BIOLOGICAL OVERVIEW

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. Employing Drosophila transgenic methods for overexpression and RNA interference (RNAi), the role of Spastin in vivo was investigated in vitro. Drosophila Spastin (DSpastin) 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, it has been found 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. It is concluded that 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 (Trotta, 2004).

Hereditary Spastic Paraplegia disease causes dysfunction in the corticospinal tract, causing progressive weakness and spasticity in the lower extremities (Reid, 2003). The age of HSP onset ranges from the first to the sixth decade, and symptoms also range widely in HSP patients, making clinical data difficult to interpret. HSP inheritance has been linked to at least 20 genetic loci (Reid, 2003), but less than half of these genes have been molecularly identified. The characterized HSP-linked genes encode a diverse range of products, including cell adhesion molecules [L1-CAM, Jouet, 1994], myelination proteins [DM20, PLP (Suagier-Veber, 1994)], and microtubule motor proteins [KIF5A; (Reid, 2002)]. However, approximately 40% of HSP disease cases are linked to mutations of the SPG4 locus, which encodes Spastin (Fonknechten, 2000; Hazan, 1999). Spastin is an ATPase that contains a microtubule-interacting domain (Ciccarelli, 2003), suggesting an active role in cytoskeleton interactions. Interestingly, a number of other HSP-linked genes have similar molecular features, including Spartin and Paraplegin (Zhao, 2001; DeMichele, 1998; Casari, 1998. In vitro experiments have confirmed that Spastin binds polymerized microtubules through its N terminus and demonstrate a requirement for Spastin ATPase activity to release it from microtubules (Patel, 2002). These data suggest that Spastin normally plays some role involving the microtubule cytoskeleton in neurons. Loss of this Spastin function leads to progressive neuronal dysfunction, culminating in axonal degeneration (Trotta, 2004 and references therein).

Microtubules affect multiple facets of neuronal development and function, with pronounced roles in growth cone motility and synaptic elaboration of both pre- and post-synaptic structures being documented in a range of vertebrate and invertebrate systems. The Drosophila neuromuscular junction (NMJ) synapse has been a particularly useful genetic system in which to assay the role of the microtubule cytoskeleton and model its role in inherited neurological disease. For example, mutations in microtubule-interacting Futsch (MAP1b) and VAP-33A alter synaptic morphology, thereby decreasing bouton number and increasing bouton size. These data have produced the hypothesis that decreased microtubule stability leads to growth and elaboration of the presynaptic terminal. In addition, the delivery of proteins and organelles required for neurotransmission, both pre- and post-synaptically, depends on microtubules and associated motor proteins. Acute pharmacological perturbation of microtubules at the Drosophila NMJ has not revealed major defects in synaptic vesicle cycling. In contrast, loss or gain of expression studies of the Drosophila fragile X-related (dfxr) gene have shown that it regulates Futsch to cooperatively modulate both synaptic architecture and neurotransmission strength. These studies indicate that microtubules play key functions in the control of synaptic structure and function and that mutations perturbing microtubule dynamics are causatively linked with inherited neurological disorders (Trotta, 2004 and references therein).

Drosophila contains a highly conserved spastin homolog (Dspastin) (Kammermeier, 2003). The Drosophila NMJ system therefore provides an attractive opportunity to model HSP and to test the in vivo function of D-Spastin. Drosophila Spastin is shown to be localized at the NMJ and specifically enriched in presynaptic boutons. The dosage of Dspastin was altered in a targeted fashion in the nervous system with transgenic RNA interference (RNAi) and overexpression techniques, and the consequence was assayed with electrophysiology and confocal imaging. Knockdown of D-Spastin (i.e., reduction of Dspastin mRNA through RNAi) in neurons results in synaptic undergrowth. Conversely, loss of D-Spastin in neurons causes much stronger neurotransmission, whereas D-Spastin overexpression weakens synaptic function. Microtubule stability was assayed locally within the synapse by using antibodies specific to modified Tubulin, which label stable and long-lived microtubules. Knockdown of D-Spastin results in accumulation of stabilized Tubulin, whereas D-Spastin overexpression conversely reduces stable microtubules. Direct pharmacological manipulation of microtubule stability, to counter the observed defects, rescues the functional synaptic defects in both classes of Dspastin mutant. Together, these data provide the first in vivo evidence that Spastin regulates microtubule stability and show that this function of Spastin strongly modulates both synaptic architecture and neurotransmission strength (Trotta, 2004).

