EP2968317A1 - Targeting des mtor-pfades bei neurologischen krankheiten - Google Patents
Targeting des mtor-pfades bei neurologischen krankheitenInfo
- Publication number
- EP2968317A1 EP2968317A1 EP14762460.5A EP14762460A EP2968317A1 EP 2968317 A1 EP2968317 A1 EP 2968317A1 EP 14762460 A EP14762460 A EP 14762460A EP 2968317 A1 EP2968317 A1 EP 2968317A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cntnap2
- mice
- kinase inhibitor
- mtor
- mtor kinase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
Definitions
- the present invention relates to methods of treating various neurodevelopmental and neuropsychiatric diseases which employ inhibition of the mTOR pathway, particularly using mTOR kinase inhibitors.
- the CNTNAP2 encodes Contactin-associated protein-like 2 (CNTNAP2), a member of the Neurexin family of proteins.
- CNTNAP2 functions as a cell adhesion protein in the vertebrate nervous system, and mediates interactions between neurons and glia cells during nervous system development.
- the loss of CNTNAP2 - known in the mouse as Casprl - results in the abnormal migration of neurons, reduction in the number of intemeurons, and abnormal neuronal network activity (Penagarikano et al., 2011).
- CNTNAP2 is critical for proper potassium ion channel clustering to the juxtaparanode region of myelinated axons, and for formation of functionally distinct domains in neurons important for saltatory conduction of nerve impulses (Poliak et al., 2001, 2003).
- CNVs intragenic copy number variations
- CNTNAP2 is a strong candidate gene for neuropsychiatric diseases, primarily ASD, SCZ and seizure disorder, and provides an excellent opportunity to model core aspects of neuropsychiatric phenotypes in mice.
- the present invention relates to methods of treating various neurodevelopmental and neuropsychiatric diseases which employ inhibition of the mTOR pathway, particularly using mTOR kinase inhibitors. It is based, at least in part, on extensive phenotypic characterization of a knock-out mouse model of Caspr2, the murine ortholog of CNTNAP2, which indicates that the mechanism via which CNTNAP2 deficits lead to neuropsychiatric disorders is overactivation of the mTOR pathway.
- the present invention provides for methods of treating subjects suffering from neurodevelopmental and/or neuropsychiatric disorders comprising administering, to the subject, an agent that inhibits the mTOR pathway.
- the inhibitor of the mTOR pathway is a mTOR kinase inhibitor such as, but not limited to, a ATK-competitive inhibitor such as WYE125132, Torin 2, AZD2014, and analogous compounds.
- subjects may be tested to determine whether they have a copy number variation or mutation in CNTNAP2 and where such a copy number variation or mutation is present treatment with a mTOR pathway inhibitor may be initiated.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease the level of S6 phosphorylation.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase expression of a glutamate receptor subunit, for example, a GluR2 receptor subunit, an NR2A receptor subunit, or combinations thereof.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease expression of a glutamate receptor subunit, for example, a GluRl receptor subunit, an NRl receptor subunit, an NR2B receptor subunit, an mGLURl receptor subunit, an mGLUR5 receptor subunit, or combinations thereof.
- a glutamate receptor subunit for example, a GluRl receptor subunit, an NRl receptor subunit, an NR2B receptor subunit, an mGLURl receptor subunit, an mGLUR5 receptor subunit, or combinations thereof.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase social interaction and/or cognition.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease the subject's susceptibility to seizures. In certain embodiments, the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase the subject's threshold for seizures, for example, increasing the subject's threshold to a seizure stimulant such as, for example, pilocarpine.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to reduce a symptom of a psychiatric disorder.
- a subject having a neurodevelopmental or psychiatric disorder is identified as likely to benefit from treatment with an inhibitor of the mTOR pathway, for example a mTOR kinase inhibitor, by determining that the subject exhibits one or more of the following, for example, as demonstrated in a cell sample from the subject: increased phosphorylation of S6, decreased GluR2 receptor subunit, decreased NR2A receptor subunit, increased GluRl receptor subunit, increased NRl receptor subunit, increased NR2B receptor subunit, increased mGLURl receptor subunit, and/or increased mGLUR5 receptor subunit, relative to a normal control subject.
- an inhibitor of the mTOR pathway for example a mTOR kinase inhibitor
- FIGURE 1 Prepulse inhibition (PPI). Mice heterozygous for the Caspr2 mutant allele showed a decrease in PPI compared to mice homozygous for the mutant allele as well as wild-type littermates.
- FIGURE 2 Abnormalities in social interaction.
- Caspr2 ⁇ ' ⁇ HOM mice spent less time socially investigating than Caspr2 +/ ⁇ /- HET mice which spent less time socially investigating than WT littermates. When dividing the 10- minute interval of time spent socially investigating into individual minutes, a statistically significant reduction in the HOMs compared to WTs is evident in the early 1-min intervals of the experiment.
- B When examining time spent sniffing as a social measure, we found that HOMs have significantly shorter bouts of sniffing compared to WT littermates (C).
- FIGURE 3 Pyramidal cells in Casprl mutant mice (A-C) and human subjects with homozygous CNTNAP2 frameshift mutations. YFP fluorescence of pyramidalcells in layer 5 of FC of 2-month-old (A) WT, (B) Caspr2 ⁇ ' ⁇ HOM and (C) Caspr2 +/ ⁇ HET mice at 200x, demonstrating increased pyramidal soma size (white arrows) in both HOM and HET mice compared to WT littermates.
- FIGURE 4 Quantification of pyramidal cell size in Caspr2 mutant mice. 200x images were taken using an epifluorescent microscope of
- somatosensory and auditory cortex were traced on Photoshop to quantify pixels. Pixels were converted to square microns.
- Left panel demonstrates a statistically significant difference in cell size between WTs and HETs (P ⁇ 0.0001) and WTs and HOMs (P ⁇ 0.0001).
- the right panel shows individual pyramidal cell measurements ordered from smallest to largest cell size, with each group plotted as a line graph. The smallest pyramidal cells are the same size in all three 3 genotypes, but the largest pyramidal cells are much larger in HOMs
- FIGURE 5 Phosphorylation of the ribosomal S6 in the hippocampus of the Caspr2 mutant mice. Hippocampal homogenates from adult mice were probed with an antibody directed against the S6 S235/236
- FIGURE 6 Pyramidal cells in the TC of human subjects with homozygous CNTNAP2 mutation. Co-staining for cresyl violet (blue) and phosphorylated S6 (pS6; brown). Right panel demonstrates an age -matched normal control with minimal staining for pS6 whereas the left panel demonstrates a dramatic pS6 staining in significantly enlarged pyramidal cells in the subjects with the homozygous CNTNAP2 mutation.
