Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery

 

Background

The human nervous system comprises an intricate network of trillions of synapses, the functional junctures between apposing neurons. Although the formation of new synapses (synaptogenesis) is well-described (Colón-Ramos 2009; Qi et al. 2022), the cellular mechanisms that predetermine where new synapses form are poorly understood.

Early in synaptogenesis, synaptic components accumulate at prospective pre- and postsynaptic sites (Prokop et al. 1996; Matteoli et al. 1992; Sabo, Gomes, and McAllister 2006). Along the axon, presynaptic precursors are deposited at naïve en passant synapses, where they then undergo maturation (Rizzoli 2014). Situated some distance from the cell body, synaptic components must necessarily be trafficked to these sites via microtubules. Intriguingly, localised microtubule growth at en passant synapses affects their ability to receive synaptic vesicle precursors (SVPs) in rat neurons (Guedes-Dias et al. 2019). However, what mechanism and which factors dictate microtubule growth at these sites and hence their presynaptic identity is unknown.

In this study, Aiken and Holzbaur identify the microtubule-severing enzyme spastin as responsible for promoting en passant synapse pre-patterning. This is achieved through the enhancement of local microtubule growth, which facilitates SVP delivery. These findings mark an advancement in our understanding of the steps preceding synapse formation and will help us unravel the role of spastin in various synaptopathies (Lopes et al. 2020; Chelban et al. 2017).

 

Key Findings

 

SVP pausing and microtubule polymerisation share hotspots along axons

Previously, the authors determined that in rat neurons, microtubule growth influences SVP delivery to en passant synapses (Guedes-Dias et al. 2019). Here, the authors first attempted to validate that same mechanism in human iPSC-derived i³neurons (Boecker et al. 2020). Expressing the fluorescently-labelled SVP protein synaptophysin in these neurons revealed bidirectional trafficking of SVPs along the axon with distinct pausing sites (SVP+ sites; Fig. 1). The authors then co-expressed a microtubule plus end marker, GFP-MACF43, to follow microtubule growth over time which can be detected as fluorescent “comets”. Indeed, 8.8x as many comet events occurred at SVP+ sites compared to sites where SVPs do not pause, spatially correlating microtubule growth and sites of SVP pausing.

 

Fig. 1. Representative time lapse of bidirectional trafficking of fluorescently-tagged Synaptophysin. Upper panels display mScarlet-Syp signal along an axon, lower panels are kymographs with SVP+ sites highlighted.

 

Altering spastin expression affects location of microtubule growth and SVP pausing, and their dynamics

Given that the microtubule-severing enzyme spastin directly influences microtubule dynamics and is suspected to regulate synaptic development (Errico et al. 2002; Butler et al. 2010; Sherwood et al. 2004), it presented itself as a good candidate for the regulation of presynaptic patterning. To explore this experimentally, the authors modulated spastin expression and evaluated microtubule growth and SVP pausing. Firstly, both spastin knockdown and overexpression perturbed microtubule comet frequency and speed. The localisation of comet events was also perturbed – whilst spastin knockdown caused fewer comet events at SVP+ sites, with no effect outside of these regions, overexpression in contrast promoted microtubule polymerisation outside of SVP+ sites. As such, spastin-mediated spatial regulation of microtubule growth along the axon could be confirmed.

Although SVP speed and directional flux were not significantly altered upon spastin knockdown, anterogradely there were fewer instances of SVP pausing. The duration of pause, however, remained unperturbed, meaning that a lack of nascent plus ends does not impinge on SVP re-engagement with microtubules after pausing.

The failure of SVPs to pause upon loss of spastin might reflect defects in delivery to SVP+ sites. To examine this possibility, pausing behaviour was analysed in relation to SVP+ sites. Indeed, anterograde SVPs pause over 50% less at SVP+ sites upon spastin knockdown compared to control neurons. As such, a new role for spastin was uncovered in the delivery of synaptic precursors to distinct sites punctuating the axon.

 

Spastin enrichment is an early feature of synaptic development and persists in mature synapses

Despite the popularity of i³neuron culture as a model system, the formation of their synapses is largely uncharted territory. To address this, the authors analysed the identity of i³neuron synapses by performing Synapsin (presynapse), PSD-95 (postsynapse), and MAP2 (somatodendritic compartment) immunocytochemistry (Fig. 2). This revealed that the vast majority of presynapses are not apposed by postsynapses and thus correlate with early structures in synaptic development (‘protosynapses’). Subsequent co-culture with primary rat astrocytes increased the abundance of mature synapses. Presence or absence of astrocytes therefore provides a platform to study early and mature synapses in i³neurons.

 

Fig. 2. Representative Z stacks of Syn/PSD-95/MAP2 immunocytochemistry in i³neuron monoculture (top) and with rat astrocytic co-culture (bottom). Light pink inset displays a protosynapse, dark pink insets display mature synapses.

 

Utilising this system, Aiken and Holzbaur then evaluated spastin presence at protosynapses and mature presynapses, as well as microtubule plus ends (EB3 marker). The presence of EB3 puncta supports new microtubule plus end polymerisation, and spastin was found to be enriched at both early and mature presynapses, and postsynaptically. Consequently, spastin enrichment is an early characteristic of synaptogenesis.

 

Spastin regulates presynaptic identity in human heterologous synapses

Since spastin was also observed postsynaptically at mature synapses, the authors then sought to analyse the role of spastin independently at mature presynapses. To do so, a novel heterologous model of synapse formation was established. In this assay, HEK cells expressing postsynaptic adhesion molecule Neuroligin-1 are introduced to i³neurons, successfully promoting the formation of heterologous synapses enriched in various presynaptic markers. After showing that spastin and EB3 were present at these presynapses, spastin was also shown to be required for both Synapsin and Synaptobrevin accumulation.

 

In Conclusion

In this preprint, Aiken and Holzbaur utilise three models of synapse formation (i³neuron culture alone, with astrocytic co-culture, and a heterologous synapse assay) to identify the microtubule-severing enzyme spastin as an early factor required for broad presynaptic component deposition at protosynapses (Fig. 3). Spastin is shown to spatially govern sites of plus end polymerisation, thereby promoting SVP dissociation from microtubules. The next significant question concerns the factors that lie upstream of spastin and govern its own localisation along the axon.

 

Fig. 3. Model of spastin function at SVP+ sites. In wild type neurons, the microtubule-severing activity of spastin produces new microtubule plus ends. Here, KIF1A transporting presynaptic cargo detaches, causing accumulation of presynaptic components and protosynapse formation. In spastin-deficient neurons, fewer microtubule plus ends are found at SVP+ sites. KIF1A fails to detach, SVPs no longer pause and presynaptic components no longer accumulate.

 

Significance of this work

The accumulation of presynaptic components is an established step in synaptogenesis. Here, Aiken and Holzbaur identify spastin as an early component of presynaptic patterning. The assays developed here encourage the identification of additional required factors.

I chose this preprint because it enhances our capacity to study early stages of presynaptic patterning and its required factors in human iPSC-derived i³neuron culture, significantly aided by the establishment of a novel heterologous synapse assay. This assay has the potential to support further studies on the contribution of the presynapse to synaptogenesis. It may be used, for example, to study the early progression of various synaptopathies, focussing on the presynaptic compartment.

 

Questions for the authors

1. Are there any proposed factors that determine spastin’s own punctuated localisation along the axon?
2. Since SVPs are synthesised in the cell body and are transported anterogradely, what is the purpose of SVPs travelling retrogradely? Also, why does SVP cycling between protosynapses occur?
3. You showed spastin is found at mature presynapses. Have you tried conditional knockdown of spastin once mature synapses have formed to determine if it is required for sustained presynapse function?

