Both mitochondria and lysosomes are essential for maintaining cellular homeostasis, and dysfunction of both organelles has been observed in multiple diseases1–4. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network, which drives cellular metabolism 5. Lysosomes similarly undergo constant dynamic regulation by the RAB7 GTPase 1, which cycles from an active GTP-bound state into an inactive GDP-bound state upon GTP hydrolysis. Here we have identified the formation and regulation of mitochondria–lysosome membrane contact sites using electron microscopy, structured illumination microscopy and high spatial and temporal resolution confocal live cell imaging. Mitochondria–lysosome contacts formed dynamically in healthy untreated cells and were distinct from damaged mitochondria that were targeted into lysosomes for degradation 6,7. Contact formation was promoted by active GTP-bound lysosomal RAB7, and contact untethering was mediated by recruitment of the RAB7 GTPase-activating protein TBC1D15 to mitochondria by FIS1 to drive RAB7 GTP hydrolysis and thereby release contacts. Functionally, lysosomal contacts mark sites of mitochondrial fission, allowing regulation of mitochondrial networks by lysosomes, whereas conversely, mitochondrial contacts regulate lysosomal RAB7 hydrolysis via TBC1D15. Mitochondria–lysosome contacts thus allow bidirectional regulation of mitochondrial and lysosomal dynamics, and may explain the dysfunction observed in both organelles in various human diseases.

Mitochondrial and lysosomal dysfunction have been implicated in substantia nigra dopaminergic neurodegeneration in Parkinson’s disease (PD), but how these pathways are linked in human neurons remains unclear. Here we studied dopaminergic neurons derived from patients with idiopathic and familial PD. We identified a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidized dopamine accumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity, lysosomal dysfunction, and α-synuclein accumulation. This toxic cascade was observed in human, but not in mouse, PD neurons at least in part because of species-specific differences in dopamine metabolism. Increasing dopamine synthesis or α-synuclein amounts in mouse midbrain neurons recapitulated pathological phenotypes observed in human neurons. Thus, dopamine oxidation represents an important link between mitochondrial and lysosomal dysfunction in PD pathogenesis.

Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for amyotrophic lateral sclerosis (ALS) and glaucoma, diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species. Parkin's E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with ubiquitinated mitochondria via its ubiquitin binding domain; in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein double FYVE-containing protein 1 (DFCP1) transiently localizes to damaged mitochondria to initialize autophagosome formation and the recruitment of microtubule-associated protein light chain 3 (LC3). Optineurin then induces autophagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria and inhibits mitochondrial degradation. These defects are rescued by expression of siRNA-resistant wild-type optineurin, but not by an ALS-associated mutant in the ubiquitin binding domain (E478G), or by optineurin with a mutation in the LIR domain. Optineurin and p62/SQSTM1 are independently recruited to separate domains on damaged mitochondria, and p62 is not required for the recruitment of either optineurin or LC3 to damaged mitochondria. Thus, our study establishes an important role for optineurin as an autophagy receptor in parkin-mediated mitophagy and demonstrates that defects in a single pathway can lead to neurodegenerative diseases with distinct pathologies.amaged mitochondria are selectively turned over in eukaryotic cells via mitophagy, a process in which double-membraned autophagosomes sequester and ultimately degrade mitochondria via lysosomal fusion (1, 2). This process is regulated by phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1) and parkin, two proteins linked to hereditary forms of Parkinson's disease (3, 4). PINK1 is stabilized on the outer membrane of damaged mitochondria and recruits the E3 ubiquitin ligase parkin, which ubiquitinates proteins on the outer mitochondrial membrane (OMM) (5-13). Parkin-mediated ubiquitination of damaged mitochondria is followed by autophagosome formation and engulfment of mitochondria (1, 2). However, the proteins involved in dynamically recruiting autophagic machinery to ubiquitinated damaged mitochondria still remain elusive.Optineurin is an autophagy receptor, characterized by its ability to bind ubiquitin via its ubiquitin binding in ABIN (A20 binding and inhibitor of NF-κB) and NEMO (NF-κB essential modulator) (UBAN) domain (14) and the autophagosome-associated protein LC3 (microtub...

