Axonal transport: Driving synaptic function

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Science  11 Oct 2019:
Vol. 366, Issue 6462, eaaw9997
DOI: 10.1126/science.aaw9997


  • Axonal transport drives cargo through extreme geometries.

    Neurons in the human central nervous system display highly complex axonal arbors that can branch thousands of times, reach hundreds of meters in total length, and contain hundreds of thousands of presynaptic sites distributed “en passant.” The axonal transport machinery supports synaptic function by delivering new synaptic vesicles to and removing aged organelles from presynaptic sites.

  • Fig. 1 Axonal microtubule organization and polarity.

    Stabilization of the microtubule “plus-end out” arrangement in the axon initial segment (AIS) is critical for axonal identity and likely acts as a template to establish the uniform polarity of the axonal microtubule network along the mid-axon.

  • Fig. 2 Axonal microtubule diversity.

    From numerous tubulin isoforms to distinct nucleotide states and several PTMs, microtubule lattices can in theory display a large number of combinatorial arrangements. This diversity can presumably be shown either at the single-microtubule level (multitude of tubulin isoforms, nucleotide states, and PTMs distributed across the same microtubule lattice) or at the microtubule population level (uniformity of tubulin isoforms, nucleotide state, and PTM distribution across microtubule populations, possibly conferring these a specific function).

  • Fig. 3 Axonal transport is regulated at multiple levels.

    (A and D) Despite having distinct biophysical properties, kinesin (A) and dynein (D) undergo similar transport phases. From an inactive unbound state, the motors bind to cargo and are recruited to the microtubule where they initiate active transport. MAPs, such as Tau or MAP7, can differentially affect motors and induce transport termination in a motor-specific manner. Kinesin-3 can sense the nucleotide state of the microtubule lattice and rapidly detaches upon encountering a dynamic GTP microtubule plus-end. (B and C) Initiation (B) and termination (C) of synaptic vesicle precursor (SVP) transport along an axon of a live hippocampal neuron in culture expressing synaptophysin-mScarlet. SVPs are transported mostly in the anterograde direction by kinesin-3 and pause preferentially at presynaptic sites, which are enriched in dynamic GTP-rich microtubule plus-ends. (E and F) Initiation (E) and termination (F) of autophagosome transport along the axon of a live cultured hippocampal neuron expressing LC3-GFP. Autophagosomes are mostly generated distally in the axon and driven by dynein in the retrograde direction toward the soma.

  • Fig. 4 Range of imaging approaches to address questions in the field of axonal transport.

    A wide range of imaging assays can be used to investigate the dynamics of biological processes in systems with different levels of biological complexity. Examples: (A) Combining in vitro reconstitution assays with total internal reflection fluorescence (TIRF) microscopy allows the assessment of the dynamic behavior of single motors on microtubules. The ability to easily manipulate the system by adding or removing components enables the elucidation of how single factors (e.g., MAPs) affect motor activity or what minimum components are required in the system to observe a specific dynamic behavior. Panels show the basic setup of an in vitro reconstitution assay and TIRF microscopy imaging of polymerization of fluorescently labeled tubulin. (B) Primary rodent CNS neurons in culture undergo a well-described polarized developmental process akin to what is observed in vivo. Neurons develop an axon and multiple dendrites, establish synaptic contacts with their neighbors, and fire action potentials spontaneously. The combined ability to easily manipulate the neurons genetically and pharmacologically, express fluorescent reporters, and use imaging techniques with high spatial and temporal resolution (such as spinning-disk confocal microscopy) makes this system ideal to investigate the mechanisms underlying organelle dynamics in a physiologically relevant context. Panels show a basic protocol to image primary neuron cultures and a confocal microscopy image of hippocampal axons in culture expressing fluorescently tagged synaptophysin. (C) The development of Cre mouse lines, improved adeno-associated viral vectors, and the advent of CRISPR/Cas9 gene-editing techniques have enabled more straightforward strategies of genetically manipulating mice, generating transgenic mouse lines, and expressing fluorescent reporters even in a neuron subtype–specific manner. Innovative surgical techniques, including spinal cord exposure and cortical window implementation, allow long-term optical access to the spinal cord and superficial layers of the cortex. By using two-photon microscopy techniques, it is thus possible to interrogate organelle dynamics and synaptic function in neurons in their native environment. Panels show a simple strategy to perform in vivo imaging assays, an image of axonal bundles in the spinal cord of a Thy1-YFP mouse, and a cholinergic axon in cortical layer I/II of a ChAT-Cre ROSA26-EGFPf mouse. (D) TIRF imaging of a single dimeric KIF1A motor labeled with tetramethylrhodamine actively moving on a fluorescently labeled microtubule in an in vitro reconstitution assay. (E) Two-photon microscopy imaging of mitochondrial dynamics in spinal cord axons of a Thy1-MitoCFP mouse. Synaptic vesicle precursors (labeled with synaptophysin-mScarlet) move mostly in the anterograde direction, whereas autophagosomes (labeled with LC3-GFP) show robust retrograde movement along the axons of live neurons in culture. (F) VGLUT1-pHluorin signal in a primary hippocampal neuron before and after electrical-field stimulation. An increased repertoire of fluorescent pH and Ca2+ indicators are allowing the study of how a variety of processes and mechanisms regulating axonal transport affect synaptic function and neuronal activity.

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