CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Chd8 haploinsufficiency impairs early brain development and protein homeostasis later in life.

BACKGROUND: Chromodomain helicase DNA-binding protein 8 (Chd8) is a high-confidence risk gene for autism spectrum disorder (ASD). However, how Chd8 haploinsufficiency impairs gene expression in the brain and impacts behavior at different stages of life is unknown. METHODS: We generated a mutant mouse line with an ASD-linked loss-of-function mutation in Chd8 (V986*; stop codon mutation). We examined the behavior of Chd8 mutant mice along with transcriptional changes in the cerebral cortex as a function of age, with a focus on one embryonic (E14.5) and three postnatal ages (1, 6, and 12 months). RESULTS: Chd8V986*/+ mutant mice displayed macrocephaly, reduced rearing responses and reduced center time in the open field, and enhanced social novelty preference. Behavioral phenotypes were more evident in Chd8V986*/+ mutant mice at 1 year of age. Pup survival was reduced in wild-type x Chd8V986*/+ crosses when the mutant parent was female. Transcriptomic analyses indicated that pathways associated with synaptic and neuronal projections and sodium channel activity were reduced in the cortex of embryonic Chd8V986*/+ mice and then equalized relative to wild-type mice in the postnatal period. At 12 months of age, expression of genes associated with endoplasmic reticulum (ER) stress, chaperone-mediated protein folding, and the unfolded protein response (UPR) were reduced in Chd8V986*/+ mice, whereas genes associated with the c-MET signaling pathway were increased in expression. LIMITATIONS: It is unclear whether the transcriptional changes observed with age in Chd8V986*/+ mice reflect a direct effect of CHD8-regulated gene expression, or if CHD8 indirectly affects the expression of UPR/ER stress genes in adult mice as a consequence of neurodevelopmental abnormalities. CONCLUSIONS: Collectively, these data suggest that UPR/ER stress pathways are reduced in the cerebral cortex of aged Chd8V986*/+ mice. Our study uncovers neurodevelopmental and age-related phenotypes in Chd8V986*/+ mice and highlights the importance of controlling for age when studying Chd8 haploinsufficient mice.

PIK3CA missense mutations promote glioblastoma pathogenesis, but do not enhance targeted PI3K inhibition.

BACKGROUND: Glioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent PIK3CA missense mutations (PIK3CAmut) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients. METHODS: We used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two PIK3CAmut from each of 3 mutated domains and induced their expression in NHA with (NHARAS) and without mutant RAS using lentiviral vectors. We then examined the role of PIK3CAmut in gliomagenesis in vitro and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors in vitro. RESULTS: PIK3CAmut, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHARAS in vitro. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHARAS tumorigenesis in vivo. PIK3CAmut status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHARAS harboring ABD or helical mutations. CONCLUSION: PIK3CAmut promoted differential gliomagenesis based on the mutated domain. While PIK3CAmut did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of PIK3CAmut promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. METHODS: We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. RESULTS: PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. CONCLUSION: Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

Genomic profiles of low-grade murine gliomas evolve during progression to glioblastoma.

BACKGROUND: Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. METHODS: We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. RESULTS: Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. CONCLUSION: These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.

Tumor-homing cytotoxic human induced neural stem cells for cancer therapy.

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSCTE), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSCTE in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSCTE that were nestin+ and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSCTE rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSCTE delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSCTE thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSCTE-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSCTE-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSCTE is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.

Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model.

BACKGROUND: Surgical resection is a universal component of glioma therapy. Little is known about the postoperative microenvironment due to limited preclinical models. Thus, we sought to develop a glioma resection and recurrence model in syngeneic immune-competent mice to understand how surgical resection influences tumor biology and the local microenvironment. METHODS: We genetically engineered cells from a murine glioma mouse model to express fluorescent and bioluminescent reporters. Established allografts were resected using image-guided microsurgery. Postoperative tumor recurrence was monitored by serial imaging, and the peritumoral microenvironment was characterized by histopathology and immunohistochemistry. Coculture techniques were used to explore how astrocyte injury influences tumor aggressiveness in vitro. Transcriptome and secretome alterations in injured astrocytes was examined by RNA-seq and Luminex. RESULTS: We found that image-guided resection achieved >90% reduction in tumor volume but failed to prevent both local and distant tumor recurrence. Immunostaining for glial fibrillary acidic protein and nestin showed that resection-induced injury led to temporal and spatial alterations in reactive astrocytes within the peritumoral microenvironment. In vitro, we found that astrocyte injury induced transcriptome and secretome alterations and promoted tumor proliferation, as well as migration. CONCLUSIONS: This study demonstrates a unique syngeneic model of glioma resection and recurrence in immune-competent mice. Furthermore, this model provided insights into the pattern of postsurgical tumor recurrence and changes in the peritumoral microenvironment, as well as the impact of injured astrocytes on glioma growth and invasion. A better understanding of the postsurgical tumor microenvironment will allow development of targeted anticancer agents that improve surgery-mediated effects on tumor biology.

Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation but resistant to temozolomide.

BACKGROUND: Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.

Modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis.

BACKGROUND: Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. METHODS: Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. RESULTS: Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. CONCLUSIONS: These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies.

