In a complex nervous system, neuronal functional diversity is reflected in the wide variety of dendritic arbor shapes. Different neuronal classes are defined by class-specific transcription factor combinatorial codes. The combination of the transcription factors Knot and Cut is particular to Drosophila class IV dendritic arborization (da) neurons. Knot and Cut control different aspects of the dendrite cytoskeleton, promoting microtubule- and actin-based dendritic arbors, respectively. Knot delineates class IV arbor morphology by simultaneously synergizing with Cut to promote complexity and repressing Cut-mediated promotion of dendritic filopodia/spikes. Knot increases dendritic arbor outgrowth through promoting the expression of Spastin, a microtubule-severing protein disrupted in autosomal dominant hereditary spastic paraplegia (AD-HSP). Knot and Cut may modulate cellular mechanisms that are conserved between Drosophila and vertebrates. Hence, this study gives significant general insight into how multiple transcription factors combine to control class-specific dendritic arbor morphology through controlling different aspects of the cytoskeleton (Jinushi-Nakao, 2007).
To understand the mechanism by which Knot promotes dendrite outgrowth, attempts were made to identify Knot-regulated genes in da neurons. Knot promotes formation of a microtubule-based dendritic arbor cytoskeleton. Therefore, candidate genes for regulation by Knot were chosen based on annotation in the Gene Ontology database indicating their association with microtubule biogenesis and function. The small GTPases rac1, cdc42, and rho, were also analyzed. Ectopic (classes I-III) or endogenous (class IV) Knot activity promotes da neuron dendritic arbor outgrowth. With this in mind, the relative expression of candidate genes was compared between wild-type and ectopic knot-expressing da neurons. To do this the RluA1-Gal4 line was used that drives UAS-mCD8::GFP expression solely in all da and some ES neurons. Purifying Gfp-positive cells from RluA1-Gal4, UAS-mCD8::GFP embryos gave a highly enriched population of da neurons for analysis. Gfp-positive cells were sorted from control (RluA1-Gal4, UAS-mCD8::GFP) and ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) embryos by Fluorescence-Activated Cell Sorting (FACS). Total RNA was isolated from the purified cells and the relative expression of candidate genes was compared between these populations by Reverse Transcription Polymerase Chain Reaction (RT-PCR). mRNA expression levels of all candidates were normalized against gapdh, and as a positive control analyzed knot levels were analyzed. Between the wild-type and ectopic knot-expressing cells, knot mRNA expression was upregulated by ratio of 2.6. Of the candidates analyzed, only spastin had an altered expression level; it was strongly upregulated by a ratio of 3.3 (Jinushi-Nakao, 2007).
These RT-PCR findings were confirmed by examining upregulation of Spastin protein via Knot ectopic expression. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) family, all of which have a related protein structure. To avoid cross-reactivity between family members, a Spastin-specific antibody, which was additionally preabsorbed against the other AAA family members, was used. Spastin protein levels were examined in western blots of protein extracts from sorted Gfp-positive cells. Ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) da neurons showed a very large upregulation in Spastin protein content as compared with those prepared from wild-type (RluA1-Gal4, UAS-mCD8::GFP) da neurons (Jinushi-Nakao, 2007).
If spastin is a bona fide target of Knot in class IV neurons, then spastin expression may be enriched in class IV da neurons versus other da neuron classes. To examine spastin expression whole-mount embryonic spastin mRNA in situ experiments were carried out. The results confirmed those of previous studies that show that spastin is ubiquitously expressed, with a higher-than-background expression level in nervous system tissues. However, the high ubiquitous background expression level made it impossible to compare levels of spastin in specific da neuron classes. To get around this problem, FACS purified a mixed population of all da neurons (RluA1-Gal4, UAS-mCD8::GFP) and a pure population of class IV neurons (ppk-Gal4, UAS-mCD8::GFP) were examined. spastin expression in these two populations was compared. spastin expression levels were clearly enriched (62% more) in the pure population of class IV neurons as compared with the mixed population of da neurons (Jinushi-Nakao, 2007).
Spastin has microtubule-severing activity in cultured cells and in vitro. It was asked if Spastin is also able to alter microtubule structure in the dendritic arbor of da neurons. To do this, spastin was ectopically expressed in class I ddaE neurons (Gal42-21, UAS-mCD8::GF8, UAS-spastin). Then the entire dendritic arbor was visualized at the wandering third-instar larva stage by staining with an anti-Gfp antibody, and examined the microtubule cytoskeleton was simultaneously by staining the arbor with antibodies to detect Futsch. In wild-type class I neurons, Futsch was present throughout the dendritic arbor. When spastin was expressed ectopically in the class I neuron, a loss of Futsch from the dendritic arbor was observed and disruption of arbor morphology. Therefore, high levels of Spastin disrupt microtubule organization within the arbor, a finding consistent with Spastin's microtubule-severing activity (Jinushi-Nakao, 2007).
Next, whether Spastin activity is required to promote class IV dendritic arbor complexity, as would be expected if it is part of the program controlled by Knot activity, was investigated. To do this class IV neurons were marked with ppk-Gal4, UAS-mCD8::GFP in the background of a null spastin5.75 allele. ppk-Gal4 was used to express an RNAi construct directed against spastin (UAS-spastinRNAi) along with UAS-mCD8::GFP to selectively knock down spastin class IV neurons. The morphology of these neurons was examined at the wandering third-instar larva stage (Jinushi-Nakao, 2007).
Reduction of spastin levels in either heterozygous null background or spastin RNAi-mediated knockdown background lead to large gaps both between neighboring class IV dendritic arbors and within the arbor of an individual neuron. Such gaps were not seen in wild-type control larvae, but were also seen in loss- or reduction-of-function knot mutants. To quantify this effect, dendrite coverage was measured by drawing a 34 × 34 grid of 10 μm × 10 μm squares over the central portion of the neuron. The number of squares that did not contain any portion of a dendrite branch was counted. This analysis provides an approximate measure of the amount of area that is not covered by the dendritic arbor. spastin RNAi knockdown had 17% more uncovered area than wild-type ddaC neurons; spastin5.75/+ mutants had 43%, and kn1/knKN2 mutants had 59% (Jinushi-Nakao, 2007).
Spastin is upregulated by Knot in da neurons and is required for class IV neuron dendritic arbor outgrowth. To confirm that Spastin is part of the program by which Knot mediates dendritic arbor outgrowth, the outcome was investigated of spastin RNAi in either a wild-type or an ectopic knot-expressing class I neuron. UAS-spastinRNAi was crossed to Gal42-21, UAS-mCD8::GFP or UAS-kn; Gal42-21, UAS-mCD8::GFP, and class I ddaE dendritic arbor shape was assayed at wandering third-instar larva stage. The spastinRNAi construct had no effect on either branching or dendrite length when expressed in a wild-type class I neuron. However, the UAS-spastinRNAi construct strongly reduced both branching and total dendrite length (by 18% and 19%, respectively) when expressed in an ectopic knot-expressing class I neuron. Therefore, Spastin activity is an essential part of the program by which ectopic knot expression mediates an increase in dendritic arbor complexity (Jinushi-Nakao, 2007).
This study has shown that Knot and Cut act simultaneously in the class IV neuron to promote dendritic arbor outgrowth and branching. However, the loss-of-function phenotypes for Knot and Cut are different, which demonstrates that each transcription factor works through a dissimilar mechanism. Indeed, ectopic expression experiments show that Knot and Cut regulate different aspects of the cytoskeleton. Knot expressed ectopically in the class I neuron promotes arbor extension that is microtubule-positive. Conversely, ectopic expression of Cut in the class I neuron leads to arbor extension that is F-actin-positive but microtubule deficient. When Cut and Knot are expressed together in the class I neuron, they have a synergistic effect on dendritic arbor area and branching. However, the effect of Cut and Knot coexpression on dendritic arbor total length is additive: both the microtubule-positive and microtubule-negative regions of the arbor are increased. This overall arbor organization mimics that of class IV neurons. The majority of the dendritic arbor of the class IV neuron contains microtubules, but the highest-order branches are microtubule deficient (Jinushi-Nakao, 2007).
When Cut levels are increased and Knot levels are reduced in a class IV neuron, its dendritic arbor takes on characteristics similar to those of class III. This transformation from a class IV to a class III shape is not absolute. Hence, it is likely that other factors are also required to fully control all aspects of class IV dendritic arbor morphology versus those of other da neuron classes. Overall, however, the data suggest that class IV-specific Knot expression demarcates arbor shape via multiple mechanisms. Knot synergizes with Cut in promoting dendrite length, branching, and area. Additionally it represses the ability of Cut to mediate filopodia/spike formation. Finally, Knot induces symmetry in the dendritic arbor of the class IV neuron, as opposed to class I–III neurons, which have asymmetric dendritic arbor shapes (Jinushi-Nakao, 2007).
Suppression of Cut-mediated filopodia/spike formation by Knot does not occur through repression of Cut protein levels and therefore acts either downstream of Cut or in parallel. Interestingly, though, the absolute level of Knot protein is controlled by the level of Cut in the cell. Tuning the level of Knot to the level of Cut protein in each neuron could be a mechanism by which Knot acts to repress only specific aspects of the Cut-driven morphogenesis program (Jinushi-Nakao, 2007).
Knot and Cut also interact very differently with Rac1. A major function of Rac1 is to promote reorganization of the actin cytoskeleton. Filopodia/spikes are rich in F-actin and deficient in microtubules, and indeed Rac1 significantly enhances the ability of Cut to promote filopodia/spike formation. Ectopic coexpression of Knot and Rac1 in class I neurons leads to large increases in the length of the short, thorn-shaped projections that are induced by expression of Rac1 alone. Rac1 has been shown to form focal F-actin in the distal edge of axonal growth cones, which acts as a site of microtubule capture during outgrowth. Perhaps similar processes are occurring in the Rac1-mediated thorn-shaped projections. Knot activity could then promote microtubule invasion and outgrowth at these points of Rac1-mediated F-actin reorganization (Jinushi-Nakao, 2007).
Knot promotes microtubule-mediated dendritic arbor outgrowth by inducing Spastin expression. A large amount of the extra arbor outgrowth induced by ectopic Knot expression is suppressed by reducing Spastin function. Therefore, Spastin is a primary component of the mechanism by which Knot promotes arbor outgrowth (Jinushi-Nakao, 2007).
Spastin acts as a microtubule-severing protein and may function by producing new seeds for microtubule polymerization. Maintenance of a population of dynamic microtubules is important for axonal extension, branching, and growth cone guidance. In vivo, Spastin has been shown to be required for growth of synaptic terminals at the Drosophila neuromuscular junction and for axon outgrowth in zebrafish. This study shows that Spastin activity can also destabilize microtubules in the dendritic arbor, and that Spastin is itself required for class IV da dendritic arbor outgrowth (Jinushi-Nakao, 2007).
The human spastin gene (SPG4) is mutated in over 40% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. SPG4 mutation usually causes pure spastic paraplegia of the lower limbs due to degeneration of the corticospinal tract axons. However, in some families SPG4 mutation is associated with additional neurological symptoms that cannot be explained by dysfunction of the corticospinal tract axons alone. This study shows that Spastin is also required for complex dendritic arbor development; hence, defects in dendrite as well as axon development and function may be part of the pathology of some AD-HSP cases (Jinushi-Nakao, 2007).
Accumulating evidence suggests that mechanisms of dendritogenesis are closely conserved between Drosophila and other species. For example, actin binding proteins, rac/rho GTPases, and calcium/calmodulin-dependent protein kinase II (CaMKII) all control dendrite branching and filopodia/spine morphogenesis in Drosophila and in vertebrates. knot (Ebf1, 2, and 3) and cut (Cux1 and 2) homologs are expressed in the developing mouse nervous system and may overlap in some subsets of neurons, e.g., in the spinal cord and cerebellum. In vertebrates, it is possible that these genes may also regulate dendrite morphology. Both human CUX1 and mouse Ebf2 can phenocopy cut and knot, respectively, when ectopically expressed in class I neurons. Interestingly, Ebf2 is involved in the migration and differentiation of Purkinje neurons. However, a specific role for Ebf2 in controlling the highly complex dendritic arbor shape of these neurons remains to be assayed (Jinushi-Nakao, 2007).
This study has elucidated mechanisms of transcription factor-mediated control of dendritogenesis. It was found that Knot and Cut function to control Drosophila class IV da sensory neuron dendritic arbor morphogenesis through different aspects of the cytoskeleton. Further analysis of Knot and Cut targets will provide a powerful entry point into understanding dendritic arbor morphogenetic mechanisms that are potentially conserved between Drosophila and vertebrate species (Jinushi-Nakao, 2007).
Dendrites and axons are two major neuronal compartments with differences that are critical for neuronal functions. To learn about the differential regulation of dendritic and axonal development, a genetic screen was conducted in Drosophila and the dendritic arbor reduction 1 (dar1) mutants were isolated that display defects in dendritic but not axonal growth. The dar1 gene encodes a novel transcription regulator in the Kruppel-like factor family. Neurons lacking dar1 function have severely reduced growth of microtubule- but not F-actin-based dendritic branches. In contrast, overexpression of Dar1 dramatically increased the growth of microtubule-based dendritic branches. These results suggest that Dar1 promotes dendrite growth in part by suppressing the expression of the microtubule-severing protein Spastin. This study thus uncovers a novel transcriptional program for microtubule regulation that preferentially controls dendrite growth (Ye, 2011).
Dendritic and axonal compartments have distinct morphological features that are fundamental to neuronal functions. During development, one neurite of the postmitotic neuron is specified as the axon, and then the remaining neurites are specified as dendrites. Subsequently, the developing dendrites and axons follow separate paths to form two compartments that are distinct in structure and function. The past several years have seen substantial progress in the elucidation of the molecular mechanisms underlying axon specification (Wiggin, 2005; Tahirovic, 2009). However, how the dendritic and axonal compartments of a neuron diverge in their development after the postmitotic neuron is polarized remains mostly unknown (Ye, 2011).
Both the microtubule (MT) cytoskeleton and the intracellular membrane system have been proposed to be important for the differential development of dendrites and axons. Microtubules are oriented differently in dendrites and axons. In the axons, microtubules are oriented uniformly with their plus-ends pointing distally, whereas there are microtubules with either orientation in dendrites. As microtubules are essential both for transporting molecules and organelles and for extending neurites, such a differential organization between dendrites and axons is likely to have profound impact on separating dendrite and axon development (Ye, 2011).
The secretory and endocytic pathways of the intracellular membrane system also contribute to the distinction between dendrite and axon growth. The rate of endocytosis in dendrites is much higher than that in the axon (Ye, 2007). This leads to a greater demand of membrane supply since the vast majority of the endocytosed plasma membrane are returned to the soma. Indeed, when the secretory pathway function is compromised as a result of mutations in key regulators such as Sar1, the dendritic growth is preferentially reduced (Ye, 2007). Furthermore, dendritic but not axonal growth is altered by disruption of PKD (protein kinase D) function, which regulates membrane protein exit from trans-Golgi network (Yeaman, 2004), via overexpression of a dominant-negative form. It thus seems likely that the membrane and membrane proteins required for dendritic or axonal growth are sorted in the trans-Golgi network (Ye, 2011).
How the microtubule cytoskeleton and the intracellular membrane system are regulated to contribute to the differential development of dendrites and axons is unknown. Through a genetic screen in Drosophila, the dendritic arbor reduction 1 (dar1) complementation group, which displays specific defects in dendrite development (Ye, 2007), was isolated. The dar1 gene encodes a novel member of the Krüppel-like factor (KLF) family, which regulates gene transcription. This study show that dar1 is a critical regulator of dendritic microtubule cytoskeleton. The results also suggest that Dar1 promotes dendrite growth in part by suppressing the expression of the microtubule-severing protein Spastin. These findings lend support to the notion that dendrite and axon development are controlled by partly non-overlapping genetic programs (Ye, 2011).
The da neurons in Drosophila PNS offer a wealth of features for analyzing different aspects of dendrite development, including different compositions of microtubule and actin cytoskeleton in different types of dendritic branches. The class III da neurons have characteristic dendritic filopodia (also termed dendritic spikes), which are distributed along major dendrites and are usually straight and devoid of additional branching. These dendritic spikes are enriched with filamentous actin (F-actin) and are essentially free of microtubules. In contrast, the major dendrites of the same neurons are enriched with microtubules and contain F-actin at a level much lower than that in the dendritic spikes. The separation of F-actin- and microtubule-based dendritic branches allowed investigation of the effect of dar1 mutations on these two types of cytoskeleton (Ye, 2011).
Although the major dendrites were dramatically reduced by ~75% in the class III da neurons mutant for the dar1 gene, the density of dendritic spikes was much less affected. This raised the possibility that dar1 preferentially regulates microtubule cytoskeleton. To test this hypothesis, Rac1 was overexpressed in class I da neurons mutant for dar1. Rac1 is a small GTPase that regulates actin cytoskeleton. Overexpression of Rac1 (OE Rac1) in many neuron types, including the da neurons, leads to hyperbranching of dendrites. The dar1 mutations did not block the branching activity of Rac1, suggesting that Dar1 is not required for regulating actin-cytoskeleton (Ye, 2011).
To further test whether Dar1 is capable of promoting MT growth, transgenic lines were generated to express Dar1 in da neurons. Overexpressing Dar1 (OE Dar1) with the driver GAL44-77 caused dramatic overgrowth of dendrites in the class IV da neurons in early third-instar larvae. Control neurons had 350.2 ± 14.0 branch points, excluding the dendrites around the segmental borders where dendrites of different neurons intermingle. In contrast, neurons overexpressing Dar1 had 930.7 ± 17.3 branch points. Similarly, the total dendritic length of control neurons, excluding the dendrites around the segmental borders, was 9159.0 ± 217.0 microm, whereas that of neurons overexpressing Dar1 was 18,044.0 ± 278.8 microm. These results suggest that Dar1 is not only necessary but also sufficient for promoting dendritic growth. Sholl analysis (Sholl, D.A., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat. 87: 387-406) showed that the increase in branch number and length took place throughout the dendritic fields rather than being restricted to particular regions. No change in the size of dendritic fields or dendritic tiling was observed (Ye, 2011).
The Flip-out technique was used to study the effects of overexpressing Dar1 on class IV da neuron axons. In contrast to its effects on dendrites, overexpression of Dar1 did not significantly affect axon growth. The axon terminal length of wild-type class IV neurons was 68.4 ± 4.2 microm, and that of class IV neuron overexpressing Dar1 was 77.7 ± 5.7 microm in dar1 mutant neurons (Ye, 2011).
Overexpressing Dar1 in class I da neurons also dramatically increased dendrite growth. Strikingly, when Dar1 was overexpressed in the class III da neurons, an increased number of long curvy terminal branches was observed that are reminiscent of microtubule-containing branches (Ye, 2011).
To directly visualize microtubule and F-actin in the class III da neurons overexpressing Dar1, use was made of tubulin-GFP and the F-actin marker GMA. GMA is a chimeric protein with the actin binding domain of Drosophila moesin fused to the C terminus of GFP and is enriched at the dendritic spikes of class III da neurons. Overexpressing Dar1 in class III da neurons led to the presence of MT in the ectopic terminal branches. No enrichment of F-actin was observed in these branches, suggesting overexpression of Dar1 promoted the growth of microtubule-containing major dendrites but not the F-actin-enriched filopodia (Ye, 2011).
Together, these results suggest that dar1 preferentially regulates microtubule cytoskeleton to mediate its specific control of dendrite growth (Ye, 2011).
The microtubule-severing protein Spastin has been found to regulate dendrite development in Drosophila da neurons. Because Dar1 preferentially regulates microtubule-based dendritic growth, it was asked whether Dar1 could regulate Spastin expression. Currently, no antibody against Drosophila Spastin provides sufficient sensitivity to detect endogenous Spastin protein level. Therefore determined the levels of Spastin transcripts was determined in dar1 mutant neurons by quantitative real-time PCR (qPCR). The qPCR technique was preferred to other techniques, such as in situ hybridization, for both consistency in quantification and sensitivity. Total RNA was extracted from purified da neurons of dar1 mutant embryos as well as from those of wild-type embryos. Real-time PCR was used to compare the amounts of Spastin transcripts between wild-type and dar1 mutant da neurons. The levels of Spastin transcripts were significantly elevated in dar1 mutant neurons. Transcripts from dar1 mutant neurons on average took 1.44 less PCR cycles to reach the threshold cycle (Ct) for amplifying Spastin than those from wild-type neurons, suggesting a 3.2-fold increase in Spastin transcript level in dar1 mutant neurons. In contrast, there is no difference in the Ct for amplifying the chromatin modifying protein 1 (Chmp1) between wild-type and dar1 mutant neurons (Ye, 2011).
Consistent with the reduced dendrite phenotype in dar1 mutants, upregulation of Spastin with the EP insertion T32 (SpaT32), which is known to cause a modest overexpression, led to a dramatic reduction in both total dendritic length and branch number. Whereas the total dendritic length in wild-type class IV neurons was 17,789 ± 444.5 microm, it was reduced to 10,582 ± 580.3 microm in neurons overexpressing SpaT32. Similarly, the number of branch points was reduced from 767.9 ± 22.8 in wild-type to 403.3 ± 43.5 in SpaT32-overexpressing neurons (Ye, 2011).
These results together suggest that Dar1 restricts the expression of Spastin either directly or through a transcription repressor, allowing dendrite growth. Different from dar1 mutants, Spastin upregulation by expressing SpaT32 also resulted in a mild yet significant reduction in axonal growth from 68.4 ± 4.2 microm in wild-type class IV neurons to 55.2 ± 2.1 microm in SpaT32-expressing neurons (Ye, 2011).
The Collier/Olf1/EBF family transcription factor Knot has been proposed to regulate Spastin expression and promotes microtubule-based dendrite growth in class IV da neurons (Jinushi-Nakao, 2007). This study asked whether Dar1 and Knot genetically interact to control dendrite development. Overexpressing Knot (OE Knot) in class I da neurons, which have the simpler dendritic arbors, leads to an increase in dendrite growth. Knot was expressed in single class I da neurons using the MARCM technique and significant increase was observed in total dendrite length. In contrast, when Knot was expressed in neurons with dar1 mutation, the dendrites were severely reduced. To test whether Knot regulates Dar1 expression, Dar1 protein levels were examined by immunostaining in class I neurons and no difference was found between control neurons and neurons overexpressing Knot. The mean intensity of Dar1 immunofluorescence was 88.1 ± 5.7 (arbitrary unit) in control neurons and 80.6 ± 5.7 in neurons overexpressing Knot. Therefore, it is unlikely that Knot controls class IV dendrite development by regulating Dar1 expression (Ye, 2011).
This study has shown that a novel molecule in the KLF family of transcriptional regulators, Dar1, regulates the microtubule cytoskeleton during the differential development of dendrites and axons. Dar1 is specifically required for dendritic growth. Neurons lacking dar1 function exhibit reduced growth of microtubule-based dendritic branches. However, overexpression of Dar1 in neurons increased the growth of microtubule-based dendritic branches. Dar1 suppresses the expression of the microtubule-severing protein Spastin, either directly or indirectly. Upregulation of Spastin expression leads to a dendrite phenotype similar to that observed in dar1 mutant neurons (Ye, 2011).
Substantial progress has been made in the past several years on the specific regulation of axon growth. The anaphase-promoting complex (APC) and its activator Cdh1 form a multiunit complex of ubiquitin ligase in the nucleus of cerebellar granule cells to specifically restrict axon growth. This function of Cdh1-APC requires the transcriptional repressor SnoN, which is a target of the ubiquitin-dependent degradation mediated by Cdh1-APC (Stegmuller, 2006). SnoN in turn requires a scaffold protein Ccd1 to promote axon growth. The TGFβ-regulated signaling protein Smad2 plays an important role in regulating the Cdh1-APC-SnoN (Ye, 2011).
Neural activities can specifically induce dendrite growth by activating transcription factors. In cerebellar granule cells, knockdown of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) or the transcription factor NeuroD results in reduced growth of dendrites but not axons. CaMKIIα phosphorylates NeuroD in response to neural activities. Moreover, synaptic activity-induced dendritic growth can be blocked by reducing NeuroD level, suggesting that CaMKII and NeuroD mediate the dendritic growth promoted by synaptic activity (Ye, 2011).
This study has identified a novel transcriptional program that is specifically involved in the development of dendrites. Whereas whether Dar1 plays a role in activity-dependent dendritic growth remains to be determined, Dar1 is likely to be part of the activity-independent transcription program that controls dendrite growth in early development before the exposure to sensory inputs or the establishment of neuronal circuits, because the dendrite defects were observed soon after the initiation of dendritic growth in embryo. The fact that dar1 is both necessary and sufficient for dendrite growth suggests that it may determine multiple aspects required for dendrite development. Indeed, although dar1 is specifically involved in dendrite growth and is likely a regulator of Spastin expression, Spastin is involved in both dendrite and axon development. This raises the possibility that Dar1 also controls the expression of other molecules which suppresses Spastin function in the axon. It will be interesting to identify such mechanisms by systematically identifying downstream molecules of Dar1 (Ye, 2011).
Microtubule severing by Katanin and Spastin is important for axon and dendrite development, possibly by keeping microtubules sufficiently short to be efficiently transported into developing neurites or by creating more free ends of microtubules to interact with other proteins in developing processes. The Spastin gene, which encodes a microtubule-severing protein, is mutated at high frequency in autosomal dominant hereditary spastic paraplegia. RNA interference (RNAi)-mediated knockdown of Spastin leads to reduced axon length in cultured hippocampal neurons from rats and reduced dendrite growth in Drosophila da neurons in vivo. Overexpression of Spastin in cultured hippocampal neurons has no effect in total axon length although results in increased branch numbers. In Drosophila neuromuscular junction, loss-of-function mutant of spastin exhibits an increase in synaptic bouton number but a reduction in bouton size. In the present studies, it was found that overexpressing Spastin dramatically reduced dendrite growth in Drosophila da neurons. The fact that both RNAi knockdown and overexpression of Spastin lead to reduced dendrite growth is consistent with the notion that proper Spastin levels are important for neurite growth. A slight reduction was observed in axonal length in da neurons on overexpressing Spastin. The in vivo technique does not provide enough resolution to determine whether there is an increase in the number of fine branches of the axons. It is possible that PNS and CNS neurons require distinct levels of Spastin for proper axon development. Alternatively, there might be a difference between mammalian and fly neurons in their requirement of Spastin for axon development (Ye, 2011).
The different phenotypes in axon development between dar1 mutants and Spastin overexpression may be explained by additional factors regulated by Dar1, which inhibits Spastin functions in the axon. It is known that microtubule-associated proteins (MAPs), such as Tau, can shield microtubules from being severed by the severing proteins. In addition, microtubule-severing abilities of the severing proteins are also influenced by posttranslational modifications of tubulin. It is possible that Dar1 also regulates MAPs and/or posttranslational modification of tubulin in concert with Spastin expression (Ye, 2011).
Little is known about how the expression of Spastin is controlled. The current results suggest that Dar1, either directly or indirectly, suppresses the transcription of Spastin. Since proper Spastin levels are important for neurite growth (Riano, 2009), it is likely that Dar1 is required for maintaining proper levels of Spastin in neurons for dendrite growth. In Drosophila, the Collier/Olf1/EBF (COE) family transcription factor Knot, which is expressed in the class IV but not other classes of da neurons, has been proposed to positively regulate Spastin expression as overexpressing Knot increases Spastin transcription (Jinushi-Nakao, 2007). Ectopic expression of Knot in the class I da neurons, which normally have the simplest dendritic arbors among the four classes of da neurons, leads to dendrite overgrowth. However, overexpressing Spastin in class I neurons did not result in dendrite overgrowth that resembles that caused by Knot overexpression. This raises the possibility that, in the class I neurons, Knot induces the expression of other factors together with Spastin to cause dendrite overgrowth. The current results show that the effects of Knot overexpression in class I neurons requires Dar1. Knot overexpression might require factors that are positively regulated by Dar1 to cause dendrite overgrowth. Alternatively, the absence of Dar1 function in that scenario probably results in Spastin levels that are too high to allow dendrite growth (Ye, 2011).
The KLF family of transcriptional regulators has been implicated in a variety of biological processes. At least 17 members of the KLF family have been identified in mammals to date. In contrast, the Drosophila genome encodes only three KLFs: Luna and two predicted molecules, CG12029 and CG9895. Luna has been reported to be a potential fly homolog of KLF6 and 7. RNAi and overexpression studies found that luna is required for embryonic development and cell differentiation in adult eyes; no luna mutant is currently available for additional genetic analysis (Ye, 2011).
Several KLFs are known to be involved in neurite development. KLF7 is required for dendrite and axon development. In the CNS of KLF7-null mice, both dendrite growth and axon growth are reduced (Laub, 2005). In the PNS of these mice, TrkA expression is reduced in the DRG neurons (Lei, 2005). Consequently, NGF-dependent nociceptive neurons undergo increased apoptosis. In trigeminal ganglion neurons, KLF7 cooperates with the POU homeodomain protein Brn3a to control TrkA expression (Lei, 2006). KLF9 also serves important functions in the nervous system. Its expression is strongly induced by thyroid hormone. Furthermore, it is required for neurite growth. It remains to be determined whether KLF9 differentially regulates dendrite or axon development. KLF4 has recently been identified as a suppressor of neurite growth in mammalian CNS neurons. Overexpression of KLF4 reduces both dendritic and axonal length. Consistently, axon length is increased both in cultured neurons and in injured optic nerve of KLF4 knock-out mice (Ye, 2011).
The closest mammalian homolog of Dar1 is KLF5. Overexpression of KLF5 in cultured retinal ganglion cells leads to a modest reduction of neurite length (Moore, 2009); the functions of KLF5 in neuronal development have not been explored using loss-of-function studies or in vivo studies. KLF5 has been reported to be expressed in many tissues, including the brain, and is downregulated in schizophrenia patients. KLF5 homozygous mutant mice die before embryonic day 8.5 and the heterozygous mutant mice have defects in angiogenesis, cardiovascular remodeling, gut development, and adipogenesis. In light of the involvement of Dar1 in Drosophila dendrite development, it will be interesting to examine these KLF5 mutant mice with respect to neural development (Ye, 2011).
It is unknown whether any of the mammalian KLFs function specifically in either dendrite or axon development. Whereas the simplest scenario of such evolutionary conservation is that one of the mammalian KLFs is the ortholog of Dar1, it is equally possible that multiple KLFs coordinate their activities (as proposed by Moore, 2009) to perform the equivalent of Dar1 function (Ye, 2011).
In summary, this study has identified a novel transcription factor and demonstrated its requirement for dendritic but not axonal growth in Drosophila. Future studies that elucidate the regulatory mechanisms of Dar1 and its downstream effector genes will shed light on the genetic program that differentiates dendrite and axon development (Ye, 2011).
Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) has been identified, and D-spastin RNAi-treated or genetic null flies were shown to exhibit neurological defects; protein overexpression decreases the density of cellular microtubules. Elucidating spastins function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. This study has investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities (Roll-Mecak, 2005).
D-spastin from a Drosophila Schneider (S2) cell cDNA library, GFP was fused to its C terminus, and it was expressed in S2 cells in order to establish its subcellular distribution and in vivo activity. Previous localization studies of mammalian spastin have yielded diverse and often conflicting results, including nuclear localization, a mixture of nuclear and cytoplasmic distributions, and endosomal localization. At low expression levels, this study found that D-spastin-GFP was localized to discrete punctate structures that were distributed throughout the cytoplasm, suggestive of membrane vesicle association. Strikingly, cells expressing even relatively low levels of D-spastin-GFP showed diminished microtubule staining and many small microtubule fragments. Cells expressing higher levels of D-spastin-GFP displayed a nearly complete absence of cytoplasmic microtubules. These results are consistent with previous studies showing that spastin overexpression results in microtubule disassembly and with a recent report showing that spastin is targeted to membrane vesicles (Roll-Mecak, 2005).
Spastins are composed of three domains: an N-terminal region containing a putative transmembrane-spanning sequence (TM), a microtubule interacting and trafficking (MIT) domain that is well conserved in the spastin family, and an ATP binding AAA domain (AAA). Because the functions of these domains remain poorly understood, various truncated D-spastin-GFP constructs were expressed in S2 cells. A construct lacking the transmembrane-containing N terminus (ΔTM D-spastin-GFP) was diffusely distributed in the cytoplasm, indicating that the TM domain is required for the vesicular staining pattern observed for the full-length construct. In contrast, a construct that lacked the AAA domain (TM + MIT) still localized to punctate structures but did not affect the microtubule cytoskeleton. The MIT or AAA domains alone were diffusely localized and did not disassemble microtubules. Immunoblot analysis confirmed that these GFP fusion constructs expressed intact proteins. These results indicate that the TM/MIT region is involved in membrane targeting and the MIT/AAA region has activity on the microtubule cytoskeleton (Roll-Mecak, 2005).
The cellular expression do not prove that spastin is a microtubule disassembly agent because such effects might be indirect or require additional cellular cofactors. Therefore whether purified D-spastin is capable of microtubule disassembly in vitro was tested. D-Spastin lacking the TM region was expressed in E. coli as a GST fusion protein and was purified to higher than 95% homogeneity. The purified D-spastin was tested for ATPase activity and it was found to hydrolyze 1.8 ATP/subunit/s, similar to the maximal microtubule-stimulated ATPase rate reported for katanin. Thus far, no stimulation of the ATPase rate by taxol-stabilized microtubules has been observed. When purified D-spastin and ATP were applied to taxol-stabilized, rhodamine-labeled microtubules bound to a glass surface, the microtubules were completely disassembled after 2 min. At earlier time points, microtubules developed discrete breaks along their length. Severing of non-taxol-stabilized microtubules was also observed when D-spastin was added to interphase Xenopus extracts containing self-assembled microtubules. Thus, like katanin, D-spastin is capable of severing microtubules. The microtubule-severing reaction requires ATP hydrolysis; microtubules remained intact if ATP was omitted from the reaction or if the nonhydrolyzable ATP analog ATPγS was added instead of ATP. Microtubules became resistant to spastin-mediated severing when the negatively charged C-terminal peptide of tubulin was cleaved by subtilisin digestion. Subtilisin-treated microtubules are also resistant to severing by katanin (Roll-Mecak, 2005).
These experiments demonstrate that D-spastin alone can couple ATP hydrolysis to microtubule disassembly, and they confirm the cellular results that the MIT-AAA region is sufficient for this activity. It has been reported that the N-terminal TM region in human spastin is crucial for microtubule binding because truncated spastin lacking this region did not cosediment with microtubules. Although it remains possible that the TM region contributes to MT binding affinity, the current data show that the N terminus of D-spastin is not required for microtubule binding and severing (Roll-Mecak, 2005).
Attempts were made to express full-length spastin and the AAA domain in bacteria but they were found to be unstable and formed large aggregates. However, the MIT domain could be expressed and purified. This domain did not sever microtubules, and in cosedimentation binding experiments, it bound to microtubules much more weakly than the ΔTM construct in the absence of nucleotide. The lack of high-affinity binding is consistent with the finding that the MIT domain expressed in S2 cells did not colocalize with microtubules. Thus, high-affinity microtubule binding appears to require the combination of the MIT and the AAA domain or at least additional sequence beyond the minimal conserved-MIT domain investigated in this study (Roll-Mecak, 2005).
More than 140 spastin gene mutations have been isolated from HSP patients. Nonsense, frameshift, or splice site mutations are mostly scattered throughout the gene, whereas missense mutations are located almost exclusively in the AAA domain, underscoring the importance of this ATP binding module for spastin’s function. The 28 known missense mutations provide an array of tools with which to probe spastin’s function. Three such mutations were examined: a critical Walker A residue (K488R) that is predicted to impair ATP binding and mutations situated at the N- (S462C) and C-terminal (D655N) ends of the AAA domain. On the basis of structure-based sequence alignments, D655 resides in the C-terminal helical subdomain of the AAA module that contributes to nucleotide-dependent conformational changes. In addition to these known disease mutations, a mutation was also investigated in a key Walker B residue (E542A) known to be involved in ATP hydrolysis in other AAA+ ATPases (Roll-Mecak, 2005).
When full-length (FL) D-spastin constructs harboring these mutations were expressed in S2 cells, it was found that they localized to distinct punctate structures, as seen with wild-type D-spastin. However, for the Walker A (K488R) and Walker B (E542A) mutants, microtubule disassembly was not observed; instead, at moderate to high expression levels, microtubule bundling was observed around the perinuclear region. Surprisingly, the S462C and D655N mutants still severed microtubules when transfected into cells, although their activity was not as robust as wild-type D-spastin because higher levels of expression appear to be needed to disrupt the microtubule network. This result differs from a study showing that expression of the equivalent serine-to-cysteine human disease mutant did not cause microtubule destabilization (Roll-Mecak, 2005).
In the ΔTM constructs, the Walker A and B site mutants showed very different subcellular localizations. At low expression levels, the ATP-binding-deficient mutant K488R was diffusely distributed, and at high levels it coated and bundled microtubules. In contrast, the ATP-hydrolysis-deficient mutant E542A colocalized with and bundled only a subset of microtubules, perhaps suggestive of cooperative binding or binding to a specific subpopulation of microtubules. As noted for the full-length constructs, the expressed ΔTM S462C and D655N mutant proteins also caused partial microtubule disassembly (Roll-Mecak, 2005).
The biochemical activities of purified D-spastin mutant proteins were examined. The Walker A and B mutations, as expected, displayed neither detectable ATPase activity nor in vitro microtubule-severing activity. In contrast, the S462C and D655N mutant proteins both showed ATPase activity but were 40% and 80% decreased in maximal activity when compared with the wild-type protein. Surprisingly, the S462C and D655N mutants displayed a greater impairment in severing taxol-stabilized microtubules than might be expected on the basis of their ATPase activities. Whereas wild-type spastin almost completely disassembled taxol-stabilized microtubules on a surface within 2 min, the S462C mutant protein generated a comparable degree of disassembly only after >10 min, and the D655N mutant generated only a few microtubule breaks after a 20 min incubation. These data suggest that the S462C and D655N mutations impair ATPase activity but also produce a defect in the coupling of ATPase activity to microtubule destabilization (Roll-Mecak, 2005).
In summary, this study shows that D-spastin severs and disassembles microtubules both in cells and in vitro. These results provide the first direct biochemical evidence of spastin's ATPase and microtubule-destabilizing activities. It is interesting to compare spastin to katanin, another AAA+ ATPase that severs microtubules. Spastin's AAA domain is highly homologous to katanin, yet these proteins share no sequence similarity in their N-terminal regions. Thus, spastin and katanin appear to have evolved distinct microtubule binding domains that communicate with a similar motor module to bring about the severing reaction. Unlike katanin, which appears to be largely soluble or centrosome associated, spastin appears to associate with vesicular membranes through its N-terminal domain containing a predicted transmembrane-spanning sequence. This raises the possibility that spastin may be involved in remodeling the microtubule cytoskeleton near membrane surfaces, which may be important for spastin's function in synaptic architecture and transmission. Further investigations linking spastin's enzymatic activity to its cellular function will be required to better understand spastin's role in the nervous system as well as the mechanism by which spastin mutations give rise to the pathology of hereditary spastic paraplegias (Roll-Mecak, 2005).
Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of γ-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing γ-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden the understanding of the molecular machinery used to move chromosomes (Zhang, 2007).
The results of this study show that three closely related MT severing enzymes, Dm-Kat60, Spastin, and Fidgetin, are important for mitosis in D. melanogaster S2 cells. Interestingly, the activity of these proteins is segregated both spatially and temporally, allowing them to perform complementary functions throughout the spindle. This is most apparent during anaphase A, when all three are integrated into the Pacman-flux machinery used to move chromosomes (Zhang, 2007).
Spastin and Fidgetin emerge from this study as new regulators of poleward MT flux. Specifically, inhibition of either protein results in a significant reduction in flux velocity. In addition, it was found that both proteins similarly promote the turnover of γ-tubulin at spindle poles and γ-tubulin at centrosomes. In sum, these data are consistent with a general model for flux and chromosome motility, in which Spastin and Fidgetin function to release MT minus ends from their nucleating γ-TuRCs, which are believed to cap and stabilize MT ends. In turn, severing exposes minus ends to depolymerization by spindle pole-associated Kinesin-13 (KLP10A in D. melanogaster), which has been shown to also contribute to flux. During anaphase A, the MT minus-end depolymerization of flux 'reels in' chromosomes to the poles (Zhang, 2007).
Based on the proposal that Spastin and Fidgetin work in concert with KLP10A to promote flux, one would expect many similarities in the phenotypes resulting from the inhibition of these proteins. Indeed, as in Spastin or Fidgetin RNAi-treated cells, depletion of KLP10A also inhibits flux and slows anaphase A. A notable difference, however, is that KLP10A RNAi induces spindle elongation, whereas Spastin or Fidgetin RNAi does not. One possible explanation for this apparent inconsistency stems from the fact that spindles probably elongate as a result of continued plus-end polymerization and MT sliding when minus-end depolymerization (i.e., flux) is decreased after KLP10A RNAi. Thus, the puzzling absence of spindle elongation after Spastin or Fidgetin RNAi might be explained by the observation that plus-end polymerization is also significantly decreased in these cells (Zhang, 2007).
Although these data demonstrate roles for Spastin and Fidgetin in regulating flux and MT-centrosome interaction (i.e., catalyzing the turnover of γ-tubulin and regulating abnormal spindle-mediated attachments with MT minus ends), it is currently unclear whether centrosomes are the sole or even primary site of action of these proteins in the spindle. Indeed, the presence of centrosomes is not required for spindle assembly, flux, and chromosome segregation in some D. melanogaster cell types and other systems such as oocyte spindles. Additionally, even in centrosome-containing cells, the majority of MT minus ends are often positioned at a distance from centrosomes, and many spindle MTs are thought to arise from noncentrosomal sources (e.g., chromosomes/kinetochores). These MTs may still be capped by cytoplasmic γ-TuRCs, and it is conceivable that severing within the spindle (i.e., away from centrosomes) is required for their normal dynamics and flux. Thus, although this model depicts Spastin and Fidgetin as functioning only at centrosomes (where these proteins concentrate), this may be an oversimplification (Zhang, 2007).
Why both Spastin and Fidgetin would be used for the same task is unclear. At present, there is no clear evidence for a functional or physical interaction between these proteins. Each protein might sever a distinct subset of centrosomal MTs, but this would be unprecedented, and it is therefore considered unlikely. Unfortunately, coinhibition of these proteins by RNAi causes a high degree of cell death, making it difficult to assess this possibility. Alternatively, a degree of functional redundancy may explain why a small portion of D. melanogaster carrying null mutations in the spastin gene survive to adulthood. Genetic analysis of the relationship between these proteins should be revealing and may help answer this question (Zhang, 2007).
It is notable that Dm-Kat60 also localizes to centrosomes but performs no obvious function there, at least based on the assays used in this study. However, Dm-Kat60 RNAi does impact the mitotic index, which is likely indicative of subtle preanaphase Dm-Kat60 activities, which are beyond the sensitivity of current visualization techniques (Zhang, 2007).
Although Dm-Kat60 does not appear to function at centrosomes, the data indicate that this protein plays an important role in moving anaphase chromosomes (McNally, 1993). Dm-Kat60, which localizes to both chromosome arms and kinetochores, functions during anaphase to stimulate the depolymerization of MT plus ends, thereby moving chromosomes by a Pacman mechanism. It is proposed that Dm-Katanin functions in this regard by uncapping MT plus ends -- much the same as Spastin and Fidgetin do at minus ends -- and exposing them to depolymerization by centromere/kinetochore-associated Kinesin-13, which is also required for Pacman. Although Pacman-inhibiting plus-end caps have not been identified, several MT-stabilizing microtubule-associated proteins (such as the plus-end tracking proteins CLASP, EB1, and CLIP-190) associate with kinetochore-associated MT plus ends. Whether the association of these proteins with plus ends inhibits depolymerization by Kinesin-13s is unknown (Zhang, 2007).
Additionally, severing by Katanin could uncap plus ends associated with chromosome arms. A vertebrate kinesin, XKLP1, which targets to chromosome arms, has been shown to bind and stabilize MT plus ends and would probably resist Pacman motility. The D. melanogaster genome encodes several potential XKLP1 homologues, and it will be interesting to see whether Katanin has an antagonistic relationship with any of these (Zhang, 2007).
The possibility cannot be ruled out that D. melanogaster Katanin directly stimulates the depolymerization of kinetochore-associated MT plus ends. Indeed, it could conceivably supplant chromosome-associated Kinesin-13s in some systems, potentially explaining why the Kinesin-13 KLP59C does not appear to play a direct role in chromosome motility in S2 cells even though it drives Pacman in D. melanogaster embryos. However, another Kinesin-13 that is needed for Pacman in S2 cells has been identified (unpublished data of Zhang, 2007), making it unlikely that Dm-Katanin directly depolymerizes plus ends (Zhang, 2007).
It is notable that Spastin and Fidgetin also target to chromosomes before anaphase, where they may function similarly to Dm-Katanin. FRAP analysis indicates that both proteins normally enhance the turnover of chromosome-associated plus ends on preanaphase spindles. Why the chromosome activity of these proteins is down-regulated at the onset of anaphase while Katanin, which associates with chromosome throughout mitosis, is up-regulated is unclear. The loss of Spastin and Fidgetin from chromosomes may result from the underlying dependence of this targeting on MTs. Both are released from chromosomes in the presence of colchicine, and alterations in MT dynamics that accompany the onset of anaphase may have the same effect. Alternatively, Katanin's activity may be up- or down-regulated by phosphorylation. Indeed, the primary sequence of Dm-Kat60 contains several putative CDK1 phosphorylation motifs. Finally, Katanin's severing activity may be negatively regulated by MT-coating microtubule-associated proteins (Zhang, 2007).
Interestingly, Katanin does not appear to target to chromosomes or kinetochores in many cell types. In fact, the only system besides D. melanogaster in which a Katanin homologue has been reported to associate with chromosomes is C. elegans, which does not use Katanin for mitosis. This raises the question of whether the mitotic functions of MT severing proteins, particularly Katanin, are conserved throughout phylogeny. In this regard, it is noted that several additional Katanin p60 homologues whose functions have not yet been analyzed have been identified within vertebrate and invertebrate genomes. Any of these could target to chromosomes and stimulate Pacman-based anaphase A. Moreover, a recent yeast two-hybrid study has shown that Fidgetin associates with the protein kinase A anchoring protein, AKAP95, which targets to chromosomes throughout mitosis. D. melanogaster contain no obvious AKAP95 homologue, perhaps explaining why Fidgetin does not impact Pacman in this system. Future studies to examine the possible mitotic functions of vertebrate Fidgetin and Katanin homologues would address this question (Zhang, 2007).
