Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila.

Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, we have developed an optogenetic loss of function "Split-Miro" allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function.

Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons.

Defective transport of mitochondria in axons is implicated in the pathogenesis of several age-associated neurodegenerative diseases. However, the regulation and function of axonal mitochondrial motility during normal ageing is poorly understood. Here, we use novel imaging procedures to characterise axonal transport of these organelles in the adult Drosophila wing nerve. During early adult life there is a boost and progressive decline in the proportion of mitochondria that are motile, which is not due to general changes in cargo transport. Experimental inhibition of the mitochondrial transport machinery specifically in adulthood accelerates the appearance of focal protein accumulations in ageing axons, which is suggestive of defects in protein homeostasis. Unexpectedly, lowering levels of Lissencephaly-1 (Lis1), a dynein motor co-factor, augments axonal mitochondrial transport in ageing wing neurons. Lis1 mutations suppress focal protein accumulations in ageing neurons, including those caused by interfering with the mitochondrial transport machinery. Our data provide new insights into the dynamics of mitochondrial motility in adult neurons in vivo, identify Lis1 as a negative regulator of transport of these organelles, and provide evidence of a link between mitochondrial movement and neuronal protein homeostasis.

Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or Aß production.

Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-ß peptide (Aß) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aß production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aß in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aß production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease.

Calsyntenin-1 mediates axonal transport of the amyloid precursor protein and regulates Aß production.

Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-ß (Aß) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aß. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aß levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aß production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aß. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease.

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria.

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca(2+)]c); elevated [Ca(2+)]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca(2+)]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca(2+)]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca(2+) insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca(2+) homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

Phosphorylation of kinesin light chain 1 at serine 460 modulates binding and trafficking of calsyntenin-1.

Kinesin light chain 1 (KLC1) binds to the intracellular cytoplasmic domain of the type-1 membrane-spanning protein calsyntenin-1 (also known as alcadein-α) to mediate transport of a subset of vesicles. Here, we identify serine 460 in KLC1 (KLC1ser460) as a phosphorylation site and show that mutation of KLC1ser460 influences the binding of KLC1 to calsyntenin-1. Mutation of KLC1ser460 to an alanine residue, to preclude phosphorylation, increased the binding of calsyntenin-1, whereas mutation to an aspartate residue, to mimic permanent phosphorylation, reduced the binding. Mutation of KLC1ser460 did not affect the interaction of KLC1 with four other known binding partners: huntingtin-associated protein 1 isoform A (HAP1A), collapsin response mediator protein-2 (CRMP2), c-Jun N-terminal kinase-interacting protein-1 (JIP1) and kinase-D-interacting substrate of 220 kDa (Kidins220). KLC1ser460 is a predicted mitogen-activated protein kinase (MAPK) target site, and we show that extracellular-signal-regulated kinase (ERK) phosphorylates this residue in vitro. We also demonstrate that inhibition of ERK promotes binding of calsyntenin-1 to KLC1. Finally, we show that expression of the KLC1ser460 mutant proteins influences calsyntenin-1 distribution and transport in cultured cells. Thus, phosphorylation of KLC1ser460 represents a mechanism for selectively regulating the binding and trafficking of calsyntenin-1.

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.

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression.

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

Cdk5/p35 phosphorylates lemur tyrosine kinase-2 to regulate protein phosphatase-1C phosphorylation and activity.

Cyclin-dependent kinase-5 (cdk5)/p35 and protein phosphatase-1 (PP1) are two major enzymes that control a variety of physiological processes within the nervous system including neuronal differentiation, synaptic plasticity and axonal transport. Defective cdk5/p35 and PP1 function are also implicated in several major human neurodegenerative diseases. Cdk5/p35 and the catalytic subunit of PP1 (PP1C) both bind to the brain-enriched, serine-threonine kinase lemur tyrosine kinase-2 (LMTK2). Moreover, LMTK2 phosphorylates PP1C on threonine-320 (PP1Cthr³²°) to inhibit its activity. Here, we demonstrate that LMTK2 is phosphorylated on serine-1418 (LMTK2ser¹4¹8) by cdk5/p35 and present evidence that this regulates its ability to phosphorylate PP1Cthr³²°. We thus describe a new signalling pathway within the nervous system that links cdk5/p35 with PP1C and which has implications for a number of neuronal functions and neuronal dysfunction.

High-Resolution Imaging of Mitochondria and Mitochondrial Nucleoids in Differentiated SH-SY5Y Cells.

Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.

Detailed Imaging of Mitochondrial Transport and Precise Manipulation of Mitochondrial Function with Genetically Encoded Photosensitizers in Adult Drosophila Neurons.

Precise distribution of mitochondria is essential for maintaining neuronal homeostasis. Although detailed mechanisms governing the transport of mitochondria have emerged, it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. How alteration in mitochondrial functionality may trigger changes in organellar dynamics also remains unclear in this context. Therefore, the use of genetically encoded tools to perturb mitochondrial functionality in real time would be desirable. Here we describe methods to interfere with mitochondrial function with high spatiotemporal precision with the use of photosensitizers in vivo in the intact wing nerve of adult Drosophila. We also provide details on how to visualize the transport of mitochondria and to improve the quality of the imaging to attain super-resolution in this tissue.

Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aß42 toxicity.

Mitochondria-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid ß42 (Aß42). Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

Riluzole protects against glutamate-induced slowing of neurofilament axonal transport.

Riluzole is the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS) but its precise mode of action is not properly understood. Damage to axonal transport of neurofilaments is believed to be part of the pathogenic mechanism in ALS and this has been linked to defective glutamate handling and increased phosphorylation of neurofilament side-arm domains. Here, we show that riluzole protects against glutamate-induced slowing of neurofilament transport. Protection is associated with decreased neurofilament side-arm phosphorylation and inhibition of the activities of two neurofilament kinases, ERK and p38 that are activated in ALS. Thus, the anti-glutamatergic properties of riluzole include protection against glutamate-induced changes to neurofilament phosphorylation and transport.

Temporal Control of Axonal Transport: The Extreme Case of Organismal Ageing.

A fundamental question in cell biology is how cellular components are delivered to their destination with spatial and temporal precision within the crowded cytoplasmic environment. The long processes of neurons represent a significant spatial challenge and make these cells particularly dependent on mechanisms for long-range cytoskeletal transport of proteins, RNA and organelles. Although many studies have substantiated a role for defective transport of axonal cargoes in the pathogenesis of neurodevelopmental and neurodegenerative diseases, remarkably little is known about how transport is regulated throughout ageing. The scale of the challenge posed by ageing is considerable because, in this case, the temporal regulation of transport is ultimately dictated by the length of organismal lifespan, which can extend to days, years or decades. Recent methodological advances to study live axonal transport during ageing in situ have provided new tools to scratch beneath the surface of this complex problem and revealed that age-dependent decline in the transport of mitochondria is a common feature across different neuronal populations of several model organisms. In certain instances, the molecular pathways that affect transport in ageing animals have begun to emerge. However, the functional implications of these observations are still not fully understood. Whether transport decline is a significant determinant of neuronal ageing or a mere consequence of decreased cellular fitness remains an open question. In this review, we discuss the latest developments in axonal trafficking in the ageing nervous system, along with the early studies that inaugurated this new area of research. We explore the possibility that the interplay between mitochondrial function and motility represents a crucial driver of ageing in neurons and put forward the hypothesis that declining axonal transport may be legitimately considered a hallmark of neuronal ageing.