- FIGURE 7 Representative traces of spontaneous postsynaptic currents (PSCs) recorded from pyramidal neurons in FC layer 5. Plotted data of recordings from FC layer 5 pyramidal neurons, across all three genotypes: WT (blue), Caspr2 ⁇ ' ⁇ HOM (red), and Caspr2 +
- HET HET mice.
- the top graph shows the amplitude of spontaneous EPSCs for the three genotype groups, whereas the bottom graph shows the amplitude of spontaneous IPSCs.
- FIGURE 8 Transcranial in vivo two-photon imaging of Caspr2 mutant mice compared to WT littermates.
- Panel (A) demonstrates a reduction in spine formation in the Caspr2 mice whereas panel (B) shows a significant increase in spine elimination.
- FIGURE 9 Electron microscopy examination of synaptic structure in Caspr2 ⁇ ' ⁇ compared to WT littermates. The left panel shows both perforated
- FIGURE 10 FDG Micro-PET-MRI imaging of baseline glucose metabolism in Caspr2 mutant mice. SPM results reveal areas of significant activation in mutants compared to the WT littermates group; areas of activation are shown in red (see arrows). Panel (A) demonstrates clearly the cortical and subcortical areas of FDG hypermetabolism in the Caspr2 +! ⁇ HET mice compared
- Panel (B) shows a similar pattern of cortical and subcortical FDG hypermetabolism in the Caspr2 '/" HOM mice compared to WT littermates.
- FIGURE 11 High resolution MRI of Caspr2 mutants.
- A-C The figure (A) shows the location of the segmented occipital lobe, while the bar graphs (B and C) show the difference in size of the occipital lobe in absolute terms (in mm3; B) and in relevant terms as a percentage of total brain volume (C). The relative volume differences held up to the multiple comparisons, with FDR values of 0.08 for the HET and 0.01 for the HOM. D and E show the areas of decrease within the occipital lobe. FDR is between 10% and ⁇ 15%.
- FIGURE 12 depicts a summary of the data for vehicle- and
- FIGURE 13 Spine elimination and spine formation in wild-type mice and mice homozygous or heterozygous for Caspr2, treated with WYE 12 ⁇ 132 or vehicle.
- FIGURE 14A-F El ecrophysiologic recordings from iCaspr2 imutants and wild-type litermates treated with vehicle or drug.
- A,C,E Long-term potentiation in wild-type mice, knockout mice treated with vehicle, and knockout mice treated with drug, respectively.
- B, D, F0 shows E:I correlations.
- FIGURE 15A-E Behavioral and Neuroimaging Phenotypes of Cntnap2 Mutant Mice.
- Cntnap2 mutant mice show social inhibition in the first minute (upper and middle panel) as well as a decrease social interaction over the whole 10-minute interval.
- Cn.tn.ap2 '1' mice were found to have a lowered seizure threshold upon pilocarpine administration as indicated by a higher seizure stage reached by Cntnap2 ⁇ ' ⁇ mice (right panel) as well as more time spent in stage 4 seizures by Cntnap2 ⁇ ' ⁇ mice (left panel).
- Bars represent, left to right, wild type, Cntnap +I' and Cntnap2 ' ' ⁇ .
- C MRI analysis demonstrated grossly normal mesoscopic neuroanatomy in Cntnap2 mutant mice. Regional differences found in the Cntnap2 ' ⁇ mouse brain were specific to the occipital cortex. Bar graphs representing these differences are shown for both absolute volume (in mm 3 ; bar graph to the left) and relative volume (% total brain volume; bar graph to the right). Error bars represent 95% confidence intervals and significance is indicated as a measure of false discovery rate (FDR) with Representing an FDR of less than 10% and ** representing an FDR of less than 5%.
- FDR false discovery rate
- Cntnap2 + ' ⁇ (upper panel) and Cntnap2 ' ' ' (lower panel) mice In Cntnap2 +I ⁇ mice, significant hypermetabolism is shown in red across location and corresponding numbered coronal plates in the following regions: OB (1,2), PFC (2), M1/M2 (3,5,6), Cg/RSC (3,5, 7-9), CPu (4,5), IC (4), Pir (4), V1/V2 (7-9), S1/S2 (5,6), cc (5,8), eg (6-8), GP (6), IntC (6) ThalN (6- 8), HPC (7,8), Aul/AuV (7), Pretectal Nucleus (8), PAG (8,9), Midbrain Nuclei (8-10), Colliculi (9-10), Cerebellar Cortex (11), Cerebellar Nuclei (10-12).
- hypometabolism clusters are shown in blue and were fewer and smaller in size.
- hypermetabolism clusters are located in the following regions: OB (1,2), PFC (2), M1/M2 (3,4), Cg/RSC (3-7), PRhC (5), Pir (5), V1/V2 (6,7), S1/S2 (3-5), ThalN (5,6), Amyg (5), Hb (5), HPC (6,7), PAG (6), Midbrain Nuclei (6,7), Colliculi (6,7), Cerebellar Cortex (8), Cerebellar Nuclei (7,9).
- FIGURE 16A-D Abnormal neuronal network activity and plasticity in Cntnap2 mutant mice.
- FIGURE 17A-F Cellular, synaptic and molecular abnormalities in
- Cntnap2 mutant mice (A) Enlarged pyramidal cells in wild type (WT) and Cntnap2 mutants. Left panel shows YFP fluorescence of pyramidal cells in layer V of the frontal cortex of 2-month-old WT (upper image), Cntnap2 ⁇ ' ⁇ (middle image), and Cntnap2 +/ ⁇ mice (right image). There is an increased pyramidal soma size in both Cntnap2 + ' ⁇ and Cntnap2 "/" mice compared to WT littermates (at 200x magnification).
- B and C Show Cntnap2 dosage effect on pyramidal cell soma size (B) (CntnapT 1 - ("(HM")> Cntnap2 +I - (“HT”)> WT). Cumulative frequency distribution plots (C) show individual pyramidal cell measurements ordered from smallest to largest cell size within each genotype. Significant variability in cell size can be observed in all 3 genotypes.
- D Electron microscopic image demonstrates a perforated postsynaptic density (PSD) in layer I of the cortex from a Cntnap2 ⁇ l ⁇ mouse.
