Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus

Abstract

Most cases of autosomal-dominant hereditary spastic paraplegia are linked to mutations in SPG4 encoding spastin, a protein involved in microtubule dynamics and membrane trafficking. In pyramidal neurons of the motor cortex and in immortalized motor neurons, spastin is localized to the synaptic terminals and growth cones. However, in other neurons and in proliferating cells spastin is prevalently nuclear. The mechanisms that determine targeting of spastin to the nucleus or the cytoplasm are unknown. We show here that the SPG4 mRNA is able to direct synthesis of two spastin isoforms, 68 and 60 kDa, respectively, through usage of two different translational start sites. Both isoforms are imported into the nucleus, but the 68-kDa isoform contains two nuclear export signals that efficiently drive export to the cytoplasm. Nuclear export is leptomycin-B sensitive. The cytoplasmic 68-kDa spastin isoform is more abundant in the brain and the spinal cord than in other tissues. Our data indicate that spastin function is modulated through usage of alternative translational start sites and active nuclear import and export, and open new perspectives for the pathogenesis of hereditary spastic paraplegia.

Introduction

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder, characterized by weakness and spasticity of the lower limbs [1], [2]. These symptoms arise as a consequence of the degeneration of the axons of the corticospinal tracts, which convey voluntary motor impulses from pyramidal neurons in the motor cortex to spinal motor neurons [3]. Albeit genetically heterogeneous, most cases of HSP are inherited as an autosomal dominant trait and are due to mutations in the SPG4 gene, encoding spastin, a 616 amino acid molecule belonging to the AAA family of ATPases [4], [5], [6]. This family is characterized by the AAA domain, often located in the C-terminal part of the protein, and comprises a vast number of molecules residing in different cellular compartments that use ATP hydrolysis to drive very diverse biological functions [7], [8]. AAA proteins can be regarded as molecular machines involved in the disassembly of protein complexes. The specific function of a single AAA protein is often determined by its binding to molecular targets through their N-terminal region [9].

The function of spastin and its role in the pathogenesis of HSP are being actively investigated in different biological systems. Previous studies performed in our laboratory suggested a role of spastin in microtubule dynamics [10]. We showed that the microtubule network in cells expressing a considerable amount of spastin is disrupted, suggesting that spastin might be implicated in microtubule severing, as demonstrated for the highly homologous p60 katanin. Spastin microtubule severing activity has been recently confirmed using both the human and Drosophila recombinant proteins [11], [12]. Overexpressed spastin interacts with the microtubules through its N-terminal region, and this interaction is regulated through the ATPase activity of the AAA domain, such that mutants in the AAA domain display a stable association with the cytoskeleton, while wild-type spastin is likely to be rapidly released from microtubules upon ATP hydrolysis [10]. More recently, we studied spastin subcellular localization and found that spastin is enriched in cellular regions characterized by extensive remodeling of the cytoskeleton [13]. During cell division spastin is a component of the centrosome, where it interacts with the centrosomal protein NA14. In postmitotic motor neurons in vitro, spastin is enriched in the growth cones and in the branching regions [13], two sites of high dynamic events involving microtubules. Independent studies have also found spastin in the cytoplasm and synaptic terminals of pyramidal cells of the cerebral cortex and hippocampus, in Purkinje cells, and in spinal motor neurons [14].

A confirmation to the possible role of spastin in regulating the microtubule network comes from studies in Drosophila melanogaster. In this system, spastin overexpression induces a decrease of microtubule stability and strength at synaptic junctions [15], [16]. Strikingly, loss-of-function mutant larvae display a phenotype in the neuromuscular junction, with more numerous synaptic boutons and impaired neurotransmitter release, associated with fewer microtubule bundles [16]. As adults, spastin-null flies have severe movement defects [16]. These data strongly suggest that spastin may modulate synaptic strength and function, by acting as a regulator of microtubule stability.

Another line of investigation has identified a possible role of spastin in intracellular membrane trafficking. This role is linked to the presence in spastin of the MIT domain that interacts with CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex [17]. This complex is involved in targeting membrane-associated cargoes to multivesicular bodies, but the exact role of CHMP1B in this process is still unknown. Notably, the MIT domain is common to a group of proteins involved in endocytosis and trafficking, such as SKD1, and SNX15 [18], and is also present in spartin, the product of the SPG20 gene, mutations in which cause a complicated autosomal recessive form of HSP, Troyer syndrome [19].

Although all the functional data obtained to date point to roles of spastin in the cytoplasm, there is convincing evidence that spastin is also a nuclear protein. Spastin contains three predicted nuclear signals, two of which were experimentally validated [20]. Spastin nuclear localization in different cell types was detected by us and by others using different antibodies [13], [14], [21]. This notwithstanding, when spastin is overexpressed in vitro the vast majority of the protein is always found in the cytoplasm [10], [17], [22]. We now provide evidence that targeting of spastin to the nucleus or the cytoplasm is regulated through two mechanisms, the usage of alternative translational start sites and active nuclear export to the cytoplasm. These data contribute to recognize spastin as a polymorph protein, with a possible array of functions in both the nucleus and the cytoplasm.