The Role of Retrotransposons and Endogenous Retroviruses in Age-Dependent Neurodegenerative Disorders.

Over 40% of the human genome is composed of retrotransposons, DNA species that hold the potential to replicate via an RNA intermediate and are evolutionarily related to retroviruses. Retrotransposons are most studied for their ability to jump within a genome, which can cause DNA damage and novel insertional mutations. Retrotransposon-encoded products, including viral-like proteins, double-stranded RNAs, and extrachromosomal cytoplasmic DNAs, can also be potent activators of the innate immune system. A growing body of evidence suggests that retrotransposons are activated in age-related neurodegenerative disorders and that such activation causally contributes to neurotoxicity. Here we provide an overview of retrotransposon biology and outline evidence of retrotransposon activation in age-related neurodegenerative disorders, with an emphasis on those involving TAR-DNA binding protein-43 (TDP-43) and tau. Studies to date provide the basis for ongoing clinical trials and hold promise for innovative strategies to ameliorate the adverse effects of retrotransposon dysregulation in neurodegenerative disorders.

A pilot study to investigate the safety and feasibility of antiretroviral therapy for Alzheimer's disease (ART-AD).

Retrotransposons are viral-like DNA sequences that constitute approximately 41% of the human genome. Studies in Drosophila, mice, cultured cells, and human brain indicate that retrotransposons are activated in settings of tauopathy, including Alzheimer's disease, and causally drive neurodegeneration. The anti-retroviral medication 3TC (lamivudine), a nucleoside analog reverse transcriptase inhibitor, limits retrotransposon activation and suppresses neurodegeneration in tau transgenic Drosophila, two mouse models of tauopathy, and in brain assembloids derived from patients with sporadic Alzheimer's disease. We performed a 24-week phase 2a open-label clinical trial of 300 mg daily oral 3TC (NCT04552795) in 12 participants aged 52-83 years with a diagnosis of mild cognitive impairment due to suspected Alzheimer's disease. Primary outcomes included feasibility, blood brain barrier penetration, effects of 3TC on reverse transcriptase activity in the periphery, and safety. Secondary outcomes included changes in cognition and fluid-based biomarkers of neurodegeneration and neuroinflammation. All participants completed the six-month trial; one event of gastrointestinal bleeding due to a peptic ulcer was reported. 3TC was detected in blood and cerebrospinal fluid (CSF) of all participants, suggestive of adherence to study drug and effective brain penetration. Cognitive measures remained stable throughout the study. Glial fibrillary acidic protein (GFAP) (P=0.03) and Flt1 (P=0.05) were significantly reduced in CSF over the treatment period; Aß42/40 (P=0.009) and IL-15 (P=0.006) were significantly elevated in plasma. While this is an open label study of small sample size, the significant decrease of some neurodegeneration- and neuroinflammation-related biomarkers in CSF, significantly elevated levels of plasma Aß42/40, and a trending decrease of CSF NfL after six months of 3TC exposure suggest a beneficial effect on subjects with mild cognitive impairment due to suspected Alzheimer's disease. Feasibility, safety, tolerability, and central nervous system (CNS) penetration assessments further support clinical evaluation of 3TC in a larger placebo-controlled, multi-dose clinical trial.

New approaches for understanding the potential role of microbes in Alzheimer's disease.

Alzheimer's disease (AD) involves a complex pathological process that evolves over years, and its etiology is understood as a classic example of gene-environment interaction. The notion that exposure to microbial organisms may play some role in AD pathology has been proposed and debated for decades. New evidence from model organisms and -omic studies, as well as epidemiological data from the recent COVID-19 pandemic and widespread use of vaccines, offers new insights into the "germ hypothesis" of AD. To review new evidence and identify key research questions, the Duke/University of North Carolina (Duke/UNC) Alzheimer's Disease Research Center hosted a virtual symposium and workshop: "New Approaches for Understanding the Potential Role of Microbes in Alzheimer's disease." Discussion centered around the antimicrobial protection hypothesis of amyloid accumulation, and other mechanisms by which microbes could influence AD pathology including immune cell activation, changes in blood-brain barrier, or direct neurotoxicity. This summary of proceedings reviews the content presented in the symposium and provides a summary of major topics and key questions discussed in the workshop.

m6A-dependent circular RNA formation mediates tau-induced neurotoxicity.

Circular RNAs (circRNAs), covalently closed RNA molecules that form due to back-splicing of RNA transcripts, have recently been implicated in Alzheimer's disease and related tauopathies. circRNAs are regulated by N6-methyladenosine (m6A) RNA methylation, can serve as "sponges" for proteins and RNAs, and can be translated into protein via a cap-independent mechanism. Mechanisms underlying circRNA dysregulation in tauopathies and causal relationships between circRNA and neurodegeneration are currently unknown. In the current study, we aimed to determine whether pathogenic forms of tau drive circRNA dysregulation and whether such dysregulation causally mediates neurodegeneration. We identify circRNAs that are differentially expressed in the brain of a Drosophila model of tauopathy and in induced pluripotent stem cell (iPSC)-derived neurons carrying a tau mutation associated with autosomal dominant tauopathy. We leverage Drosophila to discover that depletion of circular forms of muscleblind (circMbl), a circRNA that is particularly abundant in brains of tau transgenic Drosophila, significantly suppresses tau neurotoxicity, suggesting that tau-induced circMbl elevation is neurotoxic. We detect a general elevation of m6A RNA methylation and circRNA methylation in tau transgenic Drosophila and find that tau-induced m6A methylation is a mechanistic driver of circMbl formation. Interestingly, we find that circRNA and m6A RNA accumulate within nuclear envelope invaginations of tau transgenic Drosophila and in iPSC-derived cerebral organoid models of tauopathy. Taken together, our studies add critical new insight into the mechanisms underlying circRNA dysregulation in tauopathy and identify m6A-modified circRNA as a causal factor contributing to neurodegeneration. These findings add to a growing literature implicating pathogenic forms of tau as drivers of altered RNA metabolism.

Nanopore-based DNA long-read sequencing analysis of the aged human brain.

Aging disrupts cellular processes such as DNA repair and epigenetic control, leading to a gradual buildup of genomic alterations that can have detrimental effects in post-mitotic cells. Genomic alterations in regions of the genome that are rich in repetitive sequences, often termed "dark loci," are difficult to resolve using traditional sequencing approaches. New long-read technologies offer promising avenues for exploration of previously inaccessible regions of the genome. Using nanopore-based long-read whole-genome sequencing of DNA extracted from aged 18 human brains, we identify previously unreported structural variants and methylation patterns within repetitive DNA, focusing on transposable elements ("jumping genes") as crucial sources of variation, particularly in dark loci. Our analyses reveal potential somatic insertion variants and provides DNA methylation frequencies for many retrotransposon families. We further demonstrate the utility of this technology for the study of these challenging genomic regions in brains affected by Alzheimer's disease and identify significant differences in DNA methylation in pathologically normal brains versus those affected by Alzheimer's disease. Highlighting the power of this approach, we discover specific polymorphic retrotransposons with altered DNA methylation patterns. These retrotransposon loci have the potential to contribute to pathology, warranting further investigation in Alzheimer's disease research. Taken together, our study provides the first long-read DNA sequencing-based analysis of retrotransposon sequences, structural variants, and DNA methylation in the aging brain affected with Alzheimer's disease neuropathology.

Novel avenues of tau research.

INTRODUCTION: The pace of innovation has accelerated in virtually every area of tau research in just the past few years. METHODS: In February 2022, leading international tau experts convened to share selected highlights of this work during Tau 2022, the second international tau conference co-organized and co-sponsored by the Alzheimer's Association, CurePSP, and the Rainwater Charitable Foundation. RESULTS: Representing academia, industry, and the philanthropic sector, presenters joined more than 1700 registered attendees from 59 countries, spanning six continents, to share recent advances and exciting new directions in tau research. DISCUSSION: The virtual meeting provided an opportunity to foster cross-sector collaboration and partnerships as well as a forum for updating colleagues on research-advancing tools and programs that are steadily moving the field forward.

A randomized placebo-controlled trial of nicotinamide riboside in older adults with mild cognitive impairment.

Nicotinamide riboside (NR) increases blood levels of NAD+, a cofactor central to energy metabolism, and improves brain function in some rodent models of neurodegeneration. We conducted a placebo-controlled randomized pilot study with the primary objective of determining safety of NR in older adults with mild cognitive impairment (MCI). Twenty subjects with MCI were randomized to receive placebo or NR using dose escalation to achieve, and maintain, a final dose of 1 g/day over a 10-week study duration. The primary outcome was post-treatment change from baseline measures of cognition (Montreal Cognitive Assessment, MoCA). Predefined secondary outcomes included post-treatment changes in cerebral blood flow (CBF); blood NAD+ levels; and additional neurocognitive, psychometric, and physical performance tests. DNA methylation was assessed in peripheral blood mononuclear cells (PBMCs) as an exploratory outcome. The target NR dose was safely achieved as evidenced by a 2.6-fold increase in blood NAD+ in the NR group (p < 0.001, 95% CI [17.77, 43.49]) with no between-group difference in adverse event reporting. MoCA and other neurocognitive and psychometric metrics remained stable throughout the study. NR reduced CBF in the default mode network (DMN) with greatest differences observed in the left inferior parietal lobe (IPL) (DMN p = 0.013, µ = 0.92, 95% CI [0.23, 1.62]; left IPL p = 0.009, µ = 1.66, 95% CI [0.5, 2.82]). Walking speed in the placebo group significantly improved across the study duration suggestive of a practice effect but did not change in the NR group (p = 0.0402 and p = 0.4698, respectively). Other secondary outcome measures remained stable. Global methylation analyses indicated a modest NR-associated increase in DNA methylation and concomitant reduction in epigenetic age as measured by PhenoAge and GrimAge epigenetic clock analyses. In summary, NR significantly increased blood NAD+ concentrations in older adults with MCI. NR was well tolerated and did not alter cognition. While CBF was reduced by NR treatment, statistical significance would not have withstood multiple comparisons correction. A larger trial of longer duration is needed to determine the potential of NR as a strategy to improve cognition and alter CBF in older adults with MCI. ClinicalTrials.gov NCT02942888.

Alzheimer's disease and related tauopathies: disorders of disrupted neuronal identity.

Postmitotic neurons require persistently active controls to maintain terminal differentiation. Unlike dividing cells, aberrant cell cycle activation in mature neurons causes apoptosis rather than transformation. In Alzheimer's disease (AD) and related tauopathies, evidence suggests that pathogenic forms of tau drive neurodegeneration via neuronal cell cycle re-entry. Multiple interconnected mechanisms linking tau to cell cycle activation have been identified, including, but not limited to, tau-induced overstabilization of the actin cytoskeleton, consequent changes to nuclear architecture, and disruption of heterochromatin-mediated gene silencing. Cancer- and development-associated pathways are upregulated in human and cellular models of tauopathy, and many tau-induced cellular phenotypes are also present in various cancers and progenitor/stem cells. In this review, I delve into mechanistic parallels between tauopathies, cancer, and development, and highlight the role of tau in cancer and in the developing brain. Based on these studies, I put forth a model by which pathogenic forms of tau disrupt the program that maintains terminal neuronal differentiation, driving cell cycle re-entry and consequent neuronal death. This framework presents tauopathies as conditions involving the profound toxic disruption of neuronal identity.

Selective neuronal vulnerability to deficits in RNA processing.

Emerging evidence indicates that errors in RNA processing can causally drive neurodegeneration. Given that RNA produced from expressed genes of all cell types undergoes processing (splicing, polyadenylation, 5' capping, etc.), the particular vulnerability of neurons to deficits in RNA processing calls for careful consideration. The activity-dependent transcriptome remodeling associated with synaptic plasticity in neurons requires rapid, multilevel post-transcriptional RNA processing events that provide additional opportunities for dysregulation and consequent introduction or persistence of errors in RNA transcripts. Here we review the accumulating evidence that neurons have an enhanced propensity for errors in RNA processing alongside grossly insufficient defenses to clear misprocessed RNA compared to other cell types. Additionally, we explore how tau, a microtubule-associated protein implicated in Alzheimer's disease and related tauopathies, contributes to deficits in RNA processing and clearance.

Moesin is an effector of tau-induced actin overstabilization, cell cycle activation, and neurotoxicity in Alzheimer's disease.

In Alzheimer's disease, neurons acquire phenotypes that are also present in various cancers, including aberrant activation of the cell cycle. Unlike cancer, cell cycle activation in post-mitotic neurons is sufficient to induce cell death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimer's disease and related "tauopathies." Here we combine network analyses of human Alzheimer's disease and mouse models of Alzheimer's disease and primary tauopathy with studies in Drosophila to discover that pathogenic forms of tau drive cell cycle activation by disrupting a cellular program involved in cancer and the epithelial-mesenchymal transition (EMT). Moesin, an EMT driver, is elevated in cells harboring disease-associated phosphotau, over-stabilized actin, and ectopic cell cycle activation. We further find that genetic manipulation of Moesin mediates tau-induced neurodegeneration. Taken together, our study identifies novel parallels between tauopathy and cancer.

Pathogenic tau decreases nuclear tension in cultured neurons.

Neurodegenerative tauopathies, including Alzheimer's disease, are pathologically defined by the presence of aggregated forms of tau protein in brains of affected individuals. Previous studies report that the negative effects of pathogenic tau on the actin cytoskeleton and microtubules cause a toxic destabilization of the lamin nucleoskeleton and formation of nuclear invaginations and blebs. Based on the known function of the nucleus as a mechanosensor, as well as the high incidence of nuclear pleomorphism in human Alzheimer's disease and related tauopathies, we investigated the effects of pathogenic tau on nuclear tension. We first find that tau-dependent nuclear envelope invagination and relocalization of LInker of Nucleoskeleton and Cytoskeleton (LINC) complex components are conserved in a newly-developed neuroblastoma cell line that features doxycycline-inducible expression of a tau mutant associated with autosomal dominant frontotemporal dementia. We next determine that a Förster resonance energy transfer (FRET)-based sensor of nuclear tension responds to cytoskeletal stabilization and destabilization when expressed in neuroblastoma cells. Using this nuclear tension sensor, we find that induced expression of pathogenic tau is sufficient to decrease nuclear tension. This work provides the initial proof-of-concept evidence that pathogenic forms of tau alter nuclear tension, paving the way for the future study of altered nuclear mechanosensing in the context of tau-mediated neurodegenerative disorders.

Pathogenic tau-induced transposable element-derived dsRNA drives neuroinflammation.

Deposition of tau protein aggregates in the brain of affected individuals is a defining feature of "tauopathies," including Alzheimer's disease. Studies of human brain tissue and various model systems of tauopathy report that toxic forms of tau negatively affect nuclear and genomic architecture, identifying pathogenic tau-induced heterochromatin decondensation and consequent retrotransposon activation as a causal mediator of neurodegeneration. On the basis of their similarity to retroviruses, retrotransposons drive neuroinflammation via toxic intermediates, including double-stranded RNA (dsRNA). We find that dsRNA and dsRNA sensing machinery are elevated in astrocytes of postmortem brain tissue from patients with Alzheimer's disease and progressive supranuclear palsy and in brains of tau transgenic mice. Using a Drosophila model of tauopathy, we identify specific tau-induced retrotransposons that form dsRNA and find that pathogenic tau and heterochromatin decondensation causally drive dsRNA-mediated neurodegeneration and neuroinflammation. Our study suggests that pathogenic tau-induced heterochromatin decondensation and retrotransposon activation cause elevation of inflammatory, transposable element-derived dsRNA in the adult brain.

Tau-Induced Elevation of the Activity-Regulated Cytoskeleton Associated Protein Arc1 Causally Mediates Neurodegeneration in the Adult Drosophila Brain.

Alzheimer's disease and other tauopathies are neurodegenerative disorders pathologically defined by aggregated forms of tau protein in the brain. While synaptic degradation is a well-established feature of tau-induced neurotoxicity, the underlying mechanisms of how pathogenic forms of tau drive synaptic dysfunction are incompletely understood. Synaptic function and subsequent memory consolidation are dependent upon synaptic plasticity, the ability of synapses to adjust their structure and strength in response to changes in activity. The activity regulated cytoskeleton associated protein ARC acts in the nucleus and at postsynaptic densities to regulate various forms of synaptic plasticity. ARC harbors a retrovirus-like Gag domain that facilitates ARC multimerization and capsid formation. Trans-synaptic transfer of RNA-containing ARC capsids is required for synaptic plasticity. While ARC is elevated in brains of patients with Alzheimer's disease and genetic variants in ARC increase susceptibility to Alzheimer's disease, mechanistic insight into the role of ARC in Alzheimer's disease is lacking. Using a Drosophila model of tauopathy, we find that pathogenic tau significantly increases multimeric species of the protein encoded by the Drosophila homolog of ARC, Arc1, in the adult fly brain. We find that Arc1 is elevated within nuclei and the neuropil of tau transgenic Drosophila, but does not localize to synaptic vesicles or presynaptic terminals. Lastly, we find that genetic manipulation of Arc1 modifies tau-induced neurotoxicity, suggesting that tau-induced Arc1 elevation mediates neurodegeneration. Taken together, our results suggest that ARC elevation in human Alzheimer's disease is a consequence of tau pathology and is a causal factor contributing to neuronal death.

Tau-induced deficits in nonsense-mediated mRNA decay contribute to neurodegeneration.

INTRODUCTION: While brains of patients with Alzheimer's disease and related tauopathies have evidence of altered RNA processing, we lack a mechanistic understanding of how altered RNA processing arises in these disorders and if such changes are causally linked to neurodegeneration. METHODS: Using Drosophila melanogaster models of tauopathy, we find that overall activity of nonsense-mediated mRNA decay (NMD), a key RNA quality-control mechanism, is reduced. Genetic manipulation of NMD machinery significantly modifies tau-induced neurotoxicity, suggesting that deficits in NMD are causally linked to neurodegeneration. Mechanistically, we find that deficits in NMD are a consequence of aberrant RNA export and RNA accumulation within nuclear envelope invaginations in tauopathy. We identify a pharmacological activator of NMD that suppresses neurodegeneration in tau transgenic Drosophila, indicating that tau-induced deficits in RNA quality control are druggable. DISCUSSION: Our studies suggest that NMD activators should be explored for their potential therapeutic value to patients with tauopathies.

Exploration and analysis of R-loop mapping data with RLBase.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. In 2012, Ginno et al. introduced the first R-loop mapping method. Since that time, dozens of R-loop mapping studies have been conducted, yielding hundreds of publicly available datasets. Current R-loop databases provide only limited access to these data. Moreover, no web tools for analyzing user-supplied R-loop datasets have yet been described. In our recent work, we reprocessed 810 R-loop mapping samples, building the largest R-loop data resource to date. We also defined R-loop consensus regions and developed a framework for R-loop data analysis. Now, we introduce RLBase, a user-friendly database that provides the capability to (i) explore hundreds of public R-loop mapping datasets, (ii) explore R-loop consensus regions, (iii) analyze user-supplied data and (iv) download standardized and reprocessed datasets. RLBase is directly accessible via the following URL: https://gccri.bishop-lab.uthscsa.edu/shiny/rlbase/.

TauLUM, an in vivo Drosophila sensor of tau multimerization, identifies neuroprotective interventions in tauopathy.

Tau protein aggregates are a defining neuropathological feature of "tauopathies," a group of neurodegenerative disorders that include Alzheimer's disease. In the current study, we develop a Drosophila split-luciferase-based sensor of tau-tau interaction. This model, which we term "tauLUM," allows investigators to quantify tau multimerization at individual time points or longitudinally in adult, living animals housed in a 96-well plate. TauLUM causes cell death in the adult Drosophila brain and responds to both pharmacological and genetic interventions. We find that transgenic expression of an anti-tau intrabody or pharmacological inhibition of HSP90 reduces tau multimerization and cell death in tauLUM flies, establishing the suitability of this system for future drug and genetic modifier screening. Overall, our studies position tauLUM as a powerful in vivo discovery platform that leverages the advantages of the Drosophila model organism to better understand tau multimerization.

