The hereditary spastic paraplegia gene, spastin, regulates microtubule stability to modulate synaptic structure and function

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a devastating neurological disease causing spastic weakness of the lower extremities and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans; however, mutations in one gene, spastin (SPG4), are the cause of >40% of all cases. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) protein family, and contains a microtubule interacting and organelle transport (MIT) domain. Previous work in cell culture has proposed a role for Spastin in regulating microtubules. RESULTS: Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), we have investigated the role of Spastin in vivo. We show that Drosophila Spastin (D-Spastin) is enriched in axons and synaptic connections. At neuromuscular junctions (NMJ), Dspastin RNAi causes morphological undergrowth and reduced synaptic area. Moreover, Dspastin overexpression reduces synaptic strength, whereas Dspastin RNAi elevates synaptic currents. By using antibodies against posttranslationally modified alpha-Tubulin, we find that Dspastin regulates microtubule stability. Functional synaptic defects caused by Dspastin RNAi and overexpression were pharmacologically alleviated by agents that destabilize and stabilize microtubules, respectively. CONCLUSIONS: Loss of Dspastin in Drosophila causes an aberrantly stabilized microtubule cytoskeleton in neurons and defects in synaptic growth and neurotransmission. These in vivo data strongly support previous reports, providing a probable cause for the neuronal dysfunction in spastin-linked HSP disease. The role of Spastin in regulating neuronal microtubule stability suggests therapeutic targets for HSP treatment and may provide insight into neurological disorders linked to microtubule dysfunction.     

ZFYVE27 (SPG33), a novel spastin-binding protein, is mutated in hereditary spastic paraplegia

Spastin, the most commonly mutated protein in the autosomal dominant form of hereditary spastic paraplegia (AD-HSP) has been suggested to be involved in vesicular cargo trafficking; however, a comprehensive function of spastin has not yet been elucidated. To characterize the molecular function of spastin, we used the yeast two-hybrid approach to identify new interacting partners of spastin. Here, we report ZFYVE27, a novel member of the FYVE-finger family of proteins, as a specific spastin-binding protein, and we validate the interaction by both in vivo coimmunoprecipitation and colocalization experiments in mammalian cells. More importantly, we report a German family with AD-HSP in which ZFYVE27 (SPG33) is mutated; furthermore, we demonstrate that the mutated ZFYVE27 protein shows an aberrant intracellular pattern in its tubular structure and that its interaction with spastin is severely affected. We postulate that this specific mutation in ZFYVE27 affects neuronal intracellular trafficking in the corticospinal tract, which is consistent with the pathology of HSP.     

Pleiotropic effects of spastin on neurite growth depending on expression levels

Hereditary spastic paraplegia (HSP) is characterized by weakness and spasticity of the lower limbs, owing to degeneration of corticospinal axons. The most common form is due to heterozygous mutations in the SPG4 gene, encoding spastin, a microtubule (MT)-severing protein. Here, we show that neurite growth in immortalized and primary neurons responds in pleiotropic ways to changes in spastin levels. Spastin depletion alters the development of primary hippocampal neurons leading to abnormal neuron morphology, dystrophic neurites, and axonal growth defects. By live imaging with End-Binding Protein 3-Fluorescent Green Protein (EB3-GFP), a MT plus-end tracking protein, we ascertained that the assembly rate of MTs is reduced when spastin is down-regulated. Spastin over-expression at high levels strongly suppresses neurite maintenance, while slight spastin up-regulation using an endogenous promoter enhances neurite branching and elongation. Spastin severing activity is exerted preferentially on stable acetylated and detyrosinated MTs. We further show that SPG4 nonsense or splice site mutations found in hereditary spastic paraplegia patients result in reduced spastin levels, supporting haploinsufficiency as the molecular cause of the disease. Our study reveals that SPG4 is a dosage-sensitive gene, and broadens the understanding of the role of spastin in neurite growth and MT dynamics.     

The AAA ATPase spastin links microtubule severing to membrane modelling

In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing.     

Human spastin has multiple microtubule-related functions

Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.     

Centrosome‐mediated microtubule remodeling during axon formation in human iPSC‐derived neurons

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule‐organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human‐induced pluripotent stem cell (iPSC)‐derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule‐associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live‐cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus‐end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC‐derived neurons, thereby laying the foundation for further axon development and function.     

Direct evidence for axonal transport defects in a novel mouse model of mutant spastin-induced hereditary spastic paraplegia (HSP) and human HSP patients

Mutations in spastin are the most common cause of hereditary spastic paraplegia (HSP) but the mechanisms by which mutant spastin induces disease are not clear. Spastin functions to regulate microtubule organisation, and because of the essential role of microtubules in axonal transport, this has led to the suggestion that defects in axonal transport may underlie at least part of the disease process in HSP. However, as yet there is no direct evidence to support this notion. Here we analysed axonal transport in a novel mouse model of spastin-induced HSP that involves a pathogenic splice site mutation, which leads to a loss of spastin protein. A mutation located within the same splice site has been previously described in HSP. Spastin mice develop gait abnormalities that correlate with phenotypes seen in HSP patients and also axonal swellings containing cytoskeletal proteins, mitochondria and the amyloid precursor protein (APP). Pathological analyses of human HSP cases caused by spastin mutations revealed the presence of similar axonal swellings. To determine whether mutant spastin influenced axonal transport we quantified transport of two cargoes, mitochondria and APP-containing membrane bound organelles, in neurons from mutant spastin and control mice, using time-lapse microscopy. We found that mutant spastin perturbs anterograde transport of both cargoes. In neurons with axonal swellings we found that the mitochondrial axonal transport defects were exacerbated; distal to axonal swellings both anterograde and retrograde transport were severely reduced. These results strongly support a direct role for defective axonal transport in the pathogenesis of HSP because of spastin mutation.     

Modeling axonal defects in hereditary spastic paraplegia with human pluripotent stem cells

Background

Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders including hereditary spastic paraplegia (HSP). Since the discovery of the first HSP gene, SPAST that encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remain largely unknown. The development of induced pluripotent stem cell (iPSC) technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathological processes including axonal defects.

Methods

In this article, we will first review the pathology and pathways affected in the common forms of HSP subtypes by searching the PubMed database. We will then summarize the findings and insights gained from studies using iPSC-based models, and discuss challenges and future directions.

Results

HSPs, a heterogeneous group of genetic neurodegenerative disorders, exhibit similar pathological changes that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport, recapitulating disease-specific axonal defects.

Conclusions

These patient-derived neurons offer a unique tool to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy.

     

Role of spastin and protrudin in neurite outgrowth

Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder characterized by retrograde axonal degeneration that primarily affects long spinal neurons. The gene encoding spastin has a well-established association with HSP, and protrudin is a known binding partner of spastin. Here, we demonstrate that the N-terminal domain of protrudin mediates the interaction with spastin, which is responsible for neurite outgrowth. We show that spastin promotes protrudin-dependent neurite outgrowth in PC12 cells. To further confirm these physiological functions in vivo, we microinjected zebrafish embryos with various protrudin/spastin mRNA and morpholinos. The results suggest that the spinal cord motor neuron axon outgrowth of zebrafish is regulated by the interaction between spastin and protrudin. In addition, the putative HSP-associated protrudinG191V mutation was shown to alter the subcellular distribution and impair the yolk sac extension of zebrafish, but without significant defects in neurite outgrowth both in PC12 cells and zebrafish. Taken together, our findings indicate that protrudin interacts with spastin and induces axon formation through its N-terminal domain. Moreover, protrudin and spastin may work together to play an indispensable role in motor axon outgrowth.     

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

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.     

Identification of nuclear localisation sequences in spastin (SPG4) using a novel Tetra-GFP reporter system

Mutations in the human spastin gene (SPG4) cause the most prevalent form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterised by progressive weakness and spasticity of the lower limbs. We address the question of intracellular localisation of spastin. Using polyclonal antibodies against N-terminal spastin sequences, we find that the native protein is localised in both the perinuclear cytoplasm and the nucleus. To identify structural motifs within the protein that can explain entry into the nucleus, we developed a reporter system to test nuclear localisation sequence (NLS)-functionality based on four in-frame fused copies of green fluorescent protein. Using this novel tool we demonstrate that spastin carries two NLSs located in exons 1 and 6. Both are independently functional in mediating nuclear entry.     

SPG20 Protein Spartin Is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1–7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin–Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis.     