In closing, this study suggests a general mechanism in which appropriately positioned and tightly regulated MT severing proteins provide a means to rapidly create free MT ends, which are then exposed to the actions of other regulatory proteins. During anaphase, such an activity works in close coordination with Kinesin-13s, stimulating poleward chromatid motility by a combined Pacman-flux mechanism. In other instances, the creation of free ends could have a very different impact on MT behaviors. Future analyses examining the interactions between severing proteins and Kinesin-13s, as well as other regulators of MT dynamics, will help test this proposal (Zhang, 2007).
Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). This study shows that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin is sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolishes LD targeting. Increased levels of spastin-M1 expression reduce the number but increase the size of LDs. Expression of a mutant unable to bind and sever MTs causes clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila leads to bigger and less numerous LDs in the fat bodies and increases triacylglycerol levels. In contrast, Dspastin overexpression increases LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreases LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduces triacylglycerol levels in the larvae. Moreover, reduced amount of fat stores were found in intestinal cells of worms in which the spas-1 homologue is either depleted by RNA interference or deleted. Taken together, these data uncover an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP (Papadopoulos, 2015).
Studies using expression systems of wild-type and mutated mammalian spastin with reporter sequences in different cell lines have suggested an involvement of spastin in microtubule dynamics (Errico, 2002). The spatial and temporal expression patterns of Drosophila spastin were analysed by in situ hybridization. For this, the EST clone AT01057 that covers spastin was used to generate digoxigenin-labeled sense and antisense RNA probes. These were hybridized to wild-type whole-mount embryos. Maternally contributed spastin transcripts characterized early embryogenesis with high expression levels seen until blastoderm stage. At the cell formation stage, expression was strongest near the basal part of the cell layer underlying the surface. During germband extension and stomodeal plate formation, expression was seen in the ventral head and trunk ectoderm, as well as in cells near the cephalic furrow and in the invaginating hindgut and midgut primordia. After germband retraction and delamination of neuroblasts at stage 13, transcripts were observed in subsets of cells in all neuromeres of the CNS including those of the supraesophageal and subesophageal ganglia. In later embryonic stages, marked expression of spastin was observed in cell clusters throughout the supraesophageal ganglion, with pronounced expression also seen in the subesophageal ganglion. In the ventral nerve cord (VNC), transcripts were seen in two broad longitudinal stripes located laterally, and weaker expression was observed in some midline cells. In addition to expression in the CNS, spastin transcripts were also observed in some sensory head organs of the peripheral nervous system (PNS), most probably the Bolwigs organs and/or the dorsal organs. Thus, during embryogenesis, expression of Drosophila spastin is mainly restricted to the central nervous system, in contrast to the ubiquitous expression (Hazan, 1999) of the vertebrate spastin gene (Kammermeier, 2003).
Spastin protein is highly enriched within the nervous system of mammals. Recent work has shown that the Dspastin message is similarly enriched in the nervous system during Drosophila embryonic development (Kammermeier, 2003). To determine the endogenous D-Spastin protein expression pattern at maturity and, in particular, to define the subcellular localization of D-Spastin protein in neurons, a polyclonal antibody was generated against Drosophila Spastin. The specificity of this antibody was determined by Western blot analyses, which revealed a band of the predicted size for D-Spastin (84 kDa). Focus was placed specifically on D-Spastin expression in the larval neuromusculature by using immunohistochemistry with the D-Spastin antiserum (Trotta, 2004).
D-Spastin protein is present in the cytoplasm of both neurons and muscles but is not detectably expressed in nuclei. While the protein is present at a low level throughout the neuromusculature, muscles, and neuronal axons, D-Spastin is particularly enriched at NMJs throughout the body-wall muscles. NMJs on the well-characterized four ventral longitudinal muscles (12, 13, 6 and 7) all exhibit similar levels of D-Spastin immunoreactivity. The protein appears enriched in punctate domains of synaptic boutons and relatively excluded from interbouton axonal connectives. Colabeling with D-Spastin and the integral synaptic vesicle protein Synaptotagmin indicates that D-Spastin colocalizes with the synaptic vesicle pools and is not appreciably concentrated outside of the presynaptic domain. Neuronal-specific overexpression of D-Spastin with the Elav-GAL4 driver greatly enhances NMJ protein expression and further emphasizes the protein enrichment specifically within presynaptic boutons. Elav-GAL4 driven Dspastin RNAi greatly reduces D-Spastin immunoreactivity at the NMJ, again suggesting that the protein is enriched only presynaptically and that the RNAi transgene is effective in reducing protein levels (Trotta, 2004).
In order to alter D-Spastin expression in a targeted fashion within the nervous system, multiple, independent transgenic lines were constructed for both (1) the overexpression of the wild-type protein, and (2) RNAi-mediated knockdown of the protein. For overexpression, the Dspastin coding region was cloned downstream of the UAS responder element (UAS-Dspastin) and stably transformed into the Drosophila genome to generate multiple, independent transgenic lines. For inducible RNAi knockdown, a Dspastin cDNA fragment was fused with the corresponding genomic sequence. This sequence was then placed under the control of a UAS promoter (UAS-RNAi) and stably transformed multiple times into the Drosophila genome. UAS transgenes were driven in tissue-specific patterns utilizing established GAL4 lines including Tubulin-GAL4 (ubiquitous expression), Elav-GAL4 (nervous system-specific expression; presynaptic at the NMJ), and Myosin heavy chain (MHC)-GAL4 (muscle-specific expression; postsynaptic at the NMJ) (Trotta, 2004).
The efficacy of transgenic RNAi was assayed in two ways: (1) RT-PCR of total mRNA harvested from animals expressing UAS-RNAi ubiquitously driven by the Tubulin-GAL4 driver, and (2) anti-D-Spastin immunocytochemistry on animals expressing UAS-RNAi in tissue-specific patterns. Ubiquitous expression of the RNAi construct decreases Dspastin mRNA levels as assayed by RT-PCR. Amplification of Dspastin mRNA in all control lines results in a robust product of the expected size (2 kB), whereas UAS-RNAi; Tubulin-GAL4 individuals show no detectable Dspastin mRNA band, but only a product amplified from genomic DNA. This genomic fragment contamination persists when template RNA is pretreated with DNase I and also appears when reverse transcriptase is omitted from the RT-PCR reaction mixture. These results indicate that the UAS-Dspastin-RNAi transgene effectively and specifically reduces Dspastin mRNA (Trotta, 2004).
Ubiquitous expression of Dspastin UAS-RNAi results in pupal lethality, suggesting that Dspastin is an essential gene. An important caveat to this conclusion, however, is that RNAi-mediated knockdown of D-Spastin protein levels are analyzing and not a null Dspastin mutant. The highest levels of pan-neuronal RNAi expression do not result in preadult lethality. However, animals expressing Dspastin RNAi in the nervous system display compromised adult coordination and locomotory behavior (Trotta, 2004).
Transgenic overexpression of a number of independent UAS-Dspastin lines, driven either with ubiquitous or neuronal GAL4 drivers, resulted in 100% lethality during embryonic or very early larval stages. A single copy of the UAS-Dspastin transgene and a single copy of the GAL4 driver were sufficient to cause this early lethality in most cases. Targeted expression of UAS-Dspastin in subsets of the adult nervous system (the eye, for example) causes severe neurodegeneration. Given the deleterious effect of Dspastin overexpression on neuronal viability, a particularly weakly expressing UAS-Dspastin transgenic line that permits full viability was selected for further experiments (Trotta, 2004).
Previous in vitro studies indicate that mammalian Spastin associates with microtubules. Regulation of the microtubule cytoskeleton has been well documented to control growth and elaboration of the presynaptic terminal at the Drosophila NMJ. Synaptic morphology was assayed in Dspastin transgenic animals by confocal imaging with an antibody against HRP to label neuronal membranes and the area of the synaptic region was calculated by using confocal software (Trotta, 2004).
The overall morphology of the NMJ synapse was not significantly altered in any of the genetic control lines. Calculation of synaptic terminal area at the muscle 6/7 NMJ showed 826 ± 60 μm2 in Elav-GAL4/+ driver animals alone, with similar synaptic areas in UAS-Dspastin/+ (808 ± 64 μm2) and UAS-RNAi/+ (679 ± 67 μm2) (no significant change). In contrast, neural-specific Dspastin RNAi expression causes a severe reduction of total synaptic area compared to all controls. For example, at the muscle 6/7 NMJ, Dspastin RNAi (Elav-Gal4; UAS-RNAi) reduces the area of the presynaptic terminal by 50%, from 826 ± 60 μm2 in control to 429 ± 67 μm2 in mutant (p < 0.005). Neural overexpression of D-Spastin conferred no significant effect on gross synaptic area, with the overexpression line (Elav-GAL4; UAS-Dspastin) exhibiting a mean area of 782 ± 72 μm2 compared to 808 ± 64 μm2 in controls (no significant change). These data indicate that D-Spastin may play a regulatory role in synaptic growth (Trotta, 2004).
Electrophysiological studies of neural function in HSP patients have been limited by the techniques available and small sample sizes. Since D-Spastin localizes to presynaptic boutons and regulates synaptic architecture at the Drosophila NMJ, synaptic function was examined in animals with altered Dspastin dosage. This glutamatergic NMJ has been the subject of extensive morphological and physiological studies. Changes in synaptic efficacy sometimes appear to compensate for changes in synaptic area and in other cases functional strength parallels changes in synaptic area, and therefore the relationship between these parameters is variable. A two-electrode voltage-clamp recording configuration was employed to record synaptic currents at the NMJ in animals with altered D-Spastin expression specifically within the presynaptic terminal (Trotta, 2004).
In normal animals, stimulation of the motor nerve with a suction electrode elicits large, highly reproducible glutamate-gated synaptic currents in the voltage-clamped (-60 mV) muscle. All genetic control animals exhibited indistinguishable mean excitatory junctional current (EJC) amplitudes similar to wild-type in the range of 81-86 nA. These results show that the presence of GAL4 driver or UAS transgenes alone does not alter basal synaptic function. In contrast, altering the D-Spastin level in the presynaptic terminal has dramatic effects on synaptic efficacy. Neural overexpression of D-Spastin (Elav-GAL4; UAS-Dspastin) causes a highly significant reduction in mean EJC amplitude, down to 47.3 ± 8.5 nA in mutants compared to 85.3 ± 3.8 in control (p < 0.005). On the contrary, loss of D-Spastin in neurons (Elav-GAL4; UAS-RNAi) had the opposite effect of increasing synaptic currents by >50%. The mean EJC amplitude in RNAi lines is 138.9 ± 17.3 compared to 85.3 ± 3.8 in control (p < 0.05) (Trotta, 2004).
Axon length has been postulated as a cellular determinant for spastin-mediated neural dysfunction. Therefore EJC amplitudes in an anterior abdominal segment (A3, above) with a short motor nerve was compared to EJC amplitudes in a posterior segment (A6) with a much longer motor nerve. It was predicted that the severity of neurotransmission defects might correlate with motor axon length. In contrast to this prediction, neurotransmission defects were observed in A6 that were indistinguishable from those in A3. In both cases, D-Spastin overexpression caused comparable suppression of neurotransmission, and Dspastin RNAi caused comparable enhancement of neurotransmission. Thus, within the limits of this system, no correlation was found between axon length and the manifestation of Spastin-dependent alterations in synaptic efficacy or neurotransmission strength (Trotta, 2004).
Spastin regulates microtubule stability in the presynaptic terminal: In mammals, Spastin has been shown to dismantle the microtubule cytoskeleton, with an activity similar to its AAA-family relative, Katanin (Errico, 2992; Hartman, 1998). The capacity of D-Spastin to regulate microtubule stability locally at the synapse was examined by using anti-Tubulin antibodies as well as antibodies specific for both acetylated α-Tubulin and glutamylated β-Tubulin, posttranslational modifications that occur only in structurally stable microtubules. Western analyses with antibodies against each of these specific forms of Tubulin reveal a single band of the predicted molecular weight, indicating specificity (Trotta, 2004).
The dosage level of D-Spastin appears to affect the abundance and distribution of acetylated α-Tubulin within neurons. Neural-specific overexpression of D-Spastin (Elav-GAL4; UAS-Dspastin) causes a reduction of acetylated α-Tubulin within neurons and specifically at NMJ presynaptic terminals. After D-Spastin overexpression, bundled acetylated α-Tubulin in axons appears thinned and shortened and, often, the stabilized Tubulin network is no longer detectable within interbouton connectives of the NMJ. In contrast, D-Spastin knockdown in the neuron (Elav-GAL4; UAS-RNAi) results in an accumulation of acetylated α-Tubulin within the neuron and specifically at the NMJ presynaptic terminal. In the Dspastin RNAi lines, acetylated α-Tubulin immunoreactivity is more prominent in the neuronal axon and within the NMJ. After Dspastin RNAi, stabilized microtubules form loops or twists in NMJ boutons, a rare feature in normal synaptic boutons. These data suggest a role for Dspastin in destabilizing the microtubule cytoskeleton in neurons, including synaptic bouton regions responsible for presynaptic function during neurotransmission (Trotta, 2004).
It is difficult to resolve the fine structure of the microtubule cytoskeleton within the presynaptic terminal itself. Therefore the Dspastin transgenes were expressed under the control of the MHC-GAL4 driver to alter Dspastin levels in the much larger muscle cells. In muscles, individual microtubules can be visualized by confocal imaging with Tubulin and acetylated α-Tubulin antibodies. These microtubules overlap in a loose meshwork, as opposed to microtubules in neurons, that are arranged in parallel in long bundles. Overexpression of D-Spastin in the muscle (MHC-GAL4; UAS-Dspastin) results in a decrease in the acetylated α-Tubulin staining intensity, suggesting that microtubules are less stable. Although the effect is less than in the neuronal terminal, the number of acetylated α-Tubulin microtubules appears to be fewer and the density of the stabilized microtubule mesh is reduced throughout the muscle cell. In contrast, Dspastin RNAi in muscles (MHC-GAL4; UAS-RNAi) has the opposite effect of increasing the acetylated α-Tubulin staining intensity, suggesting that microtubules have become more stable. Again the effect is less pronounced than in the neuronal terminal, but there is a clear increase in the number of acetylated α-Tubulin microtubules, resulting in a thick mesh-like network throughout the multinucleate muscle cell. These data recapitulate the observations made in the NMJ and corroborate the notion that D-Spastin destabilizes microtubules (Trotta, 2004).
To extend these studies in the synaptic terminal, subcellular colocalization of total Tubulin was examined in colabeling studies with both postranslationally modified acetylated and glutamylated forms of Tubulin. In control animals, total Tubulin immunoreactivity is similar to modified forms, both in localization and intensity. After normalizing fluorescence intensity of microtubule signal to HRP signal intensity, no significant quantifiable change was found between their immunoreactivities at wild-type synapses. In overexpressing animals, signal intensities are significantly reduced for Tubulin, glutamylated Tubulin, and acetylated forms of Tubulin compared to fluorescence intensities in control animals. Also consistent with previous observations, neurons expressing D-Spastin RNAi exhibit increased levels of postranslationally modified forms of Tubulin. These changes are accompanied by a reduction in total Tubulin immunoreactivity at the synapse. This finding was unexpected, and was initially suspected to be an artifact due to preferential binding of the modified Tubulin antisera competing with the Tubulin antibody. However, by using a different Tubulin antisera, both alone and coincubated with modified Tubulin antibodies, these observations were confirmed, suggesting that reduced D-Spastin causes a dramatic decrease in the amount of free Tubulin dimers unincorporated into filaments (Trotta, 2004).
D-Spastin localizes to synaptic boutons, colocalizing with the synaptic vesicle marker anti-synaptotagmin, and altering D-Spastin dosage changes the stability of the microtubule cytoskeleton within the presynaptic arbor. To further visualize how D-Spastin regulates microtubule stability at the synapse, individual NMJ presynaptic terminals were triple labeled with antibodies against Synaptotagmin, D-Spastin, and acetylated α-Tubulin and examined the local synaptic domains in neurons following over- and underexpression of D-Spastin. In control animals, D-Spastin localizes within synaptic boutons, subcellular regions where stable microtubules are absent, exhibiting reduced acetylated Tubulin immunoreactivity. Overexpression of D-Spastin causes a thinning, and at times disappearance, of stabilized microtubules; however, exclusion of D-Spastin from interbouton connectives is maintained. In D-Spastin knockdown animals, bundled microtubules appear more stable, more prominent, and take up more of the presynaptic compartment. Subcellular compartments of the synaptic vesicle/D-Spastin domain are distinct from the stabilized microtubule domain in control animals, while this compartmentalization is compromised with the loss of D-spastin. To quantify these data, mean pixel intensity was recorded for all three channels within the Syt-positive region of the bouton. Control animals exhibit relative intensities of 67.7% ± 5.2% for D-Spastin and 49.0% ± 2.4% for acetylated Tubulin within synaptic boutons. D-Spastin exhibits a quantifiable increase in synaptic localization during overexpression (85.9% ± 5.1%) and a corresponding decrease in intensity when RNAi is expressed in neurons (20.2% ± 3.1%). Acetylated Tubulin immunoreactivity exhibits a complimentary pattern compared to D-Spastin, showing increased intensity in Syt+ bouton regions of RNAi expressing animals (71.6% ± 2.3%) and a reduction during D-Spastin neural overexpression (30.7% ± 2.2%). These results suggest that D-Spastin may play a role in spatially restricting stabilized microtubules in the presynaptic terminal (Trotta, 2004).
Synaptic function is rescued by restoring microtubule stability: The above results show that D-Spastin regulates synaptic structure and function, and locally regulates the stability of the microtubule cytoskeleton within the NMJ synapse. A pharmacological approach was used to test for a causal relationship between synaptic properties and microtubule stability. Tubulin monomers are stably polymerized by taxol while microtubules are rapidly disassembled by nocodazole. Taxol treatment should therefore counter the effect of Dspastin overexpression, and nocodazole treatment should counter the effect of Dspastin RNAi. These pharmacological agents were acutely (1 hr) applied on dissected larval preparations of these transgenic animals and their consequences were assayed on synaptic function. Similar approaches have provided verification of synaptic mechanisms in the Drosophila NMJ system (Trotta, 2004).
Previous studies have suggested that acute pharmacological treatment with drugs that alter microtubule dynamic instability dynamics does not significantly alter synaptic vesicle cycling at the wild-type NMJ. Similarly, it was found that treatment of control animals with either taxol or nocodazole produced no significant change in mean EJC amplitudes. Neural overexpression of D-Spastin causes decreased microtubule stability and decreased EJC amplitude. These animals (Elav-GAL4; UAS-Dspastin) were therefore pretreated with taxol to stabilize microtubules and then assayed for rescue of synaptic transmission. Taxol treatment elevated EJC amplitudes from a mean of 23.1 ± 6.5 nA in mock-treated animals to a mean of 49.8 ± 8.7 nA in taxol-treated animals. RNAi-mediated knockdown of D-Spastin causes increased microtubule stability and increased EJC amplitude. These animals (Elav-GAL4; UAS-RNAi) were therefore pretreated with nocodazole to destabilize microtubules and then assayed for rescue of the EJC defect. Synaptic currents in mock-treated animals averaged 75.6 ± 6.4 nA, whereas nocodazole-treatment decreased EJC amplitudes to 41.1 ± 8.6, which is statistically indistinguishable from controls. Converse pharmacological experiments were also performed in an effort to enhance the microtubule stability phenotypes previously described. Taxol pretreatment in RNAi-expressing animals resulted in a slight, but statistically irrelevant, change in synaptic currents (92.5 ± 19.7 nA), suggesting that these animals are not sensitive to additionally stabilized microtubules. D-Spastin-overexpressing animals exposed to nocodoazole also exhibit no net change in EJC amplitudes compared to mock-treated controls. These data show that synaptic function can be rescued by pharmacologically restoring, as appropriate, the stability of microtubules in animals with altered levels of neuronal D-Spastin and further suggest that these trangenic conditions cannot be enhanced pharmacologically, conferring maximal effects on synaptic transmission (Trotta, 2004).
The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, the opposite phenotype is observed: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that is observed in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord (Sherwood, 2004).
The AAA domain of Spastin is quite similar to that of Katanin-60, which is a microtubule-severing protein. To determine whether Spastin might also sever or otherwise alter microtubules in vivo, the protein was overexpressed in embryonic and larval muscles. Strikingly, this overexpression erases or greatly reduces the microtubule network. These data are consistent with the finding that overexpression of human Spastin in transfected mammalian cells causes microtubule disassembly (Sherwood, 2004).
Having demonstrated that Spastin can cause disassembly of microtubules in vivo, how its absence affects the synaptic microtubule cytoskeleton was examined. Based on the overexpression phenotype, one might have expected that microtubules would be more stable or more numerous in spastin LOF mutants. However, the observations indicate the opposite: microtubule bundles are depleted in NMJ boutons when Spastin is absent (Sherwood, 2004).
At the wild-type muscle 4 NMJ, boutons are arranged along linear axes. Continuous microtubule bundles run along the axes and connect to larger bundles within the innervating axon. Microtubules within boutons are typically arranged in loops and swirls. In spastin-null mutants, boutons are arranged in clumps, and the distal boutons of these clumps often lack any detectable tubulin staining. Looped microtubule structures are present within some proximal boutons, however, and the bundles connecting the NMJ to the axon are still present. These results suggest that the absence of Spastin selectively affects the construction of the presynaptic microtubule cytoskeleton, and that the severity of the microtubule defects in a bouton are correlated with its distance from the NMJ's axonal branchpoint (Sherwood, 2004).
These defects were quantitated using an antibody against the microtubule-associated Futsch protein, which defines a subpopulation of stable neuronal microtubule bundles. In wild-type larvae, Futsch staining forms continuous lines along the main branches of the NMJ. Some individual boutons have Futsch loops, while others display only diffuse staining. A comparison of wild-type and spastin-null larvae shows that the distribution of Futsch within boutons shifts from organized structures (bundles and loops) toward diffuse patterns or the absence of detectable staining. This effect is most pronounced at terminal boutons, and is rescued by neuronal expression of Spastin (Sherwood, 2004).
If Spastin's function in vivo is to disassemble microtubules, as suggested by the overexpression experiments, why does its absence produce a paradoxical reduction in microtubules within the NMJ? One possibility is that microtubule severing is required for movement of microtubules into or within the presynaptic region. Some evidence for this idea has been published. In one study, injection of function-blocking anti-Katanin-60 antibody into cultured sympathetic neurons reduced process outgrowth, and microtubules were 4- to 5-fold longer in antibody-injected neurons than in control cells. Recent work demonstrated that expression of dominant-negative Katanin-60 reduces axonal outgrowth (Karabay, 2004). These results were interpreted as indicating that Katanin is required for severing microtubules to a length that allows their transport along the axon to its growing tip. When Katanin is inhibited, microtubule segments may be too long to be efficiently transported, and this results in a reduction in axon outgrowth (Sherwood, 2004).
Based on these findings, it is suggested that the depletion of microtubules in the distal boutons of spastin mutant NMJs arises because severing of axonal microtubules by Spastin is necessary to generate microtubule polymers that are short enough to be efficiently moved into and through the presynaptic terminals. Perhaps Spastin normally excises sections of microtubules at branchpoints where NMJ branches leave the axon trunk, and these severed microtubule segments (or individual tubulin dimers) are then moved distally into the boutons of the NMJ as it grows (Sherwood, 2004).
Is Spastin also involved in axon outgrowth or guidance, as suggested by its embryonic gain-of-function phenotype? Clearly loss of Spastin activity in Drosophila does not strongly affect outgrowth, since the embryonic CNS axon ladder develops in a normal manner and motor axons reach their appropriate targets in spastin mutants. Furthermore, axonal and muscle microtubules are not detectably altered in spastin-null embryos. Severing of microtubules in vivo, however, may usually involve the actions of multiple severing proteins. In addition to Spastin, the Drosophila genome encodes three AAA ATPases whose AAA domains are closely related to that of vertebrate Katanin-60. These are Katanin-60, CG1193, and an ortholog of mammalian Fidgetins, CG3326. None of these proteins have been genetically characterized. Of these four proteins, Spastin is most distant from vertebrate Katanin-60, yet this study has shown that Spastin overexpression causes microtubule disassembly in vivo. Thus, the results suggest that all four fly proteins are microtubule-severing enzymes or proteins that otherwise facilitate disassembly of microtubule networks. Perhaps each is dedicated to severing microtubules in particular cellular and subcellular contexts, and their functions may be partially redundant. If so, generation of severe phenotypes in which microtubule networks are disrupted might require loss of two or more of these AAA ATPases. In mammals, Katanin and Spastin are both expressed in CNS neurons (Wharton, 2003; Karabay, 2004), consistent with the idea that they could have overlapping functions (Sherwood, 2004).
After this manuscript was submitted for initial review, a paper appeared on perturbation of Drosophila spastin using transgenic RNAi techniques (Trotta, 2004). In direct contrast to the current results, this paper concluded that (1) spastin is an essential gene (since crossing spastin RNAi flies to a ubiquitous GAL4 driver line was reported to produce lethality), (2) spastin RNAi larvae have reduced NMJs and an increase in synaptic transmission, and (3) loss of Spastin from neurons produces an increase rather than a decrease in stable microtubules in the NMJ (Sherwood, 2004).
The conclusions in the current paper are based on phenotypic analysis of spastin mutations that delete part or all of the coding region and on rescue of null mutant phenotypes by neuronal expression from a transgene. The results show that spastin is not an essential gene: spastin-null flies can even eclose and live for several days, and spastin hypomorphs, which would be expected to more closely resemble most RNAi-perturbed flies, eclose at normal rates and have lifespans and behavior that do not greatly differ from wild-type. The 17-7 mutation, which removes more than one-third of the coding region, produces no detectable alterations in bouton number or NMJ microtubules and slightly decreases synaptic transmission, while spastin-null mutants have more boutons than wild-type larvae, a reduction in NMJ microtubule bundles, and more severely reduced transmission (Sherwood, 2004).
In most transgenic RNAi work in Drosophila, different transgenic lines yield phenotypes that range from hypomorphic to near-null, and transgenic RNAi does not completely eliminate expression of the target protein. In the Trotta (2004) paper, it is unclear whether more than one transgenic RNAi line was analyzed, but RNAi is described as reducing the level of Spastin protein expression by less than 4-fold. The current findings on Spastin hypomorphic phenotypes imply that such RNAi larvae would not have morphological or microtubule bouton phenotypes and that adult flies would be relatively healthy. The origin of the discrepancies between the two sets of results is not understood (Sherwood, 2004).
Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases characterized by progressive weakness and spasticity of the lower limbs. Dominant mutations in the human SPG4 gene, encoding spastin, are responsible for the most frequent form of HSP. Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin (Dspastin), has been shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. This paper report the generation of a spastin-linked HSP animal model and shows that in Drosophila, neural knockdown of Dspastin and, conversely, neural overexpression of Dspastin containing a conserved pathogenic mutation both recapitulate some phenotypic aspects of the human disease, including adult onset, locomotor impairment, and neurodegeneration. At the subcellular level, neuronal expression of both Dspastin RNA interference and mutant Dspastin cause an excessive stabilization of microtubules in the neuromuscular junction synapse. In addition, evidence is provided that administration of the microtubule targeting drug vinblastine significantly attenuates these phenotypes in vivo. These findings demonstrate that loss of spastin function elicits HSP-like phenotypes in Drosophila, provide novel insights into the molecular mechanism of spastin mutations, and raise the possibility that therapy with Vinca alkaloids may be efficacious in spastin-associated HSP and other disorders related to microtubule dysfunction (Orso, 2005).
Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Loss of the Drosophila ortholog of Atl-1 (Atlastin) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. This study provides evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and α-spectrin. Evidence is provided that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, this study demonstrates that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, these results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability (Lee, 2009).
During Drosophila larval development, there is a coordinated growth of the muscle and NMJ. The current results suggest that these developmental processes are impaired in the absence of Atl. In atl mutant larvae, NMJ boutons are smaller and more numerous than in wild-type larvae. In addition, the size of body-wall muscles is significantly reduced. These phenotypes are rescued by muscle-specific expression of Atl, suggesting that Atl functions primarily in the postsynaptic muscle to control NMJ synapse and muscle growth. This conclusion is further supported by the observation that targeted expression of a dominant-negative form of Atl (AtlR192Q) in muscles, but not in neurons, mimics atl mutations to increase bouton number per muscle surface area (Lee, 2009).
In Drosophila, type I NMJ boutons are surrounded by the subsynaptic reticulum (SSR), which is highly elaborated infoldings of the muscle membrane. The SSR begins to form during the first larval instar and increases in size and complexity throughout late larval life. Ultrastructural analysis indicates that the SSR is less extensive and disorganized in atl mutants. Consistent with this finding, defects were observed in the levels and distribution of Dlg and spectrin, the synaptic scaffold proteins that play critical roles for SSR expansion and organization. Thus, these results suggest that Atl is required for the formation and/or maintenance of normal postsynaptic structure (Lee, 2009).
In mammals, there are three members of the atlastin subfamily of large GTPases, each with a distinct tissue distribution. In particular, Atl-1 is primarily enriched in the CNS, while Atl-2 and Atl-3 are highly expressed in muscle tissues. This expression profile of mammalian atlastins suggests the possibility that they may play an essential role in both pre- and postsynaptic cells. In Drosophila, the single ortholog of mammalian atlastins (Atl) is expressed in the adult brain and is required for maintaining dopaminergic neurons. This study investigated the cellular function of Atl in the developing postsynaptic muscle (Lee, 2009).
In muscle cells, loss of Atl produces abnormal accumulation of stable microtubules, while its overexpression has the opposite effect. Thus, these genetic experiments reveal that Atl is involved in normal disassembly of microtubules. However, Atl is not homologous to any known microtubule-associated proteins, raising the key question as to how Atl regulates microtubule stability. Genetic and biochemical data obtained from this study and others support the model in which Atl acts through Spastin to disassemble microtubules. First, phenotypic overlap between atl and spastin is very extensive in Drosophila. atl and spastin mutants display similar adult phenotypes: short life span, impairments in locomotor activity, and age-dependent neurodegeneration. spastin mutations are also shown to cause an increase in bouton number and satellite bouton formation at larval NMJs. First, this study demonstrates that NMJ morphology is similarly affected by atl mutations. Moreover, both mutations lead to a decrease in expression of ER and Golgi markers in muscle cells. Second, this study demonstrates that Atl and Spastin can physically bind each other in vitro. Finally, it was shown that spastin-null mutation suppresses the loss of stable microtubules induced by atl overexpression (Lee, 2009).
How is Atl involved in Spastin-mediated microtubule disassembly? Proteins in the AAA family have been shown to maintain their substrate specificities often by biochemical interactions with adaptor proteins that can bring them to specific subcellular compartments or molecular targets. Potential adaptor proteins for mammalian Spastin include a centrosomal protein NA14 and an endosomal protein CHMP1B, suggesting that Spastin may function at multiple subcellular compartments. This study shows that Atl in the Drosophila muscle localizes largely to the ER and partially to the Golgi apparatus. Together with biochemical interactions between Spastin and Atl, this result raises the possibility that Atl may function as an ER- and Golgi-specific adaptor for Spastin in the muscle. However, the possiblity cannot be excluded that Atl may modulate the microtubule-severing activity or stability of Spastin. Future work is needed to address this issue (Lee, 2009).
Dominant-negative or pathogenic forms of mammalian atlastins interfere with the formation, migration, or fusion of vesicles in the ER/Golgi interface in cultured cells and disrupt the morphology of these organelles. This study provides in vivo evidence that Drosophila Atl is required for normal formation of the ER and Golgi organelles. Thus, data obtained from mammalian cells and Drosophila support the role for Atl and its mammalian homologs in membrane trafficking from the ER and morphogenesis of the ER and Golgi apparatus. What is then the relationship between microtubule stability and morphogenesis of the ER and Golgi morphogenesis? Two lines of evidence suggest that Atl-dependent microtubule disassembly is critical for normal morphogenesis of those organelles. First, spastin-null mutants display ER and Golgi defects similar to those in atl mutants. Second, the ER and Golgi phenotype in atl is significantly rescued by administration of the microtubule-destabilizing drug vinblastine. Thus, accumulation of stable microtubules appears to be a causative mechanism to account for defects in ER and Golgi morphogenesis in atl. This conclusion is compatible with the previously reported roles for the microtubule cytoskeleton in membrane trafficking between the ER and the Golgi apparatus, as well as in the organization and distribution of those secretory organelles (Lee, 2009).
The postsynaptic defects of atl mutants are also rescued by pharmacologically reversing microtubule accumulation, raising an important question as to how the lack of Atl-mediated microtubule regulation induces abnormalities in postsynaptic structure. One possibility is that defects in vesicle trafficking induced by atl mutations could impair the delivery of synaptic membrane components, which in turn would produce an underdeveloped and disorganized SSR. If this is the case, Atl-mediated microtubule regulation may be critical for 'Golgi-bypass' secretory pathways since anterograde ER-to-Golgi protein trafficking appears to be normal in cells expressing dominant-negative forms of atlastins. Such pathways include a Rab1-positive route that connects the pre-Golgi intermediate compartment (IC) directly with the cell periphery. In this regard, it will be interesting to address in the future whether synaptic components critical for SSR development are transported to the plasma membrane through the Rab1-mediated or related pathways in the muscle. An alternative and additional possibility is that loss of Atl could alter local microtubules surrounding the postsynaptic area, thereby affecting SSR structure. At the Drosophila NMJ, muscle microtubules are excluded from the peribouton area (i.e. SSR) that can be defined by the spectrin/actin network. It has been proposed that the extent of the SSR is strongly correlated with postsynaptic aPKC activity, which decreases the stability of muscle microtubules. By analogy with aPKC, Atl could support the expansion of the SSR by decreasing the stability of local microtubules surrounding the SSR. At this time, however, other mechanisms by which misregulation of muscle microtubules may alter the postsynaptic structure cannot be excluded, as observed in the postsynaptic side of atl mutant NMJs (Lee, 2009).
At present, it is still unclear how postsynaptic Atl regulates presynaptic growth. Since over-activation of retrograde BMP signaling also produces an NMJ phenotype characterized primarily by increased satellite bouton formation at the NMJ, it is an open question whether microtubule misregulation in atl inhibits the activity or the secretion from postsynaptic muscles of BMP and other retrograde growth signals (Lee, 2009).
The HSP proteins implicated in microtubule organization and/or microtubule-dependent processes include microtubule motor protein KIF5A and other molecules containing the microtubule interacting and trafficking (MIT) domain (e.g., Spartin and Spastin). Previous functional characterization of these proteins has centered on their roles in axons and presynaptic terminals. For example, Spastin has been shown to be expressed at the presynaptic compartment of the Drosophila larval NMJ, and to regulate synaptic structure and function by regulating the stability of presynaptic microtubules. However, the Spastin protein is also expressed in the postsynaptic muscle in Drosophila and humans. At present, it is unknown to what extent microtubule misregulation in the postsynaptic cell contributes to the presynaptic phenotypes observed in Drosophila spastin-null mutants and human patients with HSP SPG4. This study has clearly demonstrated in Drosophila that misregulation of muscle microtubules caused by atl mutations produces synaptic defects, highly reminiscent of those seen with spastin loss-of-function mutations. Given the biochemical and functional links between Atl and Spastin, the current work provides a new perspective on the postsynaptic role of Spastin and other microtubule-related HSP proteins during synapse development (Lee, 2009).
