Immunosenescence in Choroidal Neovascularization (CNV)—Transcriptional Profiling of Naïve and CNV-Associated Retinal Myeloid Cells during Aging

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

A DNAmCULTURE Epigenetic Fingerprint Recapitulates Physiological Aging

Aging elicits dramatic changes to DNA methylation (DNAm), however the causes and consequences of such alterations to the epigenome remain unclear. The utility of biomarkers of aging based on DNAm patterns would be greatly enhanced if in vitro models existed that recapitulated physiological phenotypes such that modulation could garnish mechanistic insights. Using DNAm from serially passaged mouse embryonic fibroblasts, we developed a marker of culture aging and asked if culture phenotypes, like exhaustive replication, are epigenetically analogous to physiological aging. Our measure, termed DNAmCULTURE, accurately estimated passage number and was shown to strongly increase with age when examined in multiple tissues. Furthermore, we observed epigenetic alterations indicative of early cultured cells in animals undergoing caloric restriction and in lung and kidney fibroblasts re-programmed to iPSCs. This study identifies culture-derived alterations to the methylome as physiologically relevant and implicates culture aging as an important feature in known epigenetic aging phenomena.

Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice

Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss of function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at ~20 weeks. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65-70 weeks, with approximately 40% of male and 75% of female LmnaL648R/L648R having near-normal lifespans of almost 2 years. Starting at ~10 weeks of age, LmnaL648R/L648R mice of both sexes have lower body masses and body fat than controls. By ~20-30 weeks of age, they exhibit detectable cranial, mandibular and dental defects similar to those observed in Zmpste24-/- mice, and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a robust model to study the effects of farnesylated prelamin A during physiological aging.

Clonal Expansion of Tet2 +/- hematopoiesis Is Driven By Inflamm-Ageing Associated IL-1 Increase in Mice

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is defined as the presence of an expanded somatic blood cell clone carrying a mutation in genes that are drivers of hematologic malignancy including DNMT3A, TET2, ASXL1, TP53, JAK2, and SF3B1, at a variant allele frequency (VAF) of at least 2% in the absence of other hematological abnormalities. CHIP has a prevalence of about 10% in the 70-80 year old population, further increases with ageing and associates with an increased risk of hematological malignancies, cardiovascular disease and all-cause mortality (Genovese et al. NEJM 2014; Jaiswal, S. et al. NEJM 2014). Recent studies indicate that higher pre-malignant clonal size and mutational burden increase the chances of malignant transformation in individuals carrying CHIP (Desai, P. Nat. Med. et al., 2018; Abelson, S. et al. Nature, 2018). While age is the best predictor of CHIP development and correlates directly with pre-malignant clonal size, the specific cellular-extrinsic factors promoting CHIP clonal expansion in the context of physiological aging are still unclear. We hypothesized that ageing associated low-grade inflammation (termed "inflamm-ageing") is a driver of CHIP clonal expansion. We used standard bone marrow (BM) chimera models and developed a novel, irradiation independent, hematopoietic specific and tamoxifen inducible genetic mosaicism mouse model of Tet2 +/- driven CHIP (HSC-Scl-Cre-ERT; Tet2+/flox; R26 +/floxstop-EGFP triple transgenic mice) to determine the contribution of inflamm-aging factors to Tet2 +/- hematopoieticclonal expansion. Using these complementary models, we observe that peripheral Tet2 +/- clonal expansion rates increase with age (evident in erythroid, myeloid, lymphoid B and T lineages), which is paralleled by a significant expansion of Tet2 +/- hematopoietic stem and progenitor cell (HSPCs) populations in aged mice (12-14 months old). Importantly, Tet2 +/- clonal expansion associates with increased levels of inflammatory cytokine IL-1 in aged mice, which derives partially from Tet2 +/- mutant mature hematopoietic cells. To test the contribution of IL-1 to Tet2 +/- clonal expansion, we administered IL-1 (0.5ug/day for 14 days) to young CHIP carrying mice (2-4 months of age) and observed an IL-1R1-dependent expansion of Tet2 +/- hematopoietic mature lineages and HSPCs. Dissection of the cellular mechanisms operating downstream of IL-1/IL-1R1 revealed that Tet2 +/- clonal expansion results from increased multilineage differentiation and associates with increased HSPC cell-cycle progression (while not depending on IL-1-mediated effects on HSPC viability). Moreover, Tet2 +/- HSPCs show a higher in vitro and in vivo repopulation capacity in response to prolonged IL-1 exposure compared to their WT counterparts. Finally, to directly test the contribution of IL-1 to drive Tet2 +/- clonal expansion in the context of physiological aging, we set up genetic (BM chimeras using donor BM from Tet2 +/-; Il-1r1-/- compound mutants) or pharmacological inhibited IL-1 signaling (Anakinra, hIL-1ra) during mouse ageing. Strikingly, both approaches prevented ageing-dependent Tet2 +/- clonal expansion, thus confirming IL-1 as key "inflamm-ageing" driver of Tet2 +/- clonal expansion. Overall, our data provide proof-of-concept that IL-1 production derived from aged BM cells is a relevant and targetable driver of Tet2 +/- clonal expansion in aged mice. Disclosures Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