- FIGURE 18A-B Differential expression of glutamate receptor subunits in the prefrontal cortex (PFC) of Cntnap2 mutant mice.
- PFC prefrontal cortex
- FIGURE 19A-C Cellular, synaptic, and molecular changes in cortical tissue of individuals carrying homozygous CNTNAP2 mutations.
- Cresyl violet (CV; blue) staining demonstrates enlarged pyramidal cells throughout the cortex of a patient with homozygous CNTNAP2 mutations (upper left panel) whereas double staining for CV and pS6 (blue and brown, respectively; upper middle panel) shows that pyramidal cells are strongly positive for pS6 compared with a normal control with only minimal pS6 staining (upper right panel).
- Lower panel shows a representative section from a patient with
- Bar graph indicates a reduction in length in perforated PSDs in homozygous mutation carriers versus controls (bottom panel).
- C Staining for MAP-2 (red) and GluRl (green; left panel) and mGluR5 (green; right panel) demonstrates an increase of both glutamate receptor subunits in pyramidal cells throughout the cortex of patients with homozygous CNTNAP2 mutations. Cell nuclei are visualized with DAPI stain (blue). Scale bars in A and C represent 20 microns.
- FIGURE 20 Treatment with WYE125132 rescues cellular, synaptic and molecular abnormalities in Cntnap2 mutants.
- A Reversal of increased cell size by treatment with WYE125132. Panels demonstrate pyramidal cells throughout the cortex of both Cntnap2 +I ⁇ (HT) and Cntnap2 ⁇ ' ⁇ (HM) mice compared with WT littermates, which is entirely rescued with treatment with WYE125132 or vehicle.
- B Bar graphs (top) indicate that the dramatic increase in phosphorylation
- FIGURE 21 A-E. Treatment with WYE 125132 rescues abnormalities in synaptic plasticity and the excitatory-inhibitory balance as well as alterations in dendritic spine dynamics and behavioral deficits in Cntnap2 mutants.
- A Representative recordings showing measurements of excitation ("E") and inhibition ("I") at different stimulus intensities (normalized to maximum intensity of 15 V) from WT vehicle-treated (top; linear correlation coefficient r: 0.79), Cntnapl '1' vehicle-treated (middle; r: 0.07), and WYE125132-treated Cntnap2 ' ' ⁇ mice (bottom; r: 0.72) mice.
- B Example whole-cell recordings from layer V pyramidal neurons of adult auditory cortex from WT vehicle-treated (upper;
- WYE 125132 rescues excessive spine elimination in both Cntnap2 +I ⁇ and Cntnap ' mice.
- D Rescue of social interaction deficits. Treatment with WYE125132 corrects the social interaction deficits in the first minute of testing as well as the total frequency of social interactions over the whole 10-minute testing interval in Cntnap2 mutant mice.
- E Rescue of cognitive deficits. WYE125132-treated mutant mice demonstrate a rescue of a deficit in the Novel Object Recognition test.
- FIGURE 22 Western blot of cortical extracts from Cntnap2 mutant or wild-type mice treated with vehicle or various mTOR pathway inhibitors, showing staining for the presence of phosphorylated S6. Individual mice were tested, and are represented in the lanes as follows.
- the present invention relates to methods of treating
- a subject may be a human subject or a non-human subject such as, but not limited to, a non-human primate, a dog, a cat, a horse, a pig, a cow, a sheep, a goat, a mouse, a rat, a hamster, a guinea pig, fowl, a cetacean, etc.
- neurodevelopmental and/or neuropsychiatric disorders which may be treated include, but are not limited to, schizophrenia (SCZ), autism spectrum disorder (ASD) (such as, for example, but not limited to, autistic disorder, Asperger's syndrome, pervasive developmental disorder not otherwise specified, Rett's syndrome, childhood disintegrative disorder), bipolar disorder, attention-deficit hyperactivity disorder (ADHD), Gilles de la Tourette disorder, obsessive-compulsive disorder, depression, mood
- SZ schizophrenia
- ASD autism spectrum disorder
- ADHD attention-deficit hyperactivity disorder
- Gilles de la Tourette disorder obsessive-compulsive disorder, depression, mood
- Active 15270201.2 disorders seizure disorder, cognitive dysfunction and/or mental retardation.
- Treatment is achieved when a subject exhibits improvement in a symptom or sign of the disorder.
- treatment may be reflected by an improvement in the Global Assessment of Functioning score of the subject, for example, but not by way of limitation, which is sustained over a period of at least one month, at least 3 months, at least six months, or at least one year.
- the disorder is an autism spectrum disorder, it is not associated with a genetic defect in TSC, FXS, or PTEN and/or an associated clinical syndrome, e.g. tuberous sclerosis or Fragile X syndrome.
- Non-limiting examples of mTOR pathway inhibitors which may be used according to various embodiments of the invention include everolimus, ridaforolimus, ARmTOR26 (Array BioPharma Inc.), BN107 (Bionovo, Inc.), CU906 (Curis, Inc.), EC0565 (Endocyte, Inc.), XL388 (Exelixis Inc.), HL152B (HanAli Biopharma Co. Ltd.), NV128 (MEI Pharma Inc., Novogen, Ltd.), sirolimus, SXMTR1 (Serometrix L.L.C.), X480 (Xcovery), X414 (Xcovery),
- RG7422 F. Hoffman La Roche Ltd.
- DS3078 Daiichi Sankyo Co. Ltd.
- OS1027 Astellas Pharma US Inc.
- AZD2014 AstraZeneca Pic
- AZD8055 (Astrazeneca Pic), GDC0068 (Array BioPharma Inc.), CC223 (Celgene Corp.), CC115 (Celgene Corp.), zotarolimus, umirolimus (Terumo Corp.), tacrolimus, TOP216 (Topotarget AS), BC210 (Pfizer Inc.), PF04691502 (Pfizer Inc.), WYE125132 (Pfizer Inc.), TAFA93 (Isotechnika Pharma Inc.), LOR220 (Lorus Therapeutics Inc.), nPT-mTOR (Biotica Technology), AP23841 (ARIAD Pharmaceuticals Inc.), AP24170 (ARIAD Pharmaceuticals Inc.), and Torin2 (Tocris).
- the mTOR pathway inhibitor is rapamycin or a rapamycin analog ("rapalog").
