NUCLEAR LAMINA EROSION-INDUCED RESURRECTION OF ENDOGENOUS RETROVIRUSES UNDERLIES NEURONAL AGING

Kinh Tế - Quản Lý - Khoa học xã hội - Kiến trúc - Xây dựng Article Nuclear lamina erosion-induced resurrection of endogenous retroviruses underlies neuronal aging Graphical abstract Highlights d Multi-omics profiling of primate frontal lobe (FL) aging d Neuronal-specific nuclear lamina erosion as a hallmark and driver of FL aging d Consequent ERV activation induces neuronal senescence and inflammation d The NRT inhibitor mitigates human neuronal senescence and mouse brain aging Authors Hui Zhang, Jiaming Li, Yang Yu, ..., Jing Qu, Weiqi Zhang, Guang-Hui Liu Correspondence wangsixwh.ccmu.edu.cn (S.W.), qujingioz.ac.cn (J.Q.), zhangwqbig.ac.cn (W.Z.), ghliuioz.ac.cn (G.-H.L.) In brief Zhang et al. establish a granular view of the molecular alterations underlying primate FL aging through multi-omics profiling, neuropathological examination, and in vitro modeling. It reveals that, during neuronal aging, nuclear lamina erosion induces the reactivation of ERVs and contributes to neuronal degeneration. Zhang et al., 2023, Cell Reports 42 , 112593 June 27, 2023 ª 2023 The Authors. https:doi.org10.1016j.celrep.2023.112593 ll Article Nuclear lamina erosion-induced resurrection of endogenous retroviruses underlies neuronal aging Hui Zhang, 1,6,29 Jiaming Li, 3,6,29 Yang Yu, 11,12,29 Jie Ren,3,4,6,29 Qiang Liu,13,29 Zhaoshi Bao, 15,16,29 Shuhui Sun, 1,4,7 Xiaoqian Liu, 2,4,7 Shuai Ma, 1,4,7 Zunpeng Liu, 2,6 Kaowen Yan, 1,4,7 Zeming Wu, 1,4,7 Yanling Fan, 3,6 Xiaoyan Sun, 3,6 Yixin Zhang, 2,6 Qianzhao Ji, 1,6 Fang Cheng,6,17 Peng-Hu Wei, 20,21,22 Xibo Ma, 21,22 Shiqiang Zhang,23 (Author list continued on next page) SUMMARY The primate frontal lobe (FL) is sensitive to aging-related neurocognitive decline. However, the aging- associated molecular mechanisms remain unclear. Here, using physiologically aged non-human pri- mates (NHPs), we depicted a comprehensive landscape of FL aging with multidimensional profiling en- compassing bulk and single-nucleus transcriptomes, quantitative proteome, and DNA methylome. Conjoint analysis across these molecular and neuropathological layers underscores nuclear lamina and heterochromatin erosion, resurrection of endogenous retroviruses (ERVs), activated pro-inflamma- tory cyclic GMP-AMP synthase (cGAS) signaling, and cellular senescence in post-mitotic neurons of aged NHP and human FL. Using human embryonic stem-cell-derived neurons recapitulating cellular aging in vitro , we verified the loss of B-type lamins inducing resurrection of ERVs as an initiating event of the aging-bound cascade in post-mitotic neurons. Of significance, these aging-related cellular and molecular changes can be alleviated by abacavir, a nucleoside reverse transcriptase inhibitor, either through direct treatment of senescent human neurons in vitro or oral administration to aged mice. INTRODUCTION The frontal lobe (FL) of the primate brain evolved to control ex- ecutive functions and cognitive skills. 1 The FL is one of the brain regions with the most decreased volume with aging, 2,3 and its neuroanatomic and neurophysiological changes un- derlie the development of frontotemporal dementia and Alz- heimer’s disease (AD). 4 However, cognitive aging presumably occurs years before the onset of neurodegenerative diseases is diagnosed in elderly individuals, presenting challenges to early diagnosis and therapeutic development. 5,6 Therefore, an in-depth understanding of the cellular and molecular changes associated with FL aging may help uncover check- points that can be targeted therapeutically, either at earlier stages or preemptively, to delay the progression of neurocog- nitive decline. In the gray matter (GM) of FL, as well as embedded within the white matter (WM), billions of highly interconnected and func- tionally diverse neurons and large populations of glial and other non-neuronal cells constitute a vastly heterogeneous cell popu- lation. 7–10 Moreover, a growing number of studies have reported that extensive and complex cellular structural and functional al- terations are involved in FL aging and neuronal degenera- tion.11,12 Coupled with the diversity of regulatory factors involved at the epigenetic, transcriptional, and translational levels, our un- derstanding of the cellular and molecular drivers of FL aging, especially in primates, remains very limited. To this end, recently developed single-nucleus RNA sequencing (snRNA-seq) makes 1 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 2 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 3 CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences and China National Center for Bioinformation, Beijing 100101, China 4 Institute for Stem Cell and Regeneration, CAS, Beijing 100101, China 5 Advanced Innovation Center for Human Brain Protection, and National Clinical Research Center for Geriatric Disorders, Xuanwu Hospital Capital Medical University, Beijing 100053, China 6 University of Chinese Academy of Sciences, Beijing 100049, China 7 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China 8Aging Translational Medicine Center, International Center for Aging and Cancer, Beijing Municipal Geriatric Medical Research Center, (Affiliations continued on next page) Cell Reports 42, 112593, June 27, 2023 ª 2023 The Authors. 1 This is an open access article under the CC BY-NC-ND license (http:creativecommons.orglicensesby-nc-nd4.0). ll OPEN ACCESS it possible to study the cellular and molecular mechanisms of heterogeneous FL aging with high accuracy, 13–17 and, combined with multi-layered omics approaches, 18 it will serve as a valuable resource to thoroughly delineate the intricate regulatory mecha- nism of FL aging. Similar to humans, the non-human primate (NHP) frontal cor- tex also increases in size and evolves into an extremely elaborate neocortical region during phylogenetic development. 19,20 In addition, NHPs are highly similar to humans in neuroanatomical, physiological, and neuropathological aspects and experience neuronal deterioration and cognitive impairment in later life, similar to humans, 15,21–23 thus serving as a clinically relevant model to investigate aging-associated mechanisms underlying FL dysfunction. Here, we revealed that nuclear lamina attrition, heterochro- matin erosion, and consequent resurrection of ERVs are intrinsic to aged neurons, thereby initiating innate immune responses and ultimately neuron degeneration in aged NHP and human FL in vivo, as well as in an in vitro model. Notably, we demonstrated that pharmacological treatment with the nucleoside reverse tran- scriptase (NRT) inhibitor abacavir, either directly supplemented to the human neuron model or orally administered to physiolog- ically aged mice over a 12-month time course, inhibits neuronal senility. RESULTS Multifaceted phenotypes of neurological degeneration in the aged primate FL We first obtained FL tissues from young (4–6 years old, equiva- lent to 16–20 years of human age) and aged (18–21 years old, equivalent to  65–70 years of human age) cynomol- gus monkeys without apparent morphological anomalies (Figures 1A and S1A). The overall anatomical structures and the integrity of cortical stratification in the aged FL were compa- rable with those in the young counterparts (Figures S1B and S1C). However, distinct from their younger counterparts, we found that a spectrum of neurological degeneration indicators accu- mulated in the aged FL, especially in the GM of the FL. First, in the aged FL, we observed increased areas with senescence- associated b-galactosidase (SA-b -Gal) staining, a classic senes- cence marker 24 (Figure 1B). Consistent with earlier work docu- menting the loss of proteostasis in the aged cortex, 25,26 we found increased protein aggregates and amyloid-b (Ab ) de- posits, marked by Ab (4G8), the major component of senile pla- ques, in the GM of aged FL (Figures 1C and 1D). In addition, we detected increased lipofuscin pigment in aged FL, which usually accumulates progressively with age (Figure 1E). Consistent with Zhengwei Xie,24 Yuyu Niu,25,28 Yan-Jiang Wang, 26,27,28 Jing-Dong J. Han, 23,28 Tao Jiang, 14,15,16,28 Guoguang Zhao, 18,19,20,28 Weizhi Ji, 25,28 Juan Carlos Izpisua Belmonte, 10,28 Si Wang, 5,8,9, Jing Qu,2,4,6,7, Weiqi Zhang,3,4,6, and Guang-Hui Liu 1,4,5,6,7,8,30, Xuanwu Hospital, Capital Medical University, Beijing 100053, China 9 The Fifth People’s Hospital of Chongqing, Chongqing 400062, China 10 Altos Labs, Inc., San Diego, CA, USA 11 Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology and Key Laboratory of Assisted Reproduction, Ministry of Education, Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China 12 Clinical Stem Cell Research Center, Peking University Third Hospital, Beijing, China 13 Department of Neurology, Tianjin Medical University General Hospital, Tianjin 300052, China 14 Beijing Neurosurgical Institute, Beijing 100070, China 15 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China 16 Chinese Glioma Genome Atlas Network Asian Glioma Genome Atlas Network, Beijing 100070, China 17 National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 18 Department of Neurosurgery, Xuanwu Hospital Capital Medical University, Beijing 100053, China 19 Clinical Research Center for Epilepsy Capital Medical University, Beijing 100053, China 20 Beijing Municipal Geriatric Medical Research Center, Beijing 100053, China 21 MAIS, State Key Laboratory of Multimodal Artificial Intelligence Systems, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China 22 School of Artificial Intelligence, University of Chinese Academy of Sciences, Beijing 100049, China 23 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Center for Quantitative Biology (CQB), Peking University, Beijing 100871, China 24 Peking University International Cancer Institute, Peking University Health Science Center, Peking University, Beijing 100191, China 25 State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China 26 Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing 400042, China 27 State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China 28 Senior author 29 These authors contributed equally 30 Lead contact Correspondence: wangsixwh.ccmu.edu.cn (S.W.), qujingioz.ac.cn (J.Q.), zhangwqbig.ac.cn (W.Z.), ghliuioz.ac.cn (G.-H.L.) https:doi.org10.1016j.celrep.2023.112593 2 Cell Reports 42, 112593, June 27, 2023 Article ll OPEN ACCESS A B C D E GF Figure 1. Phenotypic insights into aging-related alterations in the primate FL (A) Experimental scheme of aging phenotype analysis, sequencing approaches, and mechanistic studies. (B) SA-b-Gal staining of the GM and WM sections of FL from young and aged monkeys. The SA-b -Gal-positive area is quantified as fold changes (aged vs. young). (C) Aggresome staining of the GM and WM sections of FL from young and aged monkeys. The number of positively stained cells is quantified as fold changes (aged vs. young). (D) Ab (4G8) immunofluorescence staining of the GM and WM sections of FL from young and aged monkeys. The number of Ab (4G8)-positive cells is quantified as fold changes (aged vs. young). (E) Lipofuscin accumulation in the GM and WM sections of FL from young and aged monkeys. The number of lipofuscin-positive cells is quantified as fold changes (aged vs. young). (F) gH2A.X immunohistochemical staining of the GM and WM sections of FL from young and aged monkeys. The signal intensity of g H2A.X is quantified as fold changes (aged vs. young). (G) MMP-9 immunofluorescence staining of the GM and WM sections in FL from young and aged monkeys. The number of MMP-9-positive cells is quantified as fold changes (aged vs. young). Scale bars, 50 mm and 10 mm (zoomed-in images) in (B); 20 mm and 10 m m (zoomed-in images) in (C)–(G). White arrowheads indicate the corresponding positive- staining cells. Young, n = 8; aged, n = 8 monkeys. Data are represented as the mean ± SEM. Two-tailed t test. Cell Reports 42, 112593, June 27, 2023 3 Article ll OPEN ACCESS A B C D E F G (legend on next page) 4 Cell Reports 42, 112593, June 27, 2023 Article ll OPEN ACCESS the notion that genomic instability is a hallmark of brain ag- ing,27,28 we observed an elevated DNA damage response (marked by g H2A.X foci formation) in aged FL (Figure 1F). Further, we detected evidence of brain inflammation, a critical player in the development of cognitive dysfunctions, as two important pro-inflammatory matrix metalloproteinases (MMPs), MMP-9 and MMP-3, 29,30 both accumulated in the aged FL (Figures 1G and S1D). Cortical neurons are prominently vulnerable in primate FL aging To dissect relationships between the multifaceted phenotypes of FL aging and underlying molecular mechanisms, we analyzed tissue-level RNA sequencing (RNA-seq) and quanti- tative proteomic analysis of