Patent Application
20110165174
This invention relates generally to learning and memory, and methods for detecting gene targets associated with these processes, methods of modulating learning and memory function, as well as to the gene targets themselves.
The term “memory” subsumes many different processes and requires the function of many different brain areas. Research in recent years has provided information necessary to understand many of the various components of memory and has identified associated brain regions. A newly acquired experience initially is susceptible to various forms of disruption. With time, however, the new experience becomes resistant to disruption. This observation has been interpreted to indicate that a labile, working, short-term memory is consolidated into a more stable, long-term memory.
Various mechanisms have been proposed to account for the formation of long-term memory. A wide range of observations suggest an evolutionarily conserved molecular mechanism involved with the formation of long-term memory. These include increased release of neurotransmitters, increased number of synaptic receptors, decreased KD of receptors, synthesis of new memory factors either in the presynaptic or postsynaptic element, sprouting of new synaptic connections, increase of the active area in the presynaptic membrane and many others. Synaptic plasticity, the change in the strength of neuronal connections in the brain, is thought to underlie long-term memory storage.
When acquired initially, newly learned information is held in a fragile state becoming robust over time (McGaugh et al., 2000). This consolidation process is driven by the hippocampus and requires de novo RNA translation and protein synthesis (Stork et al., 1999; Igaz et al., 2002), processes that provide the materials to allow the necessary structural changes in synaptic connections between neurons (Lamprecht and LeDoux, 2004). In the hippocampus, such synaptic plasticity involves three key phases: synaptic loosening, synaptic reorganization and synaptic selection. Synaptic loosening occurs when adhesive elements, such as neural cell adhesion molecule (NCAM), are selectively internalized and degraded synaptic curvature, size of synaptic elements and synaptic perforations (Marrone and Petit, 2002). For example, quantitative electron microscopic analysis of the rat hippocampus dentate gyms has revealed an increase in dendritic spine and synaptic density, 6-9 hours following either avoidance conditioning or spatial learning (O'Malley et al., 1998, 2000; Eyre et al., 2003). This increase in synaptic number is transient, however, returning to basal levels by 24-48 hours post-training These latter observations are indicative of a period of synaptic selection and elimination thought to be essential for the retention of only relevant connections in the memory circuit (Regan, 2004).
Several groups have used the large-scale screening of mRNA by microarrays to assess which genes may be involved in learning and memory at a transcriptional level (Cavallaro et al., 2002, D'Agata et al., 2003; Ressler et al., 2002; Leil et al., 2003; Igaz et al., 2004; O'Sullivan et al., 2007). These changes detected by microarray are usually considered indicative of transcriptional regulation but it must be noted that they could be due to altered RNA processing and post-transcriptional control (Mata et al., 2005). These studies have identified lists of genes that could be grouped into functional families including; transcription/translation, signal cascade enzymes, structural proteins and neural transmission. Although complex, these data begin to offer some insight into which genes may be involved in memory consolidation.
Despite continued efforts to develop methods to identify gene targets relevant for memory consolidation, and to identify genes and gene products important in regulating learning and memory processes, there is still a great need for methods that can detect such gene targets, so they can be utilized in screening therapeutics, in diagnosing learning and memory disorders, and in treating individuals with learning and memory disorders.
Many diseases of the brain are underpinned by deficits in cognition-related synaptic plasticity. In the adult mammalian brain, the hippocampus is known to mediate the consolidation of contextual and spatial memories. Identifying mediators of learning and memory, and memory consolidation is essential for developing treatments for diseases or conditions associated with neurological impairment resulting in memory loss due to reasons such as Alzheimer's disease, senile dementia of the Alzheimer's type, senile dementia, brain trauma, age-associated memory impairment, amnesia, central nervous system ischemia, degeneration, Parkinson's disease, and stroke. Identification of such mediators is also essential for understanding mechanisms underlying synapse formation which may be useful for developing treatments or preventing one or more of the conditions stated above.
In accordance with these and other objectives, this invention provides methods for identifying gene targets associated with learning and memory, with memory consolidation, methods for modulating memory function, methods for diagnosing memory disorders, methods for identifying compounds that modulate memory function, as well as systems comprising one or more of these methods. Accordingly, this invention also provides isolated nucleic acid molecules, the molecules encoded by them as well as methods and compositions utilizing these learning and memory associated molecules to modulate various aspects of learning and memory.
In one aspect of the invention, a method is provided for identifying gene targets associated with learning and memory, and with memory consolidation. Transcriptional regulation is assessed over time in animals after the animals have been administered a first learning task. From this assessment, a cohort of transcripts is then detected from these animals. The cohort of transcripts has expression regulated in an identifiable temporal pattern following the first learning task. Another group of animals is administered a second learning task, and transcriptional regulation over time is assessed in animals after this second task. Following this second learning task, a cohort of transcripts is detected, that cohort having expression regulated in an identifiable temporal pattern following the second learning task. The identifiable temporal pattern of regulated expression from the cohort detected following the first learning task is then compared with the identifiable temporal pattern of regulated expression following the second task. Based on this comparison, a new cohort of transcripts is detected, and this cohort of transcripts is regulated in an identifiable pattern following each task. The new cohort of transcripts detected contains at least one transcript in common from the cohorts detected after each task, though it may contain many transcripts in common, or all transcripts in common. The identifiable pattern following each task may be a similar pattern, or may be a different pattern. Transcripts which are identified to be regulated in both cohorts are identified as being targets associated with learning and memory and memory consolidation.
Systems for carrying out all of the methods described herein are also provided, and optionally comprise a computer system and related software. For example, as relates to a plurality of learning, memory, and/or memory consolidation tasks and a computer system comprising software, said computer system configured to (a) detect a cohort of transcripts of which expression is regulated in an identifiable temporal pattern following at least two of the learning tasks; (b) compare the identifiable temporal patterns of regulated expression from the cohorts detected in (a); and (c) detect a cohort of transcripts from (b) wherein said transcripts are temporally regulated in tissue from animals administered a different learning, memory, and/or memory consolidation task, and which cohort comprises transcripts present in (b), which exhibits an identifiable temporal pattern of regulated expression following at least two of the learning tasks. Optionally, the computer system may output a result from (c) which is indicative of gene targets associated with learning, memory, and/or memory consolidation.
In a further aspect of the invention, a method is provided of identifying gene targets associated with learning and memory, and memory consolidation. Transcriptional regulation over time is assessed in animals after a plurality of learning tasks. The animals are comprised of at least two groups, and each group of animals is administered a different learning task. A cohort of transcripts is detected from each group. The cohort from each group has expression that is regulated in an identifiable temporal pattern following the learning task that each group is exposed to. The temporal identifiable pattern of regulated expression from each cohort detected following each learning task is then compared. Based on this comparison of cohorts, a new cohort of transcripts is detected, and this cohort of transcripts is regulated in an identifiable, temporal pattern following each task. The new cohort of transcripts detected contains at least one transcript in common from the cohorts detected after each task, though it may contain many transcripts in common, or all transcripts in common. The identifiable pattern following each task may be similar patterns, or may be different patterns. These regulated transcripts are also identified as gene targets associated with learning and memory and memory consolidation. Systems for carrying out the foregoing methods are also provided, as described above.
By following the temporal expression pattern according to the methods of this invention, transcripts may be identified having a high probability of being associated with learning and memory, and memory consolidation. Different patterns of expression may be informative. Thus, common expression patterns may suggest the contemporaneous involvement of a plurality of gene products, whereas sequential expression may suggest a dependent on, the expression of more earlier expressed gene products. The time points at which certain genes are regulated may also be informative of the specific mechanisms important for learning and memory processes.
