LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A in vivo.

Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in Caenorhabditis elegans Posterior Lateral Mechanosensory neurons. The synaptic vesicle anterograde kinesin motor UNC-104/KIF1A regulates this vesicle behavior at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behavior at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight toward the asynaptic end. This suggests that the neuron optimizes UNC-104 levels on the cargo surface to maximize the fraction of vesicles entering the branch and minimize the fraction going to the asynaptic end.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Transport of synaptic vesicles is modulated by vesicular reversals and stationary cargo clusters.

Stationary clusters of vesicles are a prominent feature of axonal transport, but little is known about their physiological and functional relevance to axonal transport. Here, we investigated the role of vesicle motility characteristics in modulating the formation and lifetimes of such stationary clusters, and their effect on cargo flow. We developed a simulation model describing key features of axonal cargo transport, benchmarking the model against experiments in the posterior lateral mechanosensory neurons of Caenorhabditis elegans. Our simulations included multiple microtubule tracks and varied cargo motion states, and account for dynamic cargo-cargo interactions. Our model also incorporates static obstacles to vesicle transport in the form of microtubule ends, stalled vesicles and stationary mitochondria. We demonstrate, both in simulations and in an experimental system, that a reduction in reversal rates is associated with a higher proportion of long-lived stationary vesicle clusters and reduced net anterograde transport. Our simulations support the view that stationary clusters function as dynamic reservoirs of cargo vesicles, and reversals aid cargo in navigating obstacles and regulate cargo transport by modulating the proportion of stationary vesicle clusters along the neuronal process.

Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes.

Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin-α to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin-α acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.

Transport-dependent maturation of organelles in neurons.

Some organelles show a spatial gradient of maturation along the neuronal process where more mature organelles are found closer to the cell body. This gradient is set up by progressive maturation steps that are aided by differential organelle distribution as well as transport. Autophagosomes and endosomes mature as they acquire lysosomal membrane proteins and decrease their luminal pH as they are retrogradely transported towards the cell body. The acquisition of lysosomal proteins along the neuronal processes likely occurs through fusion or membrane exchange events with Golgi-derived donor transport carriers that are transported anterogradely from the cell body. The mechanisms by which endosomes and autophagosomes mature might be applicable to other organelles that are transported along neuronal processes. Defects in axonal transport may also contribute to the accumulation of immature organelles in neurons. Such accumulations have been seen in neurons of neurodegenerative models.

Synaptic branch stability is mediated by non-enzymatic functions of MEC-17/αTAT1 and ATAT-2.

Microtubules are fundamental elements of neuronal structure and function. They are dynamic structures formed from protofilament chains of α- and ß-tubulin heterodimers. Acetylation of the lysine 40 (K40) residue of α-tubulin protects microtubules from mechanical stresses by imparting structural elasticity. The enzyme responsible for this acetylation event is MEC-17/αTAT1. Despite its functional importance, however, the consequences of altered MEC-17/αTAT1 levels on neuronal structure and function are incompletely defined. Here we demonstrate that overexpression or loss of MEC-17, or of its functional paralogue ATAT-2, causes a delay in synaptic branch extension, and defective synaptogenesis in the mechanosensory neurons of Caenorhabditis elegans. Strikingly, by adulthood, the synaptic branches in these animals are lost, while the main axon shaft remains mostly intact. We show that MEC-17 and ATAT-2 regulate the stability of the synaptic branches largely independently from their acetyltransferase domains. Genetic analyses reveals novel interactions between both mec-17 and atat-2 with the focal adhesion gene zyx-1/Zyxin, which has previously been implicated in actin remodelling. Together, our results reveal new, acetylation-independent roles for MEC-17 and ATAT-2 in the development and maintenance of neuronal architecture.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

Imaging Intracellular Trafficking in Neurons of C. elegans.

Axonal transport is an essential component of neuronal function. Several neurodegenerative disorders have been associated with defects in cargo transport. Thus, studying axonal transport is important to understand such disorders. Live imaging of fluorescently labeled cargo is a prevailing technique to study properties of axonal transport. C. elegans is both transparent and genetically amenable, making it an excellent model system to study axonal transport. In this chapter, we describe protocols to live image several neuronal cargo in vivo in C. elegans neurons.

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons.

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16's inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.

Tissue-specific targeting of DNA nanodevices in a multicellular living organism.

