Role of tunneling nanotubes in neuroglia

Abstract

Tunneling nanotubes are crucial structures for cellular communication and are observed in a variety of cell types. Glial cells, the most abundant cells in the nervous system, play a vital role in intercellular signaling and can show abnormal activation under pathological conditions. Our bibliometric analysis indicated a substantial increase in research on tunneling nanotubes over the past two decades, highlighting their important role in cellular communication. This review focuses on the formation of tunneling nanotubes in various types of glial cells, including astrocytes, microglia, glioma cells, and Schwann cells, as well as their roles in cellular communication and cargo transport. We found that glial cells influence the stability of the neural system and play a role in nerve regeneration through tunneling nanotubes. Tunneling nanotubes facilitate the transmission and progression of diseases by transporting pathogens and harmful substances. However, they are also involved in alleviating cellular stress by removing toxins and delivering essential nutrients. Understanding the interactions between glial cells through tunneling nanotubes could provide valuable insights into the complex neural networks that govern brain function and responses to injury.

Introduction

Intercellular communication is essential for proper functioning of the nervous system (Khattar et al., 2022; Lian et al., 2024; Cong et al., 2025), including information transmission, maintenance of homeostasis, inflammatory and immune responses, neural development, and neural plasticity (Caruso Bavisotto et al., 2019; Zhang et al., 2022; Dagar and Subramaniam, 2023). Excited neurons transmit electrical signals and release neurotransmitters at synapses (Liu et al., 2023), which regulate the activity of neighboring cells. This precise process enables efficient information processing and supports complex behaviors and cognitive functions (Allen and Eroglu, 2017).

The nervous system maintains homeostasis and adapts to different conditions through intercellular communication mechanisms (Sharma and Burre, 2023). Neurotransmitter secretion adjusts the strength and speed of neuronal connections, while hormone release regulates physiological processes such as metabolism and emotions, thereby maintaining an internal balance.

Neuro-immune regulation is essential to maintain immune equilibrium (Chavan et al., 2017; Deczkowska and Schwartz, 2018). The nervous system releases hormones and neurotransmitters to control inflammation and coordinate responses. Nerve fibers communicate directly with immune cells through synapse-like connections, influencing immune responses during infection and injury while preventing potentially detrimental excessive inflammation.

These aspects highlight the importance of intercellular communication in maintaining nervous system integrity and responding to various challenges. During neural injury, damaged neurons and the surrounding cells communicate through various signaling mechanisms that activate intracellular pathways essential for neural regeneration as well as for cell migration and proliferation. Understanding these mechanisms can provide new avenues for therapeutic interventions in various neurological conditions, including neurodegenerative diseases.

Cells communicate in various ways, including direct communication via gap junctions with neighboring cells; synaptic transmission through neurotransmitters; communication with adjacent cells through exosomes; communication with cells that are far away through neuromodulators and other chemical messengers; transmission of electrochemical signals forming action potentials; and cell–cell connections via tunneling nanotubes (TNTs) (Sahu et al., 2018).

First described in 2004, TNTs are thin, F-actin-based membrane protrusions that allow direct communication between distant cells (Rustom et al., 2004). Over the last two decades, a growing body of evidence has characterized various aspects of TNTs. A variety of cell types can communicate over long and short distances using TNTs, including neuronal cells, astrocytes, microglia (Scheiblich et al., 2021), glioma, tumor cells (Sahu et al., 2018), myeloid cells (Dufrancais et al., 2021), mesenchymal cells, pericytes, and immune cells (Zhu et al., 2021). TNTs play an important role in interactions between neurons and glia, including communication between similar cell types and other types, such as neurons and astrocytes, neurons and pericytes, and neurons and microglia (Chakraborty et al., 2023).

As the most abundant cells in the nervous system, neuroglia communicate with nearby and distant cells to maintain homeostasis and support neuronal function. Glial cells play a crucial role in intercellular communication within the nervous system. They act as intermediaries, transmitting signals through calcium waves between glial cells to coordinate their activities and influence the activity of nearby neurons (Vicario et al., 2020). Additionally, glial cells release extracellular vesicles to communicate with neurons and other glial cells, modulating cellular functions and gene expression.

Due to their diverse and complex roles in intercellular communication, glial cells are essential for normal operation and adaptive responses of the nervous system. Thus, understanding glial cell functions in intercellular communication can provide better insights into the overall functioning of the nervous system and offer new approaches for addressing neurological diseases. This review will describe the evidence and research progress for the role of TNTs in communication between neuroglia, discuss the existing challenges, and propose ideas for further research in this field.

Search Strategy

To gain a comprehensive understanding of the field and a detailed overview of the research process and key areas of focus since the concept of TNT was first introduced, we began with a bibliometric analysis and a thorough review of the English-language literature on TNTs. As of August 30, 2024, we identified 873 publications related to TNTs. Our search was conducted using the Core Collection databases in Web of Science with the search formula TS= (“tunneling nanotubes”) AND LA= (English) and a timespan from January 1, 1990, to August 30, 2024. We retrieved a total of 873 literature, comprising 483 articles and 290 reviews. No duplicated records were included. We first browsed all article fields based on the Web of Science categories. After manually screening the abstracts related to interdisciplinary fields, we found that none of the retrieved articles could be excluded due to their relevance to biology. As a result, all were included in the subsequent analysis. We collected full records and cited references of each record for further analysis. Bibliometric features were analyzed using the R package ‘bibliometrix’, while VOSviewer was used for co-occurrence analysis and visualization, and Citespace was used to calculate citation burst keywords.

After a general glance of TNTs, we focus on the progress of TNT research in glial cells. Next, we performed a search on PubMed using the following search expression: ((tunneling nanotubes) OR (TNTs)) AND ((nervous system) OR (neuroglia) OR (glia) OR (astrocytes) OR (microglia) OR (glioma) OR (Schwann cell)). Inclusion criteria: (1) original articles or reviews in English; (2) study involving TNTs in glial cells; (3) studies on the cross between glial cells and other types of cells in TNTs. Exclusion criteria: (1) keywords of other interpretation; (2) non-English publications. After reviewing the abstracts of all the retrieved articles, irrelevant studies were manually excluded, and the remaining literature was systematically analyzed and summarized.

Research on Tunneling Nanotubes: Milestones in Studies on Intercellular Communication

Here, we present a timeline showing the discovery of TNTs and other advancements in research on TNTs (Figure 1). We divided the research progress into four stages based on the milestone studies and the emergence of new research topics. The first records describing TNTs in humans can be dated back to 2004. Since these first descriptions, numerous studies have endeavored to unravel the complexities of TNTs. TNTs were found to promote the exchange of various cellular signals and components between cells in 2008 (Davis and Sowinski, 2008; Wang and Gerdes, 2012). TNTs were also shown to be involved in the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions to uninfected cells (Sowinski et al., 2008; Gousset et al., 2009). The molecular signaling functions involved in the regulation of TNT formation and activation were widely studied during this period (Hase et al., 2009; Wang et al., 2011). Another significant finding was that TNTs could help transfer apoptosis regulators between apoptotic and healthy cells (Arkwright et al., 2010). Moreover, TNTs have been shown to be involved in the rescue of damaged cells from mesenchymal stem cells by mitochondrial transfer (Cselenyak et al., 2010). Thus, this stage was marked by studies providing basic knowledge of TNTs and their cellular signaling. In 2015, Wang and Gerdes further discovered that TNTs mediate mitochondrial transfer between different homologous cells in different states. Abounit et al. (2016b) found that TNTs inside lysosomal vesicles facilitated efficient transfer of alpha-synuclein (α-SYN) fibrils from donor to acceptor cells. Consequently, investigations into the role of TNTs in mediating the transfer of autophagosomes and mitochondria within the leukemia microenvironment have been gaining attention. In recent years, researchers have gained insights into the various cargoes that TNTs transport, as well as their functions in various pathological conditions. Over the past five years, the literature on TNTs has rapidly expanded, leading the way to in vivo research (Alarcon-Martinez et al., 2020, 2022; Scheiblich et al., 2021). Recent advancements in techniques such as multiphoton fluorescence imaging, ultrastructural observations, and both direct and indirect TNT tracking have greatly improved our understanding of these structures and the microenvironments in which they form. These advancements have contributed substantially to our knowledge of TNTs.

Figure 1.

Timeline showing the discovery and development of tunneling nanotube (TNT) research.

SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Between 2007 and 2019, the number of annual publications on TNTs increased dramatically from 2 to 80 (Figure 2A). Over the past four years, the number of publications has shown steady fluctuations, possibly due to challenges in detection technologies or in vivo studies. As illustrated in Figure 2B, TNT-related publications appeared in 403 different journals. The International Journal of Molecular Sciences published the most papers (n = 40, 4.6%), followed by Scientific Reports (n = 30, 3.4%), Cancers (n = 22, 2.5%), and Frontiers in Cell and Developmental Biology (n = 16, 1.8%). Among the top 15 journals, Cancer Research had the highest impact factor (IF = 12.5).

Figure 2.

Overall trend and sources of publications about tunneling nanotubes (TNTs).

(A) The overall trend in the growth of publications about TNTs from 2004 to 2024. (B) Most relevant sources of TNT-related publications.

We also analyzed the national publication output. A total of 57 countries published articles in this field, with the United States of America (USA) contributing the most publications (n = 289; 33.1% of the total). These papers have been cited 8680 times. In Figure 3A, we analyze international collaborations among the top 30 countries with the most publications. The width of the rectangles represents the number of publications; line thickness between countries represents the weight of their collaboration frequency; and line color stands for the different sources. We identified 965 different institutions. In Figure 3B, we list the top 10 most prolific institutions. Universite Paris Cite contributed the most publications (104) in this field, followed by Centre National De La Recherche Scientifique, University of Minnesota System. Notably, institutions in France account for the most relevant affiliations while those in the USA account for the largest column of publications. Despite the notable contributions from both countries, this disparity in the pattern of contributions may be attributable to specific research teams in France that have consistently contributed to this field.

Figure 3.

International collaboration among the top 30 countries and contribution of affiliations in tunneling nanotube publications.

(A) The top 30 countries with the most publications and their collaboration frequency. (B) The top 10 most prolific institutions.

The co-occurrence analysis included 148 keywords, each appearing at least 10 times, as illustrated in Figure 4A. The size of the dots represents keyword frequency, while the color indicates keyword clusters. Keywords with closer correlations were grouped into the same cluster. Four distinct clusters were identified: Cluster 1 (red) contained 47 keywords primarily focused on the structure and activation of TNTs. Examples include “actin,” “filopodia,” “mechanism,” “infection,” “plasma membrane,” and “plasmodesmata.” Cluster 2 (green) contained 40 keywords centered around cancer research, such as “cancer,” “breast cancer,” “tumor cells,” “glioblastoma,” “tumor microenvironment,” “invasion,” along with various therapies and mechanisms related to TNTs, including “angiogenesis,” “microvesicles,” “exosomes,” and “migration.” Cluster 3 (blue) contained 37 keywords primarily related to mitochondrial transfer and mesenchymal stem cells, which are considered a primary source for mitochondrial transplantation. Important keywords in this group include “mitochondrial dysfunction,” “organelle transfer,” “stem cells,” and “stromal cells.” Cluster 4 (purple) included 24 keywords related to neurodegenerative diseases, such as “Parkinson’s disease,” “Alzheimer’s disease,” “alpha-synuclein,” “amyloid-beta,” “tau,” and “astrocytes.” Keywords across these clusters demonstrate various interconnections.

Figure 4.

Contribution of keywords in tunneling nanotube publications.

(A) The network visualization map of co-occurrence keywords. The size of the dots represents keyword frequency, and the color indicates keyword clusters. Keywords with closer correlations are grouped into the same cluster. Line thickness between nodes shows the frequency of co-occurrence. (B) The top 25 keywords with the strongest citation bursts.

The top 24 keywords with the highest citation bursts are shown in Figure 4B. The keyword “immune cells” appeared the earliest and has maintained sustained attention over time. Keywords such as “mesenchymal stromal cells,” “progenitor cells,” “trafficking,” and “tumor microenvironment” have emerged since 2020, expanding the scope of TNT research. Additionally, keywords such as “fibrillar alpha-synuclein” and “miro1” have gained prominence in the past two years, reflecting current and future research trends.

Tunneling Nanotubes Are Key to Intercellular Communication in Neuroglia

The growing body of TNT-related publications reflects an increasing interest in TNTs as a crucial mechanism of intercellular communication, underscoring the importance of understanding their roles in health and disease. This rise in research is closely tied to notable advancements in technology and research methodologies. Improvements in live-cell imaging, super-resolution microscopy, and electron microscopy have enabled more precise visualization of TNTs, facilitating a deeper understanding of their structure and function. Nevertheless, despite the substantial body of research on TNTs in glial cells, a comprehensive review on this topic remains lacking. In the following sections, we will focus on exploring the specific roles of TNTs in glial cells.

Key roles of tunneling nanotubes in intercellular communication

TNTs are dynamic cell–cell contacts that primarily rely on actin polymerization. This dynamic adaptability allows for the direct transport of cellular cargo between cells without the need for secretion into the extracellular milieu, providing TNTs a unique role in intercellular communication. TNTs connect various cell types and transfer different types of cargo, including small molecules, proteins, vesicles, pathogens, nucleic acids, ions, and organelles. In particular, these cargoes can serve as signals for survival or death, as well as for physiological or pathological processes.

TNTs are highly effective at transporting proteins, particularly those prone to aggregation, such as α-SYN, tau, prions, and similar inclusions, across cells in the nervous system. While the precise cellular mechanisms driving TNT-mediated propagation remain largely unclear, several common patterns have been identified (Lagalwar, 2022). The density of TNTs initially increases due to the selective production of aggregation-prone protein types. Subsequently, protein loading and unloading onto the TNTs appear to be facilitated by endo-lysosomal processes. Lastly, TNT assembly leads to the spontaneous development of protein aggregates in “acceptor” cells, indicating that TNTs are essential for both transporting these inclusions and seeding new aggregates in naïve cells. This positions TNTs as a key factor in the spread of aggregation-prone protein inclusions in neurodegenerative diseases. The transportation of various organelles from healthy to damaged cells through TNTs has also been shown to partially mitigate pathological changes in neurodegenerative diseases. Because of their remarkable capabilities, TNTs play a crucial role in the spread of various microbial pathogens and diseases like cancer and neurodegenerative disorders and may serve as a potential mechanism for disseminating therapeutic drugs between cells, positioning them as a new target for nanomedicine. Furthermore, TNTs have made the co-cultivation of mesenchymal stem cells or healthy cells a promising treatment approach.

Neuroglia serve as the foundation of nervous system formation and function

Neurons and glial cells play essential roles in maintaining the homeostasis of the nervous system through various mechanisms of intra- and intercellular signaling. The development and functioning of the nervous system depend on intricate and precisely balanced forms of bidirectional communication. While neurons—highly specialized, electrically active cells—dominate these functions, glial cells, which account for roughly half of the cells in the nervous system, were long considered passive bystanders in its development and functioning. However, with the development of neural circuits, different types of glia play crucial roles in synaptic connections, plasticity, homeostasis, and network-level activity, dynamically monitoring and adjusting the nervous system’s structure and function (Allen and Lyons, 2018).

Neuroglial cells consist of five main types: radial glia, astrocytes, oligodendrocyte progenitor cells (OPCs, also known as NG2 cells), oligodendrocytes, and microglia. Radial glia serve as progenitors in the central nervous system (CNS), giving rise to most neurons and glial cells directly or through intermediate progenitors. Astrocytes are star-shaped cells with extensive processes that interact with various cell types in the CNS, playing key roles in the development of the nervous system and regulation of its activity. OPCs, the most proliferative cell type in the CNS, generate oligodendrocytes that provide myelination throughout life. Mature oligodendrocytes form myelin sheaths, which accelerate nerve impulse conduction and supply metabolic support to axons. Microglia, the resident macrophages of the CNS, are increasingly recognized for their roles not only in immune defense but also in various stages of nervous system development and function.

Bonds between neuroglia and tunneling nanotubes

TNTs play pivotal roles in the interactions between neurons and neuroglia, significantly influencing both the central and peripheral nervous systems. Neuroglial cells use TNTs to transmit information and transport cargo, facilitating communication between different glial cell types and neurons. This cooperation is essential for processes such as neurogenesis, neuroregeneration, and myelination. Dysregulation of TNT function has been implicated in the pathogenesis of various disorders, including obsessive-compulsive disorder (Soto et al., 2023), neurodegenerative diseases (Zhou et al., 2019), epilepsy (Ma et al., 2022), and stroke (Lee et al., 2023), contributing to nervous system dysfunction.

The existing studies on the existence and function of TNTs among neural cells (Chakraborty et al., 2023; Lee et al., 2023; Lin et al., 2024b) primarily focused on cultured cells for the convenience of detecting the ultrastructure of TNTs. Research on TNTs in glial cells has mainly examined mitochondrial transport (Xi et al., 2024), tau protein transport (Eltom et al., 2024), changes in TNT quantity caused by different stimuli, antigen presentation (Rostami et al., 2020), and virus transport (Abounit et al., 2016a). TNTs exert their effects on glial cells through various signaling pathways. We have presented a comprehensive summary of some mechanisms through which substances are transported by different glial cells, including astrocytes, microglia, and Schwann cells (SCs), through TNTs (Figure 5).

Figure 5.

Summary of mechanisms for substance transport by various cells via TNTs.

This figure was drawn by Figdraw (ID:ISTWWc4cd2). Akt: Protein kinase B; Aβ: amyloid-β; FP: fluorescent protein; MAPK: mitogen-activated protein kinase; mtDNA: mitochondrial DNA; mTOR: mammalian target of rapamycin; PI3K: phosphoinositide 3-kinase; Rab: ras-related protein; ROCK2: rho associated coiled-coil containing protein kinase 2; Tau: tubulin associated unit; TNTs: tunneling nanotubes; αSYN: alpha-synuclein.

Neuroglia constitute approximately half of all neural cells in the mammalian CNS and primarily provide nutritional and trophic support to neurons in the brain. Given their abundant quantity and functional diversity as well as their close relationships with neuronal cells, glial cells can be considered to provide nutritional support and communication for neuronal cells, regulate the number and function of these cells, and maintain the stability of the nervous system. The existing body of research on glial cells is extensive, and research on their relationship to TNTs is also rich, making them a primary focus for exploring new modes of intercellular communication in the CNS. In the subsequent section, we will thoroughly explore the TNT-based communication between different types of glial cells within the nervous system; these findings are summarized in Table 1.

Table 1.

Summary of cargo transport between various cell types via TNTs in diverse models and disease conditions, including detection approaches and markers

Reference	Neurodegenerative disease	Cell type	Stimulus condition	Transshipment substance	Culture	Transportation	Detected method	Inhibitor	Detected marker
Zhu et al., 2005	Under pressure	Astrocytes	H2O2	/	In vitro	One-way	Confocal microscopy	SB203580	F-actin, phalloidin
Wang et al., 2011	Under pressure	Astrocytes and neurons	H2O2 or serum depletion	Aβ, EGFP, RFP	In vitro	One-way	Confocal microscopy	Latrunculin A, cytochalasin D	Alexa488
Sun et al., 2012	Under pressure	Astrocytes and neurons	H2O2	EGFP or RFP	In vitro	One-way	Confocal microscopy	/	EGFP, RFP
Wang et al., 2012	/	Astrocytes and neurons	/	Tau fibrils	In vitro	One-way	Confocal microscopy	/	Cell tracker, phalloidin
Zhang and Zhang, 2015	Gliomas	Astrocytes and neuroblastoma cell	H2O2	/	In vitro	/	Immunofluorescence; live-cell imaging	Latrunculin A, knockdown of p53	RFP, GFP
Abounit et al., 2016	Neurodegenerative disease	Mouse neuron-like CAD cells	recombinant human tau fibrils	Tau fibrils	In vitro	One-way	Fluorescence microscopy	/	WGA, Rhodamine
Zhu et al., 2016	Peripheral nerve injury	Schwann cells	/	RNA, EGFP, RFP	In vitro	Two-way	Immunofluorescence; live-cell imaging	Knockdown of Rab8a/Rab11a	RFP, EGFP, DIO, DIL, F-actin
		Schwann cells and neurons	/	/	In vitro	/	Immunofluorescence	/	F-actin
Victoria et al., 2016	Prion diseases	Astrocytes and neurons	22L prion infection	PrP (C)	In vitro	One-way	Confocal microscope	/	WGA
Babenko et al., 2018	Ischemia/reoxygenation	Astrocytes and MMSC	OGD	Mitochondria	In vitro	One-way	Confocal microscopy	/	Rhodamine
Civita et al., 2019	Brain tumor	Neuroblastoma cells and astrocytes	/	Mitochondria	In vitro	One-way	Confocal laser-scanning microscope	/	Phalloidin
Chastagner et al., 2020	AD	Mouse neuron‐like CAD cells, neuroblastoma cells and neurons	Tau aggregation	Tau fibrils	In vitro and in vivo	Two-way	Confocal microscopy	/	Phalloidin, DID
Dagar et al., 2021	/	Astrocytes and neurons	/	GFP or EGFP	In vitro	Two-way	Confocal microscopy	/	Phalloidin, GFP, EGFP
Rostami et al., 2020	PD	Astrocytes	α-SYN-F, α-SYN-M and IFNγ	α-SYN	In vitro	One-way	Confocal microscopy	/	WGA
Scheiblich et al., 2021	PD	Microglia	α-SYN	α-SYN	In vitro	One-way	Immunofluorescence; EM	Y-27632, Blebbistatin, Cytochalasin D	F-actin, Tubulin
		Microglia and neighboring cells	α-SYN	α-SYN	In vivo	One-way	Two-photon imaging	/	GFP
Rostami et al., 2021	PD	Astrocytes and microglia	α-SYN	α-SYN	In vitro	One-way	Immunofluorescence	/	Vimentin, Iba1, WGA
Du et al., 2021	Gliomas	Microglia and neuroblastoma cell	Liposomes	Drug	In vitro	One-way	Immunofluorescence	Cytochalasin D	DiO, DiL
Chen and Cao, 2021	/	Homozygous ROSA26 GNZ knock-in mice	/	GFP	In vitro	One-way	Immunoelectron microscopy	Cytochalasin B	GFP
Zheng et al., 2021	/	Astrocytes and neuroblastoma cells	CoNPs explosure	Mitochondria	In vitro	One-way	EM; confocal microscopy	Latrunculin B	F-actin, DiL
Pisani et al., 2022	Ischemia/reperfusion	Microvascular endothelial cells, astrocytes and microvascular pericytes	OGD	Mitochondria	In vitro	One-way	Live immunofluorescence	Cytochalasin D	F-actin, CellMask
Chakraborty et al., 2023	PD	Microglia and neuroblastoma cell	α-SYN	α-SYN	In vitro	Two-way	Immunofluorescence; live-cell imaging	/	WGA, F-actin, α-Tubulin
		Microglia and neuroblastoma cell	α-SYN	Mitochondria	In vitro	One-way	Immunofluorescence; live-cell imaging	/	WGA, F-actin, α-Tubulin
Xi et al., 2024	OGD/R	Astrocytes and neurons	OGD/R, mild hypothermia	Mitochondria	In vitro	One-way	Fluorescence microscopy	Cytochalasin D	Actin-Tracker Green
Eltom et al., 2024	AD	Astrocytes and neurons	AD fibrils	Tau fibrils	In vitro	One-way	Time-lapse microscopy; fluorescence microscopy	/	Vimentin, Tubulin, Synaptophysin
Boyineni et al., 2024	Brain tumor	RG cells and BTICs	Hypoxic conditions	Mitochondria	In vitro	One-way	Confocal microscope	/	Astrocyte markers
Lin et al., 2024	Nanomaterial-induced neurotoxicity	Astrocytes and neuroblastoma cells	Nanomaterial exposure	Mitochondria	In vitro	One-way	Confocal microscope	N-Acetylcysteine, mitoquinone	F-Actin

AD: Alzheimer’s disease; Aβ: amyloid-β; CAD: Cath.a-differentiated; DiD; DiIC18 (5); DiI: DiIC18 (3); DiO: DiOC18 (3); EGFP: enhanced green fluorescent protein; EM: electron microscope; GFP: green fluorescent protein; H2O2: hydrogen peroxide; IFNγ: interferon gamma; MMSC: multipotent mesenchymal stem cells; OGD/R: oxygen-glucose deprivation/reperfusion; OGD: oxygen-glucose deprivation; PD: Parkinson's disease; PrP (C): cellular prion protein; RFP: red fluorescent protein; RG: radial glia; TNTs: tunneling nanotubes; WGA: wheat germ agglutinin; α-SYN: alpha-synuclein; “/”: undescribed.

Detection of Tunneling Nanotubes

TNTs are thought to be composed of cytoskeletal components and cell membranes, either containing microtubules, actin, or both (Hase et al., 2009; Wang et al., 2012; Antanavičiūtė et al., 2014). Thus, any dye capable of binding to actin, microtubules, or cell membrane antibodies can theoretically be used to label TNTs, and this approach has also been utilized to detect other cell–cell junctions (Resnik et al., 2019). TNT is not the only tubular structure that can connect two or more cells, and specific exploration methods for TNTs are currently lacking. Here, we summarize the detection methods for TNTs and briefly describe several features that distinguish TNTs from other intercellular junctions. We present a comprehensive summary of the contemporary approaches for detection of TNTs and provide a comparison of the advantages and disadvantages of each approach (Table 2).

Table 2.

Advantages and disadvantages of various methods for detecting tunneling nanotubes

Detected method	Advantage	Disadvantage
Immunofluorescence	(1) The sample is readily preparable; (2) The detection is rapid and convenient.	(1) Absence of specific markers; (2) Inadequate resolution; (3) Frequently used to observe static structures.
Live-cell imaging	(1) Facilitated for observation; (2) Capable of observing the dynamic alterations of live cells; (3) Intact structure.	Inadequate resolution.
Electron microscopy	Ultra-high resolution.	(1) The process of sample preparation is intricate; (2) It is challenging to preserve integrity; (3) Only static specimens can be observed.
Two-photon imaging	It is feasible to conduct dynamic observations both in vivo and ex vivo.	Stringent requirements for equipment and technology.

Tunneling nanotubes are labeled by labeling the cytoskeleton and cell membrane

Actin

Actin is a crucial cytoskeletal polymer within cells. Filamentous actin (F-actin) is formed through polymerization of globular actin (G-actin) monomers, which are then assembled into a highly structured actin network. This network is essential for driving changes in cell shape, including the formation of intercellular protrusions (Ljubojevic et al., 2021; Golub et al., 2023). F-actin is present in most TNTs and is uniformly distributed along their entire length, making it an important labeling target in TNT imaging (Resnik et al., 2019; Scheiblich et al., 2021). The initiation, polymerization, and stabilization of actin filaments occur in a tightly controlled manner within the cell. Thus, interfering with actin formation can significantly alter the formation of TNTs. F-actin inhibitors, such as cytochalasin D and Latrunculin A, can effectively inhibit intercellular TNTs (Wang et al., 2011; Zhang and Zhang, 2015). Phalloidin is a commonly used F-actin marker. Fluorescent derivatives of phalloidin are useful for localizing actin filaments in fixed cells and for visualizing individual actin filaments. Furthermore, fluorescence intensity measurements obtained with saturated amounts of fluorescent phalloidin can be used to quantify the amount of filamentous actin present in cells (Cooper, 1987).

Wheat germ lectin

The deformation and extension of the cell membrane are fundamental to the formation of TNT structures. WGA is a member of the lectin family and is commonly used to label the cell membrane of mammalian cells due to its ability to bind to the N-acetyl-β-d-glucosamine residues and N-acetyl-β-d-glucosamine oligomers present on cell membranes (Nicolson et al., 1975). WGA labeling of TNTs reveals a distinct cytoplasmic tunnel for transportation of translocated cargo (Bénard et al., 2015). WGA and phalloidin complement each other in illustrating the structure and function of cells. Interestingly, we also observed that unmodified WGA can induce TNT formation by promoting the redistribution of F-actin, which may be linked to the altered localization of CD31 (Pedicini et al., 2018).

Tubulin

The role of tubulin in TNTs is controversial. Some studies have used tubulin positivity to label TNTs (Eltom et al., 2024; Chakraborty et al., 2023), while other studies have considered actin-positive and tubulin-negative profiles as true markers of TNTs (Tardivel et al., 2016). Due to the uncertainty regarding tubulin levels in TNTs, most articles do not designate tubulin as a separate marker. Instead, they often use it in combination with other labeling methods to provide complementary information (Scheiblich et al., 2021).

Vimentin

Vimentin, a type III intermediate filament protein, was previously shown to be a non-essential cytoskeletal protein (Colucci-Guyon et al., 1994). However, recent studies (Hol and Pekny, 2015; Ridge et al., 2022) have demonstrated that vimentin plays a key role in coordinating transduction, signaling pathways, locomotion, and inflammatory responses. Among the markers of TNTs, vimentin has also been used as a marker of the cytoskeleton. Although its presence has been reported in TNTs, its application is not as widespread as that of other markers (Dubois et al., 2018).

DiI, DiD, and DiO

DiI, DiD, and DiO are fluorescent probes used for staining cell membranes. One major advantage of these probes is their ability to stain live cells, in contrast to protein immunofluorescence, which is typically performed with fixed, dead cells. These dyes allow observations of cell fusion, adhesion, and migration, facilitating real-time analysis of dynamic membrane changes (Honig and Hume, 1989). They also serve as excellent tracers in the nervous system and have been employed to capture the morphology of TNTs in glial cells (Zhu et al., 2016; Du et al., 2021).

Tunneling nanotubes are labeled by cell-specific antibodies and fluorescent proteins

In studies that used TNT labeling, the visualization and identification of different cell types could help delineate cells, with a key factor being the use of antibodies that specifically target protein biomarkers expressed within these cells (Hol and Pekny, 2015; Jurga et al., 2021). The most common structural marker protein for astrocytes is glial fibrillary acidic protein (GFAP), which is present in most CNS astrocytes but is expressed differently in cells from various brain regions, as well as in neuronal stem cells and peripheral glial cells (Middeldorp and Hol, 2011). GFAP plays a crucial role in maintaining mechanical strength and cell shape, and changes in GFAP expression can influence the proliferative ability of astrocytes as well as other features of astrocyte transformation (Hol and Pekny, 2015). Among cytoskeletal proteins, GFAP is a widely used marker of astrocyte activation due to its significantly increased expression in most pathological conditions, including neurodegeneration and injury (Jurga et al., 2021). Iba1 is suitable for the identification of monocyte-macrophage lineage cells, including microglia, and is a commonly used marker for microglia in TNT studies (Rostami et al., 2021; Scheiblich et al., 2024). The gene encoding Iba1 is located in a major histocompatibility complex class III region and may be involved in the immune function of microglia (Ito et al., 2001). In terms of cell morphology, Iba1 was found to accumulate with F-actin in the cell membrane folds and participate in plasma membrane alterations in cellular phagocytosis, but it did not affect the formation of filopodia (Imai and Kohsaka, 2002).

Various fluorescent proteins are widely used to characterize the shapes of TNTs through transfection of fluorescent protein genes. These proteins are particularly effective at distinguishing between different cell populations, especially within the same cell. Researchers are also exploring the delivery of these fluorescent proteins via TNTs, which appears to be an easy and interesting detection method (Zhu et al., 2016; Sun et al., 2019). However, approaches employing fluorescent proteins should take into account the potential interference across different channels, necessitating the development of better methods to enhance the separation of distinct fluorescent channels (Bénard et al., 2024).

Non-fluorescent labeling: Identification of tunneling nanotubes by electron microscopy and live-cell imaging

Detection of TNTs using fluorescence microscopy offers convenience in sample preparation; however, it inevitably lacks the accuracy and integrity of electron microscopy (EM). EM can better demonstrate the ultrastructure of TNTs, with its ultra-fine resolution allowing for more detailed analysis of their structure (Zheng et al., 2021; Djurkovic et al., 2023). This enhanced resolution has revealed features that distinguish TNTs from other cell–cell junctions. EM analyses indicate that while the diameter of TNTs shows variability, TNTs are typically narrower than tumor microtubules (TMs) (with the average diameter of TNTs being less than 1 μm and that of TMs being 1.0–2.0 μm). TNTs also exist in two forms: TNTs with two-way openings and those with one-way closures. Lateral branches, which help stabilize the position and orientation of long TNTs or facilitate cell migration, have also been identified (Zhang et al., 2021). Additionally, Anna et al. integrated cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for correlation analysis and observed that actin filaments within TNTs are arranged in parallel and bound together by N-cadherin complexes. Some vesicle structures were also discernible within TNTs, and have been potentially implicated in cargo transport (Sartori-Rupp et al., 2019a). These characteristics clearly demarcate TNTs from filamentous pseudopodia, which are typically isolated, do not form interconnected bundles, and lack vesicle structures.

Similar methods to improve signal transmittance and stability by enhancing instrument performance include stimulated emission depletion nanoscopy (Blom and Widengren, 2017; Bénard et al., 2024). A disadvantage of these methods is that breakage of TNTs during sample preparation can affect observations, precluding analyses of dynamic changes. Live-cell imaging eliminates the need for cell fixation, allowing for observation of changes in the original structure of cells and facilitating studies of dynamic substance transport within TNTs (Alarcon-Martinez et al., 2022). However, live-cell imaging typically requires a longer observation time, and its resolution is generally insufficient for detailed examination of TNTs.

Two-photon laser-scanning microscopy is a state-of-the-art, high-resolution live-cell imaging technique. This technique enables dynamic observation of TNTs in vivo, even after cell staining (Zhao et al., 2023), and allows for the monitoring of surrounding cellular changes, such as electrical signaling and variations in blood flow, which are mediated by TNT-mediated cellular communication (Alarcon-Martinez et al., 2020, 2022; Scheiblich et al., 2021).

The simultaneous use of different detection methods can significantly enhance the accuracy and versatility of TNT testing. In conclusion, with advancements in knowledge and detection technologies, TNT detection methods are flourishing, offering more opportunities to explore the structure and function of TNTs.

Astrocytes and Tunneling Nanotubes

A growing body of literature has revealed various aspects of TNTs in astrocytes. This section will provide a detailed overview of the progress made in this area. We will cover the activation and formation mechanisms of TNTs in astrocytes, their roles in pathological processes, and the interactions of TNTs among astrocytes, neurons, and other cells. Since mitochondrial transport has been widely studied in various cells and diseases, we will also specifically address the progress in the research on mitochondria in TNT-mediated intercellular communication. Additionally, the influence of nanomaterials on TNTs will be further elucidated.

Astrocytes are vital to the nervous system

Astrocytes, which constitute approximately 20% of all glia, are one of the most abundant cell populations in the brain and play multiple roles in both health and disease (Sofroniew, 2020). Multiple studies have highlighted the importance of astroglia in maintaining physiological homeostasis in the brain and in responding to pathological stimuli. Under physiological conditions, astrocytes perform various functions, such as regulating metabolic processes, providing structural support, facilitating brain development, offering neuroprotection, defending the brain, and enabling intercellular communication. In pathological conditions, astrocytes exhibit greater resistance to reductions in oxygen and glucose concentrations (Zhou, 2018), which allows them to preserve anaerobic glycolysis and adenosine triphosphate (ATP) production in areas of brain damage and thereby support neuronal metabolic functions (Cichorek et al., 2021). They also participate in various pathological processes, such as synaptogenesis, metabolic transformation, scar formation, and blood–brain barrier repair. Astrocytes connect distant cells and different types of cells in the nervous system, demonstrating greater potential in pathological circumstances, which may even facilitate both pathological processes and protective responses. This underscores their privileged status in intercellular communication.

Mechanisms underlying the activation and formation of tunneling nanotubes in astrocytes

TNTs in astrocytes were first studied in 2005 by Zhu et al. (2005). They observed that hydrogen peroxide induced the formation of TNTs between primary astrocytes, which was associated with increased co-localization of myosin Va and F-actin. This finding supported the hypothesis that TNTs may be involved in the cellular response to harmful signals, transferring cellular substances or energy to neighboring cells under stress (Wang et al., 2011). The researchers also reported that hydrogen peroxide altered the membrane and cytoskeleton of astrocytes by activating the p38 mitogen-activated protein kinase pathway. Additionally, p53 was found to be crucial for TNT formation in both rat hippocampal astrocytes and neurons. Among the products of genes activated by p53, the epidermal growth factor receptor was identified as an important regulator of TNT development. Furthermore, signaling pathways involving protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), and the mammalian target of rapamycin (mTOR) were also implicated in TNT induction.

Subsequent studies have explored the role of S100A4 in guiding the growth direction of TNTs in primary neurons and astrocytes. Sun et al. (2012) demonstrated that in stress-activated cells, p53 activation induces TNT formation alongside increased caspase-3 activity. Caspase-3 cleaves intracellular S100A4, creating a chemical gradient with a relatively high concentration of extracellular S100A4 around target cells. This gradient then directs the TNTs toward the target cells. Additional research on the mechanisms underlying TNT formation showed that the RNA-binding protein nucleolin, which interacts with the well-known TNT-inducing protein MSec, plays a key role in TNT development across various mammalian cell types, including neurons and astrocytes. Nucleolin, in collaboration with 14-3-3ζ, forms a signaling axis that promotes the phosphorylation and inactivation of cofilin, an F-actin depolymerization factor, thereby facilitating TNT formation (Dagar et al., 2021).

Most studies on TNTs (Chakraborty et al., 2023; Lee et al., 2023; Lin et al., 2024b) were performed using in vitro cell cultures due to the transient nature of TNTs, which made it difficult to observe their ultrastructure in vivo. In 2021, Chen and Cao investigated the transport process of adeno-associated virus (AAV-GFAP-EGFP-p2A-cre) via TNTs in the mouse cerebral cortex. Ten days after injection, enhanced green fluorescent protein (EGFP) was transferred from astrocytes in layers I-III to neurons in layer V. Microscopic observations revealed that the intercellular transport of EGFP through contact connections was dependent on F-actin, confirming that EGFP was transported from astrocytes to cortical neurons through TNTs.

Pathological processes of tunneling nanotubes in astrocytes

In pathological conditions such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), astrocytes utilize TNT-mediated intercellular transport to deliver mitochondria and substrates and to facilitate protein reprocessing at axonal sites distant from the neuronal soma (Engel, 2014). Tau accumulation in the form of neurofibrillary tangles is a defining feature of tauopathies, including AD. The transfer of tau aggregates relies on direct cell contact, and such aggregates have been observed within TNTs connecting neuronal cells, as well as between neurons and astrocytes in organotypic cultures (Chastagner et al., 2020).

Rostami et al. (2020) observed that human astrocytes derived from embryonic stem cells actively transfer aggregated α-SYN to healthy astrocytes through direct contact and TNTs, rather than degrading the aggregates. These stressed astrocytes internalize large amounts of oligomeric α-SYN, and the resulting α-SYN accumulation disrupts their lysosomal function and causes mitochondrial damage. In response, the astrocytes extend TNTs to facilitate the intercellular transfer of α-SYN to healthy astrocytes. Importantly, astrocytes also play critical roles in antigen presentation and T-cell activation within the PD brain. The findings of this study further highlight the transfer of α-SYN/MHC-II deposits between astrocytes via TNTs, suggesting that this mechanism not only contributes to the spread of toxic protein aggregates but also facilitates the propagation of inflammation in the PD brain.

Tunneling nanotubes between astrocytes and other cells

Neurons, which are cells specialized for information processing, delegate many of their support functions to neuroglia, particularly astrocytes. While the cooperation between neurons and astrocytes is well-documented, the mechanisms underlying the initiation of these interactions remain poorly understood. Wang et al. (2012) demonstrated that immature hippocampal neurons form short protrusions toward astrocytes, leading to the formation of TNTs with an average lifespan of 15 minutes. Thus, growing neurons may establish electrical connectivity and exchange calcium signals with astrocytes via TNTs during a brief maturation period. This process is correlated with increased connexin 43 expression in neurons. After exposure to cobalt nanoparticles (CoNPs), TNT-like structures were also observed between neurons and astrocytes (Zheng et al., 2021). Furthermore, the findings obtained using co-culture systems involving primary infected astrocytes and granule neurons or neuronal cell lines have provided direct evidence that PrP (SC) can transfer via TNTs from astrocytes to neurons in prion diseases. Additionally, TNTs containing PrP (SC) are often colocalized with endolysosomal vesicles (Victoria et al., 2016).

The crosstalk between astrocytes, endothelial cells, and pericytes is considered to represent the function of the blood–brain barrier at the cellular level. Upon exposure to ischemia/reperfusion, the formation of TNTs was upregulated, and astrocytes received functional mitochondria from pericytes and endothelial cells through these structures. Thus, TNT-mediated mitochondrial transfer from pericytes helps protect astrocytes from apoptosis caused by ischemia/reperfusion (Pisani et al., 2022). We have discussed the studies describing mitochondrial transport through TNTs (Lee et al., 2023; Xi et al., 2024) in more detail in the following sections.

Mitochondrial traffic in astrocytes

Replacement of non-functional organelles appears to be an appealing therapeutic strategy. Since its discovery in 2006, intercellular mitochondrial transport has been thoroughly investigated across various cellular models as a potential foundation for cell therapy (Babenko et al., 2018). In one previous study, treatment with mitochondria derived from rat primary astrocytes improved cell survival and repaired neurons damaged by hydrogen peroxide in a rat model of middle cerebral artery occlusion (Lee et al., 2023).

TNTs have been identified between co-cultured cells, and these TNTs have been shown to play a role in the movement of mitochondria between astrocytes and neurons. Subsequent investigations showed that mild hypothermia enhanced the transport of astrocytic mitochondria into injured neurons via TNTs, elevated the mitochondrial membrane potential, and reduced neuronal mortality and injury during oxygen-glucose deprivation/reperfusion (Xi et al., 2024). Babenko reported that mitochondria were transferred from multipotent mesenchymal stem cells to astrocytes when the astrocytes were exposed to ischemic damage associated with elevated reactive oxygen species (ROS) levels (Babenko et al., 2018). In this process, Miro1 played a key role by promoting mitochondrial transfer. Such mitochondrial translocation improved the bioenergetics of the recipient cells and stimulated cell division.

By reprogramming nearby cells, cancer stem cell mitochondrial transfer can significantly accelerate overall tumor growth within the tumor microenvironment. Human neural stem cells (NSCs) and brain cancer stem cells, often referred to as brain tumor-initiating cells (BTICs), have shown intercellular mitochondrial transfer to astrocytes in co-culture experiments (Boyineni et al., 2024). Both NSC and BTIC mitochondria trigger similar transcriptome changes upon transplantation into recipient astrocytes. However, after transplantation, the mitochondria from BTICs caused a significantly greater increase in the number of recipient astrocytes than NSC mitochondria. Mitochondrial transport can thus facilitate tumor growth and contribute to the repair of injured target cells as they progress. Non-neoplastic cells can rescue damaged target cancer cells by transferring mitochondria along TNTs (Civita et al., 2019).

Human astrocytes can form TNT connections with glioblastoma (GBM) cells, indicating that the association of TNT-mediated delivery of non-neoplastic mitochondria may be related to proliferation, migration, and response to GBM treatment.

Tunneling nanotubes and nanomaterials in astrocytes

The increasing use of and demand for man-made nanoparticles have raised concerns about their potential toxicity to the CNS. CoNPs, titanium dioxide nanoparticles (TiO₂NPs), and multi-walled carbon nanotubes are three commonly used synthetic nanomaterials that have been shown to induce TNT production in primary astrocytes and U251 cells (Lin et al., 2024b). Although TNT synthesis inhibits ATP generation and cell death, it provides protection against nanomaterial-induced neurotoxicity. Furthermore, activation of the downstream PI3K/AKT/mTOR pathway and the involvement of ROS/mtROS have been identified as common mechanisms regulating mitochondrial translocation and TNT production. Zheng et al. (2021) demonstrated that TNTs facilitate mitochondrial translocation between neurons and astrocytes on exposure to hazardous levels of CoNPs. The actin-depolymerizing drug Latrunculin B, which inhibits intercellular mitochondrial transport via TNTs, exacerbates the cellular and mitochondrial toxicity induced by CoNPs in neuronal cells.

Microglia and Tunneling Nanotubes

Microglia play a vital role in central nervous system homeostasis and diseases

Since the first proper visualization and description of microglia in 1919 (Prinz et al., 2019), our understanding of microglia has been constantly challenged and updated because of their uniqueness and mystery. Microglia have been shown to continuously monitor the CNS. They are now regarded as the archetypal tissue-resident macrophage-like innate immune cells in the CNS, possessing memory-like abilities to enable context-dependent reactions. Recent research has shown that microglia are not merely bystanders of CNS pathologies, but also serve determinants of diseases. Microglia have a highly proliferative and activated phenotype during embryonic and early postnatal development. They show multifunctional connections with neurons, astrocytes, and oligodendrocytes (Prinz et al., 2019). They regulate neuronal apoptosis and regeneration by secreting growth and complement factors and shaping neuronal circuits, and they also play an important role in the process of myelin formation of oligodendrocytes. Microglia in the adult brain supervise the CNS environment and sense the changes in the microenvironment. They are effective in removing dead and superabundant cells by secreting immune mediators to coordinate other cells participating in neuroinflammation. However, the activation of microglia is a double-edged sword. Chronic neuroinflammation and neuronal damage may occur if microglia are persistently activated and arouse an inflammatory response.

Tunneling nanotubes between microglia

When exposed to stimuli, the microglial population as a whole forms an on-demand functional network in response to attack. This process employs multiple modes of communications within the cell population, including TNTs.

The accumulation of α-SYN fibers is one of the main causes of neurodegenerative diseases such as PD and AD, and the mechanisms underlying their transmission and accumulation have received much attention. Scheiblich et al. (2021) focused on how primary microglia handle and cope with α-SYN fibrils and the role TNTs play in α-SYN transportation. They discovered that microglia formed a TNT network of multiple lengths and diameters that contained α-SYN, and α-SYN appeared to prompt the formation of those TNTs. Unlike short and thick membrane projections, small α-SYN clusters can move between microglia within approximately 3 min. When α-SYN fibrils were present, Rho signal transduction was elevated. Treatment with rho associated coiled-coil containing protein kinase (ROCK) inhibitors caused the TNT network to develop, while CytD mostly prevented it. Remarkably, this α-SYN exchange induction significantly decreased ROS release from donors and downregulated the inflammatory response signal, indicating that inflammatory cytokine-induced microglial functional gap junctions are essential for understanding inflammatory responses (Scheiblich et al., 2021). Subsequent investigations showed that ROCK2 knockout considerably increased α-SYN exchange between microglia rather than ROCK1 knockout. Notably, ROCK2 controls actin polymerization, while ROCK1 mainly affects the actomyosin complex. These findings suggested that when α-SYN are present in the environment, microglia form the TNT network to share the aggregated α-SYN with neighboring cells that do not have this pathogenic aggregate, thereby supporting each other, reducing the individual degradation burden, and helping the cell community to quickly and efficiently degrade misfolded and aggregated proteins.

Chakraborty et al. (2023) used the HMC3 microglia cell line to explore the role of TNTs. Under normal growth conditions, 39.82% of HMC3 cells were connected by TNTs. They co-cultured the human neuroblastoma cell line SH-SY5Y with HMC3 microglia to study TNT exchange within and between the two cell populations. TNT connections between the two populations were observed, and an increase in TNT formation between the same cells or between both cell types was detected following the administration of α-SYN to either cell type. In this study, the nerve cells and microglia that emitted TNT to transfer α-SYN were designated as donors, while the cells that received the transfer were termed acceptors. α-SYN transmission between donor and acceptor cells was not uniform; specifically, the number of α-SYN puncta per acceptor cell was 3.04-fold higher in microglia than in neuronal cells, and the proportion of microglia acting as acceptors was 2.7-fold greater than that of neuronal cells. One of the major pathological implications of α-SYN accumulation is its detrimental effect on mitochondria within nerve cells. The researchers then investigated whether microglia could replenish mitochondria to SH-SY5Y cells loaded with α-SYN. As anticipated, mitochondrial transfer to α-SYN-loaded cells was 6.27-fold higher than that in wild-type (WT) SH-SY5Y cells, with α-SYN-loaded neurons receiving 1.68 times as many mitochondrial organelles. Interestingly, the researchers also observed mitochondria moving from the microglia to neuronal cells along the same TNTs, alongside α-SYN aggregates moving from neuronal cells to microglia. These findings suggest that burdened neuronal cells primarily utilize TNT-mediated transfer to actively transport α-SYN aggregates to microglia while supplying damaged neuronal cells with mitochondria for metabolic support.

Tunneling nanotubes between microglia and astrocytes

The maintenance of brain homeostasis is highly dependent on effective communication and collaboration among various cell types, particularly glial cells, which play a crucial role in chronic neuroinflammation. The interactions between astrocytes and microglia have garnered significant attention, which has primarily focused on the roles of growth factors, neurotransmitters, gliotransmitters, cytokines, and chemokines. Astrocytes are generally considered to be quiescent cells within the brain parenchyma, while microglia are actively surveillant, constantly monitoring their environment. In the context of disease, both astrocytes and microglia shift to an activated inflammatory phenotype and work in concert. For instance, activated microglia secrete pro-inflammatory molecules such as interleukin 1-alpha, tumor necrosis factor-alpha, and complement component 1q in response to injury. This secretion can drive astrocytes to adopt a neurotoxic phenotype, contributing further to neuroinflammation (Liddelow et al., 2017; Yun et al., 2018). In a study focused on the retina, microglia actively regulated the population of astrocytes through a process of engulfment, highlighting a direct mechanism of interaction between these cell types (Puñal et al., 2019). Additionally, in the context of neuropathic pain, astrocytes release C–X–C motif chemokine ligand 12 (CXCL12), which activates C–X–C motif chemokine receptor 4 (CXCR4) on microglia. This interaction leads to the secretion of additional pro-inflammatory cytokines and prostaglandins, exacerbating the inflammatory response (Luo et al., 2016). These findings underscore the complex and dynamic crosstalk between astrocytes and microglia, particularly during pathological conditions, and highlight the importance of their interactions in the progression of neuroinflammation and associated neurological disorders.

The role of TNTs in the transmission of α-SYN and amyloid-β between glial cells has been highlighted in recent studies (Rostami et al., 2021). During the progression of neurodegenerative diseases such as AD and PD, lysosomal and mitochondrial deficiencies lead to the accumulation of α-SYN aggregates. When exposed to α-SYN, astrocytes accumulate this protein over time; however, their levels remain consistently lower than those observed in microglia. Notably, microglia appear to be more effective at degrading aggregated α-SYN than astrocytes. Interestingly, co-culturing astrocytes with microglia resulted in a reduction of overall α-SYN accumulation in both cell populations, indicating a synergistic effect in degradation. One study meticulously detailed the spatial relationship between TNTs and the cargo transfer of α-SYN between astrocytes and microglia. Specifically, aggregated α-SYN deposits within astrocytes were observed to relocate toward the interacting microglia, moving from areas near the nucleus into the extensively formed TNTs connecting the two cell types. Microglia are typically positioned in the annular region surrounding astrocytes and are tethered to the astrocyte membrane through elongated protrusions. They receive α-SYN from astrocytes via these TNTs, and degradation of α-SYN aggregates appears to begin almost immediately after transfer from astrocytes to microglia. In conclusion, under pathological conditions, astrocytes and microglia tend to establish complex TNT structures that facilitate the transfer of α-SYN to microglia, where the α-SYN is subsequently degraded. This process plays a critical role in reducing the overall α-SYN content, potentially mitigating the toxic effects associated with α-SYN accumulation in neurodegenerative diseases.

Both astrocytes and microglia are significant yet controversial players in the pathology of neurodegenerative diseases. Existing studies suggest that TNTs may mediate the transfer of protein aggregates between microglia and other cells. Microglia have the ability to absorb and degrade α-SYN fibrils released by neurons, which is intended to halt neurodegeneration. However, this process can potentially exacerbate neuronal death.

Additionally, TNTs are involved in the transport of mitochondrial particles. Research has shown that transferring mitochondria from healthy cells to damaged, UV-irradiated cells can rescue the phenotype of the latter, protecting them from apoptosis. This mechanism presents a promising avenue for restoring the metabolic health of α-SYN-loaded cells (Wang and Gerdes, 2015).

Tunneling Nanotubes Between Other Glial Cells

Tunneling nanotubes between glial cells and glioma cells

Gliomas are CNS tumors that account for approximately 80% of malignancies that occur in the brain. GBMs are the most aggressive form of gliomas, and they account for more than 50% of gliomas. The role of the tumor microenvironment has recently drawn considerable attention in GBM research, and peritumoral tissues may be involved in regulating tumorigenesis and treatment resistance to GBM. Glia can be recruited by multiple cytokines within the glioma microenvironment and, in turn, these glia promote the proliferation and invasion of GBM by releasing cytokines and immune adaptogens. In this regard, TNTs represent one of the most pivotal research directions currently under investigation. TNTs have been observed in several types of cancer, where they are believed to drive a more malignant phenotype by allowing intercommunication of cancer cells and exchanging mitochondria with healthy cells in the tumor microenvironment. The mechanisms by which glial cells interact in GBM and whether they have different properties and perform various functions remains unclear (Yang et al., 2022).

Does the structure of TNTs differ between normal cells and tumor cells under pathological conditions? Formicola et al. (2019) thought the answer was yes. They compared TNTs produced by normal human astrocytes (NHA), a model of healthy cells, with those produced by U87-MG cells, a model of GBM cells. They observed that NHA produced shorter and thinner TNTs, while U87-MG cells almost exclusively produced thick and lengthy protrusions. Since thick TNTs are more efficient in the transport of vesicles and organelles, these findings demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy (Pinto et al., 2021).

Microglia account for 13%–34% of the tumor mass, but they constitute only 10%–15% of normal non-neoplastic brain specimens (Hambardzumyan et al., 2016). Considering the abundance of microglia in GBM, researchers designed engineered microglia (BV2 cells) for use as transport vectors to deliver drug granules for the treatment of glioma (Du et al., 2021). Investigators used blank DiO-BV2 cells cultured with DiI-stained GL261 or U87 MG cells and found that in DiI-stained cells, DiO co-localization increased for 2–6 hours. However, this phenomenon was not observed when BV2 was co-cultured with nerve cells, indicating different predispositions for normal and tumor cells. TNTs were also observed between BV2 and glioma cells, and the uptake of drug-loaded liposomes by U87 MG and GL261 cells was significantly inhibited after co-treatment with an actin polymerization inhibitor cytochalasin D and a neutral sphingomyelinase inhibitor GW4869. Thus, GW4869 and cytochalasin D obstructed cargo transportation from BV2 cells to glioma cells, indicating that both exosomes and TNT play a key role in cargo transport in glioma.

Astrocytes are the most abundant peripheral cells in the glioma microenvironment in vivo and constitute 40%–50% of brain cells (Guan et al., 2018). They exhibit a reactive phenotype upon contact with tumor cells, expressing high concentrations of GFAP and further stimulating the uncontrolled proliferation of glioma cells by expressing mitochondrial membrane potential-2, stromal cell-derived factor 1, and monocyte chemotactic protein-4 (Guan et al., 2018). Zhang and Zhang (2015) hypothesized that TNTs between glioma cells and astrocytes could provide a communication pathway between glioma cells and astrocytes, thus influencing their properties. They transfected rat astrocytes with red fluorescent protein (RFP) and C6 glioma cells with EGFP and mixed the two cells in a 1:1 ratio to establish a co-culture system. They found that most TNTs originated from astrocytes rather than glioma cells, and they observed mitochondrial transport from astrocytes to C6 glioma cells via TNTs. Consistent with previous studies (Wang et al., 2011; Sun et al., 2012), the induction of TNT initiation by astrocytes is also dependent on p53. Hydrogen peroxide augmented the quantity of TNTs in the original system, subsequently followed by co-culture with the F-actin-depolymerizing agent Latrunculin A, which suppressed the formation of TNTs and concurrently influenced the proliferative potential of C6 cells.

Using advanced measurement techniques, Valdebenito et al. (2021) investigated the role of TNTs between glioma cells and astrocytes under hypoxic conditions. They found that mitochondrial transport via TNTs occurred from GBM cells to astrocytes, thereby altering the metabolism of the latter. The researchers utilized the U87-GBM cell line to create a co-culture system with human cortical primary astrocytes. EM analysis of the top optical sections of GBM cells revealed a significant increase in mitochondrial size and enhanced interactions between mitochondria and liposomes with the endoplasmic reticulum in regions where TNTs were formed. To further examine mitochondrial transfer, the team stained live U87 cells with mitochondrial tracking orange and observed that mitochondria followed TNTs from U87 cells into primary astrocytes. To evaluate the metabolic effects of TNT-mediated mitochondrial transfer from U87 cells to primary astrocytes, the researchers identified distinct organelle fingerprints of the two cell types through mitochondrial DNA (mtDNA) sequencing. They observed that the mitochondria from tumors differed from those already present in primary astrocytes. Subsequently, laser-capture microdissection of co-cultures of U87 and primary astrocytes, both in the presence and absence of TNT blockers, successfully identified GBM mtDNA in primary astrocytes. The use of TNT inhibitors, however, blocked mitochondrial sharing and the diffusion of GBM mtDNA into primary astrocytes. Overall, TNT-mediated transfers altered the metabolism of primary astrocytes, enabling them to adapt to glutamine metabolism, which they had not previously exhibited.

Tunneling nanotubes and Schwann cells

TNTs are also involved in the development of peripheral neurons. The glial cells in the peripheral nervous system are known as SCs. Communication and interactions between SCs and neurons are essential for the development and functioning of myelinated axons, with neurons providing critical signals that influence the functioning of SCs, thereby promoting neuronal survival and enabling efficient transduction of action potentials. Dysregulation of neuron-SC interactions often leads to developmental abnormalities and disease. Zhu et al. (2016) observed the formation of TNTs between SCs isolated from neonatal EGFP and RFP transgenic rats. They found that TNTs facilitated the transfer of red and green lipophilic components, yielding a yellowish or lighter color phenotype. In vivo, extensive fluorescent phalloidin labeling was observed in the injured proximal nerve stump in comparison with normal nerves, indicating enhanced TNT formation and cargo transfer between SCs and axons, including newly synthesized RNA. The deletion of the small GTPases Rab8a and Rab11a inhibited the formation of TNTs in SCs and further reduced SC cell migration. Additionally, the deletion of Rab8a and Rab11a in SCs inhibited the transport of neurotrophic factors from SCs to co-cultured neurons via TNTs, such as brain-derived neurotrophic factor, thereby suppressing axonal outgrowth from the neurons.

Overall, these findings suggest that Rab8a and Rab11a may play roles in the formation of TNTs in SCs, and that TNTs may influence the regeneration of peripheral nerves by regulating communication between nerve cells.

Limitations

Absence of rigorous definitions and standards

Despite the substantial advancements in research on TNTs, numerous questions regarding TNTs remain unresolved. While visualization of TNTs in cells by labeling the cytoskeleton is not seemingly difficult, TNTs show diverse and complex structures, ranging from TNTs containing only individual actin filaments to those containing both actin filaments and microtubules. They also show variability in lengths and thicknesses, with no strict standards to distinguish them from similar structures like filopodia and invadopodia (Delage et al., 2016; Melwani and Pandey, 2023). Consequently, the criteria used in the existing studies were mostly defined by individual research groups. Moreover, TNTs are highly sensitive to mechanical stress, chemical fixatives, and light, hindering efforts to determine their true structure in their natural state (Panasiuk et al., 2018).

Mechanisms underlying the formation of tunneling nanotubes remain ambiguous

Although TNTs are known to increase in response to stimulation, the specific stress stimuli that trigger their production and the underlying mechanisms remain unclear (Yamashita et al., 2018). For instance, the formation and origin of TNTs are heterogeneous. Existing literature suggests that glial cells are the primary originators of active TNTs, showing a greater propensity to emit TNTs than neurons or tumor cells (Du et al., 2021; Scheiblich et al., 2021; Chakraborty et al., 2023). Among glial cells, microglia produce TNTs more readily than astrocytes, consistent with their active role in the nervous system (Rostami et al., 2021). This difference may be attributed to differences in physiological characteristics, labeling methods, or secretory factors, although these aspects are not yet clearly understood (Zhang and Zhang, 2015).

Technical constraints in studies of tunneling nanotubes

Monitoring TNTs between glial cells is hindered by several technical limitations. In vitro research on cargo transfer between cells has primarily focused on mitochondria, while the transport of other substances remains poorly understood. This discrepancy may stem from the relative ease of detecting mitochondria in comparison with other materials (Shanmughapriya et al., 2020; Zampieri et al., 2021).

The use of in vivo disease models also presents substantial challenges for real-time visualization of the dynamics of glial morphology and its relationships with neurons, blood vessels, or infiltrating immune cells. The sensitivity and fragility of TNTs require extremely high-resolution imaging (Pinto et al., 2021). Additionally, glial infiltration often occurs in hard-to-reach areas, with limited observation windows and difficult access to the CNS (Yang and Zhou, 2019). Despite advancements in two-photon calcium imaging and EM techniques, technical constraints still hinder the widespread use of these techniques for providing a comprehensive description of glial cell morphology in disease models (Sartori-Rupp et al., 2019b). Other methods aimed at mimicking a simpler in vivo environment, such as cultures of primary cells and organoids, have been increasingly adopted to address these limitations. However, this shift also implies that changes occurring in the more complex in vivo environment of TNTs remain largely unknown (Salaud et al., 2020; Pinto et al., 2021). Future advancements in research methods may yield new insights into TNTs and lead to improved solutions for neurological diseases.

Clinical application remains premature at present

Given the vast potential and inherent uncertainties in TNT research, direct greater attention using in vivo studies focusing on the mechanisms of TNT formation and their functions are essential. While some studies have shown that pathological states can influence TNT formation and that metabolic products and drugs can be transported via TNTs, more in vivo experiments are needed to support the therapeutic efficacy of TNTs. Additionally, these studies should primarily focus on directing TNTs to enhance disease treatment by influencing the direction and efficiency of the transport of toxic substances and energy rather than facilitating the spread of pathological states.

Discussion

Glial cells influence the stability of the neural system through tunneling nanotubes

Within the nervous system, long-distance cell–cell communication is facilitated by various forms of cellular crosstalk, which protect the brain from damage caused by external stimuli. This communication occurs through various mechanisms, including direct cell–cell contact, secreted proteins, and gap junctions (Fagotto and Gumbiner, 1996; Borsini et al., 2015). The patterns of these communication systems are complementary in multicellular organisms, each offering unique advantages that collectively influence neural activity (Melwani and Pandey, 2023). Therefore, assessment of changes in nerve cell communication is essential for understanding the development of the nervous system and the progression of neurological diseases.

Among various communication methods, TNTs represent a relatively new and not fully understood type of cell–cell interaction. A detailed understanding of TNT mechanisms could open new avenues for neurotherapy. Research on TNTs has increased exponentially over the past two decades since they were first described in detail (Hodneland et al., 2006). TNTs have been widely reported in a variety of cell types, including endothelial cells (Charreau, 2021; Lin et al., 2024a), pericytes (Alarcon-Martinez et al., 2020; Pisani et al., 2022), immune cells (Sowinski et al., 2011; Yang et al., 2020), and tumor cells. They have been shown to facilitate the transfer of endocytic vesicles, lysosomes, mitochondria, membrane-bound proteins, and even viruses (Kumar et al., 2017; Okafo et al., 2017; Mittal et al., 2019), allowing for more direct and rapid cargo transport in comparison with other methods (Barcia et al., 2012).

In the nervous system, glial cells respond reactively to injuries by forming a defense system that prevents microorganisms and toxins from invading surrounding tissues (Yang and Zhou, 2019). Recent studies (Doetsch et al., 1999; Valny et al., 2017; Kiaie et al., 2022) have also highlighted the potential of glial cells as stem and progenitor cells. However, once activated, some reactive glial cells secrete neuro-suppressive factors that inhibit neuronal growth, ultimately leading to glial scarring and neuroinflammation within the brain (Guo et al., 2014). Glial cells generally perform multifunctional roles and establish TNT connections with surrounding cells, with these connections increasing under stimulation. In this review, we focused on TNT-mediated cellular communication and the transport of substances within or between glial cells and other cells in various neurological diseases, such as AD, PD, and glioma, with the aim of providing new insights into TNTs.

Various types of glial cells can influence neural regeneration through tunneling nanotubes

During the growth and development of neurons, immature neurons extend TNTs to astrocytes, establishing electrical coupling and facilitating the exchange of calcium signals (Wang et al., 2012). Microglia are the first to be activated in response to stimulation, followed by astrocytes, which undergo complex changes in morphology, gene expression, and function. These glial cells often transmit beneficial substances or energy to neurons and other cells through TNT-like structures when stimulated (Formicola et al., 2019; Chastagner et al., 2020; Rostami et al., 2020). Glial cells tend to absorb toxic substances through TNTs and degrade them more efficiently by coordinating an “on-demand” degradation network, allowing them to better cope with external stimuli as a collective unit (Rostami et al., 2021; Scheiblich et al., 2021; Chakraborty et al., 2023). Additionally, SCs generate TNTs to promote the regeneration of peripheral nerves. In the context of gliomas, TNTs from astrocytes or microglia appear to play a role in diminishing the viability of tumor cells. Overall, astrocytes, oligodendrocytes, microglia, and SCs play important and complex roles in influencing neuronal activity through glial-neuronal interactions mediated by TNTs.

New insights into tunneling nanotubes and new understanding of diseases

A thorough understanding of TNT communication networks can provide new insights into previously unexplained phenomena. For instance, ROCK inhibitors may prevent microglial activation by enhancing TNT formation among microglia, which in turn, attenuates α-SYN aggregation and restores the number of dopaminergic neuronal cells in MPTP mouse models of PD (Barcia et al., 2012; Tatenhorst et al., 2016). Targeting TNT production can reshape disease perception and treatment strategies, e.g., by facilitating the transfer of healthy mitochondria from undamaged glial cells, multipotent mesenchymal stem cells, NSCs, or BTICs to damaged cells. This transfer can help maintain homeostasis and mitigate the toxic effects of nanomaterials (Zhang and Zhang, 2015; Valdebenito et al., 2021). Further research is needed to unravel the complexities surrounding TNTs to explore novel therapeutic approaches for various diseases.

Funding Statement

Funding: This work was supported by the National Natural Science Foundation of China, No. 82101115 (to JY); the Wuhan University Independent Innovation Fund Youth Project, No. 2042021kf0094 (to JY).

Data availability statement:

Not applicable.