Mutations in the spastin (SPG4) gene are the leading cause of Hereditary Spastic Paraplegia (HSP), a progressively debilitating neurological disease characterized by dysfunction of corticospinal tract neurons, followed by eventual axonal degeneration (Reid, 2003). The Spastin protein is an ATPase that associates with microtubules in an ATP-dependent manner, leading to the hypothesis that loss of Spastin may cause microtubule-related defects in neuronal function required for axonal maintenance. Drosophila has a single, highly conserved spastin homolog (Dspastin) containing both ATPase and microtubule-interacting domains (Kammermeier, 2003). The Drosophila neuromusculature has previously proven important for assaying microtubule mechanisms relevant to neuronal function and neurological disease, making it a logical choice for modeling the neurological role of Spastin in vivo. A targeted transgenic approach for RNAi knockdown and overexpression of Spastin in the nervous system was used to assay the in vivo requirement for Spastin specifically in neurons (Trotta, 2004).

Drosophila Spastin is widely expressed in many cell types, including both muscles and neurons, where it is present throughout the cell but undetectable in the nucleus. These findings support the conclusion that Spastin is a cytoplasmic protein, in contrast to some previous reports that mammalian Spastin is a nuclear protein (Charvin, 2003). Consistent with recent RNA in situ data in the Drosophila embryo (Kammermeier, 2003), D-Spastin protein is highly enriched in neurons, and the protein is particularly abundant in the presynaptic bouton compartments in synaptic connections. Strongly driven Dspastin RNAi results in pupal lethality, showing that the gene is essential for viability. Strongly driven Dspastin overexpression causes cell death and neurodegeneration, showing that Dspastin gene dosage must be carefully controlled. Up- or down-regulation of Dspastin dosage results in striking alterations in synaptic morphology and function. Targeted removal of D-Spastin from neurons causes the loss of synaptic area but, paradoxically, strengthened neurotransmission. In contrast, overexpression of D-Spastin in neurons reduces synaptic efficacy. As predicted by previous in vitro studies in mammals (Errico, 2002), Drosophila Spastin closely regulates the stability of the neuronal microtubule cytoskeleton in vivo. Loss of D-Spastin leads to a dramatic increase in stabilized microtubules, which, in the synaptic terminal, inappropriately ramify beyond their normal axonal compartments into the synaptic boutons. Overexpression of D-Spastin causes an erosion of the stabilized microtubule network. These results indicate that D-Spastin acts as a negative regulator of microtubule stability. The relationship between neuronal function and microtubule stability was investigated in an effort to identify a causative mechanism to account for spastin-mediated HSP disease. Most importantly, misregulation of synaptic function in both the loss and overexpression of Dspastin is rescued by pharmacologically modifying microtubule stability, as appropriate, to counter the observed changes in microtubule stability in both mutant conditions. Taken together, these results strongly suggest that D-Spastin acts locally in the synapse to control microtubule stability and the cytoskeletal compartmentalization of functional synaptic domains, and that this regulation plays a critical role both in maintaining the synapse and in modulating the synaptic efficacy dictating neurotransmission strength (Trotta, 2004).

The elaboration of synaptic architecture, determining both the maintenance and extent of synaptic area, is well documented to be microtubule dependent. At the Drosophila NMJ, multiple known regulators of microtubule stability have been shown to modulate the size and complexity of the terminal arbor. The current study shows that D-Spastin has a positive role in maintaining the synapse by encouraging growth through increasing the dynamic instability of microtubules. This conclusion is consistent with previous results showing that synaptic terminals with stabilized microtubules are less prone to branching and budding. The hypothesis is that proteins that stabilize microtubules should inhibit synaptic growth, whereas proteins that destabilize microtubules should facilitate synaptic growth. D-Spastin would therefore be predicted to promote synaptic growth by destabilizing microtubules. The biochemical action of Spastin in destabilizing microtubules, which has been shown previously in mammalian cell culture (Errico, 2002), and the results here that D-Spastin destabilizes microtubules within individual Drosophila synaptic terminals in situ, strongly corroborates this hypothesis (Trotta, 2004).