Alterations in α-synuclein dosage lead to familial Parkinson's disease (PD), and its accumulation results in synucleinopathies that include PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Furthermore, α-synuclein contributes to the fibrilization of amyloid-b and tau, two key proteins in Alzheimer's disease, which suggests a central role for α-synuclein toxicity in neurodegeneration. Recent studies of factors contributing to α-synuclein toxicity and its disruption of downstream cellular pathways have expanded our understanding of disease pathogenesis in synucleinopathies. In this Review, we discuss these emerging themes, including the contributions of aging, selective vulnerability and non-cell-autonomous factors such as α-synuclein cell-to-cell propagation and neuroinflammation. Finally, we summarize recent efforts toward the development of targeted therapies for PD and related synucleinopathies.

Autophagy is an essential cellular pathway for degrading defective organelles and aggregated proteins. Defects in autophagy have been implicated in the neurodegenerative disorder Huntington's disease (HD), in which polyglutamine-expanded huntingtin (polyQ-htt) is predominantly cleared by autophagy. In neurons, autophagosomes form constitutively at the axon tip and undergo robust retrograde axonal transport toward the cell body, but the factors regulating autophagosome dynamics and autophagosome maturation are not well understood. Here, we show that both huntingtin (htt) and its adaptor protein huntingtin-associated protein-1 (HAP1) copurify and colocalize with autophagosomes in neurons. We use live-cell imaging and RNAi in primary neurons from GFP-LC3 transgenic mice to show that htt and HAP1 control autophagosome dynamics, regulating dynein and kinesin motors to promote processive transport. Expression of polyQ-htt in either primary neurons or striatal cells from HD knock-in mice is sufficient to disrupt the axonal transport of autophagosomes. Htt is not required for autophagosome formation or cargo loading. However, the defective autophagosome transport observed in both htt-depleted neurons and polyQ-htt-expressing neurons is correlated with inefficient degradation of engulfed mitochondrial fragments. Together, these studies identify htt and HAP1 as regulators of autophagosome transport in neurons and suggest that misregulation of autophagosome transport in HD leads to inefficient autophagosome maturation, potentially due to inhibition of autophagosome/lysosome fusion along the axon. The resulting defective clearance of both polyQ-htt aggregates and dysfunctional mitochondria by neuronal autophagosomes may contribute to neurodegeneration and cell death in HD.

Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis.

Mitochondrial and lysosomal function are intricately related and critical for maintaining cellular homeostasis, as highlighted by multiple diseases linked to dysfunction of both organelles. Recent work using high-resolution microscopy demonstrates the dynamic formation of inter-organelle membrane contact sites between mitochondria and lysosomes, allowing for their direct interaction in a pathway distinct from mitophagy or lysosomal degradation of mitochondrial-derived vesicles. Mitochondria-lysosome contact site tethering is mechanistically regulated by mitochondrial proteins promoting Rab7 GTP hydrolysis, and allows for the bidirectional crosstalk between mitochondria and lysosomes and the regulation of their organelle network dynamics, including mitochondrial fission. In this review, we summarize recent advances in mitochondria-lysosome contact site regulation and function, and discuss their potential roles in cellular homeostasis and various human diseases.

The discovery of genetic forms of Parkinson’s disease (PD) has highlighted the importance of the autophagy/lysosomal and mitochondrial/oxidative stress pathways in disease pathogenesis. However, recently identified PD-linked genes, including DNAJC6 (auxilin), SYNJ1 (synaptojanin 1), and the PD risk gene SH3GL2 (endophilin A1), have also highlighted disruptions in synaptic vesicle endocytosis (SVE) as a significant contributor to disease pathogenesis. Additionally, the roles of other PD genes such as LRRK2, PRKN, and VPS35 in the regulation of SVE are beginning to emerge. Here we discuss the recent work on the contribution of dysfunctional SVE to midbrain dopaminergic neurons’ selective vulnerability and highlight pathways that demonstrate the interplay of synaptic, mitochondrial, and lysosomal dysfunction in the pathogenesis of PD.