Mitophagy In Neurons Research Articles

Shedding light on mitophagy in neurons: what is the evidence for PINK1/Parkin mitophagy in vivo?

Neurons are highly specialised cells with a large bioenergetic demand, and so require a healthy mitochondrial network to function effectively. This network is compromised in many neurological disorders, in which damaged mitochondria accumulate. Dysfunctional mitochondria can be removed via an organelle-specific autophagic pathway, a process known as mitophagy. The canonical mitophagy pathway is dependent on the actions of PINK1 (PTEN-induced putative kinase 1) and Parkin and has been well studied in immortalised cells and cultured neurons. However, evidence for a role of this mitophagy pathway in the brain is still limited, and studies suggest that there may be important differences in how neurons respond to mitochondrial damage in vitro and in vivo. Here, we first describe the evidence for a functional PINK1/Parkin mitophagy pathway in neurons, and review how this pathway is affected in disease models. We then critically evaluate the literature by comparing findings from in vitro models and more recent in vivo studies in flies and mice. The emerging picture implicates that alternative mitophagy pathways operate in neurons in vivo. New mouse models that employ fluorescent biosensors to monitor mitophagy in vivo will be instrumental to understand the relative role of the different clearance pathways in the brain under physiological and pathological conditions.

Protocols for assessing mitophagy in neuronal cell lines and primary neurons.

Mitochondria are organelles that regulate essential eukaryotic functions including generating energy, sequestering excess calcium, and modulating cell survival. In order for neurons to thrive, mitochondria have to be continuously replenished by maintaining autophagic-lysosomal mediated degradation of mitochondria (mitophagy) and mitochondrial biogenesis. While a plethora of image- and biochemical-based techniques have been developed for measuring autophagy (macroautophagy) in eukaryotic cells, the molecular toolbox for quantifying and assessing mitophagy in neurons continues to evolve. Compared to proliferating cells, quantifying mitophagy in neurons poses a technical challenge given that mitochondria are predominantly present in neurites (axons and dendrites) and are highly dynamic. In this chapter, we provide a brief overview on mitophagy and provide a list of validated fluorescence- and biochemistry-based techniques used for assessing mitophagy in neuronal cells and primary neurons. Secondly, we provide comprehensive guidelines for interpreting steady-state levels of mitophagy and mitophagic flux in neurons using modern fluorescence- and biochemistry-based techniques. Finally, we provide a comprehensive list of common pitfalls to avoid when assessing mitophagy and offer practical solutions to overcome technical issues.

Efficient induction of dopaminergic neuron differentiation from induced pluripotent stem cells reveals impaired mitophagy in PARK2 neurons

Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184high/CD44- fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.

OPA1 haploinsufficiency induces a BNIP3-dependent decrease in mitophagy in neurons: relevance to Dominant Optic Atrophy.

Dominant optic atrophy (DOA) is because of mutations in the mitochondrial protein OPA1. The disease principally affects retinal ganglion cells, whose axons degenerate leading to vision impairments, and sometimes other neuronal phenotypes. The exact mechanisms underlying DOA pathogenesis are not known. We previously demonstrated that the main role of OPA1, as a mitochondrial fusogenic and anti-apoptotic protein, are inhibited by interaction with the stress inducible pro-apoptotic BNIP3 protein. Because BNIP3 was recently reported to participate in autophagy and mitophagy, we tested the involvement of these processes in DOA pathogenesis. Using an invitro neuronal model of DOA, we identified a BNIP3 down-regulation that reduced autophagy and mitophagy. Restoring BNIP3 had a biphasic effect, first rescuing autophagy and mitophagy levels but later leading to cell death. Similarly, in an invivo mouse model of DOA, we showed that BNIP3 levels are decreased in young adult mice and increase to normal levels upon aging, paralleling disease progression. Altogether, our results indicate that the relationship between OPA1 and BNIP3 may have important bearings on DOA pathogenesis.

Bioenergetic failure, mitochondrial dysfunction and mitophagy.

