Mitochondrial Transcription Factor a (tfam): a Novel Brain Intercellular Signaling Molecule and Microglial Activator

Microglia are a subtype of the non-neuronal glial cells that participate in the immune responses of the central nervous system (CNS). Evidence suggests that microglial activation contributes to neuronal death in neurodegenerative diseases. Microglia can be activated by a variety of exogenous stimuli, including toxins or infectious agents, as well as by endogenous pathology-associated molecules such as α-synuclein in Parkinson's disease or amyloid-β protein in Alzheimer's disease. Studies suggest that some endogenous molecules, which do not have a pathological origin, can also activate microglia. Damage associated molecular patterns (DAMPs) are an example of such endogenous molecules. When released from damaged or dying cells, DAMPs can trigger microglial activation through specific receptors, generating an inflammatory response. Mitochondrial transcription factor A (TFAM) has recently been implicated as a possible DAMP that is detected by peripheral immune cells. However, the effects of extracellular TFAM on CNS glial and neuronal cells have not been explored. Our study implicates TFAM as an activator of human peripheral blood monocytes, human THP-1 monocytic cells and human primary microglia isolated from adult brain tissues. Interaction of TFAM with these cell types leads to induction of pro-inflammatory cytokines and cytotoxic secretions, which may contribute to neuronal death. In this brief review, we describe endogenous signaling molecules capable of microglial activation with a specific focus on the potential involvement of TFAM. Elucidating the role of endogenous intercellular signaling molecules, such as TFAM, in CNS cell communication could expand the fundamental knowledge of glial-neuronal cell interactions and provide new insights into the mechanisms underlying glial cell activation. Such studies could facilitate the identification of novel therapeutic targets for a variety of pathologies that involve sterile neuroinflammatory processes. To cite this article: Stephanie M. Schindler, et al. Mitochondrial transcription factor a (TFAM): a novel brain intercellular signaling molecule and microglial activator. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited.


Introduction
Microglial cells, which originate from the myeloid lineage, invade the central nervous system (CNS) prior to formation of the blood brain barrier [1] .They are the primary immune effector cells in the CNS, representing the innate immune system, and thus provide the first line of defense against injury and invading pathogens.Despite making up only 5-12% of the total cell population in the RESEARCH HIGHLIGHT brain, microglia play a fundamental role in normal cell maintenance and homeostasis [2,3] .In the absence of adequate regulation or under some pathological stimulatory conditions, however, microglia can take on a more destructive role.
It is now widely accepted that microglia exist in two basic states: resting and reactive [4] .Previously, microglia in the resting state were considered to be relatively inactive.More recent studies have revealed, however, that microglia are continuously surveying their environment [4] .In this role they exercise a neuroprotective function, 'patrolling' their surroundings, prepared to react to any foreign agents or damage.
In the presence of certain stressors or immunological stimuli, microglia become activated and undergo a morphological transformation, expressing an increased number of surface receptors, including major histocompatibility complex (MHC) [3,5] .Once activated, they serve a variety of beneficial functions essential to neuronal survival, including clearing toxic cellular debris and secretion of neurotrophic factors [3,4] .However, it is the dysregulation and over activation of microglia, termed microgliosis, which has been implicated as a contributing factor to the neuronal damage observed in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD).Under these chronic neuroinflammatory conditions, microglia can produce factors, such as excessive amounts of pro-inflammatory cytokines, superoxide anion radical (O2 • ) and nitric oxide [6-9]   , which can be neurotoxic.The stimuli capable of inducing microglial activation are diverse; ranging from environmental toxins and infectious agents to neuropathology-associated and non-pathological endogenous molecules.

Microglial activation by exogenous pathogens
Microglial activation is commonly induced in cell culture models using lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria [6,10,11] .Initial studies on microglial-mediated dopaminergic neurotoxicity determined that LPS was only toxic to dopaminergic neurons in the presence of microglia [10] .Infusion of LPS into the rat brain, as well as the addition of LPS to rat mesencephalic neuron-glia cultures triggered microglial activation followed by degeneration of dopaminergic neurons.This study further determined that O2 • generated by microglial NADPH-oxidase was primarily responsible for the induced neurotoxicity [10] .Others have demonstrated that the combination of LPS and interferon (IFN)-γ is a particularly powerful stimulator of microglial-mediated neurotoxicity [6,11] .Acting synergistically, LPS and IFN-γ induced activation of human monocytic THP-1 cells and primary human microglial cells, resulting in increased toxicity towards SH-SY5Y neuronal cells [11] .Such neurotoxicity could be attenuated by, for example, inhibitors of the cytosolic phospholipase A2 (cPLA2), cGMP-selective phosphodiesterases and 5-lipoxygenase, thus providing potential therapeutic targets for inhibiting microglial activation [11,12] .
Bacterial components, such as LPS, are probably one of the most studied activators of microglia (for comprehensive reviews see [3,13,14] ); however, viruses, such as the human immunodeficiency virus-1 (HIV-1), have also been shown to induce microgliosis.HIV-associated dementia (HAD) is characterized by cognitive, behavioral and motor dysfunction; it develops during the later stages of acquired immunodeficiency syndrome (AIDS) [15] .The pathological hallmarks of HAD are very similar to AD, including significant neuronal loss, brain atrophy, as well as over activation of not only microglia but also astrocytes [15] .Specifically, the viral glycoproteins gp120 [16] , gp41 and Tat [17] have been implicated as inducers of microglial neurotoxicity.
Increased expression and circulating levels of proinflammatory cytokines, such as interleukin (IL)-6 have been reported in HIV-1 patients suggesting a possible link between cytokines and HAD [18] .Several studies using mixed glial-neuron cell culture models have demonstrated increased IL-6 expression in response to exogenously added gp120 [19,20] .Shah et al. [16] showed that incubation of human SVGA astrocytic cells with gp120 induced the expression of IL-6 by activating the nuclear factor-kappa B (NF-κB) signaling pathway, and confirmed these results using primary human fetal astrocytes.Sheng et al. [17] demonstrated that stimulation with gp41 and Tat protein induces cytokine, chemokine and O2 • production by primary human fetal microglia.Tumor necrosis factor (TNF)-α, IL-1β, IL-6 and macrophage inflammatory protein (MIP)-1α production all showed a dose-dependent increase in response to gp41 treatment [17] .Overall, these results provide further insight into the involvement of glial cells in the neuroinflammation that contributes to the neuropathology of HAD.

Microglial activation in sterile inflammatory conditions
Microglia can also be activated in the absence of any external infection or immunological insult.This process, which is termed "sterile" inflammation [21] , can be induced by an activating stimulus that has a pathological origin or may be in response to specific endogenous molecules.

Microglial activation mediated by pathology-associated molecules
Increasing evidence supports the involvement of glial cells and neuroinflammation in the onset and progression of several neurodegenerative diseases, including AD and PD [3,22,23] .Autopsies of AD brains reveal the presence of large numbers of active microglia associated with the amyloid-beta (Aβ) plaques [24] .It has been demonstrated that Aβ causes microglia to take on the reactive state.Activated microglia secrete multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α and monocyte chemotactic protein-1 (MCP-1), as well as reactive oxygen species (ROS) and nitric oxide (NO) [25,26] , which contribute to neuroinflammation and neurotoxicity.
Lewy bodies, the pathological hallmarks of PD, are comprised of aggregated α-synuclein.These pathological aggregates induce recruitment and activation of resident microglia, similar to what is observed in AD [27] .Studies using primary rat and mouse neuron-glia cultures demonstrated that aggregated α-synuclein induced microglial activation, as characterized by the release of a variety of neurotoxic factors.It has been demonstrated that microglial secretion of O2 • , ROS and prostaglandin E2 is upregulated in response to α-synuclein treatment [28] .It has also been shown that α-synuclein enhances the secretion of TNF-α and IL-1β from primary human microglial and human THP-1 cells [29] .Some of the effects of α-synuclein are further enhanced in the presence of IFN-γ [29] .It has been suggested that the damaged neurons, in turn, could release factors that could activate microglia, thus contributing to the self-propagating cycle of microglial activation and neuronal death, which is one of the common characteristics of chronic inflammation observed in neurodegenerative diseases such as AD and PD [3,23,25,30,31] .

Microglial activation mediated by adenosine 5'triphosphate (ATP)
Apart from pathology-associated molecules, microglia can also be activated by endogenous molecules, which behave as signaling molecules once released into the extracellular space.The naturally occurring adenosine 5'triphosphate (ATP) is an example of such an endogenous molecule.ATP is present in every living cell of the human body and is primarily responsible for driving energy metabolism; however, extracellular ATP has been shown to play a role in inflammation by modulating immune cell functions [32][33][34] .In response to cellular stress, ATP is released and can act as an intercellular signaling molecule by binding to the purinergic P2X7 receptor, which is predominantly expressed on macrophages and microglia [35,36] .Activation of the P2X7 receptor on primary mouse microglia by extracellular ATP enhanced NADPH-oxidase activity, resulting in increased ROS production [35] .Bartlett et al. [36] showed that ATP-induced activation of the P2X7 receptor in murine EOC13 microglial cells resulted in significant ROS production by the microglia.These findings provide further insight into microglial-mediated neuronal death, as P2X7 receptors are upregulated in a transgenic mouse model of AD [37] .