 

References

Boecker, Clemens Alexander, Mara A. Olenick, Elizabeth R. Gallagher, Michael E. Ward, and Erika L. F. Holzbaur. 2020. “ToolBox: Live Imaging of Intracellular Organelle Transport in Induced Pluripotent Stem Cell-Derived Neurons.” Traffic  21 (1): 138–55.

Butler, Richard, Jonathan D. Wood, Jennifer A. Landers, and Vincent T. Cunliffe. 2010. “Genetic and Chemical Modulation of Spastin-Dependent Axon Outgrowth in Zebrafish Embryos Indicates a Role for Impaired Microtubule Dynamics in Hereditary Spastic Paraplegia.” Disease Models & Mechanisms 3 (11-12): 743–51.

Chelban, Viorica, Arianna Tucci, David S. Lynch, James M. Polke, Liana Santos, Hallgeir Jonvik, Stanislav Groppa, Nicholas W. Wood, and Henry Houlden. 2017. “Truncating Mutations in SPAST Patients Are Associated with a High Rate of Psychiatric Comorbidities in Hereditary Spastic Paraplegia.” Journal of Neurology, Neurosurgery, and Psychiatry 88 (8): 681–87.

Colón-Ramos, Daniel A. 2009. “Synapse Formation in Developing Neural Circuits.” Current Topics in Developmental Biology 87: 53–79.

Errico, Alessia, Andrea Ballabio, and Elena I. Rugarli. 2002. “Spastin, the Protein Mutated in Autosomal Dominant Hereditary Spastic Paraplegia, Is Involved in Microtubule Dynamics.” Human Molecular Genetics 11 (2): 153–63.

Guedes-Dias, Pedro, Jeffrey J. Nirschl, Nohely Abreu, Mariko K. Tokito, Carsten Janke, Maria M. Magiera, and Erika L. F. Holzbaur. 2019. “Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse.” Current Biology: CB 29 (2): 268–82.e8.

Lopes, André T., Torben J. Hausrat, Frank F. Heisler, Kira V. Gromova, Franco L. Lombino, Timo Fischer, Laura Ruschkies, et al. 2020. “Spastin Depletion Increases Tubulin Polyglutamylation and Impairs Kinesin-Mediated Neuronal Transport, Leading to Working and Associative Memory Deficits.” PLoS Biology 18 (8): e3000820.

Matteoli, M., K. Takei, M. S. Perin, T. C. Südhof, and P. De Camilli. 1992. “Exo-Endocytotic Recycling of Synaptic Vesicles in Developing Processes of Cultured Hippocampal Neurons.” The Journal of Cell Biology 117 (4): 849–61.

Prokop, A., M. Landgraf, E. Rushton, K. Broadie, and M. Bate. 1996. “Presynaptic Development at the Drosophila Neuromuscular Junction: Assembly and Localization of Presynaptic Active Zones.” Neuron 17 (4): 617–26.

Qi, Cai, Li-Da Luo, Irena Feng, and Shaojie Ma. 2022. “Molecular Mechanisms of Synaptogenesis.” Frontiers in Synaptic Neuroscience 14 (September): 939793.

Rizzoli, Silvio O. 2014. “Synaptic Vesicle Recycling: Steps and Principles.” The EMBO Journal 33 (8): 788–822.

Sabo, Shasta L., Raquel A. Gomes, and A. Kimberley McAllister. 2006. “Formation of Presynaptic Terminals at Predefined Sites along Axons.” The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 26 (42): 10813–25.

Sherwood, Nina Tang, Qi Sun, Mingshan Xue, Bing Zhang, and Kai Zinn. 2004. “Drosophila Spastin Regulates Synaptic Microtubule Networks and Is Required for Normal Motor Function.” PLoS Biology 2 (12): e429.

 

 

 

 

 

 

 

 

 

 

Tags: brain, culture, development

doi: https://doi.org/10.1242/prelights.36074

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