INTERRELATION OF microRNAS AND TRANSPOSONS IN AGING AND CARCINOGENESIS

Активация мобильных генетических элементов играет роль в канцерогенезе и физиологическом старении человека, которое ассоциировано с развитием злокачественных новообразований. В данной статье представлены результаты анализа данных о наличии единых эпигенетических изменений при старении и канцерогенезе, обусловленных динамикой экспрессии происходящих от транспозонов микроРНК. Было выявлено, что при старении нарушается экспрессия происходящей от транспозонов 21 специфической микроРНК, уровень которых меняется также при злокачественных новообразованиях. Ранее подобных свидетельств о взаимосвязи механизмов старения и канцерогенеза на эпигенетическом уровне не было представлено. Полученные результаты позволяют предположить, что одним из ключевых механизмов старения является дисбаланс в запрограммированной активации мобильных генетических элементов, что отражается на изменении эпигенетической регуляции организма и приводит к повышенному риску развития рака. Так как прекурсоры микроРНК могут транслироваться с образованием функциональных молекул, применяемые в геронтологии пептиды могут быть рассмотрены как потенциальные противоопухолевые препараты. Dysregulation of transposable elements plays a key role in human carcinogenesis. Physiological aging in humans is also caused by deregulation of transposons. Moreover, aging is associated with the development of cancer. We presented the results of an analysis of data on the presence of common epigenetic changes during aging and carcinogenesis, associated with changes in the expression of microRNAs derived from transposons. We found that aging is characterized by changes in the expression of 21 specific transposon-derived microRNAs associated with the development of malignant neoplasms. Before us, evidence similar to ours on the relationship between the mechanisms of aging and carcinogenesis at the epigenetic level has not been presented. We hypothesized that one of the key mechanisms of aging is an imbalance in the programmed activation of mobile genetic elements, which is reflected in changes in the body’s epigenetic regulation and leads to an increased risk of cancer. Since miRNA precursors can be translated with the formation of functional molecules, peptides used in gerontology can be considered as potential anticancer drugs.

Abstract P358: Cardiac Innervation Remodeling And Impaired Brain Derived Neurotrophic Factor (bdnf) Levels In Physiological Aging Vivo Model

Aging is a multifactorial process associated with gradual loss of function and decay involving several neurohormonal systems, such as the autonomic nervous system (ANS). Progressive remodeling of ANS, induces a circulating catecholamines spillover and cardiac autonomic fibers depletion with raising both morbidities and mortality risk. Neurotrophic factors (NF) play a pivotal role in modulating neuronal function and are impaired in cardiovascular disorders. Whether and how physiological aging impacts these neurobiomarkers and cardiac innervation remains still unclear. Therefore, we investigated the impact of aging on neurotrophins (such as BDNF and NGF) production and secretion and its consequences, on cardiac nervous system homeostasis. In vivo, we used young (age: 3 months; n=10) and old (age: 24 months; n=11) male Fisher rats. In vitro, human neuroblastoma cells (SH-SY5Y) were stimulated with serum withdrawn from both experimental groups. Old rats showed a significant reduction in overall ANS fiber density, sympathetic (marked by dopamine β-hydroxylase, dβh) and cholinergic compartment (evidenced by vesicular acetylcholine transporter, VaChT) compared to the young group, assessed by immunohistochemical staining. In addition, we observed a marked downregulation of GAP-43 and BDNF protein levels in left ventricle total lysates via immunoblot analysis, in aged hearts as opposed to young ones. Conversely, no changes were observed in NGF protein expression. To further investigate the autocrine effect of aging on autonomic nerve fibers, we treated SH-SY5Y cells in vitro, with blood serum obtained by young or old rats. Both stimuli induced a remarkable increase in neuronal sprouting, as evidenced via crystal violet assay. Nevertheless, we found a bulky drop in the neuronal function of cells stimulated with old rat serum. Interestingly, this effect was accompanied by a sizeable blunt in GAP-43 and BDNF protein levels, compared to cells treated with young rat serum. Taken together, our data suggest that neuronal function impairment aging-induced associated with significant BDNF impoverishment, might favor maladaptive remodeling of cardiac ANS.