An additional aspect of the invention provides isolated nucleic acid molecules identified according to the methods of this invention. In one embodiment, this invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
In yet another aspect of the invention, a method is provided of modulating memory function in an animal. Memory function is modulated by enhancing or reducing the gene expression product of at least one nucleic acid sequence identified to be associated with learning or memory consolidation according to the method of this invention. Accordingly, through the analysis of temporal expression patterns, gene targets are identified that may serve as targets for modulating memory either by increasing or decreasing the function of the gene target. In certain embodiments of this invention it may desirable to increase memory function, whereas in other embodiments decreasing memory may be desired. Included among the gene targets for modulating memory are nucleic acid sequences selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
In another aspect of the invention, a method is provided of modulating memory function in an animal. Memory function is modulated by enhancing the gene expression of a target or targets identified according to methods of the invention, and may include at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
In yet another aspect of the invention, a method is provided of modulating memory function in an animal, which comprises reducing the gene expression product of a target or targets identified according to methods of the invention, and may include at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
In yet another aspect of the invention, a method is provided of modulating memory function in an animal, which comprises downregulating the gene expression of a target or targets identified according to methods of the invention, and may include at least ID NO:11.
In an additional aspect of the invention, a method is provided of diagnosing a memory disorder in an animal. The memory disorder is diagnosed by determining the gene expression profile of at least one nucleic acid sequence identified from the temporal expression pattern detected according to the methods of this invention, following, or throughout a learning and memory task. Examples of such nucleic acid sequences are those selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11. The gene expression profiles of normal and test subjects can be compared, and through this examination, it can be determined what nucleic acids or their products are not responding normally to tasks that elicit learning and/or memory processes.
In an additional aspect of the invention, a method is provided of identifying a compound that modulates memory function. The compound can be identified by determining the gene expression profile of at least one nucleic acid sequence identified from the temporal expression pattern detected according to the methods of this invention, following or throughout a learning and memory task. Examples of such nucleic acid sequences are those selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11. Compounds that increase or decrease the activity of these targets can be identified, which would be useful in modulating different aspects of learning and memory function.
These and other aspects of the invention may be more clearly understood by reference to the following detailed description of the invention and the appended claims.
This invention relates to methods of identifying gene targets associated with learning and memory processes and with memory consolidation. This invention also relates to nucleic acid molecules and their products which are identified according to the methods of this invention for identifying gene targets associated with learning and memory consolidation. Among such inventions are specific nucleic acid molecules that have been discovered to be associated with learning and memory processes and memory consolidation. Accordingly, the invention also relates to methods of modulating learning and memory function, methods of diagnosing memory disorders, and methods of identifying compounds that modulate memory function via manipulations of the nucleic acid molecules and their products.
An object of the invention is to provide a method of identifying gene targets associated with learning and memory processes. The inventors have observed that particular patterns of gene expression following exposure to learning and memory tasks are predictive of certain genes or gene fragments being involved in mechanisms relating to learning and memory.
The terms “learning” and “memory” subsume many different processes and require the function of many different brain areas. Memories are processed sequentially through several different phases that appear mechanistically and can be anatomically distinct. As contemplated by the invention, memory can include short-term memory and long-term memory. Short-term memory is rapidly formed and can last for seconds, minutes, hours, or days, and long-terms memory generally lasts from hours to days, weeks, or even years. Also contemplated by the invention, memory can include working memory, declarative (explicit) and procedural (implicit) memory. The terms “learning” and “memory” are often used in the their use in the art. As such, as used herein, these terms each apply equally to all aspects of the invention, and may be used interchangeably.
Any detectable change that one of skill in the art can measure which represents a change in any learning or memory processes is encompassed by the invention. The methods of this invention are generally useful for detecting spatial and temporal expression of genes and gene products associated with various aspects of learning and memory, including, but not limited to memory acquisition, consolidation, reconsolidation, and storage. Accordingly, embodiments of the invention may relate to any aspects of learning and memory. In certain embodiments, this invention relates to gene and gene products associated with memory acquisition, in other embodiments with memory storage, in yet other embodiments with memory consolidation, and in other embodiments with memory reconsolidation. As used herein the term “memory consolidation” means the process by which learned information is transformed into stable modifications. In some embodiments, the invention relates to memory re-consolidation. As used herein, the term “memory re-consolidation” relates to the process of reactivating consolidated memories, returning them to a labile state, so that they then may go through an additional consolidation process which may trigger molecular events similar or different from the original consolidation.
In one embodiment of the invention, a method is provided for identifying gene targets associated with learning and memory, or memory consolidation in which transcriptional regulation is assessed over time in a group of animals after the animals have been administered a first learning task. A cohort of transcripts demonstrating regulated expression in an identifiable temporal pattern is detected from this group of animals associated with a first task.
An additional group of animals is then administered a second learning task, which may be the same task or a different task, and a cohort of transcripts exhibiting regulated expression in an identifiable temporal pattern is then identified from this group of animals associated with the second task. This second cohort of transcripts is detected in the same manner as the first cohort. In some embodiments, the same group of animals is administered both tasks.
In one embodiment of the invention, more than two learning and memory tasks, or a plurality of learning and memory tasks, are utilized, and a separate group of animals is administered more than one of the tasks, or is administered all of the tasks. A comparison of all the identifiable temporal patterns is then made, to yield one cohort of transcripts that is regulated following the learning and memory tasks, containing transcripts that are related to learning and memory processes.
The identifiable temporal pattern of expression after the first task is then compared with the temporal pattern of expression after the second task, and if a plurality of tasks are used, then the identifiable temporal patterns of expression associated with all of the tasks are compared.
The comparison of the patterns of gene expression associated with each task includes determining which transcripts are regulated in an identifiable pattern following two learning and memory tasks or a plurality of learning and memory tasks. A new cohort of transcripts which exhibits an identifiable temporal regulation of expression following each task is then detected as being important for learning and memory processes. In one embodiment, the new cohort of transcripts includes at least one transcript which is common to both tasks, or which is common to a plurality of tasks. In another embodiment, more than one transcript is common to both tasks, or common to a plurality of tasks, and in yet other embodiment, all of the transcripts are common to both tasks, or common to a plurality of tasks. Also encompassed by the invention are cohorts of transcripts which include only a portion of the transcripts that are common to both tasks, or common to a plurality of tasks.
The identifiable patterns that are detected following the two or more tasks may be similar across tasks, having similar patterns, or the patterns may be inverse, and in some embodiments, they may have no relation to one another. Depending on the tasks utilized and the patterns assessed, the similarity or differences based on the comparisons of the detected identifiable patterns may be informative of the role that the regulated genes are playing in learning and memory processes. For example, without being bound by theory, regulated gene expression may show a trough in its pattern at a particular time point (or time points) following a task that requires an animal to avoid something, as in a passive avoidance task, and regulated gene expression may peak at a particular time point (or time points) following active learning, such as the Morris water maze task.
Transcriptional regulation can be assessed following the administration of the learning and memory tasks, although in some embodiments, groups of animals may be animals may also be assessed for transcriptional regulation at different points throughout the learning and memory task. These time points may be useful to serve as baseline measures, as well as for measuring processes taking place earlier in learning and memory formation. Such time dependent measurements provide for temporal assessments of transcriptional events during learning and memory processes and memory consolidation.