The effects on synaptic growth may have a compounding influence on synaptic function. However, an inverse relationship is observed between changes in synaptic area and synaptic transmission strength after manipulation of D-Spastin dosage. Knockdown of D-Spastin results in much smaller (area reduced 50%) but much stronger (EJC amplitude increased 50%) NMJ synapses. The increase in synaptic function may be an overcompensation for the synaptic size, or these two synaptic parameters may be misregulated completely independently; however, the interdependency of these data is unclear. Independent regulation of synaptic growth and efficacy has been shown repeatedly during the course of analyzing Drosophila mutations in several genes. The fact that opposing defects in synaptic transmission caused by either Dspastin knockdown or overexpression can be rescued with acute pharmacological treatments to counter the misregulation of microtubule dynamics suggests that D-Spastin plays a direct role in modulating synaptic efficacy, independent from a separable role in modulating synaptic growth and architecture (Trotta, 2004).

Synaptic function appears sensitive to the stability of the microtubule cytoskeleton. The exact nature of this dependency is unclear, since stabilized microtubules are normally tightly restricted to axons and interbouton connectives and excluded from presynaptic boutons. One obvious possibility is that alteration of microtubule stability influences the microtubule-dependent transport of proteins or organelles required for maintained synaptic vesicle cycling and neurotransmitter release (Kammermeier, 2003). D-Spastin protein is normally tightly restricted to synaptic boutons, which lack stabilized microtubules, and absent from interbouton connectives, which contain stabilized microtubules. It is tempting to speculate that the D-Spastin domains determine the domains of stabilized versus unstabilized Tubulin within the synaptic arbor. Changes in D-Spastin levels not only regulate the abundance of stabilized microtubules but also determine their synaptic localization; loss of D-Spastin causes stabilized microtubules to inappropriately invade synaptic boutons. The defects in neurotransmission in both loss and gain of D-Spastin conditions were strikingly rescued by pharmacologically shifting microtubule dynamics away from the aberrant condition. Interestingly, the microtubule-modulating drugs that effectively restore aberrant neurotransmission in both Dspastin loss and overexpression mutants do not significantly alter basal synaptic function in wild-type synapses. This shows that alteration in D-Spastin levels is first required to prime the system for responsiveness. It appears that alterations in Dspastin dosage lead to a constitutive shifts in the microtubule equilibrium, which makes the synapse acutely sensitive to additional changes in microtubule stability. Why this should be the case is presently unclear (Trotta, 2004).

Hereditary Spastic Paraplegias are linked to multiple genetic loci-encoding proteins apparently involved in disparate cellular activities. However, one prominently affected cellular mechanism appears to be alteration in microtubule cytoskeleton formation, stability, and/or function. Specifically, well over half of all HSP disease cases are linked to microtubule motor proteins (e.g., KIF5A) or other proteins with microtubule-interacting domains (e.g., Spartin, Paraplegin, and Spastin). The most commonly mutated gene to cause HSP is Spastin, making it the obvious choice to model the mechanistic defects caused by loss of function of this suite of related microtubule-interacting proteins. This study shows for the first time that Spastin is enriched at the synapse, controlling both functional processes driving synaptic transmission, as well as developmental pathways determining synaptic growth and maintenance. Alteration in microtubule stability locally at the synapse appears to be a primary consequence that follows changes in Spastin dosage, consistent with the hypothesis that Spastin is a negative regulator of the microtubule cytoskeleton stability. By extension, it is likely that misregulation of microtubule stability is the primary cause of HSP disease, both in the 40% of disease cases directly associated with mutations in the spastin (SPG4) gene as well as cases linked to Atlastin, Spartin, Paraplegin, and related proteins. This mechanistic insight suggests that therapeutic targets aimed at the mechanism of microtubule dynamic instability are likely to prove effective in the treatment of Hereditary Spastic Paraplegia disease (Trotta, 2004).

Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin

Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. This study reports the X-ray crystal structure of the Drosophila spastin AAA domain and provides a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, these data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. This work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients (Roll-Mecak, 2008).