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs, and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell types in vivo. Here, we show that by exploiting either endogenous or synthetic receptor-ligand interactions and leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in Caenorhabditis elegans, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

Tracking Mitochondrial Density and Positioning along a Growing Neuronal Process in Individual C. elegans Neuron Using a Long-Term Growth and Imaging Microfluidic Device.

The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and subcellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show interanimal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in subcellular events such as organelle positions, we developed a microfluidic device to feed and immobilize Caenorhabditis elegans for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process in vivo Subcellular imaging of an individual neuron in multiple animals, over 36 h in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles (SVs) at synapses. Long-term imaging of individual C. elegans touch receptor neurons (TRNs) shows that the addition of new mitochondria takes place along the entire neuronal process length at a rate of ∼0.6 mitochondria/h. The threshold for the addition of a new mitochondrion occurs when the average separation between the two preexisting mitochondria exceeds 24 µm. Our assay provides a new opportunity to move beyond simple observations obtained from in vitro assays to allow the discovery of genes that regulate positioning of mitochondria in neurons.

Molecular mechanisms governing axonal transport: a C. elegans perspective.

Axonal transport is integral for maintaining neuronal form and function, and defects in axonal transport have been correlated with several neurological diseases, making it a subject of extensive research over the past several years. The anterograde and retrograde transport machineries are crucial for the delivery and distribution of several cytoskeletal elements, growth factors, organelles and other synaptic cargo. Molecular motors and the neuronal cytoskeleton function as effectors for multiple neuronal processes such as axon outgrowth and synapse formation. This review examines the molecular mechanisms governing axonal transport, specifically highlighting the contribution of studies conducted in C. elegans, which has proved to be a tractable model system in which to identify both novel and conserved regulatory mechanisms of axonal transport.

Neuronal control of lipid metabolism by STR-2 G protein-coupled receptor promotes longevity in Caenorhabditis elegans.

The G protein-coupled receptor (GPCR) encoding family of genes constitutes more than 6% of genes in Caenorhabditis elegans genome. GPCRs control behavior, innate immunity, chemotaxis, and food search behavior. Here, we show that C. elegans longevity is regulated by a chemosensory GPCR STR-2, expressed in AWC and ASI amphid sensory neurons. STR-2 function is required at temperatures of 20°C and higher on standard Escherichia coli OP50 diet. Under these conditions, this neuronal receptor also controls health span parameters and lipid droplet (LD) homeostasis in the intestine. We show that STR-2 regulates expression of delta-9 desaturases, fat-5, fat-6 and fat-7, and of diacylglycerol acyltransferase dgat-2. Rescue of the STR-2 function in either AWC and ASI, or ASI sensory neurons alone, restores expression of fat-5, dgat-2 and restores LD stores and longevity. Rescue of stored fat levels of GPCR mutant animals to wild-type levels, with low concentration of glucose, rescues its lifespan phenotype. In all, we show that neuronal STR-2 GPCR facilitates control of neutral lipid levels and longevity in C. elegans.

Role of actin in organelle trafficking in neurons.

Actin is a major cytoskeletal element involved in multiple cellular processes. Actin-rich regions present along the neuronal process aid in neuronal function, mediating multiple events involved in organelle trafficking. Actin is involved in organelle biogenesis, transport, and anchoring at specific locations. These functions can potentially be regulated by actin in a myosin-dependent or myosin-independent manner. The actin network could aid in membrane remodeling through membrane constriction, motor dependent transport, polymerization-based transport, cargo anchoring, and halting of cargo by acting as a physical barrier. Additionally, actin dynamics is perturbed in some neurodegenerative diseases where it could impact organelle biogenesis, transport, or anchoring thereby contributing to progression of disease phenotypes. The role of actin and myosin in organelle trafficking is the primary focus of this review.

Crowd Control: Effects of Physical Crowding on Cargo Movement in Healthy and Diseased Neurons.

High concentration of cytoskeletal filaments, organelles, and proteins along with the space constraints due to the axon's narrow geometry lead inevitably to intracellular physical crowding along the axon of a neuron. Local cargo movement is essential for maintaining steady cargo transport in the axon, and this may be impeded by physical crowding. Molecular motors that mediate active transport share movement mechanisms that allow them to bypass physical crowding present on microtubule tracks. Many neurodegenerative diseases, irrespective of how they are initiated, show increased physical crowding owing to the greater number of stalled organelles and structural changes associated with the cytoskeleton. Increased physical crowding may be a significant factor in slowing cargo transport to synapses, contributing to disease progression and culminating in the dying back of the neuronal process. This review explores the idea that physical crowding can impede cargo movement along the neuronal process. We examine the sources of physical crowding and strategies used by molecular motors that might enable cargo to circumvent physically crowded locations. Finally, we describe sub-cellular changes in neurodegenerative diseases that may alter physical crowding and discuss the implications of such changes on cargo movement.

