Genome wide association studies have identified multiple loci that mediate the risk of developing late-onset Alzheimer’s Disease (LOAD). The gene WW-domain containing oxidoreductase ( WWOX ) has been identified in recent LOAD risk meta-analyses, yet its function in the brain is poorly understood. Using Drosophila, we discovered that knockdown of the highly conserved Wwox gene impacts longevity and sleep, having roles in both neuronal and glial subtypes. In an amyloid beta 42 (Aβ42) transgenic model of AD, RNAi-mediated knockdown of Wwox significantly decreased both lifespan and locomotion whilst elevating soluble Aβ42. Transcriptomic and metabolomic analyses revealed that these effects were accompanied by elevated lactate dehydrogenase ( Ldh ) mRNA and lactate levels, downstream of an increase in the key unfolded protein response protein Atf4. Strikingly, we found that upregulation of Wwox in the Aβ42 model through CRISPR activation significantly reduced amyloid load, improved longevity and locomotion. Multi-omics analysis revealed Wwox upregulation partially reversed several key Aβ42-induced transcriptional pathways in the brain and reduced levels of L-methionine and associated enzymes. These findings support a role for reduced WWOX levels in the genetic risk of developing LOAD via pyruvate metabolism and point towards WWOX activation as a protective therapeutic strategy. ### Competing Interest Statement The authors have declared no competing interest. Data analysis code is available at . Raw reads and feature counts for the transcriptomics experiment can be accessed on Array Express, for both the knockdown (E-MTAB-14948) and overexpression (E-MTAB-14949) experiments. The metabolomics data have been deposited to MetaboLights ([60][1]) repository with the study identifier MTBLS12344. Medical Research Council, https://ror.org/03x94j517, MC\_PC\_16030/1, MC\_PC\_16030/2 Wellcome Trust, 108891/B/15/Z Leverhulme Trust, RPG-2020-369 Alzheimer’s Research UK, ARUK-IRG2019B-003 Biotechnology and Biological Sciences Research Council, BB/W000865/1 [1]: #ref-60

Acyl-CoA binding domain-containing proteins (ACBDs) perform diverse but often uncharacterised functions linked to cellular lipid metabolism. Human ACBD4 and ACBD5 are closely related peroxisomal membrane proteins, involved in tethering of peroxisomes to the ER and capturing fatty acids for peroxisomal β-oxidation. ACBD5 deficiency causes neurological abnormalities including ataxia and white matter disease. Peroxisome-ER contacts depend on an ACBD4/5-FFAT motif, which interacts with ER-resident VAP proteins. As ACBD4/5-like proteins are present in most fungi and all animals, we combined phylogenetic analyses with experimental approaches to improve understanding of their evolution and functions. Notably, all vertebrates exhibit gene sequences for both ACBD4 and ACBD5, while invertebrates and fungi possess only a single ACBD4/5-like protein. Our analyses revealed alterations in domain structure and FFAT sequences, which help understanding functional diversification of ACBD4/5-like proteins. We show that the Drosophila melanogaster ACBD4/5-like protein possesses a functional FFAT motif to tether peroxisomes to the ER via Dm_Vap33. Depletion of Dm_Acbd4/5 caused peroxisome redistribution in wing neurons and reduced life expectancy. In contrast, the ACBD4/5-like protein of the filamentous fungus Ustilago maydis lacks a FFAT motif and does not interact with Um_Vap33. Loss of Um_Acbd4/5 resulted in an accumulation of peroxisomes and early endosomes at the hyphal tip. Moreover, lipid droplet numbers increased and mitochondrial membrane potential declined, implying altered lipid homeostasis. Our findings reveal differences between tethering and metabolic functions of ACBD4/5-like proteins across evolution, improving our understanding of ACBD4/5 function in health and disease. The need for a unifying nomenclature for ACBD proteins is discussed.

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.

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.

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.

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.

Significance Mitochondrial dysfunction has been associated with many age-dependent neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease, yet understanding how a neuron maintains a pool of functional mitochondria in sufficient density and location throughout axons in vivo still remains enigmatic. Through an unbiased in vivo forward genetic screen, we identified an endosomal sorting complexes required for transport component, TSG101, as a modulator of mitochondrial number and size in neurons and provide evidence to place TSG101 as a negative regulator of mitochondrial biogenesis. We further find that TSG101 does not contribute to Parkin/Pink1-mediated mitophagy and discovered that macroautophagy regulators ATG1 and ATG6 are dispensable in axonal mitochondrial number regulation. Through in vivo screening, it is possible to unravel translatable mechanisms of axonal mitochondrial maintenance.

Key points A moderate increase in (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. AbstractCx26 hemichannels open in response to moderate elevations of CO2 ( 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 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 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.

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

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 deficientDrosophila melanogasterwere investigated for mitochondrial phenotypesin vitroandin vivo. We find that a loss of function allele or RNAi knockdown of theDrosophilaKMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably,cinnabargenetically interacts with the Parkinson’s disease associated genesPink1andparkin, as well as the mitochondrial fission geneDrp1, 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.

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

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.

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.

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 alpha 7 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.

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.

Background Circadian rhythms are daily rhythms that control every aspect of our behaviour and physiology in a 24 h fashion. They are common to nearly all living organisms from cyanobacteria to plants and from flies to humans. The underlying molecular mechanisms that control circadian rhythms are well understood and flies are an excellent model to study them as their clock is very similar to ours. Additionally we have high-throughput techniques to analyse the fly’s locomotor activity rhythms, which are equivalent to our sleep/wake cycles. Circadian rhythms are also very sensitive physiological markers of wellbeing and can be easily disturbed by life style changes such as inter continental travel or shift work. Circadian problems can also be the direct or indirect result of brain pathologies as in the case with Huntington’s disease (HD). Aims We are characterising a new fly model of HD that is inducible, tuneable and cell type specific. Our aim is to understand early cellular and physiological processes that lead to neuronal dysfunction by using the circadian clock as a readout. Our work may shed light on several under explored areas. For instance: Is HD pathology directly caused by progressive accumulation of mutated huntingtin (HTT) protein over time or by the ageing cell not being able to cope with the mutant protein? Can we reverse HD pathology by turning off the mutated gene? If so, when is the critical point of no return? Methods Using the molecular toolkit available for flies, we can over-express mutant human HTT protein in the brain targeting the clock neurons. We can then analyse rhythms of locomotor activity, looking for anomalies in period, phase or amount of locomotion. Turning the mutant HTT gene on or off throughout the life span of the fly we can probe the effects of ageing on rhythmic phenotypes. Results Here we show that over expression of mutant human HTT in clock neurons causes arrhythmic behaviour in flies and that this effect is modulated by ageing. Conclusions Using circadian rhythms as a tool and fruit flies as a model we have a powerful experimental system to study the pathology caused by the mutated HD protein. In the future, we will focus on the cellular mechanisms leading to neuronal dysfunction/ neurodegeneration in HD and define the critical point in the progression of the disease before which intervention should be made.