WO1999037672A1 - Method of screening for neuropharmaceuticals using the drosophila gene volado and its mutants - Google Patents

Abstract

A new memory factor Volado is described along with the corresponding gene, protein sequences, and two mutants identified as Vol1 and Vol2. The locus encodes two isoforms of a novel α-integrin expressed preferentially in mushroom body cells. Volado mutants display an impairment of olfactory memories within 3 minutes after training, indicating an essential role for the integrin in short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. The present invention provides a novel method for screening for cognitive enhancers using the volado and integrin proteins. This procedure involves inserting a gene sequence and coding for a volado or integrin protein into test cells in culture under conditions where said gene sequence expresses the volado or integrin protein in said test cell, adding a test compound to the cell culture or cell homogenate, and measuring the effect of test compound and the activity of the volado or integrin proteins. This screening procedure can also be used in organisms such as Drosophila flies.

Description

METHOD OF SCREENING FOR NEUROPHARMACEUTICALS USING THE DROSOPHILA GENE VOLADO AND ITS MUTANTS

The work herein was supported by grants from the United States Government. The United States Government may have certain rights in this invention.

Field of the Invention

The present invention relates generally to the field of screening for pharmacological agents and drugs which can be used as cognitive enhancers. More specifically, it relates to the field of screening for modulators of integrin function as a screen for cognitive enhancers.

Background of the Invention

The ability to acquire and process information about the environment (learning) and to store and retrieve this information over time (memory) is fundamental for many organisms. Learning and memory are expressed as modifications of animal behavior (conditioning) which emerge from the function of molecules within neurons, the integrated action of many neurons comprising neural circuits, and from the engagement of multiple circuits.

Two broad phases of memory have been distinguished from behavioral and cellular studies: short-term memory and long-term memory. Short-term memory, which lasts from minutes to hours, is thought to occur through changes in synaptic efficacy produced by rapid and transient biochemical alterations in the relevant neurons. Byrne, J.H. et al., in Advances in Second Messenger and Phosphoprotein Research Shenolikar, S. & Nairn, A.C. (eds.) 47-107 (1993); Chetkovich, D.M. et a , Proc. Natl. Acad. Sci. USA 88:6467-6471 (1991); Ghirardi, M. et al., Neuron 9:479-489 (1992); Davis, R. L., Physiological Reviews 76:299-317 (1996); Hawkins, R.D. eta , Annual Rev. Neurosa. 16:625-665 (1993). In contrast, long-term memory, which lasts from days to years, is thought to occur through changes in synaptic efficacy produced by the restructuring of synapses due to altered gene expression. Davis, H.P. & Squire, L.R., Psychol. Bull. 96:518-559(1984); Montarolo, P.G. et /.,Sc/e«ce 234: 1249-1254 (1986); Tully, T. etal., Cell 79:35-47 (1994); Schacher, S. etal., Science 240: 1667-1669 (1988); Bailey, CH. & Kandel, E.R., Annual Rev. Physiol. 55, 397-426 (1993). The formation of long-term memory, but not short-term memory, has therefore been thought to rely upon moφhological restructuring of synapses using mechanisms similar to those used for brain development.

In Drosophila, the formation of olfactory memories is scripted in cyclic AMP (cAMP) signaling in neurons of the mushroom bodies. Davis, R.L., Physiological Reviews 76:299-317 (1996); Davis, R.L., Neuron 11:1-14 (1993); Davis, R.L. & Han, K.-A., Current Biology 6: 146-148 (1996). A significant series of studies linking cAMP signaling, mushroom bodies, and olfactory learning demonstrated that three genes required for normal learning - dunce (dnc), rutabaga (rut), and DCO (the genes for cAMP phosphodiesterase, adenylyl cyclase, and the catalytic subunit of protein kinase A (PKA), respectively) - are all expressed preferentially in mushroom bodies. Davis, R.L., Neuron 11, 1-14 (1993). Moreover, the characterization of two other learning genes of Drosophila is consistent with a dominant role for cAMP in modulating the physiology of neurons that mediate behavioral plasticity. The amnesiac gene encodes a peptide similar to PACAP (pituitary adenylyl cyclase activating peptide) Feany, M. S. & Quinn, W.G., Science 268, 869-873 ( 1995) and dCREB2 encodes a transcription factor that may mediate cAMP-dependent gene expression. Yin, J.C.P. etal., Cell 79 49-58 ( 1994). A leading hypothesis that has emerged from these studies is that mushroom bodies function as the integration and memory center for olfactory learning by employing the cAMP signaling system. Davis, R.L., Physiological Reviews 76:299-317 (1996); Davis, R.L., Neuron 11 : 1-14 (1993). Mushroom bodies are bilateral clusters of about 2500 neurons situated in the dorsal and posterior cortex of each brain lobe. Davis, R.L. & Han, K.-N, (1996). Current Biology 6: 146-148 (1996). These cells extend dendrites into a neuropil (calyces) just ventral to the cell bodies where inputs arrive from the antennal lobes and other centers conveying sensory information. The axons of mushroom body cells fasciculate to form the peduncle that projects anteriorly to the anterior of the brain. There it bifurcates, with some processes extending medially to comprise the neuropil region known as the β and γ lobes, and others extending dorsally to comprise the α lobe Strausfeld, N.J., Atlas of an Insect Brain (1976). Although the mushroom bodies receive inputs from many sensory modalities through the calyces and lobes and are required for olfactory learning, they are not required for olfaction per se (Heisenberg, M. et al, Neurogenetics 2: 1-30 (1985); Menzel, R. et al. The Behavior and Physiology of Bees (L.J. Goodman & R.C. Fisher eds.) (1991); DeBelle, S.J. & Heisenberg, M., Science 263:692-695 (1994).

Despite the coherent evidence pointing to the cAMP signaling system, many different types of molecules must be engaged during learning to effect the overall physiological changes in the relevant neurons. Indeed, an assortment of protein kinases, transcription factors, enzymes involved in neurotransmitter biosynthesis, neuropeptides, and other factors have been suggested to play important roles. Hawkins, R.D. etal., Annu. Rev. Neurosci. 16:625-665 (1993); Grant, S.G. & Silva, A.J., Trends in Neurosciences 17:71-75 (1994); Alberni, CM. et al, Cell 76:1099-1114 (1994); Mello, CN. & Clayton, D.F., J. Neurobiol. 26:145-161 (1995); Huston, J.P. & Hasenohrl, R.U., Behav. Brain Res. 66, 117-127 (1995); Zhuo, M. et al, Nature 368:635-639 ( 1994). The instant invention has isolated a new Drosophila memory gene, Volado (Vot), that encodes a novel α-integrin, a type of cell surface receptor known to dynamically mediate cell adhesion and signal transduction. Hynes, R.O., Cell 69: 11-25 (1992). "Volado" is a Chilean colloquialism with no English counteφart, but is loosely translated as "forgetful" or "absent-minded." In Chile, it is often used in reference to professors and scientists.

Lesions in Vol have a dominant effect upon short-term memory following olfactory conditioning. Remarkably, conditional expression of Vol just before training rescues the memory deficit of Vol mutants. This rescue is reversible, supporting a dynamic role for integrins in neuronal and behavioral plasticity. These data indicate that integrin-mediated signaling or synaptic restructuring underlie the formation, stability, or retrieval of short-term memory.

There is a pressing need for the development of new cognitive enhancers. Our abilities to learn, and to forget, are human attributes often taken foi granted because they operate in the background of our everyday tasks. Their importance surfaces when these abilities are compromised by head trauma, psychiatric or neurological disease, aging, alcoholism, or from other causes. As many as 5% of school-aged children experience grave difficulties with learning to read and spell. Memory disorders are common among the aged, affecting an estimated 12% of the population over age 65. The personal and financial costs of these disorders are staggering, with the cost of Alzheimer's disease alone at between 80-90 billion dollars each year. Therefore, it is incredibly important to discover effective treatments and cures for the numerous types of cognitive disease.

Despite this obvious need, there are few drugs on the market or in development that have a significant impact upon learning and memory cognitive processes. This is because the mechanisms underlying cognitive processes are complex and the many possible molecular targets have yet to be identified. This perhaps explains the diversity of diseases that affect cognitive processes. It also explains why the pharmaceuticals available are now directed toward enhancing the biological activity of only a handful of molecules. Given the likelihood that perhaps one hundred different molecules are involved in cognitive processes, any of these, once identified, could potentially be modulated to enhance cognition with appropriate pharmaceuticals.

This discovery deals with the identification of a new molecule involved in cognitive processes. Using a novel methodology to identify genes and molecules involved in Drosophila cognitive processes, molecules of the volado family of proteins which are essential for normal cognitive processes formation were discovered. These proteins work by participation in signal transduction cascades and principally, in modulating the activity of tyrosine kinase.

This discovery now makes possible the use of expression systems of volado genes to identify pharmaceuticals that increase or decrease their activity.

SUMMARY OF THE INVENTION

An object of the present invention is a screening system for determining cognitive enhancers using the Vol protein in test cells.

An additional object of the present invention is a method for screening for cognitive enhancers using Vol or its corresponding mutants.

An additional object of the invention is using integrins or other analogous proteins as substitutes for use of the Vol proteins.

Thus, in accomplishing the foregoing objects, there is provided in accordance with one aspect of the present invention, a method for detecting cognitive enhancers comprising the steps of inserting a gene sequence encoding for Vol protein into test cells in culture under conditions where said gene sequence expresses the Vol protein in said test cells; adding a test compound to the cell cultures or cell homogenates; and measuring the effect of the test compound on the activity of the Vol protein. In specific embodiments of the present invention, the test cells are selected from a group of invertebrate cells and vertebrate cells. More specifically, they can be mammalian cells selected from the group consisting of human embryonic kidney cells, COS cells or CHO cells, or insect cells selected from Drosophila S2 or Spodoptera SF9 cells with baculovirus vectors.

The specific methods for measuring the activity on Vol protein include cell adhesion assays to ligands applied to solid surfaces such as plastic microtiter wells, or to ligands expressed on other cells, in which case the activity can be measured by cell aggregation. Additional specific embodiments use increases in tyrosine phosphorylation within cells expressing the integrin as a measure of integrin activity.

In another specific embodiment of the present invention, the cognitive enhancers are screened by using volado mutant Drosophila flies.

Other and further objects, features, and advantages will be apparent from the following description of the presently preferred embodiments of the invention, which are given for the puφose of disclosure, when taken in conjunction with the accompanying drawings.

Description of the Drawings

Figure 1 shows the Vol gene structure, transcripts and mutations. Figure 1A shows an EcoRI (R) restriction map of the locus with the position of the Vol¹ enhancer detector element indicated by a triangle. The direction of transcription of the lacL reporter in the enhancer detector element is indicated by the arrow. Two transcription units, Fo/-long ( Vol-\) and o/-short ( Vol-s), were deduced by comparing cDNA sequences with genomic sequences. The first exon of each transcription unit is spliced to a common 2nd exon. Filled boxes represent the open reading frame. The 816 base pair deletion in Vol² is indicated by the line spanning the first exon of Vol-s. Figure IB shows blots of adult head RNA showing the 4.6 and 4.4 kb transcripts of Vol-l and Vol-s, respectively, in Canton-S (cs) and ry animals. Figure 1C shows reverse transcriptase (RT)-PCR analyses of total head RNA from rosy (ry), Vol¹ and Vol¹ adults. Each graded bar represents increasing amounts (from left to right) of a single RT reaction added to the subsequent PCR. Both Vol-l and Vol-s were present in ry; however, the expression of VolA was dramatically reduced in Vol¹ and expression of Vol-s was undetectable in Vol¹. The internal control using PKA primers allowed quantitative comparisons to be made between the various RT-PCR reactions. RNase (+) added prior to the RT reaction abolished all signals.

Figures 2A and 2B show Vol preferentially expressed in mushroom bodies. Figure 2A shows a frontal section of a Vol adult head stained for β-galactosidase activity. Staining (blue signal) was observed within the mushroom body perikarya (mb). The β-galactosidase encoded by the enhancer detector element carried a nuclear targeting sequence which explains the nuclear localization of the histochemical stain. Figures 2B-2D show frontal sections of Canton-S adults after immuno-staining with an affinity-purified antiserum raised against the carboxy-terminus of Vol. Figure 2B shows expression (dark brown signal) observed in the cell bodies (mb) and calyces (c). Figure 2C shows the peduncle (p). Figure 2D shows the α, β, and γ lobes (α, β, γ).

Figure 3 shows memory deficits in Vol mutants. Figure 3 A shows the decay curve of conditioned odor avoidance for two Vol mutants (Vol¹ and Vol¹) and the control strain (ry). N = 8 to 9 for all groups. The mean performance index ± SEM is shown for each genotype at several time points after training. The performance of Vol¹ and VoP was significantly less than ry at all time points. Figure 3B shows the performance of homozygous and heterozygous Vol mutants at 3 and 15 minutes after training. N = 8 to 11 for all groups. There were no significant differences between the homozygous mutant strains and the corresponding heterozygous strains at either time point. Figure 4 shows the lack of neuroanatomical defects in Vol mutants. R and VoP adult frontal sections are shown at the level of the mushroom body perikarya (mb) and calyces (c) after staining with hematoxylin and eosin (H&E) or with an antibody against the nuclear antigen D-mef2, and at the level of the mushroom body lobes (α, β and γ) after staining with anti-fasll or ti-leonardo antisera. No differences between the genotypes were observed in either mutant. Slight differences seen here were due to the plane of sectioning. The posterior to anterior arrangement of sections is from top to bottom.

Figure 5 shows rescue of the Vol memory defect by conditional expression of Vol-s. Figure 5A shows three minute memory without heat shock (NO HS) or 3 h after heat shock (HS 3h) in ry, Vol¹, VS-T2 and VS-T3. Heat shock was for 15 minutes at 37°C N = 6 for all groups. Rescue of the mutant phenotype was exhibited by both VS-T2 and VS-T3; in addition, VS-T2 exhibited some constitutive rescue. Figure 5B shows RT-PCR analyses of Vol-s expression. RT-PCR for ry, VS-T2 and VS-T3 without (-) or 30 minutes after HS (+). Figure 5B upper panels show ry control: HS had no effect on expression of Vol-s or PKA in ry animals (compare duplicate lanes 1 and 2 with lane 7, all of which are from PCR reactions containing equivalent amounts of input cDNA). Quantitation using a BetaGen blot analyzer demonstrated that the signals for both Vol-s and PKA were linear with the mass of input cDNA (graded bar) amplified by PCR. Figure 5B lower panels show Vol transgenics: Vol-s RNA was nearly undetectable in both VS-T2 and VS-T3 in the absence of HS (-). Thirty minutes after HS (+) there was a marked induction of the transgene. Lanes 1, 2 and 7 in the upper panels and all lanes in the lower panels are from PCR reactions seeded with equivalent amounts of input cDNA. RNase treatment (+) prior to RT eliminates all signals. Data are representative of three independent experiments. Figure 5C shows Vol protein was induced after HS in VS-T3. Immunoblotting was performed on extracts from whole flies without (Oh) or 3 and 24 hours after HS. Western blots containing

8 0.5 fly equivalents per lane were incubated with an affinity-purified antiserum generated against the carboxy-terminus of the Vol integrin. This antiserum recognizes both the full-length protein (-135 kDa) as well as the light chain (doublet at -26 kDa). These data, confirmed by detection with an antiserum generated against the extracellular domain (not shown), are representative of 2 experiments. Figure 5D shows three minute memory without HS (NO HS) or 3 (HS 3h) and 24 h (HS 24h) after HS. N = 6 for all groups. VS-T3 showed a behavioral deficit without heat shock, normal performance with HS 3 h prior to training, but a deficit again when HS was given 24 h prior to training. Figure 5E shows RT-PCR analyses of Vol-s RNA expression without (Oh) or 0.5 and 21.5 h after HS. Figure 5E upper panels show expression of Vol-s was not changed after HS in ry animals. Figure 5E lower panels show Vol-s RNA was dramatically elevated in VS-T3 0.5 h after HS, and returns to a low level at 21.5 h after HS. As in Figure 5B PKA expression was not changed by HS in either strain. All lanes are from PCR reactions seeded with equivalent amounts of input cDNA. RNase treatment prior to RT eliminates all signals. Data, from a single experiment performed in duplicate, are representative of two independent experiments.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned as well as those inherent therein. The volado and integrin proteins along with the DNA and protein sequences, methods, procedures, assays, molecules and specific compounds described herein are presently representative of the preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the claims. Detailed Description of the Invention

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The term "cognitive processes" as used herein refers to all aspects of intellectual ability, including the elements of problem solving, memory, levels of consciousness, orientation, attention and mental tracking, comprehension, judgment, calculations, reasoning, perception, planning, and constructional ability.

The term "cognitive enhancers" as used herein refers to any agent (for example a compound, composition or drug) that modifies the efficacy of one or more of the elements of the cognitive processes.

The term "mutant" as used herein refers to an alteration of the primary sequence of volado thus that it differs from the wild type or naturally occurring sequence. In the nucleic acid sequence, mutant can be any change in the sequence, for example changed base, deletion, or addition which results in an altered protein. In the amino acid sequence, the mutant is a peptide or protein whose sequence is altered from the native sequence.

The term "expression system" as used herein refers to a vector, plasmid or cell that contains all the information necessary to produce a protein from the volado gene sequence in Drosophila or its corresponding sequence from a mammalian species.

The term "transfection/transfected" as used herein describes the process of directly introducing the DNA into cells including vertebrate cells, invertebrate cells, bacteria and yeast. This includes introducing DNA by transfection of insect cells and mammalian cells.

10 As used herein, the term "transform/transformation" refers to the result of introducing DNA into a cell where the presence of the DNA genotypically and phenotypically alters a cell in a heritable manner.

The term "reporter" refers to the insertion of a nucleotide sequence downstream from a promoter such that when the promoter is activated the nucleotide sequence is produced in the cell. To be an effective reporter the nucleotide sequence must produce a peptide, protein or other change which can be monitored. For example, it could produce a protein which causes the cells to change color or can be linked to some type of enzyme or antibody reaction in order to detect the presence of the reporter. A skilled artisan readily recognizes that a variety of reporter genes are available for use in the present invention.

The term "Volado" as used herein refers to the gene in Drosophila which encodes the proteins identified in SEQ ID NOS. 1 and 2. A schematic of the gene and certain mutants is shown in Figure 1.

The terms " Vol¹" and " Vol¹" as used herein refer to the two Volado mutants. A schematic of these two mutants is shown in Figure 1.

One aspect of the present invention is the use of Volado proteins to screen for compounds, compositions or drugs to be used as cognitive enhancers. Any agent that increases the biochemical activity of Volado proteins in neurons could make cognitive processes more efficient, especially in cases of disease in with the activity of Volado proteins is compromised. Clones for the Drosophila Volado protein or its mammalian counteφarts are used in expression systems for screening new agents that alter the biochemical function of Volado proteins.

One skilled in the art readily recognizes that a variety of expression systems can be used. These expression systems can be selected from either invertebrate cells, vertebrate cells, bacteria or yeast.

11 For example, the expression system can be comprised of mammalian cells. Examples of mammalian cells which are useful in the present invention include mammalian cells in culture, such as human embryonic kidney cells, COS cells, or CHO cells. Examples of invertebrate cells which can be useful in the present invention include insect cells in culture, such as Drosophila S2 cells or Spodoptera SF9 cells with baculovirus vectors.

Agents that alter the activity of volado or integrins are screened for in several different ways. After expression of a volado or integrin gene in cultured cells, agents are applied and their ability to modulate volado or integrin function is determined by: (1) cell adhesion assays since integrins mediate adhesive functions of cells. These include the binding of expressing cells to ligands deposited on plastic surfaces in microtitre wells or other solid surfaces, or cell aggregation assays in which integrin expressing cells adhere to cells expressing ligands for the integrins, (2) increases in tyrosine phosphorylation, since activation of integrins leads to increased levels of tyrosine kinase. Drosophila mutants that have lowered volado activity sufficient to cause lethality, poor viability, or learning/memory deficiency are used to screen for agents that alter the volado or integrin activity. Agents with this property rescue the viability defects and/or the defects in learning/memory Drosophila mutants that have a lowered volado activity sufficient to cause lethality, poor viability, or learning/memory deficiency are used to screen for agents that alter the volado or integrin activity. Agents with this property rescue the viability defects and/or the defects in learning/memory.

The predicted amino acid sequence of Vol-\ and Vol-s are indicated as SEQ ID NO. 1 and SEQ ID NO. 2 respectively. The complete amino acid sequence for Vol-Ϊ is contained in SEQ ID NO. 1. The first 63 amino acids of Vol-s are listed as SEQ ID NO. 2.

12 Example 1 The Vol Locus Encodes a Novel α-integrin

Approximately 6000 enhancer detector lines were constructed and screened for preferential expression of the lacZ reporter in brain structures as is well known in the art. (See e.g., Han, P.-L. et al, J. Neurobiol. 31 :88-102 (1996)). About 100 lines with preferential mushroom body expression were isolated, including insertions at the dnc, rut, DCO and leonardo (leo) genes. Line 1116 (Vol¹) from this screen also expressed lacZ in mushroom bodies; the enhancer detector element in this line was mapped to cytological position 5 IE.

The region flanking the enhancer detector element was isolated along with wild type genomic and cDNA clones for the locus. The locus is organized into two transcription units, Vol-long (Vol-Ϊ) and o/-short (Vol-s), which encode RNAs of 4.6 and 4.4 kb, respectively (FIG. 1). The Vol-\ RNA is expressed selectively in heads, whereas Vol-s is expressed in both head and body tissues. Mapping experiments showed that the Vol¹ enhancer detector element resides within the first intron of Vol-\ and within the 5' flanking region of Vol-s (FIG. 1 A). Imprecise excision of the element led to the isolation of Vof, an allele with an 816 nucleotide deletion of genomic sequence that removes the first exon of Vol-s (FIG. 1A). Reverse-transcriptase (RT)-PCR analyses of head RNA revealed that expression of the Vol-l transcript was greatly reduced in Vol¹, while the Vol-s transcript was unaffected (FIG. 1C). Conversely, the VoP lesion eliminated the Vol-s transcript without discernible changes in Vol-l (FIG. 1C). Neither allele affected the expression of PKA, the internal control in these experiments (FIG. 1C). The effects of the alleles on expression of the two transcripts, as confirmed by RNA blotting experiments are consistent with the nature of the physical lesions at the gene (FIG. 1A). Thus, the Vol¹ and Vol¹ alleles disrupt Vol-l and Vol-s expression, respectively.

13 The cDNAs for Vol-l and Vol-s predict novel α-integrins of 1115 amino acids differing only in the first 63 amino acids (Fig. IN SEQ ID NO. 1, SEQ ID NO. 2). The Vol proteins contain many hallmarks of other α-integrins. The Vol proteins are 23-28% identical in amino acid sequence with known α-integrins and contain a single transmembrane domain near the carboxy-terminus. The proteins begin with 24 residues of a hydrophobic, putative signal peptide, have 11 potential glycosylation sites [NXT(S)] in the extracellular region, and have three repeats in the extracellular region that match the consensus DX(D N)X(D/N)GXXD, which is well known in the art to be a domain found in proteins that bind divalent cations. (See e. .,Kretsinger, R.H., CRC Critical Reviews in Biochemistry 8: 119-74 (1980)). Moreover, the Vol sequence has a cleavage recognition site (RKRR) in the extracellular domain, a site required for signal transduction by some α-integrins. After cleavage at these sites, the amino-terminal and carboxy-terminal integrin fragments are held together by disulfide bonds. Furthermore, the cytoplasmic domain of Vol contains the consensus sequence, KXFF[K/R]R, which is known in theartto bind calreticulin (See e. ., Dedhar, S., Trends in Biochem. Sci. 19:269-307 (1994)) and regulate integrin affinity for ligand.