By an “identifiable pattern,” it is meant that of the gene transcripts assessed, at least two of the time points assessed exhibit a change in gene expression in the same direction, up-regulated or down-regulated, though in some embodiments, one or more of the time points assessed can reveal no change in gene expression. In a preferred embodiment, of the gene transcripts assessed, and considered to be in an “identifiable pattern,” three or more of the time points assessed exhibit a change in gene expression in the same direction, up-regulated or down-regulated, though in some embodiments, one of the time points assessed can reveal no change in gene expression. Any detectable change in amplitude of gene expression is included in the meaning of identifiable pattern. The change in amplitude of gene expression may be a change of 1%, 10%, 50%, or 100%, and in some embodiments the change in amplitude of gene expression may be 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater. In some embodiments, no change in gene expression at one or more of the assessed time points may be meaningful, and can be encompassed as part of the identifiable pattern, and is within the scope of this invention. The change in expression can be either an increase or a decrease from baseline expression, or it may be an increase or decrease in gene expression relative to the gene expression at other time points within the temporal pattern. The gene transcripts exhibiting an identifiable temporal pattern are indicative of gene targets that are associated with learning and memory function. Compounds that increase or decrease the activity of the genes, gene fragments, and gene products identified as targets associated with learning and memory function may also be drug candidates for modulating learning and memory.
Changes in gene expression over any period of time are within the scope of this invention and may vary greatly depending on the species that is evaluated. In one embodiment, the gene expression is regulated within 24 hours of the learning and memory task. In another embodiment, the gene expression is regulated one hour, two hours, four hours, six hours, or twelve hours after the learning and memory task. In another embodiment, the gene expression is regulated within one week of the learning and memory leaning and memory task. In yet another embodiment, the gene expression is regulated within two months of the learning and memory task. In some embodiments the gene expression is regulated within one year of the learning and memory task, within five years of the learning and memory task, or within ten or more years of the learning and memory task. In some embodiments, the regulation of gene expression is regulated at time points throughout the administration of the task or tasks, and in some embodiments, the gene expression is regulated prior to the administration of the task or tasks.
In some embodiments of the invention, transcriptional regulation is assessed from brain tissue. Many regions within the brain that are important for regulating learning and memory functions, including but not limited to the hippocampus, the cortex, prefrontal cortex, neocortex, amygdala, striatum, and cerebellum can be assessed for transcriptional regulation prior to, during, and after learning and memory tasks. It is also contemplated by the invention that transcriptional regulation can be assessed from blood, plasma, lymph, urine, mucus, sputum, saliva, or tissue from other organs of the body.
In one embodiment of the invention, regulated transcripts are detected following a learning and memory task, which correspond to genes that have been previously identified to be associated with learning and memory processes. The identifiable pattern of temporal gene expression of the previously identified gene or genes is then compared with the other transcripts, those transcripts which are unknown, to yield a cohort of transcripts that is regulated in an identifiable temporal pattern with the gene or genes previously identified to be associated with learning and memory. In one embodiment, the average temporal pattern of the genes previously identified as associated with learning and memory is used as a “seed pattern” and all regulated genes are ranked for their similarity to this pattern based on the time points and amplitude as described herein, and are then considered a cohort having regulated expression in an identifiable temporal pattern. This seed pattern may include 2 or more previously identified genes. In some embodiments of the invention, the seed pattern may include 5 or 10 identified genes, and in some embodiments in may include 20, 30, 50, 100, 200, 500 or more previously identified genes. In one embodiment, the top 100 matches to the seed pattern are chosen as the transcripts with the strongest relevance for learning and memory function. In another embodiment, the top 50 matches to the seed pattern are chosen as the transcripts with the strongest relevance for learning and memory function. In yet the strongest relevance for learning and memory function.
In another embodiment of the invention, less than, all, or none of the transcripts demonstrating regulated expression following the learning and memory task or tasks have been previously identified to be involved in learning and memory processes. In this embodiment, some or all of the regulated genes following the learning and memory task have not been previously associated with learning and memory, but exhibit regulated expression in an identifiable temporal pattern.
In an embodiment of the invention, one or more of the transcripts exhibiting temporal regulation is a gene or combination of genes or gene fragments chosen from among any genes that have been previously identified to be associated with learning and memory, or chosen from the genes or gene fragments identified herein. For example, the genes or gene fragments can be chosen from translation initiation factors, heterogeneous nuclear ribonucleoproteins, RNA binding motif protein 9, ribosomal protein L5, translation elongation factors, synuclein alpha, tenascin R, smooth muscle alpha-actin, myelin-associated oligodendrocytic basic protein, low density lipoprotein receptor-related protein 3, dynamin I0like protein, ADP-ribosylation factor, kinesin family member, SNAP25, NSF, synaptoargnin IV, Atpla1, AtblbZ, calcium/calmodulin-dependent protein kinase II, MAPK1, MAPKK1, p38 MAPK, MAPK phosphatase, Ania-3, MAP2, protein kinase (cAMP-dependent catalytic and regulatory, beta), Ube2i, calnexin, cathepsinD, casolin-containing protein, Dnajb9, ATP citrate lyase, brain acyl-CoA hydrolase, sterol-coenzyme A denaturase 2, lipoprotein lipase, BDNF, IGF-2, IGF binding protein 2, fibroblast growth factor receptor 1, CD9 cell surface glycoprotein, Thy cell surface glycoprotein, G-protein coup led receptor, muscarinic receptor m2, chemokine receptor 5, GABA A, synaptogamins 7 and 8, syntaxin 2, 5, and 8, glutamate, serotonin transporters, inducible nitric oxide synthase, catenin, C-CAM2a, neurexin 1, connexin 43, contactin 1, chondroitin sulfate proteoglycan 3, myelin-associated glycoprotein, axonal glycoprotein, PSD-95NMDA-R2A, GluR6, GluR5-2, potassium channel subunits, BOD-L, homer 1a, syntaxin 1a, ERK2, PKB, mGLUR7, TRKB, VGF, CREM, gephyrin, N-cadherin, Rheb2, ARPP-21, EGR4, c-fos, c-jun, CRH, midkine, ttransthyretin, the bone morphogenetic antagonist, USAG-1, and eNNP2.
According to the invention, gene expression products include any products which have been or may be determined to be associated with, or be capable of modulating to either increase or decrease learning or memory, but are not limited to proteins, peptides, or transcription or translation.
Gene expression levels may be detected by methods known to those skilled in the art and may be obtained, for example using any apparatus that can measure gene expression levels which are widely known in the art. The nucleic acid molecule levels measured can be derived directly from the gene or, alternatively, from a corresponding regulatory gene. All forms of gene expression products can be measured, including, for example, spliced variants. Similarly, gene expression can be measured by assessing the level of protein or derivative thereof translated from mRNA. This may, however, also reflect posttranslational modifications and other forms of processing If the gene expression assessed is at the mRNA level, it can, for example, also be measured by in situ hybridization, Northern blot analysis, dot-blot hybridization analysis, microarray analysis, or by PCR. Such methods are described in detail, for example, in Ausubel et al., Current Protocols In Molecular Biology (New York: John Wiley & Sons) (1998); and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (New York: Cold Spring Harbor University Press (1989).
In another embodiment of the invention, gene expression levels can be obtained by contacting the sample of interest with a suitable microarray, and determining the extent of hybridization of the nucleic acid in the sample to the probes on the microarray. It is also contemplated by the invention that gene expression assessed by methods such as microarray can be validated by using quantitative real-time PCR analysis at the time points determined to be relevant from the temporal profile of gene expression observed. It will be apparent to those skilled in the art that any methodology that can be utilized for measuring gene expression will be suitable for use in the invention. Microarray analysis and PCR analysis can be carried out according to the Examples described herein, or by any of the available methods known in the art.
If the gene expression product is a protein or polypeptide, transcriptional regulation can be measured using techniques for protein detection and quantification that are known in the art. Antibodies, for example, can be generated which are specific to the protein using routine methods known in the art, which specifically bind to the protein of interest so that they can be detected and measured. Protein measurement can be carried out by any methods known in the art, including histochemistry, immunoblot analysis, in vitro binding studies, radioimmunoassay, and ELISAs.
and which are detected as a cohort of transcripts important in learning and memory processes are expressed sequence tags (ESTs). ESTs are short single-pass sequence reads from mRNA (cDNA). They may be of various lengths. For use in this invention, it is preferred, but not required, that they be of sufficient length to identify a unique expressed sequence. Typically, they are about 300-500 by in length. However, sequences as short as about 16 bases may be sufficient to identify a specific sequence. ESTs represent a snapshot of genes expressed in a given tissue and/or at a given developmental stage. They are tags (some coding, others not) of expression for a given cDNA library. There are now well over one million of these sequences in the publicly available database, and these sequences are believed to represent more than half of all human genes. The ESTs of, and for use with the invention, however, are not meant to be limited by what is available in public databases, and also may be novel ESTs that are generated.
The ESTs detected in the invention are useful for elucidating the genes and gene products responsible for regulating learning and memory processes, and therefore for understanding the mechanisms underlying disorders of learning and memory function. The ESTs detected according to the methods of this invention demonstrate specific regulation at time points expected to be involved in the learning and memory process, including memory consolidation. Such involvement may be to contribute, or be required, to allow for learning or memory maintenance or consolidation to occur. This is the case when less than all, or none of the transcripts are unknown, and even more so when the temporal patterns of the transcripts correspond to the temporal patterns of genes previously identified to be associated with learning and memory, during at least one of the time points assessed.
Once these ESTs are identified as being associated with learning or memory, at least one of the components of learning, acquisition, consolidation, or others, they are clearly useful for detecting or diagnosing learning and memory dysfunctions, as well as in modulating memory function by either enhancing or inhibiting gene expression or the gene expression product. Similarly, the ESTs for use with the invention are useful for development of new pharmaceutical agents that can be used to help those with diseases or disorders that influence learning and memory functions, as well as with enhancing learning and memory processes in people without dysfunction of learning and memory processes.
Some embodiments of the invention utilize learning and memory tasks in the methods to identify gene targets associated with learning and memory processes. “Task” as scenario which is capable of eliciting learning and memory processes. The invention is not limited by the means of eliciting and assessing learning and memory, and is meant to include anything which can cause learning and memory processes to take place, and which can be used to assess these processes.
The learning and memory tasks can be any such tasks that involve or elicit learning and memory processes. Depending on the specific type of learning and memory of interest, one may choose different tasks which yield data that are representative of different stages in the learning and memory process, different types of learning and memory, and which may represent the use or activation of different regions within the brain responsible for mediating those actions. One task contemplated by the present invention is the Morris water maze, which is one of the best-validated models of learning and memory. The test is a simple spatial learning task in which the animal is placed in tepid water, which may be opaque due to the addition of powdered milk. The animals learn the location of the platform relative to visual cues located within the maze and the testing room. Another important test contemplated for use in the invention is the passive avoidance test, including the one-trial, step-through, light-dark version of the passive avoidance paradigm as described previously (Fox et al., 1995, J Neurochem 65, 2796-2799). The odor-reward association paradigm is also contemplated for use with the invention. Yet another learning and memory test for use in the invention is the Y-maze test, which is based on visual discrimination. Other learning and memory tests which are contemplated as useful in the invention are eye blink conditioning, fear conditioning, and delayed match-to-position tasks. Also contemplated by the invention is the prepulse inhibition of startle response (PPI), which evaluates working memory. Any number of other tasks which elicit learning and memory processes known to the skilled practitioner will also be useful in the invention.
In some embodiments of the invention, the animals are humans. Accordingly, the tasks utilized in the invention would be appropriate for assessing learning and memory processes in humans, the appropriateness of which will be known to the skilled practitioner. Such tasks contemplated by the invention include accepted rating scales and standardized performance tests for cognitive function including, but not limited to CANTAB (Cambridge Neuropsychological Test Automated Battery), BEHAVE-AD (Behavioral Pathology in Alzheimer's Disease Rating Scale), Blessed Test, CERAD (The Consortium to Establish a Registry for Alzheimer's Disease), Clock Draw Test, Cornell Scale for Depression in Neuropsychiatric Inventory (NPI), The 7 Minute Screen, Children's Memory Scale (CMS), Continuous Recognition Memory Test (CMRT), Denman Neuropsychology Memory Scale, the Learning and Memory Battery (LAMB), Memory Assessment Clinic Self-Rating Scale (MAC-S), a Memory Assessment Scales (MAS), Randt Memory Test, Recognition Memory Test (RMT), and other clinical and neuropsychological tests.
The methods of detecting gene targets associated with learning and memory are generally carried out in animals other than humans. Once gene targets associated with learning and memory are identified according to the methods of the invention in animals other than humans, other aspects of the invention may be applied to other animals, including humans. In some embodiments, once the gene targets have been identified, it can be determined if those genes are present in humans, through, for example, sampling the blood or CSF. In some embodiments, it will be useful to determine if the detected gene targets, or their human equivalents are disrupted in humans, and if these disruptions are associated with alterations in learning and memory function, which can be assessed from a variety of tasks as described herein. If the genes, or targets, are present in humans, then one can modulate learning and memory function as described herein by modulating the activity of the identified genes or gene products, and confirming whether there has been a change in learning or memory function via various means of assessment. To assess a change in learning or memory function after modulation of an identified gene or gene product in animals other than humans, any of the tasks described herein which elicit learning and memory function may be used. To assess learning and memory function in humans, there are many tasks which may be utilized, known to those skilled in the art.
Systems for carrying out all of the methods described herein are also provided, and optionally comprise a computer system and related software. For example, as relates to the foregoing method, the system may comprise groups of mice that have been administered a plurality of learning, memory, and/or memory consolidation tasks and a computer system comprising software, said computer system configured to (a) detect a cohort of transcripts of which expression is regulated in an identifiable temporal pattern following at least two of the learning tasks; (b) compare the identifiable temporal patterns of regulated expression from the cohorts detected in (a); and (c) detect a cohort of transcripts from (b) wherein said transcripts are temporally regulated in tissue from animals administered a different learning, memory, and/or memory consolidation task, and which cohort comprises transcripts present least two of the learning tasks. Optionally, the computer system may output a result from (c) which is indicative of gene targets associated with learning, memory, and/or memory consolidation.
The targets obtained according to the methods of the invention, described above, can be used to modulate learning and memory function by being either upregulated or downregulated, or by increasing of decreasing their products. The invention is also useful for screening drug candidates which modulate the activity of the targets obtained according to the methods of the invention, and are therefore useful to modulate learning and memory. The invention is also useful for identifying possible mechanisms of learning or memory deficiency. It can be determined whether targets identified according to the methods of the invention are involved in such conditions.
In yet another embodiment, the invention provides methods of modulating memory function by enhancing the gene expression or the gene expression product of at least one of the nucleic acid sequence identified to be associated with learning and memory according to the methods of this invention. Accordingly, through the temporal analysis of expression patterns, gene targets are identified that may serve as targets for modulating memory, either by increasing or decreasing the function of the gene of interest, or a product of the gene of interest. In certain embodiments, it may be desirable to increase learning and memory function, whereas in other embodiments, it may be desirable to decrease the function of the gene of interest of a product of the gene of interest. Included among the gene targets for modulating memory function are nucleic acid sequences selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
In another embodiment, the invention provides methods of modulating memory function by downregulating or reducing the gene expression or the gene expression product of at least one of the nucleic acid sequence identified to be associated with learning and memory according to the methods of this invention. Included among the targets for decreasing gene expression are at least one of the nucleic acid sequences of SEQ ID NO:1 through SEQ ID NO:11. Downregulating or inhibiting learning or memory is useful where it is desirable for an individual not to remember or recall an experience or event, as described below.
limited to proteins, peptides, or nucleic acid molecules (e.g., mRNA, tRNA, rRNA, or cRNA) that are involved in transcription or translation.