Automated, high-dimensional evaluation of physiological aging and resilience in outbred mice

Behavior and physiology are essential readouts in many studies but have not benefited from the high-dimensional data revolution that has transformed molecular and cellular phenotyping. To address this, we developed an approach that combines commercially available automated phenotyping hardware with a systems biology analysis pipeline to generate a high-dimensional readout of mouse behavior/physiology, as well as intuitive and health-relevant summary statistics (resilience and biological age). We used this platform to longitudinally evaluate aging in hundreds of outbred mice across an age range from 6 months to 3.4 years. In contrast to the assumption that aging can only be measured at the limits of animal ability via challenge-based tasks, we observed widespread physiological and behavioral aging starting in early life. Using network connectivity analysis, we found that organism-level resilience exhibited an accelerating decline with age that was distinct from the trajectory of individual phenotypes. We developed a method, Combined Aging and Survival Prediction of Aging Rate (CASPAR), for jointly predicting chronological age and survival time and showed that the resulting model is able to predict both variables simultaneously, a behavior that is not captured by separate age and mortality prediction models. This study provides a uniquely high-resolution view of physiological aging in mice and demonstrates that systems-level analysis of physiology provides insights not captured by individual phenotypes. The approach described here allows aging, and other processes that affect behavior and physiology, to be studied with sophistication and rigor.

GENDER DIFFERENCES IN THE VOLUME OF BRAIN STRUCTURES IN THE ASPECT OF PHYSIOLOGICAL AGING

Целью работы являлось уточнение гендерных и возрастных различий в строении мозговых структур у здоровых добровольцев в связи с задачей разграничения визуальных паттернов физиологического старения и патологических процессов головного мозга. Методы исследования включали проведение структурной МРТ головного мозга у 131 условно-здорового добровольца 20-70 лет (из них 72 женщины и 59 мужчин). С помощью метода магнитно-резонансной воксель-базированной морфометрии проводили измерение объёма головного мозга в целом, а также его отделов с последующим сравнением полученных данных в возрастных подгруппах и между полами. Полученные в ходе исследования данные свидетельствуют о том, что наибольшее уменьшение размеров после 60 лет претерпевают правый и левый таламусы, левое хвостатое ядро, правая скорлупа, левый бледный шар, оба гиппокампа. Наибольшие изменения в гиппокампах претерпевает объём СА3 полей Бродмана. Установлено, что процесс физиологического старения головного мозга с изменением объёма его различных отделов имеет ряд гендерных особенностей, которые необходимо учитывать на диагностическом этапе медицинской помощи. The aim of the work was to clarify the gender, age and variable differences in the structure of brain structures in healthy volunteers in connection with the task of distinguishing between physiological aging and pathological processes of the brain. The study methods included performing structural MRI of the brain in 131 conditionally healthy volunteers aged 20 to 70 years (72 of them were women and 59 men). Using the method of MR voxel-based morphometry, the total volume of the brain as a whole, as well as its segmented parts, was measured, followed by a comparison of the data obtained in age subgroups and between the sexes. The data obtained in the course of the study indicate that the right and left thalamuses, the left caudate nucleus, the right shell, the left pale ball, and both hippocampus undergo the greatest reduction in size after 60 years. The greatest changes in the hippocampus are the volumes of CA 3 Brodman fields. It is established that the process of physiological aging of the brain.