young and aged NHP FL. By per- forming differential expression analysis, we identified a total of 497 upregulated and 524 downregulated differentially ex- pressed genes (DEGs), as well as 120 upregulated and 39 downregulated differentially expressed proteins (DEPs), dur- ing FL aging (Figures S2A–S2C; Table S1). Joint analysis of the aging-related DEGs and DEPs identified pathways involved in neuron functions, including synapse assembly and transsynaptic signaling, that were downregulated at both the RNA and protein levels in the aged FL (Figures 2A and S2D). Upregulated aging-related DEGs and DEPs were mainly related to chronic inflammation, such as leukocyte activation and granulocyte chemotaxis pathways, indicative of prominent neuroinflammation in the aged FL (Figures 2A and S2D). Furthermore, a subset of upregulated proteins in aged FL also overlapped with the upregulated DEPs that were reported to accumulate in the plasma of elderly individ- uals, 31 including galectin-1, a neuroinflammation-related pro- tein encoded by LGALS132 (Figure S2E), and therefore may serve as a circulatory indicator for FL aging. To further investigate cell-type-specific alterations during FL aging, we conducted snRNA-seq and obtained 111,698 high- quality single-nucleus transcriptomes from FL tissues of young and aged monkeys (Figures 2B, S2F, and S2G). Based on this dataset, we annotated 10 cell types with specific classic markers and gene expression signatures reflecting biological functions for each cell type, including neurons (inhibitory neuron InN; excitatory neuron ExN), glial cells (microglia; oligodendrocyte OL; immune OL ImmuOL; astrocyte), pro- genitor cells (OL progenitor cell OPC; committed OPC COP), vascular cells (endothelial cell EC), and meningeal cells (meninge) (Figures 2B, S2H, and S2I; Table S2). Among these cell types, we noticed that ExNs and InNs harbored more DEGs detectable at the population level, indicating their high susceptibility and critical importance to FL aging (Fig- ure 2C; Table S1). Indeed, neuronal functions, such as transsy- naptic signaling, ion transmembrane transport, and neuron pro- jection development, might be compromised during aging, as shown by enriched Gene Ontology (GO) terms of downregu- lated aging-associated DEGs (cluster 11) (Figures 2C, 2D, and S3A). Conversely, upregulation of neuron-specific aging DEGs was enriched for cytokine-cytokine receptor interactions (IL15, LTB, TNFRSF25, GDF15, CXCL14 ) (clusters 4 and 5), implying an intrinsic activation of inflammatory pathways in the neurons themselves (Figures 2C and 2D). We also noticed that aging-related degenerative (i.e., accumu- lation of aggresomes and Ab deposits) and pro-inflammatory (i.e., MMP-9 escalation) features were overrepresented in the neuron-enriched GM region of the FL (Figures 1C, 1D, and 1G), consistent with the molecular profiling data. Indeed, aggresome staining was primarily detected in NeuN-positive neuronal cells, which was increased in the aged FL (Figure S3B). Similarly, over 90 of SA-b -Gal-positive cells were NeuN-positive neuronal cells (Figure 2E). In particular, morphological analyses by Golgi staining revealed decreased dendritic length, arborization, and spine density in the aged FL (Figures 2F and 2G), indicative of a functional deficit and validating that FL neurons are particularly vulnerable to aging. Figure 2. Integrative analysis of transcriptomic, proteomic, and single-nucleus transcriptomic datasets underscores neuroinflammation during primate FL aging (A) Network plots showing the enriched pathways for differentially expressed genes (DEGs) and proteins (DEPs) (aged vs. young). Shapes of the nodes indicate that components of the pathways are dysregulated at both the RNA and protein levels or in an RNAprotein-specific manner. Edge colors from light to redblue indicate the Jaccard index from low to high. Node sizes from small to large indicate the number of DEGs or DEPs in the enriched terms from low to high. Left, upregulated pathways; right, downregulated pathways. (B) t-SNE plot showing the annotated cell types in the single-nucleus RNA-seq (snRNA-seq) data of the monkey FL. Samples were collected from young and old monkeys (n = 8 monkeys per group). (C) Heatmap showing the averaged gene