in affected individuals (Table 1) were all above average, ranging from the 75^th (high average ability) to the 99^th centile (superior ability): scores were lower in unaffected members. The inventors tested the significance of the association between affected status and every measured cognitive phenotype following a null hypothesis approach that accounts for the observed kindred structure (SI). The overall difference in phenotype values between affecteds and non-affecteds was significant (P=O.006; see SI). For differences in each cognitive phenotype, all except those for immediate story recall, delayed story recall and semantic fluency had P values less than 5%: vocabulary (P=O.014), digit span (P=0.020), similarities (P=0.039), phonemic fluency (P=0.050), cognitive estimates (P=0.012), immediate story recall (P=0.112), delayed story recall (P=0.087), immediate verbal learning (P=O.048), delayed verbal learning (P=O.042), semantic fluency (P=0.220) and Hayling test (P=0.010). As expected, given that the three subtests composing VIQ were all significant, when the inventors separately tested VIQ itself this was also significant (P=0.014). These measures are not all correlated when tested in a separate cohort of individuals with visual impairment due to PAX6 mutation: that uncorrelated measures differ significantly between affecteds and unaffecteds suggests that multiple facets of mental processes are being affected independently by the mutation. Despite their more severe and earlier-onset central visual impairment, the PAX6 affected individuals performed significantly less well than the RIMSl affected individuals (VIQ, Mann- Whitney PO.0005): therefore visual impairment alone does not - and would be unlikely to - explain the inventors' findings.

In this specification references to the Arg844His and Arg820His mutations should be understood to refer to the same mutation. The mutation is described in Johnson et al, Genomics 81, 304-314 (2003). In that document, a G to A point mutation was identified in the second position of what the authors referred to as codon 844. This numbering, however, assumes the presence of exon 3 of the rat and other indels in the rat and human sequences. SEQ ID NOs: 1 and 2, however, set out the human RIMSl sequence in the absence of the rat exon 3, such that the codon referred to at position 844 by Johnson et al. {supra) appears at position 820 in SEQ ID NOS: 1 and 2. The skilled person would readily understand therefore that Arg844His (in Johnson et al., supra) and Arg820His (in SEQ ID NOs: and 2) refer to the same mutation.

The present invention thus relates to the use of a modulator of the Rab3A- interacting molecule gene, RIMSl, in the enhancement of cognition and, also, to the use of the RIMSl gene as a target for identifying such modulators.

A modulator of the RIMSl gene may be a substance which reduces/attenuates/decreases or eliminates expression and/or activity of that gene or a polypeptide product thereof. A modulator of the RIMSl gene may alternatively be a substance which stimulates/potentiates/increases expression and/or activity of that gene or a polypeptide product thereof. References herein to "modulators of the RIMSl gene" should, therefore, be understood to encompass modulators which act at the level of transcription, translation and/or at the polypeptide level. References to "modulators of RIMSl" and "modulators of RIMSl" should, similarly, be understood to encompass modulators which act at the level of transcription, translation and/or at the polypeptide level.

The Arg844His/Arg820His mutation is thought to produce a gain-of function effect: knock-out mice for the RIMSl gene have learning and memory problems, suggesting a positive effect is likely to be gain-of-function. Thus, modulators which are stimulators/activators of the RIMSl gene are preferred for use in the invention.

However, RIMSl is a multidomain protein and inhibitory effects on one or more domains of the RIMSl protein may lead to an enhancement of cognition. Consequently, modulators which are inhibitors of the RIMSl gene also fall within the scope of the invention.

Expression of the RIMSl gene in this context is used to refer to any of the steps of transcription and translation. Activity of a polypeptide product of the RIMSl gene in this context is used to refer to the ability of such a polypeptide to act as an enzyme and/or the ability of the polypeptide to bind other polypeptides. Thus, a modulator of the RIMSl gene may also be a substance which reduces/attenuates/decreases or eliminates or stimulates/potentiates/increases RIMSl activity by modulating the ability of RIMSl to interact with one or more of its targets and/or substrates.

Such a modulator may disrupt the ability of RDVISl to interact with one or more of its targets and/or substrates by, for example, breaking down the target and/or substrate, by binding to the target and/or substrate such that RIMSl is unable to bind to it or by binding to RIMSl so that it cannot bind to the target and/or substrate. Alternatively, a suitable modulator potentiate the increase the ability of RIMSl to interact with one or more of its targets and/or substrates by, for example, by binding to RDVISl in such a way that the affinity of RIMS 1 for a target/substrate is increased, may increase the availability of a target/substrate for binding by RIMS 1 or may increase the amount of a RDVISl target/substrate. Thus, a modulator suitable for use in the invention may exert its effect(s) via any mechanism. Any suitable modulator of expression and/or activity of the RIMSl gene may be employed in the present invention.

Typically, a suitable modulator will be capable of specifically modulating

RIMSl. Specific modulators of RIMSl are modulators which modulator RIMSl to a substantially greater degree than any other gene/gene product, in particular other

Rab3a-interacting molecule genes/gene products.

In particular, a specific modulator will modulate RIMSl to a degree of about

2 to 1000 times, for example about 10 to 500 times, in particular about 50 to 100 times that to which it inhibits any other gene/gene product, such as a Rab3a- interacting molecule genes/gene product. A specific modulator may be one which modulates RIMSl, but which substantially does not inhibit any other Rab3a- interacting molecule gene/gene product or indeed any other gene/gene product. Generally, a modulator suitable for use in the invention is one which is capable of modulating a mammalian RIMSl, in particular a human RIMSl. The amino acid sequence of human RlMSl is set out in SEQ ID NO: 2. The coding sequence of human RIMSl is set out in SEQ ID NO: 1. The Genbank accession number for the human RIMSl gene is NM_014989.

A modulator of the expression of RIMSl may act by binding directly to the promoter of the gene, thus modulating the initiation of transcription. Alternatively, a modulator could bind to a polypeptide/polypeptide complex which is associated with the promoter and is required for transcription. This may result in altered levels of transcription.

A modulator may alter expression of RIMS 1 by binding directly to the untranslated region of the RIMSl mRNA. This may alter the initiation of translation. Alternatively, a modulator may bind to a polypeptide/polypeptide complex associated with the untranslated region and alter the way in which that polypeptide/polypeptide complex associates with the untranslated region.

A modulator of RIMSl may act by binding to the RIMSl gene product and inhibiting/stimulating any enzymatic activity in that way. Such modulation may be reversible or irreversible. An irreversible modulator dissociates very slowly from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or non-covalently. Reversible modulation, in contrast with irreversible modulation, is characterised by a rapid dissociation of the enzyme-modulator complex.

A modulator which inhibits/stimulates REvIS 1 activity may be a competitive modulator, hi competitive modulation, the enzyme can bind substrate (forming an enzyme-substrate complex) or modulator (enzyme-modulator complex) but not both. Many competitive inhibitors resemble the substrate and bind the active site of the enzyme. The substrate is therefore prevented from binding to the same active site. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. The modulator may be a non-competitive inhibitor. In non-competitive modulation, which is also reversible, the modulator and substrate can bind simultaneously to an enzyme molecule. This means that their binding sites do not overlap. A non-competitive modulator acts by increasing or decreasing the turnover number of an enzyme rather than by diminishing or increasing the proportion of enzyme molecules that are bound to substrate.

The modulator can be a mixed modulator. Mixed modulation occurs when a modulator affects both the binding of substrate and alters the turnover number of the enzyme.

A modulator of the RIMSl gene may act by binding to its substrate or to one of more the polypeptides with which it forms a complex. The substance may itself catalyze a reaction of the substrate or one or more polypeptides with which RIMSl forms a complex, so that the substrate or other polypeptide(s) is not available to RIMSl. Alternatively, the modulator may simply prevent the substrate or one or more polypeptides with which RDVISl forms a complex binding to RIMSl (by binding either with RDVISl or with the substrate other polypeptide). A modulator may also act to increase the availability of a substrate RJMS1 binding polypeptide or increase the amount of such a substrate/RHVISl binding polypeptide.

The human RIMSl gene contains a number of different protein domains. There is an N-terminal Rab3 A-GTP binding site, followed by a zinc-finger domain that contains a pair of Cys₄ zinc fingers, and a PDZ domain, a domain frequently found in synaptic proteins. The two C-terminal C₂ domains (C₂A and C₂B) are separated by a SH3-binding domain. As demonstrated in the rat, both C₂ domains mediate binding of RHVISl to a number of other synaptic proteins.

The proposed role for RIMSl in neurotransmitter release was based initially on its interaction with Rab3 A, a protein known to regulate synaptic vesicle exocytosis by limiting the extent of Ca²⁺-triggered membrane fusion. The process of exocytosis involves the targeting of docking synaptic vesicles containing neurotransmitter to the presynaptic plasma membrane, priming of these vesicles to make them fusion-competent, and the subsequent fusion of vesicles with the presynaptic membrane in response to a Ca²⁺ signal. The fusion of synaptic vesicles to the presynaptic membrane requires the formation of a highly stable core or SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex composed of synaptobrevin/VAMP (vesicle-associated membrane protein) on the synaptic vesicle and SNAP-25 (synaptosomal-associated protein of 25kDa) and syntaxin on the presynaptic membrane. These three SNAREs form a four-helix bundle that is sufficient to mediate fusion of lipid bilayers in vitro. At synapses, full zippering of this helical bundle is thought to be blocked until a Ca²⁺ signal is sensed by synaptotagmin, a synaptic vesicle-associated Ca²⁺ sensor. The function of RlMSl in this process is that it interacts through the Rab3 A-GTP binding site and the N- terminal zinc finger with GTP -bound Rab3 on the surface of synaptic vesicles. It may then interact with other members of the synaptic protein complex, especially synaptotagmin through the C₂ domains of the protein. In addition, two distinct types of REVI-binding proteins have also been identified. Proteins termed ERCl and ERC2 that are found in the active zones of neurons as well as more generally as components of the intracellular membrane trafficking process in all cells, bind to the PDZ domain of RJDVI Sl, and a group of proteins termed RBPs bind to the PXXP motif in the pore-forming subunits (αl) of L- and N-type Ca²⁺ channel proteins.