Example 2 Expression of Vol in Mushroom Bodies

The Vol¹ mutant preferentially expressed the lacL reporter in the nuclei of mushroom body neurons (FIG. 2A). To determine if the enhancer detector reflected authentic Vol protein expression, immunohistochemical analyses with an antiserum made against the carboxy-terminus of the protein were performed. The Vol antigen was found to be concentrated in the mushroom body perikarya and calyces (FIG. 2B), peduncles (FIG. 2C), and α, β, and γ lobes (FIG. 2D). The calyces, peduncles, and lobes contain the mushroom body dendrites, axons, and axon terminals,

14 respectively. The distribution of the antigen was not noticeably altered in either the Vol¹ or Vol¹ mutants (data not shown), suggesting that both Vol-l and Vol-s isoforms are globally co-expressed in the mushroom bodies. Enriched expression was also observed in the ellipsoid body (not shown), a region of the central complex thought to be involved in the coordination of motor behaviors. The distribution of Vol in the mushroom body calyces and lobes - regions in which mushroom body neurons form synapses with other neurons suggests that the Vol integrins could regulate synapse function.

Example 3 Mutations in Vol Produce a Memory Deficit

The expression pattern of Vol, coupled with preliminary behavioral experiments, suggested that this gene is important for olfactory memory. To test this hypothesis, Vol mutants were assayed for aversive olfactory classical conditioning. Populations of animals were administered electric shock (unconditioned stimulus, US) in the presence of one odor, the conditioned stimulus (CS+), and were subsequently presented a second odor (CS-) without shock. To evaluate discriminative avoidance behavior, the trained animals were allowed to distribute between converging CS+ and CS- odors carried in air currents within a T-maze.

Animals homozygous for the Vol¹ insertion or the Vol¹ deletion performed poorly relative to ry at all time points after training (FIG 3 A; genotype, P = .0001 ; retention interval, P = .0001 ; genotype x retention interval, NS). The effects of these mutations on memory were indistinguishable, suggesting that the two integrin isoforms are functionally redundant. It had been shown previously that neither the enhancer detector itself, nor the expression of lacZ in mushroom bodies per se, have any significant effect upon performance. The performance deficits in Vol mutants were present at the earliest testable time point after training (3 minutes), indicating

15 that the formation, stability, or retrieval of short-term memory is dependent upon integrin function.

To further examine the effects of the Vol alleles on early memory and to investigate their recessive or dominant nature, the performance of animals heterozygous or homozygous for the two lesions of the gene was trained and tested. The Vol.¹ and VoP animals exhibited memory deficits at both 3 and 15 minutes after training (FIG. 3B; 3 minutes, P = .0001; 15 minutes, P = .0001), confirming the results in Figure 3 A. The performance index (PI) of the VoI^l/+ and VoPl+ heterozygous animals was similarly reduced relative to ry, but was not significantly different from the corresponding homozygous mutants (FIG. 3B; Vol¹ vs. ry at 3 minutes, P = 0001; at 15 minutes, P = .0001; Vol¹ vs. ry at 3 minutes, P = .00015; at 15 minutes, P = .0001; Vol¹ vs. Vol^l/+ at 3 minutes, NS; at 15 minutes, NS; Vol² vs. Vol²/+ at 3 minutes, NS; at 15 minutes, NS). Trans-heterozygous animals, Vol^llVoP, also exhibited a PI equivalent to FO/V+ or VoP/+. Thus, as with dnc, rut, turnip, radish, and cabbage, mutations in Vol have a dominant effect on memory. The dominant effect is particularly noteworthy for Vol alleles, since three out of the four transcription units were preserved in animals heterozygous for Vol-l or Vol-s lesions. These data support the existence of a threshold requirement for Vol expression in the processes underlying memory, making them acutely sensitive to decreased expression of this gene.

Example 4 Evaluation of Vol Sensorimotor Processes

To eliminate the possibility that the poor performance of Vol mutants was due to defects in sensorimotor processes, their ability to sense and avoid electrical shock pulses and the odors used for conditioning was tested. The avoidance behavior of Vol mutants and control animals to electrified grids and odors used for conditioning at multiple strengths of these stimuli was

16 indistinguishable. For example, the avoidance indices to 0.8 ml octanol were 63±4, 68±4, and 65±5 for ry, Vol¹ and Vol¹, respectively. The moφhology of the brain was explored with a particular emphasis on mushroom bodies to determine whether the poor performance was attributable to defects in brain structure. Serial paraffin sections of control and mutant brains failed to reveal any discernible differences in moφhology when stained with Hematoxylin and Eosin (H&E); an antibody against the nuclear antigen D-mef2, which reveals a subset of mushroom body cell nuclei; an antibody against the leo gene product, which delineates the mushroom body calyces, cell bodies, peduncles, and lobes, or an antibody against fasll, which reveals a subset of the mushroom body lobes (FIG. 4). Therefore, neither sensorimotor or gross neuroanatomical defects can account for the memory deficit of Vol mutants.

Example 5 Conditional Rescue of the Vol Memory Deficit

Direct evidence for a role of the integrin in physiological processes underlying memory is obtained through the conditional expression of a Vol transgene. Four transgenic lines were generated that harbored the Vol-s cDNA under the control of the hsp70 promoter in the Vol¹ background (Vol-s mutant). Animals were heat-shocked for 15 minutes at 37° C, rested for 3 hours to allow for recovery and expression of the transgene, and subsequently trained and tested for 3 minute memory. Two of the transgenic lines failed to show any evidence of heat-dependent rescue in pilot experiments, presumably due to genomic position effects, and were not analyzed further. Two other lines, VS-T2 and VS-T3, were analyzed extensively for olfactory memory.

Normal olfactory memory of ry control animals and the residual memory in Vol² mutants was unaffected by heat shock (FIG. 5 A; for ry, NO HS vs. HS 3h, NS; for Vol², NO HS vs. HS 3h, NS). In the absence of heat shock, VS-T3 transgenic animals exhibited mutant levels of

17 performance, but VS-T2 transgenic animals showed partial rescue of memory, possibly due to elevated basal expression of the transgene in mushroom bodies (FIG 5 A; NO HS, VS-T3 vs. ry, P = .0001; VS-T3 vs. Vol², NS; VS-T2 vs. ry, P = .0008; VS-T2 vs. Vol², P = .0003). However, the 3 minute memory of VS-T2 and VS-T3 animals when tested 3 hours after heat shock, was significantly improved over that after no heat shock and was indistinguishable from the ry control (FIG. 5A; HS vs. NO HS, VS-T2, P = .0045; VS-T3, P = .0005; with HS, ry vs. VS-T2, NS; ry vs. VS-T3, NS). Therefore, conditional expression of Vol-s just before behavioral training was sufficient to fully rescue the mutant phenotype. This rescue cannot be attributed to altered sensorimotor abilities, since avoidance behavior to electric shock and odors by the control and transgenic animals was indistinguishable, with or without heat shock. These data provide compelling evidence that the defective α-integrin expression in Vol mutants is responsible for the memory deficits, and that the Vol integrin participates in the physiological processes underlying memory.

To determine whether the behavioral rescue was paralleled by the induction of the Vol transgene, Vol RNA and protein levels were assayed before and after heat shock. As assayed by RT-PCR- heat shock had no effect on the quantity of Vol-s RNA in ry control animals (FIG. 5B), but produced a -100-fold and -1000-fold increase in the level of Vol-s RNA in the VS-T2 and VS-T3 transgenic lines, respectively (FIG. 5B). The level of PKA RNA served as an internal control and was unaffected by Vol mutation (FIG. IB), Vol transgene expression, or heat shock (Fig. 5B). Western blotting was used to measure Vol protein using an affinity-purified antiserum raised against the carboxy-terminus of Vol that recognized the intact Vol protein (Mr = 125 kDa), as well as the carboxy terminal cleavage fragment produced by proteolysis (Mr = 21 kDa). This antiserum identified a band (sometimes a doublet) of 26 kDa in ry that was not found in Vol¹ mutants or in non-heat shocked transgenics animals (FIG. 5C). This band represents the

18 carboxy-terminal cleavage fragment. The full-length protein was not detected in ry extracts, presumably due to reduction of the disulfide bond that links the heavy and light chains. In contrast to the ry control, a large increase in the expression of both the Vol fiill-length protein and light chain was found in VS-T3 extracts obtained three hours after heat shock (FIG. 5C). Detection of the intact molecule suggests that the protease is limiting after over-expression of Vol. Induction of Vol protein was also observed in VS-T2. Thus, there was a marked elevation of the Vol α-integrin in the VS-T2 and VS-T3 transgenics 3 hours after heat shock. These RNA and protein analyses demonstrated that Vol was conditionally expressed at the time of behavioral assay, confirming that replacement of the Vol integrin in adulthood rescued the memory deficit.