expression of the upregulated and downregulated DEGs (aged vs. young) detected in different cell types in the monkey FL. Each row represents one cell type, and each column denotes the expression of one DEG. The color key from blue to red indicates gene expression (scaled by column) from low to high. (D) Bar plots showing the enriched pathways of upregulated (clusters 4 and 5 in C) and downregulated (cluster 11 in C) DEGs in neurons (InN and ExN) of the monkey FL. Color keys from light to bluered indicate the Log 10 P from low to high. (E) SA-b-Gal and NeuN immunostaining in FL from young and aged monkeys. Upper right, the number of SA-b -Gal-positive neurons in the GM sections is quantified as fold changes (aged vs. young). Lower right, pie plots showing the percentages of neurons and other cells to total SA-b -Gal-positive cells in the GM of young and aged monkey FL. Black arrowheads indicate SA-b -Gal-positive neurons. (F) Golgi staining in the GM sections in FL from young and aged monkeys. Left, representative images and trajectory tracking of dendrites. Middle, the average length of dendrites is quantified as fold changes (aged vs. young). Right, Sholl analyses showing reduced dendritic intersections (ranging from 60 to 140 m m) of aged neurons. (G) Golgi staining in the GM sections of FL from young and aged monkeys. The apical dendritic spine density is quantified as fold changes (aged vs. young). Red arrowheads indicate the dendritic spines. Scale bars, 20 mm and 10 mm (zoomed-in images) in (E); 50 mm in (F); and 5 mm in (G). Young, n = 8; aged, n = 8 monkeys. Data are represented as the mean ± SEM. Two-tailed t test. Cell Reports 42, 112593, June 27, 2023 5 Article ll OPEN ACCESS A B C D E F G H I J NMLK (legend on next page) 6 Cell Reports 42, 112593, June 27, 2023 Article ll OPEN ACCESS Disrupted nuclear lamina and derepressed retrotransposable elements in the aged FL In human mitotic cells, we and others have shown in vitro that disruption of nuclear lamina organization and augmentation of heterochromatin erosion can lead to the derepression of retro- transposons therein and enhance inflammatory responses. 33–40 Next, we asked whether aging-associated nuclear lamina erosion and epigenetic changes might underlie the dysfunction and inflammation of post-mitotic neurons in vivo . By analyzing the snRNA-seq data, we found that transcript levels for two ma- jor structural proteins of the nuclear lamina, LMNB1 and LMNB2 , were decreased in neurons (Figure 3A). As expected, we observed discontinuous morphology of the nuclear lamina with lower expression of Lamin B1 and Lamin B2, as well as reduced LAP2b , a heterochromatin-related inner nuclear membrane pro- tein predominantly in cortical neurons (but not in glial cells) in the aged primate FL, while other nuclear envelope proteins remained unchanged (such as Emerin, SUN1, and SUN2) (Figures 3B–3D and S4A–S4J). Moreover, the 3D conformation of nuclear morphology based on the H3K9me3 and LAP2b sig- nals and transmission electron microscopy (TEM) visualization of the nuclear structure revealed compromised integrity of the nuclear lamina along with diminished heterochromatin under- neath the nuclear membrane in the aged neurons (Figures 3D– 3F and S4K). Next, we sought to dissect changes in genome-wide DNA methylation, an important epigenetic modification that locks down heterochromatin to prevent aberrant transcription.41,42 We analyzed aging-related DNA methylation changes in both pro- tein-coding regions and regions harboring various repetitive ele- ments encapsulated in dense heterochromatin, such as ERVs belonging to retrotransposable elements.36,37,39,43–45 Although no apparent alterations in DNA methylation levels of protein-cod- ing regions were observed (Figure S4L), we revealed reduced methylation levels of ERV in the aged FL (Figure 3G). In accor- dance, nearly 20 of repetitive elements detected were transcrip- tionally upregulated, with ERVs as the top-ranking families, while very few repetitive elements were downregulated (Figure 3H; Table S3). Accordingly, elevated ERV-Env levels, the protein prod- ucts of ERV transcripts, were present in the aged FL and specif- ically detected in cortical neurons but not non-neuronal cells (Figures 3I and S4M). It is