Pathogenic Mutation of Spastin Has Gain-of-Function Effects on Microtubule Dynamics

Abstract

Mutations to the SPG4 gene encoding the microtubule-severing protein spastin are the most common cause of hereditary spastic paraplegia. Haploinsufficiency, the prevalent model for the disease, cannot readily explain many of its key aspects, such as its adult onset or its specificity for the corticospinal tracts. Treatment strategies based solely on haploinsufficiency are therefore likely to fail. Toward developing effective therapies, here we investigated potential gain-of-function effects of mutant spastins. The full-length human spastin isoform called M1 or a slightly shorter isoform called M87, both carrying the same pathogenic mutation C448Y, were expressed in three model systems: primary rat cortical neurons, fibroblasts, and transgenic Drosophila. Although both isoforms had ill effects on motor function in transgenic flies and decreased neurite outgrowth from primary cortical neurons, mutant M1 was notably more toxic than mutant M87. The observed phenotypes did not result from dominant-negative effects of mutated spastins. Studies in cultured cells revealed that microtubules can be heavily decorated by mutant M1 but not mutant M87. Microtubule-bound mutant M1 decreased microtubule dynamics, whereas unbound M1 or M87 mutant spastins increased microtubule dynamics. The alterations in microtubule dynamics observed in the presence of mutated spastins are not consistent with haploinsufficiency and are better explained by a gain-offunction mechanism. Our results fortify a model wherein toxicity of mutant spastin proteins, especially mutant M1, contributes to axonal degeneration in the corticospinal tracts. Furthermore, our results provide details on the mechanism of the toxicity that may chart a course toward more effective treatment regimens.

Cytoplasmic Dynein Transports Axonal Microtubules in a Polarity-Sorting Manner

“…The numbers of mobile MTs observed were higher than in earlier studies with GFP tubulin (He et al, 2005), which is consistent with tdEOS-tubulin more thoroughly incorporating into MTs (Solowska et al, 2014). The identity of the mobile structures as MTs (rather than membranous vesicles that incorporated the tdEOS-tubulin) was confirmed by treatment with FCPT, a compound that halts MT transport by inducing kinesin-5 (a motor that does not transport vesicles) to become a rigor complex (Rao et al, 2016).…”

Building the Neuronal Microtubule Cytoskeleton

“…Consistent with the axonal defects in various model systems, spastin mutations have been directly linked to axonal pathologies in the human neurodegenerative disease called hereditary spastic paraplegia (HSP) (Fink, 2013). Although most disease mutations in spastin either inactivate or downregulate severing activity (Evans et al, 2005), some recent studies report gain-of-function effects on MT dynamics (Solowska et al, 2014). Because MT severing by spastin could either reduce MT mass through destruction of pre-existing MT or increase MT mass through creating of novel seeds that can then elongate, a direct comparison between spastin activity and the number of neuronal MTs is ambiguous.…”

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury

“…Many genetic mutations that cause hereditary peripheral neuropathies as well as chemotherapy drugs that lead to painful neuropathies target components of the cytoskeleton (Edvardson et al, 2012; Gentil et al, 2013; Park et al, 2013; Solowska et al, 2014), and such cytoskeletal disruption can activate the DLK pathway. For example, the chemotherapeutic vincristine destabilizes microtubules and causes peripheral neuropathy, and in mouse models DLK is required for vincristine-induced axonal degeneration (Miller et al, 2009).…”