WO2014172490A1 - Models for apc related diseases and disorders, methods of diagnosing and treating, and methods for identifying therapeutic agents for treating apc related diseases and disorders - Google Patents

Abstract

Described herein is a mouse model with a conditional knockout of adenomatous polyposis coli protein function. The mouse model is used to identify molecular diagnostic determinants for diagnosing APC-related diseases and disorders, therapeutic agents useful for treating APC-related diseases and disorders, methods of treating APC-related diseases and disorders, as well as kits and reagents useful for diagnosing and treating APC-related diseases and disorders.

Description

MODELS FOR APC-RELATED DISEASES AND DISORDERS, METHODS OF DIAGNOSING AND TREATING, AND METHODS FOR IDENTIFYING THERAPEUTIC AGENTS FOR TREATING APC-RELATED

DISEASES AND DISORDERS RELATED APPLICATION

This application claims the benefit of U.S. provisional application No: 61/812,493, filed April 16, 2013, the entire contents of each of which are herein incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant R01 NS021725-23S1 , R01 NS21725, P30 NS047243 and T32-NS061764 awarded by the National Institute of

Neurological Disorders and Stroke (NINDS), and R01 DC008802 awarded by the National Institute on Deafness and Other Communication (NIDCD). The government has certain rights in the invention.

BACKGROUND

Neurocognitive disorders such as, for example, autism spectrum disorders and intellectual disabilities, are traditionally behaviorally related disorders that are difficult to diagnose and treat, as the molecular determinants of such disorders have not been clearly identified. Additionally, these disorders frequently have co-morbidities such as seizures, gastrointestinal symptoms and impaired sensory information processing. Autism spectrum disorders, for example, can include a number of behavioral symptoms, making precise diagnosis difficult and often inconsistent. As diagnosis can often be equivocal, so too can methods of treating such disorders, as therapeutic agents useful for treating one disorder might not be effective in treating other disorders, even though the disorders might be classified similarly based on a subject's behavior.

New and improved methods for diagnosing and treating neurocognitive and

neurodegenerative disorders would assist in ameliorating the effects of potentially devastating behavioral disorders.

SUMMARY

One embodiment is directed to a transgenic mouse comprising a conditional knockout of the adenomatous polyposis coli protein, wherein the adenomatous polyposis coli protein is knocked out predominantly in the forebrain after the birth of the mouse or during a desired developmental stage. In a particular embodiment, the conditional knockout is caused by a transcript under the control of a Ca²7calmodulin-dependent protein kinase II promoter. In a l particular embodiment, the transcript is a LoxP transcript. In a particular embodiment, the adenomatous polyposis coli gene is engineered to comprise one or more site-specific Cre recombination sites. In a particular embodiment, the adenomatous polyposis coli protein function is knocked out in response to an external stimulus. In a particular embodiment, the external stimulus is a physical stimulus or a chemical stimulus.

One embodiment is directed to a method of diagnosing an APC-related disease or disorder, comprising a) contacting a sample from a subject with an assay assembly wherein the assay assembly determines the expression level of one or more informative genes; and b) comparing the expression level of the one or more informative genes with one or more reference expression profile comprising expression levels for the one or more informative genes, wherein the one or more expression profiles correspond to an APC-related disease or disorder or to a phenotype that does not exhibit an APC-related disease or disorder, wherein statistical similarity to a particular reference expression profile indicates subject providing the sample has a phenotype associated with the phenotype corresponding to the reference expression profile. In a particular embodiment, the APC-related disease or disorder is selected form the group consisting of: neurodevelopmental diseases, autism, viral infection, perinatal hypoxic encephalopathy, Aicardi syndrome, seizures, an autism spectrum disorder, a neurocognitive disorder, intellectual disability, hearing loss, infantile spasms, West syndrome, inflammatory responses, gastrointestinal (Gl) disorders and co-morbid disorders. In a particular embodiment, the one or more informative genes are selected from the group consisting of cytokines, chemokines, stress hormones and micro-RNAs. In a particular embodiment, the one or more informative genes are selected from the group consisting of: neurogenesis markers, DCX, homer, TAO kinases, microglia proteins, Iba1 , β-catenin, N-cadherin and components of this synaptic adhesion complex, δ-catenin, γ-catenin (plakoglobin), oc-catenin, canonical and non-canonical Wnt signaling pathway components, disheveled, LRP5/6, Frizzleds, Ryk, GSK3 beta and alpha, endogenous regulators of Wnt signaling, Dkk1 , axins, SFRPs, connexin 43, PPARs, Tcf, Lef, Foxgl , Arx, neurotrophic factors and their receptors, BDNF, NT3, Trks, scaffold proteins, S-SCAM, Magi2, PSD-95 protein family members, scribble, homer, GRIP, Pick, gephyrin, ion channels, sodium and potassium subunits and accessory proteins, adhesion molecules, neuroligins 1 , 2 and 3, a- and β-neurexins, Ephs, Ephrins, SynCAMs, guidance molecules, semaphorins, plexins, LRRTM, LAR proteins, neuregulins, Erbs, presenilin, rictor, DOCKs, PAR3, PAR6, aPKC, CASK, Veli, Mint, synapse-associated signaling and plasticity proteins, ERK, PP2A, Akt, PTEN, PI3K, MAPK, CDK5, CAMKII, IQGAPs, Asef, tuberous sclerosis complex proteins, mTOR, S6K, FMRP, CREB, formins, mDia, regulators of local mRNA translation, cytoskeleton regulators, EB1 , MAPI B, Rac, Ras, Rho, Cdc42, CDKL5, MEF2C, SPTAN1 , SLC25A22, neurotransmitter receptor subunits, glutamatergic (ionotropic and metabotropic), GABAergic, nicotinic acetylcholine, dopaminergic and serotoninergic, MACF, Acf7, Lis1 , astrocyte proteins, active zone proteins, synaptic vesicle proteins, STXB1 , glutamate transporters, neuronal cytoplasmic calcium binding proteins, and inhibitory interneuron proteins. In a particular embodiment, the sample is a blood sample. In a particular embodiment, the assay assembly determines the expression level of two or more informative genes. In a particular embodiment, the assay assembly determines the expression level of three or more informative genes. In a particular embodiment, the statistical similarity of the sample expression levels with one or more reference expression profiles is determined by a computing device.

One embodiment is directed to a kit for diagnosing an APC-related disease or disorder comprising hybridization probes for two or more informative genes that exhibit differential expression between an afflicted sample and a non-afflicted sample.

One embodiment is directed to a method of treating a neurodegenerative or

neurocognitive disorder, comprising administering a therapeutically effective amount of a therapeutic compound identified as being effective for treating a neurodegenerative or neurocognitive disorder. In a particular embodiment, the therapeutic compound is identified as being effective for treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder by a method comprising: contacting a cell with the test compound and comparing the expression profile of the cell of a sample obtained from an animal comprising the cell, wherein a statistical similarity to an expression profile of a non-afflicted subject indicates the compound is effective for treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder. In a particular embodiment, the compound is ECGC. In a particular embodiment, the compound is decitabine.

One embodiment is directed to a method of identifying a therapeutic agent for treating or ameliorating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder, comprising: a) contacting an animal comprising a conditional APC knockout, a tissue sample from an animal comprising a conditional APC knockout or a cell obtained from an animal comprising a conditional APC knockout with a test agent; and b) comparing a sample expression profile obtained from the conditional APC knockout animal that had been contacted with the test therapeutic agent with a reference expression profile indicative of a non-afflicted state, wherein a statistically relevant similarity of the sample expression profile to the reference expression profile indicates the test agent is a therapeutic agent for treating or ameliorating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows characteristics of APC conditional knockout in mouse forebrain neurons.

The top panel is a Western blot showing APC protein levels are dramatically decreased in hippocampal, cortical and striatal, but not cerebellar, lysates of APC cKO mice at 3 months of age. Similar decreases were seen at 1 month. APC deletion is driven by CAMKII promoter- dependent expression of Cre recombinase in forebrain postmitotic excitatory neurons and striatal inhibitory neurons. The bottom panel is a histogram of decreased APC levels in the indicated brain region lysates of APC cKO mice, relative to control littermate levels. Signals are normalized to GAPDH as a loading control. ^*p<0.05, ^**p<0.01 , Student's t-test, n = 5 APC cKO mice and 4 control littermates.

FIGS. 2A-K show impaired learning and memory in APC cKO mice. FIG. 2A shows, in the novel object recognition task, control littermate mice displayed a preference for the novel object, whereas APC cKO mice showed equal interest towards the novel and familiar objects. ^*p<0.05 compared to a hypothetical 50%, n = 1 1 cKOs and controls (FIGS. 2B-E). In the Barnes maze task, control littermates showed rapid improvement and learned the location of the goal hole after 2 days, as measured by latency to find the goal (FIG. 2B, ^*p < 0.01 repeated measures ANOVA) number of errors committed before reaching the goal (FIG. 2C), and path efficiency (FIG. 2D). APC cKOs showed slower improvement, (FIG. 2B) requiring 4 days to learn the location of the goal hole, (FIG. 2C) committing more errors and (FIG. 2D) taking less efficient paths to the hole throughout the test period. No differences were found in (FIG. 2E) average speed between the cKOs and control littermates during the task. FIG. 2F: Probe trial on day 5 shows that both cKOs and controls display a strong preference for the goal location, as measured by the number of visits to the goal hole. FIG. 2G: Probe trial on day 12 shows that cKOs do not retain preference for the goal location, whereas controls do. APC cKOs (FIG. 2H) take longer to reach the goal location, (FIG. 2I) commit more errors, and (FIG. 2J) take a less efficient path, relative to control littermates, suggesting impaired long-term memory formation. ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , Student's t-test, n = 12 APC cKOs and 14 control littermates. FIG. 2K: In the continuous spontaneous alternation task to assess working memory, control littermates showed a preference to alternate, whereas APC cKOs did not; they alternated close to the chance rate of 50%. ^*p < 0.05, Student's t-test. n = 7 APC cKOs and 6 control littermates. FIGS. 3A-G show repetitive behaviors and low anxiety in APC cKO mice (FIG. 3A). In the marble burying assay, APC cKO mice buried 1 .6 times more marbles than control littermates. n = 12 APC cKOs and 14 control littermates. FIG. 3B: In the elevated plus maze assay, APC cKO mice, compared with control littermates, spent significantly more of the time (36.6% vs. 8.3%, respectively) in the open arms, suggesting reduced anxiety-like behavior. ^**p < 0.01 , Student's t-test. FIG. 3C: APC cKOs and control siblings traveled a similar distance over the course of the trial, suggesting no hyperactivity, n = 9 APC cKO mice and 8 control littermates. FIGS. 3D-G: In the repetitive four novel object contact task, (FIG. 3D) APC cKOs and controls showed equal levels of preference for the different objects. FIG. 3E: APC cKOs made a greater number of visits to the objects over the test period, suggesting that controls habituated, whereas cKOs did not. FIG. 3F: APC cKOs repeated a particular preferred sequence of visits to the four objects more frequently, after normalizing for the total number of object visits, p = 0.0042, Student's t-test. FIG. 3G: The cKOs preserved their direction of movement, such as clockwise or counterclockwise, significantly more than control littermates, based on differences in the slope of log plots of the directionality of movement, normalized for the total number of object visits (p = 0.001345, linear regression analysis, n = 8 APC cKOs and 10 controls. For comparison, the plot shows the slope for total preservation of movement in one direction (top dotted line) versus totally random directional movements to visit objects (bottom dotted line).