- the mTOR pathway inhibitor is not rapamycin or a rapamycin analog; for example, but not by way of limitation, the mTOR pathway inhibitor may be a so-called mTOR kinase inhibitor such as an ATP-competitive inhibitor of mTOR kinase (see Yu et al., "Beyond Rapalog Therapy: Preclinical pharmacology and antitumor activity of WYE- 125132, an ATP-competitive and
- the mTOR kinase inhibitor is a pyrazolopyrimidine ATP-competitor and specific inhibitor of mTORCl and/or mTORC2.
- the mTOR kinase inhibitor is a pyrazolopyrimidine substituted with a bridged morpholine ATP-competitor and specific inhibitor of mTORCl and/or mTORC2.
- the mTOR pathway inhibitor is WYE-125132 (also sometimes referred to as "WYE- 132”) or an analog thereof.
- WYE-125132 also sometimes referred to as "WYE- 132"
- the mTOR kinase inhibitor has the general formula I:
- R is a substituted or unsubstituted aromatic, for example a substituted or unsubstituted phenyl, where when present the one or more substituent may be, independently, a halogen such as fluorine, chlorine or bromine, a hydroxyl, a Q - C 4 alkoxy, or a substituted or unsubstituted amide where a substituent may be, for example, Ci - C 4 alkyl.
- R may be
- mTOR kinase inhibitors which may be used according to the invention include compounds disclosed in US20110281857, EP2382207, US20120165334, EP2398791, US20090311217, EP2300460, US20120134959, and EP2419432.
- the mTOR kinase inhibitor has the general formula II:
- Ri may be H, or Cj - C 4 alkyl, or substituted or unsubstituted amino, or a 3- 6 member aliphatic or aromatic ring, which may optionally be a heterocycle comprising at least one N where said ring may be substituted or unsubstituted, where when present the one or more substituent may be, independently, a halogen such as fluorine, chlorine or bromine, a hydroxyl, a Q - C 4 alkoxy, or a substituted or unsubstituted amide; and where R 2 is a substituted or unsubstituted amine, a halogen such as fluorine, chlorine or bromine, a hydroxyl, or a Q - C 4 alkoxy, where a substituent may be, for example, Q - C 4 alkyl.
- the specific compound having general formula II is Torin 2, having the structure:
- an effective dose of a pyrazolopyrimidine substituted with a bridged morpholine ATP-competitor and specific inhibitor of mTORCl and/or mTORC2, of which WYE- 125132 is a non- limiting example may be, for treatment of a human subject, between 0.5 and 100 mg/kg, or between about 1 and 50 mg/kg, or between about 5 and 25 mg/kg, or about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 1 1 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about
- Active 15270201.2 20 mg/kg, about 21 mg kg, about 22 mg/kg, about 23 mg/k, about 24 mg/kg, or about 25 mg/kg.
- an effective dose of AZD2014 or a related compound having general formula I may be, for treatment of a human subject, between about 0.2 and 5 mg/kg, or between about 0.5 and 2 mg/kg, or between about 1 and 2 mg/kg, or greater than 1 mg/kg and less than 3 mg/kg, or about 0.5 mg/kg, or about 0.75 mg/kg, or about 1 mg/kg, or about 1.25 mg/kg, or about 1.5 mg/kg, or about 1.75 mg/kg, or about 2 mg/kg.
- an effective dose of Torin2 or a related compound having general formula II may be, for treatment of a human subject, between about 0.2 and 5 mg/kg, or between about 0.5 and 2 mg/kg, or between about 1 and 2 mg/kg, or greater than 1 mg/kg and less than 3 mg/kg, or about 0.5 mg/kg, or about 0.75 mg/kg, or about 1 mg/kg, or about 1.25 mg/kg, or about 1.5 mg/kg, or about 1.75 mg/kg, or about 2 mg/kg.
- An effective dose of other mTOR pathway inhibitors may be a dose calculated to achieve a concentration in the central nervous system of a subject to be treated, where said concentration, in cell culture, inhibits intracellular phosphorylation of S6K relative to untreated cells, preferably by at least about 20 percent.
- the mTOR pathway inhibitor may be administered according to methods known in the art, including but not limited to oral, sublingual, nasal, by inhalation, transdermal, subcutaneous, intradermal, intramuscular, intravenous , intraperitoneal, intrathecal, etc..
- a pyrazolopyrimidine substituted with a bridged morpholine ATP-competitor and specific inhibitor of mTORCl and/or mTORC2, of which WYE- 125132 is a non- limiting example, may be administered orally.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease the level of S6 phosphorylation.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase expression of a glutamate receptor subunit, for example, a GluR2 receptor subunit, an NR2A receptor subunit, or combinations thereof.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease expression of a glutamate receptor subunit, for example, a GluRl receptor subunit, an NR1 receptor subunit, an NR2B receptor subunit, an mGLURl receptor subunit, an mGLUR5 receptor subunit, or combinations thereof.
- a glutamate receptor subunit for example, a GluRl receptor subunit, an NR1 receptor subunit, an NR2B receptor subunit, an mGLURl receptor subunit, an mGLUR5 receptor subunit, or combinations thereof.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase social interaction and/or cognition.
- the inhibitor of the mTOR pathway is administered to a subject in an amount effective to decrease the subject's susceptibility to seizures. In certain embodiments, the inhibitor of the mTOR pathway is administered to a subject in an amount effective to increase the subject's threshold for seizures, for example, increasing the subject's threshold to a seizure stimulant such as, for example, pilocarpine.
- a subject suffering from a neurodevelopmental and/or neuropsychiatric disorder may be tested to determine whether a copy number variation or other mutation or variation in CNTNAP2 is present, and if a copy number variation, mutation or variation in CNTNAP2 is found, then the subject may be treated with a mTOR pathway inhibitor.
- a test may be performed using methods known in the art, including but not limited to nucleic acid based testing, for example, using nucleic acid primers followed by amplification and sequencing, and/or microarray analysis, SNP analysis, use of nucleic acid probes, for example in FISH analysis, etc., or protein based testing such as antibody based analysis, Western blotting, etc.
- the present invention provides for kits for making such determination.
- a subject suffering from a neurodevelopmental and/or neuropsychiatric disorder may be tested to determine whether the subject exhibits a hyperactivation in the mTOR pathway, and if hyperactivation of the mTOR pathway is found, then the subject may be treated with a mTOR pathway inhibitor.