noteworthy that, consistent with the neuron-specific nuclear deformation and ERV activation in aged monkey FL, a similar upregulated expression of ERV-Env proteins is observed in the GM, but not the WM, of the FL of monkeys with Hutchinson-Gilford progeria syndrome (HGPS) (Figures S4N and S4O), an early-onset aging disorder characterized by carrying a heterozygous mutation of the LMNA gene.39 We also found a higher DNA content of ERVs in the aged FL, implying that their reverse transcripts increased andor underwent active genomic transposition (Figure 3J). Additionally, an increased number of retrovirus-like particles (RVLPs) was detected in the aged FL (Fig- ure 3K), which is consistent with our recent observation that RVLPs appear in senescent mitotic cells.39 We further calculated the co-expression gene network and identified DEGs whose expression fluctuation synchronized with that of ERVs. These DEGs were highly relevant to viral infection pathways and antigen processing-cross presentation pathways (Figures 3L–3N; Table S4). These results raise the possibility that insufficient nu- clear lamina proteins lead to epigenetic instability with resurrected ERV retrotransposons that may cause aging-related pro-inflam- matory phenotypes in the NHP FL. Figure 3. Epigenetic landscape reveals disruption of nuclear architecture and derepression of retrotransposable elements in the aged primate FL (A) Violin plots showing the expression levels of LMNB1 and LMNB2 in snRNA-seq data of neurons (InN and ExN) from the indicated group of the monkey FL. (B) Immunofluorescence staining of Lamin B1Lamin B2 and NeuN in FL from young and aged monkeys. The fluorescence intensity of Lamin B1Lamin B2 in neurons in the GM sections is quantified as fold changes (aged vs. young). Young, n = 8; aged, n = 8 monkeys. (C) 3D reconstruction of Lamin B1 (top) and Lamin B2 (bottom) immunofluorescence images in neurons. (D) Immunofluorescence staining of LAP2b and H3K9me3 in the GM sections of FL from young and aged monkeys. Left, 3D reconstruction of LAP2b and H3K9me3 immunofluorescence images in neurons. Right, the number of cells with abnormal nuclear lamina in the GM sections is quantified as fold changes (aged vs. young). White arrowheads indicate the abnormal nuclear lamina. Young, n = 8; aged, n = 8 monkeys. (E) TEM analysis of the heterochromatin architecture at the nuclear periphery in the GM sections in FL from young and aged monkeys. The percentages of neurons with heterochromatin loss at the nuclear periphery are presented below the images. Red arrowheads indicate heterochromatin-loss regions. Young, n > 200 cells; aged, n > 200 cells. (F) Immunofluorescence staining of H3K9me3 and NeuN in FL from young and aged monkeys. The fluorescence intensity of H3K9me3 in neurons in the GM sections is quantified as fold changes (aged vs. young). Young, n = 8; aged, n = 8 monkeys. (G) Metaplots showing the loss of CG methylation (mCG) levels (mCGCG) at ERVs in the monkey FL during aging. (H) Top, pie and bar plots showing the percentage of different types of differentially expressed repetitive elements (aged vs. young). Gray indicates unchanged repetitive elements. Bottom, distribution density of the log 2 FC value of all ERV members in aged monkey FL. (I) Immunofluorescence staining of ERV-Env and NeuN in FL from young and aged monkeys. Left, schematic diagram of ERV-RVLP production process. Middle, representative images. Right, the fluorescence intensity of ERV-Env in neurons in the GM sections is quantified as fold changes (aged vs. young). Young, n = 8; aged, n = 8 monkeys. (J) qPCR analysis of the relative ERV genomic DNA content in the FL from young and aged monkeys. Young, n = 8; aged, n = 8 monkeys. (K) TEM analysis of the putative RVLPs in the GM sections of FL from young and aged monkeys. The number of RVLPs per cell is quantified. The red arrowhead indicates the putative RVLP. Young, n = 80 cells from four samples; aged, n = 80 cells from four samples. (L) Dot plots showing the log 2 FC of DEGs (aged vs. young) and their Pearson’s correlation coefficients with the expression levels of ERV retrotransposable elements. DEGs with high positive or negative correlations are shown in red (correlation coefficient >0.7) or blue (correlation coefficient