C₂ domains are composed of -130 residues and characteristically bind Ca²⁺ and phospholipids. Most proteins with C₂ domains are involved in signal transduction or membrane trafficking. These include the synaptotagmins, rabphilin- 3, and Muncl3-1. The Ca²⁺-binding sites within C₂ domains are formed by five aspartate side chains, one serine side chain, and three carbonyl groups. Both C₂ domains of rat and human RIMSl lack this binding consensus, so it is unlikely that they bind Ca²⁺. This sets RJMS1 apart from other vesicular C₂ domain proteins such as rabphilin and synaptotagmin. The C₂ domains of RIMSl also interact with the αl subunits of N- and L-type Ca²⁺ channels, with SNAP-25, with synaptotagmin I and with α-liprins. The binding of synaptotagmin I to RIMSl is Ca²⁺-dependent, with binding strongly reduced in its presence. The C₂B domain of RIMSl also binds a- liprins, which are adaptor proteins in the presynaptic active zone in C. elegans.

A modulator suitable for use in the invention may be capable of modulating one or more of the interactions described above. Such a modulator may thus be able to stimulate or inhibit any such interaction. A single modulator may affect more than one, for example, two, three, four, five or more of the interactions set out above. Thus, a modulator may modulate the interaction of RHVIS 1 with Rab3A,

Munc 13-1, N-/L-type Ca²⁺ channel proteins, SNAP-25, synaptotagmin I, an α-liprin or any other RIMSl -binding protein. A modulator may affect one or more, for example, two, three, four, five or more of any such interactions.

Suitable modulators may be antibody products (for example, monoclonal or polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR-grafted or humanised antibodies) which are, for example, specific to RIMSl. Defined chemical entities, peptide and peptide mimetics, aptamer oligonucleotides or natural products may also be suitable modulators. A suitable modulator may be a chemical compound, for example a small molecule.

A modulator of RIMSl may act via an antiseiise mechanism or via an RNA interference mechanism (RNAi).

A modulator of RIMSl which acts via an antisense mechanism may comprise a polynucleotide which has substantial complementarity to all or part of the mRNA of RIMSl. A polynucleotide which has substantial sequence complementarity to all or part of the mRNA of RIMSl is typically one which is capable of hybridizing to that mRNA. If the inhibitor has substantial complementarity to a part of the mRNA of RIMSl, it generally has substantial complementarity to a contiguous set of nucleotides within that mRNA.

There are, generally speaking, two antisense approaches which maybe used in the invention. In one approach, a vector is used which allows for the expression of a polynucleotide which has substantial sequence complementarity to all or part of the mRNA of RIMSl (i.e. a polynucleotide which can hybridize to that mRNA). This results in the foπnation of an RNA-RNA duplex which may result in the direct inhibition of translation and/or the destabilization of the target message, by rendering it susceptibility to nucleases, for example. The vector will typically allow the expression of a polynucleotide which hybridizes to the ribosome binding region and/or the coding region of the RIMSl mRNA.

Alternatively, an oligonucleotide may be delivered which is capable of hybridizing to the RIMSl mRNA. Antisense oligonucleotides are postulated to inhibit target gene expression by interfering with one or more aspects of RNA metabolism, for example processing, translation or metabolic turnover. Chemically modified oligonucleotides may be used and may enhance resistance to nucleases and/or cell permeability.

In the first approach, the vector is capable of expressing a polynucleotide which has substantial sequence complementarity to all of part of the RIMSl mRNA. Such a polynucleotide will be capable of hybridizing to the mRNA. Typically, such a polynucleotide will be an RNA molecule. Such a polynucleotide may hybridize to all or part of the RIMSl mRNA. Generally, therefore the polynucleotide will be complementary to all of or part of such an mRNA. For example, the polynucleotide may be the exact complement of such an mRNA. However, absolute complementarity is not required and preferred polynucleotides which have sufficient complementarity (i.e. substantial complementarity) to form a duplex having a melting temperature of greater than 40⁰C under physiological conditions are particularly suitable for use in the present invention. The polynucleotide may be a polynucleotide which hybridises to the RIMSl mRNA under conditions of medium to high stringency, such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50⁰C to about 6O⁰C.

It is preferred that the polynucleotide hybridizes to a coding region of the RIMSl mRNA. However, a polynucleotide may be employed which hybridises to all or part of the 5'- or 3 '-untranslated region of such an mRNA. The polynucleotide will typically be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400, 500, 600 or 700 nucleotides in length or even up to a few nucleotides, such as five or ten nucleotides, shorter than the RIMSl full- length mRNA.

The polynucleotide, (i.e. the "antisense" polynucleotide), maybe expressed in a cell from a suitable vector. A suitable vector is typically a recombinant replicable vector comprising a sequence which, when transcribed, gives rise to the polynucleotide (typically an RNA). Typically, the sequence encoding the polynucleotide is operably linked to a control sequence which is capable of providing for the transcription of the sequence giving rise to the polynucleotide. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a sequence giving rise to an antisense RNA is ligated in such a way that transcription of the sequence is achieved under conditions compatible with the control sequences.

The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for transcription to occur and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of antisense RNA, or used to transfect or transform a host cell. The vector may also be adapted for used in vivo, for example in a method of gene therapy.

Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, mammalian promoters, such as beta-actin promoters, may be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters. All these promoters are readily available in the art. Preferred promoters are tissue specific promoters, for example promoters driving expression specifically within vascular tissue. Vectors may further include additional sequences, flanking the sequence giving rise to the antisense polynucleotide, which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination.

Examples of suitable viral vectors include retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. Gene transfer techniques using such viruses are will known to those skilled in the art. Retrovirus vectors, for example, may be used to stably integrate the polynucleotide giving rise to the antisense RNA into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. In the antisense oligonucleotide approach, a suitable oligonucleotide will typically have a sequence such that it will bind to the RIMSl mRNA. Therefore, it will typically have a sequence which has substantial complementarity to a part of such an rnRNA. A suitable oligonucleotide will typically have substantial complementarity to a contiguous set of nucleotides within the RIMSl mRNA. An antisense oligonucleotide will generally be from about 6 to about 40 nucleotides in length. Preferably it will be from 12 to 20 nucleotides in length.

Generally the oligonucleotide used will have a sequence that is absolutely complementary to the target sequence. However, absolute complementarity may not be required and in general any oligonucleotide having sufficient complementarity (i.e. substantial complementarity) to form a stable duplex (or triple helix as the case may be) with the target nucleic acid is considered to be suitable. The stability of a duplex (or triplex) will depend inter alia on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity between the antisense oligonucleotide and the target sequence. The system can tolerate less complementarity when longer oligonucleotides are used. However oligonucleotides, especially oligonucleotides of from 6 to 40 nucleotides in length, which have sufficient complementarity to from a duplex having a melting temperature of greater than 40°C under physiological conditions are particularly suitable for use in the present invention. The polynucleotide may be a polynucleotide which hybridises to under conditions of medium to high stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.

Antisense oligonucleotides may be chemically modified. For example, phosphorothioate oligonucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3T5'- phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.

Alternatively mixed backbone oligonucleotides (MBOs) may be used. MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides. MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or T- O-alkyloligoribonucleotides.

An inhibitor suitable for use in the invention may act via an RNA interference (RNAi) mechanism. Such an inhibitor is typically a double-stranded RNA and has a sequence substantially similar to part of the RJMSl mRNA. Preferred inhibitors of this type are typically short, for example 15mers to 25mers, in particular 18mers to 23mers.

The use of short inhibitors of the type described above is preferred because such inhibitors do not appear to trigger viral defence mechanisms of higher organisms. Such inhibitors can be used to inhibit translation of the mRNA.

Alternatively, small fragments of sequence encoding the RIMSl gene product (or a sequence substantially similar thereto) may be provided, cloned back to back in a suitable vector. The vectors described above are suitable for expression of such back to back sequences. Expression of the sequence leads to production of the desired double-stranded RNA.

The invention provides methods for the identification of agents which can binds to or which can modulate RIMSl (as defined above). Such substances may be used in the enhancement of cognition.

Thus, the invention also provides methods for identifying a substance for use in enhancing cognition. Such a method typically comprises determining whether or not a test substance may bind to or is a modulator of RIMSl (as defined above). Typically, a method of the invention will comprise determining whether or not the test substance is a modulator of the expression and/or activity of the RIMSl gene or a polypeptide product thereof. The invention provides a method for identifying an agent for use in the enhancement of cognition, which method comprises:

(a) providing the RIMSl polypeptide, a polypeptide substantially similar thereto, or a fragment of either thereof;

(b) contacting a test substance with the polypeptide or fragment under conditions that, in the absence of the test substance, would permit activity of the polypeptide or fragment; and (c) determining whether or not the test substance is capable of binding to or modulating the activity of the polypeptide or fragment.

In this way, an agent suitable for use in the enhancement of cognition may be identified. Activity of the polypeptide or fragment may be determined by, for example, the ability of the polypeptide or fragment to coprecipitate with Rab3a or any other polypeptide to which, in the absence of the test substance, the RIMSl polypeptide or fragment would bind.

