Identifying, Targeting, and Exploiting a Common Misfolded, Toxic Conformation of SOD1 in ALS: A Dissertation

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (\u3e170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis

ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 7 (2017): 115, doi:10.1038/s41598-017-00091-1.Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.We are grateful for funding from NIH/NINDS (R01 NS078145, R01 NS090352, and R21 NS091860 to D.A.B., R01 NS066942A and R21 NS096642 to G.M., R01NS023868 and R01NS041170 to S.T.B.), the ALS Therapy Alliance/CVS Pharmacy (to D.A.B. and G.M.) and the ALS Association (to C.F. and J.M.)

Parity-related molecular signatures and breast cancer subtypes by estrogen receptor status

INTRODUCTION: Relationships of parity with breast cancer risk are complex. Parity is associated with decreased risk of postmenopausal hormone receptor–positive breast tumors, but may increase risk for basal-like breast cancers and early-onset tumors. Characterizing parity-related gene expression patterns in normal breast and breast tumor tissues may improve understanding of the biological mechanisms underlying this complex pattern of risk. METHODS: We developed a parity signature by analyzing microRNA microarray data from 130 reduction mammoplasty (RM) patients (54 nulliparous and 76 parous). This parity signature, together with published parity signatures, was evaluated in gene expression data from 150 paired tumors and adjacent benign breast tissues from the Polish Breast Cancer Study, both overall and by tumor estrogen receptor (ER) status. RESULTS: We identified 251 genes significantly upregulated by parity status in RM patients (parous versus nulliparous; false discovery rate = 0.008), including genes in immune, inflammation and wound response pathways. This parity signature was significantly enriched in normal and tumor tissues of parous breast cancer patients, specifically in ER-positive tumors. CONCLUSIONS: Our data corroborate epidemiologic data, suggesting that the etiology and pathogenesis of breast cancers vary by ER status, which may have implications for developing prevention strategies for these tumors

Environment And Genetics in Lung cancer Etiology (EAGLE) study: An integrative population-based case-control study of lung cancer

Background: Lung cancer is the leading cause of cancer mortality worldwide. Tobacco smoking is its primary cause, and yet the precise molecular alterations induced by smoking in lung tissue that lead to lung cancer and impact survival have remained obscure. A new framework of research is needed to address the challenges offered by this complex disease. Methods/Design: We designed a large population-based case-control study that combines a traditional molecular epidemiology design with a more integrative approach to investigate the dynamic process that begins with smoking initiation, proceeds through dependency/smoking persistence, continues with lung cancer development and ends with progression to disseminated disease or response to therapy and survival. The study allows the integration of data from multiple sources in the same subjects (risk factors, germline variation, genomic alterations in tumors, and clinical endpoints) to tackle the disease etiology from different angles. Before beginning the study, we conducted a phone survey and pilot investigations to identify the best approach to ensure an acceptable participation in the study from cases and controls. Between 2002 and 2005, we enrolled 2101 incident primary lung cancer cases and 2120 population controls, with 86.6% and 72.4% participation rate, respectively, from a catchment area including 216 municipalities in the Lombardy region of Italy. Lung cancer cases were enrolled in 13 hospitals and population controls were randomly sampled from the area to match the cases by age, gender and residence. Detailed epidemiological information and biospecimens were collected from each participant, and clinical data and tissue specimens from the cases. Collection of follow-up data on treatment and survival is ongoing. Discussion: EAGLE is a new population-based case-control study that explores the full spectrum of lung cancer etiology, from smoking addiction to lung cancer outcome, through examination of epidemiological, molecular, and clinical data. We have provided a detailed description of the study design, field activities, management, and opportunities for research following this integrative approach, which allows a sharper and more comprehensive vision of the complex nature of this disease. The study is poised to accelerate the emergence of new preventive and therapeutic strategies with potentially enormous impact on public health

Chronic Obstructive Pulmonary Disease and Altered Risk of Lung Cancer in a Population-Based Case-Control Study

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has been consistently associated with increased risk of lung cancer. However, previous studies have had limited ability to determine whether the association is due to smoking. METHODOLOGY/PRINCIPAL FINDINGS: The Environment And Genetics in Lung cancer Etiology (EAGLE) population-based case-control study recruited 2100 cases and 2120 controls, of whom 1934 cases and 2108 controls reported about diagnosis of chronic bronchitis, emphysema, COPD (chronic bronchitis and/or emphysema), or asthma more than 1 year before enrollment. We estimated odds ratios (OR) and 95% confidence intervals (CI) using logistic regression. After adjustment for smoking, other previous lung diseases, and study design variables, lung cancer risk was elevated among individuals with a history of chronic bronchitis (OR = 2.0, 95% CI = 1.5-2.5), emphysema (OR = 1.9, 95% CI = 1.4-2.8), or COPD (OR = 2.5, 95% CI = 2.0-3.1). Among current smokers, association between chronic bronchitis and lung cancer was strongest among lighter smokers. Asthma was associated with a decreased risk of lung cancer in males (OR = 0.48, 95% CI = 0.30-0.78). CONCLUSIONS/SIGNIFICANCE: These results suggest that the associations of personal history of chronic bronchitis, emphysema, and COPD with increased risk of lung cancer are not entirely due to smoking. Inflammatory processes may both contribute to COPD and be important for lung carcinogenesis

Detectable clonal mosaicism and its relationship to aging and cancer

In an analysis of 31,717 cancer cases and 26,136 cancer-free controls from 13 genome-wide association studies, we observed large chromosomal abnormalities in a subset of clones in DNA obtained from blood or buccal samples. We observed mosaic abnormalities, either aneuploidy or copy-neutral loss of heterozygosity, of >2 Mb in size in autosomes of 517 individuals (0.89%), with abnormal cell proportions of between 7% and 95%. In cancer-free individuals, frequency increased with age, from 0.23% under 50 years to 1.91% between 75 and 79 years (P = 4.8 × 10(-8)). Mosaic abnormalities were more frequent in individuals with solid tumors (0.97% versus 0.74% in cancer-free individuals; odds ratio (OR) = 1.25; P = 0.016), with stronger association with cases who had DNA collected before diagnosis or treatment (OR = 1.45; P = 0.0005). Detectable mosaicism was also more common in individuals for whom DNA was collected at least 1 year before diagnosis with leukemia compared to cancer-free individuals (OR = 35.4; P = 3.8 × 10(-11)). These findings underscore the time-dependent nature of somatic events in the etiology of cancer and potentially other late-onset diseases

An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s) of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1) protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS

Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage

Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue