Browsing by Subject "Mouse models"

Gliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in IDH1 and IDH2 as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study.

IDH1/2 mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to α-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant IDH1 or mutant IDH2. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant IDH1/2 .

Having observed mutations in IDH1/2 in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in IDH1/2, lending less credence to the first hypothesis. To elucidate whether mutations in IDH1/2 can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant IDH2 in different populations of cells in the brain, either alone or in combination with TP53 deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant IDH2 developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both IDH2 mutation and TP53 deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant IDH2 and the cell-of-origin for progressive gliomas.

The RAS family is a group of small GTPases that can become constitutively activated by point mutations that are found in about 30% of all cancer patients. There are three well-characterized RAS family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains, but have divergent C-terminal membrane-binding regions that impart different subcellular localization and subtle differences in signaling. Although the role of constitutively activated oncogenic RAS has been well established to play a role in cancer, recent work has suggested that wild-type RAS signaling may also be important in tumorigenesis. Wild-type RAS proteins have been shown to be activated in the presence of oncogenic KRAS. However, the consequences of this activation are context-dependent, as signaling through the wild-type RAS proteins has been shown to both suppress neoplastic growth and promote tumorigenesis under different circumstances.

I sought to investigate the role of the wild-type RAS proteins in two clinically –relevant models of cancer: pancreatic, the type of cancer most frequently associated with KRAS mutations, and lung cancer, the cancer in which KRAS mutations affect the highest number of patients. First, I tested whether a loss of wild type Hras altered tumorigenesis in a mouse model of pancreatic cancer driven by oncogenic Kras. Hras homozygous null mice (Hras-/- ) exhibited more precancerous lesions of the pancreas as well as more off-target skin papillomas compared to their wild type counterparts, indicating that Hras suppresses early Kras-driven pancreatic tumorigenesis. Loss of Hras also reduced the survival of mice engineered to develop aggressive pancreatic cancer by the additional disruption of one allele of the tumor suppressor p53 (Trp53R172H/+). However, this survival advantage was lost when both alleles of Trp53 were mutated, suggesting that wild-type HRas inhibits tumorigenesis in a p53-dependant manner.

Next, I investigated the role that wild-type Hras and Nras play in a chemical carcinogen-induced model of lung cancer. In mice treated with urethane, a carcinogen that induces Kras-mutation positive lung lesions, Hras-/ mice once again developed more tumors than wild-type mice. Interestingly, however, this effect was not observed in mice lacking wild-type Nras. Mice lacking both Hras and Nras alleles developed approximately the same number of tumors as Hras-/- mice, thus the additional loss of Nras does not appear to enhance the tumor-promoting effects of loss of Hras. In summary, signaling through wild-type Hras, but not Nras, suppresses tumorigenesis in a carcinogen-induced model of lung cancer.

The tumor-suppressive effects of wild-type Ras signaling were traced to the earliest stages of pancreatic tumorigenesis, suggesting that wild-type Ras signaling may suppress tumorigenesis as early as the time of initiation. These findings suggest that differences in expression of the wild-type Ras isoforms could potentially play a role in an individual’s predisposition for developing cancer upon oncogenic insult.

Radiation therapy can cause acute toxicity and long-term side effects in normal tissues. Because part of the acute toxicity of radiation is due to p53-mediated apoptosis, blocking p53 during irradiation can protect some normal tissues from acute radiation injury and might improve the therapeutic ratio of radiation therapy. However, the mechanisms by which p53 regulates late effects of radiation are not well understood. Here, I utilized genetically engineered mouse models to dissect the role of p53 in regulating two of the most clinically significant late effects of radiation: radiation-induced carcinogenesis and radiation-induced myocardial injury.

It has been well characterized that mice with one allele of p53 permanently deleted are sensitized to radiation-induced cancer. Therefore, temporary inhibition of blocking p53 during irradiation could promote malignant transformation. Experiments with mice lacking functional p53 in which p53 protein can be temporarily restored during total-body irradiation (TBI) suggest that the radiation-induced p53 response does not contribute to p53-mediated tumor suppression. Here, I performed reciprocal experiments and temporarily turned p53 off during TBI using transgenic mice with reversible RNA interference against p53. I found that temporary knockdown of p53 during TBI not only ameliorated acute hematopoietic toxicity, but in both Kras wild-type and tumor-prone KrasLA1 mice also prevented lymphoma development. Mechanistic studies show that p53 knockdown during TBI improves survival of hematopoietic stem and progenitor cells (HSPCs), which maintains HSPC quiescence and prevents accelerated repopulation of surviving cells. Moreover, using an in vivo competition assay I found that temporary knockdown of p53 during TBI maintains the fitness of p53 wild-type HSPCs to prevent the expansion of irradiated mutant cells. Taken together, our data demonstrate that p53 functions during TBI to promote lymphoma formation by facilitating the expansion of irradiated HSPCs with adaptive mutations.