The invention further provides a further method for identifying a modulator of RIMSl, and thus a substance suitable for use in the treatment of enhancing cognition, which method comprises: providing, as a first component, a polypeptide encoded by the RIMSl gene, a polypeptide substantially similar thereto or a fragment of either thereof; providing, as a second component, a polypeptide to which the RIMSl polypeptide is known to bind, a polypeptide substantially similar thereto or a fragment of either thereof; contacting the two components with a test substance under conditions that, in the absence of the test substance, would permit the two components to interact; and determining whether the test substance is capable of inhibiting the interaction between the first and second components.

The skilled person can thereby readily determine whether or not the test substance is a modulator of the RIMSl gene and therefore whether or not it is suitable for use in the enhancement of cognition.

A fragment of a polypeptide to which the RDVIS 1 polypeptide is known to bind may be used in the assay described above. Typically, the fragment will retain similar activity (enzymatic, binding or other) to the full-length polypeptide from which is it derived (or a polypeptide substantially similar thereto) and preferably retain the ability to coprecipitate with RDVIS 1. Alternatively, the fragment will retain activity similar to one or more of the domains of the full-length RIMSl polypeptide. A polypeptide substantially similar to the RIMSl polypeptide is one which shares sequence similarity with that polypeptide and also retains similar activity thereto. Similarly, a fragment suitable for use in the assay will also retain similar activity to the RIMSl polypeptide (or a polypeptide substantially similar thereto).

Fragments of RIMSl suitable for use in the assay described above may comprise any one of the domains set out above, a sequence substantially similar to any thereto or a combination of any of the above sequences. Thus, the assay described above may be carried out with the N-terminal Rab3 A-GTP binding site or one of the two C-terminal C₂ domains.

Polypeptides substantially similar to RIMSl may be used in the assays of the invention. Such polypeptides will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98 or at least 99% sequence identity with REVISl, calculated over the full length of those sequences. However, the identity may be calculated over a shorter length, for example 20, 50, 100 or more amino acids. If identity is calculated over a shorter length, it is typically done so over a contiguous length of amino acids. The UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, ρ387-395). The PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://wvvvv.ncbi.nlm.nih.gov/).

A polypeptide substantially similar to RIMSl may be a naturally occurring sequence, such as an allelic variant of RIMSl. An allelic variant will generally be of human or non-human mammal, for example bovine or porcine, origin.

Alternatively, a polypeptide which is substantially similar to RJDVISl may have a non-naturally occurring sequence. A non-naturally occurring polypeptide which is substantially similar to REVISl may be a modified version of one of those polypeptides, obtained by, for example, amino acid substitution, deletion or addition. Up to 1, up to 5, up to 10, up to 50 or up to 100 amino acid substitutions or deletions or additions, for example, maybe made. Thus, a fragment of REVISl maybe used in an assay of the invention. Typically, if substitutions are made, the substitutions will be conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

Suitable polypeptides or fragments thereof for use in the assays described above can be obtained, for example, recombinantly by any method known to those skilled in the art. Alternatively, polypeptides may be chemically synthesized. Synthetic techniques, such as a solid-phase Merrifield-type synthesis, may be preferred for reasons of purity, antigenic specificity, freedom from unwanted side products and ease of production. Suitable techniques for solid-phase peptide synthesis are well known to those skilled in the art (see for example, Merrifield et ah, 1969, Adv. Enzymol 32, 221-96 and Fields et a!., 1990, Int. J. Peptide Protein Res, 35, 161-214). In general, solid-phase synthesis methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain.

Polypeptides (or fragments or variants thereof) suitable for use in the invention may be fused to a carrier polypeptide. Thus, additional amino acid residues may be provided at, for example, one or both termini of RTMSl or a functional variant of either thereof for the purpose of providing a carrier polypeptide, by which the polypeptide can be, for example, affixed to a label, solid matrix or carrier. Thus a polypeptide for use in a method of the invention may be in the form of a fusion polypeptide which comprises heterologous sequences. Indeed, in practice it may often be convenient to use fusion polypeptides. This is because fusion polypeptides may be easily and cheaply produced in recombinant cell lines, for example recombinant bacterial or insect cell lines. In addition, fusion polypeptides may be easy to identify and isolate. Typically, fusion polypeptides will comprise a polypeptide sequence as described above and a carrier or linker sequence. The carrier or linker sequence will typically be derived from a non-human, preferably a non-mammalian source, for example a bacterial source. This is to minimize the occurrence of non-specific interactions between RIMSl and a polypeptide which binds with RIMS 1 if a two component assay is to be used.

Polypeptides maybe modified by, for example, addition of histidine residues, a T7 tag or glutathione S-transferase, to assist in their isolation. Alternatively, the carrier polypeptide may, for example, promote secretion of the polypeptide from a cell or target expression of the polypeptide to the cell membrane. Amino acids carriers can be from 1 to 400 amino acids in length or more typically from 5 to 200 residues in length. The polypeptide may be linked to a carrier polypeptide directly or via an intervening linker sequence. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid or aspartic acid. Suitable polypeptides for use in the methods of the invention may be chemically modified, for example, post translationally modified. For example they may be glycosylated or comprise modified amino acid residues. Polypeptides can be in a variety of forms of polypeptide derivatives, including amides and conjugates with polypeptides i.e. RTMSl or a polypeptides substantially similar thereto may be so-modified. Chemically modified polypeptides also include those having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized side groups include those which have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups and formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.

In the method of the invention, a fragment of a polypeptide encoded by the RIMSl gene or a polypeptide substantially similar thereto may comprise an N- terminal Rab3 A-GTP binding site, a zinc finger domain which comprises two Cys₄ zinc fingers and a PDZ domain, a C₂A domain, a C₂B domain or a SH3 -binding domain. The method may be carried out with a polypeptide substantially similar to any one thereto. A polypeptide encoded by the RTMSl gene may comprise two or more, for example The method of the invention may be carried out in the presence of Rab3 a,

Munc 13-1, aN/L-type Ca²⁺ channel protein, SNAP-25, a RDVl-binding protein, synaptotagmin I, an ce-liprin, CAST (Takao-Rikitsu et ah, J. Cell Biol. 164(21 301- 11 (2004)), PKA (Lonart et ah, Cell 115(1), 49-60 (2003)) or 14-3-3 (Sun et ah, J. Biol. Chem. 278f40\ 38301-9 (2003)) or a polypeptide substantially similar to any thereof or a fragment of any thereof (as defined with reference to the discussion set out above). The method of the invention may be carried out in the presence of two or more of the above polypeptides/fragments, for example in the presence of two, three, four or five or the above polypeptides/fragments.

A modulator of the invention may modulate expression of the RIMSl gene. A method suitable for identifying such a modulator may comprise:

(a) providing a polynucleotide construct comprising a promoter of the RIMSl gene, or functional equivalent thereof, operably linked to a coding sequence;

(b) contacting a test substance with the polynucleotide construct under conditions that, in the absence of the test substance, would permit expression of the polypeptide encoded by the coding sequence; and

(c) determining the level of expression of the polypeptide encoded by coding sequence, thereby to determine whether or not the test substance is a modulator of the expression of the RIMSl gene and, therefore, whether or not the test substance is useful in the enhancement of cognition.

A functional equivalent of a promoter of the RIMSl gene is a promoter having a sequence similar to that of a wild-type RIMSl promoter and which retains ability to drive transcription. Typically, a functional equivalent will have a sequence substantially similar to that of a wild-type RIMS 1. Substantial similarity is defined above in relation to nucleotide and amino acid sequences above. The functional equivalent may be a promoter from a homolog, ortholog or paralog. Typically, the coding sequence encodes a reporter polypeptide. The reporter polypeptide may be any suitable reporter polypeptide, for example /3-glucuronidase (GUS), the lacZ gene product (|3-galactosidase) or a green fluorescent protein (GFP).

In a typical assay, a cell harbouring a promoterreporter construct is used as follows: - a defined number of cells are inoculated, in for example 1 OOμl of growth medium, into the wells of a plastics micro-titre plate in the presence of a test substance; optical density (OD) at 590nm may be measured as may expression of the reporter polypeptide according to any method appropriate for the reporter polypeptide being used; the micro-titre plates are covered and incubated at 37°C in the dark; and the OD is read again and expression of the reporter polypeptide assayed at convenient time intervals.

If GUS is used as the reporter polypeptide, GUS expression may assayed by measuring the hydrolysis of a suitable substrate, for example 5-bromo-4-chloro-3- indolyl-β-D-glucoronic acid (X-gluc)or 4-methylumbelliferyl-β-glucuronide (MUG). The hydrolysis of MUG yields a product which can be measured fluorometrically. GFP is quantified by measuring fluorescence at 590nm after excitation at 494nm. These methods are well known to those skilled in the art. Alternatively the coding sequence may be the RIMSl coding sequence itself.

The expression of RIMSl may be followed by, for example, Northern/RNA blotting, Western/antibody blotting or biochemical assay.

For all of the methods set out above, control experiments can be carried out, for example in which the test substance is omitted. Also, substances so-identified may be tested with other known promoters (to exclude the possibility that the test substance is a general inhibitor of gene expression) and other known polypeptides (to exclude the possibility that the test substance is a general inhibitor of enzyme activity, for example a protease).