Despite the arguments presented above for a physiological role for Vol, it seemed plausible that the α-integrin might be required for a final step in synapse formation that occurs normally during development, and that the induced expression of the integrin during adulthood simply allows completion of this terminal step. In other words, the presence of the integrin might be essential for synapse formation but not for synapse stability. If so, the induction of Vol expression might cause a long-lasting or permanent rescue of memory. If, on the other hand, Vol participates in a non-developmental, acute aspect of neuronal function, the rescue of memory produced by induction of the Vol transgene would be expected to be transient and reversible, persisting only as long as adequate levels of the Vol integrin are present.

To distinguish between these possibilities, whether induction of Vol produced a permanent or a reversible restoration of memory was explored. As before (FIG. 5 A), heat shock treatment 3 hours prior to training and testing dramatically improved the performance of VS-T3 animals (FIG. 5D; for VS-T3, NO HS vs. HS 3h, P = .0001). This rescue was completely reversible. The memory in heat-shocked VS-T3 transgenic animals returned to mutant levels when the animals

19 were trained and tested 24 hours after heat shock (Fig. 5D, for VS-T3, no HS vs. HS 24h, NS, HS 3h vs HS 24h, P 0001, for HS 24h, ry vs VS-T3, P = 0001, Vol² vs VS-T3, NS).

Fo/RNA and protein expression in the transgenic animals, which reflect abundance in all cells, were markedly elevated at early time points after heat shock (0.5 and 3 hours, respectively), and decreased to low levels at late time points (21.5 and 24 hours, respectively) (FIGS. 5C, 5E). Thus, the induction and ensuing decline of Vol expression correlated well with the behavioral rescue and subsequent return to a state of memory impairment. The temporal parallels in RNA level, protein expression, and memory argue strongly that Vol mediates a physiological process that is critical to memory formation, stability, or retrieval

Collectively, these results support three important points First, reduced expression of the Vol integrin produces an impairment in memory without altering sensorimotor abilities or neuroanatomy Second, this phenotype is rescued by the expression of the integrin just before training in the adult animal, demonstrating an adult role for this adhesion molecule. Third, the reversibility of the memory rescue indicates that the Vol integrin mediates a dynamic process underlying memory

Example 6 Integrins, Synaptic Plasticity, Mushroom Bodies and Memory

The results of the identification, isolation, and characterization of Vol properties of Vol similar to those seen in studies of four other learning genes with similar expression patterns: dnc, rut, DCO and leo (For examples of other learning genes see e.g., Davis, R.L., Neuron 11.1-14

(1993), Skoulakis, E.M C & Davis, R L , Neuron 17 931-44 (1996) Nighorn, A etal, Neuron

6:455-467 (1991), Han, P -L et al, Neuron 9 619-627 (1992), Skoulakis, E et al, Neuron

11:197-208 (1993 )) The discovery of another memory mutant in which the underlying gene is

20 expressed preferentially in mushroom bodies reinforces the conclusion that these cells play a crucial role in olfactory learning and memory. The mushroom bodies may serve as centers for the reception and integration of many different forms of sensory information, including information about odors and electric shock presented during olfactory classical conditioning. The converging sensory information is thought to alter the physiology of mushroom body cells to encode memory, employing the cAMP signalling system as well as other types of molecules. The results with Vol demonstrate that integrins are included in the family of molecules required for memory formation.

Integrins have diverse biological roles in apoptosis, cell cycle regulation, cell migration, blood clotting and leukocyte function. They function as αβ heterodimers, mediating adhesive interactions of cells with the extracellular matrix or with counter-receptors displayed by other cells. Most interestingly, they dynamically transduce information across cell membranes bi-directionally. Ligand binding to integrins induces a variety of signalling events within cells, and agonist activation of classical signal transduction pathways can alter the affinity of integrins for their ligands within a time-frame of a few minutes.

The dynamic adhesion role for integrins offers a hypothesis for how the Vol integrin, and integrins in general, underlie alterations in synaptic plasticity and behavior. It is envisioned that release of a modulatory neurotransmitter upon a mushroom body neuron might mobilize the intracellular events leading to an altered binding of integrins displayed at another synapse made by that cell. For example, protein kinase C or ras activation is known to activate integrin binding. This could produce a rapid (within minutes) alteration in the structure and efficacy of that synapse. The modulation of integrin affinity for ligands might also underlie the construction or pruning of existing synapses, or the activation of silent synapses during learning or memory encoding. Thus, the formation of short-term memory may employ synaptic rearrangements like long-term memory, but through an integrin-dependent, and protein synthesis-independent

21 mechanism. Alternatively, it is possible that integrins modulate neuronal function through ligand binding followed by activation of intracellular signalling events. For example, integrins are known to stimulate a number of signal transduction pathways in many types of cells, including Ca²⁺ mobilization, tyrosine kinase activation, and induction of protein kinase C. Integrin-dependent stimulation of these pathways in the relevant neurons may be fundamental to learning and memory.

The results demonstrating a role for integrins in behavioral plasticity mesh well with studies showing integrin-dependent modulation of synaptic plasticity. Notably, peptide inhibitors of integrin binding have no effect upon the formation of long-term potentiation, but block the maintenance of this form of synaptic plasticity. In addition, the enhancement of neurotransmitter release from motor nerve terminals due to muscle stretch is blocked by the peptide inhibitors. Psychological studies coupled with these behavioral studies, support a model in which integrins mediate dynamic processes at synapses underlying memory formation or stability.

EXAMPLE 7 Cloning, Mutagenesis and Transgenic Animals

Genomic sequences flanking the Vol¹ insertion were isolated by plasmid rescue. Wild-type genomic clones were isolated from a Canton-S library made in lambda DASHII; cDNA clones were isolated from libraries prepared from Drosophila head RNA. The 4.6 kb Vol-l RNA sequence is represented by a cDNA of -4600 residues. The 4.4 kb Vol-s RNA is represented by a 3366 bp cDNA.

The VoP- excision was isolated after dysgenesis. Flies carrying the Vol¹ enhancer detector element were crossed to Xcs; CyO/2cs; ry Sb P[ry+,D2-3,99B]/TM6,Tb. ("cs" denotes chromosomes derived from a wildtype Canton-S stock.) Dysgenic progeny carrying CyO were

22 crossed to Xcs; CyO/leo¹³⁷⁵; ry⁵⁰⁶-iso animals. CyO; ry^-iso progeny were selected for stocks, ry^-iso is an isogenic ry⁵⁰⁶ chromosome. Excision derivatives were characterized by Southern blotting, extensive PCR analyses, and sequencing of PCR products that cross deletion break points.

Due to the nonspecific behavioral effects of mini-white vectors, a new P-factor vector (pCy-20-dbhsp) for driving genes behind the hsp70 promoter was constructed with ry+ as the selectable marker. This vector, containing a Mlul-Kpnl fragment of the Vol-s cDNA was injected into VoP embryos. Chromosomal localization of the transgenes and the generation of homozygotes for the transgenes were performed by standard crosses. The presence of the VoP allele in the transgenic animals was confirmed by PCR analyses of genomic DNA. The Vol transgene resides on the X and 2nd chromosome, respectively, in VS-T2 and VS-T3.

Flies were collected in clean food vials, transferred to pre-warmed food vials, and immersed in a 37° C water bath for 15 minutes. Following heat-shock, flies were transferred to room-temperature food vials and stored until testing.