p53 functions in the heart to promote myocardial injury after multiple types of stress, including ischemic injury, pressure overload and doxorubicin-induced oxidative stress. However, how p53 regulates radiation-induced myocardial injury, which develops after radiation therapy, is not well understood. Here, I utilized the Cre-loxP system to demonstrate that p53 functions in endothelial cells to protect mice from myocardial injury after a single dose of 12 Gy or 10 daily fractions of 3 Gy whole-heart irradiation (WHI). Mice in which both alleles of p53 are deleted in endothelial cells succumbed to heart failure after WHI due to myocardial necrosis, systolic dysfunction and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density. Mechanistic studies using primary cardiac endothelial cells (CECs) irradiated in vitro indicate that p53 signals to cause a mitotic arrest and protects CECs against radiation-induced mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, are also sensitized to myocardial injury after 12 Gy WHI. Together, our results demonstrate that the p53/p21 axis functions to prevent radiation-induced myocardial injury in mice. Our findings raise the possibility that when combining radiation therapy with inhibitors of p53 or other components of the DNA damage response that regulate mitotic arrest, patients may experience increased radiation-related heart disease.

Taken together, our results demonstrate crucial but distinct roles of p53 in regulating late effects of radiation: p53-mediated apoptosis promotes radiation-induced lymphomagenesis, but p53-mediated cell cycle arrest prevents radiation-induced myocardial injury. These findings indicate that p53 may generally play a protective role from radiation, particularly at high doses, in cells where p53 activation is uncoupled from the induction of the intrinsic pathway of apoptosis. Therefore, selectively inhibiting p53-mediated apoptosis may be a promising approach to ameliorate acute radiation toxicity without exacerbating late effects of radiation.

Developmental disabilities, including intellectual disability (ID), attention-deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD), affect approximately 1 in 6 children in the United States. Attempts to produce treatment for developmental disabilities have been hampered by our current lack of understanding of the molecular mechanisms underlying these disorders. Advancements in genome sequencing and animal modeling technologies have proven to be an invaluable resource in the elucidation of potential disease mechanisms, with recent studies reporting novel mutations of the Ptchd1 and Shank3 genes in patients with developmental disabilities. Though these two genes have been proposed to play important roles in neural development, their function in the normal brain and defective behavioral output are poorly understood.

In this dissertation, I characterize the circuit and behavioral dysfunction of the genetically-engineered Ptchd1 and Shank3 knockout mice. With respect to Ptchd1, I found that expression is developmentally enriched in the thalamic reticular nucleus (TRN), which is a group of GABAergic neurons serving as the major source of inhibition for thalamo-cortical neurons. Slice and in vivo electrophysiological experiments revealed that deletion of this gene in mice disrupts SK2 currents and burst firing mechanisms in the TRN, a region that has previously been shown to play an important role in sleep, attention, and cognition. Consistent with clinical findings, Ptchd1 knockout mice display behavioral phenotypes indicative of hyperactivity, attention deficits, motor dysfunction, hyperaggression, and cognitive impairment. Interestingly, attention-like deficits and hyperactivity are rescued by SK2 pharmacological enhancement, suggesting a potential molecular target for developing treatment.

Shank3 knockout mice display ASD-like phenotypes, including social interaction deficits and repetitive behaviors. In addition, biochemical, electrophysiological, and morphological abnormalities were discovered in the medium spiny neurons (MSNs) of these mice. However, the exact neural circuits and cell types responsible for the autistic-like behaviors have not been identified. To address this important question, I developed a new conditional Shank3 knockout mouse. Importantly, the behavioral abnormalities reported in the original Shank3 knockout mice were recapitulated in this novel conditional Shank3 knockout mouse, indicating that this mouse may be useful for future pathway-specific dissections of ASD-like behaviors. Together, these two sets of studies not only provide mouse models for dissecting the function of PTCHD1and SHANK3 in normal and abnormal neural development, but also demonstrate a critical role for PTCHD1 in TRN neurons and SHANK3 in MSN cells and in the case of PTCHD1, identify potential cellular and circuit pathway targets for much-needed pharmacological intervention.

