Since its original characterisation in cancer cells, the concept of ferroptosis has instigated growing efforts to explicitly detect and measure its footprint in neurodegeneration (Guiney et al., 2017; Morris et al., 2018). In this regard, more direct evidence for the role of ferroptosis in neurodegeneration has recently been generated. Neuronal cells are sensitive to erastin and RSL3 toxicity in vitro. This toxicity is associated with mitochondrial impairments, and is rescued by ferroptosis inhibitors or a mitochondrially targeted ROS scavenger (Neitemeier et al., 2017; Jelinek et al., 2018). Genetic models demonstrate the substantial reliance of neurons on Gpx4 to prevent toxic lipid peroxidation. Whereas global deletion of Gpx4 is embryonic lethal (Imai et al., 2003), mice with targeted mutation of Gpx4 selenocysteine to cysteine (sensitive to inactivation), or knockout of Gpx4 specifically in neurons are viable but exhibit selective loss of CA3 hippocampal interneurons, resulting in seizures and early death (Seiler et al., 2008; Ingold et al., 2018). Targeting Gpx4 knockout to photoreceptor neurons results in death of these cells within 21 days of birth (Ueta et al., 2012). Postdevelopment, conditional knockout of Gpx4 in adult mice results in loss of CA1 hippocampal neurons and rapid death, indicating neurons are specifically sensitive to Gpx4 deletion (Yoo et al., 2012). Conditional ablation of Gpx4 targeted to neurons results in dramatic degeneration of motor neurons that rapidly progresses to paralysis and death (Chen et al., 2015). Targeting conditional knockout of Gpx4 to forebrain neurons of adult mice causes cognitive impairments and hippocampal degeneration reminiscent of Alzheimer's disease (Hambright et al., 2017). These models are all accompanied by lipid peroxidation and mitochondrial impairments, consistent with ferroptosis. Conditional Gpx4 knockout can partially rescued by ferroptosis inhibitors, indicating the involvement of ferroptosis (Chen et al., 2015; Hambright et al., 2017).

Ferroptosis inhibitors have also been investigated in explicit models of neurodegneration. Ferroptosis inhibitors prevent the toxicity of Huntington's disease-associated mutated Htt in brain slice cultures (Skouta et al., 2014). Ferroptosis inhibitors are also protective in dopaminergic cultured cells, organotypic slice cultures, and in an MPTP mouse model of Parkinson's disease (Do Van et al., 2016). Furthermore, ferroptosis is implicated in hemorrhagic and ischemic stroke based on the ability of ferroptosis inhibitors to protect against neuronal death in both in vivo and in vitro models (Li et al., 2017; Tuo et al., 2017; Zille et al., 2017). Ischemic stroke is also strongly modulated by brain iron levels (Tuo et al., 2017). Knockout of glutathione synthesis accelerates the disease phenotype in motor neuron disease-model mice, and ultrastructural analysis of spinal cord tissue reveals mitochondrial swelling, rupture and decreased cristae, all consistent with ferroptosis (Vargas et al., 2011).

Ferroptosis also appears to mechanistically overlap with other cell death modalities in neurodegeneration. Consistent with a distinct form of cell death, it was initially reported that ferroptosis induced by erastin does not involve mitochondrial genes implicated in other cell death pathways, nor the release of cytochrome c (Dixon et al., 2012). However, recent studies show that ferroptosis induces the pro-apoptotic translocation of BH3 interacting-domain death agonist (BID) to mitochondria in neuronal cells (Neitemeier et al., 2017; Jelinek et al., 2018). Oxytosis is described as glutamate-induced inhibition of cystine uptake via system xc − leading to glutathione depletion and subsequent lipoxygenase-dependent toxic lipid peroxidation (Tan et al., 2001). This is clearly very similar to ferroptosis and has led to the recent proposal that perhaps ferroptosis and oxytosis are in fact the same pathway (Lewerenz et al., 2018). Accordingly, oxytosis in neurons causes mitochondrial morphological changes similar to ferroptosis (Lee et al., 2011), and the toxicity of glutamate mirrors that of erastin or RSL3 in neuronal cells, and are all amenable to prevention by ferroptosis inhibitors (Liu et al., 2015; Neitemeier et al., 2017; Jelinek et al., 2018) or necrostatin-1 (Xu et al., 2007). Furthermore, oxytosis/ferroptosis in neurons involves the translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus, prevention of which alleviates cell death (Xu et al., 2010; Neitemeier et al., 2017; Jelinek et al., 2018). To further complicate the mechanisms of cell death, mitochondrial AIF release is the penultimate stage of parthanatos, a poly ADP-ribose polymerase 1 (PARP-1) mediated form of cell death (Fricker et al., 2018; Galluzzi et al., 2018). Parthanatos can be induced by oxidative stress, and is also implicated in neurodegeneration (Fricker et al., 2018; Galluzzi et al., 2018). Hence both initiating events and late stages are shared by ferroptosis/oxytosis and parthanatos.

Together these studies show that many features facilitating ferroptosis are present in neurodegeneration, and mounting evidence indicates that targeting ferroptosis with specific inhibitors is a valid therapeutic strategy. However, overlapping mechanisms highlight the need for more comprehensive delineation of the cellular mechanisms of cell death, and the potential for new or repurposed treatments. An alternate approach to attenuate ferroptosis is to augment the endogenous anti-ferroptotic mechanisms within cells. To this end, the role of the transcription factor Nrf2 will now be discussed.

In terms of endogenous cellular mechanisms preventing ferroptosis, the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is exquisitely positioned to modulate the onset and outcomes of ferroptosis. Nrf2 is responsible for regulating hundreds of antioxidant genes (Gao et al., 2014). Under normal conditions, Nrf2 resides in the cytosol bound to its negative regulator Kelch-like ECH-associated protein 1 (Keap1). Keap1 constitutively targets Nrf2 for ubiquitination and proteasomal degradation, thereby maintaining Nrf2 signaling capacity at a low level. However,

#### TABLE 1 | Ferroptosis-related genes that are transcriptionally regulated by Nrf2.

states of increased oxidative stress facilitate the dissociation of Nrf2 from Keap1, and promote the nuclear translocation of Nrf2. In the nucleus, Nrf2 interacts with antioxidant response elements (AREs) in the promoter region of target genes resulting in their transcriptional activation (Yamamoto et al., 2018).

Importantly in the context of ferroptosis, almost all genes thus far implicated in ferroptosis are transcriptionally regulated by Nrf2 (**Table 1**). These include genes for glutathione regulation (synthesis, cysteine supply via system xc −, glutathione reductase, glutathione peroxidase 4), NADPH regeneration which is critical for Gpx4 activity (glucose 6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malic enzyme), and iron regulation (including iron export and storage, heme synthesis and catabolism) (Sasaki et al., 2002; Lee et al., 2003; Wu et al., 2011; Kerins and Ooi, 2017). In addition, Nrf2 is involved in the regulation of lipids via the ligand-mediated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Nrf2 and PPARγ are reciprocally regulated, with activation of either upregulating the other (Cai et al., 2017; Lee, 2017). PPARγ is a major regulator of lipid metabolism (Cai et al., 2017), and can be activated by oxidized lipids relevant to the initiation of ferroptosis (Itoh et al., 2008). Hence Nrf2 indirectly modulates the lipids whose abundance contributes to the sensitivity to ferroptosis (Doll et al., 2017) (**Figure 1**).

Nrf2 also plays important roles in modulating mitochondrial function. Nrf2 can be physically bound to mitochondria and thus monitor and respond to changes in mitochondrial function (Lo and Hannink, 2008). Nrf2 also regulates mitochondrial dynamics, including biogenesis (Piantadosi et al., 2008; Merry and Ristow, 2016) via interaction with peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) (Dinkova-Kostova and Abramov, 2015; Navarro et al., 2017), and mitophagy via a P62-dependent, PINK1/Parkin-independent mechanism (East et al., 2014). Mitochondria in Nrf2 knockout mice have impaired function, whereas activation of Nrf2 enhances mitochondrial function and resistance to stressors (Greco and Fiskum, 2010; Neymotin et al., 2011; Holmstrom et al., 2016).

Therefore, by virtue of its direct and indirect regulation of many genes central to ferroptosis and its regulation of mitochondrial function, Nrf2 is positioned to be a key player in ferroptosis. Despite this, the role of Nrf2 in ferroptosis has received very little attention thus far, limited mainly to in vitro studies using cancer cells.  Genetic modulation of Nrf2 expression, including knockdown and overexpression, can finetune the sensitivity of glioma cells to the ferroptosis inducers erastin and RSL3 (Fan et al., 2017). This may contribute to the

cancer-promoting and chemoresistance effects of elevated Nrf2 present in many cancers (Fan et al., 2017; Taguchi and Yamamoto, 2017).

Nrf2 activation is sensitive to shifts in cellular redox status, hence it follows that the lipid peroxidation accompanying ferroptosis should activate Nrf2. Indeed, the ferroptosis inducers erastin and sorafenib are sufficient to activate Nrf2 in hepatocellular carcinoma cells (Houessinon et al., 2016; Sun et al., 2016b). Furthermore, the protective action of the mitochondrially targeted antioxidant, MitoQ, involves Nrf2 activation (Hu et al., 2018).

Given the pervasive lipid peroxidation evident in neurodegeneration, it would be expected that Nrf2 would be elevated in neurodegenerative diseases. While there is some evidence for Nrf2 activation in neurodegeneration, it appears to be relatively mild and clearly insufficient to prevent neuronal dysfunction (Liddell, 2017). In some cases, particularly for motor neuron disease, Nrf2 signaling appears to be impaired (Moujalled et al., 2017). This is in contrast to the strong activation of Nrf2 evident when genetic or pharmacological Nrf2 inducers are applied to models of neurodegeneration (Liddell, 2017). These treatments are robustly neuroprotective in animal models of disease. While several drugs targeting Nrf2 are currently under clinical investigation, to date, dimethyl fumarate (Tecfidera) remains the only clinically approved drug for the treatment of a neurodegenerative disease (relapsing-remitting multiple sclerosis) in which Nrf2 activation clearly contributes to its mechanism of action (Havrdova et al., 2017).

The above mentioned studies in cancer cells provide empirical in vitro support for the anti-ferroptotic action of Nrf2. However, direct in vivo evidence for an anti-ferroptotic effect of Nrf2 induction does not yet exist, and it is currently unknown whether and to what extent the demonstrated neuroprotective efficacy of Nrf2 activation in models of neurodegeneration involve attenuation of ferroptosis. Evaluation of ferroptosis in vivo is currently hindered by the lack of specific markers of ferroptosis. Studies examining the effect of Nrf2 activation in explicit in vivo models of ferroptosis (e.g., Gpx4 knockout) would provide some insight.

#### CONCLUSION AND FUTURE PERSPECTIVES

Ferroptosis is an iron-dependent form of regulated cell death instigated by impaired glutathione metabolism, culminating in

#### REFERENCES

mitochondrial failure and toxic lipid peroxidation. Emerging evidence implicates ferroptosis in neurodegeneration, both in the molecular and biochemical signatures of neurodegeneration, and in terms of functional abrogation of neuron death via specific ferroptosis inhibitors. Elucidating how ferroptosis provokes neurodegeneration will expose new therapeutic opportunities to treat these diseases. To this end, targeting the antioxidant transcription factor Nrf2 is an attractive option. Nrf2 signaling is involved in regulating mitochondrial function and impacts almost all identified molecular aspects of ferroptosis. Treatments targeting Nrf2 have been demonstrated to exert anti-ferroptotic effects in the context of cancer cells, and are beneficial in many models of neurodegeneration. The protective mechanism of Nrf2 activation in these models may involve attenuating ferroptosis via upregulation of the endogenous anti-ferroptotic machinery, however, direct evidence for this is currently lacking. The myriad of Nrf2 actions described here suggest that targeting Nrf2 is an exciting therapeutic option to attenuate ferroptosis.

Several questions remain unanswered. The discovery of bonafide biomarkers of ferroptosis will be invaluable to unequivocally probe its involvement in neurodegeneration and facilitate the development of therapeutic treatments targeting ferroptosis. The continued evaluation of ferroptosis inhibitors and comparison to inhibitors of other cell death modalities in further models of neurodegeneration will help elucidate the key pathways involved. Finally, whether Nrf2 activation is directly alleviating ferroptotic stress.

Taken together, the arguments presented in this review elucidate a coherent network that links Nrf2 signaling to mitochondrial function and ferroptotic cell death, and proposes the targeting of Nrf2 as a rational line of therapy for ferroptotic neurodegeneration.

# AUTHOR CONTRIBUTIONS

JL conceived the review. MA prepared the first draft. RL, KK, and TM reviewed literature and contributed to writing. JL prepared the final manuscript.

#### FUNDING

JL was supported by an Australian National Health and Medical Research Council Peter Doherty Fellowship. We gratefully acknowledge funding from the Academy of Finland and The Sigrid Juselius Foundation, Finland.

ferroptosis. Free Radic. Biol. Med. 117, 45–57. doi: 10.1016/j.freeradbiomed. 2018.01.019

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Abdalkader, Lampinen, Kanninen, Malm and Liddell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Mitochondria and Calcium Regulation as Basis of Neurodegeneration Associated With Aging

#### Marioly Müller1,2, Ulises Ahumada-Castro1 , Mario Sanhueza3 , Christian Gonzalez-Billault1,4,5, Felipe A. Court1,3,5 and César Cárdenas1,4,6,7 \*

1 Geroscience Center for Brain Health and Metabolism, Santiago, Chile, 2 Department of Medical Technology, Faculty of Medicine, Universidad de Chile, Santiago, Chile, 3 Center for Integrative Biology, Faculty of Sciences, Universidad Mayor, Santiago, Chile, 4 Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile, 5 The Buck Institute for Research on Aging, Novato, CA, United States, 6 Anatomy and Developmental Biology Program, Institute of Biomedical Sciences, University of Chile, Santiago, Chile, 7 Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA, United States

#### Edited by:

Victor Tapias, Cornell University, United States

#### Reviewed by:

Rebecca Jane Rylett, University of Western Ontario, Canada Martin Lothar Duennwald, University of Western Ontario, Canada Jeremy Michael Van Raamsdonk, Van Andel Institute, United States

> \*Correspondence: César Cárdenas<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 26 March 2018 Accepted: 20 June 2018 Published: 13 July 2018

#### Citation:

Müller M, Ahumada-Castro U, Sanhueza M, Gonzalez-Billault C, Court FA and Cárdenas C (2018) Mitochondria and Calcium Regulation as Basis of Neurodegeneration Associated With Aging. Front. Neurosci. 12:470. doi: 10.3389/fnins.2018.00470 Age is the main risk factor for the onset of neurodegenerative diseases. A decline of mitochondrial function has been observed in several age-dependent neurodegenerative diseases and may be a major contributing factor in their progression. Recent findings have shown that mitochondrial fitness is tightly regulated by Ca2 + signals, which are altered long before the onset of measurable histopathology hallmarks or cognitive deficits in several neurodegenerative diseases including Alzheimer's disease (AD), the most frequent cause of dementia. The transfer of Ca2 + from the endoplasmic reticulum (ER) to the mitochondria, facilitated by the presence of mitochondria-associated membranes (MAMs), is essential for several physiological mitochondrial functions such as respiration. Ca2 + transfer to mitochondria must be finely regulated because excess Ca2 + will disturb oxidative phosphorylation (OXPHOS), thereby increasing the generation of reactive oxygen species (ROS) that leads to cellular damage observed in both aging and neurodegenerative diseases. In addition, excess Ca2 + and ROS trigger the opening of the mitochondrial transition pore mPTP, leading to loss of mitochondrial function and cell death. mPTP opening probably increases with age and its activity has been associated with several neurodegenerative diseases. As Ca2 + seems to be the initiator of the mitochondrial failure that contributes to the synaptic deficit observed during aging and neurodegeneration, in this review, we aim to look at current evidence for mitochondrial dysfunction caused by Ca2 + miscommunication in neuronal models of neurodegenerative disorders related to aging, with special emphasis on AD.

Keywords: mitochondria, MAMS, calcium, neurodegeneration, ROS, MPTP, aging, endoplasmic reticulum

# INTRODUCTION

In the last century, the population aged over 60 years old has rapidly increased around the world (Beard et al., 2016). Aging is the major risk factor for many chronic diseases such as cancer, diabetes, hypertension, and neurodegenerative disorders (Kennedy et al., 2014). In particular, aging has been correlated with the occurrence of several types of dementia, affecting 5–10% of people over 65, and

about 50% of people over 85 years old according to the Alzheimer's Disease (AD) International (Prince et al., 2015). AD, the most common and still incurable form of dementia, shares several similar cellular alterations with brain aging including mitochondrial dysfunction, oxidative stress, Ca2+ dysregulation, and impaired proteostasis (Leuner et al., 2007; Kern and Behl, 2009; Rodrigue et al., 2009; Martinez et al., 2017). Most cases of AD are sporadic (SAD) and characterized by a late onset of symptoms, such as a decline of intellectual and cognitive functions and irreversible memory loss as major features. Several genes have been found to increase the risk of SAD, with the gene for apolipoprotein E (APOE) being the most studied, specifically, the polymorphism that produces the ε4 allele of the APOE, APOE4 variant of the protein (Allen et al., 2012). In addition, nearly 1% of the cases of AD that are dominantly inherited present an early development known as familial AD (FAD) characterized by mutations in presenilin-1 (PS1) and -2 (PS2) or in the amyloid precursor protein (APP; Sherrington et al., 1995). Both SAD and FAD are characterized by neuronal cell death and assumed to be similar to some extent (Hardy and Selkoe, 2002), but the key events prior to cell death are still unclear.

Mitochondria are central organelles in neuronal physiology integrating several crucial functions such as cell respiration, energy metabolism, Ca2+ homeostasis, and reactive oxygen species (ROS) generation, all of which have been found to be dysregulated in aging, AD, and other neurodegenerative disorders such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) disease (Winklhofer and Haass, 2010; Schon and Przedborski, 2011; Itoh et al., 2013; Manfredi and Kawamata, 2016). Here we present an overview of selected findings regarding mitochondrial dysfunction in neurodegenerative disease and discuss their potential as therapeutic targets.

#### ER-MITOCHONDRIA COMMUNICATION AND CA2+ REGULATION IN AGE-ASSOCIATED NEURODEGENERATIVE DISEASES

Communication between organelles allows cells to function and adapt in a changing cellular environment. The endoplasmic reticulum (ER) and mitochondria couple at specific sites termed mitochondria-associated membranes (MAMs), which integrate and coordinate several cellular functions, including synthesis and exchange of phospholipid, apoptosis, mitochondrial dynamics, and Ca2+ signaling (Liu and Zhu, 2017; **Figure 1A**). Remarkably, all these processes are affected early during aging, AD pathogenesis, and other neurodegenerative conditions, suggesting a role for MAMs in the pathogenesis of these diseases (De Vos et al., 2012; Hedskog et al., 2013; Gautier et al., 2016; Area-Gomez et al., 2018). For example, the overexpression of both wild-type and familial ALS/FTD mutant TDP-43 in HEK293, CV-1, and NSC34 cell lines reduces ER–mitochondria associations and Ca2+ exchange between these two organelles (Stoica et al., 2014; **Figure 1C**). Likewise, loss of Sigma 1 receptor (which is responsible for some familial forms of ALS/FTD) has been shown to interfere with ER–mitochondria associations (Bernard-Marissal et al., 2015; **Figure 1C**). Conversely, an increase in the lipidic enzymatic function of MAMs and their inter-organelle extension has been described in fibroblasts from patients with SAD, in human SAD brains, and in AD mouse models (Area-Gomez et al., 2012; Hedskog et al., 2013). Remarkably, one of the most common and validated risk factors for SAD, the presence of APOE4 (Holtzman et al., 2012), has recently been associated with an increase in the ER–mitochondrial communication and MAM enzymatic activity (Tambini et al., 2016; **Figure 1B**). Furthermore, MAMs are highly enriched in PS1 and PS2 proteins (Area-Gomez et al., 2009) which when mutated, as in fibroblasts from patients with FAD, also increase the lipidic enzymatic function of the MAMs and ER–mitochondria communication (Area-Gomez et al., 2012), through a mechanism that involves an interaction between the mutated form of PS2 and mitofusin-2 (Mfn2; **Figure 1B**), a key protein in the formation of MAMs (Filadi et al., 2016).

Conditions that increase or decrease the extension of MAMs will affect the transfer of Ca2+ from the ER to mitochondria, resulting in either a mitochondrial Ca2+ overload, or a lack of Ca2+ . If the transfer of Ca2+ is excessive, cell death occurs (Schinder et al., 1996). If Ca2+ transfer to mitochondria is too low, a bioenergetics crisis occurs, also resulting in cell death (Cardenas et al., 2010). Importantly, Ca2+ is dysregulated in the aged brain and in AD (Landfield and Pitler, 1984; Gibson and Peterson, 1987; Khachaturian, 1987). Upregulation of Ca2+ levels can both initiate and accelerate several AD features, from amyloid deposition to synapse loss (Stutzmann et al., 2007). Several mechanisms have been proposed to explain the upregulation of cytoplasmic Ca2+ levels in AD including overexpression of the ryanodine receptor (RyR; Chakroborty et al., 2012), or β-amyloid (Aβ)-triggering release of Ca2+ from both extracellular and intracellular sources (Demuro and Parker, 2013). Another mechanism involves an increase of Ca2+ leak from the ER through sensitization of the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ channel by directed interaction with FAD-linked PS mutants (Cheung et al., 2008, 2010) or indirectly by interaction of FAD-linked PS mutants with the SERCA pump (Green et al., 2008). In agreement with the latter, it has been demonstrated that overexpression of the FAD-linked PS2 mutant leads to an increase in the generation of cytosolic Ca2+ hot spots, ER–mitochondria tethering, and mitochondrial Ca2+ uptake (Zampese et al., 2011). This in turn may result in mitochondrial Ca2+ overload and could explain the metabolic dysfunction and cell death observed in AD (**Figure 1B**). On the other hand, decreasing intracellular Ca2+ overload, specifically through a reduction of the InsP3R protein expression by 50%, normalizes FAD PS-associated Ca2+ signaling and rescues the biochemical, electrophysiological, and behavioral phenotypes observed in two different PS1-FAD animal models (Shilling et al., 2014; **Figure 1B**). Altogether, the above findings highlight the importance of MAMs and the transfer of Ca2+ from the ER to mitochondria in AD pathogenesis and their potential as a therapeutic target.

The role of MAMs in aging has just begun to be unveiled. Similar to what has been observed in AD models, aging increases the ER to mitochondria Ca2+ transfer in long-term culture of hippocampal neurons, with correlates with an increase of the mitochondrial Ca2+ uniporter (MCU; Calvo-Rodriguez et al., 2016; **Figure 1B**). Interestingly, a decrease in Ca2+ transfer to mitochondria and a dissociation of the MAMs have been described in cardiomyocytes from old mice suggesting that the type of modification that MAMs undergo with aging might be cell specific (Fernandez-Sanz et al., 2014).

#### THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE (MPTP) FORMATION IN AGE-ASSOCIATED NEURODEGENERATIVE DISEASES

Under conditions of Ca2+ and/or ROS overload, formation of the mitochondrial permeability transition pore (mPTP) takes place, which corresponds to a non-selective channel formed by a protein complex spanning the outer and inner mitochondrial membranes (Bernardi et al., 2006). In physiological conditions, transient opening of the mPTP can regulate Ca2+ levels in the mitochondrial matrix (Ichas et al., 1997). However, dysregulated mPTP opening triggers the release of most matrix metabolites such as ROS, Ca2+, and NAD + , leading to loss of the mitochondrial membrane potential, inhibition of oxidative phosphorylation (OXPHOS), and mitochondrial swelling (Elrod and Molkentin, 2013; Rottenberg and Hoek, 2017). Even though several proteins are known to participate in mPTP formation [anion channel VDAC, adenine nucleotide translocator (ANT), mitochondrial ATP synthase (F0F1), phosphate carrier (PiC), and cyclophilin D (CypD; Bernardi et al., 2006; Rao et al., 2014), its detailed structural configuration is not yet entirely known.

The mPTP has been linked to neurodegeneration in vitro and in vivo. In neural progenitor cells, Aβ-amyloid exposure leads to mPTP opening and a decrease in mitochondrial membrane potential, release of cytochrome C, and cell death (Hou et al., 2014). In human AD brains, Aβ-amyloid binds CypD in mitochondria (Du and Yan, 2010), and CypD deficiency improves mitochondrial function, memory, and learning in an AD mouse model (Du et al., 2008). Aβ-induced neurotoxicity in vitro was

also attenuated pharmacologically by inhibition of the mPTP using cyclosporine A (CsA) on neural stem cells (Chen et al., 2016). Interestingly, it has been shown that CypD knock-out mice exhibit delayed axonal degeneration, a common feature of diverse neurodegenerative diseases (Barrientos et al., 2011; Catenaccio et al., 2017; Salvadores et al., 2017). Indeed, genetic deletion of CypD delays disease progression in other mouse models of neurodegenerative disorders, including ALS (Martin et al., 2009), PD (Thomas et al., 2012), and multiple sclerosis (Forte et al., 2007). Therefore, novel compounds that inhibit mPTP opening are currently been developed, including sanglifehrin A, N-Me-Ala-6-cyclosporin A, and antmanide (Rao et al., 2014).

Aging also modifies the opening probability of the mPTP (Rottenberg and Hoek, 2017). During aging, the probability of the mPTP opening increases due to higher expression levels of CypD and the CypD-activator p53 (Priami et al., 2015). Furthermore, the expression of HSP90, a chaperone that binds CypD to trigger its degradation, is decreased in aged cells (Lam et al., 2015), which could also increase the mPTP opening probability. This evidence is further supported by a faster Ca2+ -induced mitochondrial swelling in purified liver mitochondria obtained from aged mice (Goodell and Cortopassi, 1998). Interestingly, CypD is inactivated by the deacetylase SIRT3 (Hafner et al., 2010), a known modulator of longevity in diverse species (Jasper, 2013). The reported decline in SIRT3 activity during aging (Brown et al., 2013) may lead to a greater activation of the mPTP, underscoring the role of mitochondria in longevity and onset of age-dependent neurodegenerative diseases. Interestingly, several modulators of longevity, including metformin, mitochondrial UPR, and caloric restriction inhibit the activation of the mPTP (Bhamra et al., 2008; Altieri, 2013; Amigo et al., 2017), and may contribute to lifespan extension (Rottenberg and Hoek, 2017). A key role for mitochondria in age-related disorders has been associated to broad damaging events including increased ROS production and defects in the regulation of intracellular Ca2+ levels, which are directly associated to mPTP activation with profound negative consequences for cell survival. Therefore, mPTP emerges as a potential target for neuroprotection in agerelated neurodegenerative conditions.

#### MITOCHONDRIA, ROS, AGING, AND NEURODEGENERATION

Reactive oxygen species are chemical species that are produced by most cell types. The group of molecules that fulfill the criteria for ROS includes hydrogen peroxide, and the highly reactive species superoxide anion and hydroxyl radical (Wilson et al., 2017). The production of ROS in cells is controlled by enzymatic or non-enzymatic mechanisms. The main source for ROS production in terms of quantitative production is the mitochondria (Holmstrom and Finkel, 2014). Mitochondria produces superoxide anion, a by-product of the inefficient transfer of electrons by the electron transport chain (ETC) during OXPHOS, that is quickly converted into hydrogen peroxide by the action of the superoxide dismutases 1–3 (SOD1–3; Quinlan et al., 2013). Of note, despite mitochondria being the main source of ROS in cells, hydrogen peroxide can be produced by more than 30 different enzymes (Go et al., 2015).

While a huge amount of work in the past focused on the deleterious roles for ROS species in cells and organisms, including the "free radical" or "oxidative stress" theory of aging (Harman, 1956) supported by many studies (Harman, 1992; Cadenas and Davies, 2000; Golden et al., 2002), there is growing evidence in the last decades that ROS may serve physiological functions (Zuo et al., 2015; Sies et al., 2017; Wilson et al., 2017). Related to aging, other studies show that unbalanced ROS production does not modify lifespan in mice under tightly controlled conditions (Van Remmen et al., 2003; Ran et al., 2007). Moreover, it was demonstrated that there is no increased oxidative damage with age (Barja and Herrero, 2000; Kauppila et al., 2017). Currently, it has been proposed that adaptive or hormetic production of ROS is required to maintain several cellular mechanisms including stem cell proliferation and fate determination in the brain (Sena and Chandel, 2012; Chaudhari et al., 2014).

In terms of neurodegeneration associated to aging, it has been reported in AD that the Aβ peptide interacts with the mitochondrial protein termed amyloid binding alcohol dehydrogenase (ABAD) in AD mouse models and in postmortem samples derived from AD human patients. The functional consequence of such an interaction is an increase in ROS production due to abnormal mitochondrial membrane permeability (Lustbader et al., 2004). Altered OXPHOS increases the generation of ROS (Koopman et al., 2013) and is indeed a hallmark for early AD abnormalities in humans. In fact, samples from human subjects show that mitochondrial-encoded OXPHOS genes are altered in aging, mild cognitive impairment, and AD (Mastroeni et al., 2017). Similarly, AD mouse models have shown that both Aβ and tau protein can induce alterations in mitochondrial proteins involved in OXPHOS (Caspersen et al., 2005; Rhein et al., 2009; Eckert et al., 2010), causing an aberrant ROS generation leading to cellular damage. In addition, ROS are known to cause mitochondrial fragmentation (Wang et al., 2014), which reduces mitochondrial performance (Westermann, 2012) favoring the generation of more ROS and cellular damage associated to it.

#### MITOCHONDRIAL DYSFUNCTION AND SYNAPTIC DEFICITS

Synapses are neuronal structures in which mitochondria are fundamental (Li et al., 2004) by providing large amounts of ATP required to fuel synaptic vesicle physiology and by acting as a Ca2+ buffer modulating cytoplasmic Ca2+ signal and hence, neurotransmission (Ghosh and Greenberg, 1995; Verstreken et al., 2005; Gunter and Sheu, 2009; Wan et al., 2012). Synaptic mitochondria are more vulnerable to cumulative damage showing impaired Ca2+ uptake capacity and increased propensity to undergo mPTP compared to nonsynaptic mitochondria (Scheff et al., 2006). Likewise, in an APP/PS1 AD mouse, synaptic mitochondrial function was significantly more affected than non-synaptic mitochondria (Dragicevic et al., 2010). Synaptic deficit is an early event in the

Müller et al. Mitochondria in Aged Neuron

pathogenesis of several neurodegenerative disorders including AD and worsens with disease progression and age (Wilcox et al., 2011). The extent of cognitive decline in AD patients is tightly associated with the extent of synapse loss in specific brain regions including cortex and hippocampus (Scheff et al., 2006; Mattson, 2010). Post-mortem hippocampus from AD patients shows a considerable decrease in dendritic spine density (Ferrer et al., 1990) and transgenic mouse models of AD show agedependent reduction in spine density before plaque deposition (Lanz et al., 2003). Along these lines, in aged synapses and from AD models, a decline in mitochondrial respiration and signs of mitochondrial damage such as reduced antioxidant contents and increased oxidative stress markers has been described (Du et al., 2010; Quiroz-Baez et al., 2013). Proteomic analysis of aged synaptic mitochondria reveals changes in ETC proteins, antioxidants, and proteins related to mitochondrial dynamics (Stauch et al., 2014). Just recently, through the use of cytoplasmic hybrid ("cybrid") technology, Yu et al. (2017) were able to recapitulate mitochondrial structural and functional changes observed in AD-affected brains. In this model, their findings demonstrate that AD-affected mitochondria elicited detrimental effects on synaptic development (Yu et al., 2017). How ER vesicles found in the synaptic region and the transfer of Ca2+ contribute to the impairment of mitochondria and synaptic formation remains to be explored, but given the dysregulation of Ca2+ observed during aging, AD, and other neurodegeneration, an important role is expected. Elucidating the factors that underlie early synaptic dysfunction will be key to prevent the widespread neurodegeneration associated with aging.

#### CONCLUSION

Aging continues to be the most relevant risk factor for AD, the most common form of dementia in the elderly, and other neurodegenerative diseases. Both aging and neurodegeneration are accompanied by a loss in the ability of the cells to adjust and rewire their metabolic networks to keep a tight balance between energy production and expenditure in an ever-changing

#### REFERENCES

environment. Mitochondria work as an adaptable metabolic control, a "rheostat," that integrates inputs from the intra and extracellular environment to generate functional outputs that adjust cell behavior and energy production and consumption. Several lines of evidence suggest that mitochondrial function deteriorates with increasing age and the progression of several neurodegenerative diseases. This supports the notion that aging and the neurodegenerative diseases such as AD may share a common root, the failure of the rheostat program. Since Ca2+ is also altered in both conditions and can either energize or overload the rheostat depending on the concentration, understanding how MAM formation is regulated is important. Identifying the players that participate in the regulation to assure a proper Ca2+ transfer to mitochondria is critical in order to determine the real potential of this intracellular signaling platform as an intervention candidate to improve aging and hinder the onset of neurodegenerative disease such as AD.

#### AUTHOR CONTRIBUTIONS

MM, UA-C, and CC designed and outlined the structure and contents of the review. MM, UA-C, MS, FC, CG-B, and CC contributed to the literature review, discussion, and writing of the manuscript. All authors contributed equally to the draft revisions and final approval of the version to be published.

# FUNDING

This work was supported by Geroscience Center for Brain Health and Metabolism (FONDAP-15150012; FC, CG-B, and CC) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) #1160332 (CC), 1150766 (FC) and #1180419 (CG-B).

#### ACKNOWLEDGMENTS

The authors want to thank Dr. Alenka Lovy for her feedback and useful comments.

impairment in Alzheimer's transgenic mice. J. Alzheimers Dis. 20(Suppl. 2), S535–S550. doi: 10.3233/JAD-2010-100342

in the pathobiology of ALS mice. Exp. Neurol. 218, 333–346. doi: 10.1016/j. expneurol.2009.02.015

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Müller, Ahumada-Castro, Sanhueza, Gonzalez-Billault, Court and Cárdenas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Excitatory Dendritic Mitochondrial Calcium Toxicity: Implications for Parkinson's and Other Neurodegenerative Diseases

#### Manish Verma1 , Zachary Wills2 and Charleen T. Chu1,3,4,5,6,7 \*

1 Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2 Department of Neurobiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 3 Department of Ophthalmology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 4 Pittsburgh Institute for Neurodegenerative Diseases, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 5 McGowan Institute for Regenerative Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 6 Center for Protein Conformational Diseases, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 7 Center for Neuroscience, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States

#### Edited by:

Victor Tapias, Cornell University, United States

#### Reviewed by:

Carlos M. Opazo, University of Melbourne, Australia Francisco José Pan-Montojo, Ludwig-Maximilians-Universität München, Germany

> \*Correspondence: Charleen T. Chu<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 28 February 2018 Accepted: 12 July 2018 Published: 02 August 2018

#### Citation:

Verma M, Wills Z and Chu CT (2018) Excitatory Dendritic Mitochondrial Calcium Toxicity: Implications for Parkinson's and Other Neurodegenerative Diseases. Front. Neurosci. 12:523. doi: 10.3389/fnins.2018.00523 Dysregulation of calcium homeostasis has been linked to multiple neurological diseases. In addition to excitotoxic neuronal cell death observed following stroke, a growing number of studies implicate excess excitatory neuronal activity in chronic neurodegenerative diseases. Mitochondria function to rapidly sequester large influxes of cytosolic calcium through the activity of the mitochondrial calcium uniporter (MCU) complex, followed by more gradual release via calcium antiporters, such as NCLX. Increased cytosolic calcium levels almost invariably result in increased mitochondrial calcium uptake. While this response may augment mitochondrial respiration, limiting classic excitotoxic injury in the short term, recent studies employing live calcium imaging and molecular manipulation of calcium transporter activities suggest that mitochondrial calcium overload plays a key role in Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and related dementias [PD with dementia (PDD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD)]. Herein, we review the literature on increased excitatory input, mitochondrial calcium dysregulation, and the transcriptional or post-translational regulation of mitochondrial calcium transport proteins, with an emphasis on the PD-linked kinases LRRK2 and PINK1. The impact on pathological dendrite remodeling and neuroprotective effects of manipulating MCU, NCLX, and LETM1 are reviewed. We propose that shortening and simplification of the dendritic arbor observed in neurodegenerative diseases occur through a process of excitatory mitochondrial toxicity (EMT), which triggers mitophagy and perisynaptic mitochondrial depletion, mechanisms that are distinct from classic excitotoxicity.

Keywords: mitochondrial calcium uniporter, PINK1, LRRK2, calcium overload, Parkinson Disease/Lewy body dementia, Alzheimer Disease, FTD-ALS, dendrite degeneration

# INTRODUCTION

fnins-12-00523 July 31, 2018 Time: 17:33 # 2

Neuronal function is dependent upon the formation, maintenance, and activity-regulated remodeling of multiple synaptic contacts supported by extensive axo-dendritic arborization. The primary excitatory neurotransmitter is glutamate. Glutamate binds to calciumpermeable ionotropic receptors that are also activated by N-methyl-D-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). These NMDA receptors (NMDAR) and AMPA receptors (AMPAR) are present predominantly in dendritic spines, but also exist in perisynaptic regions. NMDA receptors are composed of NR1 and NR2 subunits; the four NR2A-NR2D subunits confer different kinetic properties, channel open probabilities, ion conductance, and effects on synaptic plasticity. Excitatory synaptic activity engages NMDAR subsets that contain the NR2A subunit, resulting in activation of Akt, ERK1/2, and CREB (Lau and Zukin, 2007). In addition to ligand-gated channels, L-type voltage gated calcium channels are also involved in activating ERK1/2 and CREB to regulate activity-dependent transcription (West et al., 2002). Transient activation of these signaling pathways is implicated in neuronal survival as well as in synaptic plasticity.

Due to its essential role in signaling both pre-synaptic and post-synaptic processes, as well as cellular processes of differentiation, cell death, vesicular transport, and cytoplasmic motility, calcium undergoes exquisitely precise regulation in neurons that allow simultaneous engagement of multiple spatially separated calcium-dependent processes. Dendritic spines function as physical compartments that isolate and concentrate calcium signals arising from synaptic activity (Koch et al., 1992). Following depolarization or ligand-stimulated calcium uptake, calcium signal recovery is mediated by channel inactivation, plasma membrane sodium-calcium exchangers (NCX) that extrude calcium, and sequestration of calcium into mitochondria, endoplasmic reticulum, and other intracellular stores. The mitochondrion plays a key role in rapid, poststimulatory calcium recovery by taking up massive amounts of calcium into its matrix (White and Reynolds, 1997), while also fueling ATP-dependent pumps on other membranes (Budd and Nicholls, 1996).

While increased excitatory stimulation has been extensively studied in the context of acute neuronal injury and cell death, it has become clear in recent years that increased neuronal calcium handling may also play a pathogenic role in chronic neurodegenerative diseases. Shortening and simplification of the dendritic arbor and spine loss, often accompanied by loss of dendritic mitochondria (Cherra et al., 2013; Dagda et al., 2014) are observed in post-mortem studies of Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) (Hammer et al., 1979; Patt et al., 1991; Baloyannis et al., 2004; Stephens et al., 2005) or their experimental models (MacLeod et al., 2006; Wu et al., 2010; Dagda et al., 2014; Fogarty et al., 2016). Although inhibiting calcium uptake from the extracellular space is frequently neuroprotective (Ilijic et al., 2011; Cherra et al., 2013; Esposito et al., 2013; Plowey et al., 2014), the mechanism(s) by which sublethal increases in cytosolic calcium fluxes trigger dendritic retraction have been unclear. A series of recent studies discussed below implicate increased mitochondrial calcium stress as a key factor by which increased excitatory neuronal activity triggers mitochondrial depletion from and retraction of dendritic structures. Moreover, several mitochondrial calcium transporters are regulated by genes mutated in familial PD, causing functional changes that increase susceptibility to this neurodegenerative mechanism, which we have termed excitatory mitochondrial toxicity (EMT). Genetic and aging- or disease-related signaling alterations may also predispose to EMT in sporadic PD, AD, and the ALS-frontotemporal dementia (FTD) spectrum.

# A BRIEF SUMMARY OF EXCITOTOXICITY

Over the past 40–50 years, it has become well recognized that excessive glutamatergic neurotransmission leads to neuronal cell death, which was first described by Olney (1971). While this has been studied most extensively in the context of brain ischemia from stroke or trauma, excitotoxic cell death has also been implicated in epilepsy and to a lesser extent in AD (Tannenberg et al., 2004), PD (Caudle and Zhang, 2009), and ALS (Shi et al., 2010).

In classic excitotoxicity, a transient episode of ischemia causes the extracellular concentrations of glutamate to rise. This results in widespread stimulation of both synaptic and extrasynaptic NMDARs, resulting in massive influx of sodium and calcium (**Figure 1A**). Ischemia induced neuronal damage is attenuated by pretreatment with an NMDAR antagonist, implicating glutamate toxicity (Simon et al., 1984). Apart from glutamate, earlier studies also implicated kainate and N-methyl-DL-aspartate in calcium dependent neuronal cell death (Berdichevsky et al., 1983). Whereas sodium may mediate the initial, reversible swelling of neurons, irreversible excitotoxic injury is believed to be mediated primarily by elevated calcium levels (Choi, 1995). The data suggest that transient elevations of intracellular calcium is tolerated by the cell and is reversible, whereas sustained calcium overload causes activation of intracellular enzymes (Choi, 1987) and a wave of mitochondrial collapse propagating to the cell body (Greenwood et al., 2007) to cause cell death. The initial glutamate stimulated calcium influx also triggers secondary increases in cytosolic calcium through other mechanisms, which are tightly correlated with neuronal cell death (Randall and Thayer, 1992). Classic excitotoxicity thus involves multiple calcium-dependent pathways initiated in the cytosolic compartment.

In addition to activating calcium-dependent degradative enzymes, such as calpains, phospholipases, and endonucleases, engagement of extrasynaptic NMDARs shuts off CREB signaling (Hardingham et al., 2002), while activating death associated protein kinase 1 (DAPK1) and neuronal nitric oxide synthase (nNOS) bound to the NR2B cytosolic tail (Tu et al., 2010; Martel et al., 2012). Calpain inhibitors confer dose-dependent protection from excitotoxic cell death, as well as preventing mitochondrial permeability transition and release of prodeath factors (Lankiewicz et al., 2000). In turn, inhibiting mitochondrial calcium uptake confers at least partial protection

FIGURE 1 | Pathogenic mechanisms in excitotoxicity and dendritic EMT. (A) In classic excitotoxicity an insult, such as ischemia or trauma, causes increased release of excitatory neurotransmitters (glutamate) leading to post-synaptic uptake of calcium through channels (AMPAR, α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; LTCC, L-type calcium channel; NMDAR, N-methyl-D-aspartate receptor; VGCC, voltage gated calcium channel). Falling ATP levels impair calcium pumps, contributing to cytosolic calcium overload. Sustained intracellular calcium elevation activates a variety of degradative enzymes (calpains, endonucleases, proteases, and lipases), ER stress and mitochondrial permeability transition (MPT), leading to cell death within hours of initiation of excitotoxicity. (B) In neurodegenerative diseases (PD/PDD/DLB, ALS/FTD, and AD), various changes including enhanced glutamate neurotransmission contribute to increased cytosolic calcium flux. In PD/PDD/DLB, aggregated α-synuclein (SNCAagg) can insert into the membrane forming calcium permeable pores. Mutations in the LRRK2 gene contribute to more frequent excitatory post-synaptic potentials and increased calcium influx via NMDAR, AMPAR, and/or LTCC. In AD, soluble amyloid beta (Aβ) can either directly stimulate VGCC or aggregates to form calcium permeable pores. Chronically elevated calcium transients, while insufficient in magnitude to trigger calcium-dependent cell death, results in greater calcium uptake by mitochondria. In addition to elevating cytosolic calcium, mutations in LRRK2 (R1441C/G2019S) transcriptionally upregulate MCU through activation of ERK1/2. On the other hand, loss of function (LOF) mutations in PINK1 result in impaired activation of NCLX-mediated calcium efflux from the mitochondria. Dysregulation of mitochondrial calcium handling, whether driven by increased excitatory cytosolic calcium uptake or changes in the function of mitochondrial calcium transporters, results in mitochondrial injury sufficient to trigger mitophagy. Depletion of dendritic mitochondria precedes subsequent shortening and simplification of the dendritic arbor. Inhibiting NMDAR, LTCC, MCU or autophagy/mitophagy, or stimulating the activity of NCLX, each confers protection against excitatory dendritic mitochondrial toxicity.

against cell death, depending on the severity of injury (Stout et al., 1998; Qiu et al., 2013), and mitochondrial permeability transition further amplifies calpain as well as caspase activation (Ferrand-Drake et al., 2003). However, adult neurons dying from ischemic/hypoxic injuries do not exhibit classic apoptotic morphology, most likely due to calpainmediated inactivation of procaspase-9 (Volbracht et al., 2005). These data implicate m-calpain activation as a major factor in both mitochondrial and non-mitochondrial mechanisms of excitotoxic cell death.

# CALCIUM DYSREGULATION IN CHRONIC NEURODEGENERATION

In recent years, it has become clear that calcium dysregulation also contributes to chronic neurodegeneration in relation to AD (Lopez et al., 2008; Anekonda and Quinn, 2011), ALS (Joo et al., 2007), and PD (Cali et al., 2014) and its related dementias: Parkinson disease with dementia (PDD) (Verma et al., 2017) and Dementia with Lewy Bodies (DLB) (Overk et al., 2014), which by convention are distinguished by the relative timing of cognitive and motor symptoms. A variety of clinical studies have converged on the possible neuroprotective role of calcium channel inhibitors for PD. In particular, both substantia nigra pars compacta neurons and cortical neurons express L-type voltage-gated channels (Guzman et al., 2009). Interestingly, L-type calcium channel inhibitors confer protection of SN and cortical neuron types in both toxic (Ilijic et al., 2011) and genetic (Cherra et al., 2013) models of PD. Moreover, use of centrally acting dihydropyridine L-type calcium channel blockers for hypertension treatment may reduce the risk of PD (Becker et al., 2008; Ritz et al., 2010). These studies emphasize the importance of understanding how neuronal calcium dysregulation contributes to structural and functional changes early in the neurodegenerative process.

In the remainder of this review, we summarize data that supports the concept of a new pathway of sublethal excitatory injury focused near sites of calcium entry, which contributes to dendrite retraction rather than propagating to the soma to cause cell death. We propose the term EMT, to emphasize the key role of mitochondrial calcium dysregulation in this pathway of neurodegeneration. Dysregulation of post-synaptic calcium handling may be triggered by several mechanisms involving proteins implicated in PD, ALS, or AD. The resultant elevations in cytosolic and mitochondrial calcium result in mitochondrial calcium injury, mitochondrial autophagy (mitophagy) and depletion of mitochondria from dendrites (**Figure 1B**).

In contrast to excitotoxicity, which is predominantly triggered by excessive extracellular glutamate release, this pathway may also be triggered by post-synaptic changes in excitability, calcium buffering/recovery, and mitochondrial calcium influx or efflux.

#### CALCIUM, MITOCHONDRIAL CONTENT/DISTRIBUTION, AND NEURONAL ARBORIZATION

Mitochondria play a key role in buffering and shaping cytosolic calcium transients, as well as a critical permissive role for neurite outgrowth, maintenance, and remodeling of axo-dendritic extensions. Before considering disease-linked alterations, basic mechanisms underlying these processes are briefly summarized below.

#### Regulation of Mitochondrial Calcium Handling

Mitochondrial act to buffer intracellular calcium levels through high capacity, low affinity uptake by the mitochondrial calcium uniporter (MCU) complex (Baughman et al., 2011; De Stefani et al., 2011). As such, disease-associated changes in excitatory activity or other sources of increased cytosolic calcium will invariably affect mitochondria. Fine-tuning of MCU function is mediated by accessory proteins MICU1 (Perocchi et al., 2010), MICU2 (Plovanich et al., 2013), EMRE (Sancak et al., 2013), MCUR1 (Mallilankaraman et al., 2012), and MCUb (Raffaello et al., 2013). Mitochondrial calcium uptake is balanced by the activity of sodium/calcium antiporters, such as NCLX (Palty et al., 2010), which release calcium back into the cytosol. Another mitochondrial inner membrane protein LETM1 may act to mediate calcium uptake in response to moderate increases in cytosolic calcium as well as acting in calcium extrusion from the matrix (Doonan et al., 2014), although this latter effect is controversial (De Marchi et al., 2014). Changes to the numbers or function of ER-mitochondrial contact sites may also affect mitochondrial calcium homeostasis (Raffaello et al., 2016), and this process may be regulated by Parkin (Cali et al., 2013), whose mutations cause recessive PD.

From a physiological perspective, calcium uptake into the mitochondrial matrix results in enhanced respiratory function, tuning mitochondrial function to synaptic activity (Bianchi et al., 2004; Vos et al., 2010). However, with massive or sustained calcium stress, this response may result in mitochondrial injury from calcium overload. Following classic excitotoxic glutamate stimulation, excess mitochondrial calcium uptake results in ROS production (Reynolds and Hastings, 1995), collapse of membrane potential and opening of the permeability transition pore (Li et al., 2009), and induction of neuronal cell death (Stout et al., 1998). Indeed, MCU overexpression exacerbates NMDARmediated mitochondrial depolarization and excitotoxic cell death (Qiu et al., 2013). As discussed below, calcium uptake via MCU may also contribute to sublethal pathways of mitochondrial injury sufficient to trigger mitophagy and subsequent dendritic remodeling.

# Calcium, Mitochondria and Dendritic Remodeling

Mitochondria play a key role in the maintenance of dendritic integrity in neurons. Neurons are heavily dependent on the proper function and distribution of mitochondria to stay healthy (Gusdon and Chu, 2011). These accumulate or move toward regions of high energy demand, such as the growth cones of developing neurons (Morris and Hollenbeck, 1993) or regions of higher synaptic activity (Chang et al., 2006). The density and distribution of dendritic mitochondria regulates dendritic morphology, spinogenesis, and the plasticity of spines and synapses (Li et al., 2004). Moreover, in genetic models of neurodegeneration, loss of dendritic mitochondria precedes dendritic retraction (Cherra et al., 2013). This may relate to the requirement for sufficient mitochondrial densities not only to support synaptogenesis during development (Ishihara et al., 2009), but also for maintenance of dendritic arbors in mature neurons (Lopez-Domenech et al., 2016). Depletion of dendritic mitochondria may occur through reduced mitochondrial biogenesis, increased mitochondrial degradation, or alterations in mitochondrial transport.

Two important signals affect mitochondrial movement within a neuronal cell. (i) The energy status of the neuron modulates the transport of mitochondria, wherein high ATP levels increases mobility and high ADP concentration causes either slowing or total arrest of mitochondrial movement. Interestingly, mitochondrial velocity is also decreased in the close vicinity of a spine (Mironov, 2007). (ii) Changes in intracellular calcium levels regulate mitochondrial motility, wherein high cytosolic calcium levels decrease mitochondrial mobility. This may account for the tendency of mitochondria to accumulate near glutamate receptors, where they are situated to provide ATP and to buffer incoming intracellular calcium. Activity dependent mitochondrial movement was elegantly shown by Li et al. (2004), with the number of mitochondria in dendritic protrusions increased by repetitive KCl depolarization. Although the majority of mitochondria were present in the dendritic shaft, a small fraction of mitochondria was observed in the spine itself (Li et al., 2004). Interestingly, even under basal conditions, levels of mitochondria derived oxidative stress is higher in dendrites than in the soma, consistent with an increased bioenergetic demand associated with buffering calcium near a synapse (Dryanovski et al., 2013).

The stimulation of NMDA receptors leads to the activation of protein kinases, such as Ca(2+) /calmodulin-dependent protein kinase (Ojuka, 2004), AMP kinase, and mitogen activated protein kinases, such as ERK1/2 (Yun et al., 1999). Whereas AMP kinase (Ojuka, 2004) and ERK1/2 (Wang et al., 2014) show opposite effects on mitochondrial biogenesis, activation of either signaling pathway serves to promote autophagy or mitophagy (Pattingre et al., 2003; Meijer and Codogno, 2007; Dagda et al., 2008; Bootman et al., 2018). The ability of neuronal cells to undergo mitochondrial biogenesis regulates the outcome of mitophagy

stimulation (Zhu et al., 2012). It is reasonable to surmise that an imbalance in the rates of mitochondrial degradation by mitophagy and replacement by biogenesis/transport will similarly determine the degree of mitochondrial depletion from dendrites.

#### SUBLETHAL EXCITATORY CALCIUM DYSREGULATION IN CHRONIC NEURODEGENERATION

In contrast to classic excitotoxicity, in which massive, acute elevations in glutamatergic neurotransmission results in both non-mitochondrial and mitochondrial pathways of cell death, functional impairment and shrinkage of the synaptic-dendritic arbor likely occur long before cell death in chronic neurodegenerative diseases. Interestingly, sublethal stimulation of NMDA receptors decreases dendrite outgrowth in immature neurons (Monnerie et al., 2003), but the impact on dendritic integrity in mature neurons is less understood. Nevertheless, there are a growing number of studies implicating chronic elevations in excitatory post-synaptic potentials and cytosolic calcium in models of neurodegenerative diseases, which may be due to either pre-synaptic or post-synaptic changes.

#### Parkinson's Disease

The movement symptoms that characterize PD result from degeneration of dopaminergic substantia nigra neurons in the midbrain, which project to the striatum. In addition, PD patients frequently experience olfactory and autonomic dysfunction, mood disorders, and cognitive/executive dysfunction. In addition to playing a key role in cortical neuron function, glutamate plays an important role in modulating dopaminergic neurotransmission, acting on both pre-synaptic and postsynaptic sides. Dopaminergic midbrain neurons express both synaptic and extrasynaptic glutamate receptors (Wild et al., 2015) and are susceptible to classic NMDA excitotoxicity (Kikuchi and Kim, 1993). Excitatory cortical input also modulates striatal neurotransmission in both direct and indirect basal ganglia pathways (Stocco, 2012). While dementia may represent a late stage development in some forms of PD, early cognitive-executive dysfunction represents the defining feature of DLB as well as in several forms of familial PD.

Mutations in the LRRK2 gene, which encodes leucine-rich repeat kinase 2, represent the most frequent known cause of PD (Gandhi P.N. et al., 2009). Recent studies using cultured primary neurons transfected with disease-linked G2019S and R1441C mutations of LRRK2 implicate increased excitatory neurotransmission as one of the earliest pathogenic changes, preceding subsequent dendritic degeneration (Plowey et al., 2014). EPSP frequency was elevated basally, and neurons showed enhanced responses to NMDA and AMPA. Interestingly, memantine, a partial NMDA antagonist conferred protection against subsequent dendritic simplification and loss, implicating an excitatory pathogenesis (Plowey et al., 2014). Increased post-synaptic excitatory neurotransmission was also observed in hippocampal slice cultures of LRRK2-G2019S transgenic mice (Sweet et al., 2015). Interestingly, LRRK2-G2019S knockin mice exhibit an early stage of hyperactivity, accompanied by increased striatal glutamate and dopamine neurotransmission (Volta et al., 2017). Thus, primary neuron cultures, slice cultures and in vivo studies all support an early role for increased excitatory synaptic activity in several mutant LRRK2 models.

Other mechanisms may also contribute to increased intracellular calcium in mutant LRRK2-expressing neurons. As mentioned above, L-type voltage-gated channels contribute to Ca2+ influx during an action potential. Expression of either the G2019S or the R1441C mutation in LRRK2 dysregulates intracellular calcium homeostasis in response to KCl depolarization (Cherra et al., 2013). Calcium chelators or inhibitors of L-type calcium channels confer protection in this system. Calcium release from lysosomal stores has also been implicated in mutant LRRK2 pathogenesis (Gomez-Suaga et al., 2012; Hockey et al., 2015).

Oligomeric α-synuclein, implicated in both dominant familial and sporadic PD/DLB, elicits increased cytosolic calcium uptake through effects on AMPARs (Huls et al., 2011). This creates increased susceptibility to MPP+ toxicity (Lieberman et al., 2017). Interestingly, α-synuclein oligomers can act to increase intracellular calcium levels by forming pores in the plasma membrane (Pacheco et al., 2015). Furthermore, the neurite retraction and increased intracellular calcium elicited by the A53T mutation in α-synuclein are exacerbated by concurrent expression of PINK1-W437X (Marongiu et al., 2009), implicating a mechanistic convergence between dominant and recessive forms of PD. It has been proposed that the reduced mitochondrial membrane potential often observed in PINK1 knockdown/knockout cells (Exner et al., 2007; Dagda et al., 2011, 2014; Huang et al., 2017) may serve to limit mitochondrial calcium uptake, exacerbating excitotoxic injury (Heeman et al., 2011). When post-synaptically expressed, Parkin participates in pruning excitatory synapses (Helton et al., 2008). This may represent another point of convergence between dominant and recessive PD, as either loss of Parkin function or dominant LRRK2 mutations would tend to increase excitatory synapses, conferring enhanced vulnerability to excitatory injury. Taken together, dysregulated neuronal calcium handling resulting in increased cytosolic levels forms a common theme in multiple forms of familial Parkinsonism.

#### Alzheimer's Disease

Alzheimer's disease is the most common age-related neurodegenerative disease, characterized by memory deficits and the pathological hallmarks of neuritic plaques and neurofibrillary tangles. Proteins that are pathologically implicated in AD include the amyloid beta peptides (Aβ) and the microtubule associated protein tau. Calcium mishandling has been implicated in AD and elevated serum calcium levels are well correlated with cognitive decline in aging (Lopez et al., 2008; Popugaeva et al., 2017). Oligomeric Aβ, proteolytic products of the amyloid precursor protein (APP) that is mutated in familial AD (fAD), are enriched

in the plaques that typify the disease and are primary culprits for initiating this calcium dysregulation. Neurons exposed to Aβ oligomers elicit elevations in somatic, dendritic, and synaptic calcium in neurons (Arbel-Ornath et al., 2017; Zhao et al., 2017) and contribute to excitotoxic neuron death (Mattson et al., 1992; Shankar et al., 2008). In mice engineered to co-express fAD mutations in APPswe and Presenilin 1(PS1G384A), a gamma secretase that cleaves APP to generate Aβ peptides, hyperactive neurons are observed in the hippocampus and cortex of young animals, prior to formation of plaques (Busche et al., 2012). Mutations in presenilins alone have also been reported to elicit endoplasmic reticulum calcium overload, with post-translational modification of neuronal ryanodine receptors further promoting calcium leakage into the cytosol (Lacampagne et al., 2017; Popugaeva et al., 2017). Emerging theories suggest long-term Aβ-dependent calcium dysregulation may trigger a cascade of deficits in homeostatic machinery that result in loss in neural network activity (Frere and Slutsky, 2018). For example, elevated calcium plays a key role in promoting tau pathology (Zempel et al., 2010), a component of neurofibrillary tangles that characterize an intermediate step in AD progression, through activation of numerous kinases thought to mediate tau's effects (Mairet-Coello et al., 2013).

# ALS-FTD

Amyotrophic lateral sclerosis is a debilitating disorder affecting upper and lower motor neurons in the cortex, brainstem, and spinal cord (Rowland and Shneider, 2001). Similar to other neurodegenerative diseases, most of the ALS cases are sporadic and 10% of the cases are familial. There is both genetic and pathological overlap between ALS and forms of FTD characterized by accumulations of TAR DNA binding protein-43 (TDP-43) and/or with mutations in C90rf72 (Ling et al., 2013). Motor neurons in ALS are vulnerable to excitotoxic injury as these neurons highly express AMPAR calcium channels (Williams et al., 1997; Corona and Tapia, 2007), accompanied by low expression of calcium buffering proteins (Alexianu et al., 1994; Jaiswal, 2013). In addition, mitochondrial dysfunctions have been reported in post-mortem brain tissues of ALS patients (Sasaki and Iwata, 1996; Kong and Xu, 1998) as well as in animal models of ALS (Nguyen et al., 2009; Santa-Cruz et al., 2016). Given the susceptibility of these neurons to calcium overload induced toxicity, mitochondria play important calcium buffering roles in these neurons (Smith et al., 2017). Excessive exposure to glutamate can lead to glutamate induced excitotoxicity (Stout et al., 1998). Increased glutamate toxicity could be due to enhanced synaptic activity (Milanese et al., 2011) or dysfunctional reuptake by neighboring glial cells (Fray et al., 1998), which can cause persistent activation of AMPAR and increased cytosolic calcium burden leading to mitochondrial calcium overload (Goodall and Morrison, 2006). Interestingly, increased excitatory activity and dendritic spine numbers are observed in early presymptomatic stages of the TDP-43(Q331K) model of ALS (Fogarty et al., 2016). Thus, genetic mouse models of all three diseases, PD, AD, and ALS indicate an early phase of excitatory hyperactivity.

# EXCITATORY MITOCHONDRIAL TOXICITY (EMT) IN CHRONIC NEURODEGENERATIVE DISEASES

In this section, we discuss how PD-linked changes in mitochondrial calcium transport proteins act in concert with sublethal elevations in excitatory neurotransmission to elicit mitochondrial injury and mitochondrial depletion from dendrites. Mitochondrial depletion then contributes to retraction and simplification of the dendritic arbor. In contrast to excitotoxicity, which rapidly results in the classic red, dead neuron observed in stroke, calcium injury triggered autophagy/mitophagy plays a key role in dendritic simplification observed in several models of PD. Given that dendritic pathology is observed in post-mortem studies of PD (Patt et al., 1991), AD (Brizzee, 1987), and ALS (Genc et al., 2017), the review closes with a discussion of the potential implications of the EMT mechanism for sporadic PD and other neurodegenerative diseases.

# EMT in the LRRK2 Model

Shrinkage of the dendritic arbor represents one of the most frequently reported phenotypes exhibited by neurons expressing disease-linked mutations in LRRK2 (MacLeod et al., 2006; Ramonet et al., 2011; Winner et al., 2011; Cherra et al., 2013; Reinhardt et al., 2013; Plowey et al., 2014; Verma et al., 2017). This may be related to effects on microtubule dynamics, endosomal trafficking and/or autophagy [Reviewed in Ref. (Verma et al., 2014)]. Among the earliest changes exhibited by primary cortical neurons transfected with LRRK2-G2019S or LRRK2-R1441C are increased activity-dependent calcium influx through glutamate receptors and L-type calcium channels (Cherra et al., 2013; Plowey et al., 2014). This is followed by loss of mitochondria specifically from the dendritic compartment, which precedes subsequent neuritic retraction (Cherra et al., 2013). The loss of mitochondria can be blocked by inhibiting autophagy or expressing a phosphomimicking mutation of the autophagy protein LC3 (Cherra et al., 2013), which is predicted to impair the cardiolipin pathway of mitophagy (Chu et al., 2014). Mitochondrial fission is often required for efficient mitophagy (Twig et al., 2008; Dagda et al., 2009). Interestingly, mutant LRRK2 regulates Drp1-dependent mitochondrial fission as well as activating ULK1 to mediate mitophagy (Zhu et al., 2012; Su and Qi, 2013). The mechanism that leads to mitophagy of dendritic mitochondria downstream of mutant LRRK2-induced cytosolic calcium uptake was recently delineated using primary neurons transfected with genetically encoded calcium sensors (Verma et al., 2017). As expected, LRRK2-G2019S and -R1441C increased intracellular calcium uptake in response to stimulation, and this was accompanied by increased mitochondrial calcium uptake in dendrites. The increased dendritic mitochondrial calcium uptake persisted even in permeabilized neurons exposed to the same calcium concentrations, implicating increased mitochondrial calcium transport capacity in dendrites of mutant-LRRK2 expressing neurons. Further investigation revealed that mutant LRRK2-transfected neurons, as well as fibroblasts from

PD patients with the G2019S and R1441C mutations, showed increased mRNA and protein expression of MCU and MICU1, with no changes in MICU2 or NCLX expression (Verma et al., 2017). Neurons treated with MCU inhibitors exhibited decreased mitophagy and were protected from dendritic simplification induced by mutant LRRK2. These data implicate calciumdependent injury to mitochondria within dendrites, and their subsequent mitophagic elimination, as mechanisms linking increased excitatory input with dendritic simplification.

#### EMT in the PINK1 Model

The recessive PD-linked gene PINK1, which is targeted to mitochondria via a classic N-terminal mitochondrial targeting sequence, has also been implicated in regulation of mitochondrial calcium homeostasis. As mentioned above, decreases in mitochondrial membrane potential are likely to have multiple consequences including decreased cytosolic calcium buffering and the loss of mitochondria due to mitophagy. Indeed, PINK1-deficient systems exhibit impaired calcium recovery (Heeman et al., 2011) and elevated basal mitophagy in neuronal cells (Dagda et al., 2009; Chu, 2010) and in pancreatic beta cells in vivo (McWilliams et al., 2018), evidently through one of several PINK1- and Parkin-independent mechanisms (Chu et al., 2013; Strappazzon et al., 2015; Bhujabal et al., 2017). In particular, mitochondrial calcium overload has been implicated, as inhibiting mitochondrial calcium uptake, in cells co-expressing α-syn A53T and Pink1 W437X, restores 19m and rescues neurite outgrowth (Marongiu et al., 2009).

Like primary neurons expressing mutant LRRK2, neurons cultured from PINK1 knockout mice to model recessive PD pathogenesis exhibit reduced dendritic arbors (Dagda et al., 2014). Interestingly, PINK1 was shown to regulate calcium efflux via NCLX, with PINK1 deficiency causing mitochondrial calcium overload (Gandhi S. et al., 2009). Indeed, it has recently been shown that PINK1 promotes PKA-dependent phosphorylation and activation of NCLX (Kostic et al., 2015). PINK1-deficient neurons are susceptible to dopamine toxicity, and expression of the NCLX-S258D phosphomimic mutant restores mitochondrial calcium efflux and confers neuroprotection. LETM1, another mitochondrial calcium transporter, represents a direct phosphorylation target of PINK1 (Huang et al., 2017). As LETM1 mediates both calcium influx and efflux (Doonan et al., 2014), the effects of LETM1 activation or loss of function are difficult to predict. Nevertheless, impaired mitochondrial calcium efflux, in a parallel pathway to the effects of PINK1 deficiency on NCLX activity, appears to represent the key pathogenic factor.

#### Converging Mechanisms in Neuroprotection

Whereas episodic calcium entry into the mitochondrial matrix stimulates respiration to adjust mitochondrial output to bioenergetic needs, chronically elevated cytosolic calcium oscillations elicit mitochondrially derived ROS, elevated mitophagy and decreased basal mitochondrial content in dopaminergic substantia nigra neurons (Liang et al., 2007; Guzman et al., 2018). While this occurs under normal conditions for pacemaking cells, such as substantia nigra neurons (Guzman et al., 2018), the mitochondrial response to cytosolic calcium influx is exaggerated in disease states, triggering mitophagy and loss of dendritic mitochondria, followed by a delayed degeneration of dendritic processes (Cherra et al., 2013; Verma et al., 2017). Inhibiting cytosolic calcium influx through NMDA receptors (Plowey et al., 2014) or L-type calcium channels (Cherra et al., 2013; Guzman et al., 2018) prevents the elevated dendritic mitophagy and restores mitochondrial density and dendrite lengths. Inhibiting mitochondrial calcium uptake via MCU confers complete restoration of dendrite lengths (Verma et al., 2017), supporting a central role for mitochondrial calcium mishandling in EMT.

It is likely that increased mitochondrial calcium uptake in the mutant LRRK2 model represents, at least initially, a compensatory response to increased excitatory input. Indeed, MCU and MICU1 are transcriptionally upregulated in mutant LRRK2-expressing neurons and fibroblasts through activation of the ERK1/2 signaling pathway (Verma et al., 2017), which has been proposed to mediate several effects of mutant LRRK2 (Carballo-Carbajal et al., 2010; Reinhardt et al., 2013). Interestingly, a similar elevation in phospho-ERK2, MCU, and MICU1 expression is observed in cortical brain samples from patients with sporadic PD/PDD (Verma et al., 2017, suggesting that enhanced susceptibility to mitochondrial calcium overload could contribute to sporadic disease as well.

One of the models of sporadic disease involves complex I inhibitors, such as MPP+ and rotenone, as PD patients exhibit systemically decreased complex I activity (Greenamyre et al., 2001). Mitochondrial calcium overload has been implicated specifically in the substantia nigra, but not the relatively resistant ventral tegmental area, in the MPP+ model of parkinsonian complex I deficiency (Lieberman et al., 2017). Downregulating autophagy through PKA-mediated phosphorylation of LC3 confers protection against neurite retraction in this model (Cherra et al., 2010), although the potential protective role of modulating MCU or NCLX activities remains unexplored.

While stimulated mitochondrial calcium uptake is either unchanged (Kostic et al., 2015) or slightly decreased (Huang et al., 2017) in PINK1-deficient cells, future studies are needed to determine whether or not there may be concurrent disruption of mitochondrial efflux mechanisms in the mutant LRRK2 model. Irregardless, mutant forms of NCLX that mimic phosphorylation at the PINK1/PKA-regulated NCLX-S258 site (Kostic et al., 2015) confer protection from mutant LRRK2-mediated dendritic simplification to the same extent as inhibiting MCU (Verma et al., 2017). Likewise, inhibition of MCU is neuroprotective in a zebrafish model of PINK1 deficiency (Soman et al., 2017), indicating that reducing the likelihood of mitochondrial calcium overload through either the influx or efflux pathways may be effective irregardless of the original predisposing mechanism.

#### Implications for Other Diseases

It is known that inhibiting mitochondrial calcium uptake via MCU is beneficial in protecting against neuronal cell death after

stroke (Abramov and Duchen, 2008) or during NMDA induced excitotoxic neuronal cell death (Qiu et al., 2013). Recent studies have also shown neuroprotective effects of inhibiting of MCU on Aβ induced microglial cell death (Xie et al., 2017), loss of hippocampal neurons in pilocarpine induced status epilepticus (Wang et al., 2015) or ischemia/reperfusion injury (Zhao et al., 2013). Interestingly, inhibition of MCU protects against neuronal ischemia-reperfusion injury by inhibiting excess mitophagy (Yu et al., 2016), similar the mechanism described in the mutant LRRK2 model (Verma et al., 2017). However, the potential role of sublethal mitochondrial injuries in triggering EMT has been much less studied outside of PD.

In particular, it is unknown if changes in MCU or NCLX expression or post-translational modifications may contribute to sensitivity to excitatory injury in these other diseases. It would be important to delineate whether or not the activities of kinases that regulate mitochondrial calcium transporters are altered in susceptible neurons. In addition to being elevated in patient brains with familial LRRK2 mutation (Verma et al., 2017), elevated ERK1/2 is also observed in sporadic PD/PDD/DLB (Zhu et al., 2002; Verma et al., 2017), AD (Perry et al., 1999), hypoxia-ischemia (Wang et al., 2003), and in organotypic spinal cord culture models of ALS-related TDP-43 pathology (Ayala et al., 2011). As ERK1/2 drives the changes in MCU and MICU expression observed in familial PD patient cells and models, it is possible that EMT contributes to dendritic retraction and simplification in a spectrum of neurodegenerative conditions.

While experiments involving the inhibition of autophagy support the conclusion that mitophagy contributes to mitochondrial depletion from dendrites, another key question to be considered is to understand why aging or diseased neurons fail to replace the degraded mitochondria. These factors may include age- or disease-related decline in mitochondrial biogenesis as observed in PD (Zheng et al., 2010; Zhu et al., 2012) or alterations in mitochondrial transport in neuronal processes as implicated in AD (Calkins and Reddy, 2011) and ALS (Magrane et al., 2012). Mitochondrial depletion would persist only if mitophagy is not balanced by mechanisms to replace the degraded mitochondria. It is thus conceivable that therapies targeting mitochondrial biogenesis or transport may also rescue the ill effects of dendritic EMT.

# CONCLUSION

Dendritic simplification is observed in mutant LRRK2-expressing neurons, in PINK1 knockout neurons, in post-mortem sporadic

#### REFERENCES

PD patient neurons and in other neurodegenerative and neuropsychiatric conditions. We propose that dominant, recessive and sporadic contributions to altered mitochondrial calcium homeostasis converge on a process of EMT to mediate degeneration of the dendritic arbor observed in many neurodegenerative diseases. This may result from effects on the MCU complex itself, on mitochondrial calcium extrusion mechanisms and/or any change that result in greater cytosolic calcium levels. While an alteration in mitochondrial calcium handling on its own may be insufficient to cause neuronal injury, when combined with increased post-synaptic calcium fluxes that accompany excitatory synaptic activity, EMT leads to dendritic shortening and simplification by triggering unbalanced mitophagy and perisynaptic mitochondrial depletion, mechanisms that are distinct from classic excitotoxicity. Moreover, while dominant and recessive contributions to dendritic EMT occur through different mechanisms, interventions that either reduce mitochondrial calcium uptake via MCU or that target NLCX to enhance mitochondrial calcium release are reciprocally effective in both systems (Kostic et al., 2015; Soman et al., 2017; Verma et al., 2017). Future studies to determine whether increased excitatory activity observed in AD and ALS-FTD is also linked to dendritic simplification and spine loss via mitochondrial calcium overload will help determine whether or not therapies targeting EMT may have even broader applicability.

# AUTHOR CONTRIBUTIONS

MV reviewed the literature, designed figures, and wrote sections of the manuscript. ZW contributed to concept development and edited the manuscript. CC developed the conceptual framework, wrote, and edited the manuscript.

# FUNDING

The Chu Laboratory is supported in part by the National Institutes of Health (AG026389, NS065789, and NS101628), a Pilot Grant from the University of Pittsburgh Medical Center (UPMC) Stimulating Pittsburgh Research in Geroscience (SPRIG) initiative, and the Helen Mendel Fund. CC holds the A. Julio Martinez Chair in Neuropathology at the University of Pittsburgh. The Wills Laboratory is supported in part by R21-MH107966.

ERK1/2 dysfunction: implications in ALS. Acta Neuropathol. 122, 259–270. doi: 10.1007/s00401-011-0850-y

mitochondrial Ca2+ uptake that regulates cellular metabolism. Nat. Cell Biol. 14, 1336–1343. doi: 10.1038/ncb2622

disease links oxidative stress to abnormal phosphorylation. Neuroreport 10, 2411–2415. doi: 10.1097/00001756-199908020-00035

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Verma, Wills and Chu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Oldies but Goldies mtDNA Population Variants and Neurodegenerative Diseases

Patrick F. Chinnery1,2 and Aurora Gomez-Duran1,2 \*

1 Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge, United Kingdom, 2 Medical Research Council-Mitochondrial Biology Unit, Cambridge Biomedical Campus, Cambridge, United Kingdom

mtDNA is transmitted through the maternal line and its sequence variability, which is population specific, is assumed to be phenotypically neutral. However, several studies have shown associations between the variants defining some genetic backgrounds and the susceptibility to several pathogenic phenotypes, including neurodegenerative diseases. Many of these studies have found that some of these variants impact many of these phenotypes, including the ones defining the Caucasian haplogroups H, J, and Uk, while others, such as the ones defining the T haplogroup, have phenotype specific associations. In this review, we will focus on those that have shown a pleiotropic effect in population studies in neurological diseases. We will also explore their bioenergetic and genomic characteristics in order to provide an insight into the role of these variants in disease. Given the importance of mitochondrial population variants in neurodegenerative diseases a deeper analysis of their effects might unravel new mechanisms of disease and help design new strategies for successful treatments.

Keywords: mtDNA, haplogroups, PD, LHON, neurodegenerative diseases

#### MITOCHONDRIA AND OXPHOS

Mitochondria, from ancient Greek, mito (thread), and chondros (grain), are highly dynamic organelles in continuous communication with the rest of the cell, that mediate several key cellular functions (Wai and Langer, 2016). These include being the primary source of cellular energy, in the form of adenosine triphosphate (ATP), regulating levels of calcium (Grishanin et al., 1996), reactive oxygen species (ROS) (Boveris and Chance, 1973) and apoptosis (Green and Reed, 1998). Mitochondria play a central role in several fundamental metabolic pathways including the tricarboxylic acids cycle (TCA), fatty-acid β-oxidation, and the pyrimidine biosynthesis (Attardi and Schatz, 1988).

Mitochondria generate the vast majority of cellular energy through the oxidative phosphorylation system (OXPHOS), combining respiration with the synthesis of ATP. Cellular respiration is an ordered chain of redox reactions using reducing equivalents [Nicotinamide adenine dinucleotide (NADH) and Flavin adenine dinucleotide (FADH2)] produced from the degradation of carbohydrates to convert oxygen into water. These reactions are carried out by four multi-protein enzymes: the complexes of the electron transport chain (ETC) I, II, III, and IV and two "shuttles": ubiquinone (coenzyme Q10) and cytochrome c (Saraste, 1999; Smeitink et al., 2000). The energy released in this process is used to pump protons (H+) through complex I (4 H+), III (4H+), and IV (2H+), into to the inter-membrane space,

#### Edited by:

Victor Tapias, Weill Cornell Medicine, United States

#### Reviewed by:

Anat Ben-Zvi, Ben-Gurion University of the Negev, Israel Richard G. Boles, The Center for Neurological and Neurodevelopmental Health (CNNH), United States Petr A. Slominsky, Institute of Molecular Genetics (RAS), Russia

\*Correspondence:

Aurora Gomez-Duran<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 22 May 2018 Accepted: 10 September 2018 Published: 12 October 2018

#### Citation:

Chinnery PF and Gomez-Duran A (2018) Oldies but Goldies mtDNA Population Variants and Neurodegenerative Diseases. Front. Neurosci. 12:682. doi: 10.3389/fnins.2018.00682

**183**

generating a positive electrochemical gradient which drives the transport back to the matrix through the complex V or ATP synthase. This complex acts as a proton channel that returns the protons to the mitochondrial matrix. The proton flux provides the energy needed to bind adenosine di-phosphate (ADP) and inorganic phosphate into ATP (Mitchell, 1961).

# MITOCHONDRIAL GENOME

One of the principal features of mitochondria is that they contain their own genetic system. In humans this is a double chain circular molecule, 16.6 Kb long, which codes for 13 proteins of the OXPHOS system, and the 22 tRNAs and 2 rRNAs required for their expression (Taanman, 1999). Seven of the 13 subunits contribute to complex I (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6), one to the complex III (CYB), three to the complex IV (CO1, CO2, and CO3), and two to the complex V (ATP6 and ATP8). mtDNA is composed of 2 chains, heavy (H) and light (L) with different density based on the G/C composition. Most of the genes are coded by the heavy chain including 2 rRNAs, 14 tRNAs and 12 polypeptides), while 8 tRNAs and only one polypeptide (ND6) are encoded by the light chain (Montoya et al., 1982).

mtDNA is polyplasmic, with each mitochondria containing several copies. A somatic cell can contain between hundreds to thousand mtDNA copies depending on the cell type (Robin and Wong, 1988). Usually all of the molecules are identical (homoplasmy). The presence of more than one type or allele of mtDNA in a cell is known as heteroplasmy (DiMauro et al., 1993). The proportion of a heteroplasmic mutation can vary from cell to cell and selective pressures can influence both of these processes (Taylor and Turnbull, 2005; Stewart and Chinnery, 2015; Burr et al., 2018).

Mitochondrial DNA is strictly maternally inherited (Giles et al., 1980; Pyle et al., 2015) and has a mutation rate, 5–10 times higher than the nuclear genome (nDNA) (Brown et al., 1995). These factors have led to the accumulation of a wide range of polymorphisms across the mtDNA sequence that are restricted to geographically isolated populations throughout the globe. Given that these genetic variants are inherited from mother to offspring without any recombination, they sequentially accumulate along the radiating maternal lineages (**Figure 1**). This had generated phylogenetically related haplotypes (**Figure 1**). The most common (>1% frequency in the population) are known as mitochondrial haplogroups (Wallace et al., 1999; Wallace, 2015).

#### ANCIENT POLYMORPHISMS MTDNA HAPLOGROUPS

The human mtDNA phylogeny clusters into a three with "unique" ancestor, known as the "Mitochondrial Eve," rooted in Africa about 150,000 years before present (YBP) (Cann et al., 1987). From this root, four lineages specific for sub-Saharan Africa: L0, L1, L2, and L3 were generated about 100,000 YBP. Then, 60,000 YBP the African haplogroup L3 diverged into the two

recent macro-haplogroups M and N, which define populations which left Africa to populate the rest of the world (Soares et al., 2012). During this migration, the haplogroup N was directed to Eurasia and Asia and America, while the M went exclusively to Asia giving place to the haplogroups A, B, C, D, G, and F. In Europe, the haplogroup N 60,000 YBP gave rise to the haplogroup R (Gandini et al., 2016), which is the root of the "European" haplogroups U (60,000 YBP), J (40,000 YBP), T (20,000 YBP), H (30,000–5,000 YBP) (Achilli et al., 2004), and V (15,000 YBP) (Torroni et al., 1996) (**Figure 2**). With the implementation of the next generation sequencing techniques (NGS) there has been a huge increase in the number of mtDNA genomes sequenced and the branches of the tree have diverged into many sub-haplogroups (Brotherton et al., 2013). The current global mtDNA phylogenetic tree contains more than 4,000 different haplogroups (van Oven and Kayser, 2009).

In Europe, 90% of the population belongs to the macro-haplogroups HV, U, and JT (Macaulay et al., 1999; Torroni et al., 2000). The macro-haplogroup HV represents more than 50% of the population, it comprises the haplogroups H and V and HV∗ . Among them, the haplogroup H an extremely widely distributed and has the highest frequency reaching 45% in Europe, 20% in Turkey and the Caucasus, and around 10% in Gulf countries (Roostalu et al., 2007). It is formed by more than 90 sub-haplogroups (van Oven and Kayser, 2009), where the H1 is the major one having two peaks of high frequency in Scandinavian Peninsula and Southern Iberian Peninsula. The second most frequent haplogroup is the H3, commoner in South of Europe, particularly in France and Spain. The rest of the sub-haplogroups, as for example H2 and H6, are more frequent in the Caucasus and Eastern Europe (Pereira et al., 2005). On the other hand, haplogroup V is found in 4% of the population, principally in European populations, but also present in the north of Africa (Coudray et al., 2009).

The other two macro-haplogroups are the JT and U which account 40% of the European population. Haplogroup U is divided into several sub-haplogroups that make up 20% of the Caucasian population, whereas the subhaplogroups U5 and Uk comprise 9% (Montiel-Sosa et al., 2006). This clade is widely spread from Portugal to India and North of

Africa (Achilli et al., 2005). The macro-haplogroup JT includes haplogroups J and T (Ruiz-Pesini et al., 2004). Haplogroup T is divided also in several haplogroups and embody 8% of the population (SanGiovanni et al., 2009). Haplogroup J is found in 9% of Europeans, and is divided into 2 principal sub-haplogroups J1c and J2 (Carelli et al., 2006).

#### MTDNA POLYMORPHISMS AND NEUROLOGICAL DISEASES

Over the last 20 years, many studies have found associations between inherited mtDNA population variants and neurological diseases. Since the first association of Leber Hereditary Optic Neuropathy (LHON) with the mitochondrial haplogroup J in the late 90s (Torroni et al., 1996; Carelli et al., 1997; Hofmann et al., 1997; Lamminen et al., 1997), many other mitochondrial disorders along with classic neurodegenerative diseases like Parkinson (PD), Alzheimer (AD), Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS).

#### Mitochondrial Diseases

Mitochondrial diseases are a large group of heterogeneous disorders caused by mutations in the mtDNA and the nuclear DNA (nDNA). Although, their phenotypes are hugely variable, some of them overlap with clinical phenotypes observed in neurodegenerative diseases (Betts et al., 2004; Swerdlow, 2009). For example, neurodegeneration in the cerebellar purkinje layer and cortical neurons in the occipital and parietal lobes has been observed in patients with Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) (Betts et al., 2004). Leber Hereditary Optic Neuropathy (LHON) disorder is characterized by neurodegeneration of the retinal ganglion cell (RGC) layer and optic nerve (Yu-Wai-Man et al., 2011). In addition, a small sub-set of LHON patients develop similar signs to multiple sclerosis, in a disease known as Harding's disease (Pfeffer et al., 2013; Bargiela and Chinnery, 2017) and signs of parkinsonism have also been observed in LHON pedigrees (Simon et al., 1999; Vital et al., 2015).

#### Leber Hereditary Optic Neuropathy

LHON is a mitochondrial neurodegenerative disorder characterized by RGC dysfunction and rapid visual loss. In Caucasian population, approximately 90% of LHON cases are caused by a mutation in the MT-ND genes subunits encoding for the mitochondrial complex I; m.11778G > A:MT-ND4 (60%), m.3460G > A:MT-ND1 (15%) and m.14484T > C:MT-ND6 (15%). The remaining 10% of the LHON cases harbor rarer mutations (Achilli et al., 2012; Maresca et al., 2014; Caporali et al., 2018). Although the primary genetic cause of LHON is known, the presence of a mtDNA mutation is not enough on its own to cause the blindness. Many factors such as gender, with higher penetrance in males than females, genetic factors including the mitochondrial haplogroups, and other environmental factors have been related with the clinical penetrance of the disease (Kirkman et al., 2009; Caporali et al., 2017).

The role of mtDNA population variants in LHON has been widely studied. Initial studies described the variants m.4216C > T:MT-ND1 and m.13708G > A:MT-ND5, m.15257G > A:MT-CYB, m.15812G > A:MT-CYB defining J and J2 haplogroup, respectively, as "secondary" LHON mutations (Johns and Berman, 1991; Johns and Neufeld, 1991; Huoponen et al., 1993; Oostra et al., 1994; Brown et al., 1995; Harding et al., 1995; Howell et al., 1995; Lodi et al., 2000), before the association was described with haplogroup J (Carelli et al., 1997; Hofmann et al., 1997; Lamminen et al., 1997; Torroni et al., 1997). Studies with bigger cohorts in Italian population (86 cases) narrowed down the associations to specific LHON mutations, where the m.11778G > A:MT-ND4 mutation was over-represented in subhaplogroups J1c and J2b defined by variants in the MT-CYB; and m.14484T > C: MT-ND6 was over-represented in haplogroup J1 (Carelli et al., 2006). The role of the MT-CYB variants was also confirmed in the most comprehensive study carried out to date. In a cohort counting with 3,613 individuals from 159 European LHON families, Hudson et al. confirmed mutation specific sub-haplogroup associations (Hudson et al., 2007). Individuals carrying the mutation m.3460G > A: MT-ND1 had an increase of risk in the haplogroup Uk [OR = 2.37, p = 0.002, CI 95% (1.36–4.13)]. Individuals carrying m.11778G > A: MT-ND4 had an increased risk in the J haplogroup [OR = 1.31, p = 0.02, CI 95% (1.03– 1.65)], and a reduced risk on a haplogroup H background

[OR = 0.79, p = 0.04, CI 95% (0.63–0.98)] (Hudson et al., 2007).

Recent studies have shown the combinations of polymorphisms may lead to a reduced OXPHOS efficiency and be sufficient to trigger LHON (Achilli et al., 2004; Caporali et al., 2018). This is in keeping with our recent findings, where we showed that some mtDNA sequences appear to influence the probability of acquiring new pathological mutations in a population specific manner (Wei et al., 2017).

#### Other Mitochondrial Diseases

Independently from LHON studies, other mitochondrial disorders such as MELAS, neuropathy, ataxia, retinitis pigmentosa (NARP)/Maternally inherited Leigh's syndrome (MILS), and age-related hearing loss (ARHL) have been associated with certain mtDNA haplogroups, but these findings remain controversial.

In the case of MELAS, a study in 142 unrelated French families carrying the m.3243A > G mutation observed a statistically significant under-representation of the mutation in haplogroup J patients [OR = 0.26, p = 0.01, CI 95% (0.08–0.83)] (Pierron et al., 2008). Analysis of the same mutation in smaller sample from Spanish population did not find any association (Torroni et al., 2003).

Mitochondrial haplogroups do not appear to play a role in non-syndromic deafness caused by the m.1555G > A (Torroni et al., 1999), although an association with haplogroup H3 was described in one small cohort of Spanish mutation carriers (Achilli et al., 2004). The analysis of 912 ARHL patients found that haplogroup U was significantly associated with moderate to severe phenotype (OR 3.02; CI 95%: 1.30–6.99); and in patients aged from 50 to 59 years sub-haplogroup Uk was associated with severe ARHL only (OR 3.02; CI 95%: 1.30–6.99) (Manwaring et al., 2007). Although it is difficult to draw firm conclusions from these small single studies, in line with these findings, a study in transmitochondrial cell lines carrying the mutation m.8993T > G: MT-ATP6 responsible NARP/MILS syndrome showed an significant increase severity of the OXPHOS defect in cell lines from the haplogroup U5b compared those belonging to haplogroup H (D'Aurelio et al., 2010).

### Parkinson Disease

The association of mtDNA polymorphisms and Parkinson's disease pathogenesis has been controversial, although recent large studies have validated the original findings in independent cohorts. Many studies have shown association between PD and particular haplogroups (Moilanen et al., 2001; Ross et al., 2003; van der Walt et al., 2003; Otaegui et al., 2004a; Ghezzi et al., 2005; Huerta et al., 2005; Pyle et al., 2005; Gaweda-Walerych et al., 2008; Khusnutdinova et al., 2008; Latsoudis et al., 2008; Georgiou et al., 2017), while others could not directly replicate these findings (Simon et al., 2010; Fachal et al., 2015). Most of the studies showed population specific associations. Indeed, an early work in Finns found a reduced risk for PD in the supercluster HVKU compared to the supercluster JTIWX which was exclusively associated with the Uk cluster (Autere et al., 2004). Studies in a Polish population of 241 PD patients and 277 control subjects, didn't find differences between the haplogroups, however, after stratification by gender, they found that haplogroup J [OR = 0.19, p = 0.0014, CI 95% (0.069–0.53)] was associated with a lower PD risk in males (Gaweda-Walerych et al., 2008). Another study in an Italian population comprising of 620 idiopathic PD patients and about 2000 controls found a role of haplogroup Uk in decreasing the penetrance of PD [OR = 0.54, p = 0.048, CI 95% (0.35–0.83)] (Ghezzi et al., 2005), while in an study on a Spanish cohort of 271 PD patients and 230 healthy controls significant association was found for the polymorphism defining the haplogroup H5 mt.4336 T>C:tRNAGln with a significantly increased frequency in PD compared to controls [OR = 4.45, p = 0.011, CI 95% (1.23–15.96)] but only in females (Huerta et al., 2005). Other study in a Tatar Russian population of 183 unrelated PD patients and 157 controls found that polymorphisms associated with haplogroup H mtDNAs increased PD risk (OR = 2.58, p = 0.0001), whereas those associated with haplogroup Uk were protective (OR = 0.38, p = 0.003) (Khusnutdinova et al., 2008). Conversely, the Irish study that included 90 Irish PD patients and 129 Irish controls, reported that haplogroups J and T increased PD risk (Ross et al., 2003).

The variability of results could have been affected by population stratification issues, small sample sizes and variations in the statistical approach used. However, three recent meta-analysis have shown evidence of the existence of both protective and risk haplogroup alleles associated with PD. The first study counted with 3,074 PD cases and 5,659 ethnically matched controls followed by meta-analysis of 6,140 PD cases and 13,280 controls. They found that two variants, m.2158T > C:MT-RNR2 and m.11251A > G:MT-ND4, which are phylogenetically linked and define the mitochondrial superhaplogroup JT and sub-haplogroup J1b, respectively, were associated with a reduced risk of PD [OR = 0.87, CI 95% (0.77–0.97)] (Hudson et al., 2013). In line with this finding, a second study from the same group, including 2,197 patients compared with three independent control groups genotyped on the same platforms (C1 = 2997, C2 = 2897, and C3 = 5841 control samples), also showed a protective effect for the variant m.10398A > G:MT-ND3, homoplastic (recurrent in several branches of the mtDNA phylogenetic tree) on haplogroups J and Uk, which was previously associated with reduced risk to PD (Ghezzi et al., 2005; Huerta et al., 2005; Hudson et al., 2014). Finally, a meta-analysis from 13 different studies combining n = 9243 patients and n = 17,999 controls found that individuals mtDNA haplogroup Uk [OR = 0.839, p = 0.004, CI 95% (0.744–0.945)], haplogroup T [OR = 0.857, p = 0.014, CI 95% (0.757–0.969)] and haplogroup J [OR = 0.876, p = 0.011, CI 95% (0.79–0.971)] had a significantly reduced risk of developing PD, while the macro-haplogroup HV act as a increased risk factor [OR = 1.091, p = 0.038, CI 95% (1.005–1.184)] (Marom et al., 2017).

#### Alzheimer Disease

The implication of mtDNA variants in AD remains contentious. Several studies have reported haplogroup associations (Chagnon et al., 1999; Carrieri et al., 2001; Fesahat et al., 2007; Santoro et al.,

2010; Maruszak et al., 2011; Coskun et al., 2012; Ridge et al., 2012) but the results have been inconsistent, and others found no evidence of an association (Chinnery et al., 2000; Hudson et al., 2012; Fachal et al., 2015; Pyle et al., 2015) (for extensive review of the data see Ridge and Kauwe, 2018). The largest study for mtDNA carried with 3,250 AD patients and 1,221 controls found no significant association between mtDNA variants and AD (Hudson et al., 2012). However, a more recent meta-analysis involving several studies did report an increased risk of AD in people belonging to haplogroup H [OR = 1.283, p = 0.016, 95% CI (1.047–1.574)] (Marom et al., 2017), but larger studies are warranted to validate these findings which only just reach conventional statistical significance.

In addition to the genetic analysis, a study from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database including 175 controls and 154 AD patients found that individuals from U5b1 and Uk1a1b haplogroups had grater rates of temporal pole atrophy, an endophenotype of AD risk (Ridge et al., 2013).

#### Multiple Sclerosis

Following the trend of the previous disorders, the data on associations between mitochondrial haplogroups and MS is contradictory. While some small studies have seen an increase in the risk associated with the haplogroup Uk in different populations (Kalman et al., 1999; Hassani-Kumleh et al., 2006; Vyshkina et al., 2008), others have shown a consistent association with haplogroup J (Kalman et al., 1999; Houshmand et al., 2005; Mihailova et al., 2007), and others failed to find any association in United Kingdom (Ban et al., 2008) and Basque population (Otaegui et al., 2004b).

Larger studies have, however, found consistent associations, mainly with the macro-haplogroup JT as a risk factor in European populations. The first meta-analysis studying more than 2500 MS samples and the same number of controls found that the variant m.13708G > A: MT-ND5, defining the haplogroup J, was significantly associated with an increased risk to MS [OR = 1.71, P = 0.0002, CI 95% (1.29–2.27)] (Yu et al., 2008). Similarly, another recent meta-analysis involving 7,391 and 14,568 controls confirmed the association between haplogroup J (OR = 1.11, p = 0.03, CI 95% (1.01–1.22)] and haplogroup T carriers [OR = 1.17, p = 0.002, CI 95% (1.06–1.29)]. In the same study, only 3 populations (Italy, United Kingdom, and Germany) showed an association between primary progressive (PP) PPMS and haplogroup J [OR = 1.49, p = 0.009, CI 95% (1.10–2.01)], but a smaller cohort with 3,720 cases and 879 controls from US did not show the same association (Tranah et al., 2015). These findings could be due to differences in the frequency of sub-haplogroups in the populations studied (Andalib et al., 2015).

# Amyotrophic Lateral Sclerosis

The larger study carried out in ALS in a cohort of 700 patients and 462 controls from two European populations did not find any association between mtDNA haplogroups and ALS (Ingram et al., 2012). Similar results were previously obtained in a cohort from UK population of 504 ALS patients and 493 controls (Chinnery et al., 2007). Conversely, in a cohort from Italian population with 222 patients with sporadic ALS (sALS) and 151 matched controls, the haplogroup I demonstrated to be associated a higher risk of ALS when compared to the common haplogroup, H [OR: 0.08, p < 0.01, CI 95% (0.04–0.4)] (Mancuso et al., 2004). Overall, studies of mtDNA haplogroups in ALS remain much smaller than other neurodegenerative diseases.

