BACKGROUND: Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive. METHODS: Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human and cDNA together with a short hairpin siRNA against (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs. RESULTS: Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres' proliferation and MET/MAPK/AKT signaling. CONCLUSIONS: Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

No description available.

The United States is currently experiencing an epidemic of opioid use, use disorder, and overdose-related deaths. While studies have identified several loci that are associated with opioid use disorder (OUD) risk, the genetic basis for the ability to discontinue opioid use has not been investigated. Furthermore, very few studies have investigated the non-genetic factors that are predictive of opioid cessation or their predictive ability. In this thesis, I studied a novel phenotype–opioid cessation, defined as the time since last use of illicit opioids (< 6 months ago as not cease, >1 year ago as cease) among persons meeting lifetime DSM-5 criteria for opioid use disorder (OUD). In chapter two, I identified novel genetic variants and biological pathways that potentially regulate opioid cessation success through a genome wide study, as well as genetic overlap between opioid cessation and other substance cessation traits. In chapter three, I identified multiple non-genetic risk factors specific to each racial group that are predictive of opioid cessation from the same individuals analyzed in chapter two by applying several linear and non-linear machine learning techniques to a set of more than 3,000 variables assessed by a structured psychiatric interview. Factors identified from this atheoretical approach can be grouped into opioid use activities, other drug use, health conditions, and demographics, while the predictive accuracy as high as nearly 80% was achieved. The findings from this research generated more hypotheses for future studies to reference. In chapter four, I performed differential gene expression and network analysis on mice with different oxycodone (an opioid receptor agonist)-induced behaviors and compared the significantly associated genes and network modules with top-ranked genes identified in humans. The pathway cross-talks and gene homologs identified from both species illuminate the potential molecular mechanism of opioid behaviors. In summary, this thesis utilized statistical genetics, machine learning, and a computational biology framework to address factors that are associative with opioid cessation in humans, and cross-referenced the genetic findings in a mouse model. These findings serve as references for future studies and provide a framework for personalizing the treatment of OUD.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic (DA) neurons and the formation of Lewy bodies. Despite most cases being idiopathic, mutations in several genes have been implicated in familial forms of PD. In particular, recessive mutations in Parkin gene (PARK2) are the most common cause of young-onset inherited parkinsonism. Parkin is an E3 ubiquitin ligase involved both in the control of mitochondrial turnover and in the proteasome-dependent degradation of proteins, two pathways that have been causally linked to PD development. Although initially described as a recessive disorder, experimental evidence suggests that heterozygous Parkin mutations can exert dominant toxic effects causing neurodegeneration. In 2012, Ruffmann and colleagues identified the first pure heterozygous R275W Parkin patient with clinical features of typical late-onset PD and a diffuse Lewy body pathology. To assess the impact of R275W Parkin, we generated the first mouse line carrying Parkin R274W mutation, which corresponds to the human R275W substitution. Unlike Parkin deficient mouse models, both homo- and heterozygous R274W mice show an age-related motor impairment, degeneration of dopaminergic neurons and neuroinflammation. We detected structural and functional mitochondrial abnormalities related to PARIS-PGC-1α axis impairment in R274W+/+ mice brain and skeletal muscle. Strikingly, we noticed signs of protein aggregation in both R274W+/- and +/+ mice, while we identified bona fide Lewy bodies only in the midbrain of heterozygous mice. Additionally, in the brains of R274W mice we discovered overt abnormalities of the glymphatic system, the main route for brain waste clearance. Our preliminary observations suggest that Parkin influences aquaporin-4 (AQP4) localization. Altogether, our data suggest that R274W Parkin substitution behaves both as a loss ofand a gain of toxic function, highlighting a link between Parkin dominant toxicity and age-dependent motor impairment, neuroinflammation, DA neurons loss, glymphatic system dysfunctions and α-synuclein aggregation in vivo. Hence, our study provides a new robust mouse model to explore PD pathogenesis and glymphatic dysfunctions, offering the possibility to test novel therapeutic strategies with great predictivity.

Advanced liver fibrosis in chronic hepatitis C infection (HCV) is associated with a generalized impaired immune system. Many immune cells are affected in chronic liver disease, including CD8+ T cells. The Crawley lab reported CD8+ T cell hyperfunction in cirrhotic HCV-infected individuals that persisted after effective antiviral therapy. To evaluate the link between CD8+ T cell dysfunction in advanced fibrosis, we adapted a hepatotoxic carbon tetrachloride (CCl4) murine model. We consistently observed severe fibrosis in CCl4-treated mice resembling fibrosis in chronic HCV infected individuals. After stimulation of PBMC, the proportion of granzyme B+, and IFN-γ+ CD8+ T cells in fibrotic mice was significantly higher than the controls, particularly naïve and central memory CD8+ T cells. This state of hyperfunction was sustained after liver insult removal and significant fibrosis regression to near normal tissue integrity. Sex differences were also studied in this model and were apparent after prolonged exposure to CCl4 and in the capacity to repair liver fibrosis. Following an ectopic challenge with cancer cells, tumor growth was significantly greater in fibrotic mice. Moreover, the response to immunotherapy was significantly delayed in CCl4-treated mice. In summary, we reported for the first time that circulating CD8+ T cells are hyperfunctional in a murine model of advanced liver fibrosis in response to a hepatotoxin. In this context, affected mice failed to control the growth of a tumor whose growth is known to be controlled by a robust CD8+ T cell response. In addition, the reduced responses to immunotherapeutic effects suggest deficiencies in antigen-specific CD8+ T cell responses. Therefore, this animal model might be useful to identify mechanistic targets with translational potential for immune restoring treatments in human chronic liver diseases with advanced liver fibrosis.