Quality-controlled R-loop meta-analysis reveals the characteristics of R-loop consensus regions.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 810 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate R-loop data quality control method, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called 'R-loop regions' (RL regions). In the process, we identified a stark divergence between RL regions detected by S9.6 and dRNH-based mapping methods, particularly with respect to R-loop size, location, and colocalization with RNA binding factors. Taken together, this work provides a much-needed method to assess R-loop data quality and offers novel context regarding the differences between dRNH- and S9.6-based R-loop mapping approaches.

Pathogenic tau accelerates aging-associated activation of transposable elements in the mouse central nervous system.

Transposable elements comprise almost half of the mammalian genome. A growing body of evidence suggests that transposable element dysregulation accompanies brain aging and neurodegenerative disorders, and that transposable element activation is neurotoxic. Recent studies have identified links between pathogenic forms of tau, a protein that accumulates in Alzheimer's disease and related "tauopathies," and transposable element-induced neurotoxicity. Starting with transcriptomic analyses, we find that age- and tau-induced transposable element activation occurs in the mouse brain. Among transposable elements that are activated at the RNA level in the context of brain aging and tauopathy, we find that the endogenous retrovirus (ERV) class of retrotransposons is particularly enriched. We show that protein encoded by Intracisternal A-particle, a highly active mouse ERV, is elevated in brains of tau transgenic mice. Using two complementary approaches, we find that brains of tau transgenic mice contain increased DNA copy number of transposable elements, raising the possibility that these elements actively retrotranspose in the context of tauopathy. Taken together, our study lays the groundwork for future mechanistic studies focused on transposable element regulation in the aging mouse brain and in mouse models of tauopathy and provides support for ongoing therapeutic efforts targeting transposable element activation in patients with Alzheimer's disease.

Current directions in tau research: Highlights from Tau 2020.

Studies supporting a strong association between tau deposition and neuronal loss, neurodegeneration, and cognitive decline have heightened the allure of tau and tau-related mechanisms as therapeutic targets. In February 2020, leading tau experts from around the world convened for the first-ever Tau2020 Global Conference in Washington, DC, co-organized and cosponsored by the Rainwater Charitable Foundation, the Alzheimer's Association, and CurePSP. Representing academia, industry, government, and the philanthropic sector, presenters and attendees discussed recent advances and current directions in tau research. The meeting provided a unique opportunity to move tau research forward by fostering global partnerships among academia, industry, and other stakeholders and by providing support for new drug discovery programs, groundbreaking research, and emerging tau researchers. The meeting also provided an opportunity for experts to present critical research-advancing tools and insights that are now rapidly accelerating the pace of tau research.

The role of nuclear Ca2+ in maintaining neuronal homeostasis and brain health.

Nuclear Ca2+ has emerged as one of the most potent mediators of the dialogue between neuronal synapses and the nucleus that regulates heterochromatin states, transcription factor activity, nuclear morphology and neuronal gene expression induced by synaptic activity. Recent studies underline the importance of nuclear Ca2+ signaling in long-lasting, activity-induced adaptation and maintenance of proper brain function. Diverse forms of neuroadaptation require transient nuclear Ca2+ signaling and cyclic AMP-responsive element-binding protein (CREB1, referred to here as CREB) as its prime target, which works as a tunable switch to drive and modulate specific gene expression profiles associated with memory, pain, addiction and neuroprotection. Furthermore, a reduction of nuclear Ca2+ levels has been shown to be neurotoxic and a causal factor driving the progression of neurodegenerative disorders, as well as affecting neuronal autophagy. Because of its central role in the brain, deficits in nuclear Ca2+ signaling may underlie a continuous loss of neuroprotection in the aging brain, contributing to the pathophysiology of Alzheimer's disease. In this Review, we discuss the principles of the 'nuclear calcium hypothesis' in the context of human brain function and its role in controlling diverse forms of neuroadaptation and neuroprotection. Furthermore, we present the most relevant and promising perspectives for future studies.