EXAMPLE 8 RNA Blots and RT-PCR Analyses

For RNA blots, polyA+ RNA was isolated after tissue homogenization in guanidinium-isothiocyanate, banding in CsCl gradients, and by batch adsoφtion to oligo-(dT) cellulose. Ten μg polyA+ RNA was fractionated per lane by formaldehyde-agarose gel electrophoresis. For RT-PCR experiments, total RNA from heads or whole flies was extracted using Trizol (Gibco-BRL) according to the manufacturer's instructions. Each RT reaction contained 3 μg total RNA, 500 ng oligo-(dT), and 200 U Superscript II (Gibco-BRL) in a total volume of 20 μl. The reactions were incubated at 42°C for 50 minutes and digested with 10 U

23 Sαw3AI and 10 U Acil at 37°C for 3 hours. RNase A treatments (10 μg) prior to RT reactions were for 1 hour at 37°C Aliquots of 0.2-5.0% of the RT reactions were amplified using PCR for 20 cycles. For amplification of Vol first-strand cDNAs, an antisense primer that anneals to the common 2nd exon of Vol (857 nucleotides 3' of translation start site) was used in combination with sense primers specific for the first exon of either Vol-l or Vol-s (84 and 118 nucleotides 5' to translation start site, respectively). For amplification of PKA, primers that anneal to the 2nd exon of DCO were used. PCR products (942, 975 and 356 bp for Vol-l, Vol-s and PKA, respectively) were electrophoresed in agarose gels, blotted, and hybridized to 32P-labelled probes.

EXAMPLE 9 Histology, Generation of Antisera, and Immunoblotting β-galactosidase staining and H&E staining was performed as is known in the art. (See e.g., Han, P.-L. etal. . Neurobio 31 :88-102 (1996); Skoulakis, E.M.C. &Davis, R. .,Neuron 17:931-44 (1996); Han, P.-L. et al, Neuron 9:619-627 (1992); Skoulakis, E.M. etal, Neuron 11:197-208 (1993); Han, K.-A. et al, Neuron 16:1127-35 (1996)).

For generation of antisera, rabbits were injected with a purified GST -Vol fusion protein containing either Vol amino acid sequence 1087-1 1 15 (carboxy-terminus) or 358-496 (extracellular domain). For immunohistochemistry using an -Vol antisera and the anti-fasll monoclonal antibody 1D4, adult heads were fixed in 4% paraformaldehyde at 4 ° C for 2 hours and incubated in 25% sucrose in Ringer's solution at 4°C overnight. Ten mm serial cryosections were incubated with affinity-purified anti- Vol or anti-fasll antibody at 4 ° C overnight. For anti-D-mef2 and anti-leonardo staining, adult heads were fixed in Carnoy's for 4 hours, embedded in paraffin, sectioned and incubated with the appropriate antiserum overnight at 23 °C. In all cases, the antigen/antibody complexes were visualized using the Elite Vectastain ABC kit (Vector

24 Laboratories). For immunoblotting, protein extracts were prepared by homogenizing whole flies in 2X Laemli's sample buffer containing 1% β-mercaptoethanol at 75 °C for 30 minutes. Fly extracts (0.5 fly equivalents per lane) were electrophoresed on SDS-polyacrylamide gels and blotted onto PVDF membranes (Millipore). Blots were incubated with affinity-purified anύ-Vol sera overnight at 4°C, HRP-conjugated goat-anti-rabbit IgG (Jackson Laboratories) for 1 hour at 23 °C, and visualized with SuperSignal Chemiluminescent substrate (Pierce).

EXAMPLE 10 Behavioral Analyses

The differential olfactory conditioning paradigm pairing the presentation of one odor with aversive shock and a second odor with the absence of shock, was used to assess learning and memory performance. Training and testing were performed blind to strain under dim red light at

23-25°C and 63-68% relative humidity using procedures well known in the art. (See e.g.,

Skoulakis, E.M.C & Davis, R.L., Neuron 17:931-944 (1996)). In each group, a performance index (PI) was calculated as the fraction of flies that avoided the CS+ minus the fraction of flies that avoided the CS-, and multiplied by 100. In practice, PI scores ranged from 0 (naive behavior) to 100 (perfect performance). Because the minimum possible time between training and testing is 3 minutes (due to handling and recovery of flies after transfer), 3 minute memory reflects the earliest testable time point. To test longer-term memory, the flies were returned as a group to their collection vials for the appropriate retention interval and then tested as above. Odor avoidance was calculated as the fraction of flies that avoided the odor in one arm minus the fraction of flies that avoided fresh air (and multiplied by 100) provided in the control arm.

Electroshock avoidance was calculated similarly.

25 EXAMPLE 11 Statistics

Statistical analyses were performed with Statview 2.0 (Abacus Concepts, Berkeley, CA).

Overall ANOVAs were followed by planned comparisons contrasting the relevant groups. Error rate due to multiple comparisons was controlled by dividing the alpha level by the number of comparisons being performed on a given set of data.

REFERENCES

All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents, publications herein are incoφorated by reference to the same extent as if each individual publication was specifically and individually indicated to be incoφorated by reference.

Byrne, J. H., Zwartjes, R., Homayouni, R., Critz, S. D. & Eskin, A., Roles of second messenger pathways in neuronal plasticity and in learning and memory, in Advances in Second Messenger and Phosphoprotein Research (eds Shenolikar, S. & Nairn, A.C) 47-107 (New York; Raven Press, 1993).

Chetkovich, D.M., Gray, R., Johnston, D., & Sweatt, J.D., N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2+ channel activity in area CAl of hippocampus. Proc. Natl. Acad. Sci. USA 88, 6467-6471 (1991).

26 Ghirardi, M., Braha, O., Hochner, B., Montarolo, P. G, Kandel, E. R. & Dale, N., Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons. Neuron 9, 479-489 (1992).

Davis, R. L., Physiology and biochemistry of Drosophila learning mutants. Physiological Reviews 76, 299-317 (1996).

Hawkins, R. D., Kandel, E. R. & Siegelbaum, S. A., Learning to modulate transmitter release: themes and variations in synaptic plasticity (Review). Annu. Rev. Neurosci. 16, 625-665 (1993).

Davis, H. P. & Squire, L. R., Protein synthesis and memory: a review. Psychol. Bull. 96, 518-559 (1984).

Montarolo, P. G., Goelet, P., Castellucci, V. F., Morgan, J., Kandel, E. R. & Schacher, S., A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. Science 234, 1249-1254 (1986).

Tully, T., Preat, T., Boynton, S. C & Del Vecchio, M., Genetic dissection of consolidated memory in Drosophila. Cell 79, 35-47 (1994).

Schacher, S., Castellucci, V. F. & Kandel, E. R., cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis. Science 240, 1667-1669 (1988).

27 Bailey, C H. & Kandel, E. R., Structural changes accompanying memory storage. Annu. Rev. Physiol. 55, 397-426 (1993).

Davis, R. L., Mushroom bodies and Drosophila learning. Neuron 11, 1-14 (1993).

Davis, R. L. & Han, K.-A., Mushrooming mushroom bodies. Current Biology 6, 146-148 (1996).

Feany, M. S. & Quinn, W.G. A neuropeptide gene defined by the Drosophila memory mutant amnesiac. Science 268, 869-873 (1995).

Yin, J. C. P., Wallach, J. S., Del Vecchio, M., Wilder, E. L., Zhou, H., Quinn, W. G & Tully, T., Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79, 49-58 (1994).

Grant, S.G.& Silva, A.J., Targeting learning. Trends in Neurosciences 17,71-75 (1994).

Alberni, C M., Ghirardi, M., Metz, R. & Kandel, E. R. C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia. Cell 76, 1099-1114 (1994).

Mello, CN. & Clayton, D.F., Differential induction of the ZEΝK gene in the avian forebrain and song control circuit after metrazole-induced depolarization. J. Νeurobiol.26, 145-161 (1995).

Huston, J. P. & Hasenohrl, R. U., The role of neuropeptides in learning: focus on the neurokinin substance P. (Review). Behav. Brain Res. 66, 117-127 (1995).

28 Zhuo, M., Hu, Y., Schultz, C, Kandel, E. R. & Hawkins, R. D., Role of guanylyl cyclase and cGMP-dependent protein kinase in long-term potentiation. Nature 368, 635-639 (1994).

Hynes, R. O., Integrins: Versatility, modulation, and signalling in cell adhesion. Cell 69, 11-25 (1992).

Han, P.-L., Meller, V. & Davis, R. L., The Drosophila brain revisited by enhancer detection. J. Neurobiol. 31, 88-102 (1996).

Skoulakis, E.M.C. & Davis, R.L., Olfactory learning deficits in mutants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron 17, 931-44 (1996).

