STUDIES OF OXIDATIVE DAMAGE, BRAIN PROTEOME, AND NEUROCHEMICAL METABOLITES IN COGNITIVE AND NEURODEGENERATIVE DISORDERS: (1) CHEMOTHERAPY-INDUCED COGNITIVE IMPAIRMENT; (2) PARKINSON DISEASE RAT MODEL

The rate of cancer patients is increasing as the development of science and technology. Twenty million cancer survivors are estimated living in the United States by 2025. However, many cancer survivors show cognitive dysfunction, negatively affecting the quality of life. These cognitive impairments are recognized as chemotherapy-induced cognitive impairment (CICI), also called chemo brain by cancer survivors, including the diminished ability of memory and learning, hard to concentrate and focus, as well as diminution of executive function and processing speed. The etiologies and pathologies of CICI are complicated, especially in most cases the anti-cancer drug cannot cross the blood-brain barrier (BBB). One of the significant candidate mechanisms underlying CICI is chemotherapy-induced, oxidative damage-mediated tumor necrosis factor-alpha (TNF-a) elevation. One of the prototypes of reactive oxygen species (ROS)-generating chemotherapeutic agents is Doxorubicin, normally used as part of multi-drug chemotherapeutic regimens to treat solid tumors and lymphomas. In this dissertation, TNF-a null (TNFKO) mice were used to investigate the role of TNF-a in Dox-induced, oxidative damage-mediated alterations in brain. Dox-induced oxidative damage in brain is ameliorated and brain mitochondrial function is preserved in brains of TNFKO mice. Both Dox-decreased levels of hippocampal choline-containing compounds and activities of brain phospholipases are partially protected in the TNFKO group. It is shown in this dissertation that Dox-targeted mitochondrial damage and levels of brain choline-containing metabolites, as well as changes in the activity of phospholipases, including both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD), are decreased in the CNS and associated with oxidative damage mediated by TNF-a. The results are discussed with respect to identifying a potential therapeutic target to protect against cognitive problems after chemotherapy and thereby improve the quality of life of cancer survivors. We also tested the effect of a chemotherapy drug adjuvant, 2-mercaptoethane sulfonate sodium (MESNA), on CICI in this dissertation research. MESNA ameliorated Dox-induced oxidative protein damage in plasma and led to decreased oxidative damage in brain. MESNA was demonstrated to rescue the memory deficits caused by Dox in the novel object recognition test. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. This study is the first evidence for demonstrating the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI. Parkinson Disease (PD) is considered as the second most neurodegenerative disease, associated with aging and gender. Although the detailed mechanisms remain unknown, inflammation and oxidative damage are two main etiological factors of PD. Certain genetic factors have been discovered related to this disease. Thus, using rodent models with relative gene mutations are the main strategies to investigate PD. However, few rodent models showed same clinical and biochemical features of PD. PTEN-induced putative kinase -1 (PINK1) knockout (KO) rat is the rodent model used in this dissertation research. The oxidative damage in the brain of PINK1 KO rats, the ventricle sizes, and neurochemical metabolite profiles in these rats as a function of age and gender were measured. Distinct gender- and age-related alterations were found, many consistent with those in PD. The proteome of brain of PINK1 KO rat as a function of age and gender also was studied. Based on the collected data, the suitability of this unique rat as a faithful model of known characteristics of PD with our results is discussed

TDP-43 Liquid-Liquid Phase Separation (LLPS) Deficiency Attenuates Amyloid Beta Deposition in the 5XFAD Transgenic Mouse Model of Alzheimer\u27s Disease

Background: TAR DNA-binding protein 43 (TDP-43) can be found within the cell nucleus in most tissues and is a fundamental component to protein production, as it works to slice and reconfigure mRNA molecules. Recently, TDP-43 inclusions have been identified as a prevalent proteinopathy in the brains of individuals diagnosed with Alzheimer\u27s Disease (AD). However, despite the growing body of evidence demonstrating the important role of TDP-43 in AD pathogenesis, whether and how TDP-43 proteinopathy and other AD pathological hallmarks interact remain largely unknown. Furthermore, TDP-43 has a high propensity to undergo liquid-liquid phase separation (LLPS), a biological process necessary for the condensation of proteins, nucleic acids, and other biomolecules. Purpose of Research: The correlation between TDP-43 LLPS and AD deposition is an intriguing, yet currently unexplored area of interest. The purpose of this study is to investigate whether and how TDP-43 and its phase separation are involved in amyloid deposition in APP transgenic mice for Alzheimer\u27s Disease. Methods: We crossed our recently generated mice expressing endogenous LLPS-deficient murine TDP-43 with the widely used 5XFAD transgenic mouse model. Different approaches were then performed to assess amyloid deposition and associated neuroinflammation. Results: When compared to 5XFAD mice, 5XFAD mice expressing LLPS-deficient TDP-43 showed significantly reduced amyloid deposition throughout the brain. Neuroinflammation, as evaluated by GFAP and Iba1 expression was also alleviated by LLPS-deficient TDP-43. Conclusion: For the first time, our study demonstrates the likely role TDP-43 LLPS plays in amyloid deposition. And, targeting TDP-43 LLPS may serve as a novel therapeutic approach to Alzheimer\u27s Disease treatment.https://digitalcommons.unmc.edu/surp2021/1036/thumbnail.jp

TDP-43 Phase Separation Does Not Likely Regulate LPS-Induced Neuroinflammation

Immunohistochemistry (IHC) was performed to assess whether Transactive response Deoxyribonucleic acid binding Protein 43 (TDP-43) liquid-liquid phase separation (LLPS) regulates lipopolysaccaride (LPS)-induced neuroinflammation. Quantification and intensity results of glia cells and cytokines indicate that TDP-43 LLPS does not likely regulate LPS-induced neuroinflammation.https://digitalcommons.unmc.edu/surp2021/1060/thumbnail.jp

Doxorubicin-Induced Elevated Oxidative Stress and Neurochemical Alterations in Brain and Cognitive Decline: Protection by MESNA and Insights into Mechanisms of Chemotherapy-Induced Cognitive Impairment ( Chemobrain )

Chemotherapy-induced cognitive impairment (CICI) is now widely recognized as a real and too common complication of cancer chemotherapy experienced by an ever-growing number of cancer survivors. Previously, we reported that doxorubicin (Dox), a prototypical reactive oxygen species (ROS)-producing anti-cancer drug, results in oxidation of plasma proteins, including apolipoprotein A-I (ApoA-I) leading to tumor necrosis factor-alpha (TNF-α)-mediated oxidative stress in plasma and brain. We also reported that co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma. In this study, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. This study also provides further functional and biochemical evidence of the mechanisms of CICI. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits, an effect that was rescued by MESNA. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline-containing compounds assessed by (Cho)/creatine ratios in the hippocampus in mice. To better elucidate a potential mechanism for this MRS observation, we tested the activities of the phospholipase enzymes known to act on phosphatidylcholine (PtdCho), a key component of phospholipid membranes and a source of choline for the neurotransmitter, acetylcholine (ACh). The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox; however, PLD activity was not protected. This study is the first to demonstrate the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine (PCho) levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI

Systems Biology of the qa Gene Cluster in Neurospora crassa

An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa

A functional genomic approach to actionable gene fusions for precision oncology

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize similar to 100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology

Comprehensive molecular characterization of gastric adenocarcinoma

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer