Astrocyte Mitochondria: Central players and potential therapeutic targets for neurodegenerative diseases and injury

Abstract

Mitochondrial function has long been the focus of many therapeutic strategies for ameliorating age-related neurodegeneration and cognitive decline. Historically, the role of mitochondria in non-neuronal cell types has been overshadowed by neuronal mitochondria, which are responsible for the bulk of oxidative metabolism in the brain. Despite this neuronal bias, mitochondrial function in glial cells, particularly astrocytes, is increasingly recognized to play crucial roles in overall brain metabolism, synaptic transmission, and neuronal protection. Changes in astrocytic mitochondrial function appear to be intimately linked to astrocyte activation/reactivity found in most all age-related neurodegenerative diseases. Here, we address the importance of mitochondrial function to astrocyte signaling and consider how mitochondria could contribute to both the detrimental and protective properties of activated astrocytes. Strategies for protecting astrocytic mitochondrial function, promoting bidirectional transfer of mitochondria between astrocytes and neurons, and transplanting healthy mitochondria to diseased nervous tissue are also discussed.

1.1. Introduction

As the body grows older and slowly moves further away from physiologic homeostasis, many changes in all of the bodily systems occur. Arguably, one of the most devastating is the changes that occur in the central nervous system- leading to loss of cognitive, motor, and emotional function that in essence make us who we are. Dementia is one such disease that progresses steadily with age, and to date there are not many effective treatments to stave off the changes that occur. Many studies have focused on salvaging neuronal function, but there are many cell types in the central nervous system that directly interact with neurons and can be a target for therapeutics, such as astrocytes.

1.2. Astrocytes

Astrocytes are the predominant glial cells found in all areas of the brain, providing uniform, non-overlapping coverage (Bushong et al., 2002) in a network of astrocytes connected by gap junctions. Pertinent to synaptic health, astrocytes guide the development and directionality of synapses (Powell and Geller, 1999), regulate transmission efficiency at pre and post-synaptic sites (Ullian et al., 2001), and modulate synapse formation and turnover (Allen et al., 2012; Bialas and Stevens, 2013; Blanco-Suarez et al., 2018; Christopherson et al., 2005; Diniz et al., 2012; Fossati et al., 2019; Gomez-Casati et al., 2010; Kucukdereli et al., 2011; Mauch et al., 2001; Vainchtein et al., 2018). Astrocytic endfeet enwrap nearly the entire cerebrovasculature creating a physical barrier known as the blood brain barrier (BBB) (Abbott et al., 2006) allowing astrocytes to shuttle metabolites from the blood to other neural cells, exchange water molecules between blood vessels and the brain parenchyma, take up potassium and glutamate to protect neurons from excitotoxicity (Nielsen et al., 1997; Price et al., 2002; Simard et al., 2003) and link elevations in neuronal activity to elevations in blood flow through a process called neurovascular coupling (NVC) (Gordon et al., 2007; Koehler et al., 2009).

It’s well established that astrocytes use sophisticated Ca²⁺ signaling networks and mechanisms to communicate directly with neurons and other astrocytes (for a more in-depth review see (Rossi, 2015; Verkhratsky, 2019) and (Rusakov, 2015)). Activation of a variety of receptors on the astrocyte cell membrane trigger Ca²⁺ release from internal stores, which can spread to neighboring astrocytes through gap junctions, creating Ca²⁺ waves across large interconnected astrocyte networks (Charles et al., 1991). Ca²⁺ waves are most prevalent in astrocytic processes (Grosche et al., 1999) creating heterogenous microdomains within the astrocyte (Panatier et al., 2011{Bindocci, 2017 #717)}. These waves can be generated spontaneously without neuronal input (Nett et al., 2002{Parri, 2001 #60)} or initiated by neurotransmitters, thus allowing neurons to influence Ca²⁺ levels in astrocytes and play an important role in synchronizing astrocytic Ca²⁺ transients (Aguado et al., 2002). Once initiated in astrocytes, Ca²⁺ signals may have a major impact on the physiology of nearby neurons and other cellular constituents of the neurovascular unit. For instance, astrocytic Ca2+ transients can be shaped by the mitochondrial sodium/Ca²⁺ exchanger, altering exocytotic release of a variety of gliotransmitters including glutamate, ATP, and D-serine, at the synapse (Parnis et al., 2013). These gliotransmitters can act upon neuronal receptors, providing a route for direct astrocyte-neuron communication (for review see (Araque et al., 2014)).

1.3. Astrocyte Activation

Astrocytes undergo many morphologic/biological changes during aging, disease, and injury, collectively referred to as “astrocyte activation” or “astrocyte reactivity”. Reactive astrocytes are often morphologically distinct from “resting” astrocytes and are usually characterized as more ramified, larger, with elongated processes (Burda et al., 2016; Wilhelmsson et al., 2006). Astrocyte activation is complex, with ongoing debates over the merits and detriments of astrocytic activation. Detrimental astrocytes appear to exhibit increased ROS and Ca²⁺ dysregulation (Ishii et al., 2017), release numerous cytokines, chemokines, and ROS involved in chronic neuroinflammation (Choi et al., 2014; Clarke et al., 2018; Farina et al., 2007; Zhu et al., 2009), impair glutamate uptake leading to excitotoxicity and neuronal degeneration (Dossi et al., 2018; Schousboe and Waagepetersen, 2005; Sompol et al., 2017), create abnormal synapses resulting in epilepsy and neuropathic pain (Boroujerdi et al., 2008), or cause the loss of synapses leading to impaired synaptic plasticity and cognitive decline (Furman et al., 2012; Furman et al., 2016; Hong et al., 2016; Shi et al., 2017; Stevens et al., 2007). In contrast, beneficial astrocytes release an array of factors that aid in synaptic development, repair, and rewiring including thrombospondin, matricellular proteins (SPARC-1 and hevin), TGF-beta, IL-33, D-serine, glypicans, BDNF, pentraxin 3, Chrd11, C3 and cholesterol to name just a few (Allen et al., 2012; Bialas and Stevens, 2013; Blanco-Suarez et al., 2018; Christopherson et al., 2005; Diniz et al., 2012; Fossati et al., 2019; Gomez-Casati et al., 2010; Kucukdereli et al., 2011; Mauch et al., 2001; Schafer et al., 2012; Vainchtein et al., 2018). Beneficial astrocytes also assist microglia in clearing dead cells, cellular debris, and/or protein aggregates (Gomez-Arboledas et al., 2018; Wakida et al., 2018; Wyss-Coray et al., 2003), and may assume greater glycogen storage capacity resulting in better memory formation and neuronal survival (Matsui et al., 2017; Waitt et al., 2017). Finally, astrocyte-containing glial scars, formed around injured tissue and protein aggregates, have long been thought to provide a physical barrier for shielding healthy tissue from damaging factors (Faulkner et al., 2004; Okada et al., 2006; Wang et al., 2018), though deleterious properties of glial scars have also been described (Liddelow and Barres, 2015; Liddelow and Barres, 2017; Perez-Nievas and Serrano-Pozo, 2018; Sofroniew and Vinters, 2010).

1.4. Mitochondrial Function in Astrocytes

Despite their heavy reliance on glycolysis for energy production (Hamberger and Hyden, 1963; Pellerin and Magistretti, 1994; Zhang et al., 2014), astrocytes are responsible for 20% of the brain’s total oxygen consumption (Bluml et al., 2002), most of which occurs during oxidative phosphorylation within astrocytic mitochondria en route to the production of adenosine triphosphate (ATP). In addition to oxidative metabolism, mitochondria also serve as storage organelles for Ca²⁺ and play an important role in intracellular Ca²⁺ sequestration and signaling. Elevations in cytosolic Ca²⁺ appear to promote the immobilization of mitochondria near highly active synapses (Stephen et al., 2015) and fine processes where Ca²⁺ transients occur most frequently (Bindocci et al., 2017). Mitochondria are strategically located in these microdomains to meet higher local energy demands (Jackson and Robinson, 2018), shape Ca²⁺ fluctuations (Agarwal et al., 2017; Gunter et al., 2004), and/or modulate Ca²⁺ -dependent processes such as gliotransmission (Parnis et al., 2013; Stephen et al., 2015). Regardless of the exact function, mitochondria loss in these microdomains is associated with neurodegeneration, suggesting an essential role in astrocyte-neuron synergy. Additionally, it was shown that a decrease in healthy mitochondria in astrocytic processes correlated to neuronal loss after ischemia, even though the number of astrocytes remained stable, indicating that the loss of mitochondria at the astrocyte-synapse junctions may play a part in neuronal loss (Ito et al., 2009). Mitochondria can also be found in cerebral blood-vessel associated astrocyte endfeet, which are also highly metabolically active and exhibit dynamic Ca²⁺ signaling (Kacem et al., 1998; Stobart et al., 2018). However, very little is known about the role of the mitochondria in these specialized microdomains.