Any suitable formats may be used for carrying out the assays. The reaction mixtures may contain a suitable buffer. A suitable buffer includes any suitable biological buffer that can provide buffering capability at a pH conducive to the reaction requirements of the enzyme in question. Typically, the assays may be adapted so that they can be carried out in a single reaction vessel and more preferably can be carried out in a single well of a plastics microtitre plate and thus can be adapted for high through-put screening. Suitable test substances include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR- grafted antibodies and humanised antibodies) which are specific for the RIMSl gene product. Furthermore, combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonucleotides and natural product libraries may be screened for activity as inhibitors of RIMSl in assays such as those described below. The candidate substances may be chemical compounds. The candidate substances may be used in an initial screen often, for example, test substances per reaction, and the test substance of those batches which show inhibition may then be tested individually. RIMSl itself and variants of RDVIS 1, i.e. sequences substantially similar to

RJDVIS 1 and, and fragments thereof, as defined above, may also be modulators of RDVISl and may therefore be used in the assays of the invention as test substances. In particular, variants comprising the Arg820His mutation may be used as test substances in the method of the invention. A suitable variant may be a fragment of RHVISl comprising the said mutation.

A substance suitable for use in the enhancement of cognition is one which produces a measurable reduction/increase in expression and/or activity of RDVIS1 in an assay described above. Preferred substances are those which stimulate/inhibit expression and/or activity of RDVIS1 by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the stimulator/inhibitor of lμg ml^"1, lOμg ml^"1, lOOμg ml^"1, SOOμg ml^"1, lnig ml^"1, lOmg ml^"1, lOOmg ml^"1. The percentage inhibition/stimulation represents the percentage decrease/increase in expression/activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define a substance suitable for use in the enhancement of cognition or other proliferative disorder. Substances having greater inhibition at lower concentrations being preferred.

Candidate substances suitable for use in the enhancement of cognition, i.e. candidate modulators of the RJMSl gene which show activity in assays such as those described above, can be tested on mammalian cell lines for the ability to modulate the activity and/or expression of the RIMSl gene.

Modulators of RIMSl, including those identified according to a method as set out above (ie. substances identified by a method of the invention), in particular modulators specific for RIMSl, may be used in a method of treatment of the human or animal body by therapy. In particular, such modulators may be used in the enhancement of cognition.

Modulators identified in the methods of the invention may be tested in animal models, for example a mouse or rat model, for ability to increase cognition. Such an animal model may comprise mutation in the RIMSl gene or may be a knock-out for the RIMSl gene.

Thus, the invention provides a modulator RTMSl, for example a modulator identified in a method of the invention, for use in a method of treatment of the human or animal body by therapy. The invention also provides use of such modulators in the manufacture of a medicament for use in the enhancement of cognition. The invention also provides a method of enhancing cognition in a host, which method comprises the step of administering to the host an effective amount of a modulator of RIMSl, for example a modulator identified in a method of the invention. The host may be a human or an animal.

The condition of a patient suffering from decreased cognition can be improved by administration of a modulator of RIMS 1 , for example a modulator identified in a method of the invention. A therapeutically effective amount of a modulator of RIMSl, for example a modulator identified in a method of the invention may be given to a patient in need thereof.

RIMSl itself and variants of RIMSl, i.e. sequences substantially similar to RDVISl, as defined above, or fragments thereof may also be modulators of RIMSl and may therefore be used in the enhancement of cognition, m particular, variants and/or fragments comprising the Arg820His mutation may be used in the enhancement of cognition. Polynucleotides encoding RBVISl and RIMSl variant modulators and expression vectors comprising such polynucleotides may similarly be used in the enhancement of cognition. Enhancement of cognition in this context may comprise an increase in any indicator/component of cognition/cognitive function. For example, enhancement of cognition according to the invention may involve one or more of the following: verbal intelligence quotient; vocabulary; digit span; similarities; phonemic fluency; cognitive estimates; immediate story recall; delayed story recall; immediate verbal learning; delayed verbal learning; semantic fluency; or performance in the Hayling test. These components of cognition are well known to the skilled person and such a skilled person would readily be able to test such components of cognition in a subject. See for example, Spreen and Strauss, E. A COMPENDIUM OF NEUROPSYCHOLOGICAL TESTS: ADMINISTRATION, NORMS AND COMMENTARY. Oxford University Press, Inc. New York (1998). Further details regarding the measurement of the indicators of cognitive function listed above are set out in the Examples.

A subject requiring enhancement of cognition may be suffering from any cognitive impairment, decline or neurological disorder. Any cause of such cognitive impairment, decline or neurological disorder may be treated using a modulator of RDVIS 1 according to the invention. The cause may be unknown, due to a genetic defect in the RIMSl gene or in an unrelated gene, causing any condition in which cognitive failure (failure to ever develop certain cognitive skills) or decline (once deficit incurred) are observed, acquired or due to an environment defect, for example traumatic brain injury, progressive or static. The neurological disorder may be, for example, Alzheimer's disease. A modulator of RIMSl, for example an inhibitor or activator identified in a method of the invention, is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in phannaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

A suitable modulator is administered to a patient. The dose of a suitable modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical dose is from about 0.1 to 500 mg. Appropriate dosages may depend on a variety of factors, for example, body weight, according to the activity of the specific antagonist, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Such a dose may be given, for example, once only, or more than once for example 2, 3, 4 or 5 times. The dose may be given, for example daily, every other day, weekly or monthly.

The antisense oligonucleotides or RNA interference (RNAi) molecules described above may be administered by direct injection into the site to be treated. Preferably, the antisense oligonucleotides or RNAi molecules are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.

The dose at which an antisense oligonucleotide or RNAi molecule is administered to a patient will depend upon a variety of factors such as the age, weight and general condition of the patient, the nature of the neurological disorder being treated that is being treated, and the particular antisense oligonucleotide or RNAi molecule that is being administered. A suitable dose may, however, be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight.

A RIMSl polynucleotide, a variant or fragment of such a polynucleotide, a polynucleotide having substantial sequence complementarity to all or part of an niRNA of the RIMSl gene or a vector capable of expressing such a polynucleotide may be administered directly as a naked nucleic acid construct. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Examples of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam and transfectam ). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition. Preferably, polynucleotide, vector or composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.

The pharmaceutical composition is administered in such a way that the polynucleotide or vector can be incorporated into cells at an appropriate area. When the polynucleotide of the invention is delivered to cells by a viral vector, the amount of virus adminstered is in the range of from 10⁴ to 10⁸ pfu, preferably from 10⁵ to 10⁷ pfu, more preferably about 10⁶ pfu for herpes viral vectors and from 10⁶ to 10¹⁰pfu, preferably from 10⁷ to 10⁹ pfu, more preferably about 10⁸pfu for adenoviral vectors. When injected, typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide of the invention is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg.

Where the polynucleotide giving rise to RIMSl, a variant thereof or antisense RNA is under the control of an inducible promoter, it may only be necessary to induce gene expression for the duration of the treatment. Once the condition has been treated, the inducer may be removed and expression of the polypeptide of the invention ceases. This will clearly have clinical advantages. Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/VP16 fusion protein.

The use of tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention. For example, several neurological disorders are due to aberrant expression of particular gene products in only a small subset of cells. It will be advantageous to be able express therapeutic genes in only the relevant affected cell types, especially where such genes are toxic when expressed in other cell types. The routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. The inventors have shown that the RIMSl gene is polymorphic at nucleotide position 2459 (at least), as defined with reference to SEQ ID NO: 1. In particular, at position 2459, a G or A residue may be present. The inventors have shown that this polymorphism is associated with increased cognitive function. In particular, the inventors results show further that subjects with an A at position 2459 (either in one or both alleles) are more likely to have an increased cognitive function as compared with normal control subjects.

Thus, such subjects are significantly more likely to exhibit increased cognitive function, for example as measured using any of the indicators/components of cognitive function described above and/or in the Example, as compared to the population mean. That is to say, such subjects are statistically likely to exhibit greater cognitive function than the population mean. The method of the invention may, therefore, be used to identify individuals who are statistically likely to exhibit greater than average cognitive function. Accordingly, the present invention provides a method for method for predicting the level of cognitive function in a subject, typically a mammal, for example a human. The method comprises typing the RIMSl gene of the subject. Accordingly, it maybe possible to determine the level of cognitive function of the subject. The subject may be asymptomatic for a known neurological or ophthalmologic disorder.

Typing of the RIMSl gene typically comprises the measurement of any suitable characteristic of the RIMSl gene to determine the level of cognitive function in the individual. In this context, the term "gene" encompasses not only the RIMSl coding sequence, but also untranslated and regulatory regions situated 5' and 3' to the coding sequence and introns.

Suitable typing may involve determining whether the RIMSl gene comprises a polymorphism which is indicative of increased or decreased cognitive function. Such typing will typically involve determining the sequence of all or part of the RIMSl gene in the subject. Typically, all of part of the sequence of the promoter of the RIMSl gene may be determined. The human RIMSl gene sequence is set out in SEQ ID NO: 1. Thus, typing according to the invention may involve determining all of part of that sequence in a subject or an allelic variant thereof. Alternatively, suitable typing in a method of the invention may involve determining the amount of expression of the RIMSl gene in the subject. Such expression typing may be carried out at the nucleotide, for example RNA level, such as by RNA blotting or reverse transcriptase-PCT, and/or at the polypeptide level, for example by use of an antibody.

The method of the invention may be carried out in vivo, although typically it is more convenient to carry it out in vitro on a sample derived from the subject. The sample typically comprises a body fluid of the individual and may for example be obtained using a swab, such as a mouth swab. The sample may be a blood, urine, saliva, cheek cell or hair root sample. Alternatively, the sample may be neural tissue.

The sample is typically processed before the method is carried out, for example nucleic acid extraction, such as extraction of genomic DNA or RNA, in particular mRNA may be carried out. RNA, such as mRNA, extracted from a sample may subsequently be converted into cDNA. The nucleic acid or protein in the sample may be cleaved either physically or chemically (e.g. using a suitable enzyme, such as a restriction endonuclease). The polynucleotide in the sample may be copied (or amplified), e.g. by cloning or using a PCR based method. hi a preferred embodiment the typing comprises identifying whether the subject has a polymorphism which is indicative of the level of cognitive function, or a polymorphism which is in linkage disequilibrium with such a polymorphism.