FIGS. 4A-F are plots showing reduced social interest in APC cKO mice. FIGS. 4A and

4C: In the social versus non-social olfaction task, both APC cKOs and controls show the ability to distinguish odors. FIG. 4A: They displayed similar levels of interest to non-social odors. FIG. 4B: They also display the typical habituation pattern, reduced interest over the three successive exposures to the same non-social odor. FIG. 4C: APC cKOs exhibited significantly less interest in social odors, compared with control littermate mice. FIG. 4D: Controls showed the typical habituation pattern over three separate exposures to the same social odor, followed by dishabituation when exposed to a novel social odor, while cKOs showed a less robust, behavior pattern. FIG. 4C: novel male cage odor, n = 8 cKOs and 10 controls; novel male cage odor, n = 1 1 cKOs and 9 controls). FIG. 4E: In the three-chambered social interaction assay, both cKOs and controls displayed a preference for a caged, novel ovariectomized female mouse, versus an empty cage, but APC cKOs spent significantly less time interacting with the novel mouse, indicating reduced social interest. FIG. 4F: APC cKOs and controls littermates made a similar number of entries to both side chambers, n = 1 1 cKOs and 7 controls, ^*p < 0.05, ^**p < 0.01 , ^***p < 0.001 , Student's t-test. FIGS. 5A-E show increased β-catenin levels and canonical Wnt target gene expression levels, and decreased presenilin 1 levels, in APC cKO forebrain neurons. Representative immunoblots of hippocampal, cortical and striatal lysates and their quantification show two-fold increased levels of β-catenin in APC cKO mice. Signals were normalized to GAPDH as a loading control. FIG. 5B Epifluorescence micrographs show increased β-catenin nuclear immunostaining in forebrain neurons of APC cKO mice, compared with control littermates, processed in parallel, consistent with enhanced canonical Wnt signaling. Bottom panels:

Immunopositive neuronal nuclei were identified by their characteristic size and shape, and by Dapi staining. FIG. 5C: Histogram showing APC cKOs display increased transcript levels of several Wnt target genes, including Sp5, a transcriptional repressor of Sp1 genes, neurogl, implicated in cortical neuronal differentiation, syn2, a synaptic vesicle phosphoprotein that functions to maintain the reserve pool of glutamatergic vesicles, predicted to be a Wnt target, and dkkl, a Wnt antagonist that functions in a feedback mechanism to regulate excessive Wnt signaling (^*p < 0.05, ^**p < 0.01 , Student's t-test; n = 5 APC cKO, 6 ctl littermate mice. FIG. 5D: Immunoprecipitation of N-cadherin showing increased association with β-catenin in the APC cKO hippocampus (n = 2 APC cKO, control). FIG. 5E: Quantitative immunoblot and histogram show unchanged levels of N-cadherin and a significant decrease in Presenilin 1 levels

(^*p < 0.05, Student's t-test, n = 5 APC cKO, 4 controls).

FIGS. 6A and 6B show APC regulates FMRP levels in the hippocampus. FIG. 6A:

Representative immunoblots of hippocampal lysates show altered levels of FMRP,

phosphorylated (activated) Akt, and PTEN in APC cKO mice. Postsynaptic density fractions showed similar results. FIG. 6B: Quantification of FIG. 6A showing decreased FMRP and pAkt, and increased PTEN. n = 5 APC cKO mice and n = 4 control littermates. Control and APC cKO bands are from the same gel. For quantifications, band densities were normalized to GAPDH or actin, or in the case of phosphorylated Akt, to total levels of that protein. ^*p < 0.05, ^**p < 0.01 , Student's t-test.

FIGS. 7A-C show APC loss leads to increased synaptic spine density and reduced structural maturation in pyramidal neurons. FIG. 7A: Representative images showing increased spines on the apical dendrite of APC cKO cortical layer 5 pyramidal neurons, (left panels) Golgi-Cox stained brightfield images; (right panels) IMARIS reconstructions of confocal stacks of apical dendrite (red) and spines (blue) from APC cKO-Thy-1 -YFP and littermate control layer 5 neurons. FIG. 7B: Histogram shows increased spine density (number of spines per unit length) on the apical dendrite of APC cKO cortical layer 5 pyramidal neurons and hippocampal CA1 pyramidal neurons in the striatum radiatum. ^* p < 0.05, ^***p <0.001 , Student's t-test. FIG. 7C: Histogram shows that less mature stub-shaped spines predominated in APC cKOs, whereas mushroom spines predominated in control littermates. ^***p < 0.001 , Student's t-test, n = 10 APC cKO, 10 Ctl neurons, average of 10 side plane view spines/neuron.

FIGS. 8A-H show basal synaptic transmission is not altered in the APC cKO

hippocampus. FIG. 8A: Representative traces of AMPAR-mediated mEPSCs measured by whole-cell recordings from CA1 neurons of control and APC cKO brain slices. FIG. 8B: APC cKO CA1 neurons show increased AMPAR mEPSC frequency. AMPAR-mediated mEPSC amplitude (FIG. 8C) and rise time (FIG. 8D) are unchanged, whereas the decay time (FIG. 8E) is decreased in APC cKO neurons. ^*p < 0.05, Student's t-test, n = 9 cKOs and 6 controls. FIG. 8F: Paired pulse ratio is unaffected at SC-CA1 synapses of APC cKOs. n = 5 cKOs and 6 controls. FIG. 8G: Representative traces of fEPSPs measured by extracellular recordings from the CA1 region in response to Schaffer collateral stimulation in freshly isolated brain slices. FIG. 8H: Quantification of traces (FIG. 8G) indicates normal input-output relationships in APC cKO hippocampal CA1 neurons, suggesting no change in basal synaptic transmission, n = 5 cKOs and 6 controls.

FIGS. 9A-D show modestly enhanced synaptic plasticity in APC cKO mice. FIGS. 9A,B: APC cKOs, compared with control littermates, show no significant change in mGluR-dependent LTD induced at Schaffer collateral-CA1 synapses by treating freshly isolated hippocampal slices with DHPG (100 μΜ) for 10 minutes, p = 0.0668, repeated measure ANOVA, n = 1 1 cKOs and 10 controls. FIGS. 9C,D: APC cKO mice show enhanced LTP induced at SC-CA1 synapses by delivering 5 trains of theta burst stimulation in hippocampal slices. ^*p = 0.0227, repeated measure ANOVA.

FIGS. 10A-D show reduced hearing in APC cKO mice. FIG. 10A: ABR thresholds are increased at all frequencies tested in APC cKOs (n = 10 APC cKOs and 9 WT littermates at 6-9 weeks old). FIG. 10B: Representative ABR traces. FIG. 10C: ABR peak 1 amplitudes at

16 kHz and 32 kHz are reduced in APC cKOs. FIG. 10D: DPOAE thresholds are shifted upward at higher frequencies, but not significantly altered, in APC cKOs (12 APC cKOs and 13 WTs at 6-9 weeks old).

FIGS. 1 1 demonstrates altered size of afferent ribbon synapses in inner hair cells (IHCs) of APC cKO mice. APC cKO IHCs show increases in ribbon synapse size, selectively on the pillar side, based on CTBP2 immunostaining, in whole mount confocal micrographs of the middle cochlear turn (n = 6 APC cKOs and 4 control littermates at 6-7 weeks old). Shown are representative flattened z stack and 5-cell-thick volume rendered images. In contrast, the total number of ribbons per IHC is not significantly altered in APC cKOs versus control littermate mice (15.6 ± 1 .4 vs. 14.8 ± 1 .2, respectively). Right panels: Volumetric analysis of CTBP2 staining shows a shift to larger ribbons per IHC on the pillar side, but not the modiolar side, of APC cKOs relative to control littermate mice. Normal hearing requires the two structurally and functionally distinct ribbon synapse types: small, apically localized ribbon synapses are necessary for low-threshold hearing, while large, basally localized ribbon synapses are necessary for high threshold hearing. Thus, in the inner ear, APC is essential for proper synapse development and normal hearing.

FIGS. 12A-L show APC regulates expression levels of FMRP and other plasticity and signaling proteins in vivo. FIG. 12A: Representative immunoblots of hippocampal postsynaptic density fractions (psds) show increases in synaptic levels of inactivated GSK-3

(phosphorylated at Ser9) and decreases in phosphorylated (Ser37) β-catenin, FMRP,

PP2A-B56, phosphorylated (activated) ERK2 and phosphorylated (activated) Akt in APC cKO mice compared with control littermates. FIG. 12B: Quantification of FIG. 12A. FIG. 12C:

Representative immunoblots of hippocampal lysates show altered plasticity protein expression levels (decreases in FMRP and PP2A-B56 regulatory subunit levels, and increases in PTEN) in APC cKOs compared with control littermates. FIG. 12D: Quantification of FIG. 12C.

Immunoblots of glutamate receptor levels in APC cKO hippocampal psds (FIG. 12E) and hippocampal lysate (FIG. 12G). FIGS. 12F,H: Quantification of FIGS. 12E,G. Immunoblots of synaptic adhesion proteins in APC cKO hippocampal psds (FIG. 121) and lysates (FIG. 12K). FIGS. 12J,L: Quantification of FIGS. 121, K. All bands are representative of an n of 5 distinct lysates from 5 separate APC cKO and control littermate mice. All bands are representative of an n of 3 psd samples, each pooled from the hippocampi of 3 mice. Control and APC cKO bands are from the same gel. For quantifications, band densities were normalized to GAPDH or actin, or in the case of phosphorylated proteins, to total levels of that protein. Statistically significant differences: ^*p < 0.05, ^**P < 0.01 , ^***P < 0.001 , Student's t-test.

FIG. 13 is a schematic showing aspects of signaling pathways downstream of APC. APC regulates Wnt/ -catenin, Presenilis /Creb pathway, FMRP, PP2A and ERK signaling pathways. These pathways regulate synapse maturation and plasticity, selected gene expression, and activity dependent protein synthesis- processes that are critical for normal learning and behavior. These signaling pathways are dysregulated in the absence of APC. Direction of change in APC cKO neurons indicated in parentheses.

FIG. 14 is a series of still images from video recordings of a litter of P5 APC cKOs (#4,6,7) and control siblings (#2,3). Early neonatal APC cKO mice display spasms (increased high amplitude spontaneous movements), compared with control littermates. FIG. 1 5 shows that acute brain slices from neonatal APC cKOs display increased spontaneous discharges and increased evoked network activity. Top Panel: Spontaneous activity- APC cKO (red) mice show spontaneous, low-amplitude neuronal activity at P8, and high amplitude discharges at P15, as compared to WT littermates (black). Inset, Increased frequency of spontaneous field discharges in APC cKO. ** p<0.01 . Bottom panel: Electrically evoked activity- Electrically evoked activity in cortical slices containing 1 μΜ GABAzine (a GABAA receptor antagonist) at 10^χ threshold stimulation. APC cKOs exhibited significantly prolonged network discharges at P1 5 compared to WT littermates. Inset, x axis=fold stimulation compared to threshold.

FIG. 1 6 are electroencephalograms (EEGs) from IS patients that show strikingly similar activity patterns to APC cKO mice. Top Panel: EEG recorded from a patient with Aicardi's Syndrome (EEG pattern of initial discharge (black) followed by electrodecrement (red) and a slow oscillation (blue). Spasm occurs (indicated by arrow)). Bottom panel: Cortical field potential recorded from acute cortical brain slice of APC cKO mouse in 1 μΜ GBZ (similar network activity).

FIG. 1 7 shows APC cKO mice recapitulate a number of phenotypes of Aicardi

Syndrome, a devastating neurodevelopmental disease associated with IS. FIG 17 shows corpus callosal and commissural agenesis in APC cKO mice (left panel) and altered eye morphology (right panel).

FIG. 1 8 shows postnatal behavioral spasms. The left panel shows observed spastic movements at P9: Flexion/extension of limbs (typically 2-4), fast twitching of legs, slow falling over and remaining on the side, curling trunk and arching back. The right panel shows observed spastic movements at P14: extremely rapid twitchy movements, high speed switching directions, head shaking side to side. Lower panels: Quantification of the increased spastic movements in APC cKO mice at P9 and P14.

FIG. 1 9 provides evidence of spontaneous seizures in adult APC cKO mice. EEG recorded from chronically implanted 2 month old APC cKO mouse. Some APC cKO mice exhibited spontaneous seizures (n = 3 out of 6 cKOs sampled for two weeks), whereas control littermate mice did not (n = 5 controls also sampled for two weeks).

FIGS. 20A-C show increased β-catenin/Wnt levels in APC cKO forebrain neurons.

FIG. 20A shows immunoblots of cortical lysates indicating a progressive reduction of APC protein during development (from P9 to P60) in APC cKO mice, with a parallel and gradual increase of β-catenin (2-fold increase at P60). FIG. 20B shows confocal micrographs showing increased β-catenin staining in specific cortical layers in APC cKO mice at P14. FIG. 20C shows significant increase of nuclear β-catenin immunostaining in layer V pyramidal neurons of APC cKO cortex.

DETAILED DESCRIPTION

The findings described herein provide a novel molecular etiology of cognitive and autistic disabilities, e.g., cognitive impairments, autistic-like disabilities, reduced hearing and seizures that resemble the childhood epilepsy syndrome of infantile spasms (IS). A new role for adenomatous polyposis coli (APC) is identified for interconnecting and regulating synaptic adhesion complexes and key signal transduction/ plasticity pathways in the mammalian brain.