Tied up: Does altering phosphoinositide-mediated membrane trafficking influence neurodegenerative disease phenotypes?

Phosphoinositides are a class of membrane lipids that are found on several intracellular compartments and play diverse roles inside cells, such as vesicle formation, protein trafficking, endocytosis etc. Intracellular distribution and levels of phosphoinositides are regulated by enzymes that generate and breakdown these lipids as well as other proteins that associate with phosphoinositides. These events lead to differing levels of specific phosphoinositides on different intracellular compartments. At these intracellular locations, phosphoinositides and their associated proteins, such as Rab GTPases, dynamin and BAR domain-containing proteins, regulate a variety of membrane trafficking pathways. Neurodegenerative phenotypes in disorders such as Parkinson's disease (PD) can arise as a consequence of altered or hampered intracellular trafficking. Altered trafficking can cause proteins such as α-synuclein to aggregate intracellularly. Several trafficking pathways are regulated bymaster regulators such as LRRK2,which is known to regulate the activity of phosphoinositide effector proteins. Perturbing either the levels of phosphoinositides or their interactions with different proteins disrupts intracellular trafficking pathways, contributing to phenotypes often observed in disorders such as Alzheimer's or PDs. Thus, studying phosphoinositide regulation and its role in trafficking can give us a deeper understanding of the contribution of disrupted trafficking to neurodegenerative phenotypes.

UNC-16/JIP3 and UNC-76/FEZ1 limit the density of mitochondria in C. elegans neurons by maintaining the balance of anterograde and retrograde mitochondrial transport.

We investigate the role of axonal transport in regulating neuronal mitochondrial density. We show that the density of mitochondria in the touch receptor neuron (TRN) of adult Caenorhabditis elegans is constant. Mitochondrial density and transport are controlled both by the Kinesin heavy chain and the Dynein-Dynactin complex. However, unlike in other models, the presence of mitochondria in C. elegans TRNs depends on a Kinesin light chain as well. Mutants in the three C. elegans miro genes do not alter mitochondrial density in the TRNs. Mutants in the Kinesin-1 associated proteins, UNC-16/JIP3 and UNC-76/FEZ1, show increased mitochondrial density and also have elevated levels of both the Kinesin Heavy and Light Chains in neurons. Genetic analyses suggest that, the increased mitochondrial density at the distal end of the neuronal process in unc-16 and unc-76 depends partly on Dynein. We observe a net anterograde bias in the ratio of anterograde to retrograde mitochondrial flux in the neuronal processes of unc-16 and unc-76, likely due to both increased Kinesin-1 and decreased Dynein in the neuronal processes. Our study shows that UNC-16 and UNC-76 indirectly limit mitochondrial density in the neuronal process by maintaining a balance in anterograde and retrograde mitochondrial axonal transport.

The C-terminal of CASY-1/Calsyntenin regulates GABAergic synaptic transmission at the Caenorhabditis elegans neuromuscular junction.

The C. elegans ortholog of mammalian calsyntenins, CASY-1, is an evolutionarily conserved type-I transmembrane protein that is highly enriched in the nervous system. Mammalian calsyntenins are strongly expressed at inhibitory synapses, but their role in synapse development and function is still elusive. Here, we report a crucial role for CASY-1 in regulating GABAergic synaptic transmission at the C. elegans neuromuscular junction (NMJ). The shorter isoforms of CASY-1; CASY-1B and CASY-1C, express and function in GABA motor neurons where they regulate GABA neurotransmission. Using pharmacological, behavioral, electrophysiological, optogenetic and imaging approaches we establish that GABA release is compromised at the NMJ in casy-1 mutants. Further, we demonstrate that CASY-1 is required to modulate the transport of GABAergic synaptic vesicle (SV) precursors through a possible interaction with the SV motor protein, UNC-104/KIF1A. This study proposes a possible evolutionarily conserved model for the regulation of GABA synaptic functioning by calsyntenins.

Cargo crowding at actin-rich regions along axons causes local traffic jams.

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1; Video S2.