2.1. Discussion

2.2. Dysfunction of astrocytic mitochondria can cause deleterious actions on neurons

As in other cell types, astrocytic mitochondria are vulnerable to aging, injury and disease. In fact, many of the same stimuli that trigger robust astrocyte activation (for example, ischemia, traumatic tissue injury, inflammation, cytokines) also lead to astrocytic mitochondrial dysfunction (Figure 1), the telltale signs of which include calcium dysregulation, MPTP, the excessive generation of reactive oxygen species (ROS) and cell death cascades. Due to the intimate relationships between astrocytes, these factors can possibly have far-reaching effects across the astrocyte network, causing further astrocyte activation and exacerbating the astrocyte activation/ mitochondria dysfunction loop. Oxidative stress in astrocytes, triggered by pathogenic factors such as amyloid-beta, has been shown to inflict extensive damage to surrounding neurons (Abramov et al., 2004). Neurotoxicity initiated by astrocytic mitochondrial dysfunction may arise from the release of a number of proinflammatory cytokines, ROS, and other inflammatory factors (Kubik and Philbert, 2015; Min et al., 2015; Nahirnyj et al., 2013).

Figure 1. Factors that activate astrocytes also influence mitochondrial health and function.

Many of the pathogenic factors (either by loss of tissue homeostasis or release of molecular signals) that are responsible for activating astrocytes can also trigger mitochondrial dysfunction. Activation of astrocytes increases the activity burden placed upon their mitochondria, which may in turn cause dysfunction and the release of an array of factors within the cell. These factors can themselves cause activation or damage to the astrocyte. Due to the nature of this bidirectional loop, it is difficult to assess whether the pathogenic factors cause astrocyte activation, which subsequently causes mitochondrial dysfunction, or vice versa.

Breakdown of the astrocytic mitochondrial membrane potential by adding fluorocitrate to primary astrocytes caused a delayed dissipation of the mitochondria membrane potential leading to reduced glutamate uptake (Voloboueva et al., 2007). Consequently, neurons co-cultured with fluorocitrate-treated astrocytes exhibited increased vulnerability to glutamate-mediated excitotoxic death. It is interesting to note that the deleterious effects of fluorocitrate on astrocytes was hastened in the presence of co-cultured neurons, suggesting that neurons actively tax the energy output of astrocytic mitochondria. Similarly, it was shown that there was increased neuronal death following ischemic injury when specifically targeting astrocytic electron transport chain for damage in vivo (Fiebig et al., 2019). These results infer that functional astrocytic mitochondria are necessary for the support functions that astrocytes provide to neurons following injury. If mitochondrial dysfunction in astrocytes is harmful to neurons, it is logical to assume that treating astrocytic mitochondria can then be beneficial to neurons.

Similar to neurons, activated astrocytes exhibit extensive Ca²⁺ dysregulation during aging, injury, and disease characterized by elevated cytosolic Ca²⁺ levels, augmented Ca²⁺ transients, and/or more frequent Ca²⁺ oscillations (Agulhon et al., 2008; Kuchibhotla et al., 2009). Excess accumulation of Ca²⁺ into the mitochondria, which often occurs when other Ca²⁺ regulatory mechanisms are challenged, depletes the mitochondrial membrane potential, leading to impaired ATP production, and, in extreme cases, generation of the mitochondrial permeability transition pore (MPTP) (Hunter et al., 1976) resulting in release of Ca²⁺, cytochrome c, ROS and other cytotoxic factors into the cytoplasm (Krajewski et al., 1999). Severe Ca²⁺ dysregulation in astrocytes likely leads to aberrant activation of Ca²⁺ -dependent enzymes, such as calpain proteases and calcineurin phosphatases (Feng et al., 2011; Norris et al., 2005; Pleiss et al., 2016; Price et al., 2018; Shields et al., 2000; Shields et al., 1998), which in turn can exacerbate mitochondrial dysfunction. Consequently, inhibition of calcineurin signaling in astrocytes under a variety of injurious conditions helps maintain protective properties of astrocytes (e.g. glutamate uptake) and reduces excitotoxic damage to neurons (Furman et al., 2012; Sompol et al., 2017), perhaps through the dampening of vicious feedback cycles between oxidative stress and Ca²⁺ dysregulation.

2.3. Bidirectional exchange of mitochondria between astrocytes and neurons

Mitochondria may also be central to many of the neuroprotective functions associated with astrocyte activation. Presumably, the release of growth factors, establishment of protective glial scars, and increased synaptic support all depend critically on the energy output of astrocytic mitochondria. Recent work has established that astrocytes can’t mount a protective proliferative response following acute brain lesions if mitochondrial function is impaired (Fiebig et al., 2019). Astrocytes, like many other cell types, dispose of their own damaged mitochondria via autophagy (mitophagy) which is elevated following acute injury (Quintana et al., 2019), perhaps to minimize the deleterious consequences of oxidative stress and Ca²⁺ dysregulation discussed above. Not only do astrocytes actively degrade their own damaged mitochondria, they can also take up and dispose of mitochondria released from other cells. This form of “transmitophagy” was first reported by Davis et al (2014), who showed that fluorescently tagged mitochondria from otherwise healthy retinal ganglion cells were present (in high numbers) in astrocytic lysosomes. It’s unclear whether astrocytes take a leading role in disposing of neuron-derived mitochondria in the context of neurodegenerative disease or following acute brain injury.

Interestingly, the intercellular transport of mitochondria in astrocyte-neuron networks appears to work in both directions. But, while neurons tend to hand off damaged mitochondria to astrocytes for degradation, it seems that activated astrocytes can donate some of their healthy mitochondria to nearby damaged neurons, which in turn, leads to greater neuronal viability. Release of mitochondria-containing vesicles from primary astrocytes was found to depend on a Ca²⁺ -CD38-cADPR signaling pathway (Hayakawa et al., 2016). Upregulation of astrocytic CD38 using a CRISPR/Cas9 system significantly augmented the release of mitochondria-containing vesicles, that, when added to oxygen-glucose starved neurons, restored neuronal energy production. Hayakawa et al subsequently validated these observations in intact mice by showing that fluorescently tagged astrocytic mitochondria are passed to neurons after a transient ischemic insult. Astrocyte-derived mitochondria appeared to fuse with neuronal mitochondria in peri-infarct neocortex and were associated with the upregulation of cell survival pathways. Moreover, blockade of CD38 in infarcted mice had a negative impact on a number of functional outcome measures, suggesting that astrocyte-mediated mitochondria release occurs naturally in the brain as long as CD38 signaling is maintained. It is presently unknown whether a similar neuroprotective mechanism is used in other, more chronic neurodegenerative conditions.

2.4. Mito-Centric Therapies

Mitochondrial-targeting strategies have long been investigated for their potential to treat brain injury and neurodegenerative disease. Administration of antioxidants, mild uncouplers, MPTP inhibitors, and alternative biofuels are among the most commonly used strategies. In the fields of neurotrauma and neurodegeneration, these treatments are typically aimed at providing neuroprotection through the improvement of neuronal mitochondrial function. However, as outlined in this review, astrocytic mitochondrial health may be just as important to overall neuronal survival and adaptation to injury/disease. By targeting the main support cells of the brain (i.e. astrocytes) many other cell types besides neurons may reap substantial benefits. For research (and possibly clinical) purposes, astrocytes can be selectively targeted with recombinant viral vectors, nanoparticles, or specialized peptides (Figure 2) (Bosson et al., 2017; Cannon et al., 2011; Furman et al., 2012; Terashima et al., 2018; Valiante et al., 2015). Valori et al. (2019) published an excellent review regarding the possible hurdles to translation of astrocyte-centered therapy as well as current approaches (Valori et al., 2019). Evidence for imparting neuroprotection through the selective targeting of astrocyte signaling pathways has already been established in several different disease models including acute cortical lesions (Wilhelmsson et al., 2004), β-amyloid expression (Ceyzeriat et al., 2018; Fernandez et al., 2016; Furman et al., 2012; Sompol et al., 2017), spinal cord injury (Gaudet and Fonken, 2018), chronic pain (Li et al., 2019), and CNS injury (Furman et al., 2016; Huang et al., 2019). Astrocyte-directed therapeutics to protect and/or maintain healthy mitochondrial function could include a variety of antioxidants, MPTP modulators, and alternative biofuels utilized in oxidative phosphorylation. Alternatively, strategies could focus on the release of healthy mitochondria from astrocytes through the stimulation of CD38-cADPR signaling.