Described herein, for example, are materials and methods related to the discovery of a new transgenic animal model for neurodegenerative, neurocognitive and autism spectrum disorders, seizures, as well as co-morbid disorders. The animal model comprises a conditional knockout (cKO) of the APC gene, wherein the APC gene function is knocked out predominantly at postnatal stages of development. The APC cKO mouse, compared to control littermates, exhibits autistic-like behaviors and cognitive deficits. The conditional knockout can be achieved, for example, by conditional deletion of critical exons of the APC gene, e.g., engineered with flanking by LoxP sequences that lead to excision of the key coding regions by Cre recombinase expressed under the control of the Ca²7calmodulin kinase II (CamKII) promoter. The APC cKO model is herein shown to be a useful animal model for autism spectrum disorders (ASD), cognitive impairments, reduced hearing, IS, immune system changes and epilepsy. Using the APC cKO animal model, methods of treating neurodegenerative, neurocognitive and autism spectrum disorders, and co-morbid disorders such as, for example, IS, epilepsy and reduced hearing, can be identified and verified, and responders and non-responders to such therapeutic treatments can also be identified. The APC CKO animal model can also be used, for example, to identify informative genes and markers that can be used to more accurately diagnose and identify neurodegenerative, neurocognitive and autism spectrum disorders and co-morbid disorders, as well as effective therapeutic interventions.

Data described herein, for example, identify APC cKO mice as a new genetic model of IS. APC cKO mice, compared to control littermates, exhibit: cognitive impairments and autistic-like behavior, increased density of excitatory synaptic spines on pyramidal neurons, spasms and high amplitude movements at P9 and P14, spontaneous seizures recorded by in vivo EEG at two months of age, and excessive levels of β-catenin/canonical Wnt signaling in the forebrain, and altered inflammatory cytokine levels in monocytes, indicating cross-talk between the brain and the immune system. APC deletion in excitatory neurons leads to deregulated Wnt and synaptogenic protein networks, and subsequently to synaptic and circuit dysfunction associated with IS.

APC

The APC gene encodes a multidomain protein that plays a major role in tumor suppression by negatively regulating the canonical Wnt signaling pathway (FIG. 13).

Inappropriate activation of this pathway through loss of APC function contributes to cancer progression, as in familial adenomatous polyposis. Although APC has been associated with, for example, colon cancer, its roles during development and in the brain have not been investigated thoroughly. As described herein, a cKO of the APC gene allows for the temporal and spatial targeted knockout of the gene function in the brain at later developmental stages.

The APC protein is an integral part of the β-catenin/Wnt signaling pathway. The Wnt signaling pathway controls cell fate and cell-cell communication in the embryo and adult. It was identified first for its role in mouse breast cancer, and separately in creating normal patterns of embryonic development. Its role in embryonic patterning was discovered when genetic mutations in critical players in this pathway produced abnormal fruit fly embryos. It is evolutionarily conserved, and functions across species ranging from the fruit fly to humans

APC-associated diseases and disorders

Although APC has been previously associated with cancer and proliferative disorders, described herein is the first direct test that shows a link between APC and social, cognitive and autistic disabilities and co-morbid disorders. cKOs of the APC gene that target deletion of the gene in selected brain cells at postnatal (late) developmental stages in mice produce animals with behavioral and cognitive phenotypes that model, for example, autism spectrum disorders (ASD), intellectual disabilities (ID) and IS, a childhood epilepsy, reduced hearing and immune system change.

IDs and ASDs link to human APC gene deletions, as described herein. APC is essential for coordinated maturation of presynaptic and postsynaptic specializations at nicotinic synapses of avian peripheral neurons. Described herein is a novel APC cKO mouse, with APC depletion targeted predominantly to excitatory neurons during synaptic differentiation, because APC is enriched at these postsynaptic sites. Use of the materials and methods described herein provides the first direct evidence of APC's role in cognition and behavior. APC cKO mice, compared with wild-type littermates, exhibit impaired learning and memory formation, increased repetitive behaviors, reduced social interactions, increased density of synaptic spines on pyramidal neurons, and altered levels of plasticity and signaling proteins in the hippocampus, cortex and striatum. Described herein is a novel role for APC- showing that it links to and regulates both fragile-X mental retardation protein (FMRP) and β-catenin/canonical Wnt and presenilinl/Creb pathways. APC cKO mice display unique, as well as shared, molecular, structural and functional changes compared with mice expressing other single gene mutations associated with cognitive and autistic disabilities in humans. These findings indicate that excessive β-catenin/canonical Wnt signaling underlies a novel molecular etiology of syndromic ID and ASD.

IS constitute a catastrophic childhood epilepsy syndrome that is poorly controlled by existing therapeutics and often leads to developmental delays. Described herein are data indicating that a mouse model with cKO of APC in excitatory neurons shows characteristics associated with IS. APC deletion causes aberrant brain development and function that increases susceptibility to IS.

APC cKO mouse pups also exhibit spasms and aberrant cortical excitatory activity that can be used to model IS in humans. IS are a specific type of seizure seen in an epilepsy syndrome of infancy and childhood known as West Syndrome. IS are associated with several neurodevelopmental diseases, autism, viral infection, perinatal hypoxic encephalopathy, Aicardi syndrome, West syndrome and others. West Syndrome is characterized by IS, developmental regression, and a specific pattern on electroencephalography (EEG) testing called

hypsarrhythmia (chaotic brain waves). The onset of IS is usually in the first year of life, typically between 4-8 months. The seizures primarily consist of a sudden bending forward of the body with stiffening of the arms and legs; some children arch their backs as they extend their arms and legs. Spasms tend to occur upon awakening or after feeding, and often occur in clusters of up to 100 spasms at a time. Infants may have dozens of clusters and several hundred spasms per day. Infantile spasms usually stop by age five, but may be replaced by other seizure types. Similarly, Aicardi Syndrome is characterized by agenesis of the corpus callosum, stereotyped changes in eye morphology, developmental delays and debilitating seizures. APC cKO mice exhibit these phenotypes.

Additional molecular and phenotypic aspects of the APC cKO mouse are demonstrated, for example, in FIG. 12.

Hearing deficits

The APC cKO mice described herein can also be used to model aspects of hearing loss. APC function in the cKO described herein is lost in brainstem olivocochlear (OC) neurons that innervate sensory hair cells and in cochlear support cells that surround the hair cells. APC is depleted from the OC neurons during synapse formation with inner hair cells at prehearing stages. This efferent OC synaptic connection is essential for proper differentiation of afferent synaptic connections between presynaptic hair cells and postsynaptic primary auditory neurons that signal sound reception to the brain.

APC is also depleted from the non-sensory support cells, at a delayed stage, compared to the OC neurons, approximately 2-3 weeks postnatal. The non-sensory support cell population is of great interest for regeneration studies in deafened mammals. Utilizing genetic manipulations in the support cells that induce them to transdifferentiate or de-differentiate and divide allows them to replace lost sensory hair cells in mammals suffering from sensorineural deafness. The data described herein identify the APC cKO model, driven by the CamKII promoter, as useful for identifying genetic manipulations in the support cell population that lead to sensory hair cell regeneration.

Normal hearing requires proper assembly of afferent ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) that signal to the brain. Differences in the size and polarized distribution of ribbon synapses have been shown to correlate with the

spontaneous rate (sensitivity) of afferent fibers, suggesting that two distinct ribbon synapse types (large basal and small apical ribbons) are required for hearing in low and high noise environs. Despite the critical role of functional ribbon synapse assembly and maintenance for hearing sensitivity, the underlying molecular mechanisms are poorly defined.

The APC cKO mouse demonstrates that APC is essential for normal cochlear afferent function, as indicated by recordings that show reduced auditory brainstem response (ABR) thresholds, and by alterations in both ribbon synapse structure (shift to significantly larger ribbon sizes on the pillar side of inner hair cells) and spatial distribution (loss of polarized localization) within IHCs based on quantitative immunofluorescent confocal microscopy analyses. These results suggest that sensitivity and polarization of ribbon synapses are not inner hair cell or spiral ganglion neuron autonomous, but rather are regulated by support cells and the transient efferent olivocochlear inputs. Thus, loss of APC disrupts intercellular signaling between cochlear hair cells, neurons and support cells. The findings described herein identify APC as a critical molecule for normal hearing and ribbon synapse maturation in the mammalian inner ear in vivo.

APC conditional knockout

Described herein is a cKO of the APC gene in mice. The cKO is conditional in that the loss of APC function is triggered by a recombinase, e.g., Cre recombinase, under the control of the oc-CamKII promoter. Recombinase target sequences introduced near or into the APC gene direct site-specific recombination events that delete, for example, critical coding sequences of the APC gene upon expression of the recombinase driven by the oc-CamKII promoter. This and related promoters drive expression chiefly in post mitotic excitatory neurons during synapse differentiation, within the first three postnatal weeks, throughout the brain, except for the cerebellum, and also drive expression in striatal inhibitory neurons (medium spiny neurons). APC function is selectively knocked out, for example, by recombinase-dependent excision of recombination site-flanked exons 1 1 and 12, leading to out-of-frame splicing of exon 10 to exon 13 and the generation of a prematurely terminated 468-amino acid APC protein that lacks all identified protein interaction domains and is unstable (Gounari, F. et al., Nat. Immunol., 6:800-9, 2005; see EXAMPLE 1 )). Recombinase expression under control of the CaMKII promoter tends toward the forebrain, so APC can be knocked out in a tissue-specific manner.

The APC cKO mouse described herein offers advantages over other transgenic mouse lines expressing distinct autism-associated gene mutations because it exhibits two features that are found in autistic humans- 1 ) co-morbid autism and hearing defects, and 2) agenesis of the corpus callosum, a structural feature often found in autistic humans that correlates to poor bimanual motor coordination and impaired social cognition. The APC cKO mouse shows dysfunction of several signaling pathways identified as convergent targets of diverse

autism-associated gene mutations. Methods for screening and treatment described herein can be defined using the mouse model.

Although the present disclosure describes a cKO of the APC gene in mice, one of skill in the art could use a variety of methods to effectively knockout the APC function in mice at a specified stage of development and in desired tissues. Recombination systems other than those described herein can be used for, example, and a variety of tissue- and developmental stage-specific promoters can be used to drive expression of a recombinase, e.g., Cre recombinase. Alternatively, one of skill in the art can use an inducible system to knockout APC function in response to a particular physical signal, e.g., a chemical signal, a physical signal such as, for example, a light pulse, UV irradiation, etc. Alternatively, one of skill in the art could achieve tissue and developmental stage-specific deletion of APC using sterotaxic injection of viral vectors that drive, for example, Cre recombinase expression.

Methods of diagnosing APC-related diseases or disorders

Informative genes can be identified to diagnose a disease or disorder or a susceptibility to a disease or disorder, associated with loss of APC function, e.g., cognitive disorders, infantile spasms, West syndrome, Aicardi syndrome, hearing loss, ASD, ID, inflammatory changes, seizures, etc. As used herein, an "informative gene" is a gene that confers a certain statistically significant level of predictive power to indicate a particular phenotype, e.g., disease, disorder, drug sensitivity or other phenotypic state. Informative genes can be, for example, genetic markers based on allele types, or expression markers based on genes that are differentially expressed in different phenotypes of interest.

Informative genes that show differential expression in the brain of the APC cKO mouse versus normal mouse include, but are not limited to, for example, neurogenesis markers (e.g., DCX), homer, TAO kinases, microglia proteins (e.g., Iba1 ), β-catenin, N-cadherin and components of this synaptic adhesion complex, γ-catenin, oc-catenin, δ-catenin, canonical and non-canonical Wnt signaling pathway components (e.g., disheveled, LRP5/6, Frizzleds, Ryk), GSK3 beta and alpha, endogenous regulators of Wnt signaling (such as, for example, Dkk1 , axins, SFRPs), connexin 43, PPARs, Tcf, Lef, Foxg l , Arx, neurotrophic factors and their receptors (e.g., BDNF, NT3, Trks), scaffold proteins (e.g., S-SCAM (Magi2), PSD-95 protein family members, scribble, homer, GRIP, Pick, gephyrin), ion channels (e.g., sodium and potassium subunits and accessory proteins), adhesion molecules (e.g., neuroligins 1 , 2 and 3, a- and β-neurexins, Ephs, Ephrins, SynCAMs), guidance molecules (e.g., semaphorins, plexins), LRRTM, LAR proteins, neuregulins, Erbs, presenilin, rictor, DOCKs, PAR3, PAR6, aPKC, CASK, Veli, Mint, CDK5, synapse-associated signaling and plasticity proteins (e.g., ERK, PP2A, Akt, PTEN, PI3K, MAPK, CAMKI I), IQGAPs, Asef, tuberous sclerosis complex proteins, mTOR, S6K, FMRP, CREB, formins (mDia), regulators of local mRNA translation, cytoskeleton regulators (e.g., EB1 , MAPI B, Rac, Ras, Rho, Cdc42), CDKL5, MEF2C, SPTAN1 , SLC25A22, neurotransmitter receptor subunits (e.g., glutamatergic (ionotropic and metabotropic),

GABAergic, nicotinic acetylcholine, dopaminergic and serotoninergic), MACF (Acf7), Lis1 , astrocyte proteins, active zone proteins, synaptic vesicle proteins, STXB1 , glutamate

transporters, neuronal cytoplasmic calcium binding proteins, inhibitory neuron proteins;

inflammatory cytokines, chemokines and their receptors; including the phosphorylation state of these proteins, as indicators of their activation state and/or of the affinity for binding partners.

The APC cKO mouse is used, for example, to obtain an expression profile comprising one or more, two or more, five or more or between two and one hundred informative genes, such expression profile being indicative of one or more conditions, disorders or phenotypic states described herein. The APC cKO-derived expression profile is then compared, for example, to an expression profile derived from a sample obtained from a mouse with the same genetic background as the APC cKO mouse, except without the APC cKO. Differentially expressed genes in the cKO-derived sample can be used as informative genes for one or more of the conditions, diseases, disorders or phenotypic states described herein. To diagnose or identify a phenotype from a patient sample, a patient expression profile is compared to the cKO-derived expression profile, the non-cKO-derived expression profile, or both. Statistical similarity to the cKO-derived expression profile indicates the sample is from a patient with the phenotype indicated by the cKO-derived expression profile.

As used herein, the term "sample" refers to biological material from a subject. The sample assayed is not limited to any particular type. Samples include, as non-limiting examples, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or supernatants or extracts of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymph tissue, lymph fluid, fibroblasts, stem cells, cerebrospinal fluid, mucous and stool samples. The sample used will vary based on the assay format, the detection method and the nature of the tumors, tissues, cells or extracts to be assayed, as determined by one of skill in the art.

A subset or all informative genes can be assayed for gene expression to generate an "expression profile" that is indicative of a disease or disorder described herein, or a particular phenotype. As used herein, an "expression profile" refers to the level or amount of gene expression of one or more informative genes in a given sample (e.g., blood sample, tissue sample, cell sample, or cultured cell sample, etc.). A "reference" expression profile is a profile of a particular set of informative genes under particular conditions such that the expression profile is characteristic of a particular condition or lack thereof- it is the expression profile to which an expression profile obtained from a sample is compared. In one embodiment, expression profiles are comprised of two or more to about fifty informative genes that exhibit differential expression between an afflicted versus an non-afflicted state (or a first phenotypic state and a second phenotypic state), and provide sufficient power to predict or diagnose with high accuracy between the two states. Other embodiments can include, for example, expression profiles comprising about 5 informative genes, about 25 informative genes, about 100 informative genes, or any number of genes in the range of about 5 to about 400 informative genes.

The informative genes that are used in expression profiles can be genes that exhibit increased expression over normal cells or decreased expression versus normal cells. The particular set of informative genes used to create an expression profile can be, for example, the genes that exhibit the greatest degree of differential expression, or they can be any set of genes that exhibit some degree of differential expression and provide sufficient power to accurately predict the state to be diagnosed. The genes selected are those that have been determined to be differentially expressed in either a disease, drug-responsiveness, or drug-sensitive cell or animal model relative to a normal cell or animal model and confer power to predict a particular phenotypic state. By comparing tissue samples from patients with these reference expression profiles, a patient's susceptibility to a particular disease, drug-responsiveness, or drug- resistance can be determined. Informative genes can include small non-coding RNAs and micro-RNAs that regulate the translation and/or stability of their target mRNAs.

As used herein, the term "differentially expressed" refers to a gene expression product that represents or corresponds to a gene that is differentially expressed in a first cell when compared with a second cell, e.g., mRNA is found at levels at least about 25%, at least about 50% to about 75%, at least about 90%, at least about 1 .5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, or at least about 50-fold or more, different (e.g., higher or lower). The comparison can be made in tissue, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also or alternatively be made between cells removed from their tissue source, or between one cell in situ and a second cell removed from its tissue source.

The generation of an expression profile requires both a method for quantitating the expression from informative genes and a determination of the informative genes to be screened. As used herein, "gene expression products" are transcription or translation products that are derived from a specific gene locus. The "gene locus" includes coding sequences as well as regulatory, flanking and intron sequences. Expression profiles are descriptive of the expression level of gene products that result from informative genes present in cells. Methods are available to one of skill in the art to quickly determine the expression level of several gene products from a sample of cells. For example, short oligonucleotides complementary to mRNA products of several thousand genes can be chemically attached to a solid support, e.g., a "gene chip," to create a "microarray." This hybridization assay allows for a rapid determination of gene expression in a cell sample. Next generation sequencing is another gene expression profiling technique available to one of skill in the art. Alternatively, methods are known to one of skill in the art for a variety of immunoassays to detect protein gene expression products. Such methods can rely, for example, on conjugated antibodies specific for gene products of particular informative genes.

Individuals, for example, with APC disruptive gene mutations, heterozygous due to de novo or inherited deletion, exhibit ID ranging from severe to mild. APC heterozygous deletion and polymorphisms also associate with ASD, albeit less prevalent than the link to ID. Further, the APC cKO targets the particular cell types, cortical glutamatergic neurons, at the

developmental ages shown to be key for convergent expression of several risk genes for both ASD and overlapping ID/ASD in the human and mouse brain (Parikshak, N. et al., Cell, 155:1008-21 , 2013; Willsey, A. et al., Cell, 155:997-1007, 2013). In particular, this expression pattern has been found for human CHD8, one of the highest confidence risk genes for sporadic ASD; CHD8 de novo loss of function mutations lead to excessive β-catenin-dependent gene expression levels, as does APC loss.

The APC cKO mouse data suggest a key role for deregulation of β-catenin and its functions in canonical Wnt target gene expression and N-cadherin synaptic adhesion

complexes, including reduced levels of presenilis . Described herein are findings that identify novel functional and molecular changes not observed previously.

Molecular diagnostic assay

Described herein is the detection of multiple molecular changes that are useful for diagnosing, for example, IDs, ASD and co-morbid disorders in patients. The changes were found in the blood and brains of two different mouse models of IDs and ASDs, APC cKO mice and transgenic mice genetically manipulated to delete fragile X mental retardation protein (FMRP), as a model of human Fragile X syndrome, the most common cause of heritable mental retardation and autistic disabilities. The molecules that showed alterations include stress hormones, pro-inflammatory and anti-inflammatory cytokines, chemokines, β-catenin, Wnt signaling pathways, regulators of the β-catenin/Wnt pathway and proteins indicative of microglia and macrophage activation.

One of skill in the art can test for further gene expression changes using qPCR, immunoblotting and other methods known in the art. The two mouse models of IDs and/or ASD, as well as other genetic mouse models of these disorders, can be used to define correlations between molecular changes and specific cognitive and behavioral phenotypes. Mouse models that selectively exhibit IDs versus ASDs are also used to expand the diagnostic correlations. Identification of genetic and expression markers in animal models can be verified in blood samples from human patients, e.g., children with diagnosed ASD and/or ID and control siblings, whenever possible.

Methods of treating

Described herein is an animal model for APC-related cognitive disorders and diseases. The animal model is useful for screening and identifying therapeutic agents useful for treating APC-related cognitive disorders and diseases, as well as disorders and diseases with shared molecular changes (convergent pathways) to those of the APC cKO mouse. The animal and/or cells and/or tissues derived from the animal model can be contacted, for example, with one or more test therapeutic agents to determine whether contact with the test therapeutic agent alleviates or ameliorates the cognitive disorder or disease. Test agents can target, for example, APC directly to alter APC activity, or a test agent(s) can target one or more downstream molecules that exhibit altered activity when APC activity is altered (e.g., β-catenin, Wnt signaling, presinilinl signaling).

"Treatment" refers to the administration of a therapeutic agent or the performance of medical procedures with respect to a patient or subject, for either prophylaxis (prevention) or to cure or reduce the symptoms of the infirmity or malady in the instance where the patient is afflicted. The compounds described herein and/or identified by screening methods described herein can be used as part of a treatment regimen in therapeutically effective amounts. A "therapeutically effective amount" is an amount sufficient to decrease, prevent or ameliorate the symptoms associated with a medical condition, e.g., an APC-related phenotype, cognitive disorder or disease, IS, ASD, hearing loss, seizure, inflammatory responses, etc.

The terms "patient" and "subject" mean all mammals including humans.

The treatment(s) described herein are understood to utilize formulations including compounds identified herein or identified through methods described herein and, for example, salts, solvates and co-crystals of the compound(s). The compounds of the present disclosure can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as, for example, water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present disclosure.

The term "pharmaceutically acceptable salts, esters, amides and prodrugs" as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, prodrugs and inclusion complexes of the compounds of the present disclosure that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the disclosure.

The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compounds of the above formula, for example, by hydrolysis in blood (T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series;

Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical

Association and Pergamon Press, 1987; both of which are incorporated herein by reference in their entirety). Activation in vivo may come about by chemical action or through the

intermediacy of enzymes. Microflora in the Gl tract may also contribute to activation in vivo. The term "solvate" refers to a compound in the solid state, wherein molecules of a suitable solvent are incorporated. A suitable solvent for therapeutic administration is

physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. Co-crystals are combinations of two or more distinct molecules arranged to create a unique crystal form whose physical properties are different from those of its pure constituents (Remenar, J. et al., 2003. J. Am. Chem. Soc, 125:8456-8457). Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19.sup.th Ed. (1995) volume 1 , page 176-177. The most commonly employed inclusion complexes are those with cyclodextrins, and all cyclodextrin complexes, natural and synthetic, with or without added additives and polymer(s), as described in U.S. Pat. Nos.

5,324,718 and 5,472,954. The disclosures of Remenar, Remington and the '718 and '954 patents are incorporated herein by reference in their entireties.

The compounds can be presented as salts. The term "pharmaceutically acceptable salt" refers to salts whose counter ion derives from pharmaceutically acceptable non-toxic acids and bases. Suitable pharmaceutically acceptable base addition salts for the compounds of the present disclosure include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N-dialkyl amino acid derivatives (e.g., Ν,Ν-dimethylglycine, piperidine-1 -acetic acid and morpholine-4- acetic acid), Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Where the compounds contain a basic residue, suitable pharmaceutically acceptable base addition salts for the compounds include, for example, inorganic acids and organic acids. Examples include acetate, benzenesulfonate (besylate), benzoate, bicarbonate, bisulfate, carbonate, camphorsulfonate, citrate, ethanesulfonate, fumarate, gluconate, glutamate, bromide, chloride, isethionate, lactate, maleate, malate, mandelate, methanesulfonate, mucate, nitrate, pamoate, pantothenate, phosphate, succinate, sulfate, tartrate, p-toluenesulfonate, and the like (Barge, S et al., 1977. J. Pharm. Sci., 66:1 -19, the entire contents of which are incorporated herein by reference).

Diluents that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, pharmaceutically acceptable inert fillers such as

microcrystalline cellulose, lactose, sucrose, fructose, glucose dextrose, or other sugars, dibasic calcium phosphate, calcium sulfate, cellulose, ethylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, saccharides, dextrin, maltodextrin or other polysaccharides, inositol or mixtures thereof. The diluent can be, for example, a water-soluble diluent. Examples of preferred diluents include, for example:

microcrystalline cellulose such as Avicel PH1 12, Avicel PH101 and Avicel PH102 available from FMC Corporation; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose DCL 21 ; dibasic calcium phosphate such as Emcompress; mannitol; starch; sorbitol; sucrose; and glucose. Diluents are carefully selected to match the specific composition with attention paid to the compression properties. The diluent can be used in an amount of about 2% to about 80% by weight, about 20% to about 50% by weight, or about 25% by weight of the treatment formulation.