In one embodiment of the invention, gene expression products can refer to epigenetic changes such as DNA methylation and chromatin remodeling. Chromatin remodeling is initiated by the posttranslational modification of the amino acids that make up histone proteins, or by the addition of methyl groups to the DNA at CpG sites to convert cytosine to 5-methylcytosine. Any epigenetic modulation known to one of skill in the art is contemplated by the invention.
Also contemplated by the invention are methods of diagnosing a learning or memory disorder or dysfunction in an animal. In one embodiment of this invention, the animal is a human. Such a disorder can be diagnosed by evaluating the expression of at least one nucleic acid sequence or its product identified to be associated with learning and memory according to the methods of the invention, either at a single time point, or over time. One can determine how such a nucleic acid sequence differs in its expression at one or more time points following any activity known to elicit learning and memory function, or its expression may be measured at baseline conditions. A learning and memory disorder may also be assessed by modulating the activity of at least one nucleic acid sequence or its product as described above, to assess if learning and memory function is altered through the modulation. Included among the gene targets for diagnosing a learning and memory disorder are nucleic acid sequences detected by the methods according to the invention, and in particular, may include, but not be limited to, those selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
Impairments and abnormalities in learning and memory processes can occur in a number of conditions or diseases. Non limiting examples of such conditions and diseases that may be treated by the methods and compositions of this invention include, but are not limited to age-related memory loss, dementia, senile dementia, Mild Cognitive Impairment, Alzheimer's disease, senile dementia of the Alzheimer's type, Multiple Sclerosis, brain injury, amnesia, neuronal toxicity, brain aneurysm, stroke, schizophrenia, epilepsy, chronic fatigue syndrome, fibromyalgia syndrome, chemotherapy (e.g., cancer chemotherapy), traumatic brain injury, Parkinson's disease, and post-traumatic stress disorder (PTSD). treatment of impairments and abnormalities in learning and memory processes through modulating the gene expression or gene expression product of at least one nucleic acid sequence identified according to the methods of this invention. In certain embodiments, the nucleic acid is selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11. The aim is to enhance learning and memory function in individuals with reductions in normal learning and memory processes, for example, such as those having any of the conditions stated above, an in particular, those with Alzheimer's disease, schizophrenia, and stroke. In those with memory abnormalities such as PTSD, in which memories of unpleasant experiences are actually enhanced, the invention contemplates downregulating or reducing relevant gene expression or gene expression products to reduce or alleviate symptoms.
Modulating the nucleic acid sequences and gene products identified through this invention may be accomplished by increasing or decreasing their activity depending on the desired result. This will vary, depending on whether the goal is to enhance or reduce learning or memory. For the conditions mentioned above, where enhancement is desired, one may seek to enhance those sequences that demonstrate an increase in expression over time in the temporal patterns observed during or following a learning and memory task, depending on the nature of the task administered, and depending on the specific time points at which increased expression is observed. One may also seek to reduce those sequences that decrease during or following a learning and memory task, with the goal of enhancing learning and memory depending on the nature of the task administered, and depending on the specific time points at which decreased expression is observed.
In one embodiment, in order to modulate the nucleic acid sequences and gene products identified through this invention, it may be desirable to enhance the gene expression or gene expression product that is observed increasing expression 2 hours, 12 hours, or 24 hours after a learning and memory task. More specifically, one may enhance the gene expression or gene expression product after training on the Morris water maze. In another embodiment, it may be desirable to enhance the gene expression or gene expression product that is observed decreasing expression 1 hour after a learning and memory task, and, more specifically, after training on a passive avoidance task. In yet other embodiments, it may be desirable to enhance or inhibit the gene expression or gene expression products identified according to the methods of the invention by intervening at different times either prior to, during, or after a learning and memory task, such as, for example, at 0, 1, 2, 12, or 24 hours following the task. Modulating the nucleic acid sequences and gene products identified through the methods of this invention may be for the purpose of treating a learning and memory disorder, for enhancing learning and memory, or in other embodiments, for identifying compounds useful for modulating learning and memory function.
In certain situations it may be desirable to reduce the learning or memory of an individual, particularly following exposure to a traumatic experience. Such an experience may be following witnessing a crime, post traumatic stress syndrome, surgical procedures, or other experiences the memory of which may cause harm to the individual. In humans where consolidations may take up to ten (10) years or more to be complete, the experience may be reconsolidated so that it is susceptible to the modulations contemplated by this invention. Where reductions in learning and memory are desired, one may seek to reduce those sequences that demonstrate an increase in expression over time in the temporal patterns observed during or following a learning and memory task depending on the nature of the task administered. One may also seek to enhance those sequences that decrease during or following a learning and memory task, with the goal of reducing learning and memory depending on the nature of the task administered.
Any means of enhancing gene expression, or the gene expression product in an individual that is known by one of skill in the art may be used to modulate gene expression or a gene expression product as contemplated for methods of treatment in the invention. For example, pharmaceutical compositions which are agonists for the gene product of interest may be used to enhance learning and memory processes.
Any means of reducing gene expression, or the gene expression product in an individual that is known by one of skill in the art may be used to reduce gene expression or a gene expression product as contemplated for methods of treatment in the invention. Non-limiting examples for use in reducing gene expression or gene expression products according to the invention include RNA interference, antisense RNA, antibodies, and pharmaceutical compositions that antagonize the activity of the gene product of interest.
In certain embodiments, antisense is used to decrease expression of a gene. Antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). Included within antisense are antisense RNA (“asRNA”) molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, promoter that permits the synthesis of a coding strand. Once introduced, this transcribed strand combines with natural mRNA to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. Regions of a nucleic acid sequences that are accessible to antisense molecules can be determined using available computer analysis methods.
In certain embodiments, an RNA interference (RNAi) molecule is used to decrease expression of a gene. RNA interference (RNAi) is defined as the ability of double-stranded RNA (dsRNA) to suppress the expression of a gene corresponding to its own sequence. RNAi is also called post-transcriptional gene silencing or PTGS. Since the only RNA molecules normally found in the cytoplasm of a cell are molecules of single-stranded mRNA, the cell has enzymes that recognize and cut dsRNA into fragments containing 21-25 base pairs (approximately two turns of a double helix). The antisense strand of the fragment separates enough from the sense strand so that it hybridizes with the complementary sense sequence on a molecule of endogenous cellular mRNA. This hybridization triggers cutting of the mRNA in the double-stranded region, thus destroying its ability to be translated into a polypeptide. Introducing dsRNA corresponding to a particular gene thus knocks out the cell's own expression of that gene in particular tissues and/or at a chosen time.
Double-stranded RNA can be used to interfere with gene expression in mammals (Wianny & Zemicka-Goetz, 2000, Nature Cell Biology 2: 70-75; incorporated herein by reference in its entirety). dsRNA is used as inhibitory RNA or RNAi of the function of the gene of interest to produce a phenotype that is the same as that of a null mutant of the gene of interest (Wianny & Zernicka-Goetz, 2000, Nature Cell Biology).