The combination of X-ray crystallography, SAXS ab initio reconstructions and structure-guided mutagenesis provides the first structural information on microtubule-severing proteins and allows the proposal of a molecular model for spastin-mediated severing. Owing to their similar domain organization and high sequence similarity, this model probably pertains to katanin as well. It is proposed that face A of the spastin AAA ring docks onto the microtubule, placing the positively charged N-terminal pore entrance in contact with the negatively charged C terminus of tubulin. The translocation from face A to face B would correspond to the direction of substrate translocation proposed for the distantly related AAA ATPases ClpX, ClpA and ClpB. The linker and MIT domains extending from the ring would make additional contacts with the microtubule, thus increasing microtubule avidity and potentially stabilizing the hexamer on the microtubule. On the basis of affinity measurements, only a subset of the six arms is likely to make strong binding interactions with the microtubule (Roll-Mecak, 2008).

It is proposed that the tubulin polypeptide is threaded through the pore, perhaps driven by nucleotide-driven conformational changes of the pore loops. However, spastin may not need to completely translocate the tubulin polypeptide substrate, but instead just grip the C-terminal tubulin tail and exert mechanical 'tugs' that might partially unfold tubulin or locally destabilize protomer-protomer interactions, leading to catastrophic breakdown of the microtubule lattice. It also remains possible that the MIT domains could participate in this nucleotide-driven process by binding and 'feeding' the C-terminal tails to the pore. Further biophysical characterization will be needed to decipher the structural details of substrate recognition and mechanical force production. The data also suggest that spastin may selectively recognize post-translationally modified tubulins ('Glu' tubulins) that are part of stable microtubules. Consistent with this idea, loss of spastin in Drosophila results in the accumulation of stabilized polyglutamylated tubulin in neurons and spastin knockout mice show axonal swellings enriched in detyrosinated, stable microtubules. The structure also provides the first glimpse into how spastin disease mutations contribute to spastin dysfunction and disease, most of which are likely to be involved in destabilizing protomer-protomer interactions, microtubule- or ATP-binding; in such cases, spastin-linked HSP is probably caused by haploinsufficiency and not a dominant negative effect. Further elucidation of the mechanistic details of how spastin interacts with particular tubulin isoforms and post-translational modifications and leads to microtubule destabilization may provide insight into the origin of spastin paraplegias and potential treatments for this disease (Roll-Mecak, 2008).

Spastin, atlastin and ER relocalization are involved in axon, but not dendrite, regeneration

Mutations in over 50 genes including spastin and atlastin lead to Hereditary Spastic Paraplegia (HSP). It was previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin and spichthyin were reduced. Impaired regeneration was dependent on genetic background, and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. Other markers were examined to identify additional changes associated with regeneration. While mitochondria, endosomes and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was only observed during axon regeneration, and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. It is proposed that the HSP proteins Spastin and Atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip (Rao, 2016).

Previous work has shown that axon regeneration is impaired when one copy of spastin is mutant. This study now shows that atlastin is also haploinsufficient for axon regeneration and that reduction of several other HSP proteins using RNAi impairs regeneration. Thus axon regeneration seems to be a postdevelopmental process that involves at minimum a subset of HSP protein (Rao, 2016).

The sensitivity of axon regeneration to partial reduction of HSP proteins, however, depends on the genetic background. In a previous study, EB1-GFP was used as a dual-purpose marker of cell shape and microtubule dynamics. This fusion protein is not, however, completely neutral. GFP fused to the C-terminus of EB1 can interfere with binding of partner proteins to EB1. Because EB1 acts as a dynamic platform at growing microtubule ends that recruits other proteins, the presence of large amounts of EB1-GFP could reduce recruitment of other plus end-binding proteins. Indeed, in Drosophila, neurons EB1 binds Apc, which in turn brings kinesin-2 to growing dendritic microtubules to help maintain minus-end-out polarity, and high levels of EB1-GFP result in mixed polarity. Because of this, this study expressed EB1-GFP at low levels, but it is still possible that there is a subtle defect in microtubule growth or organization. Under normal circumstances, this does not result in any defects in regeneration, which is indistinguishable in control neurons expressing EB1-GFP, mCD8-RFP, or Rtnl1-GFP. Only when combined with partial reduction of HSP proteins was a difference seen among neurons expressing different markers. This difference was most clearly demonstrated in spastin heterozygotes, which had a very strong reduction in regenerative growth in neurons labeled with EB1-GFP but not with mCD8-RFP. The synthetic interaction between EB1-GFP and spastin suggests that even though the early microtubule changes triggered by axon injury were normal, with reduced levels of HSP proteins, microtubules were somehow involved in the phenotype. This conclusion was supported by a similar effect of EB1-CT, a dominant-negative form of EB1, and the fact that introduction of tdEOS-αtubulin suppressed the spastin phenotype (Rao, 2016).

To probe in more depth how HSP protein function related to regenerative axon growth, several other approaches were taken. First, dendrite regeneration was examined. Complete regeneration of dendrites after removal of the entire arbor involves very rapid outgrowth, and so it was reasoned that if HSP proteins were generally involved in facilitating growth of neuronal processes, they should be required for dendrite regeneration. No defects in dendrite regeneration were observed, and this suggested that the cells were healthy and that a process specific to axon regeneration was sensitive to HSP protein reduction. Second, a catalogue of intracellular markers was examined to look for rearrangements associated with regenerative axon growth. The ER was most dramatically different in regenerating axons and accumulated near the growing tip. Moreover, this was specific to axon regeneration and not observed in dendrite regeneration (Rao, 2016).

All of these observations were assembled into a model for HSP protein function during regeneration. It is proposed that a subset of HSP proteins is involved in concentrating the ER, together with underlying microtubules, at tips of axons undergoing regenerative growth. This model makes particular sense for spastin and atlastin. Because spastin is a microtubule regulator that in flies and mammals also has a transmembrane domain and binds the ER regulatory protein atlastin, their combined action could facilitate concentration of the ER at growing axon tips. In support of this idea, reduction of either protein disrupted ER accumulation at single growing axon tips (Rao, 2016).

Although it is believed that atlastin and spastin help to concentrate ER at growing axon tips by linking the ER to microtubules, which also accumulate at growing tips, it is not known what mediates the microtubule redistribution. It is suspected, however, that microtubule polarity is involved in setting up tip accumulation of tubulin and ER. Tip accumulation is seen only during regenerative axon growth, which requires microtubules in the growing process to be plus-end-out, and not during regenerative dendrite growth, when microtubules are largely minus-end-out. In initiation of regenerative axon outgrowth in cultured Drosophila neurons, kinesin-mediated microtubule sliding is important, and so one possibility is that motors slide short pieces of microtubules out to the new tip (Rao, 2016).

It is intriguing that ER accumulates at regenerating axon but not dendrite tips. This suggests that increased amounts of local ER are specifically important for promoting maximal axon growth. Local Smooth Endoplasmic Reticulum (SER) could promote axon growth by increasing local lipid production or increasing availability of intracellular calcium. A recent study in Caenorhabditis elegans suggests that it is the calcium storage function of SER that is important in this context. In this system, release of ER calcium through ryanodine receptors is required for maximal axon outgrowth, and high levels of calcium were seen at axon tips up to 5 h (the latest time point examined) after axon injury. Thus perhaps atlastin and spastin help concentrate ER at growing axon tips to provide a local source of intracellular calcium stores, which in turn facilitate regenerative growth (Rao, 2016).

It is difficult to know how a function for atlastin and spastin, and potentially other HSP proteins, in ER localization during regenerative axon growth relates to the axon degeneration that occurs in the disease. All HSP proteins seem to have important basic cellular functions that are quite universal. For example, atlastin is biochemically an ER fusion protein, but only a subset of disease-causing atlastin mutations affect ER fusion. Perhaps the function of atlastin, spastin, and other HSP proteins important for disease is not the core function but a subtler role these proteins play in very long neurons. ER relocalization during regenerative growth of axons is worth considering as a disease-relevant function for several reasons: 1) it is important for mature neurons, 2) at least two different HSP proteins contribute to it, 3) repeated small failures of regeneration could lead to accumulated axon loss over a long time period, and 4) at least in some genetic backgrounds, the capacity for regenerative growth is reduced when only one allele of the gene is mutant. An interesting further speculation is how this function might relate to cell-type susceptibility to regeneration. The hallmark of HSP is degeneration of upper motor neurons. As in many neurodegenerative diseases, it is unclear why these cells might be more sensitive than others. One possibility raised by the data is that the suite of microtubule regulators expressed in different neurons could influence sensitivity of regeneration to reduction of HSP proteins. For example, slightly higher levels of a microtubule-stabilizing protein might eliminate the need for full HSP protein function during regeneration in the same way that tdEOS-αtubulin bypassed the requirement for spastin and atlastin for regeneration. Similarly, a different set or ration of microtubule plus end-binding proteins could make a particular neuron type more sensitive to partial reduction of HSP proteins in the same way that EB1-GFP and EB1-CT did in this study (Rao, 2016).