Other agents that can be used in the treatment formulation include, for example, a surfactant, dissolution agent and/or other solubilizing material. Surfactants that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, sodium lauryl sulphate, polyethylene stearates, polyethylene sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, polyoxyethylene alkyl ethers, benzyl benzoate, cetrimide, cetyl alcohol, docusate sodium, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, lecithin, medium chain triglycerides, monoethanolamine, oleic acid, poloxamers, polyvinyl alcohol and sorbitan fatty acid esters. Dissolution agents increase the dissolution rate of the active agent and function by increasing the solubility of the active agent. Suitable dissolution agents include, for example, organic acids such as citric acid, fumaric acid, tartaric acid, succinic acid, ascorbic acid, acetic acid, malic acid, glutaric acid and adipic acid, which may be used alone or in combination. These agents can also be combined with salts of the acids, e.g., sodium citrate with citric acid, to produce a buffer system. Other agents that can be used to alter the pH of the microenvironment on dissolution include salts of inorganic acids and magnesium hydroxide.

Disintegrants that are suitable for use in the pharmaceutical composition of the present disclosure include, for example, starches, sodium starch glycolate, crospovidone,

croscarmellose, microcrystalline cellulose, low substituted hydroxypropyl cellulose, pectins, potassium methacrylate-divinylbenzene copolymer, polyvinyl alcohol), thylamide, sodium bicarbonate, sodium carbonate, starch derivatives, dextrin, beta cyclodextrin, dextrin

derivatives, magnesium oxide, clays, bentonite and mixtures thereof.

The active ingredient of the present disclosure can be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Various excipients can be homogeneously mixed with the active agent of the present disclosure as would be known to those skilled in the art. The active agent, for example, can be mixed or combined with excipients such as but not limited to microcrystalline cellulose, colloidal silicon dioxide, lactose, starch, sorbitol,

cyclodextrin and combinations of these.

Compositions of the present disclosure can also optionally include other therapeutic ingredients, anti-caking agents, preservatives, sweetening agents, colorants, flavors, desiccants, plasticizers, dyes, and the like. Any such optional ingredient must, of course, be compatible with the compound of the disclosure to insure the stability of the formulation.

The dose range for adult humans is generally from 0.1 μg to 10 g/day orally. Tablets or other forms of presentation provided in discrete units can conveniently contain an amount of compound of the disclosure that is effective at such dosage or as a multiple of the same, for instance, units containing 0.1 mg to 500 mg, usually around 5 mg to 200 mg. The precise amount of compound administered to a patient will be the responsibility of the attendant physician. The dose employed will depend on a number of factors, including, for example, the age and sex of the patient, the precise disorder being treated, and its severity. The frequency of administration depends on the pharmacodynamics of the individual compound and the formulation of the dosage form, which is optimized by methods known in the art (e.g., controlled or extended release tablets, enteric coating etc.).

The pharmaceutical preparations of the disclosure can be administered in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Such unit dosages generally contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The term "subject" is intended to include organisms, e.g., eukaryotes, which are suffering from or afflicted with a disease, disorder or condition associated with APC. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from an APC-related phenotype. An "APC-related phenotype" includes diseases or disorders, for example, caused by the misregulation of APC, including the absence of expression, and/or the subsequent downstream effects caused by misregulated APC, altered β-catenin activity and/or canonical Wnt and presenilinl signaling. The treatments (therapies) described herein can also be part of "combination therapies." Combination therapy can be achieved by administering two or more agents, each of which is formulated and administered separately, or by administering two or more agents in a single formulation. The second active ingredient can be, for example, a second compound identified herein or through screens described herein, or active ingredients useful for treating, for example, an APC-related phenotype, symptoms associated therewith, or symptoms associated with treatment by the first active agent ("side effects"). Other combinations are also

encompassed by combination therapy. For example, two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents can be administered within minutes of each other or within any number of hours of each other or within any number or days or weeks of each other.

Using the APC cKO mouse and the methods described herein for identifying a therapeutic agent effective for treating an APC-related disease or disorder, epigallocatechin gallate (EGCG), was so identified. EGCG, also known as epigallocatechin-3-gallate, is the ester of epigallocatechin and gallic acid, and is a type of catechin. EGCG is the most abundant catechin in tea and is a potent antioxidant.

Decitabine (trade name Dacogen), or 5-aza-2'-deoxycytidine, is a drug for the treatment of myelodysplastic syndromes, a class of conditions where certain blood cells are dysfunctional, and for acute myeloid leukemia (AML). Chemically, it is a cytidine analog.

β-catenin levels are increased two-fold in brain regions of the APC cKO mouse and excess β-catenin leads to structural and functional changes that resemble those found in the APC cKO mice. Reducing the augmented β-catenin levels are used to test for amelioration of cognitive impairments, autistic behaviors, IS, seizures and reduced hearing. Daily injections of adult APC cKO mice with EGCG and decitabine for five days were found to effectively reduce excessive β-catenin levels to those resembling wild-type littermate controls. In contrast, the same drug regimen did not significantly alter β-catenin levels in wild-type littermates, indicating undesirable effects are unlikely. EGCG is also administered to APC cKO mice at early developmental ages by adding it to the drinking water of pregnant female mice, on a continuous basis up to weaning of the pups, after which decitabine is injected in the APC cKO juvenile mice and control littermates. Behavioral and cognitive tests are utilized to assess quantitatively the effectiveness of these drug treatment paradigms in ameliorating the APC cKO mouse phenotypes.

EXEMPLIFICATION

EXAMPLE 1 .

Generation of APC conditional knock-out mice.

To gain new insights into APC's brain functions, mice with cKO of APC targeted predominantly to excitatory neurons were generated (APC is enriched at these postsynaptic sites). This cKO approach is necessary because the APC global knock-out is embryonic lethal (Moser, A. et al., Dev. Dyn., 203:422-33, 1995), and its targeted deletion in all embryonic neural progenitor cells leads to severe defects in radial glia polarity, generation and migration of cortical neurons, and construction of the cerebral cortex (Ivaniutsin, U. et al., Neural Dev., 4:3, 2009; Yokota, Y. et al., Neuron, 61 :42-56, 2009). Mice carrying floxed APC alleles were crossed with CaMKII-Cre mice, which express Cre recombinase driven by the

oc-calcium/calmodulin-dependent protein kinase II (CaMKII) promoter (Rios, M. et al., Mol.

Endocrinol., 15:1748-57, 2001 ). Crosses with the Rosa26R reporter mouse line indicate that the cre transgene is expressed chiefly in postmitotic excitatory neurons during synapse differentiation, within the first three postnatal weeks, throughout the brain, except for the cerebellum. APC is selectively deleted by Cre-dependent excision of loxP flanked exons 1 1 and 12, leading to out-of-frame splicing of exon 10 to exon 13 and the generation of a prematurely terminated 468-amino acid APC protein that lacks all identified protein interaction domains and is unstable. The loss of APC was confirmed by immunoblotting of lysates from both the whole brain and selected regions of three-month old APC cKO mice. Compared to control littermates (floxed APC, Cre negative), APC expression is dramatically reduced in APC cKO hippocampus and cortex (28.4 ± 0.8% and 19.3 ± 6.5% of control littermate hippocampal and cortical levels; p = 0.0019 and p = 0.022 Student's t-test, respectively) (FIG. 1 ). The small amount of APC protein remaining in the APC cKO lanes likely originates from glial cells and inhibitory neurons, as APC is ubiquitously expressed, but the oc-CaMKII-Cre transgene is generally not expressed in these cell types.

APC levels are also reduced in the APC cKO striatum (21 .9 ± 10.1 % of control littermate levels; p = 0.0058 Student's t-test) (FIG. 1 ), however, which is predominantly composed of inhibitory neurons and corticostriatal excitatory inputs. In contrast to cortical and hippocampal interneurons, striatal inhibitory medium spiny neurons do express the oc-CaMKII-Cre transgene (Ng, J. et al., Neuroscience, 165:535-41 , 2010; Novak, G. & Seeman, P., Synapse, 64:794-800, 2010). The striatal decreases reflect loss of APC in the inhibitory neurons, as well as loss of APC that is normally bound to microtubules in the extensive excitatory inputs. In comparison, cerebellar APC levels are not altered (98.6 ± 16.4% of control littermate levels; p = 0.95

Student's t-test) (FIG. 1 ) as expected based on previous reports of the absence of Cre transgene expression in this region in this mouse line.

Impaired learning and memory formation in APC cKO mice.

Because APC gene deletions correlate with IDs in patients (Hodgson, S. et al., J. Med. Genet, 30:369-75, 1993; Barber, J. et al., J. Med. Genet., 31 :312-6, 1994; Raedle, J. et al., Am. J. Gastroenterol., 96:3016-20, 2001 ; Finch, R. et al., Dis. Colon Rectum, 48:2148-52, 2005; Heald, B. et al., Nat. Clin. Pract. Neurol., 3:694-700, 2007), altered cognitive function in APC cKO mice was examined, relative to control littermates. To assess learning and memory, the novel object recognition (NOR) task was employed, using a 24hr time window between training (exploring a chamber with two identical objects) and the test (exploring the chamber after replacing one of the familiar objects with a novel object) (Brown, M. & Aggleton, J., Nat. Rev. Neurosci., 2:51 -61 , 2001 ). Control littermates showed a clear preference for the novel object, indicating memory of the familiar object (56.4 ± 2.6 % of time spent with novel object;

significantly different, p = 0.014, Student's t-test, compared with hypothetical 50%). In contrast, APC cKO mice showed no preference, spending equal time with both objects (49.8 ± 2.6 % of time with novel object; p = 0.93 compared with hypothetical 50%) (FIG. 2A). APC cKO mice, therefore, display cognitive deficits in the NOR task, which assesses both cortical and hippocampal function.

Performance in the Barnes maze learning task was tested to assess

hippocampal-dependent spatial memory (Barnes, C, J. Comp. Physiol. Psychol., 93:74-104, 1979). APC cKO mice were slower to learn the task, based on several lines of evidence. They required three days of testing (improved by trial 6; FIG. 2B), whereas control littermates required only two days (improved by trials 3 and 4). Over the course of training, APC cKO mice took more time to reach the goal (higher latencies, p = 0.0075 repeated measures ANOVA) and were less accurate (committed more errors, p = 0.00017 repeated measures ANOVA) (FIGS. 2B,C). Further, APC cKO mice took less efficient routes to the goal (path efficiency; p = 0.00081 ) (FIG. 2D), while showing no change in average speed (p = 0.20; FIG. 2E). These results indicate that APC cKO mice have impaired hippocampal-dependent learning.

To assess memory formation, probe trials were performed on day 5, and again one week later, on day 12. On day 5, APC cKO mice showed a strong preference for the goal hole, as did control littermates (FIG. 2F). This is expected given that APC cKO mice performed the task by day 4 trials. In sharp contrast, APC cKOs performed poorly on day 12, whereas control littermates continued to display a strong preference for the correct location (FIG. 2G). In particular, APC cKOs showed no preference for the goal hole, took more than twice as long to visit the goal hole for the first time (cKO = 52.8 ± 10.9 seconds, ctl = 16.8 ± 5.6 seconds;

p = 0.0064 Student's t-test), and committed four times as many errors (cKO = 19.2 ± 4.0, ctl = 4.5 ± 1 .4; p = 0.0013, Student's t-test), while taking less efficient routes to the goal hole (path efficiency: cKO = 0.18 ± 0.07, ctl = 0.49 ± 0.10, p = 0.019 Student's t-test) (FIGS. 2H-J). These findings indicate that APC cKO mice have deficits in hippocampus-dependent long-term memory consolidation.

As a separate test of hippocampal-dependent working memory and responsiveness to novelty, the continuous spontaneous alternation Y-maze task was used (Hughes, R., Neurosci. Biobehav. Rev., 28:497-505, 2004). The number of alternations between the symmetrical arms of the Y maze was measured during an eight minute test period. APC cKO mice showed a significantly lower rate of alternation, compared to control littermates (ctl = 71 .8 ± 3.63%, cKO = 57.8 ± 4.00%, p = 0.032 Student's t-test; cKOs not significantly different from chance rate of 50%, cKO p = 0.1078, ctl p = 0.0038 One-sample t-test; FIG. 2K). There were no significant differences in total distance traveled, number of arm entries or mean velocity of movement calculated on the basis of frequency of arm entries.

To further rule out impaired locomotion and hyperactivity as potential factors that could influence the performance of APC cKOs in the diagnostic behavior tasks, two independent assays were conducted. First, no difference in overall activity or distance traveled during home cage monitoring over 30 hours was detected (p = 0.42, repeated measures ANOVA). Second, gait analysis showed no difference in stride length of APC cKOs compared to control littermates (stride length/body length, right side: ctl = 0.72 ± 0.02, cKO = 0.74 ± 0.03; p = 0.48 Student's t-test; left side: ctl = 0.73 ± 0.03, cKO = 0.74 ± 0.03; p = 0.81 ). In summary, APC cKO mice exhibit learning and memory impairments, demonstrating that APC is essential for normal cognition.