In another embodiment, as method is provided which comprises screening compounds for their ability to increase or decrease the activity of one or more of the targets identified according to the methods of the invention. Accordingly, an assay is provided which comprises administering a test compound to a group of animals, and in some embodiments, a placebo to a groups of animals, and detecting changes in the expression profile following one or more learning and memory tasks as described above. A test compound is any compound of interest, wherein one is interested in determining the compound's involvement in learning and memory. Such compounds may then be identified as drug candidates for modulating learning, memory, or memory consolidation. Compounds can also be identified by comparing the gene expression profiles to those of the gene expression profiles of those selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11. In addition to nucleic acids, such as siRNA sequences described above, other compounds that bind to the gene products either competitively or non-competitively, or otherwise affect their activity may be useful as drug candidates for modulating learning, memory, or memory consolidation.
Also contemplated by the invention are isolated nucleic acid molecules identified according to the methods of this invention. In one embodiment, this invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11.
Pharmaceutical compositions comprising the genes or gene fragments derived according to the methods of the invention, including the nucleic acids from SEQ ID NO:1 through SEQ ID NO:11 are also contemplated by the invention.
The pharmaceutical compositions of the invention can be formulated as natural or salt forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric, or phosphoric acids, or organic acids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Salts of the invention include amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the invention also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopcia Convention, Inc., Rockville, Md. (1989), the relevant contents of which are incorporated herein by reference.
in part by the specific composition administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 20th ed., 2003).
The term “pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and for use in humans. The term “carrier” can mean a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes New York, pp. 353-365 (1989); Lopez-Berestein, pp. 317-327).
In yet another embodiment of the invention, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the antibodies of the invention or fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; International Publication No. WO 99/15154; and International Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Controlled release systems are discussed in Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the invention or fragments thereof. See, e.g., U.S. Pat. No. 4,526,938, International publication No. WO 91/05548, International publication No. WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entirety.
In some embodiments of the invention, in which the composition is one or more nucleic acid molecules obtained according to the methods of the invention, the nucleic acid or nucleic acids can be administered in vivo by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.
Pharmaceutical compositions of the invention are formulated to be compatible with their intended route of administration. Examples of suitable routes of administration include, but are not limited to, parenteral (e.g., intravenous, intramuscular, intradermal, intra-tumoral, intra-synovial, and subcutaneous), oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intra-tumoral, intra-synovial, vaginal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intra-tummoral, intra synnovial, intranasal or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
If the compositions of the invention are to be administered orally, the compositions can be formulated orally in the form of, e.g., gum, tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release or sustained release of a prophylactic or therapeutic agent(s).
If the compositions of the invention are to be administered intranasally, the compositions can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compositions of the invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
compositions can be formulated in the form of, e.g., an, ointment, cream, transdermal patch, lotion, gel, oral gel, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 4.sup.th ed., Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon), or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
The compositions of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described above, the compositions of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggestive to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Unless otherwise specified, in the examples provided below, animals were maintained as follows.
Animal Maintenance
Postnatal day 80 male Wistar rats (300-350 g) were obtained from the Biomedical Facility at University College Dublin and were group housed on a 12:12 light/dark cycle, with ad libitum access to food and water. The animals were introduced into the experimental rooms 5 days prior to commencement of training On the 2 days prior to training the animals were handled, their weights monitored and spontaneous behavior assessed in an open field apparatus (620 mm long, 620 mm wide, 150 mm high). The floor of the open field was ruled into a series of squares (77×77 mm), the animal was placed in the centre and the number of lines crossed in a 5-minute period counted. Other behaviors assessed included rearing, grooming, piloerection, defecation and posture. All observations were carried out in the quiet room under low-level red illumination between 08:00 and 12:00 to minimize circadian influence. Naïve littermates were treated precisely as their trained counterparts except they were not exposed to the training environment prior to sacrifice.
Materials and Methods
Water Maze Training
The spatial learning task has been described in detail previously (Murphy et al., 1996). The water maze apparatus consisted of a large circular pool (1 m diameter, 80 cm high, temperature 26±1° C.) with a platform (11 cm diameter) submerged 1.5 cm below the water surface. Both the pool and the platform were constructed of black polyvinyl plastic and offered no intra-maze cues to guide escape behavior. The experimental room contained several extra-maze visual cues. During training the platform was hidden in the same quadrant 30 cm from the edge of the maze. Each trial started with the rat facing the wall of the maze at one of three locations. The time taken by the rat to find the hidden platform within a 60 sec period was recorded. On the first trial, rats failing to find the platform within the 60 sec period were placed on it for 10 sec. Times to the platform were measured over 5 trials in the training session with an inter-trial interval of 300 sec. To control for stress and other non-learning associated factors during water maze training each trained animal was paired with a corresponding passive control animal that was allowed to swim in the maze for a time matching its trained counterpart for each trial but in the absence of a platform.
Passive Avoidance Training
On the day of training, spontaneous behaviors were assessed prior to passive avoidance training The one-trial, step-through, light-dark version of the passive avoidance paradigm was employed as we have described previously (Fox et al. 1995). Briefly, the smaller illuminated compartment was separated from the larger dark compartment by a shutter that contained a small entrance. The floor of the training apparatus consisted of a grid of stainless steel bars that would deliver a scrambled shock (0•75 mA every 0•5 ms) for 5 seconds when the animal entered the dark chamber. Passive controls were exposed to the avoidance apparatus for a time matched to trained counterparts but never received a foot-shock.
Sample Collection
In order to determine the sequential expression of mRNA in the rat dentate gyrus during memory consolidation, animals were sacrificed at discrete time points 0, 0.5, 1, were killed by cervical dislocation, the dentate gyms rapidly dissected and snap frozen. All experimental procedures were approved by the Animal Research Ethics Committee of the Biomedical Facility at University College, Dublin, and were carried out by individuals who held the appropriate licence issued by the Minister for Health and Children.
Microarray and Real-Time Sample Preparation
Total RNA was extracted from each dentate gyms by homogenization in TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and following the TRIzol protocol. The resulting RNA samples were purified using an RNeasy mini kit (Qiagen, UK). RNA concentration was determined spectrophotmetrically, and RNA integrity was confirmed by agarose gel electrophoresis. Double-stranded cDNA was synthesised from 10 μg total RNA (Superscript System, Invitrogen, Carlsbad, Calif., USA). Briefly, the RNA was mixed with 100 pmol oligonucleotide GGC CAT GGAAT TGTAATAC GAC T CAC TATAGGGAGGC GG (dT)24 in 20 μl water, annealed at 70° C. for 10 min, and quick-chilled. Buffer, dithiothereitol, and dNTP mix were than added and incubated at 37° C. for 2 min. Second-strand synthesis was performed by adding reaction buffer, dNTPs (200M), DNA ligase (10 U), DNA polymerase (40 U), ribonuclease H (2 U), and water (to a final volume of 150 μl), and the reaction was incubated for 2 h at 16° C. This was followed by addition of 10 U T4 DNA polymerase and incubation at 16° C. for 5 min. The cDNA was purified by phenol/chloroform extraction, precipitated, and transcribed in vitro using T7 RNA polymerase. Biotinylated cRNA was generated using the BioArray HighYield RNA Transcription Kit (Enzo Diagnostics, Inc., Farmingdale, N.Y.). The cRNA was purified by RNeasy minispin columns and fragmented by incubation in 40 mM Tris (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate buffer at 94° C. for 35 min.
