Glial–neuronal mitochondrial transfer is mediated via a number of active processes including the release of extracellular vesicles, the formation of tunnelling nanotubes, and potentially other mechanisms (Figure 1, Table 1). These processes are discussed in detail below:

Extracellular vesicles (EVs) are bilayer membranous structures that are secreted into the extracellular space. There are two main types of EVs: exosomes and microvesicles. Microvesicles are distinguished from exosomes based on their larger size (typically 0.1–1 μm in diameter), whereas exosomes are much smaller (30–150 nm in diameter) [38]. Astrocytes have been shown to shed EVs containing functional mitochondria ranging from 300 nm up to 8 μm in diameter [20,39]. Similarly, microglia release EVs containing mitochondria into the extracellular space, where they are internalised by astrocytes [35]. It should be noted that EVs containing mitochondria are differentiated from previously identified EV subtypes such as mitovesicles [40] and mitochondria-derived vesicles (MDVs) [41], which are small vesicles that transport mitochondrial-derived cargo but lack several mitochondrial structures such as cristae, mitochondrial ribosomes, and proteins found in mitochondria, such as Tomm20 [40].

Tunnelling nanotubes (TNTs) were first described in 2004 by Rustom and colleagues [42], who identified membranous channels comprised of F-actin that connect two or more cells and are involved in cell-to-cell communication [43]. TNTs were shown to transfer different organelles, including mitochondria, from one cell to another but also other cargo, including proteins (e.g., α-synuclein) and nucleotides [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44]. Intercellular mitochondrial transfer via TNTs has been observed in astrocytes, microglia, and neurons. For instance, microglia have been shown to form a network of F-actin-positive intercellular membrane projections with neighbouring microglia that contain mitochondria [44]. TNT formation has also been observed between astrocytes and adjacent neurons in rat primary astrocyte and neuronal cells [33,45,46,47]. The direction of mitochondrial transfer between cells via TNTs is not fully understood; however, there is evidence to suggest that TNTs are formed in response to stress. For instance, exposure to hydrogen peroxide (H₂O₂) increased TNT formation between astrocytes in vitro [47]. Further, Wang and colleagues [45] reported that in astrocyte–neuron co-culture, the cells exposed to stressful stimuli, such as H₂O₂ or serum deprivation, develop TNTs towards the unstressed cells but not vice versa. Other studies have identified the small calcium-binding protein S100A4 and its putative receptor RAGE (receptor for advanced glycation end product) as guidance molecules that mediate the growth and direction of TNTs Wang [45]. However, further studies on the mechanistic pathway regulating TNT formation and directionality are needed. Table 1 summarizes findings of studies investigating the effects of mitochondria transfer via TNTs in different brain disease models and highlight the positive effect of this transfer [19,33,36,44,45,48,49,50].

Of note, Sartori-Rupp and colleagues found that mitochondria can be transported between neuronal cells via individual TNTs composed of a single, continuous bundle of parallel actin filaments [51]. This suggests that mitochondria directly bind to actin filaments and are transported via a microtubule-independent mechanism that remains to identified.

Although most evidence to date suggests that glial–neuronal mitochondrial transfer occurs via EVs or TNTs, a number of other intercellular transfer mechanisms have been proposed, including cell fusion and gap junctions [52]. Gap junctions are plasma membrane channels composed of connexins that have been shown to transfer small molecules [53], and more recently, whole mitochondria [54] between cells. Li and colleagues [55] observed mitochondrial transfer from bone marrow mesenchymal stem cells to motor neurons via gap junctions, and gap junction connexin32 is expressed in neurons. However, whether glia cells are capable of transferring mitochondria via gap junction has not yet been investigated. Cell fusion involves the direct fusion of two cellular membranes, which would theoretically allow for the transfer of cytoplasmic molecules, including mitochondria. However no direct evidence of cell fusion mediated mitochondrial transfer in glia or neurons has been observed. Further studies are needed to investigate whether glial–neuronal mitochondrial transfer can occur via gap junctions and cell fusion.