Autistic-like behaviors in APC cKO mice.

APC cKO mice were analyzed for autistic-like behaviors based on the association of ASD with APC heterozgous deletion and polymorphisms in humans (Barber, J. et al., J. Med. Genet., 31 :312-6, 1994; Zhou, X. et al., Am. J. Med. Genet. B Neuropsychiatr. Genet.,

144B:351 -4, 2007; Kumar, A. et al., PLoS One, 6:e28431 , 201 1 )

(http://www.mindspec.org/autdb.html; https://sfari.org/sfari-gene). Diagnostic assays specifically designed to test for an autistic-like phenotype were used in mice by evaluating repetitive behaviors and social interactions (Silverman, J. et al., Nat. Rev. Neurosci., 1 1 :490-502, 2010). Repetitive behavior was assessed using the marble-burying assay. APC cKO mice buried 1 .6 times more marbles than control littermates (APC cKO = 1 1 .5 ± 0.9, Ctl = 7.2 ± 1 .0 marbles buried; p = 0.0056, Student's t-test) (FIG. 3A).

Increased marble burying is reported to reflect repetitive digging behavior and did not correlate with anxiety-related measures (Thomas, A. et al., Psychopharmacology (Berl.), 204:361 -73, 2009). Consistent with this interpretation, APC cKO mice display decreased anxiety-like behavior in the elevated plus maze task. They spent four times more time in the open arms (cKO = 36.6 ± 7.1 %, ctl = 8.3 ± 3.1 % time in open arms; p = 0.0052, Student's t-test) (FIG. 3B) and traveled similar total distances relative to control littermates (FIG. 3C).

APC cKO mice exhibited increased repetitive behavior in the repetitive novel object contact task. This test measures the number of times mice investigate each of four novel objects, as well as the sequence in which the objects are visited during a 10 minute test period (Pearson, B. et al., Genes Brain Behav., 10:228-35, 201 1 ). Increased repetition of a pattern of sequential object investigations indicates stereotypy. The four objects were of similar general interest to APC cKOs and control littermates based on their similar ranked preferences (FIG. 3D). APC cKOs made a greater number of visits, suggesting that control littermates habituated to the objects, whereas cKOs did not (FIG. 3E). Most importantly, APC cKOs showed

"preferred" sequences, they repeated a specific sequence of 4-object visits significantly more times (p = 0.0042, Student's t-test), after normalizing for the total number of object visits (FIG. 3F). The "preferred" sequences likely derive from their distinct movement patterns, APC cKOs displayed increased preservation of their direction of movement, such as clockwise or counterclockwise during object visits (FIG. 3G). Again, APC cKOs showed low anxiety and no hyperactivity in separate tests (FIGS. 3B,C). Taken together, APC cKO mice displayed increased repetitive behaviors in two different tasks. Stereotypies are one of the three behavioral abnormalities that characterize ASD in individuals.

An evaluation of reduced social interactions, another hallmark of ASD, was conducted. Because social interactions between mice depend on olfactory cues, a social versus non-social olfaction test was performed. Using three consecutive timed exposures to each odor, the amount of time mice spent sniffing cotton swabs scented with either non-social odors (water, vanilla or banana extract) or social odors (the litter of two different non-familiar male or female mouse cages) was measured. APC cKOs and control littermates showed a similar level of interest in the non-social odors, indicating intact olfactory function (FIG. 4A). Both spent more time investigating social odors versus non-social odors. The APC cKO male mice, however, exhibited significantly less interest in social odors, from both male and female cages, suggesting reduced social interest (FIG. 4C). Further, control littermates showed a typical

habituation/dishabituation pattern, spending less time investigating the same social odor on successive trials, followed by renewed interest for a novel social odor. This normal behavior pattern is less robust in APC cKO mice, further indicating deficits in social interest.

To more directly test social interest, a classic three-chambered social interaction test was performed. The assay requires the test mouse (male) to initiate interaction with a novel mouse (an ovariectomized, wild-type female mouse) that is housed in a wire mesh cage in one side chamber, while the other side chamber contains an empty, but otherwise identical, wire cage. As expected, the control mice spent more time interacting with the cage housing the novel mouse, compared to the empty cage (FIG. 4E). APC cKOs showed a similar trend, but spent significantly less time interacting with the novel mouse (cKO = 141 ± 10s, ctl = 218 ± 16s; p = 0.00075, Student's t-test). There was no difference in overall locomotion between cKOs and controls, as both made a similar number of transitions between the three chambers (FIG. 4F). These data indicate that APC cKO mice display modestly reduced interest, relative to control littermates, in sniffing social odors and interacting with a novel mouse, whereas their interest in non-social odors is normal.

Aberrant levels of plasticity and signaling proteins in APC cKO mice.

Based on the cognitive impairments of APC cKO mice, tests were conducted for altered levels of plasticity and signaling proteins that link to APC and are known to regulate synaptic function and plasticity. In particular, APC associated proteins, β-catenin and FMRP, and the β-catenin binding partner presenilinl were assessed.

β-catenin protein levels increased two-fold in APC cKO forebrain regions, hippocampus, cortex and striatum (214.9 ± 10.3 %, 200.0 ± 22.9%, and 247.7 ± 36.9% of control littermate lysate (total protein) levels; p = 0.00022, p = 0.025, and p = 0.012, Student's t-test, respectively)

(FIG. 5A), based on quantitative immunoblotting. Similar changes were seen in cortical and hippocampal isolated postsynaptic density fractions (psds) to measure synaptic protein levels.

In contrast, ctnnbl (β-catenin) mRNA levels were not altered, as assessed by quantitative PCR, indicating posttranslational regulation. Increased β-catenin protein levels in the forebrain of APC cKOs are consistent with APC's role as a negative regulator in the canonical Wnt pathway; it is an essential component of the degradation complex that targets β-catenin for ubiquitin- mediated proteosomal degradation (Roberts, D. et ai, Mol. Biol. Cell, 22:1845-63, 201 1 ). Additionally, APC cKOs displayed dysregulation of the β-catenin/Wnt pathway.

β-catenin nuclear localization was increased in APC cKO forebrain neurons (FIG. 5B).

Additionally, several Wnt target genes showed augmented expression levels in the APC cKO hippocampus (FIG. 5C), including Sp5, a transcriptional repressor of Sp1 genes (p = 0.005, Student's t-test), neurogl , implicated in cortical neuronal differentiation (p = 0.038), syn2, a synaptic vesicle phosphoprotein that functions to maintain the reserve pool of glutamatergic vesicles, recently predicted to be a Wnt target (p = 0.021 ), and dkkl , a Wnt antagonist

(p = 0.01 1 ). Increased dkkl mRNA levels is indicative of a feedback mechanism to restrain enhanced Wnt signaling, as seen in transgenic mice with conditional augmentation of β-catenin in the forebrain (Diep, D. et al., Brain Res. Dev. Brain Res., 153:261 -70, 2004). Importantly, aberrant Wnt signaling links to cognitive disorders and abnormal synaptic plasticity (De Ferrari, G. & Moon, R., Oncogene, 25:7545-53, 2006; Salinas, P., Cold Spring Harb. Perspect. Biol., 4:a008003, 2012).

N-cadherin adhesion complexes were also altered in the APC cKO hippocampus.

β-catenin showed increased co-precipitation with N-cadherin (FIG. 5D), whereas total levels of N-cadherin were not altered (FIG. 5E), suggesting their enhanced association at the synapse. Presenilinl interacts with these synaptic components. Presenilinl is the catalytic subunit of the γ-secretase that cleaves amyloid precursor protein and N-cadherin. The N-cadherin CTF2 fragment translocates to the nucleus and regulates CREB-dependent gene transcription

(Marambaud, P. et al., Cell, 1 14:635-45, 2003). Cognitive impairments link to presenilinl cKO in forebrain neurons and human PSEN1 gene mutations link to Alzheimer's disease (Yu, H. et ai, Neuron, 31 :713-26, 2001 ). Presenilinl levels were reduced (FIG. 5E), in the APC cKO hippocampus, p = 0.021 , Student's t-test). These molecular changes identify APC as a hub that links to and regulates synaptic adhesion and signal transduction networks.

The RNA binding protein FMRP regulates local protein synthesis required for normal learning and memory. FMRP associates with APC in migrating fibroblasts; shRNA knockdown of APC disrupted the localization of FMRP-associated mRNAs at distal sites in these non- neuronal cells (Mili, S. et al., Nature, 453:1 15-9, 2008). It is shown herein that, in the

hippocampus in vivo, APC loss leads to 40% decreases in FMRP protein levels (lysate:

Ctl = 100 ± 4.6 % of Ctl, cKO = 62.9 ± 6.3; p = 0.0018. psd: Ctl = 100 ± 2.9 %, cKO = 60.3 ± 7.0; p = 0.0008, Student's t-test) (FIGS. 6A,B). Fmr1 (FMRP) mRNA levels were unaltered, indicating regulation at the posttranslational level. Phosphorylation regulates FMRP protein stability; dephosphorylated FMRP is rapidly ubiquitinated and proteosomally degraded

(Nalavadi, V. et al., J. Neurosci., 32:2582-7, 2012). Akt is a key upstream effector of the kinase that phosphorylates FMRP; FMRP levels correlate with Akt phosphorylation/activation (pAkt) in neurons (Jeon, S. et al., J. Neurochem., 123:226-38, 2012). Akt activation, in turn, is inhibited by the phosphatase, PTEN, which links indirectly to APC (Sotelo, M. et al., J. Cell. Biochem., 1 13:2661 -70, 2012). Consistent with these signaling molecules mediating the regulatory effects of APC on FMRP, Akt activation/ pAkt levels were decreased (Ctl = 100 ± 10.3%,

cKO = 75.9 ± 12.6; p = 0.044, Student's t-test) and PTEN levels were increased

(Ctl = 100 ± 9.3%, cKO = 187.7 ± 26.6; p=0.0090, Student's t-test) in the APC cKO

hippocampus (FIGS. 6A,B). In contrast, FMRP, pAkt and PTEN levels were not significantly altered in the APC cKO cortex and striatum. Distinct changes between the hippocampus and cortex have been observed for mice expressing the autism-related neuroligin-3 R451 C mutation (Etherton, M. et al., Proc. Natl. Acad. Sci. USA, 108:13764-9, 201 1 ). The findings described herein indicate brain region-specific differences in FMRP regulation. This is the first description of APC as a novel regulator of FMRP levels in the hippocampus in vivo.

Increases in synaptic spine density in APC cKO pyramidal neurons.

APC loss leads to increases in β-catenin and Wnt signaling (FIG. 5). Their

enhancement has been shown to increase synaptic spine density, as well as dendritic and axonal arborization, in cultured hippocampal neurons (Yu, X. & Malenka, R., Nat. Neurosci., 6:1 169-77, 2003; Yu, X. & Malenka, R., Neuropharmacol., 47:779-86, 2004; Kundel, M. et al., J. Neurosci., 29:13630-9, 2009; Varela-Nallar, L. et al., Proc. Natl. Acad. Sci. USA, 107:21 164-9, 2010; Ciani, L. et al., Proc. Natl. Acad. Sci. USA, 108:10732-7, 201 1 ). As shown herein, APC cKO mice exhibited significant increases in synaptic spine density (FIG. 7A) in vivo, on the apical dendrites of both layer 5 pyramidal neurons in the cerebral cortex (FIG. 7B,

175.1 ± 21 .7%; p = 0.014, Student's t-test) and CA1 pyramidal neurons in the hippocampal striatum radiatum (FIG. 7B, 160.7 ± 6.9%; p = 0.0002, Student's t-test). Further, less mature stub-shaped spines predominated in the APC cKOs (FIG. 7C), whereas mushroom spines predominated in control littermates.

Basal synaptic transmission is not altered in the APC cKO hippocampus.