- Indicators of hyperactivation of the mTOR pathway include, but are not limited to, for example, as demonstrated in a cell sample from the subject: increased phosphorylation of S6, decreased GluR2 receptor subunit, decreased NR2A receptor subunit, increased GluRl receptor
- Active 15270201.2 subunit, increased NR1 receptor subunit, increased NR2B receptor subunit, increased mGLURl receptor subunit, and/or increased mGLUR5 receptor subunit, relative to a normal control subject may be performed using methods known in the art, including but not limited to nucleic acid based testing, for example, using nucleic acid primers followed by amplification and sequencing, and/or microarray analysis, SNP analysis, use of nucleic acid probes, for example in FISH analysis, etc., or protein based testing such as antibody based analysis, Western blotting, etc.
- the testing may be performed in vivo, for example using PET scanning in conjunction with
- the level of activation of the mTOR pathway may be assessed in vitro via phosphorylation of S6, as described in the working examples below.
- the present invention provides for kits for making such determination.
- Treatment of a subject with a mTOR pathway inhibitor may be practiced in conjunction with one or more conventional treatments of the neurodevelopmental or neuropsychiatric disease being treated.
- Caspr2 leads to an abnormal cellular phenotype and overactivation of the mTOR pathway.
- Coronal sections of Caspr2/T yl -YFP/H mice were analyzed at 200x magnification utilizing epifluorescent microscope analysis of pyramidal cells in the frontal cortex (FC). Soma size was traced on Photoshop to quantify pixels.
- Caspr2 +l ⁇ /Thy 1 -YFP/H and Casprl'- /Thy- 1 -YFP/H mice were found to have pyramidal cells with a significantly enlarged soma size throughout layer 5 of the FC and temporal cortex (TC)(Fig. 3A, B).
- a similar phenotype of enlarged pyramidal cells has
- Active 1 5 270201.2 previously been described in different transgenic mouse models of ASD which all involve overactivation of the mTOR pathway, including models of tuberous sclerosis complex (Goto et al., 2011) and PTEN mutations (Zhou et al., 2009).
- CNTNAP2 leads to overactivation of the mTOR pathway (as measured by pS6), which, in turn, leads to cellular abnormalities (i.e., increased size of pyramidal cells) and neurocircuitry level deficits (i.e., abnormalities in excitation-inhibition balance, see below). It is therefore very likely that, by effectively targeting the mTOR overactivation in cells that carry a CNTNAP2 mutation, one might be able to prevent and/or reverse ASD- associated changes in cellular morphology and electrophysiology.
- Active 15270201.2 postsynaptic current (IPSC) of Casprl 1' /Thy-l-YFP/H or Caspr2 +/' /Thyl-YFP/H compared to WT littermates.
- ISC interleukin-like current
- the decrease in length of perforated PSDs may reflect the structural synaptic deficits underlying the previously characterized cognitive deficits, seizures, and the ASD-related behavioral abnormalities in Caspr2 mutant mice and human subjects with CNTNAP2 mutations.
- a template detailing regions of interest was made in accordance with the MRI used for co-registration to allow for a more simple method of detailing regional brain activation.
- SPM Statistical Parametric Mapping
- SPM analysis revealed significant activation in the Caspr2 +/ ⁇ group compared to the WT group (PO.01, Ke>50) in the following regions: OB, M1/M2, PFC, SI, CPu, NAc, st, Hb, Th, V2, RSC, PAG, and Cb. Of these regions, the greatest activation was seen in the OB (spanning from the OB through PFC) and
- MRI Magnetic resonance Imaging
- Active 15270201.2 determinants of the deformation fields are then calculated as measurements of volume at each individual voxel.
- Significant regional volume changes can then be calculated in two different ways.
- regional measurements can be calculated by registering a pre-existing classified MRI atlas on to the population atlas, which allows for the volume measurement of 62 different brain regions.
- the 62 regions in the classified atlas include the cortical lobes, large white matter structures (i.e., the corpus callosum), ventricles, cerebellum, brain stem structures, and olfactory bulbs. The regions were then assessed in all brains and volumes were calculated in mm3.
- individual voxel measurements can be calculated from comparisons of the Jacobian determinants in a specific voxel between the Caspr2 +/ ⁇ , Caspr2 ' ' and WT mice. These measures can be calculated as measures of absolute volume (in mm3) or relative volume (% total brain volume). Multiple comparisons were controlled for by using either the False Discovery Rate (FDR) for the regional comparisons, or Threshold Free Cluster Enhancement (TFCE) for the voxel-wise whole brain comparisons.
- FDR False Discovery Rate
- TFCE Threshold Free Cluster Enhancement
- WYE125132 a specific mTOR kinase inhibitor compound
- WYE125132 has the potential to prevent and or reverse the molecular-, synaptic-, cellular-, and neurocircuitry level abnormalities associated with a wide variety of neuropsychiatric disorders associated with CNTNAP2 and other gene mutations which lead to overactivation of the mTOR pathway in human subjects.
- This group of neuropsychiatric disorders potentially includes, but is not limited to schizophrenia, autism-spectrum disorders, mood disorders, attention-deficit hyperactivity disorder, OCD and Tourette's, cognitive dysfunction or mental retardation, and seizure disorder.
- WYE125132 In order to assess whether we could reverse or prevent the cellular abnormalities (i.e., enlarged pyramidal cell size), we treated with WYE125132 ('Tx' in Fig. 12) or vehicle ('veh' in Fig. 12) Caspr2 + ⁇ /Thyl-YFP/H and CasprZ 1' /Thy-l-YFP/H mice and WT littermates also positive for YFP/H for the entire period P12-P35. The mice were perfused and subsequently fixed with 4% PFA and sectioned coronally at 16 microns.
- Caspr2 ⁇ ' ⁇ /Thy-l-YFP/H mice were grouped together as 'mutant' mice (see Fig. 12).
- Fig. 12 we present a summary of the data for vehicle- and WYE125132- treated animals and soma size.
- One-way ANOVA clearly indicated that there is a significant difference across all datasets (P ⁇ 0.0001).
- There was no significant difference in pyramidal cell size between WT and mutant compound-treated indicating that treating with WYE 125132 for approximately 3 weeks completely reverses the enlarged pyramidal cell size in Caspr2 mutants.
- thinned regions were identified on the basis of the maps of the brain vasculature.
- Microsurgical blades were used to re-thin the region of interest until a clear image could be obtained.
- the area of the imaging region was 200 ⁇ ⁇ 200 ⁇ in the frontal association cortex.
- the centers of the imaging regions were as follows: +2.8 mm bregma, +1.0 mm midline.
