COPI-regulated mitochondria-ER contact site formation maintains axonal integrity.

Coat protein complex I (COPI) is best known for its role in Golgi-endoplasmic reticulum (ER) trafficking, responsible for the retrograde transport of ER-resident proteins. The ER is crucial to neuronal function, regulating Ca2+ homeostasis and the distribution and function of other organelles such as endosomes, peroxisomes, and mitochondria via functional contact sites. Here we demonstrate that disruption of COPI results in mitochondrial dysfunction in Drosophila axons and human cells. The ER network is also disrupted, and the neurons undergo rapid degeneration. We demonstrate that mitochondria-ER contact sites (MERCS) are decreased in COPI-deficient axons, leading to Ca2+ dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca2+ uptake but also ER morphology, dramatically delaying neurodegeneration. This work demonstrates an important role for COPI-mediated trafficking in MERC formation, which is an essential process for maintaining axonal integrity.

Live Imaging of Mitochondria in the Intact Fly Wing.

Detailed mechanisms governing the transport of mitochondria in neurons have recently emerged, although it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. Here, we provide a protocol to study the intracellular dynamics of mitochondria in the wing neurons of adult Drosophila in situ. The mounting and imaging procedures that we describe are suitable for use on most microscopes, and they can be easily implemented in any laboratory. Our noninvasive mounting procedures, combined with the translucency of the wing cuticle in adult animals, makes the wing nervous system accessible to advanced microscopy studies in a physiological environment. Combining the powerful genetics of Drosophila with time-lapse live imaging, users of this protocol will be able to analyze mitochondrial dynamics over time in a subset of sensory neurons in the wing. These cells extend long axons with a stereotypical plus-end-out microtubule orientation that represents a unique model to understand the logic of neuronal cargo transport, including the mitochondria. Finally, the neurons in this tissue respond to mechanical and chemical stimulation of the sensory organs of the wing, opening up the possibility of coupling the study of mitochondrial dynamics with the modulation of neuronal activity in aging Drosophila We anticipate that the unique characteristics of this in vivo system will contribute to the discovery of novel mechanisms that regulate mitochondrial dynamics within an organismal context with relevant implications for the pathogenesis of age-dependent neurological disorders.

Clonal Imaging of Mitochondria in the Dissected Fly Wing.

Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis, and mitophagy. Here, we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Most mitochondrial analysis of axons is currently performed in vitro with neurons in a developmental state. Therefore, an understanding of the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often not developed. By using a clonal system paired with fluorescent genetically encoded markers in the Drosophila wing, we can visualize individual neurons (out of the whole bundle), including their long axons and the mitochondria that they contain, using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondria. Mitochondrial morphology and health in axons are tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopathy.

Analysis of Mitochondrial Dynamics in Adult Drosophila Axons.

Neuronal survival depends on the generation of ATP from an ever-changing mitochondrial network. This requires a fine balance between the constant degradation of damaged mitochondria, biogenesis of new mitochondria, movement along microtubules, dynamic processes, and adequate functional capacity to meet firing demands. The distribution of mitochondria needs to be tightly controlled throughout the entire neuron, including its projections. Axons in particular can be enormous structures compared to the size of the cell soma, and how mitochondria are maintained in these compartments is poorly defined. Mitochondrial dysfunction in neurons is associated with aging and neurodegenerative diseases, with the axon being preferentially vulnerable to destruction. Drosophila offer a unique way to study these organelles in fully differentiated adult neurons in vivo. Here, we briefly review the regulation of neuronal mitochondria in health, aging, and disease and introduce two methodological approaches to study mitochondrial dynamics and transport in axons using the Drosophila wing system.

TSG101 negatively regulates mitochondrial biogenesis in axons.

There is a tight association between mitochondrial dysfunction and neurodegenerative diseases and axons that are particularly vulnerable to degeneration, but how mitochondria are maintained in axons to support their physiology remains poorly defined. In an in vivo forward genetic screen for mutants altering axonal mitochondria, we identified tsg101 Neurons mutant for tsg101 exhibited an increase in mitochondrial number and decrease in mitochondrial size. TSG101 is best known as a component of the endosomal sorting complexes required for transport (ESCRT) complexes; however, loss of most other ESCRT components did not affect mitochondrial numbers or size, suggesting TSG101 regulates mitochondrial biology in a noncanonical, ESCRT-independent manner. The TSG101-mutant phenotype was not caused by lack of mitophagy, and we found that autophagy blockade was detrimental only to the mitochondria in the cell bodies, arguing mitophagy and autophagy are dispensable for the regulation of mitochondria number in axons. Interestingly, TSG101 mitochondrial phenotypes were instead caused by activation of PGC-1ɑ/Nrf2-dependent mitochondrial biogenesis, which was mTOR independent and TFEB dependent and required the mitochondrial fission-fusion machinery. Our work identifies a role for TSG101 in inhibiting mitochondrial biogenesis, which is essential for the maintenance of mitochondrial numbers and sizes, in the axonal compartment.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

KEY POINTS: A moderate increase in PCO2 (55 mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. ABSTRACT: Cx26 hemichannels open in response to moderate elevations of CO2 ( PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30 mm propionate, we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics.

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson's disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.

Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Autophagic and endo-lysosomal dysfunction in neurodegenerative disease.

Due to their post-mitotic state, metabolic demands and often large polarised morphology, the function and survival of neurons is dependent on an efficient cellular waste clearance system both for generation of materials for metabolic processes and removal of toxic components. It is not surprising therefore that deficits in protein clearance can tip the balance between neuronal health and death. Here we discuss how autophagy and lysosome-mediated degradation pathways are disrupted in several neurological disorders. Both genetic and cell biological evidence show the diversity and complexity of vesicular clearance dysregulation in cells, and together may ultimately suggest a unified mechanism for neuronal demise in degenerative conditions. Causative and risk-associated mutations in Alzheimer's disease, Frontotemporal Dementia, Amyotrophic Lateral Sclerosis, Parkinson's disease, Huntington's disease and others have given the field a unique mechanistic insight into protein clearance processes in neurons. Through their broad implication in neurodegenerative diseases, molecules involved in these genetic pathways, in particular those involved in autophagy, are emerging as appealing therapeutic targets for intervention in neurodegeneration.

The Parkinson's Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration.

Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson's disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an "upstream" oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis.

Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

The original version of this Article incorrectly gave a publication date of 8 October 2018; this should have been 28 September 2018. This has now been corrected in the PDF and HTML versions of the Article.

Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). Here, we show that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, we identify Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration.

Adaptive and Behavioral Changes in Kynurenine 3-Monooxygenase Knockout Mice: Relevance to Psychotic Disorders.

BACKGROUND: Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. METHODS: In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo-/-) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. RESULTS: Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo-/- mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo-/- mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo-/- mice showed potentiated horizontal activity in the open field paradigm. CONCLUSIONS: Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders.

The kynurenine pathway and neurodegenerative disease.

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been closely linked to the pathogenesis of several neurodegenerative diseases. Tryptophan is an essential amino acid required for protein synthesis, and in higher eukaryotes is also converted into the key neurotransmitters serotonin and tryptamine. However, in mammals >95% of tryptophan is metabolized through the KP, ultimately leading to the production of nicotinamide adenosine dinucleotide (NAD(+)). A number of the pathway metabolites are neuroactive; e.g. can modulate activity of several glutamate receptors and generate/scavenge free radicals. Imbalances in absolute and relative levels of KP metabolites have been strongly associated with neurodegenerative disorders including Huntington's, Alzheimer's, and Parkinson's diseases. The KP has also been implicated in the pathogenesis of other brain disorders (e.g. schizophrenia, bipolar disorder), as well as several cancers and autoimmune disorders such as HIV. Pharmacological and genetic manipulation of the KP has been shown to ameliorate neurodegenerative phenotypes in a number of model organisms, suggesting that it could prove to be a viable target for the treatment of such diseases. Here, we provide an overview of the KP, its role in neurodegeneration and the current strategies for therapeutic targeting of the pathway.