To eat or not to eat: neuronal metabolism, mitophagy, and Parkinson's disease

Abstract

Neurons are exquisitely dependent upon mitochondrial respiration to support energy-demanding functions. Mechanisms that regulate mitochondrial quality control have recently taken center stage in Parkinson's disease research, particularly the selective degradation of mitochondria by autophagy (mitophagy). Unlike other cells, neurons show limited glycolytic potential, and both insufficient and excessive mitophagy have been linked to neurodegeneration. Kinases implicated in regulating mammalian mitophagy include extracellular signal-regulated protein kinases (ERK1/2) and PTEN-induced kinase 1 (PINK1). Increased expression of full-length PINK1 enhances recruitment of parkin to chemically depolarized mitochondria, resulting in rapid mitochondrial clearance in transformed cell lines. As parkin and PINK1 mutations cause autosomal recessive parkinsonism, potential defects in clearing dysfunctional mitochondria may contribute to mitochondrial abnormalities in disease. Given the unique features of metabolic regulation in neurons, however, mechanisms regulating mitochondrial network stability and the threshold for mitophagy are likely to vary from cells that preferentially utilize aerobic glycolysis. Moreover, removal of the entire mitochondrial complement may represent part of a neuronal cell death pathway. Future work utilizing physiological injuries that affect only a subset of mitochondria would help to elucidate whether defective recognition of damaged mitochondria, or alternatively, inability to maintain or generate healthy mitochondria, play the major roles in parkinsonian neurodegeneration.

FIG. 1.

Mitophagy depends upon coordinated upregulation of autophagy and specification of mitochondria as cargo. (A) Although it is unclear what triggers autophagy in response to mitochondrial injury, potential mediators known to induce autophagy include reactive oxygen species (ROS), calcium dysregulation, AMP kinase,and ERK1/2. The mitochondrial protein Nix has also been implicated in uncoupling-induced autophagy. These upstream signals engage the core autophagy machinery, which is necessary, but not specific for mitophagy. (B) Current studies are beginning to elucidate mechanisms by which damaged or dysfunctional mitochondria, schematized here by fission and surface alterations, may be selectively targeted for mitophagy. Whether there are also mechanisms to actively exclude functional mitochondria is unknown.

FIG. 2.

Model 1: Cargo enrichment by selective axonal transport. In this model, damaged or dysfunctional mitochondria are recognized through an unknown adapter for retrograde dynein-dependent transport to the soma. Ubiquitinated mitochondria may also undergo p62-mediated aggregation. The increased concentration of damaged mitochondria in the perinuclear region in turn facilitates their relatively selective clearance by autophagy. PINK1, which is stabilized on depolarized mitochondria, has been reported to interact with Miro (55), a transport adapter involved in both anterograde and retrograde transport. Whether this interaction has functional effects on mitochondrial transport, and whether axonal transport regulates mitophagy, remain to be determined.

FIG. 3.

Model 2: Localized cargo recognition via adapter proteins. In analogy to yeast mitophagy or autophagy of ubiquitinated protein aggregates (48), changes on the surface of damaged or dysfunctional mitochondria may be recognized by adapter proteins that link to components of the core autophagy machinery. Parkin-mediated ubiquitination of mitochondria, for example, could lead to recruitment of the ubiquitin-binding protein p62/SQSTM1, which is also capable of binding the N-terminal helical region of LC3 (57). While the roles of p62 and VDAC1 ubiquitination in FCCP/CCCP-induced mitophagy remain unresolved (19, 44), macromolecular components of the mitochondrial permeability transition pore and phospholipid oxidation represent attractive potential “eat-me” signals. The crystal structure of LC3 exhibits positively charged surface patches (61), which could hypothetically interact with phosphorylated or oxidatively modified macromolecules exposed on the mitochondrial surface. Interestingly, a recently described PKA phosphorylation site between the α1 and α2 helices of LC3 (asterisk) regulates induced, but not basal autophagy (5).

FIG. 4.

Metabolic differences between HeLa cells and primary neuron cultures. Parameters of metabolic function were compared between primary cortical neuron cultures from C57BL/6 mice and HeLa cells cultured in glucose-containing media. Dissociated E15 cortical neurons were plated at 5×10⁴/well in a poly-L-lysine coated Seahorse 24-well plate and studied at 7 DIV. The extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) were measured using a Seahorse XF24 Flux Analyzer. Data was normalized to relative protein content by Coomassie dot blot performed at the end of the assay. *p<0.01 vs. primary neurons for both OCR and ECAR.

FIG. 5.

OCR analysis reveals a robust spare respiratory (reserve) capacity in neurons, but not in HeLa cells. Following quantification of basal OCR, oligomycin (1 μM), and then FCCP (300 nM) were added to each well. Mitochondrial-specific OCR was obtained by subtracting the nonmitochondrial OCR (in the presence of antimycin A) from total OCR. *p<0.01 vs. basal primary; ^†p<0.05 vs. basal HeLa., **p<0.001 vs. basal primary & vs. FCCP HeLa.

FIG. 6.

Unlike primary neurons, ATP levels in HeLa cells are not affected by mitochondrial inhibitors. ATP levels were measured 30 min after treatment with the indicated inhibitors using a luminescence-based assay. All data are means±standard deviation. *p<0.01 vs. basal primary.