- Active 15270201.2 which the animals were euthanized and thalamocortical slices were prepared. Briefly, animals were anesthetized, decapitated and the brain quickly placed into ice-cold dissection buffer containing (in mM): 75 sucrose, 87 NaCl, 2.5 KC1, 1.25 Na3 ⁇ 4P0 4 , 0.5 CaCl 2 , 7 MgCl 2 6 H 2 0, 25 NaHC0 3 , 10 dextrose, bubbled with 95% ⁇ 3 ⁇ 4 / 5% C0 2 (pH 7.4).
- Slices 400 ⁇ were prepared with a vibratome (Leica, VT1200S), placed in 33-35°C for artificial cerebrospinal fluid (ACSF, in mM: 124 NaCl, 2.5 KC1, 1.25 NaH 2 P0 4 , 2.5 CaCl 2 , 1.5 MgS0 4 7H 2 0, 26 NaHC0 3 , and 10 dextrose) for ⁇ 30 min; then returned to room temperature >1 hr before use. Slices were then transferred to the recording chamber and perfused (2.0-2.5 ml min-1) with oxygenated ACSF at 33-35°C and given 30 min to stabilize.
- ACSF cerebrospinal fluid
- Somatic whole- cell recordings were made from layer 5 pyramidal cells in voltage and current clamp mode with a Multiclamp 700B amplifier (Molecular Devices). Selection was based on morphology and electrophysiological criteria. Patch pipettes (3-8 ⁇ ) contained a current clamp solution. Data were filtered at 2kHz, digitized at 10kHz and analyzed with Clampfit. Cells were excluded from analysis if Ri or Rs changed by >25% over the course of the recording. Excitatory post-synaptic currents or potentials (EPSCs/Ps) were evoked by extracellular stimulation (0.01-lms, 1- 10V) with a 4x1 array of electrodes placed in layer 4, and straddling the patched layer 5 neuron.
- EPCs/Ps Excitatory post-synaptic currents or potentials
- mice we compared spontaneous and evoked inhibitory responses by clamping the membrane potential of the cell to sub-threshold levels. Furthermore, we compared inhibition generated by each stimulation electrode, to the corresponding amount of excitation when the cell was held at -80mV. Lastly, we tested Spike-Timing Dependent Plasticity (STDP).
- Fig. 14 The results of this experiment are delineated in Fig. 14, where representative recordings from Caspr2 mutants and WT littermates are shown.
- LTP was induced with repetitive pre-post spike pairing as in STDP (Fig. 14A, C, E).
- WT littermates treated with vehicle ('w/t'; Fig.l4A) or WYE125132, as well as Casprl 1' /Thy-l-YFP/H mice treated with WYE125132 ('KO + drug' in Fig. 14 E) demonstrated robust LTP, whereas Casprl 1' /Thy-l-YFP/H mice treated with vehicle ('KO + veh'; Fig. 14C) did not.
- mice Cntnap2 mutant and WT mice were obtained from heterozygous crossings and were born with the expected Mendelian frequencies. The three obtained genotypes were housed together. Mice were treated with p.o. gavaging once per day with either WYE125132 (10 mg/kg) or vehicle. All procedures involving animals were performed in accordance with the Columbia University / New York State Psychiatric Institute animal research committee.
- WYE 125132 (Chemscene) was administered by a daily p.o. gavaging in a volume of 10 ml/kg.
- dams were treated from P0 to P12 after which individual pups were gavaged on a daily basis going forward. Mice also received drug treatment on the days of testing, at least 1 hour prior the experiment.
- mice were gavaged from P12 to p35, after they were euthanized.
- adult mice were gavaged for 14 consecutive days after which they were euthanized.
- ultramicrotome placed on 200 mesh copper grids and stained with lead citrate. Micrographs were taken on a FEI TecnaiTM transmission electron microscope 12 operating at 80KV.
- Cntnapl mice were crossed to Thyl-YFP in order to visualize layer V pyramidal neurons in the neocortex.
- Cntnap2+/-, Cntnap2-/- and WT mice were were cardially
- Sections containing auditory and somatosensory cortices were analyzed. Sections were re-hydrated in PBS, blocked with 10% normal serum and
- Triton-X-100 permeablized in 0.1 % Triton-X-100.
- GFP antibody (1 : 1500, Naclai-Tesque) was incubated in 0.1% Triton X-100, 4% normal serum in PBS overnight at 4°C and visualized with donkey anti-rat FITC secondary antibody (1 :1000, Jackson
- mice were anesthetized with ketamine/xylazine and intracardially perfused with 30mL of 0.1 M PBS containing lOU/mL heparin (Sigma) and 2mM ProHance (a Gadolinium contrast agent) followed by 30mL of ice cold 4% paraformaldehyde (PFA) containing 2mM ProHance (Spring et al., 2007). After perfusion, mice were decapitated and the skin, lower jaw, ears, and
- FDG-microPET/MRI Image Analysis All imaging was carried out by the staff at the Center for Molecular and Genomic Imaging (CMGI University of California, Davis) under an approved animal use protocol. The mice were anesthetized with a mixture of isoflurane and oxygen gas ( ⁇ 1-1.5%) for a short period for injection of the FDG. Animals were re-anesthetized immediately prior to scanning and secured onto an imaging bed. Image analysis was performed as previously described (Thanos et al. 2008; Pascau et al. 2009).
- mice were used in the experiments. Spine formation and elimination were examined by imaging the mouse frontal association cortex through a thinned-skull window as described previously (Lai, 2012).
- Cntnapl mutant mice show altered social interaction: Detailed measurements of interaction between pairs of mice placed together in standard cages provide insights into reciprocal social interactions (Silverman et al., 2010). We examined (i) whether juvenile Cntnapl mutant mice display decreased social interaction over a 10-minute interval and (ii) whether mutant mice have reduced tendency to approach novel social stimuli, as evidenced by social inhibition in the first minute of the tested interval (Curley et al., 2009). The first minute of this social interaction paradigm represents the phase when preference for social novelty is normally established.
- Cntnap2 mutant mice have a lower threshold to pilocarpine- induced seizures: Individuals carrying CNTNAP2 mutations commonly have seizures (Strauss et al., 2006; Friedman et al., 2008) and CNV studies have identified genetic lesions of CNTNAP2 in individuals with epilepsy (Mefford et al., 2011). Although some 6-12 mo old Cntnap2 mutant 8 mice were observed to have spontaneous generalized tonic-clonic seizures, this was a rare occurrence and we were not able to elicit a significant number of seizures by handling or audiogenic stimulation as previously reported (Penagarikano et al., 2011). We therefore utilized a pilocarpine seizure induction protocol to test whether Cntnap2 mutant mice display lower seizure threshold compared with WT littermates.