IGF-1 Participates Differently in Regulation of Severing Activity of Katanin and Spastin

Spastin and p60-katanin are AAA family proteins that participate in microtubule severing, while lipotransin, another AAA family protein is a hormone sensitive lipase interacting protein. Sequence alignment analysis suggests that lipotransin and human p60-katanin are the orthologs of each other. Studies identified that insulin may negatively regulate ATPase activity of lipotransin. To reveal the effects of insulin on regulation of severing activity of p60-katanin and spastin, hippocampal neurons over-expressing spastin and p60-katanin were treated with IGF-1. Changes in neuronal branching by considering the total process lengths and average process numbers were quantitatively analyzed. According to the results of this study, total process lengths of hippocampal neurons and average process numbers remained similar in control and p60-katanin over-expressing neurons upon IGF-1 treatment, while significant decrease was observed in spastin over-expressing neurons. This study indicated that IGF-1 participates differently in the regulation of spastin and p60-katanin in terms of neuronal branching.     

Localization of Gi alpha proteins in the centrosomes and at the midbody: implication for their role in cell division

At the plasma membrane, heterotrimeric G proteins act as molecular switches to relay signals from G protein-coupled receptors; however, G(alpha) subunits also have receptor-independent functions at intracellular sites. Regulator of G protein signaling (RGS) 14, which enhances the intrinsic GTPase activity of G(ialpha) proteins, localizes in centrosomes, which suggests the coexpression of G(ialpha). We show expression of G(ialpha1), G(ialpha2), and G(ialpha3) in the centrosomes and at the midbody. Fluorescence resonance energy transfer analysis confirms a direct interaction between RGS14 and G(ialpha1) in centrosomes. Expression of GTPase-deficient G(ialpha1) results in defective cytokinesis, whereas that of wild-type or GTPase-deficient G(ialpha3) causes prolonged mitosis. Cells treated with pertussis toxin, with reduced expression of G(ialpha1), G(ialpha2), and G(ialpha3) or with decreased expression of RGS14 also exhibit cytokinesis defects. These results suggest that G(ialpha) proteins and their regulators at these sites may play essential roles during mammalian cell division.     

ZFYVE26/SPASTIZIN and SPG11/SPATACSIN mutations in hereditary spastic paraplegia types AR-SPG15 and AR-SPG11 have different effects on autophagy and endocytosis

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis.

Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A₁; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.     

GPI‐anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI‐anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI‐anchored proteins have been found on both the axonal and somatodendritic cell‐surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication‐incompetent retroviral vector to express a model GPI‐anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high‐density monolayers, and maintained for 1–9 days under serum‐free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP‐expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP‐expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well‐polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI‐anchored protein expressed in a well‐polarized primary neuronal cell type in vitro; other signals, such asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

Protrudin Regulates Endoplasmic Reticulum Morphology and Function Associated with the Pathogenesis of Hereditary Spastic Paraplegia

Protrudin is a membrane protein that regulates polarized vesicular trafficking in neurons. The protrudin gene (ZFYVE27) is mutated in a subset of individuals with hereditary spastic paraplegia (HSP), and protrudin is therefore also referred to as spastic paraplegia (SPG) 33. We have now generated mice that express a transgene for dual epitope-tagged protrudin under control of a neuron-specific promoter, and we have subjected highly purified protrudin-containing complexes isolated from the brain of these mice to proteomics analysis to identify proteins that associate with protrudin. Protrudin was found to interact with other HSP-related proteins including myelin proteolipid protein 1 (SPG2), atlastin-1 (SPG3A), REEP1 (SPG31), REEP5 (similar to REEP1), Kif5A (SPG10), Kif5B, Kif5C, and reticulon 1, 3, and 4 (similar to reticulon 2, SPG12). Membrane topology analysis indicated that one of three hydrophobic segments of protrudin forms a hydrophobic hairpin domain similar to those of other SPG proteins. Protrudin was found to localize predominantly to the tubular endoplasmic reticulum (ER), and forced expression of protrudin promoted the formation and stabilization of the tubular ER network. The protrudin(G191V) mutant, which has been identified in a subset of HSP patients, manifested an increased intracellular stability, and cells expressing this mutant showed an increased susceptibility to ER stress. Our results thus suggest that protrudin contributes to the regulation of ER morphology and function, and that its deregulation by mutation is a causative defect in HSP.