Polymorphisms which are in linkage disequilibrium with each other in a population are typically found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40 %, at least 50%, at least 70% or at least 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus a polymorphism which is not a functional susceptibility polymorphism, but is in linkage disequilibrium with a functional polymorphism, may act as a marker indicating the presence of the functional polymorphism.

Polymorphisms which are in linkage disequilibrium with the polymorphism mentioned herein are typically located within 500kb, preferably within 400kb, within 200kb, within 100kb, within 50kb, within 10kb, within 5kb, within 1 kb, within

500bp, within lOObp, within 50bp or within lObp of the polymorphism. Typing of such a polymorphism is considered to be encompassed within the phrase "typing of the RIMSl gene" for the purposes of this invention

The polymorphism is typically an insertion, deletion or substitution with a length of at least 1, 2, 3, 4, 5, 10, 15 or more base pairs or amino acids. However, preferred polymorphisms are those where substitution of 1 base pair occurs, i.e. a single nucleotide polymorphism (SNP) is preferred. The polymorphism may be located 5' to the coding region, in the coding region, in an intron or 3' to the coding region. The polymorphism which is detected is typically a functional mutation which contributes to cognitive function, but may be a polymorphism which is in linkage disequilibrium with a functional mutation, i.e. may be a marker for a functional mutation.

Generally, the polymorphism will be associated with cognitive function, for example as can be determined in a case/control study (e.g. as discussed in the Example below). The polymorphism will generally cause a change in any characteristic of the RIMSl gene or protein encoded thereby, for example expression levels, such as transcription and/or translation rates, enzymic activity, expression of a variant, cellular localisation or the pattern of expression in different tissues.

Typically, the polymorphism is a single polynucleotide polymorphism, generally in an exon of the RIMSl gene, in particular the 14^th exon (defined with reference to the human sequence which does not comprise the third exon present in the rat sequence). A preferred polymorphism is that at 2459 of SEQ ID NO: 1. Other preferred polymorphisms are those at a position corresponding to position 2459 of SEQ ID NO: 1, in an allelic variant of SEQ ID NO: 1. The term "corresponding position" refers to a position in an allelic variant which is equivalent to a position defined with reference to SEQ ID NO: 1. Those skilled in the art will be able to determine a position in an allelic variant which corresponds to a position in SEQ ID NO: 1. Comparison of an allelic variant with the sequence set out in SEQ ID NO: 1, using for example the PILEUP program referred to above, will allow corresponding positions to be identified in an allelic variant, in particular positions in an allelic variant which correspond to position 2459 in SEQ ID NO: 1. Further preferred polymorphisms are ones which are in linkage disequilibrium with one or more of the above-mentioned polymorphisms. In the method of the invention, the identity of one of the above-mentioned nucleotides may be determined for both alleles of the subject. Polymorphism at one or both of the alleles maybe indicative of an increased level of cognitive function.

A polymorphism which can be typed to determine the level of cognitive function may be identified by a method comprising determining whether a candidate polymorphism in the RIMSl gene is: (i) associated with the cognitive function; or (ii) is in linkage disequilibrium with a polymorphism which is associated with cognitive function, and thereby determining whether the polymorphism can be typed to determine the level of cognitive function. A polymorphism to be typed according to the method of the invention may conveniently be detected by directly determining the presence of the polymorphic sequence in a RIMSl polynucleotide or protein of the subject. Such a polynucleotide is typically genomic DNA or mRNA, or a polynucleotide derived from these polynucleotides, such as a cDNA, which optionally may be in the form of a library. The detection method may be based on the detection of a difference in a characteristic between a RIMSl polynucletide or a RIMS 1 protein that carries the polymorphism and one which does not. For example, mobility of the proteins, such as mobility on a gel, may be detected. The polymorphism may be identified in a subject who has or is of abnormal cognitive function. The polymorphism is typically detected by directly determining the presence of the polymorphic sequence in a RIMSl polynucleotide or protein of the individual. Such a polynucleotide is typically genomic DNA or mRNA, or a polynucleotide derived from these polynucleotides, such as in a library made using polynucleotide from the individual (e.g. a cDNA library). The presence of the polymorphism may be determined in a method that comprises contacting a polynucleotide or protein of the subject with a specific binding agent for the polymorphism and determining whether the agent binds to a polymorphism in the polynucleotide or protein, the binding of the agent to the polymorphism indicating that the individual has an increased level of cognitive function.

Generally, the agent will also bind to flanking nucleotides and amino acids on one or both sides of the polymorphism, for example at least 2, 5, 10, 15 or more flanking nucleotide or amino acids in total or on each side. Generally, in the method, determination of the binding of the agent to the polymorphism can be done by determining the binding of the agent to the polynucleotide or protein. However, the agent may be able to bind the corresponding wild-type sequence by binding the nucleotides or amino acids which flank the polymorphism position, although the manner of binding will be different to the binding of a polynucleotide or protein containing the polymorphism, and this difference will generally be detectable in the method (for example this may occur in sequence specific PCR).

In the case where the presence of the polymorphism is being determined in a polynucleotide it may be detected in the double stranded form, but is typically detected in the single stranded form.

The agent may be a polynucleotide (single or double stranded) typically with a length of at least about 10 nucleotides, for example at least 15 or 20, up to about 25, 30, 35 or more nucleotides. The agent may be a molecule which is structurally related to polynucleotides that comprises units (such as purines or pyrimidines) able to participate in Watson-Crick base pairing. The agent may be a polypeptide, typically with a length of at least 10 amino acids, such as at least about 20, 30, 50 or more up to about 75, 100, 150, 200 or more amino acids. The agent may be an antibody, including a fragment such as of such an antibody which is capable of binding the polymorphism. Suitable fragments include Fv, F(ab') and F(ab')₂ fragments, as well as single chain antibodies. Furthermore, suitable antibodies and fragments thereof may be chimeric, CDR-grafted or humanised.

A polynucleotide agent which is used in the method will generally bind to the polymorphism and flanking sequence or wholly to the flanking sequence, of the polynucleotide of the individual in a sequence specific manner (e.g. hybridise in accordance with Watson-Crick base pairing) and thus typically has a sequence which is fully or partially complementary to the sequence of the polymorphism and/or flanking region. The partially complementary sequence shared sequence identity with the fully complementary sequence. The agent may be a probe. This may be labelled or may be capable of being labelled indirectly. The detection of the label may be used to detect the presence of the probe on (and hence bound to) the polynucleotide or protein of the individual. The binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein (and thus to separate it from a composition or solution).

All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be clear to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.

The following Example illustrates the invention:

Example

Materials and Methods

Ethics The imaging and cognitive study of the kindred was approved by the Joint

Research Ethics Committee of the Institute of Neurology and National Hospital for Neurology and Neurosurgery and the Research Ethics Committee of Moorfields Eye Hospital NHS Trust. Human embryonic/fetal material was obtained from the MRC/ Wellcome Trust Human Developmental Biology Resource with full ethics approval. The Lothian Birth Cohort study has been carried out with the consent of the Lothian Research Ethics Committee. All participating individuals provided written informed consent.

Kindred The pedigree is illustrated in Figure 1. Eight (of nine in total) affected and seven (of nine in total) unaffected adult individuals agreed to participate. The tester was unaware of individuals' status (mutation carrier/wild type) in many (11:4, 111:4, 111:9, ΓV:2-IV:7), but not all, members of the kindred. Identical tests were administered to all subjects: the tests themselves required only verbal and auditory interaction and were objective.

Imaging

MRI sequences

Coronal Tl-weighted images were acquired at 1.5T: acquisition parameters were TE= 4.2, TI= 450, TR= 15, NEX= 1, flip angle= 20, acquisition matrix 256 x 128, field of view 24cm, producing 124 contiguous slices, voxel dimension 0.9375mm x 0.9375mm x 1.5mm. Data were reformatted in multiple planes to allow careful examination of regions of interest. Additionally T2 and FastFLAlR sequences were obtained (T2 and PD sequence: TEl= 30, TE2= 120, TR= 2000, NEX= 1, acquisition matrix 256 x 128, field of view 24xl8cm, slice thickness 5mm contiguous; FastFLAlR sequence TEl= 152, TE2= 2200, TR= 10002, NEX= 1, acquisition matrix 256 x 128, field of view 24cm, slice thickness 5mm contiguous).

Quantitation

Quantitative analysis of the structural MRI data was carried out in 5 subjects. The volumetric MRI data were preprocessed and the grey and white matter extracted (Merschhemke et al, Neuroimage IS, 642-9 (2003)). Using an anatomical template image (Hammers et al., Hum Brain Mapp. 19, 224-47 (2003)) the grey and white matter volumes of 8 cerebral lobes (left and right frontal, parietal, temporal and occipital) were estimated. Volumes in the 5 subjects were compared with volumes from 50 female or 50 male control subjects collected in a previous study (Mitchell et al., Annals of Neurology 53, 658-663 (2003)). Abnormal values were those which fell outside 3SD from the mean. The thickness of cortex was assessed with a size filter. A computer algorithm filled the extracted cortical ribbon with spheres of increasing diameter. The diameter of sphere which just fits across the cortical ribbon indicates the cortical thickness. Frequency histograms of the sphere diameters were estimated for the same lobar regions as above and were compared with 50 female or 50 male control subjects (as above) and values less than the 2.5 or greater than the 97.5 percentiles were considered abnormal. Cognitive assessments

The verbal intelligence quotient (verbal IQ, VIQ) is a composite measure generated from a range of tests. In this kindred with visual impairment, VIQ was prorated from the Wechsler Adult Intelligence Scale (Revised; WAIS-R) Vocabulary, Digit Span and Similarities subtests (Thompson et ah, Neurology 62, 1216-1218 (2004); Spreen and Strauss, E. A COMPENDIUM OF NEUROPSYCHOLOGICAL TESTS: ADMINISTRATION, NORMS AND COMMENTARY. Oxford University Press, Inc. New York (1998)).