Microarray Analysis
Fragmented cRNA for each sample (n=6×8 time points×2 (trained & passive control)=96 samples per training paradigm) was hybridised to the Affymetrix rat genome RG230.02 chip using the protocol outlined in the GeneChip Expression Analysis Technical Manual (Affymetrix Inc., Santa Clara, Calif., USA). Hybridised chips were washed and stained using Affymetrix Fluidics Station 400 and EukGE-WS 1 Standard Format as recommended by the manufacturer. The staining was performed using streptacidin-phycoerythrin conjugate (SAPE; Molecular Probes, Eugene, Oreg., USA), followed by biotinylated antibody against scanned using a Hewlett-Packard GeneArray Scanner and analysed using Affymetrix MAS5.0 software. Hybridization intensities were normalised using a method featuring a pool of 11 biotin-labelled cRNA control transcripts, derived by in vitro transcription of 11 cloned Bacillus subtilis genes, which were spiked into each hybridization experiment. This normalization method has been described in detail previously (Hill et al., 2001). The 5′/3′ ratio for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and for beta-actin ranged from 0.8 to 1.1.
DNA microarrays were used to study the mRNA expression profiles of rat dentate gyms at increasing times following either water maze spatial learning or avoidance conditioning. Approximately 5500 and 2500 genes were transcriptionally regulated across the 24 h post-training period following spatial learning and avoidance conditioning, respectively. When these gene lists were compared, a substantial cohort consisting of 1500 transcripts was identified whose expression levels were regulated following both learning tasks. Collectively, these genes can be considered a core transcriptional program for memory consolidation, deployed independent of the nature of the learning task.
Analysis of the known genes in this core group revealed several transcripts of interest with respect to a potential role in memory-associated synaptic plasticity such as the novel neurotrophin, midkine (Mdk), the carrier molecule, Transthyretin (Ttr), USAG-1, and eNNP2. These transcripts shared a characteristic temporal pattern of regulation following water maze learning that of learning-specific increases in expression at the 2 and 24 h post-training times (
Clusters of unknown genes were identified that shared these temporal patterns of regulation with the known genes in both tasks (
Genes and gene fragments identified as changing significantly in animals at time 0 following water maze training relative to controls are provided in Table 2 below.
Genes and gene fragments identified as changing significantly in animals 0.5 hours following water maze training relative to controls are provided in Table 3 below.
Genes and gene fragments identified as changing significantly in animals 1.0 hour following water maze training relative to controls are provided in Table 4 below.
Genes and gene fragments identified as changing significantly in animals 2 hours following water maze training relative to controls are provided in Table 5 below.
Genes and gene fragments identified as changing significantly in animals 3 hours following water maze training relative to controls are provided in Table 6 below.
Genes and gene fragments identified as changing significantly in animals 6 hours following water maze training relative to controls are provided in Table 7 below.
Genes and gene fragments identified as changing significantly in animals 12 hours following water maze training relative to controls are provided in Table 8 below.
Genes and gene fragments identified as changing significantly in animals 24 hours following water maze training relative to controls are provided in Table 9 below.
Genes and gene fragments identified as changing significantly in animals at time 0 following passive avoidance training relative to controls are provided in Table 10 below.
Genes and gene fragments identified as changing significantly in animals 0.5 hours following passive avoidance training relative to controls are provided in Table 11 below.
Genes and gene fragments identified as changing significantly in animals 1 hour following passive avoidance training relative to controls are provided in Table 12 below.
Genes and gene fragments identified as changing significantly in animals 2 hours following passive avoidance training relative to controls are provided in Table 13 below.
Genes and gene fragments identified as changing significantly in animals 3 hours following passive avoidance training relative to controls are provided in Table 14 below.
Genes and gene fragments identified as changing significantly in animals 6 hours following passive avoidance training relative to controls are provided in Table 15 below.
Genes and gene fragments identified as changing significantly in animals 12 hours following passive avoidance training relative to controls are provided in Table 16 below.
Genes and gene fragments identified as changing significantly in animals 24 hours following passive avoidance training relative to controls are provided in Table 17 below.
Drosophila homolog) 10, splicing factor, arginine/serine-rich 10
This example provides confirmation of the pattern of Mdk regulation over time following the learning tasks, using quantitative real-time PCR as described below.
Quantitative Real-Time PCR
Real-time PCR was carried out using TaqMan technology on an ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems, UK). cDNAs, from 1 μg of DNase treated RNA from each animal (n=6 per group) were produced using SuperScript II RNase H Reverse Transcriptase Kit (Invitrogen) and 50-250 ng random primers (Invitrogen). cDNA (0.8 μl) from each sample was amplified using TaqMan® Gene Expression Assay primers and probe (Applied Biosystems, UK), Assay ID Rn—00578324_m1. Relative quantitation was determined by constructing a standard curve for each primer and probe set, using pooled DNA from all the samples. A ribosomal RNA control primer and probe set (Applied Biosystems) was used for normalization purposes.
Preparation of RNA Probes
Riboprobes were prepared from Midkine cDNA (GenBank database accession number NM—030859) by PCR amplification of a 387 by fragment (nucleotides 93-490) using a SP6 (sequence in bold) tagged forward primer 5′-GATTTAGGTGACACTATAGAAGTTTCTTCCTTCTAGCCCTTG-3′ and a T7 (sequence in bold) tagged reverse primer 5′-GTAATACGACTCACTATAGGGTCAGTCCTTTCCTTTTCCTTTC-3′. RNA probes were prepared and labeled with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase enzymes [digoxigenin RNA labeling kit (SP6/T7)] according to manufacturer's recommendations (Boehringer Mannheim, Mannheim, Germany).
The temporal pattern of mRNA expression of Mdk was confirmed at the time points of interest following water maze training (
The regulation of Mdk at message level was shown to translate into corresponding protein level regulation following passive avoidance training (
Protein Sample Preparation
The dentate gyrus (n=6) from trained and passive animals was homogenised in ice-cold 0.32M sucrose. Protein concentrations were determined according to the method of Bradford (1976). Samples, of equal protein concentrations, were prepared in reducing sample buffer [3X Blue loading buffer with 10% (v/v) dithiothreithol (DTT) (New England Biolabs)] and boiled at 100° C.
SDS-PAGE and Immunoblotting
Normalised proteins samples were separated on polyacrylamide minigels and electrophoretically transferred to nitrocellulose membranes (Bio-rad). Equal protein loading was confirmed by ponceau S staining of the membrane (not shown). The nitrocellulose was blocked in 5% non-fat milk in 10 mM Tris-HCl, 150 nM NaCl, and 0.05% (vol/vol) Tween-20 (TBS-T) for 1 h at room temperature. The monoclonal antibody to midkine (Abeam) was diluted in 5% milk and incubated overnight at 4° C. The membranes were then incubated with appropriate secondary horseradish peroxidase-linked antibodies and visualised by SuperSignal Chemiluminescent Substrate (Pierce) and exposed to X-ray film (Kodak) for appropriate times. Films were digitised and quantitative densitometry was performed using NIH Image software (Version 1.61) to determine amount of midkine protein electrophoresed per sample.
2-Dimensional Gel Electrophoresis
Protein samples were sonicated in lysis buffer (9.5 M urea, 2% CHAPS, 20 mM Tris pH 8.0). CyDye DIGE Fluors minimal labelling (GE Healthcare) that covalently bind to the ε-amino group of lysine of proteins were employed. The internal standard containing an equal amount of protein of all the samples was labelled with Cy2 and was run in all the gels in order to compensate for variation across the gels. After the labelling, 2× dilution buffer (9.5 M urea, 2% CHAPS, 2% DTT, 1.6% Pharmalyte) was added in order to unfold the protein. Strips used were Amersham Bioscience 24 cm strips pH range 4-7; the pool sample, a trained (Cy3 or Cy5) and passive (Cy5 or Cy3) sample were loaded on the same strip overnight during the dehydration step and run the day after on IPGphor (80 kVh). The second dimension was performed in 12% Acrylamide SDS-gel and the gels were scanned on the Typhoon 9410 scanner. Images were analyzed using Decyder 6.5 (GE Healthcare) in BVA mode. Prep gel was ran to cut spots in order to obtain protein ids by mass spectrometry. 300 μg of protein samples were loaded per gel and gels were stained with PlusOne silver staining kit (GE Healthcare).