Synaptic function in the hippocampus of freshly isolated brain slices was assessed. Whole cell recordings in CA1 pyramidal neurons revealed a 4-fold increase in the frequency of AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs) in APC cKOs, compared to control littermates (cKO = 0.8835 ± 0.22 Hz; ctl = 0.208 ±0.049 Hz, p = 0.033, Student's t-test) (FIGS. 8A,B). Excessive β-catenin levels lead to similar changes in mEPSC frequency in cultured hippocampal neurons. The amplitude of the AMPAR-mediated mEPSCs was not altered (cKO = 9.86 ± 0.43 pA; ctl = 8.41 ± 0.79 pA; p = 0.10) (FIG. 8C), suggesting no change in baseline postsynaptic sensitivity. The mEPSC rise time showed no change (cKO = 2.96 ± 0.08 ms; ctl = 2.88 ± 0.13 ms; p = 0.61 ) (FIG. 8D). Decay kinetics of the AMPAR-mediated mEPSCs were more rapid (cKO = 7.96 ± 0.72 ms; ctl = 10.58 ± 0.94 ms; p = 0.044) (FIG. 8E), however, consistent with AMPARs that lack GRIA2 or contain GRIA4, characteristic of immature synapses (Liu, S. & Savtchouk, I., J. Physiol., 590:13-20, 2012).

Increased mEPSC frequency suggests either an increase in the probability of release, or an increase in synapse number. Presynaptic function at Schaffer collateral (SC)-CA1 synapses was therefore assessed by measuring paired pulse facilitation (PPF). No significant change in APC cKOs compared with control littermates (FIG. 8F) was found. Increased mEPSC frequency, with no change in PPF, indicates an increase in the density of functional presynaptic terminals. Basal excitatory synaptic transmission was normal, however, as measured by extracellular recordings of field excitatory postsynaptic currents (fEPSCs) at SC-CA1 pyramidal synapses. There was no change in the ratio of stimulus intensity (input) to the slope of the fEPSCs (output) (FIGS. 8G,H). Taken together, the data indicate that APC cKOs have an increased number of immature excitatory synapses.

Modest increases in LTP in APC cKO hippocampal neurons.

Changes in synaptic plasticity were tested. FMRP levels are reduced in the APC cKO hippocampus (FIG. 6). FMRP absence in the Fragile X mouse model leads to enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at SC-CA1 synapses in hippocampal slices (Niere, F. et al., J. Neurosci., 32:5924-36, 2012). mGluR- dependent LTD at SC-CA1 synapses of APC cKOs was not significantly different from that of wild-type littermates [ctl = 32 ± 2.5% depression, cKO = 41 ± 3.3%; p = 0.0668, repeated measure ANOVA, in hippocampal slices treated with the group I mGluR agonist

3,5-dihydroxyphenylglycine (DHPG) (FIGS. 9A,B)].

APC cKO SC-CA1 synapses showed significant, modest increases in long-term potentiation (LTP) induced by five trains of theta burst stimulation (TBS) (ctl = 54 ± 5.5% potentiation, cKO = 72± 5.2%; p = 0.0227, repeated measure ANOVA) (FIGS. 9C,D). In contrast, LTP induced by high frequency stimulation was not altered (2 tetanic stimuli,

2 x 100 Hz).

APC deficiency selectively enhanced TBS-LTP. The modest plasticity change resembles that caused by excessive Wnt signaling, canonical and non-canonical, in

hippocampal slices, with facilitated induction of LTP via acute and rapid upregulation of synaptic NMDA receptor currents (Chen, J. et al., J. Biol. Chem., 283:5918-27, 2006; Cerpa, W. et al., J. Neurosci., 31 :9466-71 , 201 1 ). All together, the neuronal changes in APC cKOs are consistent with increased local synaptic connectivity and enhanced plasticity.

Discussion

APC is a large multi-functional scaffold protein that associates with several proteins implicated in neurodevelopmental brain disorders (Chow, M. et al., PLoS Genet, 8:e1002592, 2012; Kalkman, H., Mol. Autism, 3:10, 2012; Zhou, J. & Parada, L, Curr. Opin. Neurobiol., 22:873-9, 2012). However, its role in the mammalian brain is poorly defined. The findings described herein demonstrate that APC is essential for normal cognition and behavior. APC cKO mice, compared with control littermates, exhibit learning and memory deficits, autistic-like behaviors (reduced social interest and increased repetitive behaviors), increased excitatory synaptic spine density, altered synaptic function (enhanced mEPSC frequency and TBS- induced LTP), and aberrant levels of FMRP, presenilinl and β-catenin. These results identify a novel function for APC as a central player in a pathway that links to and regulates

β-catenin/Wnt, presenilin/Creb and FMRP networks. The essential roles of these networks in normal learning and behavior highlight the importance of APC function in the mammalian brain (Neale, B. et al., Nature, 485:242-5, 2012; Zoghbi, H. & Bear, M., Cold Spring Harb. Perspect. Biol., 4.pii:a009886, 2012).

β-catenin levels are increased in the APC cKO forebrain. This molecular change is consistent with the known role of APC as the major negative regulator of β-catenin levels in the canonical Wnt pathway, thereby extending this APC function to postmitotic neurons of the mammalian brain in vivo, β-catenin, in turn, has dual roles in canonical Wnt signaling and cadherin-based intercellular adhesion and both are critical for proper brain function (Kiryushko, D. et al., Ann. NY Acad. Sci., 1014:140-54, 2004; Vitureira, N. et al., Nat. Neurosci., 15:81 -9, 201 1 ). Dynamic regulation of β-catenin's interaction with N-cadherin modulates excitatory synapse density, structure and plasticity (Murase, S. et al., Neuron, 35:91 -105, 2002; Bamji, S. et al., J. Cell Biol., 174:289-99, 2006; Brigidi, G. & Bamji, S., Curr. Opin. Neurobiol., 21 :208-14, 201 1 ), referred to here as the synaptogenic pathway. Canonical Wnt signaling leads to β-catenin dependent transcription of genes required for brain maturation and plasticity, referred to here as the Wnt pathway.

During normal development of the mammalian brain, Wnt pathway components are expressed at high levels at immature ages and down-regulated with maturation (Shimogori, T. et al., J. Comp. Neurol., 473:496-510, 2004). The loss of APC perturbs the ability to developmental^ down-regulate β-catenin/Wnt signaling. While not bound by theory, from the data described herein, excessive β-catenin levels in the APC cKO brain leads to deregulation of the Wnt, presenilin/Creb and synaptogenic pathways that, in turn, aberrantly modulate synaptic density, maturation, and plasticity, and these changes likely underlie the intellectual and autistic- like disabilities.

Several lines of evidence support this model. Excessive increases in β-catenin, Wnt and

N-cadherin synaptogenic pathways in wild-type mice cause similar functional changes to those seen in APC cKO excitatory neurons. The shared phenotypes are enhanced mEPSC frequency, TBS-induced LTP, including induction, and density of excitatory synaptic spines in hippocampal neurons (Beaumont, V. et al., Mol. Cell Neurosci., 35:513-24, 2007; Cerpa, W. et al., J. Biol. Chem., 283:5918-27, 2008; Avila, M. et al., J. Biol. Chem., 285:18939-47, 2010). β-catenin functions presynaptically to augment mEPSC frequency, with no change in evoked release, via its role in regulating the localization and release of specific synaptic vesicle pools, independent of Wnt-induced transcription (Bamji, S. et al., Neuron, 40:719-31 , 2003; Sara, Y. et al., Neuron, 45:563-73, 2005). Further, excessive TBS-induced LTP in the CA1 region has been found previously to associate with impaired hippocampal-dependent spatial learning and memory (Migaud, M. et al., Nature, 396:433-9, 1998; Kim, M. et al., J. Neurosci., 29:1585-95, 2009).

Tight regulation of β-catenin levels also appears to be necessary for memory

consolidation, β-catenin levels, experimentally manipulated to be abnormally high or low in the amygdala during fear learning, disrupt fear memory consolidation (Maguschak, K. & Ressler, K., J. Neurosci., 31 :13057-67, 201 1 ). The data shown herein suggest that high β-catenin levels may impair spatial memory consolidation, based on the poor performance of APC cKOs in Barnes maze probe tests at 12 days. Similarly, mice heterozygous for APC (whole body), as a model of APC haploinsufficiency in humans, also show deficits in working memory, but also anemia and hypoactivity (Koshimizu, H. et al., Front. Behav. Neurosci., 5:85, 201 1 ).

Constitutively high or low levels of β-catenin and canonical Wnt signaling negatively impact cognitive function. Synaptic activity induces Wnt release and dynamic, transient changes in β-catenin and canonical Wnt signaling levels. The data herein suggest that an optimal range and tight regulation of both β-catenin and canonical Wnt target gene expression levels are critical for normal learning and memory formation, highlighting the importance of APC's function as a key regulator of these networks in the mammalian brain.

The data herein show that, via regulation of β-catenin levels, APC also serves to modulate cadherin-catenin synaptic adhesion complexes and thereby regulates other signal transduction pathways critical for normal cognition. APC loss leads to increased association between β-catenin and N-cadherin, and decreased levels of presenilini , that, in turn, regulates CREB-dependent gene transcription. Cognitive impairments link to presenilini cKO in forebrain neurons and human PSEN1 gene mutations link to Alzheimer's disease.

Impaired learning and memory in APC cKO mice likely stems from aberrant levels of β-catenin, canonical Wnt signaling and presenilin 1 . APC loss leads to dysregulation whereby neurons are not able to rapidly and transiently alter β-catenin/Wnt and presenilin/Creb signal transduction levels, as required for normal memory consolidation. These data have important implications for modulating the canonical Wnt pathway as a potential therapeutic target for cognitive deficits.

APC cKO mice also display reduced hippocampal levels of the RNA binding protein FMRP. Fragile-X syndrome, caused by mutational silencing of the Fmr1 gene that encodes FMRP, is the most common cause of inherited intellectual and autistic disabilities in patients (Hatton, D. et al., Am. J. Med. Genet. A, 140A:1804-13, 2006). FMRP functions as a translation brake to regulate activity-dependent local protein synthesis near synapses (Darnell, J. et al., Cell, 146:247-61 , 201 1 ). APC loss leads to reduced FMRP protein, but not mRNA, levels. FMRP protein stability is regulated by its phosphorylation state, dephosphorylated FMRP is rapidly degraded. As shown herein, APC loss modulates the levels and activation of phosphatases and kinases that function as upstream effectors in the FMRP phosphorylation pathway. APC interacts indirectly with the phosphatase PTEN. PTEN inhibits the

phosphorylation/activation of Akt; pAkt, in turn, activates S6K1 that phosphorylates and stabilizes FMRP (Bassell, G. & Warren, S., Neuron, 60:201 -14, 2008); FMRP levels correlate with those of pAkt in neurons. APC cKOs show increased PTEN and decreased pAkt levels. These molecular changes are likely responsible for the reduced FMRP protein levels. The results presented herein identify APC as a key modulator of FMRP hippocampal levels in vivo.

APC cKO and Fmr1 KO mice exhibit similar cognitive and behavioral phenotypes.

However, the functional changes differ, perhaps not surprisingly, as molecular changes do as well (Table 1 ). APC cKO mice show modestly enhanced TBS-LTP and no significant change in mGluR5-dependent LTD. In contrast, Fmr1 KO mice show augmented mGluR5-LTD, while LTP is decreased or unaffected (Zhang, J. et al., J. Neurophysiol., 101 :2572-80, 2009). As for molecular differences, APC cKOs display increased β-catenin levels, whereas Fmr1 KOs show increased APC and decreased β-catenin levels. TABLE 1 . APC gene deletions lead to a new syndrome of ID and ASD

A more relevant comparison to APC cKOs may be mice expressing Fragile X

premutations (CGG repeats in the 3'UTR) that cause reductions, but not total absence, of FMRP protein. FMRP levels are reduced to a similar extent between APC cKO and FMR1 premutation mice (lliff, A. et al., Hum. Mol. Genet., 22:1 180-92, 2013). The premutation differs, however, from both APC cKO and Fmr1 KO mice in that it causes significantly higher FMR1 mRNA levels that accumulate in the nucleus and lead, over time, to neuron degeneration (Chen, Y. et al, Hum. Mol. Genet., 19:196-208, 2010).