Tests applied to kindred

Neuropsychological tests employed had been used in earlier investigations, including one of individuals heterozygous for PAX6 gene mutations (Thompson et al., Neurology 62, 1216-1218 (2004)). Due to variable visual impairments, all tests were selected to require auditory and verbal interaction only.

Intellectual level

The Vocabulary, Digit Span, and Similarities subtests from the WAIS-R were administered and the scores were pro-rated to obtain an estimate of VIQ (Spreen and Strauss, E. A COMPENDIUM OF NEUROPSYCHOLOGICAL TESTS: ADMINISTRATION, NORMS AND COMMENTARY. Oxford University Press, Inc. New York (1998)).

Executive Functions

1). Verbal fluency: The subject had to produce as many words beginning with the letter "s" (phonemic fluency) and as many animal names (semantic fluency) both in a minute. Performance on fluency tests, particularly for the phonemic category (letter "s"), is a sensitive indicator of frontal lobe functioning (Henry & Crawford, Neuropsychology 18, 284-295 (2004)).

2). The Hayling Test is a response suppression task. The subject has to complete two series of 15 sentences each missing the last word. In the first section a sensible completion is required and, in the second, a nonsensical completion. The test yields two measures of mental processing speed and an error score for the second series. Performance on this measure has been shown to involve frontal brain regions in healthy individuals (Collette et al. Neuroimage 14, 258-267 (2001)) and to be adversely affected by frontal lobe pathology (Burgess & Shallice, THE HAYLING ISLAND AND BRIXTON TEST MANUAL. Thames Valley Test Co; Bury St Edmunds (1997)).

3) Cognitive estimates: This is a semantic reasoning task that requires the subject to provide a reasonable estimated answer to ten questions that have no exact answer based on their available semantic knowledge. The questions are of the format 'How fast do race horses gallop?'. Penalties are awarded for inaccurate responses and the higher the score the poorer the reasoning demonstrated. This test has been shown to be sensitive to frontal lobe pathology (Spreen & Strauss (1998), supra; Delia SaIa et al. J Neurol. 25_1, 156-164 (2004)).

Memory 1). Verbal Recall. This was assessed using the Story Recall subtest from the

Adult Memory and Information Processing Battery (AMIPB) (Coughlan & Hollows, THE ADULT MEMORY AND INFORMATION PROCESSING BATTERY. Psychology Dept. St James Hospital, Leeds (1986)). The subject is read a short story and then has to recall as many details as possible immediately following presentation and again following a delay of 30 minutes. Performance measures used were the immediate recall score and the % retained score (delayed recall/immediate recall x 100). 2). Verbal Learning. The List Learning test from the AMIPB was employed. The subject is presented with a list of 15 words on five occasions and following each presentation has to recall as many of the words as possible. A second list of words is then presented and following one attempt at recall of the second list, is required to recall as many words as possible from the first list (delayed recall). The total number of words remembered in the learning phase (verbal learning trials) and in the delayed recall condition (verbal learning delay) were recorded. Results

Cognitive function is enhanced in individuals with RIMl mutation

The pedigree is illustrated in Figure 1. Eight (of nine in total) affected and seven (of nine in total) unaffected adult individuals were tested. The tester was unaware of individuals' status (mutation carrier/wild type) in many (11:4, 111:4, 111:9, rV:2-rV:7), but not all, members of the kindred. Identical tests were administered to all subjects: the tests themselves required only verbal interaction and were objective. The verbal intelligence quotient (VIQ) is a composite measure generated from a range of tests, hi this kindred with visual impairment, VIQ was pro-rated from the Wechsler Adult Intelligence Scale (Revised; WAIS-R) Vocabulary, Digit Span and Similarities subtests (Spreen and Strauss, E. A COMPENDIUM OF NEUROPSYCHOLOGICAL TESTS: ADMINISTRATION, NORMS AND COMMENTARY. Oxford University Press, Inc. New York (1998)). VIQs in affected individuals (Table 1) were all above average with quotients ranging from the 75^th (high average ability) to the 99^th centile (superior ability). Centile levels for subjects 11:2 and 11:6 are likely to be an underestimate as normative data are available up to 75 years only. Some pro-rated VIQ scores eg 150 in subject 111:1, reflect a ceiling effect. On measures of memory and executive skills, performance levels were more variable with some scores falling below average. Normative data available for these measures was based on smaller sample sizes than for VIQ and, with the exception of semantic fluency, were not available for older age bands.

To investigate whether this superior cognitive performance might be due to the mutation, as opposed to being a familial trait unlinked to the mutation, unaffected members of the kindred underwent identical cognitive testing (see Figure 2).

Affected and unaffected first-degree sibs were tested in each generation (11:2, 11:6, 11:8 vs 11:4; 111:5 vs 111:7; IV:3 vs IV:4, IV:5). The mean age of affected members (53.8 years) was higher than unaffected members (41.1 years), which should act conservatively against any superior cognitive performance in the affected group, given the known effect of age (Keller, Ageing Res. Rev., Aug 3 (2005) [Epub ahead of print]). We tested the significance of the association between affected status and every measured cognitive phenotype following a randomization approach that accounts for kindred structure (Rabinowitz & Laird, Hum. Hered. 50, 211-223 (2000)). This approach randomizes RIMSl genotypes to the pedigree, following the laws of Mendelian inheritance, while keeping the phenotype values of each individual fixed. The null hypothesis therefore allows for the possibility of familial effects on phenotype that are unlinked to RIMSl, and any significant departure from the null hypothesis can only be ascribed to an effect linked to RIMSl. The eleven measured phenotypes were: vocabulary (subcomponent of verbal IQ), digit span (subcomponent of verbal IQ), similarities (subcomponent of verbal IQ), phonemic fluency, cognitive estimates, immediate story recall, delayed story recall, immediate verbal learning, delayed verbal learning, semantic fluency and Hayling Test. Each phenotype was normalised by dividing by the standard deviation of values taken from a disease-control group of fifteen visually-impaired individuals with mutation in PAX6 (Thompson et al, Neurology 62, 1216-1218 (2004)).

The perfect observed association between affected status and the RIMSl mutation allowed us to infer a dominant Mendelian disease model and also to infer the genotypes of all founders and marry-ins. We simulated genotypes, and thence the affected status, of all individuals in the pedigree based on Mendelian inheritance from the known founders, and for each normalised phenotype we calculated the difference in means between affecteds and non-affecteds (keeping phenotype values fixed among individuals). To address multiple testing issues, we generated a single score for the overall difference between the affected and non-affected groups. We first normalised all phenotypes to a common variance scale, dividing by the standard deviations estimated from a disease-control group of fifteen visually-impaired individuals with mutation in PAX6 (Thompson et ah, 2004 {supra)), so all phenotypes would have equal weight. We then used the Euclidean distance of the eleven difference-in-means values. To control for possible ascertainment bias, we further conditioned on the observed number of affecteds and non-affecteds in the pedigree by rejecting any pedigrees from the Monte Carlo process that did not match this. We carried out 10,000 simulations that matched these conditions. The single score for the overall difference in all mean phenotype values between affecteds and non-affecteds was significant (P=0.006). For individual phenotypes, all except those for immediate story recall, delayed story recall and semantic fluency had P values less than 5%: vocabulary (P=0.014), digit span (P=0.020), similarities (P=0.039), phonemic fluency (P=0.050), cognitive estimates (P=0.012), immediate story recall (P=O.112), delayed story recall (P=0.087), immediate verbal learning (P=O.048), delayed verbal learning (P=O.042), semantic fluency (P=0.220) and Hayling Test (P=0.010). As expected, given that the three sub-pheno types of verbal IQ were all significant, when we separately tested verbal IQ itself this was also significant (p=0.014).

We determined to what extent the coincidence of these associations is due to the natural correlations among these phenotypes. Table 2 presents the correlation coefficients of the eleven phenotypes, plus verbal IQ, in the disease control sample of fifteen individuals (Thompson et al., 2004 (supra)). Taking verbal IQ as a reference point, it is not surprising that the three subcomponents (vocabulary, digit span and similarities) generating VIQ score are highly correlated with this score. Phonemic fluency, verbal learning and semantic fluency are highly correlated with verbal IQ, and it is unclear whether the significant results observed in some of these variables are due simply to correlation with verbal IQ. Most interestingly, the phenotypes of cognitive and Hayling Test are poorly correlated with verbal IQ, the other variables, and each other, suggesting that multiple facets of the mental processes are being affected independently by the mutation. There was no history or genetic evidence for inbreeding (number of markers scored = 16, heterozygous average for 14 members of kindred = 11.6 (72%); equivalent scores for 5 "married-ins" = 11.4 (71%); data from Kelsell et ah, Am J Hum Genet. 63, 274-9 (1998), omitting IV:4-7).

To address the specific, but unlikely, possibility that visual impairment may enhance function in other domains, we compared identical measures (Thompson et al., 2004 (supra)) from the disease-control cohort of fifteen individuals (mean age 36 years, range 17-54 years; eight females) all of whom had congenitally symptomatic PAX6 mutations. The PAX6 subjects had earlier onset of visual symptoms and more severe central visual impairment at the time of cognitive testing than the RIMSl subjects (Michaelides et al., 2005; see Table 2). The mean VIQ of the PAX6 subjects was 103. The PAX6 subjects as a group do not differ from the RIMSl unaffected individuals: cognitive measures for both groups fall within the normal ranges (Table 3). Despite their more severe and earlier-onset visual impairment, the PAX6 affected individuals performed significantly less well than the RIMSl affected individuals (VIQ, Mann-Whitney PO.0005).