Kretsinger, R.H., Structure and evolution of calcium-modulated proteins. CRC Critical Reviews in Biochemistry. 8, 119-74 (1980).

Barr, P. J.. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66, 1-3 (1991).

Delwel, G. O., Hogervorst, F. & Sonnenberg, A. Cleavage of the a6A subunit is essential for activation of the aόAbl integrin by phorbol 12-myristate 13-acetate. J. Biol. Chem. 271, 7293-7296 (1996).

Diamond, M. S. & Springer, T. A., The dynamic regulation of integrin adhesiveness. Curr. Biol. 4, 506-517 (1994).

29 Dedhar, S., Novel functions for calreticulin: interaction with integrins and modulation of gene expression? Trends in Biochem. Sci. 19, 269-307 (1994).

Bausenwein, B., Muller, N. R. & Heisenberg. M., Behavior-dependent activity labeling in the central complex of Drosophila during controlled visual stimulation. Journal of Comparative Neurology 340, 255-68 (1994).

Nighorn, A., Healy, M. J. & Davis, R. L., The cyclic AMP phosphodiesterase encoded by the Drosophila dunce gene is concentrated in the mushroom body neuropil. Neuron 6, 455-467 (1991).

Han, P.-L., Levin, L. R., Reed, R. R. & Davis, R. L., Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects. Neuron 9, 619-627 (1992).

Skoulakis, E. M., Kalderon. D. & Davis, R. L., Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory. Neuron 11,197-208 (1993).

Han, K.-A., Millar, N., Grotewiel, M.S. & Davis, R.L., DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 16, 1127-35 (1996).

30 Sastry, S. K. & Horwitz, A. F., Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signalling. Current Opinion in Cell Biology 5, 819-31 (1993).

Clark, E. A. & Brugge, J. S., Integrins and signal transduction pathways: The road taken. Science 268, 233-239 (1955).

Zhang, Z., Vuori, K., Wang, H.-G, Reed, J. C & Ruoslahti, E., Integrin activation by R-ras. Cell 85, 61-69 (1996).

Staubli, U., Vanderklish, P. & Lynch, G. Rapid Communication: An inhibitor of integrin receptors blocks long-term potentiation. Behav. Neural Biol. 53, 1-5 (1990).

Bahr, B. A., Staubli, U., Xiao, P., Chun, D., Ji, Z.-X., Esteban, E.T. & Lynch, G, Arg-gly-asp-ser-selective adhesion and the stabilization of long-term potentiation: pharmacological studies and the characterization of a candidate matrix receptor. J. Neurosc. 17, 1320-1329 (1997).

Chen, B.-M. & Grinnell, A. D., Integrins and modulation of transmitter release from motor nerve terminals by stretch. Science 269, 1578-1580 (1995).

Jones, L.S., Integrins: possible functions in the adult CNS. TINS 19, 68-72 (1996).

31 Pirrotta, V., Cloning Drosophila genes, in Drosophila, a practical approach, (ed Roberts, D.B.) 83-110 (Oxford. IRL Press, 1986).

Zhang, S.D. & Odenwald, W.F., Misexpression of the white (w) gene triggers male-male courtship in Drosophila. Proc. Nat. Acad. Sci. USA 92, 5525-5529 (1995).

Hing, A.L. & Carlson, J.R., Male-male courtship behavior induced by ectopic expression of the Drosophila white gene: role of sensory function and age. J. Neurobil. 30, 454-64 (1996).

Lindman, H. R., Analysis of Variance in Experimental Design. Springer- Verlag: New York, 1992).

32 SEQUENCE LISTING

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Neuropharmaceuticals Using the Drosophila Gene Volado and Its Mutants

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(ii) MOLECULE TYPE: protein

33 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Asn Ala Glu Ser Thr Met Phe Pro His lie Phe Leu Ala Leu Leu 1 5 10 15

Ala Leu lie Ser His lie Glu Ala Phe Asn Phe Met Pro Arg Pro Ser 20 25 30

Arg Val lie Asn Ser Pro Lys His Leu Lys Phe His lie Asn Gin Thr 35 40 45

Arg Ser Ser Tyr Phe Gly Tyr Thr Leu Val lie Arg Gin Thr Ser lie 50 55 60 lie Val Gly Ala Pro Arg Ala Gin Ser Thr Leu Glu Ser Gin Arg Thr 65 70 75 80 lie Asn Glu Thr Gly Ala lie Tyr Arg Cys Ser Leu Thr Asn Gly Val 85 90 95

