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Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere–DDR–ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells’ senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T B NK SCID pigs through site directed CRISPR/Cas9 mutagenesis of within a naturally occurring genetic background. We confirmed pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed injections of cultured human CD34 selected cord blood cells into the fetal SCID pigs. At birth, human CD45 CD3ε cells were detected in cord and peripheral blood of injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

High-resolution karyotyping detects cytogenetic anomalies in 5-10% of cases of autism. Karyotyping, however, may fail to detect abnormalities of chromosome subtelomeres, which are gene rich regions prone to anomalies. We assessed whether panels of FISH probes targeted for subtelomeres could detect abnormalities beyond those identified by karyotyping in 104 individuals with Pervasive Developmental Disorders (PDDs) drawn from a general clinical population. Four anomalies were detected by karyotyping, while no additional anomalies were detected by subtelomere FISH or by probes targeted for 15q11.2q13 or 22q11.2 in subgroups of our sample. We conclude that while karyotyping may be more broadly indicated for autism than previously supposed, subtelomere FISH appears less likely to be a useful screening tool for unselected PDD populations.

Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (∼50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

In efforts to evaluate potential biomarkers and drug targets for Neurofibromatosis Type I (NF1) we utilized affinity mass spectrometry and global proteomics to investigate how variation within and loss of NF1 affect immortalized human Schwann cells. We used Strep tagged m Nf1 cDNAs (both wild type (WT) and variant) to affinity purity NF1 Protein-Protein interactors (PPIs) from the Schwann cells. We were able to identify 98 PPIs and show that some of these PPIs bind differentially to variant proteins. Next, we evaluated global proteomes. We identified over 1900 proteins in immortalized human Schwann cells both with and without NF1 expression. We identified 148 proteins with differential expression levels based on genotype. Following Ingenuity Pathway analysis (IPA) we found multiple pathways were altered including decreases in “oxidative phosphorylation,” increases in “mitochondrial dysfunction”, and “glycolysis”, as well as changes in “Myelination Signaling Pathway.” When we evaluated the proteome of NF1 null cells stably transfected with tagged m Nf1 cDNAs we again identified an overall trend of metabolic differences pertaining to “oxidative phosphorylation”, “mitochondria dysfunction”, and “glycolysis” in the variant cDNA expressing cells. We then validated differential expression of the following proteins: LAMC1, CYB5R3, and SOD2 that are observed in the altered pathways. Finally, consistent with our proteomics findings, we show that NF1 is required to maintain mitochondrial respiratory function in Schwann cells by stabilizing NADH-linked oxidative phosphorylation and electron transfer. Taken together, these data indicate that NF1 plays a significant role in mitochondrial metabolism that results in proteomic changes in Schwann cells and may serve as a future drug target.

Aims: Norway is experiencing a rising life expectancy combined with an increasing dependency ratio – the ratio of those outside over those within the working force. To provide data relevant for future health policy we wanted to study trends in total and healthy life expectancy in a Norwegian population over three decades (1980s, 1990s and 2000s), both overall and across gender and educational groups. Methods: Data were obtained from the HUNT Study, and the Norwegian Educational Database. We calculated total life expectancy and used the Sullivan method to calculate healthy life expectancies based on self-rated health and self-reported longstanding limiting illness. The change in health expectancies was decomposed into mortality and disability effects. Results: During three consecutive decades we found an increase in life expectancy for 30-year-olds (~7 years) and expected lifetime in self-rated good health (~6 years), but time without longstanding limiting illness increased less (1.5 years). Women could expect to live longer than men, but the extra life years for females were spent in poor self-rated health and with longstanding limiting illness. Differences in total life expectancy between educational groups decreased, whereas differences in expected lifetime in self-rated good health and lifetime without longstanding limiting illness increased. Conclusions: The increase in total life expectancy was accompanied by an increasing number of years spent in good self-rated health but more years with longstanding limiting illness. This suggests increasing health care needs for people with chronic diseases, given an increasing number of elderly. Socioeconomic health inequalities remain a challenge for increasing pensioning age.

Little is known about the individual factors that predict outcomes in Internet-administered psychological treatments. We hypothesized that greater cognitive flexibility (i.e. the ability to simultaneously consider several concepts and tasks and switch effortlessly between them in response to changes in environmental contingencies) would provide a better foundation for learning and employing the cognitive restructuring techniques taught and exercised in therapy, leading to greater treatment gains. Participants in three trials featuring Internet-administered psychological treatments for depression (n = 36), social anxiety disorder (n = 115) and tinnitus (n = 53) completed the 64-card Wisconsin Card Sorting Test (WCST) prior to treatment. We found no significant associations between perseverative errors on the WCST and treatment gains in any group. We also found low accuracy in the classification of treatment responders. We conclude that lower cognitive flexibility, as captured by perseverative errors on the WCST, should not impede successful outcomes in Internet-delivered psychological treatments.