Figure 2. Possible mitochondria-centric astrocyte therapies.

Healthy mitochondrial function is key to proper astrocyte function. Targeting mitochondria to increase astrocytic vitality and thereby support neuronal and vascular health is becoming a popular potential therapeutic. Multiple avenues to target mitochondrial health are available, including delivery of drugs specifically to the mitochondria via AAVs, peptides, and nanoparticles. Supplementing the astrocyte with healthy mitochondria, or conversely promoting the degradation of unhealthy mitochondria, are also viable potential therapies.

Endogenous transcellular mitochondrial donation has also inspired a variety of exogenous mitochondrial delivery strategies, whereby healthy mitochondria are harvested from an allogeneic or, preferably an autologous source, and then transplanted into damaged or diseased tissue. Mitochondrial transplantation has already shown considerable promise for limiting ischemic damage to the heart, especially in pediatric patients with coronary artery disease (Emani and McCully, 2018) where it is being intensively investigated in clinical trials. While there are presently no similar clinical trials underway for CNS disorders, mitochondrial transplantation has shown significant neuroprotective and/or functional benefits in diverse experimental models (Chang et al., 2016; Cowan et al., 2016; Gollihue et al., 2018; Gollihue et al., 2017; Hayakawa et al., 2016; McCully et al., 2009). When injected directly into gliomas of living mice, isolated mitochondria led to a metabolic switch from glycolysis to aerobic respiration coincident with decreased glioma growth (Sun C, 2019). In a mouse model of stroke, transplantation of mitochondria to the infarct region resulted in neuronal uptake of mitochondria. Similarly, after stroke in these mice there is evidence of mitochondrial transfer from astrocytes to neurons resulting in increased pro-survival signaling, reduced neuronal death, and ameliorated motor and neurologic deficits (Hayakawa et al., 2016). Using a modified transplant procedure, supplemented with the peptide carrier protein, PEP1, mitochondrial delivery to the medial forebrain bundle of a Parkinson’s model rats led to greater neuronal sparing in the substantia nigra and improved locomotive functional outcome (Chang et al., 2013). More recently, we found that transplant of viable mitochondria to injured rat spinal cord helped to maintain acute bioenergetics, though no long term benefits were observed for tissue sparing or the recovery of essential sensory-motor functions (Gollihue, Patel et al. 2018). Together, these studies show that exogenous mitochondrial incorporation into neurons is possible in vivo, and provide proof-of-principle that transplants can result in measurable improvements for many different physiological outcomes.

3.1. Summary and Conclusions

Astrocytes are highly metabolically active cells in the central nervous system that have a variety of functions- one of the most important being neuronal support. Mitochondria produce energy to meet the energy demands of astrocytes. Astrocytic mitochondrial dysfunction is incidentally highly detrimental, leading to insufficient energy production, Ca²⁺ dysregulation, inflammatory response induction, and glutamate dysregulation. Treating mitochondria in astrocytes can directly benefit neuronal survival because of the interdependence of neurons and astrocytes. Additionally, astrocytes have an inherent ability to donate their mitochondria to neurons in need- a process that could be exploited for neuroprotection in aging, injury, and disease. Supplying exogenous healthy mitochondria to injured or susceptible tissue could have far-reaching effects in neuroprotection, though there does not seem to be a one size-fits-all technique that is applicable in all instances of CNS injury. Further research to optimize disease-specific techniques for targeting astrocytic mitochondrial health are needed.