Beyond the ends: potential implications of telomeric repeat-containing RNA (TERRA) for CNS diseases

Abstract

Telomeric repeat-containing RNA (TERRA) is a class of non-coding RNAs emanating from telomeres and controlling telomere dynamics. Recent studies showed that TERRAs influence chromatin structure and gene expression. TERRAs can also play a crucial role in controlling inflammation, oxidative stress, DNA damage, and cellular senescence. This review discusses the significance of TERRAs in modulating these processes, particularly in the central nervous system (CNS). While our understanding of TERRAs largely stems from cancer research, their involvement in these physiologic and pathologic pathways highlights their potential as therapeutic targets for CNS disorders as well.

Introduction

In mammals and rodents, ∼80% of the genome is transcribed, and the majority of those transcripts are different classes of non-coding RNAs (ncRNAs) that exhibit high diversity in length and genomic origin (intragenic, intergenic, telomeric, and subtelomeric) (Luke and Lingner, 2009; Li and Liu, 2019; Zhang et al., 2019b; Park et al., 2022). The ncRNAs encompass diverse functional categories including housekeeping RNAs, such as transfer RNA and ribosomal RNA, regulatory RNAs, such as microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), and glycoRNA, and chromatin-associated RNAs, such as enhancer RNA, small nucleolar RNA (snRNA), and telomeric repeat-containing RNA (TERRA) (Zhang et al., 2019b; Tang et al., 2023; Yin and Shen, 2023).

Many studies showed that various ncRNAs play significant roles in health and disease by interacting with proteins and RNAs to modulate transcription, translation, chromatin dynamics, and cell signaling pathways (Barrandon et al., 2008; Han et al., 2020; Statello et al., 2021; Zhang et al., 2021; Tang et al., 2023; Palihati and Saitoh, 2024). In particular, miRNAs, lncRNAs, and circRNAs were shown to influence the pathophysiology of immune disorders, cancer, stroke, traumatic brain injury, spinal cord injury, and chronic neurological disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD) (Han et al., 2020; Mehta et al., 2020, 2024; Zhang et al., 2021; Fagan and Pfeiffer, 2022; Li et al., 2024). However, the role of some classes of ncRNAs, such as TERRA, in physiologic and pathologic states is still evolving.

TERRAs result from telomere transcription (Luke and Lingner, 2009). Telomeres, composed of repetitive DNA sequences and a protein complex called shelterin, serve as protective caps on chromosomal ends (Stewart et al., 2012; Smith et al., 2020; Lim and Cech, 2021). This structure is crucial for preventing DNA damage response (DDR) from recognizing them as sites of DNA damage. This protection ensures telomere homeostasis, maintains genome stability, and prevents cell senescence (Karlseder et al., 2004; Doksani et al., 2013).

Telomere dysfunction arises when telomeres become critically shortened or uncapped, often due to continuous loss of telomeric repeats without adequate repair, leading to decreased shelterin complex association and subsequent activation of DDR (Fagagna et al., 2003; De Lange, 2005; Xu et al., 2021; Rossiello et al., 2022). This will be exacerbated by oxidative stress, inflammation, and aging, contributing to various disease states. Specifically, shortened telomeres are linked to aging-associated diseases such as cardiovascular disease, neurodegeneration, and cancer (von Zglinicki, 2002; Collado et al., 2007; Pfeiffer and Lingner, 2013; Correia-Melo et al., 2014; Kam et al., 2021; Eppard et al., 2023).

Recent studies highlight the role of TERRAs in modulating telomere dynamics and maintaining genomic stability (Balk et al., 2013; Bettin et al., 2019; Rivosecchi et al., 2024). TERRAs interact with shelterin subunits, such as telomeric repeat binding factor 2 (TRF2), and modulate the recruitment of other subunits, such as protection of telomeres 1 (POT1), to telomeres, as well as regulate telomerase activity (Redon et al., 2010; Flynn et al., 2011; Mei et al., 2021). Furthermore, TERRAs participate in telomere dynamics by forming specialized structures with genomic DNA and altering chromatin structure and gene expression, thereby influencing genome stability (Paeschke et al., 2005; Roake and Artandi, 2017; Fernandes et al., 2021; Mei et al., 2021; Statello et al., 2021; Glousker et al., 2022; Tsai et al., 2022; Gong and Liu, 2023).

Telomere dysfunction, genomic instability, and cellular senescence are the key hallmarks of aging and are closely linked to the progression of neurodegenerative diseases such as AD and PD, as well as other age-related disorders such as cardiovascular diseases and cancer (Flanary and Streit, 2004; Ferrón et al., 2009; Eitan et al., 2014). In gliomas, TERRA expression correlates with telomere maintenance mechanisms related to tumor progression. In astrocytomas, TERRA levels are significantly lower compared to that in normal brain tissue, and the expression is associated with alternative lengthening of telomeres (ALT) and telomerase activity (Sampl et al., 2012). Beyond cancer, TERRA levels increase with age in the human brain and are upregulated during differentiation of human embryonic stem cells to neuronal phenotypes (Hsieh et al., 2024). In AD, increased TERRA levels were detected in various brain cell types, particularly excitatory neurons, during early pathological stages (Hsieh et al., 2024). Moreover, inflammatory signaling in mice mediated by tumor necrosis factor-α (TNF-α) induces telomere shortening accompanied by increased levels of TERRA (Hsieh et al., 2024), suggesting a link between neuroinflammation, telomere dysfunction, and TERRA regulation.

TERRAs have roles beyond telomere biology. They have been shown to trigger inflammatory responses (Wang et al., 2015; Wang and Lieberman, 2016) and bind to proteins such as heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and THO complex (THOC) that influence RNA processing, cellular morphology, and survival (Maeder et al., 2018; Kumar et al., 2020; Lalonde and Chartrand, 2020; Anees et al., 2021; Lim and Cech, 2021). Recent studies also suggested that TERRAs have the potential to code for dipeptide repeats (DPRs) (Al-Turki and Griffith, 2023; De Rosa and Opresko, 2023). All these molecules (hnRNPA1, THOC, and DPRs) were shown to be altered in neurodegenerative diseases (Chi et al., 2009; Maeder et al., 2018; Ségal-Bendirdjian and Geli, 2019; Kumar et al., 2020; Martins et al., 2020; Clarke et al., 2021).

This review delved into the potential implications of TERRAs in the central nervous system (CNS), drawing from their established roles in telomere biology, inflammation, oxidative stress, and cell senescence.

Transcription and structure of TERRAs

Transcription of TERRAs is a conserved feature in eukaryotes with linear chromosomes (Silva et al., 2021) and can originate from both telomeric and subtelomeric regions of most, if not all, chromosomes (Figure 1; Azzalin et al., 2007; Nergadze et al., 2009; Feuerhahn et al., 2010; Arnoult et al., 2012; Porro et al., 2014a; Negishi et al., 2015; Silva et al., 2021). TERRAs contain telomeric repetitive sequences of 5′-TTAGGG-3′ (Statello et al., 2021) vary in length from 100 bp to 9 kb (Feuerhahn et al., 2010). They are also characterized by a methyl cap (Figure 1; Luke and Lingner, 2009; Feuerhahn et al., 2010). A small fraction (7%–10%) of TERRAs are also polyadenylated (polyA⁺), which enhances their stability (Figure 1; Feuerhahn et al., 2010).

Figure 1.

Characteristics and transcription regulatory elements of TERRAs. (i) Structure of TERRA: TERRAs are composed of repeat sequences of UUAGGG, a 7-methylguanosine cap (m7G), and G4 structure. (ii) Types of TERRA: TERRA can either be polyadenylated (polyA⁺) or not (polyA⁻). TERRA lengths vary from 100 bp to several kb and harbor N⁶-methyladenosine (m⁶A). (iii) Transcription of TERRA: TERRAs are transcribed by RNA polymerase II. Type I and II promoters in subtelomeric regions regulate TERRA transcription, which are CpG-rich and contain CCCTC binding factor (CTCF) binding sites along with other TFs. Type I promoters harbor tandem repeats of 61-29-37 bp. Telomeric and subtelomeric regions are rich in repressive heterochromatin (histone methylation marks: H3K9me3 and H4K20me3). Euchromatin marks (histone acetylation: H3K27Ac) exist at low levels. DNA methyaltransferases (DNMT) 1/3B suppress TERRA expression through methylation of CpG islands. Created with Biorender.com.

Similar to other RNAs, the stability of TERRAs increases with methylation (m⁶A) (Chen et al., 2022). However, METTL3-mediated m⁶A methylation is observed only in the subtelomeric TERRAs (Chen et al., 2022). These characteristics indicate a tight regulation of TERRAs for specific cell-dependent and context-dependent physiological functions. The chromatin state at telomeric and subtelomeric regions is enriched with repressive histone methylation marks (H3K9me3 and H4K20me3) that affect the transcription of TERRAs (Figure 1; Caslini et al., 2009; Oliva-Rico et al., 2022) and euchromatin marks (H3K4me3 and H3K27Ac) that promote transcription of TERRAs (Figure 1; Barral and Déjardin, 2020). The synthesis of TERRAs is facilitated by RNA polymerase II, indicating that they are transcribed rather than being the product of post-transcriptional processing like circRNAs (Schoeftner and Blasco, 2008; Feuerhahn et al., 2010).

Subtelomeric regions are rich in repetitive elements containing CpG islands that exhibit promoter activity (Nergadze et al., 2009). Specifically, two types of promoters (I and II) have been found to drive transcription of TERRAs (Figure 1; Nergadze et al., 2009; Deng et al., 2012; Porro et al., 2014a). In type I promoters, transcription of TERRAs is controlled by DNA methylation driven by DNA methyltransferases DNMT1 and DNMT3B at the CpG islands containing 61-29-37 bp tandem repeats (Figure 1; Barral and Déjardin, 2020). Type II promoters are located 5–10 kb upstream of the telomere tract (Barral and Déjardin, 2020). The mechanisms that regulate type II promoter activity are not clear. However, the presence of two types of promoters suggests differential regulation of TERRA expression in physiological or pathological contexts.

TERRA promoters within subtelomeric regions are known to contain binding sites for transcription factors (TFs) such as nuclear respiratory factor 1, CCCTC-binding factor, tumor protein p53, heat shock factor 1 (HSF1), and chromatin remodeler (ATRX) (Nergadze et al., 2009; Deng et al., 2012; Beishline et al., 2017). Inactivation of these TFs has been shown to affect the transcription of TERRAs (Deng et al., 2012; Diman et al., 2016; Koskas et al., 2017; Feretzaki et al., 2019). Other proteins also regulate the expression of TERRAs. For instance, deletion of TRF1 in mouse embryonic stem cells increased levels of TERRAs (Marión et al., 2019). Conversely, in human cancer cells, deletion of the GAR domain of TRF2, where TERRAs bind, decreased the expression of TERRAs (Mei et al., 2021).

TERRAs may contribute to their own expression via feedback loops. For example, TERRA R-loops can recruit breast and ovarian cancer susceptibility protein 1 (BRCA1), a DNA repair-associated protein, at CpG-rich TERRA promoters to repress TERRA transcription via recruiting epigenetic modifiers (e.g. DNMT3B) (Vohhodina et al., 2021). In HeLa cells treated with the histone deacetylase inhibitor Trichostatin A, levels of TERRAs increased (Azzalin and Lingner, 2008). Additionally, the histone methyltransferase MLL was shown to increase levels of TERRAs in human fibroblasts (Caslini et al., 2009). Furthermore, loss of heterochromatin protein 1 (HP1), essential in maintaining H3K9me3, also disrupts the expression of TERRAs in various human cell types (Arnoult et al., 2012; Lim and Cech, 2021).

TERRAs are mainly detected in nuclear fractions with partial colocalization with telomeres (Feuerhahn et al., 2010; Yamada et al., 2016; Barral and Déjardin, 2020). They bind to telomere heterochromatin and can also be present in the nucleoplasm (Feuerhahn et al., 2010; Barral and Déjardin, 2020; Lalonde and Chartrand, 2020). Chromatin-associated TERRAs lack a polyA tail, whereas polyA⁺ TERRAs, which are more stable, colocalize in the nucleoplasm (Feuerhahn et al., 2010; Lalonde and Chartrand, 2020). Polyadenylation is also involved in the release of TERRAs from telomeres (Porro et al., 2010). Hence, the processing and maturation of TERRAs may regulate their retention at telomeres.

TERRA binding proteins also play a role in their localization. Mutated TRF2 binding site impaired TERRA localization at telomeric ends in human colorectal cancer cells (HCT116) (Deng et al., 2009). Additionally, the nonsense-mediated RNA decay (NMD) machinery was shown to regulate TERRA localization (Azzalin and Lingner, 2008). Specifically, the binding of NMD subunits, up-frameshift protein 1 and serine/threonine-protein kinase, triggers the degradation of polyA⁻ TERRAs associated with chromatin (Azzalin and Lingner, 2008, 2015). Other TERRA-binding proteins, such as hnRNPs, also displace them from the telomere (De Silanes et al., 2010; Cusanelli and Chartrand, 2015). The presence of redundant localization mechanisms provides a safety net for the function of TERRAs, while enabling precise targeting based on cellular requirements and potentially facilitating interactions with other molecules. TERRAs were also found in extracellular inflammatory exosomes in plasma (Wang et al., 2015). The presence of TERRAs in the nucleoplasm and exosomes suggests their possible interaction with non-telomeric loci and participation in cell–cell communication.

Functions of TERRAs

Roles of TERRAs in telomere dynamics

Recent studies have shown that TERRAs are essential for maintaining telomere integrity by preventing the activation of DDR pathways, modulating telomerase activity to ensure proper telomere elongation, creating R-loop structures at telomeres, and promoting the formation of heterochromatin to protect the chromosomal ends (Redon et al., 2010; Flynn et al., 2011; Montero et al., 2016; Bettin et al., 2019; Lalonde and Chartrand, 2020; Mei et al., 2021; Rivosecchi et al., 2024). Disruption of their expression, localization, or interactions with proteins, DNA, or RNA can lead to significant telomere dysfunction that is detrimental to cell survival and homeostasis, depending on the cellular context and pathological state (Feretzaki et al., 2020; Fernandes et al., 2021; Gong and Liu, 2023).

TERRAs prevent DDR by promoting the assembly of telomere binding and capping proteins. Specifically, TERRAs form G quadruplex (G4) structures that bind to the GAR domain of TRF2, which in turn binds to telomeric DNA and inhibits ATM-dependent DDR, non-homologous end joining, and homologous repair mechanisms (Karlseder et al., 2004; Denchi and De Lange, 2007; Deng et al., 2009; Okamoto et al., 2013; Mei et al., 2021; Scionti et al., 2023). The interaction of TERRAs with hnRNPA1 also facilitates POT1 binding to chromosomal ends, further strengthening the shelterin complex and preventing from recognizing these ends as DNA damage sites (Flynn et al., 2011). It has also been suggested that TERRAs form clusters with telomerase RNA component (TERC) to colocalize with telomeres during the S phase of cellular division, thereby maintaining the telomere length (Porro et al., 2010; Flynn et al., 2015; Montero et al., 2016). Conversely, depletion of TERRAs has been shown to induce telomere DNA damage and loss or duplication of telomeric sequences in mouse embryonic cells (Chu et al., 2017). Heterochromatin formation is also important in telomere homeostasis. The binding of TERRAs and TRF2 is critical for heterochromatin formation at telomeres and further maintaining DNA integrity at those ends (Mei et al., 2021). Additionally, TERRAs can recruit chromatin-modifying complexes, such as polycomb repressive complex 2 (PRC2), which catalyzes the trimethylation of H3K27, a hallmark of heterochromatin (Azzalin et al., 2007; Luke and Lingner, 2009; Deng et al., 2010; Montero et al., 2018).

TERRAs also participate in telomere dynamics via R-loop formation. TERRAs bind to telomeric DNA, forming quadruplex and R-loop structures in cis (TERRA origin of transcription) and acting in trans on other telomeres (far distant telomeres) (De Silanes et al., 2014; Montero et al., 2016). Thus, R-loops can influence the stability of the genome at telomeric ends (Paeschke et al., 2005; Fernandes et al., 2021; Mei et al., 2021; Glousker et al., 2022; Gong and Liu, 2023). In telomerase-negative cancer cells, R-loops aid telomere maintenance (Arora et al., 2014). For example, in cells with ALT mechanisms, TERRA R-loops accumulate at short telomeres and sustain telomeric DNA replication (Redon et al., 2013; Fernandes et al., 2021). ALT is a break-induced replication-based mechanism that elongates telomeres without the involvement of telomerase (Dilley et al., 2016; Zhang et al., 2019a). This mechanism was shown to be active in certain cancer cells, such as osteosarcoma, adrenocortical carcinoma, breast invasive carcinoma, colon adenocarcinoma, lung adenocarcinoma, and pancreatic adenocarcinoma (Henson et al., 2002; Sung et al., 2020). However, it was also observed in normal mammalian cells (Henson et al., 2002; Neumann et al., 2013). RAD51 recombinase, a key player in DNA repair pathways, physically binds to TERRAs and catalyzes R-loop formation (Feretzaki et al., 2020; Kaminski et al., 2022) and hence may contribute to telomere homeostasis. Moreover, methylated TERRAs form R-loops and promote homologous recombination, an essential process for the ALT mechanism (Chen et al., 2022). In contrast, TERRA-mediated R-loops were shown to induce telomere fragility in telomerase-positive cells (Fernandes and Lingner, 2023). Increased levels of TERRAs induce telomere-free ends and telomere shortening by interfering with telomere replication, inducing DNA damage and replication stress via increased R-loop formation (Petti et al., 2019; Feretzaki et al., 2020; Chen et al., 2022; Liu et al., 2023).

Aberrations in TERRA levels are also associated with telomere dysfunction. Several studies have reported that the depletion of TERRAs results in telomere dysfunction via aberrant heterochromatin at telomeres (Cusanelli and Chartrand, 2015; Chu et al., 2017). Indeed, disruption of TERRA transcription resulted in aberrant heterochromatin and dysfunctional telomeres in human osteosarcoma cells (Deng et al., 2009). Conversely, expression of TERRAs destabilizes telomeres by inhibiting telomerase and decreasing telomere length in human and mouse cells (Schoeftner and Blasco, 2008; Redon et al., 2010).

In human ALT cells, TERRA expression was shown to activate break-induced replication (Silva et al., 2021), which is necessary to initiate ALT that delays cell senescence (Cusanelli and Chartrand, 2015), whereas inhibition of TERRA expression in these cells impaired ALT activity and telomere length maintenance (Silva et al., 2021). Furthermore, elevated levels of TERRAs due to loss of DNA methylation at subtelomeric regions have been shown to increase R-loop formation, leading to DNA damage and loss at telomeres in immunodeficiency centromere instability and facial anomalies syndrome cells (Sagie et al., 2017). Deletion of the DNA damage repair protein BRCA1 was also shown to increase TERRA levels and R-loop formation, leading to telomeric DNA damage in human cancer cells (Vohhodina et al., 2021). These findings indicate a need for tight regulation of TERRA expression. Overall, the dual role of TERRAs in telomere dynamics underscores the need for a better understanding of their regulation and function.

Roles of TERRAs in the regulation of chromatin and gene expression

Chromatin structure is crucial in regulating gene expression and is modulated by ncRNAs such as miRNAs, snRNAs, and lncRNAs (Tang et al., 2023; Yin and Shen, 2023; Palihati and Saitoh, 2024). These RNAs interact with chromatin regulators and nucleosome remodeling factors, either by recruiting or sequestering them, thus influencing chromatin organization and gene expression (Francia, 2015; Han and Chang, 2015; Yin and Shen, 2023). Similarly, TERRAs also regulate chromatin organization and gene expression. The following subsections will explore the mechanisms by which TERRAs influence these.

Effects of TERRAs on chromatin status

Heterochromatin protects telomeres from degradation by nucleases and distinguishes telomeres from double-strand breaks (Kwapisz and Morillon, 2020; Warecki and Sullivan, 2022). Modifications of the heterochromatin in telomeres and subtelomeres have been linked to telomere length disturbances (Gonzalo et al., 2006; Benetti et al., 2007; Barral and Déjardin, 2020). TERRAs contribute to the maintenance of heterochromatin by binding to both telomeric and non-telomeric sites. They recruit chromatin remodelers through interactions with repetitive elements complementary to the TERRA sequences (Feuerhahn et al., 2010; Wang et al., 2015; Chu et al., 2017; Barral and Déjardin, 2020; Lalonde and Chartrand, 2020).

In mammalian cells, TERRAs are primarily associated with chromatin (Porro et al., 2010) and their downregulation changes the heterochromatin status at telomeres. For example, in osteosarcoma cells, silencing of TERRAs was shown to correlate with >70% decrease in H3K9 methylation at telomeres, leading to the formation of telomere dysfunction-associated foci, a form of DNA damage that can contribute to cellular senescence depending on the severity and cellular context (Deng et al., 2009).

TERRAs can also function as scaffolds for recruiting and organizing various transacting factors such as TRF2 and PRC2 (Rinn and Chang, 2012; Montero et al., 2018). TERRA–PRC2 complexes maintain histone trimethylation at H3K27 sites (Montero et al., 2018), while TERRA–TRF2 complexes promote heterochromatin formation (Deng et al., 2009; Mei et al., 2021). Other TERRA-binding proteins, such as ATRX chromatin remodeler (Chu et al., 2017; Tsai et al., 2022), SUV39H1 (Porro et al., 2014a), and lysine-specific demethylase 1A (LSD1) (Porro et al., 2014b), play critical roles in chromatin remodeling and histone modifications. Importantly, TERRAs sequester ATRX and prevent its allocation to telomeric DNA binding sites (Tsai et al., 2022). This sequestration controls heterochromatin formation necessary for genomic stability and telomere homeostasis (Chu et al., 2017; Teng et al., 2021). Additionally, telomeric and subtelomeric regions are enriched with histone methylation marks bound to HP1 (Nishibuchi and Déjardin, 2017). TERRAs specifically bind to HP1α isoform (Deng et al., 2009; Roach et al., 2020) that recognizes and binds to the G quadruplexes (Arnoult et al., 2012) to maintain heterochromatin structures.

In summary, the interaction of TERRAs with epigenetic modifiers modulates chromatin status. These interactions occur by either recruiting remodeling factors to specific loci or anchoring them and keeping them away. Interestingly, TERRAs were found in regions other than the telomeres (Chu et al., 2017; Montero et al., 2018), indicating that they can also regulate the chromatin states in the distant locations.

Effects of TERRAs on gene expression

Studies have shown that expression of TERRAs correlated with changes in the transcriptome. Depletion of TERRAs in mouse embryonic cells has been shown to induce significant changes in the expression of genes. Examples of these include downregulated genes associated with mTOR signaling and transcriptional regulation and upregulated genes associated with telomere capping (Chu et al., 2017). Furthermore, in ALT cells, suppression of TERRA transcription resulted in up/downregulation of genes involved in nervous system development, cell movement, cell cycle progression, and cell death (Silva et al., 2021).

TERRAs in the nucleoplasm are believed to be transacting and, hence, their effect may spread to other locations of the genome (Azzalin et al., 2007; Schoeftner and Blasco, 2008). TERRAs were also reported to target intragenic and intergenic regions of chromosomes, including binding to near transcription and terminal sites of genes (Chu et al., 2017; Montero et al., 2018; Marión et al., 2019). TERRAs can also regulate transcription by inhibiting ATRX suppression of genes. This was shown to be mediated by sequestering the ATRX protein, thus preserving the DNA G4 structure around gene transcriptional start sites in mouse embryonic cells (Tsai et al., 2022). Moreover, TERRA-depleted cells showed a decrease in DNA G structure, which correlated with altered gene expression (Tsai et al., 2022).

TERRAs also regulate gene expression through telomere positioning effect (TPE), which silences long telomeres by forming loops to induce heterochromatin in distal genomic regions, thereby repressing gene expression in subtelomeric regions (Hu et al., 2019). The impact of telomere silencing is similar to that of heterochromatin, but this phenomenon can occur in regions devoid of heterochromatin due to the high-order chromatin arrangement (Ottaviani et al., 2008; Rédei, 2008; Tennen et al., 2011; Lee et al., 2021). Associations of TERRAs with telomeres and epigenetic modifiers in subtelomeric regions suggest a potential contribution to TPE.

Potential implications of TERRA

DNA damage, oxidative stress, and neuroinflammation, which are intertwined with telomere dysfunction and cell senescence, lead to neurodegenerative diseases (Martínez-Cué and Rueda, 2020). Telomere attrition leads to genomic instability and activation of DDR, ultimately causing cell senescence (Eitan et al., 2014; Harley et al., 2023). Studies have indicated that altered telomerase activity contributes to the pathogenesis of CNS diseases such as brain tumors, PD, AD, and amyotrophic lateral sclerosis (Uzun Cicek et al., 2020; Kuan et al., 2023; Shao et al., 2024).

Neuronal function depends on healthy glial cells to provide structural, metabolic, and trophic support (Flanary, 2005; Colonna and Butovsky, 2017; Savage et al., 2019; Woollacott et al., 2020). Microglial cells, the primary immune cells in the CNS, are activated by acute injuries to the CNS, such as stroke and traumatic brain injury (TBI) (Jessen, 2004; Wolf et al., 2017). Their morphology and function also change during aging, contributing to the onset of neurodegenerative diseases (Cornejo and von Bernhardi, 2016; Colonna and Butovsky, 2017; Savage et al., 2019; Woollacott et al., 2020; Green and Rowe, 2024). Telomere shortening may also trigger the observed senescent microglial phenotype and promotes the onset and progression of PD and AD (Flanary and Streit, 2004; Cai et al., 2013; Papageorgakopoulou et al., 2024). Additionally, TBI is positively correlated with telomere shortening in microglia (Papageorgakopoulou et al., 2024), which may contribute to the development of neurodegenerative diseases. Altered microglia along with telomere shortening and increased TERRA expression may exacerbate the development of CNS diseases (Cornejo and von Bernhardi, 2016; Colonna and Butovsky, 2017; Savage et al., 2019). In gliomas, TERRA expression plays a crucial role in telomere maintenance mechanisms and tumor progression. A study on astrocytoma revealed that TERRA levels were correlated with the active ALT mechanism in high-grade astrocytoma, while telomerase-active tumors displayed lower levels of TERRA (Sampl et al., 2012). Furthermore, TERRA accumulation has been observed in rapidly proliferating neural progenitor cells and brain tumors. For example, in a mouse model of medulloblastoma, TERRA formed distinct nuclear focus in dividing cells independent of traditional DNA damage or replication stress markers (Deng et al., 2012). Notably, the Sonic hedgehog signaling pathway, which drives neural progenitor proliferation, was found to enhance TERRA expression. TERRA upregulation was also observed in highly proliferative progenitor cells during brain development, suggesting a role in neural proliferation and possibly tumorigenesis (Deng et al., 2012).

These findings suggest that TERRA plays a complex role in both cancer and neurodegeneration. Further studies are needed to clarify whether TERRA primarily acts as a protective factor or contributes to disease progression in these contexts.

Disruption of TERRA function could lead to impaired telomere dynamics, increased oxidative stress, DNA damage, and cellular senescence, all of which may drive chronic CNS disorders. The interaction of TERRA with proteins such as telomerase reverse transcriptase (TERT), hnRNPA1, and THOC, which are dysregulated in various CNS diseases, strengthens this hypothesis. Figure 2 illustrates the putative functions of TERRAs in the CNS.

Figure 2.

Putative functions of TERRAs in the CNS. TERRAs are involved in telomere dynamics. (i) Telomere shortening: homeostatic TERRA levels are necessary for maintaining telomere length. Loss of shelterin protein, oxidative stress, DNA damage, and aging lead to an imbalance in TERRA levels that promote telomere shortening. (ii) Inflammation: cell-free TERRAs in exosomes can trigger inflammation by promoting the expression of pro-inflammatory molecules like interleukin-6 (IL-6), CXC chemokine 10 (CXCL10), and TNF-α via telomere-associated molecular patterns (TAMP). (iii) Translation to DPRs: TERRAs can be translated into DPRs via repeat-associated non-ATG (RAN) translation. DPRs can contribute to inflammation and NE dysfunction. (iv) Nuclear pore and envelope deregulation: DPR-induced NE dysfunction includes nuclear pore obstruction, affecting the transport of molecules between the nucleus and the cytoplasm. TERRA–NE interaction can potentially affect telomere tethering to the NE, impacting telomere maintenance. (v) Interference with hnRNPA1 and THOC: TERRAs can interfere with hnRNPA1 and THOC function via physical binding and sequestration, subsequently altering RNA processing, neuronal morphology, and survival. When TERRA levels are low, high levels of hnRNPA1 can affect telomere length via binding to single-stranded DNA and preventing telomerase activity. (vi) Inhibition of telomere elongation: TERRAs interfere with TERT, thus inhibiting telomere length maintenance. Created with Biorender.com.

TERRAs and oxidative stress

Telomeres are particularly susceptible to DNA damage by reactive oxygen species (ROS) compared to other genomic regions due to the presence of triplet guanine in the telomeric repeats (Rhee et al., 2011; Gavia-García et al., 2021; Gao et al., 2022). Under oxidative stress, heat shock factors such as HSF1 are activated, leading to the expression of heat shock proteins (Åkerfelt et al., 2010). These proteins act as chaperones, assisting in DNA repair and preventing protein misfolding and aggregation, which collectively protect cells from oxidative damage (Åkerfelt et al., 2010; Dubrez et al., 2020).

In HeLa cells with stable HSF1 knockdown, stress-induced telomeric DNA damage was observed, highlighting the critical role of HSF1 in protecting telomeres under stress (Koskas et al., 2017). Conversely, active HSF1 binds to subtelomeric regions and increases TERRA expression, which appears to confer telomere protection (Koskas et al., 2017). Environmental stressors such as UV radiation and cold exposure induce oxidative stress and further alter TERRA expression, affecting telomere integrity and cellular stress responses (Blagojevic et al., 2011; de Jager et al., 2017; Cong et al., 2018; Wei et al., 2024). For example, UV-C radiation decreased and heat shock increased TERRA levels in mouse embryonic fibroblasts (Schoeftner and Blasco, 2008; De Silanes et al., 2014). In mice exposed to cold, levels of TERRAs increased in brown adipose tissue (Galigniana et al., 2020). In this context, oxidative stress has been shown to elevate TERRA levels, cAMP levels, and cytoskeleton dynamics (Galigniana et al., 2020). Telomere shortening can also enhance oxidative stress. For example, macrophages from TERC-deficient mice and aged mice with shorter telomeres showed an accumulation of ROS (Sebastián et al., 2009). These macrophages also exhibited reduced proliferation (Sebastián et al., 2009).

TERRAs and DNA damage

TERRAs were shown to be associated with DNA damage at telomeres that leads to neurodegeneration (Flanary and Streit, 2004; Ferrón et al., 2009; Eitan et al., 2014). Depletion of Tel20q TERRA was shown to induce DNA damage in chromosomal ends of osteosarcoma cells (Montero et al., 2016). Similarly, depletion of Tel15q TERRA was shown to promote DNA damage in gastric adenocarcinoma cells (Avogaro et al., 2018). Using live-cell imaging, depletion of MS2-tagged TERRA transcripts was shown to activate DDR in both telomeric and extratelomeric regions (Avogaro et al., 2018). Moreover, partial depletion of Tel18q TERRA in mouse embryonic cells activated DDR and resulted in telomeric DNA damage (De Silanes et al., 2014). Likewise, silencing TERRAs using siRNAs and ASL-locked nucleic acids (LNAs) resulted in the production of telomere dysfunction-induced foci due to the promotion of DNA damage (Deng et al., 2009; De Silanes et al., 2014; Chu et al., 2017). Intriguingly, expression of TERRAs due to TRF2 depletion contributed to DDR by recruiting LSD1 and SUV39H1 to dysfunctional telomeres (Porro et al., 2014b; Azzalin and Lingner, 2015). TERRAs may also indirectly affect DNA damage by modulating the expression of genes involved in telomere capping (Chu et al., 2017). Furthermore, TERRA's ability to interact with non-telomeric DNA suggests TERRA R-loop formation at distant genomic locations, potentially interfering with replication forks and leading to DNA breaks throughout the genome.

These findings suggest a critical role for TERRAs in maintaining telomere integrity and preventing DNA damage, potentially linking them to the development of neurodegenerative diseases and aging.

TERRAs and inflammation

Decreased telomeric length in leukocytes was shown to be associated with increased inflammation (Wong et al., 2014). Similarly, in telomerase-deficient mice, cells exhibit increased production of pro-inflammatory molecules via Toll-like receptor 4 activation (Bhattacharjee et al., 2010). Furthermore, telomere uncapping in human vasculature is associated with inflammation, independent of telomere length (Morgan et al., 2013). ROS trigger inflammation and intensify telomere dysfunction (Jurk et al., 2014). Dysfunctional telomeres in human lung fibroblasts were shown to activate innate immune responses through mitochondrial TERRA–ZBP1 association (Nassour et al., 2023). Similarly, a putative association between TERRAs and inflammation was reported in HeLa cells, where 80% of TERRA proximal promoters were found to harbor binding sites for NFKB1, a master inflammatory TF (Porro et al., 2014a). While most TERRAs are chromatin-associated, they were also found cell-free in the nucleoplasm and exosomes. Cell-free TERRAs (cf-TERRAs) have been shown to trigger several pro-inflammatory cytokines, including IL-6, CXCL10, and TNF-α in human peripheral blood mononuclear cells (Wang et al., 2015; Wang and Lieberman, 2016). Exosomal transport of cf-TERRAs as a telomere-associated molecular pattern and alarmin from dysfunctional telomeres to the extracellular environment was proposed to trigger an inflammatory response (Wang and Lieberman, 2016).

Involvement of TERRA in CNS diseases through its interacting partners

TERRAs were shown to bind to various RNA-binding proteins, such as telomerase, THOC, and hnRNPA1, that contribute to the CNS homeostasis and pathologies.

TERRAs and telomerase

TERT, the enzymatic unit of telomerase, maintains telomeres in dividing cells by adding DNA repeats to the 3′ ends of the chromosomes (Schrumpfová and Fajkus, 2020; Smith et al., 2020). However, recent studies suggested additional functions of TERT in the CNS (Ségal-Bendirdjian and Geli, 2019). Telomerase is present at high levels in neural stem cells, neural progenitor cells, microglia, and vascular endothelial cells of humans, with stable expression in adult brain (Liu et al., 2018). This is important for preserving telomere length and homeostasis (Villa et al., 2004). Telomerase deficiency and telomere shortening are associated with impaired neural differentiation, aberrant neurogenesis, and an increased risk of age-related neurodegenerative diseases (Harley et al., 2023).

Telomerase activity potentially plays non-canonical roles beyond telomere elongation (Liu et al., 2018; Saretzki, 2023). These non-canonical functions include shuttling the TERT protein out of the nucleus into the cytoplasm and mitochondria upon increased oxidative stress (Saretzki, 2023). The same process occurs in neuronal cells as well (Spilsbury et al., 2015). Mitochondrial TERT can protect cells from oxidative stress, DNA damage, and apoptosis (Saretzki, 2023). TERC and TERRAs function as telomerase-limiting factors to maintain telomere length and control the survival of neural stem cells in adults (Grammatikakis et al., 2014; Pereira Fernandes et al., 2018).

TERRA levels are significantly lower in glioblastomas compared to normal brain tissue, and this decrease correlates with the presence of telomerase activity, suggesting that TERRA might inhibit telomerase function. Elevated TERRA levels, along with longer telomeres and absence of telomerase activity, are associated with lower tumor grade and improved survival, highlighting TERRA's role in regulating telomere dynamics and tumor progression (Sampl et al., 2012).

As previously noted, TERRAs bind to TERT and TERC components of telomerase and play a dual role in its activity (Lalonde and Chartrand, 2020). Whether TERRA–TERT binding aids in TERT mitochondrial relocation to protect cells from oxidative stress and DNA damage warrants investigation. Further, TERT has been shown to interact with the NMD factor EST1A/SMG6, which also binds TERRAs to displace them from telomeres (Reichenbach et al., 2003; Redon et al., 2007; Azzalin and Lingner, 2015). This physical binding releases TERRAs and allows telomerase activity. Recently, TERT was shown to have a protective role in neurons by counteracting the effects of toxic neurodegenerative proteins via changes in gene expression, activation of neurotrophic factors, and protein-degrading pathways such as autophagy (Saretzki, 2023). It is possible that the binding of TERRAs to TERT may impede its protective function in neurons. These findings indicate a potential role for TERRA–telomerase interaction in neurodegenerative diseases.

TERRAs and the nuclear envelope

The nuclear envelope (NE) is essential for maintaining genome stability (Pennarun et al., 2023). NE alterations are associated with aging and diseases (Chi et al., 2009; Martins et al., 2020). Importantly, telomeres interact with the NE, a tethering that helps in maintaining the heterochromatin in these ends (Crabbe et al. 2012; Bridger et al., 2016). Proteins mediating telomere–NE tethering are thought to ensure telomere stability. In yeasts, NE attachment to telomeres is impacted by telomere length, and NE–telomere interactions negatively affect expression of TERRAs (Maestroni et al., 2020).

Another potential TERRA–NE interplay is through DPRs, which are short chains of repeating amino acids implicated in neurodegenerative diseases (Andrade et al., 2020). Interestingly, DPRs can be produced from TERRAs via RAN translation (Al-Turki and Griffith, 2023; De Rosa and Opresko, 2023). Specifically, glycine–leucine (GL) and valine–arginine are feasible from TERRA repeats (De Rosa and Opresko, 2023) with (GL)9 DPRs having the capacity to form filaments and amyloid-like networks (Al-Turki and Griffith, 2023; De Rosa and Opresko, 2023). These TERRA-derived DPRs can potentially affect the NE function by interacting and sequestering the karyopherin subunit beta 1 (KPBN1) proteins, crucial importins for nuclear pore transport and maintenance (Khalil et al., 2024). Studies have shown that DPRs such as poly-PR and arginine-rich can disrupt KPBN1 function and hinder nuclear transport (Hutten et al., 2020; Jafarinia et al., 2022). TERRA–DPR connection presents a compelling new avenue for understanding health and disease. However, it remains unclear whether other DPRs, formed from TERRA sequences, also contribute to these effects. For instance, potential functional consequences of additional TERRA-derived DPRs on NE stability and cellular homeostasis and their effects in different cell types are not yet evaluated. Also, the contribution of different TERRA-derived DPRs and their interactions with the NE to cellular stress responses and pathophysiology of neurodegenerative diseases is also not yet studied.

TERRA–THOC interaction

THOC processes RNA and coordinates transcripts for synapse development and dopamine neuron survival by facilitating their nuclear export for translation (Maeder et al., 2018). THOC mutant mice exhibited decreased presynaptic proteins and defective synapse assembly (Maeder et al., 2018). Dysfunction of the THOC is thought to be a contributing factor to the development of neurodegenerative diseases (Maeder et al., 2018; Kumar et al., 2020). While THOC does not affect transcription of TERRAs, it appears to facilitate their transport to the nucleoplasm, prevent TERRA R-loop formation, and hence maintain telomere stability in both telomerase-positive and ALT human cells (Fernandes and Lingner, 2023).

TERRA–hnRNPA1 interaction

hnRNPA1 is a TERRA-binding protein implicated in RNA processing (Lim and Cech, 2021). Its dysregulation contributes to neurodegenerative diseases (Clarke et al., 2021). A recent study showed that neurites exhibit decreased outgrowth and branching when hnRNPA1 was dysfunctional in mouse neuroblastoma cell line (Anees et al., 2021).

Moreover, depletion of hnRNAPA1 in mouse cells resulted in shorter telomeres (Lim and Cech, 2021). TERRA–hnRNPA1 interaction contributes to telomere maintenance by displacing RPA present at the telomeric single-stranded DNA and allowing the association of POT1 with telomeres in replicating cells (Flynn et al., 2011). This process is vital for telomere protection and overall genomic stability, impacting cellular aging and disease susceptibility. Furthermore, the interaction between hnRNPA1 and TERRAs releases TERRA-bound telomerase, allowing telomere length maintenance (Redon et al., 2013). Conversely, at high levels, hnRNPA1 binds to telomeric DNA, subsequently inhibiting telomerase binding to that location and thus affecting telomere length (Redon et al., 2013). While TERRA–hnPRNA1 interaction plays a crucial role in telomere maintenance by either stabilizing the shelterin complex or releasing telomerase bound to TERRAs, further research is needed to explore whether increasing TERRA levels can effectively sequester excessive hnRNPA1, allowing telomerase to function more freely.

TERRA-based therapeutic strategies in CNS diseases

Given TERRA's deregulated expression in CNS diseases (Sampl et al., 2012; Hsieh et al., 2024), along with its critical role in regulating telomere dynamics, genomic stability, and inflammatory responses, targeting TERRA expression and function presents a promising therapeutic strategy. Several approaches to modulate TERRA activity could influence diverse biological processes, including LNA-based therapies, TERRA mimics or antisense oligonucleotides, and CRISPR-based strategies. LNA-based therapies aim to stabilize TERRA RNA or inhibit its function depending on the pathological context. By modulating TERRA levels, these therapies could restore telomere maintenance and/or reduce inflammation, which play crucial roles in neurodegenerative diseases. Another strategy is to use TERRA mimics or antisense oligonucleotides, which target TERRA's interactions with RNA-binding proteins and chromatin regulators. These molecules can modulate TERRA's role in telomere protection, genomic stability, and stress response regulation, making them particularly beneficial in diseases linked to genomic instability or cellular senescence. Precisely editing TERRA expression using CRISPR-based technologies might also influence its interactions with other regulatory proteins.

Recent studies have manipulated TERRA expression, revealing significant effects on both TERRA levels and cellular phenotypes. For example, knocking down TERRA in mouse embryonic stem cells using LNA mimics resulted in the production of telomere-induced foci, promoting telomere integrity maintenance (Chu et al., 2017). Similarly, TERRA–LDS1 interaction was shown to be crucial for telomere maintenance in ALT cancer cells (Xu et al., 2024). Hence, TERRA mimics might be useful in cells with low TERRA levels to enhance these interactions, promoting telomere lengthening and maintenance. Additionally, inhibiting TERRA transcription reduced DNA replication stress and DNA damage at telomeres, impairing ALT and telomere maintenance, which suggests therapeutic potential in cancer (Montero et al., 2016). TERRA mimics and antisense oligonucleotides targeting RNA–protein interactions have also shown promise in modulating telomere protection and genomic stability (Deng et al., 2009). More recently, a CRISPR–Cas13-based system to visualize and manipulate endogenous TERRA in live cells was developed, which helps to promote genomic stability (Xu et al., 2022). While these TERRA-targeting strategies offer exciting opportunities to address telomere dysfunction, inflammation, and genomic instability in CNS diseases, further research is needed to fully evaluate their feasibility. The effectiveness of these approaches will likely depend on the specific cellular context, as TERRA's role can vary across different cell types and disease states. More studies are required to optimize these strategies for clinical applications, ensuring their potential for therapeutic use.

Conclusions and future directions

Telomere dysfunction, genomic instability, and cell senescence are hallmarks of neurodegenerative diseases, with TERRA playing a key role in these processes. Current evidence underscores TERRA's influence on telomere biology, gene expression, chromatin structure, and inflammation. However, many questions remain regarding the specific mechanisms by which TERRA influences the progression of neurodegenerative diseases. Future research should focus on elucidating how TERRA impacts telomere stability and function in both neurons and glial cells, as these cell types may exhibit distinct responses to TERRA regulation. Techniques such as single-cell RNA sequencing could provide insights into cell-type-specific TERRA expression that can help to understand the role of TERRA in various cell types in modulating functions such as neuroinflammation, cellular senescence, and neurodegeneration. Investigating the interplay between TERRA and other cellular processes, such as oxidative stress and DNA repair, will indicate their significance in protecting or damaging the CNS functions in various diseases. Furthermore, CRISPR–Cas9 gene editing could be employed to explore the consequences of TERRA depletion or overexpression in specific cell types.

Given the tight regulation of TERRA expression, developing TERRA-based therapies, such as inhibitors or mimics, holds promise as a strategy to mitigate telomere dysfunction and inflammation in the CNS. However, the success of these therapies will depend on the specific cellular context, as TERRA's role may vary across disease states and cell types. Additionally, challenges related to off-target effects and effective CNS drug delivery need to be addressed. In vivo models of neurodegeneration will be critical for assessing the efficacy of these therapeutic approaches. Further studies are required to evaluate the feasibility of these strategies in clinical settings to optimize their therapeutic potential.

Funding

The study was supported in part by the Department of Neurological Surgery, University of Wisconsin, the US Department of Veterans Affairs (I01 BX005127 and I01 BX006062), and the National Institute of Health (RO1 NS130763 and R35 NS132184). Dr Vemuganti is the recipient of a Research Career Scientist Award (IK6BX005690) from the US Department of Veterans Affairs.

Conflict of interest: none declared.