Microglial activation mediated by damage-associated molecular patterns (DAMPs)
Microglial cells are also capable of initiating an immune response following activation of their pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), or the receptor for advanced glycation end products (RAGE) [38- 40] .PRRs recognize not only pathogen associated molecular patterns (PAMPs), but also damage-associated molecular patterns (DAMPs), which are endogenous danger signals secreted by damaged or dying cells.Such stimuli are capable of triggering microglial responses in the absence of infection [21,41] .High mobility group box 1 (HMGB1) is a well-characterized DAMP.It is a ubiquitous DNA-binding protein which can play two main roles depending on its location: in the cell nucleus, HMGB1 acts as a regulator for gene transcription and homeostasis; when released extracellularly, HMGB1 acts as a signaling molecule with pro-inflammatory functions [42,43] .It has been demonstrated that HMGB1 released into the media of cultured neurons is able to activate microglia and elicit their inflammatory response characterized by the release of TNF-α and IL-1β [44] .
Mitochondrial transcription factor A (TFAM) is a structural and functional homolog of HMGB1 and has, therefore, been implicated as a potential DAMP and key trigger of inflammation in peripheral tissues.A recent study demonstrated that TFAM augments plasmacytoid dendritic cell activation by engaging RAGE and TLR9.This activation was characterized by the increased release of IFN-α and TNF-α [45,46] .Another study showed that TFAM in combination with N-formyl peptides was able to significantly increase IL-8 release from human blood monocytes [47] .
Our recent work has revealed a potential role for TFAM in CNS inflammation in addition to its effects on peripheral immune cells.We have demonstrated that extracellular TFAM in combination with IFN-γ activates human primary microglia extracted from post-mortem brain tissues.TFAM caused similar activation of human peripheral blood monocytes and human THP-1 monocytic cells, which were used as models of human primary microglia [48] .Extracellular TFAM induced the expression and release of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8.The combination of TFAM plus IFN-γ induced release of cytotoxins, which was monitored by exposing human SH-SY5Y neuronal cells to supernatants from TFAMstimulated monocytic cells [48] .Our data also indicate that c-Jun N-terminal kinase (JNK) partially mediates the induction of cytotoxicity by TFAM; however, the receptors that are engaged by TFAM causing microglial activation have not been identified yet.Since, TFAM and HMGB1 exhibit significant structural homology, it is possible that TFAM acts through receptors and cellular mechanisms similar to those used by HMGB1.Therefore, RAGE, TLRs and macrophage-1 antigen (Mac-1) are all potential TFAM receptor candidates.

Final Remarks
In summary, microglial activation and dysregulation initiated by immunological insult or injury to neurons can result in a self-propagating pro-inflammatory cycle, which can be sustained throughout the course of neurodegenerative disease.This mechanism may contribute to the cumulative loss of neurons over time.Therefore, it is essential that future research focuses on expanding the fundamental knowledge of the mechanisms underlying glial cell activation.Our in vitro study demonstrating microglial and monocytic cell activation by TFAM provides preliminary evidence supporting a potential role for TFAM in neurodegeneration.Further research should focus on molecular mechanisms engaged by TFAM during microglial activation, including the receptor(s) and signaling pathways involved.These results could be used to design therapeutic agents capable of breaking the pathogenic cycle of glial activation and neuronal death, since TFAM could be one of the molecules that are released from damaged neurons that cause glial activation.Furthermore, activity of TFAM as a CNS intercellular signaling molecule has to be confirmed in vivo by, for example, injecting TFAM into the brain and monitoring glial responses around the area of injection.In addition, changes in TFAM concentration in the extracellular space and cerebrospinal fluid could be monitored in response to an acute neurotoxic insult or in animal models of neurodegenerative diseases where neuronal death is observed.Such studies may explore the validity of TFAM as a molecular marker of neuronal death and immune activation in neurodegenerative disease.Expanding our knowledge about the triggers of microglial activation, such as TFAM, will also allow for the exploration and identification of specific novel therapeutic targets for diseases involving sterile neuroinflammatory processes.