# FROM EPIDEMIOLOGY TO BIOENERGETICS

Despite earlier contention, there is now clear evidence that mtDNA variants within haplogroups are associated with specific neurodegenerative disorders, with the most consistent evidence for Parkinson disease and LHON. This raises the question: how do these genotypes mediate their pleiotropic effects? Functional studies are technically challenging because the biochemical effect of common polymorphisms is likely to be subtle. In addition, given that many of the associated variants are found on genomes containing other polymorphisms, several linked alleles may be interacting. Unfortunately, the double mitochondrial membrane precludes site directed mutagenesis at present, so functional studies are limited to mtDNA harvested from human cells. The generation of transmitochondrial cybrids present one approach to address some of these issues (Chomyn et al., 1994). A cybrid gives the opportunity of studying the effect of a determined mtDNA in a fixed nuclear background under the same ambient conditions. Briefly, a rho0 cell line is generated by removing mtDNA (King and Attardi, 1996; Miller et al., 1996), can be fused with enucleated fibroblasts (King and Attardi, 1989) or with platelets containing the mtDNA of interest (Chomyn et al., 1994; **Figure 3**).

Cybrid technology has been widely used for the study of phenotypical effect of inherited pathogenic mutations in the mtDNA (Hayashi et al., 1991; Trounce et al., 1994; Vergani et al., 1995; Brown et al., 2001; D'Aurelio et al., 2001), but the model itself raises several concerns, including aneuploidy and an unstable nuclear background in a cancer derived cell line, the use of mutagenic agents for the generation of rho0, and biochemical properties of the original tumor (for further information see Iglesias et al., 2012). However, given the benefits shown when using this approach to elucidate disease mechanisms for mitochondrial diseases, the technique has been extensively applied for the study of the mtDNA population variants to look for subtler biochemical effects of defined genetic variants.

Cybrids from haplogroup H contain higher mtDNA levels and mtDNA encoded mRNA levels, growing faster, have a higher membrane potential, and consume more oxygen per ETC unit than cybrids from haplogroup Uk individuals (Gómez-Durán et al., 2010). Similar differences were found when characterizing cybrids from haplogroup H and T in HEK293 cells background, where H cybrids showed higher respiration capacity per molecule of mtDNA, compared to HEK293 cybrids, but lower growth rate (Mueller et al., 2012). Contrarily, in another study, Caucasian haplogroups H and T showed no differences in the mitochondrial membrane potential and the oxygen consumption per cell. Other analysis didn't find differences between the "artic" (A, C, and D)

and the "tropical" haplogroups (L1, L2, and L3) or the European haplogroups H and T (Amo and Brand, 2007; Amo et al., 2008).

Haplogroup J has been widely linked to several diseases, and therefore deeply characterized using different nuclear genetic backgrounds. Similarly, to the epidemiological studies, initial work with a small number of samples showed no functional differences between the haplogroup H, J, and T (Carelli et al., 2002). However, larger studies showed that haplogroup J cell lines have slower rate of assembly of the mitochondrial complexes (Pello et al., 2008) on a nuclear 143B background. In studies in Wal-2A cell line haplogroup J1b revealed higher mtDNA levels and TFAM binding than a cell lines from haplogroup H (Suissa et al., 2009). However, using ARPE-19 cells, the same group did not find differences in the mtDNA levels between haplogroups H and J, but found lower levels in ROS production and ATP levels in haplogroup J (Kenney et al., 2013). Similarly, J cybrids on a 143B background showed less lower ATP and ROS production than haplogroup H cybrids (Bellizzi et al., 2012). This contrasts with another study of 9 cybrid lines from the haplogroup J, which were compared to 5 from the haplogroup H, and did not find any difference in manganese superoxide dismutase (MnSOD) expression, a marker of reactive oxidative species (ROS) production. This study did, however, confirmed the previous findings of lower oxygen consumption and low total ATP levels in the haplogroup J cell lines (Gómez-Durán et al., 2012).

Besides the discrepancies among the studies, it seems that cell lines carrying the mitochondrial haplogroup J have less OXPHOS capacity and ATP levels than those from the haplogroup H. In keeping with this, in vivo studies of individuals showed that individuals from the J haplogroup were shown to have lower maximal oxygen uptake (VO2max) (Marcuello et al., 2009) and haplogroup H higher (Martinez-Redondo et al., 2010) in an Spanish cohort.

The variation observed in all studies between the haplogroups carried in vitro could be due to 2 factors; the effect in the nuclear background uses and/or to the differences between the sub-haplogroups of each haplogroup (Chen et al., 2012). Indeed, while in our studies we studied cells carrying mtDNA from the subhaplogroups of the J; J1b1 (1), J1c (4), J2 (4), H1 (3), H5 (1), and H1 (3) (Gómez-Durán et al., 2012), Suissa et al. and the Kenney et al. included J1 (1), J1b2 (1), H1 (1), H6 (2) and H∗ (2) and J1c (2), J1d1a (1), and H subhaplogroups that were not stated, respectively, (Suissa et al., 2009; Kenney et al., 2013). Thus, the observed inconsistency could be due to the variants in the younger branches of the phylogenetic tree that define the sub-haplogroups (Hudson et al., 2014). Altogether, this could also affect nDNA-mtDNA retrograde response signaling in a haplogroup dependent manner.

#### MITOCHONDRIAL SIGNALING IN MTDNA POPULATION VARIANTS

Mitochondrial nuclear crosstalk was first described in yeast after the depletion of mtDNA which induce the expression of transcription factors (Butow and Avadhani, 2004; Picard et al., 2013). Mitochondrial dysfunction also induces retrograde responses (Chae et al., 2013). The biochemical disruption alter many downstream including immune signaling (Picard et al., 2015), mTORC1 (Khan et al., 2017), AMP-activated protein kinase (AMPK) (Zheng et al., 2016) and the transcription factors ATF4 (Quiros et al., 2017) and/or ATF5 (Fiorese et al., 2016; Suomalainen and Battersby, 2017).

mtDNA population variants also have been shown to affect a variety of signaling pathways. Cybrids in 143B osteosarcoma background from haplogroup J showed higher expression levels of IL-1β and TNFR2 than the ones from haplogroup H (Bellizzi et al., 2006). In the same cell lines J haplogroup showed an increase expression of the methionine adenosyltransferase 1A (MAT1A) gene, and therefore an increase in the global methylation compared to the cells from the H haplogroup (Bellizzi et al., 2012). In ARPE-19, cybrids cell lines from the haplogroup J have reduced Complement factor H (CFH),

Complement component 3 (C3) and expression levels than those from haplogroup H (Kenney et al., 2013) and increase in apoptotic genes like RAR (Kenney et al., 2014). Another study in 143B cells showed a significantly increased expression of BBC3 in H cybrids compared with J cybrids (Fernández-Moreno et al., 2017). We have seen an increase in the expression of phosphofructokinase in the cell lines from the haplogroup J and Uk when compared to cells from the haplogroup H (Gómez-Durán et al., 2010, 2012).

In addition, a recent study has shown that the variant defining haplogroup J m.13708G > A: MT-ND5 is 2 bases upstream of a methylation RNA site at the gene MT-ND5 (13710) which severely reduces the capacity of MT-ND5 to be methylated and its predicted to have phenotypical implications (Safra et al., 2017).

# FUTURE PERSPECTIVES

In summary, the large number of studies on mtDNA population variants over the last 25 years have clearly shown that mtDNA polymorphisms defining the mitochondrial haplogroups are certainly not phenotypically neutral as previously assumed. Extensive work has shown their role in evolution, disease, bioenergetics and cell signaling. However, understanding how the same variant could be advantageous or detrimental in different contexts, and their effect in mtDNA-nDNA communication needs to be further understood. An important caveat is that many of the published studies could be false positive results, in part related to multiple significance testing. To date, there has been limited or no attempt to independently replicate the original findings. Confirmatory replication has been possible in some instances, most notably Parkinson's disease (Hudson et al., 2013; Marom et al., 2017), but there is a need for large well-powered discovery and replication studies to validate many of the findings discussed above, accounting for multiple significance testing. Another challenge is the phylogenetically related nature of

#### REFERENCES

the mtDNA which makes difficult to determine how the dependence between the variants influences the associations. The recent increase of available data in NGS for whole genome sequence studies (including mtDNA) in combination with accessible public data from other "omic" technologies such as proteomics, metabolomics and transcriptomics will definitely make a difference helping understanding which ("goldies") variants are responsible for the associations. Given the complexity of these pathologies, a combination of efforts between multidisciplinary teams combining genomics studies on patient samples in combination with wet lab studies on cell biology and biochemistry will pave the way to elucidate the mechanisms of their role in cellular metabolism and dysfunction. This will lead to enhanced understanding of mtDNA heritability in neurodegenerative diseases and open new avenues for effective treatments.

# AUTHOR CONTRIBUTIONS

AG-D designed and wrote the manuscript together with PFC.

#### FUNDING

AG-D receives support from NIHR Biomedical Research Centre pilot studies (RROI.GAAB). PFC was a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC\_UP\_1501/2), the Medical Research Council (United Kingdom) Centre for Translational Muscle Disease (G0601943), and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.

and dementia with Lewy bodies. Neurology 55, 302–304. doi: 10.1212/WNL.55. 2.302

DiMauro, S., and Hirano, M., and Merrf. (1993).

Green, D. R., and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 1309–1312. doi: 10.1126/science.281.5381.1309

variants on late-onset Alzheimer's disease risk. J. Alzheimers Dis. 27, 197–210. doi: 10.3233/JAD-2011-110710

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chinnery and Gomez-Duran. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Potential Role of Mic60/Mitofilin in Parkinson's Disease

Victor S. Van Laar1,2 \*, P. Anthony Otero2,3,4, Teresa G. Hastings1,2,5 and Sarah B. Berman1,2,6 \*

1 Department of Neurology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2 Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA, United States, 3 Division of Neuropathology, Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 4 Cellular and Molecular Pathology (CMP) Program, Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 5 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, United States, 6 Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA, United States

There are currently no treatments that hinder or halt the inexorable progression of Parkinson's disease (PD). While the etiology of PD remains elusive, evidence suggests that early dysfunction of mitochondrial respiration and homeostasis play a major role in PD pathogenesis. The mitochondrial structural protein Mic60, also known as mitofilin, is critical for maintaining mitochondrial architecture and function. Loss of Mic60 is associated with detrimental effects on mitochondrial homeostasis. Growing evidence now implicates Mic60 in the pathogenesis of PD. In this review, we discuss the data supporting a role of Mic60 and mitochondrial dysfunction in PD. We will also consider the potential of Mic60 as a therapeutic target for treating neurological disorders.

#### Edited by:

Victor Tapias, Weill Cornell Medicine, United States

#### Reviewed by:

Kim Tieu, Florida International University, United States Ruth G. Perez, Texas Tech University Health Sciences Center El Paso, United States

#### \*Correspondence:

Victor S. Van Laar<EMAIL_ADDRESS>Sarah B. Berman<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 05 October 2018 Accepted: 16 November 2018 Published: 25 January 2019

#### Citation:

Van Laar VS, Otero PA, Hastings TG and Berman SB (2019) Potential Role of Mic60/Mitofilin in Parkinson's Disease. Front. Neurosci. 12:898. doi: 10.3389/fnins.2018.00898 Keywords: Mic60/mitofilin, mitochondria, Parkinson's disease, neurodegeneration, mitochondrial dynamics

# INTRODUCTION

Parkinson's disease (PD), the most common neurodegenerative movement disorder, was first described in 1817 by James Parkinson in "An Essay on the Shaking Palsy." (Parkinson, 1817) In the 200 years that have passed since recognition of this neurological disorder, great strides have been made to characterize disease pathology, distinguish clinical symptoms, and develop a therapeutic treatment. However, the causes of PD neurodegeneration are still unknown, and there is no cure nor are there any available neuroprotective therapies to hinder disease progression. Identifying and understanding the etiology of PD progression is key to the development of new therapeutics for disease treatment.

Epidemiological studies and laboratory research have long sought to find potential causes of this prevalent disease (Khandhar and Marks, 2007; Delamarre and Meissner, 2017). Though the underlying mechanism of PD pathogenesis remains elusive, current ideology suggests that a combination of environmental exposure and genetic predisposition are responsible for most cases of PD (Horowitz and Greenamyre, 2010; Ritz et al., 2016). Genetic and epidemiological studies have identified multiple biological pathways that promote PD pathogenesis, many of which converge on the function of the mitochondria, the "powerhouses" of the cell (Schapira, 2008; Cieri et al., 2017; Ammal Kaidery and Thomas, 2018; Zanon et al., 2018).

Mitochondrial dysfunction is a known contributor to PD pathophysiology, with impaired mitochondrial respiration, morphology, and fission/fusion/transport dynamics all associated with PD (Van Laar and Berman, 2013; Bose and Beal, 2016). The connection between PD and mitochondria is reinforced by heritable forms of the disease, wherein monogenetic PD-causing

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mutations in nuclear-expressed proteins such as PINK1, Parkin, LRRK2, and alpha-synuclein have all been shown to affect mitochondrial function (Narendra et al., 2010; Sanders et al., 2014; Di Maio et al., 2016; Verma et al., 2017). In recent years, independent studies from multiple labs have associated the inner mitochondrial membrane protein Mic60, also known as mitofilin, with PD pathogenesis (Van Laar et al., 2008, 2009, 2016; Akabane et al., 2016; Tsai et al., 2018). Mic60 is a core component of the mitochondrial contact site and cristae junction organizing system (MICOS) (Zerbes et al., 2012b; Pfanner et al., 2014; Kozjak-Pavlovic, 2017). The MICOS is a large, multi-protein complex of the mitochondrial inner membrane that maintains cristae structure, forms inner-outer mitochondrial membrane contact sites, organizes respiratory complexes, and regulates protein import (Bohnert et al., 2012; Zerbes et al., 2012a; Harner et al., 2014; Pfanner et al., 2014; Friedman et al., 2015; Horvath et al., 2015; Kozjak-Pavlovic, 2017; **Figure 1A**). Growing evidence places the MICOS complex, and in particular the protein Mic60, in a central role in regulating PD-relevant mitochondrial processes. Here, we will review the role of Mic60 in mitochondrial function and will review evidence for a role for Mic60 in PD neurodegeneration and as a potential therapeutic target in PD.

#### MITOCHONDRIAL DYSFUNCTION, MITOCHONDRIAL DYNAMICS, AND OXIDATIVE STRESS IN PD