Brain structure in individuals with RIMSl mutation

In seven affected members of the kindred, high-resolution brain MRI was obtained. In two, a mother (11:2) and son (111:1), brain abnormalities were apparent on inspection (see Figure 3a,b). Cortical grey matter was preserved around widened CSF spaces: these changes would be compatible with a bilateral parasagittal polymicrogyric malformation. Intrafamilial heterogeneity of brain malformation has previously been noted in PAX6 kindreds (Mitchell et al., Annals of Neurology 53, 658-663 (2003)). Notably, the more severe visual impairment in the PAX6 affected subjects was not associated with similar findings nor with global brain atrophy (Free et al., Neuroimage 20, 2281-90 (2003)). To determine whether the cognitive findings in the RIMSl kindred had any structural correlate, quantitation of cerebral, lobar and hippocampal volumes, and cortical thickness was undertaken. No volumes were increased in comparison to controls. Only subject 11:2 had any volume reductions, in left parietal lobe grey and white matter and left and right occipital white matter and right occipital grey matter in comparison to controls. Significant areas of reduced thickness were seen in the frontal and temporal lobes of the two eldest subjects (11:2, 11:6). Subject 11:2 also had reduced cortical thickness in the right occipital lobe which is consistent with the reduction in cortical volume noted above. 11:2 was 82 years old at the time of the scan. The control subjects range in age from 17 to 45 years. The normal decrease in cerebral volume associated with aging cannot be excluded as the cause for the apparent volume reductions identified. However, 11:6 (76 years at time of scan) did not have any lobar volume reductions. In the absence of sufficient age-matched controls for thickness measures, we cannot exclude increased grey matter thickness in the older affected subjects. Structural and functional correlation: olfaction

A standardised, age- and sex-corrected test of olfaction (University of Pennsylvania Smell Identification test) was administered to four individuals: ELI had normal olfaction, 111:5 had mild microsmia, 11:2 had severe anosmia and 11:6 was anosmic. Rimla is expressed in adult mouse olfactory bulb (Figure 4a); Pax6 is also expressed here (Fig. 4b; Kohwi et ah, JNeurosci. 25, 6997-7003 (2005)). Impaired olfaction was also noted in our PAX6 cohort (Sisodiya et ah, Nat Genet. 28, 214-6 (2001)).

Discussion

Few individual genetic factors influencing or underlying the heritability of intelligence in humans have been identified (Deary et ah, 2006). Mutations in a large number of genes (eg. MCPHl, ASPM), inherited in Mendelian fashion, are associated with reduced intelligence, often associated with gross physical disability, dysmorphism or other problems (Bond et ah, Nat Genet. 32, 316-20 (2002); Evans et ah, Hum MoI Genet. 13, 1139-45, 2004)). Notably, recent variation in both MCPHl and ASPM have been subject to strong positive selection indicating ongoing adaptive evolution of the human brain (Evans et αl, 2005; Mekel-Bobrov et αh, 2005), and emphasising the importance of population genetic analyses of genes mutations in which affect human cognitive function. Data from quantitative trait analyses in populations with learning disabilities also suggest many genes with Mendelian effects when mutated are candidates to broadly influence normal variation in learning abilities in the general population (Plomin and Kovas, Psychol Bull. 131, 592-617 (2005)). To our knowledge, no human genetic mutations have yet been identified that enhance cognitive function, though discordant abilities, including superior language function, have been reported in association with chromosomal aberration (Steele et αl., Am J Med Genet B Neuropsychiαtr Genet. 134, 104-9 (2005)).

Here we show that a mutation in RIMSl, that encodes a synapse active zone protein, is associated in one kindred with significantly enhanced cognitive function in at least the verbal (likely to be related to general ability, g) and executive domains. RJMSl is an excellent candidate gene to influence cognitive function. Rim lα protein regulates synaptic-vesicle fusion, by interaction with several other active zone molecules, including alpha-liprins, Munc 13-1 (Wang et al., Nature 388, 593-8 (1997)), CAST (Takao-Rikitsu et al, J Cell Biol. 164, 301-11 (2005)), SNAP-25, synaptotagmin (Coppola et al, J Biol Chem. T76_., 32756-62 (2001)) and 14-3-3 (Sun et al, J Biol Chem. 278, 38301-9 (2003)), generating a protein scaffold in the presynaptic terminal. Rimlα greatly enhances neurotransmitter exocytosis in a Rab3- dependent manner (Wang et al, 1997, supra). Studies in Rimla. knockout mice confirm that Rimla protein is essential for maintenance of the normal probability of neurotransmitter release, for regulation of release during short-term synaptic plasticity (Schoch et al, Nature 415, 321-6 (2002)), and for presynaptic long-term potentiation (Castillo et al, Nature 415, 327-30 (2002)). Absence of Rimlα protein leads to severe impairment of learning and memory (Powell et al, Neuron 42, 143- 53 (2004)).

Could the RJMSl mutation be a chance finding, unrelated to the eye or cognitive phenotype? The intrafamilial distribution of cognitive measures argues that the detected mutation is most likely causative, especially as it segregates with both the eye phenotype (clinical symptomatic) and the cognitive enhancement. Co- mingling in each outbred generation of mutation-carrying and wild-type sibs each with respective enhanced or normal cognitive phenotype and the respective, co- segregating impaired or normal visual phenotype, renders extremely unlikely the possibility of an intrafamilial founder effect unrelated to the RIMSl mutation, as supported quantitatively by our modelling.

One interpretation of our findings is that the RIMSl mutation in this kindred alone (i.e. with this genetic background and environmental setting) is permissive of a gain of verbal cognitive abilities in response to impaired visual function, rather than directly causative. This is inherently unlikely, especially in this kindred, as with increasing age higher cognitive abilities are usually associated with better, rather than defective, sensory processing (Lindenberger et al, Psychol Aging. 16, 196-205 (2001)). In the RIMSl kindred, development of visual symptoms occurs later than the age at which values of cognitive measures (eg NART, VIQ) are set — for example, subject 111:2 did not develop retinal dystrophy until aged 42, but had VIQ 146 at age 49. In addition, visual impairment due to PAX6 mutation in unrelated subjects with earlier and more severe loss of central vision, was not associated with any gain in VIQ (but PAX6 mutations may not be "permissive"). The data suggest, therefore, that the observed genetic change is a gain-of-function mutation leading to increased performance in specific cognitive domains in affected humans, irrespective of other genetic background and unrelated to visual impairment. Domain-specific influences on cognitive function are well known (eg the antiepileptic drug topiramate specifically impairs language function (Salinsky et al, Neurology 64, 792-8 (2005)) as do mutations in FOXP2 (Vargha-Khadem et al, Nat Rev Neurosci. 6, 131-8 (2005). The RIMSl mutation studied may affect other cognitive domains - the paradigm we applied is biased to tests requiring only auditory presentation and verbal response and verbal, not visual, processing and was developed for subjects we suspected of having cognitive impairment, not cognitive enhancement. Thus only a limited cognitive assessment was undertaken, in line with our previous reports. We note that, undoubtedly, other genes must also contribute to cognitive scores in this kindred. We note also the cone-rod dystrophy observed in this kindred is of late-onset with no overt clinical features that allow its clinical diagnosis prior to symptom onset (Johnson et al, Genomics 81, 304-14 (2003); Michaelides et al, Br J Ophthalmol. 89, 198-206 (2005)). Thus self-selection or parental direction for more academic or verbal education or interests were not possible. The mutation was not identified until 2003, so that genetic data could not have influenced cognitive achievements for the kindred members studied. The data also exclude secular trends as an explanation.

RIMSl is a plausible candidate not only because of data from animal studies discussed earlier, but also in terms of the pattern of expression in humans. Thus, human RIMSl mRNA is detectable from an early age not only in retinal, but also in cerebral, anlagen and in adult human hippocampus. RIMS 1 protein is more abundant in phylogenetically newer brain regions than in older regions (Wang et al, J Biol Chem. 275, 20033-44 (2000)). However, we were not able to demonstrate accelerated evolution of RIMSl in the primate lineage, nor were genes encoding RIMSl -interacting molecules primate- fast outliers in a previous analysis (Dorus et al, Cell U9, 1027-40 (2004))

The RIMSl mutation alters brain structure, causes a retinal dystrophy, and impairs olfaction in some kindred members. The RIMSl gene shows extensive organ-specific alternative mRNA splicing (Johnson et ah, Genomics 81, 304-14 (2003)). Thus, although the mutation, an Arg844His substitution, lies in the protein C₂A domain, and is shared by the eye and brain isoforms, nevertheless the ocular neurophysiology in the kindred (Michaelides et ah, Br J Ophthalmol. 89, 198-206 (2005)) need not reflect mutant brain RIMS 1 protein function. Lack of homogeneity within a kindred for the functional consequences of gene mutation has previously been noted (Sisodiya et ah, Nat Genet. 28, 214-6 (2001); Mitchell et ah, Annals of Neurology 53, 658-663 (2003)). Our qualitative findings also implicate RIMSl as a candidate gene for bilateral parasagittal polymicrogyria (Jansen and Andermann, J Med Genet. 42, 369-78 (2005)). Unsurprisingly, our limited quantitative MRI findings do not provide a simple structural correlate of the enhanced cognition observed.