Mitophagy specifically describes autophagy of damaged or dysfunctional mitochondria and occurs in programmed cell death when mitochondria fragment and remodel their cristae. Cellular bioenergetics is entwined with mitochondrial dynamics, and mitochondrial insults, including depolarization and inhibition of electron transport chain, trigger mitochondrial fragmentation. Here we investigated mitophagy in neurons during manipulation of mitochondrial bioenergetics. Dysfunction of mitochondria was induced by pharmacological inhibition of respiratory chain complexes I-V (rotenone, 3-nitropropionic acid, antimycin A, KCN & oligomycin, respectively) in primary cultures of cerebellar granule cells. The extent of bioenergetic failure was determined by measuring [ATP], depolarization of mitochondrial membrane potential and decrease in oxygen consumption rate in the Seahorse XF24. All stressors produced mitochondrial dysfunction as shown by concentration- and time-dependent decline in [ATP] over 4-24h. Complexes I, III or IV showed rapid loss of mitochondrial membrane potential and decreases in oxygen consumption rate over 4-24h. Autophagolysosomal flux was increased as shown by increased LC3-II and labelling of acidic vacuoles with monodansylcadaverine. Immunocytochemistry for PINK1 showed translocation of PINK1 from cytoplasm to mitochondria after injury, indicating likely involvement of mitophagy during bioenergetic dysfunction. Transfection of fluorescent pH-biosensor Rosella (Rosado CJ et al. Autophagy 4: 205 (2008)) targeting mitochondria indicated the pH of the mitochondrial location dropped with the inhibition of complex I and II, implying acidification of mitochondria, presumably in acidic vesicles undergoing mitophagy. Dieback of neuronal arbor observed here paralleled that seen with a GFP-plasmid. Together these data indicate that bioenergetic dysfunction produces mitophagy in primary neurons and is likely to be involved in neuronal dynamics.

Parkin and its Role in Skeletal Muscle Function

Autophagy is a major intracellular degradation system involving the delivery of long-lived cellular constituents to lysosomes for degradation. Under basal conditions, autophagy assists in the maintenance of cellular homeostasis and is elevated when cellular energetic demands are increased. When mitochondria are dysfunctional and unable to assist in sustaining cellular requirements, mitophagy is activated to selectively degrade damaged mitochondria. Parkin has been implicated in neuronal mitophagy, but little is known about its function in muscle. Young (3 months) Parkin deficient (KO) mice were compared to wild-type (WT) animals to examine how endogenous Parkin levels mediate mitophagy and mitochondrial content in mammalian muscle. Our analysis revealed that Parkin KO mice exhibited no muscle mass differences in their quadriceps and tibialis anterior when compared to WT mice. This was accompanied by no differences in mitochondrial yield and whole muscle cytochrome c oxidase activity. When mitochondrial function was assessed, reactive oxygen species (ROS) production was similar in isolated mitochondria from the hindlimb muscles of WT and Parkin KO mice. However, Parkin KO mice exhibited a 30% reduction in State 3 (active) mitochondrial respiration. Furthermore, mitochondrial localization of ubiquitin and autophagy marker light chain 3 (LC3II) were reduced by 40% and 60%, respectively, in Parkin KO animals. Interestingly, expression of endoplasmic reticulum (ER) stress related protein CHOP was increased by 250% in KO animals. Collectively, these data suggest that mitochondrial content is preserved, mitophagy signaling is downregulated, and the ER-stress response is elevated in skeletal muscle of young animals when Parkin is absent. Supported by NSERC.

NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Long-distance autophagy

Long-distance autophagy

Glutamate excitotoxicity in neurons triggers mitochondrial and endoplasmic reticulum accumulation of Parkin, and, in the presence of N-acetyl cysteine, mitophagy

Disruption of the dynamic properties of mitochondria (fission, fusion, transport, degradation, and biogenesis) has been implicated in the pathogenesis of neurodegenerative disorders, including Parkinson's disease (PD). Parkin, the product of gene PARK2 whose mutation causes familial PD, has been linked to mitochondrial quality control via its role in regulating mitochondrial dynamics, including mitochondrial degradation via mitophagy. Models using mitochondrial stressors in numerous cell types have elucidated a PINK1-dependent pathway whereby Parkin accumulates on damaged mitochondria and targets them for mitophagy. However, the role Parkin plays in regulating mitochondrial homeostasis specifically in neurons has been less clear. We examined whether a stressor linked to neurodegeneration, glutamate excitotoxicity, elicits Parkin–mitochondrial translocation and mitophagy in neurons. We found that brief, acute exposure to glutamate causes Parkin translocation to mitochondria in neurons, in a calcium- and N-methyl-d-aspartate (NMDA) receptor-dependent manner. In addition, we found that Parkin accumulates on endoplasmic reticulum (ER) and mitochondrial/ER junctions following excitotoxicity, supporting a role for Parkin in mitochondrial–ER crosstalk in mitochondrial homeostasis. Despite significant Parkin–mitochondria translocation, however, we did not observe mitophagy under these conditions. To further investigate, we examined the role of glutamate-induced oxidative stress in Parkin–mitochondria accumulation. Unexpectedly, we found that glutamate-induced accumulation of Parkin on mitochondria was promoted by the antioxidant N-acetyl cysteine (NAC), and that co-treatment with NAC facilitated Parkin-associated mitophagy. These results suggest the possibility that mitochondrial depolarization and oxidative damage may have distinct pathways associated with Parkin function in neurons, which may be critical in understanding the role of Parkin in neurodegeneration.

BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke.

A basal level of mitophagy is essential in mitochondrial quality control in physiological conditions, while excessive mitophagy contributes to cell death in a number of diseases including ischemic stroke. Signals regulating this process remain unknown. BNIP3, a pro-apoptotic BH3-only protein, has been implicated as a regulator of mitophagy. Both in vivo and in vitro models of stroke, as well as BNIP3 wild-type and knock out mice were used in this study. We show that BNIP3 and its homologue BNIP3L (NIX) are highly expressed in a "delayed" manner and contribute to delayed neuronal loss following stroke. Deficiency in BNIP3 significantly decreases both neuronal mitophagy and apoptosis but increases nonselective autophagy following ischemic/hypoxic insults. The mitochondria-localized BNIP3 interacts with the autophagosome-localized LC3, suggesting that BNIP3, similar to NIX, functions as a LC3-binding receptor on mitochondria. Although NIX expression is upregulated when BNIP3 is silenced, up-regulation of NIX cannot functionally compensate for the loss of BNIP3 in activating excessive mitophagy. NIX primarily regulates basal level of mitophagy in physiological conditions, whereas BNIP3 exclusively activates excessive mitophagy leading to cell death.

Mild hypothermia alleviates excessive autophagy and mitophagy in a rat model of asphyxial cardiac arrest.

Mild hypothermia is an effective therapeutic strategy to improve poor neurological outcomes in patients following cardiac arrest (CA). However, the underlying mechanism remains unclear. The aim of the study was to evaluate the effect of mild hypothermia on intracellular autophagy and mitophagy in hippocampal neurons in a rat model of CA. CA was induced in Sprague-Dawley (SD) rats by asphyxia for 5 min. After successful resuscitation, the surviving rats were randomly divided into two groups, the normothermia (NT) group and the hypothermia (HT) group. Mild hypothermia (32 °C) was induced following CA for 4 h, and animals were rewarmed at a rate of 0.5 °C/h. Neurologic deficit scores (NDS) were used to determine the status of neurological function. Cytoplasmic and mitochondrial protein from the hippocampus was extracted, and the expression of LC3B-II/I and Parkin were measured as markers of intracellular autophagy and mitophagy, respectively. Of the 60 rats that underwent CA, 44 were successfully resuscitated (73 %), and 33 survived until the end of the experiment (55 %). Mild hypothermia maintained eumorphism of nuclear and mitochondrial structures and significantly improved NDS (p < 0.05). Expression of LC3B-II/I and Parkin in hippocampal nerve cells were significantly increased (p < 0.05) in the NT group relative to the control. Meanwhile, mild hypothermia reduced the level of LC3B-II/I and Parkin (p < 0.05) relative to the NT group. Mild hypothermia protected mitochondria and improved neurological function following CA and resuscitation after ischemia/reperfusion (I/R) injury, likely by reducing excessive autophagy and mitophagy in neurons.

Role of protein phosphatase 2A in modulating autophagy and mitophagy (LB220)

Protein phosphatase 2A (PP2A) is a holoenzyme composed of a scaffolding/catalytic dimer that binds to a regulatory (B) subunit which dictates subcellular targeting and substrate specificity of PP2A. The PP2A holoenzymes that modulate autophagy and mitophagy in neurons are not known to date. Bβ2 is a regulatory subunit that redirects PP2A from the cytosol to the mitochondrion to induce mitochondrial fission and apoptosis of neurons. Given that mitochondrial fission precedes mitophagy, we surmised that Bβ2 regulates mitophagy in neurons. To address this hypothesis, we used a combination of cell imaging and biochemical approaches to quantify autophagy and mitophagy in SH‐SY5Y cells transiently expressing the cytosolic regulatory subunit Bβ1 and the mitochondrial regulatory subunits Bβ2 and B56α. Western blot analyses showed that all three PP2A holoenzymes decrease the levels of the autophagosome marker microtubule‐associated protein 1 light chain 3 (LC3‐II) and prevent further increases in LC3‐II as induced by bafilomycin, a drug used to measure autophagic flux. Although all PP2A holoenzymes decrease the number of GFP tagged LC3 (GFP‐LC3) puncta per cell, confocal analyses show that Bβ2 increases the colocalization GFP‐LC3 puncta with mitochondria. Similar results were observed in COS‐7 cells. Our results suggest that PP2A decreases autophagic flux while PP2A/ Bβ2 promotes mitophagy.Grant Funding Source: P20 GM103554

Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death

Abnormal autophagy is frequently observed during dopaminergic neurodegeneration in Parkinson's disease (PD). However, it is not yet firmly established whether active autophagy is beneficial or pathogenic with respect to dopaminergic cell loss. Staurosporine, a common inducer of apoptosis, is often used in mechanistic studies of dopaminergic cell death. Here we report that staurosporine activates both autophagy and mitophagy simultaneously during dopaminergic neuronal cell death, and evaluate the physiological significance of these processes during cell death. First, staurosporine treatment resulted in induction of autophagy in more than 75% of apoptotic cells. Pharmacological inhibition of autophagy by bafilomycin A1 decreased significantly cell viability. In addition, staurosporine treatment resulted in activation of the PINK1–Parkin mitophagy pathway, of which deficit underlies some familial cases of PD, in the dopaminergic neuronal cell line, SN4741. The genetic blockade of this pathway by PINK1 null mutation also dramatically increased staurosporine-induced cell death. Taken together, our data suggest that staurosporine induces both mitophagy and autophagy, and that these pathways exert a significant neuroprotective effect, rather than a contribution to autophagic cell death. This model system may therefore be useful for elucidating the mechanisms underlying crosstalk between autophagy, mitophagy, and cell death in dopaminergic neurons.

Morphological and bioenergetic demands underlying the mitophagy in post-mitotic neurons: the pink–parkin pathway

Evidence suggests a striking causal relationship between changes in quality control of neuronal mitochondria and numerous devastating human neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Contrary to replicating mammalian cells with a metabolism essentially glycolytic, post-mitotic neurons are distinctive owing to (i) their exclusive energetic dependence from mitochondrial metabolism and (ii) their polarized shape, which entails compartmentalized and distinct energetic needs. Here, we review the recent findings on mitochondrial dynamics and mitophagy in differentiated neurons focusing on how the exceptional characteristics of neuronal populations in their morphology and bioenergetics needs make them quite different to other cells in controlling the intracellular turnover of these organelles.

Induction of Neuronal Mitophagy in Acute Spinal Cord Injury in Rats

Autophagy and up-regulation of autophagy-associated proteins microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 have been shown to occur in spinal cord injury (SCI). Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and Nip-like protein X (NIX, also known as BNIP3L) are mitochondrial BH3-only proteins that are implicated in mitophagy. In this study, we show that mitophagy is activated in the injured neurons, and hypoxia-inducible proteins BNIP3, NIX, and p53 are upregulated after SCI. Numerous autophagosomes containing damaged mitochondria (mitophagosomes) were found in the injured neurons 24h after SCI in rats by transmission electron microscopy; mitophagy, therefore, had occurred. Hypoxia-inducible proteins BNIP3, NIX, and p53 were upregulated in spinal cord neurons in both a rat model of SCI and cultured primary spinal neurons exposed to hypoxia. BNIP3 and NIX were transcriptionally regulated mainly by hypoxia-inducible factor-1α as well as p53 in cultured spinal cord neurons. This study provides direct morphological and biochemical evidence for mitophagy in the damaged neural tissue after SCI.

BNIP3 regulates mitophagy and apoptosis in delayed neuronal death in stroke

Physiological level of autophagy promotes neuronal survival, while apoptosis contributes to delayed neuronal death in ischemic stroke. In this study, we show that the proapoptotic BCL‐2 family member BNIP3 is an upstream regulator for both processes. Specifically, deletion of BNIP3 gene is neuroprotective by affecting mitophagy and apoptosis pathways. We performed IHC, western blot, Co‐ip, ELISA, and cell transfection to analyze the BNIP3's regulation on mitophagy and apoptosis in cortical neurons and ischemic brains. Both BNIP3 wild‐type and knock‐out mice were used. In primary neurons exposed to OGD/reperfusion, BNIP3 was highly expressed, with the time course and expression levels of apoptosis‐related proteins (i.e. active caspase‐3, cytochrome C, and BAX) and autophagy‐related proteins (i.e. LC3, Beclin‐1, and LAMP‐2) positively regulated. Promoting or inhibiting autophagy pharmacologically didn't affect the expression patterns of BNIP3, indicating it is an upstream regulator. We also measured the brain damage of neonatal stroke in transgenic mice. TTC staining showed the infarct volume of ischemic brains was significantly reduced in BNIP3 KO mice compared to WT mice upon 3–7 days recovery. Silence of BNIP3 gene in cortical neurons of KO mice activated a robust autophagic response and decreased apoptosis, coordinately contributing to the neuroprotection in the KO animals after stroke.Grant Funding Source: Canadian Institute of Health Research, Canadian Stroke Network, Manitoba Health Research Council, Manitoba Institute of Child Health

Parkin- and PINK1-Dependent Mitophagy in Neurons: Will the Real Pathway Please Stand Up?

Parkinson’s disease (PD) is characterized by massive degeneration of dopaminergic neurons in the substantia nigra. Whereas the majority of PD cases are sporadic, about 5–10% of cases are familial and associated with genetic factors. The loss of parkin or PINK1, two such factors, leads to an early onset form of PD. Importantly, recent studies have shown that parkin functions downstream of PINK1 in a common genetic pathway affecting mitochondrial homeostasis. More precisely, parkin has been shown to mediate the autophagy of damaged mitochondria (mitophagy) in a PINK1-dependent manner. However, much of the work characterizing this pathway has been carried out in immortalized cell lines overexpressing high levels of parkin. In contrast, whether or how endogenous parkin and PINK1 contribute to mitophagy in neurons is much less clear. Here we review recent work addressing the role of parkin/PINK1-dependent mitophagy in neurons. Clearly, it appears that mitophagy pathways differ spatially and kinetically in neurons and immortalized cells, and therefore might diverge in their ultimate outcome and function. While evidence suggests that parkin can translocate to mitochondria in neurons, the function and mechanism of mitophagy downstream of parkin recruitment in neurons remains to be clarified. Moreover, it is noteworthy that most work has focused on the downstream signaling events in parkin/PINK1 mitophagy, whereas the upstream signaling pathways remain comparatively poorly characterized. Identifying the upstream signaling mechanisms that trigger parkin/PINK1 mitophagy will help to explain the nature of the insults affecting mitochondrial function in PD, and a better understanding of these pathways in neurons will be the key in identifying new therapeutic targets in PD.

The Cell and Molecular Biology of Neurodegenerative Diseases: An Overview

The Cell and Molecular Biology of Neurodegenerative Diseases: An Overview

Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live Cortical Neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.

Bioenergetics of neurons inhibit the translocation response of Parkin following rapid mitochondrial depolarization

Recent studies delineate a pathway involving familial Parkinson's disease (PD)-related proteins PINK1 and Parkin, in which PINK1-dependent mitochondrial accumulation of Parkin targets depolarized mitochondria towards degradation through mitophagy. The pathway has been primarily characterized in cells less dependent on mitochondria for energy production than neurons. Here we report that in neurons, unlike other cells, mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone did not induce Parkin translocation to mitochondria or mitophagy. PINK1 overexpression increased basal Parkin accumulation on neuronal mitochondria, but did not sensitize them to depolarization-induced Parkin translocation. Our data suggest that bioenergetic differences between neurons and cultured cell lines contribute to these different responses. In HeLa cells utilizing usual glycolytic metabolism, mitochondrial depolarization robustly triggered Parkin-mitochondrial translocation, but this did not occur in HeLa cells forced into dependence on mitochondrial respiration. Declining ATP levels after mitochondrial depolarization correlated with the absence of induced Parkin-mitochondrial translocation in both HeLa cells and neurons. However, intervention allowing neurons to maintain ATP levels after mitochondrial depolarization only modestly increased Parkin recruitment to mitochondria, without evidence of increased mitophagy. These data suggest that changes in ATP levels are not the sole determinant of the different responses between neurons and other cell types, and imply that additional mechanisms regulate mitophagy in neurons. Since the Parkin-mitophagy pathway is heavily dependent on bioenergetic status, the unique metabolic properties of neurons likely influence the function of this pathway in the pathogenesis of PD.