- mice compared with 1.80 minutes for Cntnap2 + ' ⁇ and 41.25 minutes for Cntnapl 1' mice.
- time to onset of stage 4 seizures Independent samples Kruskal-Wallis test, P ⁇ 0.009
- duration of stage 4 seizures Kruskal-Wallis test, P ⁇ 0.025
- Cntnap2 dosage conferred increased susceptibility to pilocarpine-induced seizures, with effects on both severity and duration.
- Cntnap2 is expressed in multiple adult brain regions, primarily cerebral cortex, hippocampus, striatum, olfactory tract, and cerebellar cortex (Penagarikano et al., 2011). Utilizing a multi-channel 7.0 Tesla MRI scanner, we examined sixty-two different brain regions to determine changes
- Cortical and subcortical hypermetabolism in Cntnap2 mutant mice We compared changes in regional brain glucose metabolism in Cntnap2 mutant mice and WT littermates using micro-positron emission tomography (microPET) with [18F]2-fluoro-2-deoxy-D-glucose co-registered with structural MRI images. Both Cntnap2 +I ⁇ and Cntnap2 '/ ⁇ mutant mice show a strikingly similar spatial pattern of hypermetabolism in specific brain regions, including large areas of the cortex, thalamic nuclei, olfactory bulb, cerebellum, and hippocampus (FIGURE 15E). When mutant mice were compared with each other, Cntnap2 +/' mice demonstrated a significantly larger degree of hypermetabolism in the prefrontal cortex, thalamic nuclei, and olfactory bulb (FIGURE 15E).
- mice show a more restricted pattern of hypometabolism confined in the piriform cortex, midbrain nuclei, and brain stem (FIGURE 15E).
- Cntnapl has been shown to be important for the localization of potassium channels, we assessed the action potential characteristics and found that Cntnapl '1' mice have a more depolarized voltage threshold compared WT littermates. This voltage difference is consistent with the importance shown for Cntnapl in the clustering of voltage-gated channels in myelinated axons (Horresh et al., 2008). In contrast, other single action potential characteristics, such as the delay to first spike, the action potential half-width, the amplitude, and the after- hyperpolarization amplitude did not differ among genotypes (FIGURE 16A).
- Cntnapl mutant mice for abnormalities in spine turnover.
- Enlarged pyramidal cells soma size in Cntnapl mutant mice We characterized cellular morphology in Cntnap2 +I' l Thy 1-YFP/H and Cntnap2 ⁇ ' ⁇ I Thy- 1-YFP/H mice, and compared it to WT/Thyl-YFP/H littermates.
- perforated synapses present higher densities of glutamate receptors compared to non-perforated synapses (Lamprecht & LeDoux, 2004).
- This apparent discrepancy between increased expression of several AMPA- R and NMDA-R subunits and the decrease in the length of segmented PSDs of perforated synapses suggests that a significant proportion of these receptors might be located at extrasynaptic sites.
- iGluR ionotropic glutamate receptor subunit expression profiles, involving amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) and N-methyl-D-aspartate receptors (NMDARs).
- AMPA-Rs amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors
- NMDARs N-methyl-D-aspartate receptors
- mice showed selective increased NR1 (P ⁇ 0.05) and NR2B (P ⁇ 0.01) expression at 9-1 1 months of age.
- Enlarged pyramidal and dysplastic cells with differential expression of cell-specific markers and increased pS6 We examined whether the cellular phenotype identified in the Cntnap2 mutant mice involving enlarged pyramidal cells throughout the cortex is also evident in temporal cortex tissue surgically removed due to debilitating seizures from individuals with homozygous CNTNAP2 mutations who were also diagnosed with ASD. Cresyl violet (CV) staining, performed in three subjects and 3 age-matched controls, revealed a pattern of diffusely distributed similarly enlarged cells, which appear to have intact morphological characteristics of pyramidal cells (FIGURE 19A).
- CV Cresyl violet
- FIGURE 19A To confirm that overactivation of the mTOR pathway accompanies this cellular phenotype, we carried out co-staining for CV and pS6 (blue and brown respectively, FIGURE 19A). This analysis demonstrated strong pS6 staining in the enlarged pyramidal cells throughout the cortex in human mutation carriers, while cortical tissue from normal controls displayed only weak staining for pS6 (FIGURE 19A).
- TSC Tuberous Sclerosis Complex
- CNTNAP2 mutation is associated with altered neuronal differentiation.
- These undifferentiated enlarged cells were immunopositive for the neuronal marker SMI31 1 , but also for vimentin, an intermediate filament typically expressed in neuroglial progenitor cells, including radial glia (Weissman et al., 2003), whereas the pyramidal neurons were only immunopositive for neuronal neurofilament marker SMI311 (FIGURE 19A).
- the undifferentiated enlarged cells had a distinct rounded appearance and often presented two nuclei, resembling the TSC giant cells.
- the NeuN and MAP-2 markers which indicate the cellular commitment to the neuronal lineage, were exclusively expressed in dysplastic neurons, not in "giant cells", which again is pronounced of what is found in cortical tubers in brain tissue from patients with TSC (Talos et al., 2008).
- NR2B was also increased, especially in the dendrites, while GluR2 was undetectable in most neurons from patients, but highly expressed in the control neurons.
- These receptor profiles are consistent with altered excitation/inhibition balance due to dysregulated mTOR signaling (Bateup et al., 2013).
- these changes suggest significant alterations of synaptic plasticity in these patients, due to both increased Ca 2+ influx through AMPA-R and NMDA-R with altered subunit composition, as well as upregulated mGluR5- dependent signaling.
- Rapamycin the first mTOR inhibitor described, has been consistently utilized to achieve repression of the mTOR signaling pathway in mouse models of ASD (Delorme et al., 2013).
- rapamycin is a partial mTOR inhibitor acting through allosteric inhibition of the mTORCl -but not the mTORC2- complex, while mTORCl is also known to have some rapamycin-resistant activity raises the fundamental question as to whether rapamycin can indeed lead to a robust and sustained inhibition of mTOR (Guertin & Sabatini, 2009).