Cys Ser Pro Tyr Val Leu Asp Ser Arg Gly Asn Val Asp Ala Pro Tyr 100 105 110

Ser Glu Tyr Thr Phe Asp Ser Glu Arg Lys Asp Phe Gin Trp Leu Gly 115 120 125

Gly Ser Met Asp Gly Gly Thr Lys Asp Thr Asp Lys Leu Leu Val Cys 130 135 140

Ala Pro Arg Phe Tyr Ala Pro Ser Ser Arg Asp Asn His Leu His Gly 145 150 155 160

Val Cys Tyr Trp Val Asn Asn Thr Val Ala Ser Thr Pro Gin His Val 165 170 175

Thr Arg lie Ser Pro Leu Arg Leu Lys Ser Glu Gin Val Lys Glu Glu 180 185 190

Asp Asn Gly Asn Lys Ala Ser Phe Phe Tyr lie Met Gly Glu Leu Gly 195 200 205

Leu Ser Ala His Val Ala Asp Asp Asn Thr Lys Phe Leu lie Gly Ala 210 215 220

Pro Gly lie Asn Thr Trp Arg Gly Ser Val lie Leu Tyr Arg Gin Val 225 230 235 240

Asp Pro Val Asp Asn Pro Thr Ala Ser Arg Arg Asp Thr Ser Lys Ala 245 250 255

Leu Arg Arg Thr Tyr Arg Asp Val Asp Ser Asn Asp Tyr Thr Pro Glu 260 265 270

34 His Tyr Ala Pro Glu lie Pro Thr Pro Gly Leu Trp Gly Gin Glu Glu 275 280 285

Asp Ser Tyr Phe Gly Tyr Ala Val Ser Ser Gly Phe Phe Asp Ser Ser 290 295 300

Asn Pro Thr Lys Leu Leu Tyr Val Ala Thr Ala Pro Gin Ala Asn Lys 305 310 315 320

Gin Ser Gly Glu Ala Tyr lie Phe Asp Val Arg Gly Lys Ser lie His 325 330 335

Lys Tyr His Val Phe Arg Gly Glu Gin Phe Gly Glu Tyr Phe Gly Tyr 340 345 350

Ser Val Leu Ala Glu Asp Leu Asn Gly Asp Gly Lys Thr Asp Val He 355 360 365

Val Ser Ala Pro Gin His Ala Leu Glu Asp Ser His Asp Asn Gly Ala 370 375 380

He Tyr Val Phe He Asn Lys Gly Phe Phe Asn Phe Glu Arg Gin He 385 390 395 400

Leu Arg Ser Pro Val Glu Thr Met Ala Arg Phe Gly Thr Ala Leu Ser 405 410 415

Arg Leu Gly Asp He Asn His Asp Gly Tyr Asn Asp Val Ala Val Gly 420 425 430

Ala Pro Phe Ala Gly Asn Gly Thr Val Phe He Tyr Leu Gly Ser Glu 435 440 445

Asn Gly Leu Arg Asp Gin Pro Ser Gin Arg Leu Asp Ala Pro Ser Gin 450 455 460

Gin Pro Ser Lys Tyr Gly Ser His Met Phe Gly His Gly Leu Ser Arg 465 470 475 480

Gly Ser Asp He Asp Gly Asn Gly Phe Asn Asp Phe Ala He Gly Ala 485 490 495

Pro Asn Ala Glu Ala Val Tyr Leu Tyr Arg Ala Tyr Pro Val Val Lys 500 505 510

Val His Ala Thr Val Lys Ser Glu Ser Arg Glu He Lys Pro Glu Gin 515 520 525

Glu Lys Val Lys He Thr Ala Cys Tyr Arg Leu Ser Thr Thr Ser Thr 530 535 540

Asp Lys Leu Val Gin Glu Gin Glu Leu Ala He Arg He Ala Met Asp 545 550 555 560

Lys Gin Leu Lys Arg Val Lys Phe Thr Gin Thr Gin Thr Asn Glu He 565 570 575

35 Ser Phe Lys Val Asn Ala Asn Phe Gly Glu Gin Cys Arg Asp Phe Glu 580 585 590

Thr Gin Val Arg Tyr Ser Glu Lys Asp He Phe Thr Pro He Asp Leu 595 600 605

Glu Met His Tyr Glu Leu Thr Lys Lys Val Pro Asp Ser Glu Glu Phe 610 615 620

Cys Glu Thr Cys Ala Val Val Asp Pro Thr Glu Pro Lys Val Ser Thr 625 630 635 640

Gin Asn He He Phe Ser Thr Gly Cys Ala Thr Asp Val Cys Thr Ala 645 650 655

Asp Leu Gin Leu Arg Ser Lys Asn Val Ser Pro Thr Tyr He Leu Gly 660 665 670

Ser Ala Asp Thr Leu Arg Leu Asn Tyr Glu He Thr Asn He Gly Glu 675 680 685

Thr Ala Tyr Leu Pro Gin Phe Asn Val Thr Ser Thr Ser Arg Leu Ala 690 695 700

Phe Ala Gin Val Pro Gly Asn Cys Lys Val Val Asp Ala Val Met Val 705 710 715 720

Cys Asp Leu Asn Arg Gly Arg Pro Leu Ala Lys Gly Asp Thr Asp Ser 725 730 735

Val Thr He Ser Phe Asp Val Ser Gin Leu Ser Gly Gin Ser Leu He 740 745 750

Ser His Ala Glu Val Phe Ser Thr Gly Tyr Glu Gin Asn Pro Thr Asp 755 760 765

Asn Arg Gin Thr Asn Val He Gly Leu Lys Glu Phe Thr Glu He Asp 770 775 780

Ala Ser Gly Gly Gin Thr Asn Arg Gin He Asp Leu Glu His Tyr Ser 785 790 795 800

Asn Ser Ala Glu He Val Asn Asn Tyr Glu He Lys Ser Asn Gly Pro 805 810 815

Ser Val He Glu Gin Leu Thr Val Ser Phe Tyr He Pro He Ala Tyr 820 825 830

Lys Val Ala Gly Ser Thr Ala He He Pro He He Asn Val Thr Ser 835 840 845

Leu Lys Met Gin Ala Ser Tyr Asp Ser Gin Leu Leu Ser He Asp Leu 850 855 860

Tyr Asp Gin Asn Asn Thr Met Leu Val Val Asp Pro Val Glu Val Thr 865 870 875 880

36 Thr Thr Leu Ser Gly Gly Leu Glu Arg Thr Val He Thr Gin Asn Arg 885 890 895

Gin Ser Tyr Asp He His Thr Ser Gly His Val His Gin Thr Met Glu 900 905 910

Val Leu Asp Thr Ser Met Val Ala Thr Ala Ser Met Ser Arg Lys Arg 915 920 925

Arg Asp Leu Lys Ala Leu Thr Ala Asn Arg Glu Gin Tyr Ala Arg He 930 935 940

Ser Asn Val Lys Ala His Asp Leu Leu Ser Asp Asp Phe Lys Gly Lys 945 950 955 960

Leu Pro Val Asn Arg Thr He Val Phe Asn Cys Arg Asp Pro Glu Met 965 970 975

Thr He Cys Val Arg Ala Glu Met Arg Val His Phe Arg Pro Glu Lys 980 985 990

Ser He Asn Leu Asn Met Arg Tyr Ser Val Asp Leu Asn Glu Val Asn 995 1000 1005

Ala He Leu Val Asp Pro Trp Glu Tyr Phe Val He Leu Thr Asp Leu 1010 1015 1020

Lys Leu Gin Lys Lys Gly Asp Pro Thr Ser Thr Ser Phe Ser He Asn 1025 1030 1035 1040

Arg Arg He Glu Pro Asn He He Ser Lys His Gin Glu Thr Gly Leu 1045 1050 1055

Pro He Trp He He He Val Ser Val He Gly Gly Leu Leu Leu Leu 1060 1065 1070

Ser Ala He Ser Tyr Leu Leu Tyr Lys Phe Gly Phe Phe Asn Arg Thr 1075 1080 1085

Lys Lys Asp Glu Leu Asp Arg Leu Val Gin Gin Asn Pro Val Glu Pro 1090 1095 1100

Glu Ala Glu Asn Leu Asn Ser Gly Gly Asn Asn 1105 1110 1115

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

37 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Val Gly Gin Asp Arg Asp Phe Trp Ala Leu Leu Val Leu Gly Leu 1 5 10 15

Trp Cys Leu Ser Ser His Cys Asn Ala Phe Asn Leu Ser Pro Leu Pro 20 25 30

Asn Arg Gin He Leu Asp Pro Gin Phe Ala Thr Asn Leu Pro Lys Val 35 40 45

Arg Ala Ser Tyr Phe Gly Phe Thr Met Ser Leu Arg Pro Asn Gly 50 55 60

38

Claims

We claim:

1. A method for detecting a test compound for cognitive enhancer activity, comprising the steps of: inserting a gene sequence encoding for a protein involved in cognitive processes into test cells in culture under conditions where said gene sequence expresses the protein involved in cognitive processes in said test cell; adding the test compound to the cell culture or cell homogenates; and measuring the effect of the test compound on the activity of the protein involved in cognitive processes.

2. The method of claim 1, wherein the protein is a Volado protein.

3. The method of claim 1, wherein the protein is an integrin protein.

4. The method of claim 1, 2, or 3 wherein the test cells are selected from the group consisting of invertebrate cells and vertebrate cells..

5. The method of claim 1, 2, or 3 wherein the test cells are mammalian cells.

6. The method of claim 5, wherein the mammalian cells are selected from the group consisting of human embryonic kidney cells, COS cells and CHO cells.

7. The method of claim 1, 2, or 3 wherein the cells are insect cells.

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8. The method of claim 7, wherein the cells are selected from the group consisting of Drosophila S2 cells and Spodoptera SF9 cells with baculovirus vectors.

9. The method of claim 1 , 2, or 3 wherein the measuring step includes determining the degree of cell adhesion to ligands applied to solid surfaces.

10. The method of claim 9 wherein the solid surface is a plastic microtiter well.

11. The method of claim 1 , 2, or 3 wherein the measuring step includes determining the degree of cell adhesion to ligands expressed on other cells.

12. The method of claim 1, 2, or 3 wherein the measuring step includes biochemical assays of activity of tyrosine kinase.

13. The method of claim 2, wherein the gene expressing said volado protein is from a vertebrate or an invertebrate.

14. The method of claim 3, wherein the gene expressing said integrin protein is from a vertebrate or an invertebrate.

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5. A method of detecting a test compound for cognitive enhancer activity, comprising the steps of: feeding Drosophila flies the test compound; and testing the treated Drosophila flies for effects on their olfactory learning and memory.

16. The method of claim 15, wherein the Drosophila flies are wild type flies or volado mutants.

17. The method of claim 13, wherein the gene is from Drosophila.

18. The method of claim 13 or 14, wherein the gene is from a mammal.

19. The method of claim 16, wherein said Volado mutant is selected from the group consisting of vol¹ and voP.

20. An Antibody against a Volado protein.

21. An Antibody against an integrin protein.

22. A method of screening for mutant Drosophila flies involved in cognitive processes comprising the steps of: making a plurality of Drosophila fly line, each line containing a transposable element linked to a reporter gene;

41 crossing each such line with Drosophila flies containing specific genetic make-up, wherein during each such cross the transposable element is capable of moving the reporter gene to a new site in the genome; and screening the progeny of such cross for alteration in reporter gene activity in the mushroom bodies.

23. The protein sequence identified in SEQ ID NO. 1.

24. The protein sequence of claim 23 in which the first 63 amino acids are replaced with the 63 amino acids identified in SEQ ID NO. 2.

25. The method of claim 2, wherein the protein sequence is SEQ ID NO.1 or SEQ ID NO. 1 wherein the first 63 amino acids are replaced by SEQ ID NO. 2.

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