Our population genetic data suggest RIMSl is unlikely to have a major role in normal variation of the studied cognitive measures in a Western European population. Whilst population genetic studies can provide useful or even conclusive evidence that a gene and its encoded protein have a biological function, negative population genetic studies do not exclude such a role. The genetics of Parkinson's disease illustrate this well: rare, early-onset, Mendelian cases may be caused by mutation in PINKl (Valente et ah, Science 304, 1158-60 (2004)), but common variation in PINKl does not influence the risk of common, sporadic Parkinson's disease (Healy et ah, Ann Neurol. 56, 329-35 (2004)). Even if the observed RIMSl mutation turns out to be a private mutation enhancing cognition in affected members of this kindred only, the observation remains important because as the first such mutation identified in humans, it formally establishes the principle that genetic mutations can lead to increased, as well as reduced, cognitive function in man.

Clearly, the evolutionary advantage of this particular mutation is counterbalanced by the concomitant severe visual phenotype, albeit of late onset. Whilst our data do not provide evidence more generally that RIMSl variation influences cognitive performance, it is nevertheless of importance in understanding the genetics of cognition that specific mutations, even in genes that have not shown accelerated evolution in primates, may enhance cognitive function in man. Finally, our findings give additional support for a focus on genes encoding proteins active at the synapse in the search for genetic influences underlying human cognition. Intriguingly, slower reaction times have been posited as the mechanistic link between lower IQ and earlier death (Deary and Der, Psychol Sd. 16, 64-9 (2005)). Molecular dissection of cognition in turn is important not only for biological understanding, through the discovery of specific mechanisms that may underlie particular cognitive functions, but also because rational therapies for cognitive decline may thence emerge.

Tables

Table 1: VIQ and subtest data in members of the kindred tested

Abbreviations: AMIPB= Adult Memory and Information Processing Battery; * norms given up to 70-74 year band; Φnorms given up to 61-75 year band; Onorms given up to 31-40 year band; -φ-norms given up to 80-95 year band Table 2: Correlation coefficients of phenotypes in disease control (PAX6) sample

STORY STORY VERBAL VERBAL

DIGIT PHONEMIC COGNITIVE RECALL RECALL LEARNING LEARNING SEMANTIC HAYLING

VIQ VOCABULARY SPAN FLUENCY ESTIMATES IMMEDIATE DELAYED IMMEDIATE DELAYED FLUENCY SCORE

VlQ 1.00 0.90 0.74 0.44 -0.25 0.38 0.48 0.58 0.42 0.41 0.02

VOCABULARY 1.00 0.55 0.3S -0.27 0.33 0.50 0.43 0.35 0.42 0.03

DIGITSPAN 1.00 0.35 -0.12 -0.05 0.08 0.50 0.18 0.09 -0.35

PHONEMIC

FLUENCY 1.00 -0.03 0.52 0.66 0.44 0.44 0.69 0.13

COGNITIVE

ESTIMATE 1.00 -0.05 0.04 0.26 0.20 0.00 -0.20

STORY

RECALL

IMMEDIATE 1.00 0.90 0.18 0.45 0.53 -0.04

STORY

^ RECALL -j

DELAYED 1.00 0.30 0.55 0.64 -0.08

VERBAL

LEARNING

IMMEDIATE 1.00 0.76 0.47 0.09

VERBAL

LEARNING

DELAY 1.00 0.58 0.02

SEMANTIC

FLUENCY 1.00 0.24

HAYLING

SCORE 1.00

Table 3: JRIMSl and PAX6 group cognitive test scores

Mann-Whitney, actual P values: RIMl affected vs RIMl unaffected, *** P<0.0005; 5 ** PO.001; * PO.01; RIMl affected vs PAX6 affected, f PO.0005; ^® higher score equates to a poorer performance Table 4: Visual acuities in RIMl and PAX6 cohorts

* Individuals from Thompson et al, Neurology 62, 1216-8 (2004)

Claims

1. A method for identifying an agent suitable for use in the enhancement of cognition, which method comprises determining whether or not a test substance binds and/or modulates the Rab3A-interacting molecule (RIMSl) gene, thereby to determine whether or not the test substance is suitable for use in the enhancement of cognition.

2. A method according to claim 1, which method comprises determining whether or not the test substance is a modulator of the expression and/or activity of the RIMSl gene.

3. A method according to claim 1 or 2, which method comprises determining whether or not the test substance is a modulator of the affinity of a polypeptide encoded by the RIMSl gene for the celD-subunit of L-type Ca²⁺ channels or synaptotagmin.

4. A method according to any one of the preceding claims, which method comprises:

(a) providing a polypeptide encoded by the RIMSl gene, a polypeptide substantially similar thereto, or a fragment of either thereof;

(b) contacting a test substance with the polypeptide or fragment under conditions that, in the absence of the test substance, would permit activity of the polypeptide or fragment; and

(c) determining whether or not the test substance is capable of binding to or modulating the activity of the polypeptide or fragment, thereby to determine whether or not the test substance is useful in the enhancement of cognition.

5. A method according to claim 4, which comprises carrying out a reaction in the presence and absence of the test substance in order to determine whether or not the test substance is capable of binding to or modulating the activity of the polypeptide or fragment.

6. A method according to claim 4 or 5, wherein the fragment of a polypeptide encoded by the RIMSl gene or a polypeptide substantially similar thereto comprises an N-terminal Rab3 A-GTP binding site, a zinc finger domain which comprises two Cys₄ zinc fingers and a PDZ domain, a C₂A domain, a C₂B domain or a SH3 -binding domain.

7. A method according to any one of claims 4 to 6, wherein the method is carried out in the presence of Rab3a, Munc 13-1, a N/L-type Ca²⁺ channel protein, SNAP-25, a RM-binding protein, synaptotagmin I, an α-liprin, CAST, PKA or 14-3- 3 or a polypeptide substantially similar to any thereof.

8. A method according to any one of claims 4 to 7, wherein the polypeptide encoded by the RIMSl gene, polypeptide substantially similar thereto or fragment of either thereof comprises an arginine residue at codon 844 or the codon at a corresponding position.

9. A method according to any one of the preceding claims wherein the enhancement of cognition comprises an enhancement of verbal intelligence quotient, vocabulary, digit span, similarities, phonemic fluency, cognitive estimates, immediate story recall, delayed story recall, immediate verbal learning, delayed verbal learning, semantic fluency or performance in the Hayling test.

10. A method for the preparation of a pharmaceutical composition, which method comprises:

(a) identifying an agent suitable for use in the enhancement of cognition by a method according to any one of claims 1 to 9; and

(b) formulating the agent identified in (a) with a pharmaceutically acceptable carrier or diluent.

11. A method for the enhancement of cognition in a host, which method comprises:

(a) identifying an agent suitable for use in the enhancement of cognition by a method according to any one of claims 1 to 9;

(b) formulating the modulator identified in (a) with a pharmaceutically acceptable carrier or diluent; and (c) administering to the host a therapeutically effective amount of the pharmaceutical composition formulated in (b).

12. Use of an agent which is capable of binding to or modulating the activity of the RIMSl gene in the manufacture of a medicament for use in the enhancement of cognition.

13. Use according to claim 12, wherein the agent modulates the affinity of a polypeptide encoded by the RIMSl gene for the αlD-subunit of L-type Ca²⁺ channels or synaptotagmin.

14. Use according to claim 12 or 13, wherein the enhancement of cognition comprises an increase in verbal intelligence quotient, vocabulary, digit span, similarities, phonemic fluency, cognitive estimates, immediate story recall, delayed story recall, immediate verbal learning, delayed verbal learning, semantic fluency or performance in the Hayling test.

15. Use according to any one of claims 12 to 14 for use in the treatment of a neurological disorder.

16. Use according to claim 15, wherein the neurological disorder is characterised by any cognitive impairment or decline.

17. An agent as defined in any one of the claims 12 to 16 for use in the enhancement of cognition.

18. A pharmaceutical composition comprising an agent as defined in any one of claims 12 to 16 and a pharmaceutically acceptable carrier or diluent.

19. A method for the enhancement of cognition in a host, which method comprises modulating the RIMSl gene in the host.

20. An agent identified by a method according to any one of claims 1 to 9.

21. An agent according to claim 20 for use in the enhancement of cognition.

22. Use of an agent according to claim 20 in the manufacture of a medicament for use in the enhancement of cognition.

23. A pharmaceutical composition comprising an agent according to claim 20 and a pharmaceutically acceptable carrier or diluent.

24. A method for the enhancement of cognition in a host, which method comprises administering to the host a therapeutically effective amount of an agent according to claim 20.

25. A method for predicting the level of cognitive function in a subject, which method comprises typing the RIMSl gene of the subject.

26. A method according to claim 25, wherein the typing comprises determining whether the individual has a polymorphism in the RIMSl gene associated with cognitive function.

27. A method according to claim 26, wherein the polymorphism is a causative factor in cognitive function or is in linkage disequilibrium with such a polymorphism.

28. A method according to claim 26 or 27, wherein the polymorphism is exon 14 of the RIMSl gene.

29. A method according to any one of claims 26 to 28 wherein the typing comprises:

(i) determining the identity of the nucleotide at position 2459 of SEQ ID NO: 1;

(ii) determining the identity of a nucleotide at a position corresponding to a nucleotide as defined in (i) in an allelic variant of SEQ ID NO: 1; or (iii) determining the identity of a nucleotide in linkage disequilibrium with a nucleotide as defined in (i) or (ii).

30. A method according to claim 29, wherein the presence of A at position 2459 of SEQ ID NO: 1, or at a corresponding position in an allelic variant thereof, is indicative of increased cognitive function in the subject.

31. A method according to claim 29 or 30, wherein the identity of a nucleotide as defined in (i), (ii) or (iii) is determined for both alleles in the subject.