Although the idea that the function identified for HSP proteins during axon regeneration is appealing in many ways, it is not a perfect fit. It is not known whether axon regeneration is triggered during normal wear and tear of axons in the spinal cord, and this is a critical missing piece of information necessary to evaluate whether reduction in regeneration might lead to long-term degeneration (Rao, 2016).

Independently of potential relevance to disease, the differential effect of reduction of HSP proteins on axon and dendrite regeneration is intriguing. One might expect that proteins with core cellular functions like ER and microtubule regulation would be equally required for both types of outgrowth. Similarly, if the ER is concentrated at growing axon tips to provide an extra calcium reservoir, why is this not important for dendrite regenerative growth? It will be extremely interesting to learn what promotes dendrite regeneration in future studies (Rao, 2016).

GENE STRUCTURE

cDNA clone length - 2833

Bases in 5' UTR - 387

Exons - 6

Bases in 3' UTR - 780

PROTEIN STRUCTURE

Amino Acids - 758

Structural Domains

The human SPG4 locus encodes the spastin gene, which is responsible for the most prevalent form of autosomal dominant hereditary spastic paraplegia (AD-HSP), a neurodegenerative disorder. The predicted gene product CG5977 is identified in this study as the Drosophila homolog of the human spastin gene, with much higher sequence similarities than any other related AAA domain protein in the fly. Furthermore a new potential transmembrane domain is reported in the N-terminus of the two homologous proteins. During embryogenesis, the expression pattern of Drosophila spastin becomes restricted primarily to the central nervous system, in contrast to the ubiquitous expression of the vertebrate spastin genes. Given this nervous system-specific expression, it will be important to determine if Drosophila spastin loss-of function mutations also lead to neurodegeneration (Kammermeier, 2003).

Close inspection of the Drosophila sequence and of the alignment of Drosophila and human spastin reveals, besides the two known highly conserved domains [namely the AAA domain (70% identity) of 300 residues, and the MIT (microtubule interacting and trafficking molecules) domain (55% identity) of about 100 residues], an additional conserved region of 33 residues near the N-terminus. The three domains are also recognized with SMART domain analysis as AAA, MIT and as a putative transmembrane domain, respectively. Since the similarity in the N-terminal domain extends beyond the hydrophobic core that could be a transmembrane domain, this domain is called a TM+ domain. This domain appears to be specific for spastin proteins. MIT domains, and to a comparable degree AAA domains, also appear in unrelated proteins; one was discovered in spartin, the gene responsible for SPG20 (Patel, 2002). The association of MIT and AAA domains, however, appears to be evolutionarily old since even the highly conserved vacuolar protein sorting 4 (VPS4) subfamily shows this domain structure. The members of the AAA family most closely related to spastin, however, are not VPS4 but members of the fidgetin subfamily, which may be of neurological interest because of the shaking phenotype in the fidget mouse (Kammermeier, 2003 and references therein).

Paraplegin, the gene responsible for SPG7, however, is only distantly related to spastin, and the paraplegin protein is localized to the mitochondria and contains a peptidase domain. Since spastin has already been shown not to be targeted to the mitochondria, but appears to be localized to the nucleus (Charvin, 2003), it will be important to test whether the potential transmembrane domain with the highly conserved neighboring residues could be involved in localization of spastin to the nuclear membrane system (Kammermeier, 2003 and references therein).

Human spastin is comprised of several functional domains, all of which are conserved in the Drosophila gene (Kammermeier, 2003). Overall, Dspastin exhibits 48% identity and 60% similarity at the amino acid level with the human homolog. Both human and Drosophila proteins contain a predicted transmembrane domain (40% identity), a microtubule interacting and organelle transport (MIT) domain (55% identity) and an AAA domain (70% identity). The high degree of conservation of these motifs suggests that the function of Spastin has been tightly maintained throughout evolution (Trotta, 2004).

date revised: 12 June 2021

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