Numerous mouse models of autism spectrum disorder (ASD) have been generated to determine the molecular and circuit mechanisms underlying the condition. In this dissertation, I characterize the behavior of two mouse models mimicking some of the most common mutations found in human patients, which is an important step in establishing them as models.

One important application of mouse models is our ability to use them to test specific treatments in a preclinical setting. These treatments are primarily pharmacological in nature, but some work has also been done to test the effects of the environment on the presentation of phenotypes. In particular, environmental enrichment has been shown to prevent the manifestation of ASD-like behaviors in several different mouse models of the disorder. In this dissertation, I tested the effects of environmental enrichment on our Shank3 mouse model of ASD and found that it did not rescue any of the behavioral phenotypes we previously observed, but rather exacerbated some common comorbidities.

More recently, methods have been developed to manipulate gene expression in mice over space and time and have been utilized to gain a clearer picture of the cell types, circuits, brain regions, and developmental time periods involved in autism spectrum disorder. I utilized conditional knockout technology to test whether behavioral phenotypes associated with ASD could be induced by deleting Shank3 in adult and developing mice. We found that Shank3 may play a role in development and that inducing mutations in adult mice is insufficient to cause ASD-like behaviors. Unfortunately, technical concerns limit our interpretation of the data, and this study adds to the growing concern of the limitations inherent in this technology.

Autism spectrum disorders (ASDs) are increasingly prevalent, and the costs associated with caring for affected patients across the lifespan are immense. However, the pathophysiology and brain regions involved in characteristic behavioral impairments remain poorly defined, which hinders progress towards targeted therapeutic development. Different brain regions have been suggested from human neuroimaging studies but the circuit mechanism is not known and cannot be easily defined in human studies. Genetic studies indicate that SHANK3, a gene encoding a scaffolding protein at the postsynaptic density, is a strong ASD causative gene. Studies of various isoform-specific knockout mice support these mice as valid models to dissect the pathophysiology of ASDs and implicate differential involvement of brain regions such as hippocampus and striatum. However, none of these mice recapitulate the most frequent SHANK-related mutation found in ASD patients: a deletion of the entire SHANK3 gene.

For this reason, we have created conventional complete knockout mice by deleting almost the entire coding region of exons 4 to 22, Shank3 Δe4-22, and performed a thorough characterization of their behavioral phenotypes. Their abnormalities in complex social and communication behaviors in addition to their profound display of repetitive and restrictive behaviors in combination with comorbid anxiety, locomotor, and learning phenotypes support them as a mouse model for SHANK3-causing autism with good construct and face validity. Additional studies by collaborators identified a striatal-centered model of circuit and synaptic dysfunction. Manipulation of metabotropic glutamate receptor 5 (mGluR5) activity attenuated the excessive grooming and instrumental learning differentially in Δe4-22-/- mice. These findings show that deficiency of the autism-associated Shank3 gene can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities which underlie deficits in learning and ASD-like behaviors. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs.

However, because these and other existing Shank3 mutant mice are not region specific, causality between different brain regions and ASD-like behaviors cannot be firmly established. In order to define anatomic regions implicated in behavioral manifestations of ASD, conditional knockout mice lacking Shank3 proteins in different brain regions including forebrain excitatory neurons (NEX-Cre) and striatal inhibitory neurons (Dlx5/6-Cre), as well as distinct cell populations including direct (D1-Cre) and indirect (D2-Cre) medium spiny neurons, were generated and subjected to behavioral phenotyping. Different autism-relevant behaviors as well as comorbid behaviors were recapitulated by targeting Shank3 deletion to different brain regions or cell types. Electrophysiological and biochemical studies further identified synaptic defects resulting from region- or cell-autonomous loss of Shank3, with different biochemical pathways implicated when Shank3 deletion was targeted to the cortex and hippocampus versus the basal ganglia. This study demonstrates the impact of specific brain regions in modulating ASD-related behavior and identifies key molecular defects which are restricted to specific brain regions in SHANK3-deficient ASD, thus providing future therapeutic targets.

Lastly, as one of the major advantages of modeling ASDs in mice is their amenability for pre-clinical studies of interventions, we have tested two different cellular therapies hypothesized to modulate neuronal circuit function either through direct differentiation of stem cells into brain cells including neurons, glia, and microglia or through indirect effects on neuro-immune modulation. While neither perinatal nor young adulthood treatment with human umbilical cord blood derived stem cells affected significant improvements in the behaviors of Shank3 knockout mice, these experiments underscored the robust, reliable behavioral phenotypes of this animal model as well as supported the safety and tolerability of these treatments in a rodent pre-clinical model with a relevant genetic construct.