Selectivity, cocrystal structures, and neuroprotective properties of leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid leucettamine B.

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3ß. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease.

Genetically engineered mouse models of diffuse gliomas.

Over the last decade, genetically engineered mouse models have been extensively used to dissect the genetic requirements for neoplastic initiation and progression of diffuse gliomas. While these models faithfully recapitulate the histopathological features of human gliomas, comparative genomic analyses are increasingly being utilized to comprehensively assess their fidelity to recently identified molecular subtypes of these tumors. Future progress with these models will rely on incorporating insights not only from oncogenomics studies of cancer, but also from the developmental neuroscience and stem cell biology fields to design accurate and experimentally tractable models for use in translational cancer research, particularly for experimental therapeutics studies of molecularly defined subtypes of gliomas.

Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins.

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aß peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.

Inhibition of c-Jun kinase provides neuroprotection in a model of Alzheimer's disease.

The c-Jun N-terminal kinase (JNK) pathway potentially links together the three major pathological hallmarks of Alzheimer's disease (AD): development of amyloid plaques, neurofibrillary tangles, and brain atrophy. As activation of the JNK pathway has been observed in amyloid models of AD in association with peri-plaque regions and neuritic dystrophy, as we confirm here for Tg2576/PS(M146L) transgenic mice, we directly tested whether JNK inhibition could provide neuroprotection in a novel brain slice model for amyloid precursor protein (APP)-induced neurodegeneration. We found that APP/amyloid beta (Abeta)-induced neurodegeneration is blocked by both small molecule and peptide inhibitors of JNK, and provide evidence that this neuroprotection occurs downstream of APP/Abeta production and processing. Our findings demonstrate that Abeta can induce neurodegeneration, at least in part, through the JNK pathway and suggest that inhibition of JNK may be of therapeutic utility in the treatment of AD.

L1 and NCAM adhesion molecules as signaling coreceptors in neuronal migration and process outgrowth.

Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily engage in multiple neuronal interactions that influence cell migration, axonal and dendritic projection, and synaptic targeting. Their downstream signal transduction events specify whether a cell moves or projects axons and dendrites to targets in the brain. Many of the diverse functions of CAMs are brought about through homophilic and heterophilic interactions with other cell surface receptors. An emerging concept is that CAMs act as coreceptors to assist in intracellular signal transduction, and to provide cytoskeletal linkage necessary for cell and growth cone motility. Here we will focus on new discoveries that have revealed novel coreceptor functions for the best-understood CAMs--L1, CHL1, and NCAM--important for neuronal migration and axon guidance. We will also discuss how dysregulation of CAMs may also bear on neuropsychiatric disease and cancer.

A methyl-CpG-binding protein 2-enhanced green fluorescent protein reporter mouse model provides a new tool for studying the neuronal basis of Rett syndrome.

Rett syndrome, a pervasive X-linked neurodevelopmental disorder in young girls, is caused by loss-of-function mutations in the gene that encodes the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2). Mecp2-knockout mice phenocopy the major symptoms found in human patients and have advanced our understanding of the function of MeCP2 and mechanism of Rett syndrome. To study the behavior of the MeCP2 protein in vivo, we have generated a knock-in reporter mouse model that expresses MeCP2-enhanced green fluorescent protein (EGFP) fusion protein instead of endogenous MeCP2. Here we show that expression of the fusion protein in the brain remarkably mirrors endogenous MeCP2 expression in all temporal and spatial aspects. This mouse model may be a valuable tool for studying Rett syndrome and for developing therapies.

Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex.

Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.

Generation and characterization of brain lipid-binding protein promoter-based transgenic mouse models for the study of radial glia.

Radial glia play an essential role in the generation of the cerebral cortex through their function as neuronal precursors and as neuronal migration guides. A molecular marker for radial glia in the developing central nervous system is the brain lipid-binding protein (BLBP). To generate mouse models for the visualization and study of radial glia, we expressed EGFP, EYFP, or dsRed2 in transgenic mice under the control of the BLBP promoter. In these transgenic lines, fluorescent protein expression is restricted to radial glia in the embryonic cortex and to astrocytes in the adult brain. Electroporation of the transgenes into embryonic cortex also resulted in radial glia-specific transgene expression. These BLBP promoter driven transgenic mice and organotypic brain slices expressing different fluorescent markers in a radial glia-specific manner will be useful tools to further study the differentiation and function of radial glia in distinct regions of the developing CNS.

Reelin, integrin and DAB1 interactions during embryonic cerebral cortical development.

Extracellular matrix-like molecule reelin and cell surface adhesion receptors such as alpha3beta1 integrin can regulate neuronal migration and position in the developing cerebral cortex. Here we show that alpha3beta1 integrin binds to the N-terminal region of reelin, a site distinct from the region of reelin shown to associate with other reelin receptors such as VLDLR/ApoER2. Furthermore, Dab1, a member of the reelin signaling pathway, can complex with the cytoplasmic region of beta1 integrin in a reelin-dependent manner. Thus, alpha3beta1 integrin-reelin interactions may contribute to appropriate neuronal placement in the developing cerebral cortex.