Boys with Fragile X premutations exhibit social deficits (ASD) that are similar to, but milder than the full deletion (Cornish, K. et al, Brain Cogn., 57:53-60, 2005). Premutation mouse models, similar to APC cKO mice, display cognitive impairments (spatial memory deficits), reduced anxiety, increased spine density on pyramidal neurons, and normal basal synaptic transmission, compared with wild-type mice (Qin, M et al, Neurobiol. Dis., 42:85-98,

201 1 ) . FMRP premutation mice were less socially impaired than APC cKOs, they exhibited normal social approach and subtle deficits in social novelty. Synaptic plasticity differed between the mouse models (Table 1 ). mGluR-LTD was enhanced in Fmr1 premutations, likely because the premutation impairs activity-dependent local translation of FMRP mRNA. Additionally, it was dependent on protein synthesis and therefore mechanistically distinct from increased mGluR-LTD of Fmr1 nulls. In further contrast to APC cKOs, FMRP premutation SC-CA1 synapses show decreased induction/early phases of LTP, but normal late phases (Berman, R. et al, Epilepsia, 53 Suppl. 1 :150-60, 2010; Hunsaker, M. et al, Hippocampus, 22:2260-75,

2012) . While not bound by theory, these differences indicate that APC loss leads to syndromic intellectual and autistic disabilities with distinct molecular underpinnings. Synaptic plasticity changes in APC cKO mice more closely resemble those seen with excessive β-catenin/Wnt, rather than reduced FMRP levels. This study provides new insights into APC human gene mutations as a risk factor for IDs and ASDs. APC gene mutations in patients fall most commonly in the "mutation cluster region" and result in truncated proteins that lack most of the β-catenin binding domains. APC interactions are essential for normal β-catenin levels in the brain. Excessive β-catenin levels lead to deregulated synaptogenic and Wnt and presenilin pathways and abnormal brain function.

In rodents, prenatal exposure to valproic acid (VPA), a known environmental risk factor for ASD in humans, leads to increases in β-catenin levels, canonical Wnt signaling, axon branching suggesting aberrant neuronal connectivity, autistic-like behaviors

(repetitive/stereotypic activity) and impaired learning and memory (Wang, Z. et al., Anat. Rec. (Hoboken), 293:1947-53, 2010; Zhang, Y. et al., Neurochem. Res., 37:1409-19, 2012).

Similar to APC, CHD8 negatively regulates β-catenin-mediated gene transcription.

Human de novo loss-of-function mutations in CDH8 (chromodomain helicase DNA binding protein 8) are a high confidence risk factor for sporadic ASD, often also associated with intellectual disability (O'Roak, B. et al., Nat. Genet, 338:1619-22, 201 1 , 2012; Sanders, S. et al., Neuron, 70:863-85, 201 1 ). Further, Wnt copy number variants and mutations have been identified in patients with ASD, including a rare Wnt1 missense mutation with increased canonical Wnt pathway activation (Chow, M. et al., PLoS Genet., 8:e1002592, 2012; Martin, P. et al., Transl. Psychiatry, 3:e301 , 2013). Mutations in the human CTNNB1 (β-catenin) gene itself also link to sporadic ASD/ID (de Ligt, J. et al., N. Engl. J. Med., 367:1921 -9, 2012; Krumm, N. et. al., Trends Neurosci., 37:95-105, 2014). Further, β-catenin/Wnt signaling levels are abnormally low in a distinct cognitive disorder, Alzheimer's disease (Farias, G. et al., Dev. Dyn., 239:94-101 , 2010). These clinical associations highlight the importance of defining

pathophysiological changes caused by APC loss of function and deregulation of β-catenin networks in the mammalian brain. An optimal range of β-cateninZ Wnt is critical for normal cognitive function. APC cKO mouse studies described herein provide support in an animal model for a role of excessive β-catenin/Wnt/synaptogenic pathways in ID and ASD.

EXAMPLE 2.

Normal afferent function in the mammalian cochlea requires coordinated maturation of presynaptic ribbons in inner hair cells (IHCs) and postsynaptic AMPA receptor clusters in spiral ganglion (SG) neurons- the primary auditory neurons that signal sound reception to the brain. Noise damage leads to degradation of ribbon synapses and loss of low spontaneous rate (SR) SG neurons, greatly reducing the dynamic hearing range in noise-exposed animals. Studies have demonstrated differences in the size and polarized distribution of ribbon synapses that correlate with the SR of afferent fibers, suggesting that two distinct ribbon synapse types are required for hearing in low and high noise environs. Despite the critical role of functional ribbon synapse assembly and maintenance in hearing sensitivity, the underlying molecular

mechanisms are poorly defined. Described herein are findings indicating APC plays a key synapse-organizing role in the cochlea.

APC is a ubiquitously expressed, large, multi-functional scaffold protein; it regulates the canonical Wnt signaling pathway, cell polarity, microtubule and actin cytoskeleton dynamics, as well as neuronal axon outgrowth and maturation of presynaptic and postsynaptic

specializations. Further, APC is concentrated at nicotinic and glutamatergic postsynaptic sites in neurons. To test whether APC directs functional synapse assembly in the mammalian cochlea, the APC cKO mouse was used.

APC cKO mice, compared with control littermates, exhibit altered afferent ribbon synapse function, as indicated by reduced auditory brainstem response (ABR) thresholds. In addition, alterations in both ribbon synapse structure (shift to larger ribbon sizes only) and spatial distribution (loss of polarized localization) within IHCs, were observed based on immunofluorescent confocal microscopic analyses. These findings identify APC as a critical molecule for proper functional maturation of afferent synapses and normal hearing. The APC cKO mouse is a useful model for elucidating the specific role of ribbon size and spatial distribution in directing functional output of afferent synapses in the mammalian cochlea.

EXAMPLE 3.

Infantile spasms (IS) constitute a catastrophic childhood epilepsy syndrome that is poorly controlled by existing therapeutics and often leads to developmental delays. Elucidating the underlying pathophysiological mechanisms is essential for developing effective

interventions. Shown herein are data indicating that the APC cKO mouse displays

characteristics associated with IS. Early neonatal APC cKO mice display spasms (increased high amplitude spontaneous movements), compared with control littermates. Both spontaneous and evoked excitatory electrical activity is increased in the APC cKO cortex. Synaptic spines show increased density (number of spines per unit length) on the apical dendrite of layer 5 pyramidal neurons. Additionally, adult APC cKO mice display learning and memory deficits in cognitive behavioral assays. Molecular changes in the APC cKO brain include increased levels of β-catenin and canonical Wnt pathway activation, consistent with the role of APC as a major negative regulator of β-catenin levels in the canonical Wnt pathway, β-catenin is known to have dual functions in the N-cadherin synaptic adhesion complex and the canonical Wnt signal transduction pathway. Deregulation of both networks in the developing brain leads to altered axon guidance cues, excessive branching, and aberrant density and plasticity of excitatory synapses, all consistent with enhanced seizure susceptibility. Intriguingly, Wnt and β-catenin pathways are also regulated by other genes implicated in human IS, including FoxG1 , ARX, TSC1/2 and Magi-2/S-SCAM.

Other Embodiments

Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present disclosure are not limited to the above examples, but are encompassed by the following claims. The contents of all references cited herein are incorporated by reference in their entireties.

Claims

CLAIMS What is claimed is:

1 . A method of diagnosing an APC-related disease or disorder, comprising

a) contacting a sample from a subject with an assay assembly wherein the assay assembly determines the expression level of one or more informative genes; and b) comparing the expression level of the one or more informative genes with one or more reference expression profile comprising expression levels for the one or more informative genes, wherein the one or more expression profiles correspond to an APC-related disease or disorder or to a phenotype that does not exhibit an APC-related disease or disorder,

wherein statistical similarity to a particular reference expression profile indicates subject providing the sample has a phenotype associated with the phenotype corresponding to the reference expression profile.

2. The method of Claim 1 , wherein the APC-related disease or disorder is selected from the group consisting of: neurodevelopmental diseases, autism, viral infection, perinatal hypoxic encephalopathy, Aicardi syndrome, an autism spectrum disorder, a

neurocognitive disorder, intellectual disability, hearing loss, infantile spasms, West syndrome, seizures, inflammatory response, gastrointestinal disorders and co-morbid disorders.

3. The method of Claim 1 , wherein the one or more informative genes are selected from the group consisting of cytokines, chemokines, stress hormones and micro-RNAs.

4. The method of Claim 1 , wherein the one or more informative genes are selected from the group consisting of: neurogenesis markers, DCX, homer, TAO kinases, microglia proteins, Iba1 , β-catenin, γ-catenin, oc-catenin, N-cadherin and components of this synaptic adhesion complex, δ-catenin, canonical and non-canonical Wnt signaling pathway components, disheveled, LRP5/6, Frizzleds, Ryk, GSK3 beta and alpha, endogenous regulators of Wnt signaling, Dkk1 , axins, SFRPs, connexin 43, PPARs, Tcf, Lef, Foxgl , Arx, neurotrophic factors and their receptors, BDNF, NT3, Trks, scaffold proteins, S-SCAM, Magi2, PSD-95 protein family members, scribble, homer, GRIP, Pick, gephyrin, ion channels, sodium and potassium subunits and accessory proteins, adhesion molecules, neuroligins 1 , 2 and 3, a- and β-neurexins, Ephs, Ephrins,

SynCAMs, guidance molecules, semaphorins, plexins, LRRTM, LAR proteins, neuregulins, Erbs, one or more presenilin family members, presenilin 1 , rictor, DOCKs, PAR3, PAR6, aPKC, CASK, Veli, Mint, synapse-associated signaling and plasticity proteins, ERK, PP2A, Akt, PTEN, PI3K, MAPK, CAMKII, CDK5, IQGAPs, Asef, tuberous sclerosis complex proteins, mTOR, S6K, FMRP, CREB, formins, mDia, regulators of local mRNA translation, cytoskeleton regulators, EB1 , MAPI B, Rac, Ras, Rho, Cdc42,

CDKL5, MEF2C, SPTAN1 , SLC25A22, neurotransmitter receptor subunits,

glutamatergic (ionotropic and metabotropic), GABAergic, nicotinic acetylcholine, dopaminergic and serotoninergic, MACF, Acf7, Lis1 , astrocyte proteins, active zone proteins, synaptic vesicle proteins, STXB1 , glutamate transporters, neuronal cytoplasmic calcium binding proteins, and inhibitory interneuron proteins.

5. The method of Claim 1 , wherein the sample is a blood sample, fibroblast sample,

induced pluripotent stem cell sample, biopsy sample or postmortem tissue sample.

6. The method of Claim 1 , wherein the assay assembly determines the expression level of two or more informative genes.

7. The method of Claim 1 , wherein the assay assembly determines the expression level of three or more informative genes.

8. The method of Claim 1 , wherein the statistical similarity of the sample expression levels with one or more reference expression profiles is determined by a computing device.

9. A transgenic mouse comprising a conditional knockout of the adenomatous polyposis coli protein, wherein the adenomatous polyposis coli protein is knocked out in the forebrain after the birth of the mouse or during a desired developmental stage.

10. The transgenic mouse of Claim 9, wherein the conditional knockout is caused by a

transcript under the control of a Ca²7calmodulin-dependent protein kinase II promoter.

1 1 . The transgenic mouse of Claim 9, wherein the transcript is a LoxP transcript.

12. The transgenic mouse of Claim 1 1 , wherein the adenomatous polyposis coli gene is engineered to comprise one or more site-specific Cre recombination sites.

13. The transgenic mouse of Claim 9, wherein the adenomatous polyposis coli protein

function is knocked out in response to an external stimulus.

14. The transgenic mouse of Claim 13, wherein the external stimulus is a physical stimulus or a chemical stimulus. A kit for diagnosing a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder comprising hybridization probes for two or more informative genes that exhibit differential expression between an afflicted sample and a non-afflicted sample.

A method of treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder, comprising administering a therapeutically effective amount of a therapeutic compound identified as being effective for treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder.

The method of Claim 16, wherein the therapeutic compound is identified as being effective for treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder by a method comprising: contacting a cell with the test compound and comparing the expression profile of the cell of a sample obtained from an animal comprising the cell, wherein a statistical similarity to an expression profile of a non-afflicted subject indicates the compound is effective for treating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder.

The method of Claim 16, wherein the compound is ECGC.

The method of Claim 16, wherein the compound is decitabine.

A method of identifying a therapeutic agent for treating or ameliorating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder, comprising:

a) contacting an animal comprising a conditional APC knockout, a tissue sample from an animal comprising a conditional APC knockout or a cell obtained from an animal comprising a conditional APC knockout with a test agent; and

b) comparing a sample expression profile obtained from the conditional APC

knockout animal that had been contacted with the test therapeutic agent with a reference expression profile indicative of a non-afflicted state,

wherein a statistically relevant similarity of the sample expression profile to the reference expression profile indicates the test agent is a therapeutic agent for treating or ameliorating a neurocognitive disorder, an autism spectrum disorder or a co-morbid disorder.