TRIM46 Controls Neuronal Polarity and Axon Specification by Driving the Formation of Parallel Microtubule Arrays

Abstract

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

CDK5‐dependent activation of dynein in the axon initial segment regulates polarized cargo transport in neurons

“…In proximal dendrites, microtubules exhibit mixed polarity: they can be oriented with their plus ends towards the cell body or towards the end of the dendrite. 15,16 In contrast, within the AIS and axon, microtubules are found with uniform plus-end-out polarity 15,16,38 (Figure 2A). This organization facilitates the polarized activities of the anterograde motor kinesin and retrograde motor dynein, moving cargo either toward or away from the axon terminal, respectively.…”

“…However, the polarity of microtubules within the bundles, rather than the number of bundles, determines the effectiveness of motor protein-driven transport. 38 Thus, we tracked end-binding protein 3 (EB3), which binds to the dynamic plus-ends of microtubules, as well as to the membrane in the AIS (Figure 2A). 16 EB3 binds preferentially to the growing end of the microtubule, resulting in a comet-like appearance during live cell imaging, and short angled runs in kymographs of the resulting videos (Figure 2B, control).…”

Antibodies to TRIM46 are associated with paraneoplastic neurological syndromes

“…The following antibodies and reagents were used in this study: mouse anti‐ankyrinG (Life Technologies, Carlsbad, USA), chicken anti‐MAP2 (Abcam, Cambridge, UK), mouse anti‐GFP (Roche, Almere, Netherlands), rabbit anti‐TRIM46 (in‐house, generated as described in10), and Alexa‐405, ‐488, or ‐568‐conjugated secondary antibodies directed at human, mouse, chicken, or rabbit IgG (Thermo Fisher, Landsmeer, Netherlands). For western blotting, horseradish peroxidase‐conjugated donkey anti‐human (Calbiochem, Amsterdam, Netherlands) and swine anti‐mouse (DAKO, Heverlee, Belgium) were used.…”

“…The AIS and NOR are molecularly related axonal regions that are involved in the initiation and propagation of action potentials 9. Recently, the protein tripartite motif 46 (TRIM46) was found to be the autoantigen in a patient with paraneoplastic encephalomyelitis (PEM) and antibodies to the AIS but not the NOR of the sciatic nerve 6, 10. TRIM46 localizes specifically to the proximal axon where it bundles parallel microtubules and is important for axon specification and outgrowth during early brain development 10…”

Cytoskeletal control of axon domain assembly and function

“…Historically, the earliest recognized ultrastructural feature of the AIS (prior to the discovery of ankG) was the bundling of microtubules linked by molecularly undefined cross-bridges [15]. Recently, van Beuningen et al [•• 16] discovered that the cross-linked AIS microtubules depend on tripartite motif containing protein 46 (TRIM46), which functions to bundle microtubules into closely spaced arrays with their plus-end out (Fig. 1B).…”