No description available.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality around the world. It has multiple causative factors including sports injuries, vehicular accidents, war, and other forms of trauma. Though patients can recover, it has the potential to cause mild to severe persistent cognitive deficits. Medical treatment involves treating individual problems as they arise; this treatment is based upon clinical signs. Developing a standard of care for TBI is complex due to the difficulty in finding common cell and molecular changes in TBI variants that can be prevented or ameliorated. Tumor necrosis factor (TNF) is a prominent inflammatory cytokine present in all forms of traumatic brain injury. It is the target of multiple therapies in other disease processes. As TNF inhibitors lead to billions of dollars in worldwide sales, their use in neuropathologies is an active research area. XPro1595, a preclinical drug developed by Xencor, uniquely inhibits more than 99% of soluble TNF. However, there is only one published study to date on the effects of XPro1595 in any model of traumatic brain injury. The purpose of this study was to characterize the presence of TNF and the proinflammatory TNFR1 pathway in a controlled cortical impact (CCI) mouse model of TBI and to determine if XPro1595 could improve behavioral and neuropathological outcomes. TNF and the TNFR1 pathway have shown to be chronically present in a CCI mouse model for at least two weeks. Injured animals treated with one course of the drug did not show any improvements in spatial learning or memory. However, decreased activity in the TNFR1 pathway and changes in glial markers indicated that XPro1595 lessened neuroinflammation via this mechanism. This study suggests potential benefits of XPro1595 in TBI that could lead to a common standard of care.

As the most common motor neuron disease, Amyotrophic lateral sclerosis (ALS) affects around 4 in every 100,000 people worldwide with reports of increasing prevalence over the years. Characterized by progressive degeneration of motor neurons, ALS patients suffer impairments of movement and typically die from respiratory failure 2-5 years after diagnosis. Curiously, ALS exists on a disease continuum with Frontotemporal Dementia (FTD) where 30-50% of patients will be diagnosed with both diseases. In FTD, degeneration of cortical neurons results in diverse behavioural changes including deficits in executive and social skills as well as language and memory. A central connection between ALS and FTD is TDP-43 (encoded by TARDBP), an essential DNA/RNA binding protein known to serve critical functions in numerous cellular processes. Despite mutations in TARDBP constituting a small percentage of familial cases, TDP 43 nuclear-to-cytoplasmic mislocalization is a pathological hallmark of most ALS-FTD cases. Therefore, therapeutic targets to rectify pathology and disease may be uncovered by identifying factors that regulate TDP-43. While it is currently established TDP-43 is ubiquitinated and phosphorylated in diseased states, our lab recently found TDP-43 is SUMOylated in response to stress. Of note, perturbations in the stress response are becoming increasingly implicated in neurodegenerations. Furthermore, TDP-43 plays critical roles in the stress response which become perturbed in ALS/FTD. We developed a TDP-43 "SUMO dead" mouse allele to gain an understanding of how disrupting this may contribute to the pathogenesis of ALS-FTD. Longitudinal characterization of the model explored behavioural and histological in vivo consequences following loss of TDP-43 SUMOylation. However, the phenotypes observed in the mutant mice were less robust in comparison to established ALS/FTD mouse models. Mutant mice did not have consistent differences in tests for similar outcomes, trials of the same test, or across age. Female mutant mice presented with early hyperactivity and disinhibition along with altered social grooming behaviour. At later age, these female mice developed impairments in spatial working memory. Male mice developed apathetic behaviour and motor deficits at the middle age timepoint. Histologically, various forms of pathological TDP-43 were observed in the absence of neurodegeneration. These data reveal that TDP-43 SUMOylation may play an important role in ALS/FTD pathogenesis.

Leptin and its receptor are essential for regulating food intake, energy expenditure, glucose homeostasis and fertility. Mutations within leptin or the leptin receptor cause early-onset obesity and hyperphagia, as described in human and animal models. The effect of both heterozygous and homozygous variants is much more investigated than compound heterozygous ones. Recently, we discovered a spontaneous compound heterozygous mutation within the leptin receptor, resulting in a considerably more obese phenotype than described for the homozygous leptin receptor deficient mice. Accordingly, we focus on compound heterozygous mutations of the leptin receptor and their effects on health, as well as possible therapy options in human and animal models in this review.

No description available.