- WYE 125132 a highly potent, ATP-competitive, and specific new generation mTOR kinase inhibitor, which targets the catalytic site, inhibits both mTORCl and mTORC2 and has a good bioavailability profile. WYE 125132 is therefore capable of leading to strong and sustained mTOR inhibition in vivo (Yu et al., 2010).
- FIGURE 20 A C,D
- WYE125132 has no effect on soma size in WT animals
- FIGURE 20 A In concordance with the normalization of pyramidal cell size in the cortex of Cntnapl mutant mice, we also found that treatment with WYE 125132 reverses phosphorylation of S6 to levels observed in WT littermates (PO.001; FIGURE 20B).
- Active 1 5 270201.2 showed a significant difference between vehicle-treated WT mice and vehicle- treated Cntnap2 ⁇ ' " mice (PO.05; FIGURE 2 IE) as well as a rescue of this phenotype in WYE125132-treated Cntnap2 'L mice (PO.05; FIGURE 21E).
- FKBP12 FK506-binding protein 12
- Active 15270201.2 rescue many core molecular, synaptic, cellular, neurocircuitry, and behavioral phenotypes in the Cntnapl mutant mice confirming that the ASD-related pathophysiology in mutant mice is driven to a large extent by overactive mTOR signaling. This is one of the first efforts for novel drug discovery in non-syndromic ASD based on a gene target unequivocally linked to disease risk.
- FDG-PET imaging could be utilized as a biomarker in clinical trials utilizing mTOR inhibitors (Thomas et al., 2006).
- FDG-microPET/MRI imaging in Cntnap2 mutant mice demonstrated that mutant mice exhibit a pattern of metabolic activity alterations predominated by cortical and subcortical hypermetabolism.
- mTOR overactivation has been shown to lead to an altered balance of excitatory and inhibitory synaptic transmission which, in turn, has been associated with hippocampal hyperexcitability (Bateup et al., 2013) and impaired cellular information processing and behavioral deficits consistent with ASD-associated phenotypes (Yizhar et al., 2011 ; Gkogkas et al., 2013).
- Examination of the frontal cortex utilizing in vivo transcranial twophoton microscopy revealed an increase in elimination and decrease in formation of dendritic spines. Consistently, it was recently demonstrated that RNAi-mediated knockdown of Cntnap2 leads to abnormalities in spine development in pyramidal neurons (Anderson et al., 2012).
- Electron microscopy of the frontal cortex revealed a highly selective decrease in length of perforated PSDs.
- Perforated synapses are characterized by a discontinuity in the postsynaptic density, resulting in a hole, a slit or a complete segmentation of the postsynaptic density plate. This is thought to reflect a structural correlate of enhanced efficacy of synaptic transmission, which is believed to underlie learning (Morrison & Baxter, 2012).
- excitatory synapses in the frontal cortex are further characterized by a specific pattern of alterations in glutamate receptor subunit composition, including an increase in expression of GluRl, and NR1 , NR2B and mGluR5 while GluR2 and NR2A were both decreased in Cntnap2 mutants, highly suggestive of synaptic immaturity (Talos et al., 2006; Jantzie 2013).
- the pattern observed here is similar to one described in cortical brain tissue from patients with TSC, where pyramidal cells in tubers demonstrate an increase in GluRl and NR2B expression and a relative GluR2 deficiency (Talos et al., 2008). This suggests that the neuropsychiatric
- Active 15270201.2 mTOR kinase inhibitors will have a comparable therapeutic potential for human subjects with neuropsychiatric disorders who harbor CNTNAP2 mutations.
- CNTNAP2 Mutations in the CNTNAP2 gene may only account for a small fraction of cases, but here we show that the gene is involved in signaling pathways previously implicated in syndromic ASD and therefore having possibly far- reaching effects. Therefore, the study of CNTNAP2 has the potential for a more generalized understanding of disease mechanisms and therapies. Our findings furthered our knowledge of the cellular and neurophysiological consequences of CNTNAP2 deletions and allowed us to identify compounds targeting the mechanisms that lead from gene mutation to disease. Targeting a known genetic variation enables the generation of reliable mouse models that closely mimic the risk alleles, therefore ensuring maximal translational validity.
- Mutant CNTNAP2 mice or wild-type mice were treated for 14 consecutive days with rapamycin (LC Systems) 3mg/kg, Torin2 (Tocris, Liu et al. (2011, 2013)) lOmg/kg, AZD2014 (Chemscene LLC) lOmg/kg or vehicle, and at the conclusion of the study, the mice were sacrificed and cortical samples were evaluated for phosphorylated S6 by Western blot.
- rapamycin LC Systems
- Torin2 Tocris, Liu et al. (2011, 2013)
- AZD2014 Cyhemscene LLC
- Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development. Proc. Natl. Acad. Sci. U S A. 109, 18120-5.
- hippocampal neurons dependence on spike timing, synaptic strength, and postsynaptic cell type. J. Neurosci. 18, 10464-72.
- TAG1 regulates the endocytic trafficking and signaling of the semaphorin3A receptor complex. J. Neurosci. 32, 10370-82.
- the glutamate receptor 2 subunit controls post-synaptic density complexity and spine shape in the dentate gyrus. Eur. J. Neurosci. 27, 315-25.
- Genome-wide copy number variation in epilepsy novel susceptibility loci in idiopathic generalized and focal epilepsies.
- Active 15270201.2 Meltzer, C.C., Adelson, P.D., Brenner, R.P., Crumrine, P. ., Van Cott, A., Schiff, D.P., Townsend, D.W., Scheuer, MX. (2000). Planned ictal FDG PET imaging for localization of extratemporal epileptic foci. Epilepsia 41 ,193-200.
- Nicholson DA et al. Reduction in size of perforated postsynaptic densities in hippocampal axospinous synapses and age-related spatial learning impairments. J Neurosci 24:7648-53 (2004).
- Active 15270201.2 superfamily is localized at the juxtaparanodes of myelinated axons and associates with K+ channels. Neuron 24, 1037-47.
- a common genetic variant in the neurexin superfamily member CNTNAP2 is associated with increased risk for selective mutism and social anxiety-related traits. Biol. Psychiatry 69, 825-31.
- Tan GC et al. Normal variation in fronto-occipital circuitry and cerebellar structure with an autism-associated polymorphism of CNTNAP2. Neuroimage 53: 1030-42 (2010).
- Verkerk AJ, et al. CNTNAP2 is disrupted in a family with Gilles de la Tourette syndrome and obsessive compulsive disorder. Genomics 82:1-9 (2003).
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