When recombinant Mdk was administered into the 3rd cerebral ventricles of rats, spatial memory was enhanced following training on the Morris Water Maze. Animals were trained on the Morris Water Maze as described above. Mdk improved spatial memory on this learning task when administered to the 3rd cerebral ventricle 2 hours post training on each of the four days of training (
Mdk administered into the 3rd cerebral ventricles of rats also enhanced recall of an odor reward association. Odor reward association was carried out as described below.
Odor-Reward Association Paradigm
The training protocol employed has been described in detail previously (Foley et al., 2003). The training apparatus was a square box constructed of opaque plastic measuring 34×34×27 cm. Sponges measuring 6×7×2 cm deep had a hole of 2 cm diameter cut into the center and were placed in glass slide-holders of the same size. The food reinforcement was placed at the bottom of the opening in the sponge so the rat had to put its head inside the hole (nose poke) to obtain the chocolate rice crispy breakfast cereal reward (Chocokrispies®, Kellog's, France). On the first trial, four Chocokrispies were also placed on the corners of that sponge, which was impregnated with the target odor, as well as in the hole. The sponge with the non-targeted odor did not contain reinforcement. Sponges were placed in three corners of the box and the position of each odor within the box was changed for each trial according to a previously determined protocol. In addition, the set of sponges was changed between trials to preclude identification based on visual cues. Sponges were impregnated with an odor by placing 15 μl of essence on each corner of the sponge. Odors used were coffee, lime and almond. A video camera was fixed above the apparatus and the rat was observed on a video monitor in the same room. The sessions were recorded on a video tape for possible re-analyses off-line. The first day of pre-training session, the rats were given free access to food for 20 min in a neutral cage. The second day, rats were given free access to the reinforcement for 10 min in the same neutral cage and placed for 10 min in the experimental box without the sponges. Training was carried out in a single session, in five trials. Latency before a correct response (nose-poke into the reinforced sponge) and number of errors (nose-poke into incorrect sponges) were recorded.
All rats rapidly acquired an association between an odor and a food reward (
Prepulse inhibition of startle is used as a readout of sensorimotor processing in the prefrontal cortex. Midkine administration into the 3rd cerebral ventricle of the brain reverses the deficit observed in prepulse inhibition of startle in animals that have been reared in isolation.
Isolation Rearing (Geyer et al., 1993)
Isolation reared animals (n=7-8) were raised in single perspex cages from weaning on postnatal day (P) 25. Separate cohorts were analyzed at P30, P40, P60 and P80. All isolation reared animals are compared to aged matched social controls (raised in cages of 3-4 from weaning, n=8).
Prepulse Inhibition of Startle Response (Ellenbroek et al., 1998)
The startle apparatus consisted of a sound attenuating chamber (54.6×50.8×30.5 cm), a startle platform which measured the startle response, and an audio stimulator, controlled by the startle reflex software (MED Associates Inc.). Animals were restrained in an appropriately sized animal holder, placed on the startle platform. Animals were placed in the apparatus and allowed to acclimatize to a background noise of 70 dB[A] for 5 minutes. Subsequently, the animals received five startle trials (120 bD[A] burst of white noise lasting 20 ms). Next, the rats received five blocks of trials, consisting of two startle trials and four prepulse inhibition trials (of differing prepulse intensity), as well as a no stimulus condition. The prepulse inhibition trials consisted of a prepulse stimulus of 72, 76, 80 or 84 dB[A] burst of white noise, followed 100 ms later by the startle stimulus of 120 dB[A]. The inter-trial interval was between 10 and 20 s. The session terminated with five further startle trials. The resulting movement of the rats was measured during 100 ms after startle stimulus onset, and then rectified, amplified and analyzed by computer and the maximal response and average response over the 100 ms period determined. Baseline startle amplitude was determined as the mean response of all startle trials. The % prepulse inhibition was determined according to the following formula: 100−([startle amplitude at prepulse trial/startle amplitude at startle trial]×100%). The effect on baseline startle amplitude was analyzed by a one-way analysis of variance (ANOVA). The effect of rearing on PPI was determined by 2-way ANOVA (factors: rearing and prepulse intensity), with post-hoc Bonferroni tests.
Isolation reared animals exhibited a significant deficit in prepulse inhibition (PPI) of startle. Four daily ICV injections of midkine (2 μg/day) reversed PPI behavior back to normal compared to vehicle-treated animals (Two-way ANOVA, p<0.05 for treatment) (
This example provides confirmation of the pattern of Ttr regulation over time following the learning tasks, using quantitative real-time PCR as described above. The temporal pattern of mRNA expression of Ttr was confirmed following water maze training (
The regulation of Ttr at message level was shown to translate into a prolonged increase at the protein level following water maze training, which lasted from 6-12 hours (
When recombinant Ttr was administered into the 3rd cerebral ventricles of rats, spatial memory was enhanced following training on the Morris Water Maze. Animals were trained on the Morris Water Maze as described above. Ttr improved spatial memory on this learning task when administered to the 3rd cerebral ventricle 2 hours post training on each of the four days of training (
This example provides confirmation of the pattern of an unknown EST's regulation over time following the learning tasks, using quantitative real-time PCR as described above.
The temporal pattern of mRNA expression of the unknown EST, UK8, was confirmed at the time points of interest following water maze training (
The expression of this unknown transcript was analyzed by in situ hybridization as described below.
In Situ Hybridization
All steps prior to and during hybridization were conducted under RNase-free conditions. 40 μm fresh frozen sections were fixed in cold (4° C.) 4% paraformaldehyde in PBS before being washed in PBS and 0.1% Triton-X. Sections were incubated in a humidified chamber overnight at 60° C. with hybridization solution containing 1% of the digoxigenin-UTP labeled antisense or sense RNA probes. The slides were then washed twice for 15 mM each time with 2×sodium-saline-citrate buffer (SSC), 1×SSC and 0.1×SSC at 60° C. After washing, slides were prepared for immunodetection by incubating them in 150 mM NaCl and 100 mM Tris, pH 7.5 (Buffer 1) containing 1% normal goat serum and 0.1% triton-X for 30 mM at room temperature. The sections were then exposed to anti-digoxigenin alkali-phosphatase conjugate (Boehringer Mannheim) at 1:1000 dilution in the same buffer for 2 h at room temperature. They were then washed twice for 10 min each with Buffer 1 and Buffer 2 (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, and 50 mM MgCl2). The bound antibody was detected by incubating the slides with 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitroblue Tetrazolium (BCIP/NBT) substrate (Boehringer Mannheim), producing a purple precipitate reaction product. The reaction was stopped by incubating sections in Buffer 3 (10 mM Tris-HCl (pH 8.1) and 1 mM EDTA) and then washed with distilled water. The sections were then mounted in glycerol (Sigma) and imaged using an Aperio scanning system. The Aperio Slide Scanning system at the UCD Conway Institute was funded under a Health Research Board of Ireland Equipment Grant.
In situ hybridization analysis demonstrated that UK8 was highly localized to the hippocampus and cerebellum (
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/33732 | 2/11/2009 | WO | 00 | 2/17/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61029213 | Feb 2008 | US |
