Potential Mechanisms Driving Mitochondrial Motility Impairments in Developing Iron-Deficient Neurons

Abstract

Brain development is highly demanding energetically, requiring neurons to have tightly regulated and highly dynamic metabolic machinery to achieve their ultimately complex cellular architecture. Mitochondria are the main source of neuronal adenosine 5′-triphosphate (ATP) and regulate critical neurodevelopmental processes including calcium signaling, iron homeostasis, oxidative stress, and apoptosis. Metabolic perturbations during critical neurodevelopmental windows impair neurological function not only acutely during the period of rapid growth/development, but also in adulthood long after the early-life insult has been rectified. Our laboratory uses iron deficiency (ID), the most common nutrient deficiency, as a model of early-life metabolic disruptions of neuronal metabolism because iron has a central role in mitochondrial function. Recently, we published that ID reduces hippocampal neuronal dendritic mitochondrial motility and size. In this commentary, we delve deeper into speculation about potential cellular mechanisms that drive the effects of neuronal ID on mitochondrial dynamics and quality control pathways. We propose that understanding the basic cellular biology of how mitochondria respond and adapt to ID and other metabolic perturbations during brain development may be a key factor in designing strategies to reduce the risk of later-life psychiatric, cognitive, and neurodegenerative disorders associated with early-life ID.

COMMENT ON: Bastian TW, von Hohenberg WC, Georgieff MK, Lanier LM. Chronic energy depletion due to iron deficiency impairs dendritic mitochondrial motility during hippocampal neuron development. J Neurosci. 2019 Jan 30;39(5):802-813. doi:10.1523/JNEUROSCI.1504-18.2018. Epub 2018 Dec 6. PubMed PMID: 30523068; PubMed Central PMCID: PMC6382976.

Commentary

The brain accounts for ~60% of a human baby’s total oxygen consumption (compared with ~20% in the adult brain),¹ indicating the high metabolic requirements of brain development. Metabolic insults, including dietary macronutrient and micronutrient deficiencies, anemia, gestational diabetes, and hypoxic-ischemia, are relatively common developmental health problems and can have lasting deleterious effects on cognitive function and predispose to adult mental health diseases.^2–5 Neurodevelopmental, psychiatric, and neurodegenerative diseases/conditions (eg, autism spectrum disorder, schizophrenia, major depressive disorder, bipolar disorder, Parkinson disease, Huntington disease, Alzheimer disease) are also characterized by impaired brain energy metabolism.^6,7 Therefore, it is not surprising that there has been a recent resurgence in biomedical research on the mitochondrion and its functional role(s) in a wide array of diseases.⁸ Once thought of mainly as “cellular powerhouses,” we now have a more comprehensive understanding that mitochondria are not just “energy hubs” but rather are dynamic and multifunctional organelles with unique tissue-specific and age-specific roles. Not surprisingly, mitochondrial dynamics are particularly important in tissues/cells with high energy demands, such as in postmitotic rapidly developing neurons, where adenosine 5′-triphosphate (ATP) is mainly produced through mitochondrial oxidative phosphorylation.⁹ In developing neurons, mitochondria are trafficked between the cell soma and distal dendritic and axonal processes, regulating local ATP production, calcium signaling, iron homeostasis, apoptotic signaling, and multiple other metabolic pathways.¹⁰ Together these mitochondrial processes impinge on critical neurodevelopmental processes including axonal/dendritic growth and branching and synapse formation and activity.^6,7,10 Because of these critical and diverse roles for mitochondria in the developing brain, it is important that we further our understanding of how early-life energy metabolism perturbations alter neuronal mitochondrial dynamics.

Iron is a critical regulator of neuronal energy metabolism, providing structural and catalytic components for all 4 complexes of the electron transport chain (ETC) and multiple tricarboxylic acid (TCA) cycle enzymes.¹¹ The mitochondrion, in turn, is the intracellular site of heme and iron-sulfur cluster biosynthesis. When developing neurons have insufficient iron, they enter a hypometabolic state with impaired ETC complex activity¹² and an overall reduction of mitochondrial oxygen consumption and glycolysis.¹³ Iron deficiency (ID) is also the most common nutrient deficiency, affecting 40% to 50% of pregnant women and children, and impairs neurodevelopment.² Early-life ID increases the risk for many of the same neuropsychiatric diseases (eg, autism, schizophrenia, depression, impaired cognitive function) in which energy impairment has been implicated as part of the pathophysiology.² Thus, developmental neuronal ID provides a physiologically and clinically relevant model of the effects of early-life metabolic insufficiencies on developing neurons.

Despite iron’s well-known role in important mitochondrial functions, little is known about how insufficient iron alters mitochondrial dynamics in neurons. Our recent study¹⁴ aimed to comprehensively assess the effect of neuronal ID on mitochondrial motility in actively growing dendrite branches in relation to neuronal energy status and to begin to form hypotheses related to mitochondrial fusion, fission, and mitophagy. To do this, we used our mouse primary embryonic hippocampal neuron culture model of ID. At 11 days in vitro (DIV), we assessed mitochondrial motility in terminal dendrite segments and oxygen consumption rate, ATP levels, and expression of mitochondrial fusion/fission genes in whole cultures. We also measured fusion/fission gene expression and dendritic mitochondrial density 1 week later at 18 DIV. As the first study of neuronal ID on mitochondrial dynamics, our findings highlight several important remaining questions and areas for future investigation related to both basic mitochondrial biology and translation to human health.

The major novel finding of this study was that chronic ID in developing neurons reduces mitochondrial motility in terminal dendrite branches with a concomitant reduction in whole neuron mitochondrial respiration and ATP levels. In diving deeper, we determined that the reduced motility is not caused by reduced mitochondrial speed (in fact iron-deficient mitochondria trended toward an increased speed when moving) but rather by more frequent pausing. An important area of future investigation is determining what drives the increased mitochondrial pausing. Several potential cellular mechanisms are possible:

1. Transient local ATP depletion. This mechanism, whereby there is a transient decrease in ATP in the immediate area surrounding the paused mitochondria, was the focus of our speculation in the original paper. Experimentally induced acute, local neuronal energy depletion has been shown to cause mitochondrial pausing.^10,15 Mechanistically, the pausing may be driven by a decreased ATP to ADP ratio as the functional state of microtubule motor proteins depends on this ratio.¹⁵ Our data showing that iron-deficient neurons have impaired mitochondrial respiratory capacity and decreased ATP support this mechanism. However, the effect of neuronal ID on ATP:ADP ratios in the area immediately surrounding motile vs stationary mitochondria will need to be assessed.

2. Altered neuronal glucose metabolism. In neurons, high cytosolic glucose induces mitochondrial arrest through glycosylation of milton (mitochondrial motor adaptor protein) by O-GlcNAc transferase, which acts as a cellular nutrient sensor.¹⁶ Early-life ID increases mRNA expression levels of the BBB and neuronal glucose transporters (ie, Glut1 and Glut3) in the developing hippocampus and glucose concentrations in the developing striatum,^17–19 suggesting that iron-deficient dysregulation of neuronal glucose may play a role in mitochondrial motility impairments.

3. Altered neuronal calcium signaling. Increased cytosolic calcium levels can also induce pausing of dendritic mitochondria. Calcium binds to the EF-hand motif of Miro (a mitochondrial motor complex adaptor protein) and disrupts its interaction with Kif5 (a kinesin motor protein), causing mitochondria to dissociate from microtubules and/or bind to the anchoring protein Syntaphilin.¹⁰ This is thought to recruit mitochondria to sites of high energy demand and calcium flux such as presynaptic and postsynaptic sites. The question of whether or not the paused mitochondria have been released from the motor protein complex and microtubules is of potential importance. Our data showing that mitochondrial pause duration is similar between ID and controls suggests that pausing mitochondria in iron-deficient neuron dendrites stay on the microtubules at the same frequency, which is not consistent with the current model of a calcium-induced increase in pause frequency. This still may be a fruitful area of future research as ID impairs synaptic function^20,21 and the effect of ID on calcium signaling in developing neurons has not been assessed.

4. A combination of signals or unknown signal. Given ID’s global metabolic effects, it is certainly possible that alterations in ATP, glucose, and calcium all contribute vital aspects to the disrupted mitochondrial motility. In addition, mitochondrial motor complex-microtubule interactions can be disrupted by other signals (that may or may not also alter ATP, glucose, or calcium) including mitochondrial membrane depolarization, reactive oxygen species (ROS), and interactions with myosin and actin filaments.^10,15,22,23 Most of the work on signals regulating the pool of motile vs stationary mitochondria has been performed in axons or mixed neurite populations, and thus, there are dendrite-specific mechanisms that are still unclear. For example, Syntaphilin serves as a synaptic mitochondrial anchoring protein in axons but an equivalent dendritic anchoring protein has yet to be discovered.¹⁰ In addition, neuronal mitochondrial motility is regulated in a developmental age-specific manner, decreasing as the neuron matures. Whether the mitochondrial motor complex is differentially regulated throughout development has yet to be determined.

The second major finding of our study was that neuronal ID shifts the size distribution of mitochondria toward smaller mitochondria, with a striking increase in a population of small (<0.5 µM), round mitochondria. We also showed a corresponding decrease in messenger RNA (mRNA) expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and fission (Drp1) at this time point. These data raise several questions:

1. What is the identity/source of the small, round mitochondria population in the ID neuronal dendrites? Do they come from mitochondria with a low membrane potential? Or are they normal, small mitochondria that are just a product of increased fission/decreased fusion? ID effects on mitochondrial fusion/fission rates have not been directly assessed but our gene expression data suggest that these processes may be altered.

2. What happens to the small mitochondria? Are they targeted for mitophagy? Or, do they end up undergoing fusion with another mitochondrion?

3. Are the membrane and cristae structures of the small mitochondria altered? How metabolically functional are these small mitochondria?

It is becoming increasingly clear that mitochondrial dynamics and quality control pathways play an important role in normal brain development and the pathophysiology of multiple neurological diseases. It is also clear that programming of mitochondrial metabolic rate based on early-life environment can have long-lasting effects on brain function and risk of dysfunction.^2,5 Thus, identifying and alleviating the early-life mitochondrial impairments associated with ID and perturbations of other metabolic substrates critical for neuronal mitochondrial function may be a key factor in reducing the risk of psychiatric and neurodegenerative disorders later in life. Understanding how nutritional/metabolic regulation of mitochondrial dynamics is programmed during brain development is critical to this endeavor.