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Natural ageing is accompanied by a decline in motor, sensory and cognitive functions, all impacting life quality. Ageing is the predominant risk factor for many neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease. We need therefore to gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the long time required to age mammalian or vertebrate model animals.Here we introduce a new cellular model within theDrosophilabrain where neurons show typical ageing hallmarks known from the primate brain, including axonal swellings, cytoskeletal decay, a reduction in axonal calibre and morphological changes arising at synaptic terminals. In the fly brain, these changes occur within just a few weeks, ideal to study the underlying mechanisms. We observe that decay of the neuronal microtubule cytoskeleton clearly precedes other ageing hallmarks. We show that the microtubule-binding factors Tau, EB1 and Shot, are necessary for microtubule maintenance in axons and synapses. Their functional loss during ageing triggers microtubule bundle decay followed by the decline in axons and synapses. Genetic manipulations that improve microtubule networks, slow down other neuronal ageing hallmarks and confer aged specimens with the ability to outperform age-matched controls. Our work suggests therefore that microtubule networks are a key lesion site in ageing neurons and offer promising opportunities to improve neuronal decay in advanced age.

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require bundles of microtubules that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse orDrosophilacauses severe decay of microtubule bundles and axon growth inhibition. The underlying mechanisms are best understood forDrosophilaShort stop (Shot) and believed to involve cytoskeletal cross-linkage: the N-terminal calponin homology (CH) domains bind to F-actin, and the C-terminus to microtubules and Eb1. Here we have gained new understanding by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shot’s CH domains induces changes in Shot function - with a Lifeact-containing Shot variant causing remarkable remodelling of neuronal microtubules. In addition to actin-MT cross-linkage, we find strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. We argue that these likely involve the neuronal Shot-PH isoform, which is characterised by a large, unexplored central plakin repeat region (PRR). Work on PRRs might therefore pave the way towards important new mechanisms of axon biology and architecture that might similarly apply to central PRRs in mammalian spectraplakins.

The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila, and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.