Hypoparathyroidism, mental retardation and facial dysmorphism (HRD) is a fatal developmental disease caused by mutations in tubulin-specific chaperone E (TBCE). A mouse Tbce mutation causes progressive motor neuronopathy. To dissect the functions of TBCE and the pathogenesis of HRD, mutations were generated in Drosophila tbce, and its expression was manipulated in a tissue-specific manner. Drosophila tbce nulls are embryonic lethal. Tissue-specific knockdown and overexpression of tbce in neuromusculature resulted in disrupted and increased microtubules, respectively. Alterations in TBCE expression also affected neuromuscular synapses. Genetic analyses revealed an antagonistic interaction between TBCE and the microtubule-severing protein Spastin. Moreover, treatment of muscles with the microtubule-depolymerizing drug nocodazole implicated TBCE as a tubulin polymerizing protein. Taken together, these results demonstrate that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes microtubule formation. As defective microtubules are implicated in many neurological and developmental diseases, this work on TBCE may offer novel insights into their basis (Jin, 2009).
Microtubules (MTs), one of the major building blocks of cells, play a crucial role in a diverse array of biological functions including cell division, cell growth and motility, intracellular transport and the maintenance of cell shape. As MTs are important in all eukaryotes, it is not surprising that defects in MTs are associated with a number of severe human diseases, including Fragile X mental retardation and autosomal dominant hereditary spastic paraplegia (AD-HSP). MTs are formed by polymerization of tubulin heterodimers consisting of one α- and one β-tubulin polypeptide. The formation of α-β tubulin heterodimers is mediated by a group of five tubulin chaperones, TBCA-TBCE (see Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization). TBCA and TBCD assist in the folding of β-tubulin, whereas TBCB and TBCE facilitate the folding of α-tubulin (Jin, 2009 and references therein),
A group of rare, recessive and fatal congenital diseases, collectively called hypoparathyroidism, mental retardation and facial dysmorphism (HRD), is caused by mutations in the gene encoding TBCE (Parvari, 2002). TBCE contains three functional domains: a glycine-rich cytoskeleton-associated protein domain (CAP-Gly) that binds α-tubulin, a series of leucine-rich repeats (LRR), and an ubiquitin-like (UBL) domain; the latter two mediate protein-protein interactions. Identification of the HRD disease gene revealed a 12 bp deletion in TBCE that leads to the expression of a mutated TBCE protein lacking four amino acids in the CAP-Gly domain. The mutation causes lower MT density at the MT organizing center, perturbed MT polarity and decreased precipitable MT, while total tubulin remains unchanged. Remarkably, overexpression of TBCE in cultured cells also results in disrupted MTs. Thus, both loss-of-function mutations and overexpression of TBCE disrupt the MT network in mammalian systems (Jin, 2009 and references therein).
Two independent studies have demonstrated that a Trp524Gly substitution at the last residue of mouse TBCE results in progressive motor neuronopathy (PMN), which has been widely used as a model for human motor neuron diseases. Similar to what has been reported for cells from human HRD patients, the point mutation in mouse Tbce leads to a reduced number of MTs in axons. Isolated motor neurons from mutant mice exhibit shorter axons and irregular axonal swellings. More specifically, axonal MTs are lost progressively from distal to proximal, which correlates with dying-back axonal degeneration in mutant mice. This demonstrates a mechanistic link between TBCE-mediated tubulin polymerization and neurodegeneration (Jin, 2009 and references therein).
TBCE is well conserved across species, from yeast to human. Genetic analyses of the TBCE homolog in S. pombe, Sto1P, show that it is essential for viability and that it plays a crucial role in the formation of cytoplasmic MTs and in the assembly of mitotic spindles. S. cerevisiae mutants of the TBCE homolog PAC2 show increased sensitivity to the MT-depolymerizing agent benomyl. Similarly, tbce mutants of Arabidopsis have defective MTs, leading to embryonic lethality (Jin, 2009 and references therein).
The Drosophila genome contains a TBCE ortholog, listed as CG7861 in FlyBase, but no studies of it have been reported. To gain a mechanistic insight into the in vivo functions of TBCE, different mutations were introduced into Drosophila tbce. Drosophila tbce nulls are embryonic lethal, indicating that it is an essential gene. The developmental, physiological and pharmacological consequences with regard to neuromuscular synapses and MT formation were examined when the expression of TBCE was altered specifically in neurons or muscles using the UAS-Gal4 system. It was found that TBCE is required for the normal development and function of neuromuscular synapses and that it promotes MT formation in vivo (Jin, 2009).
How MTs functions at synapses is poorly understood. Futsch, a MT-associated protein, stabilizes MTs in presynaptic neurons, and futsch mutants show reduced bouton number and increased bouton size, whereas spastin mutants have the opposite phenotype of increased bouton number but decreased bouton size. This analyses show that TBCE plays a role at synapses. Except for the presynaptic overexpression of tbce, all other manipulations of tbce at either side of the NMJ synapse caused increased branching number, increased bouton number and decreased bouton size, demonstrating that tbce is required for normal NMJ synapse development. Given the dramatic MT alterations on both pre- and postsynaptic sides, the NMJ phenotypes appear subtle. The seemingly conflicting result that both overexpression and knockdown of tbce on the postsynaptic side led to similar phenotypes in synapse development supports an existing hypothesis that abnormal synaptic growth results from the disruption of MT dynamics, rather than from an alteration in the absolute quantity of MTs (Jin, 2009).
Increased neurotransmission, reflected in both EJP and mEJP amplitude, was observed upon presynaptic alteration of tbce expression, whereas postsynaptic manipulations of tbce showed normal neurotransmission. This suggests that synaptic neurotransmission is sensitive to pre-but not postsynaptic MT alteration, although postsynaptic alterations of tbce had a significant effect on synapse development. Interestingly, both overexpression and knockdown of tbce on the presynaptic side led to a similar increase in both EJP and mEJP amplitude. The increased EJP amplitude observed upon presynaptic alterations of TBCE might be accounted for by increased mEJP amplitude. The increase in mEJP amplitude could be caused by an increase in presynaptic vesicle size, an increase in the concentration of vesicular glutamate, or an increase in postsynaptic glutamate receptor sensitivity. It is interesting to note that the mEJP is also increased in both Fmr1-null and Fmr1-overexpression NMJ synapses. However, the exact mechanism by which TBCE, and other MT regulators, affect neurotransmission remains to be elucidated (Jin, 2009).
Genetic analyses revealed an antagonistic interaction between TBCE and Spastin. TBCE promotes MT formation, whereas Spastin severs MTs. Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a heterogeneous group of neurodegenerative disorders characterized by progressive and bilateral spasticity of the lower limbs, with specific degeneration of the longest axons in the CNS. Forty to fifty percent of all AD-HSP cases are caused by mutations in spastin. However, the MT-related pathology of human patients with spastin mutation has not been documented (Jin, 2009).
Overexpression of spastin in Drosophila neuromusculature and in cultured cells causes dramatically fragmented and reduced MTs. Surprisingly, morphologically normal muscles are present in patients with spastin mutations, although large-scale disruption of MT pathways was detected at the molecular level. No MT defects were reported in a mouse model in which the endogenous spastin is truncated. Similarly, spastin-null mutants of Drosophila show no dramatic change in MT appearance in muscles, suggesting that Spastin plays a fine-tuning role in MT dynamics. Indeed, spastin nulls are late pupal lethal with a few adult escapers, further confirming a subtle role for Spastin in MT regulation. By comparison, tbce nulls are embryonic lethal, whereas knockdown of tbce leads to a dramatically reduced MT network in Drosophila neuromusculature. Thus, in contrast to the nuanced role of endogenous Spastin, TBCE plays a crucial role in MT formation (Jin, 2009).
Although Drosophila possesses a TBCE ortholog, no previous studies of it have been reported. This work shows that tbce is essential for early neuromuscular development in Drosophila. In vivo evidence is provided demonstrating that Drosophila TBCE is both required and sufficient for MT formation, supporting early in vitro biochemical studies that showed that TBCE assists in α-β-tubulin heterodimer formation (Jin, 2009 and references therein).
Overexpression of tbce produced increased MTs. This is the first report of increased MT formation when a tubulin chaperone is overexpressed, and is contrary to reports in other systems. Overexpression of human TBCE in cultured cells leads to complete disruption of MTs, as does overexpression of a TBCE-like protein. It was further hypothesized that the UBL domains present in TBCE and the TBCE-like protein might contribute to the degradation of tubulin via the proteasomal pathway. In addition, the overexpression of other tubulin chaperones, such as TBCD, results in a similar disruption of MTs. These in vivo data are consistent with the early in vitro observation that TBCD or TBCE in excess destroys tubulin heterodimers by sequestering the bound tubulin subunit, leading to the destabilization of the freed partner subunit. It is thus believed that in addition to assisting in the folding pathway, TBCE also interacts with native tubulins to disrupt α-β-tubulin heterodimers. The discrepancy between the overexpression result and findings of others could have several explanations: (1) the use of different experimental systems: transgenic animals in this work and cultured cells in other studies; (2) different systems might have different expression levels of tbce, leading to varying effects on MTs; (3) Drosophila and human TBCE might have diverged functions. Further analyses are needed to reconcile the conflicts in the effects of TBCE overexpression in these different systems. In general, however, tbce mutant phenotypes are consistent in all species examined so far, from yeast to human, indicating that the function of TBCE in promoting MT formation has been well-conserved throughout evolution (Jin, 2009).
Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development. The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin's gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity. These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis (Chen, 2010).
In animal photoreceptor cells, the surface membrane is enlarged for the storage of opsin photopigment. Insect eyes use an actin-based structure for surface membrane enlargement, but mammalian eyes use microtubule-based structure. This study examined whether the Drosophila photoreceptor cells may have any microtubule-based structures. There is a distinctive structure which is specifically labeled by anti-acetylated-tubulin antibody in the developing photoreceptors of Drosophila. Given the specific localization of stable microtubules in developing pupal Drosophila photoreceptors, these subcellular structures might provide a functional role for photoreceptor morphogenesis (Chen, 2010).
Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin, has been shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. Genetic analysis of the spastin mutation strongly indicates that spastin not only modulates the microtubules, but also modulates the apical Crb membrane domain during rhabdomere elongation. The apical membrane modulation activity of spastin was further confirmed by spastin overexpression which caused a dramatic expansion of the apical membrane domain (Chen, 2010).
Based on the highly concentrated localization of Spastin in the apical domains of the photoreceptors, it is proposed that the apically localized Spastin might control the apical Crb domains. This apical domain-specific function of Spastin is based on the following results; (1) Apical domain localization of Spastin, (2) Loss of spastin causes apical domain defects, and (3) Overexpression of spastin causes apical domain expansions. But, another possibility of the direct modulation of stable microtubules by Spastin cannot be excluded. Any subtle changes in stable microtubules by spastin might affect potential trafficking machinery which is responsible for the apical Crb targeting. But, these two possibilities are not necessarily mutually exclusive (Chen, 2010).
Spastin has microtubule-severing activities in vitro. Therefore, microtubule-severing activity of Spastin may facilitate the apical Crb domain, since loss of spastin caused loss of Crb, and gain of spastin caused the expansion of the Crb domain. Furthermore, this facilitating activity of Spastin for the apical domain could be independent from the main stable microtubule structures which are located far beneath the apical domains. This possibility is supported by the more direct influence on apical Crb, as the stable microtubules were not dramatically changed, relatively speaking, by either spastin mutants or spastin overexpression (Chen, 2010).
During the massive growth of the rhabdomeres in the pupal retina, many membrane materials, including Crb, will be targeted into the growing apical membranes. Spastin may participate in this material transport to the apical membrane domain during rhabdomere growth, although the initial targeting is spastin-independent. The outcome of this study will provide useful information for understanding the molecular genetic mechanism of spastic paraplegia. Although the spastin mutation subtly affects the main microtubules, this genetic approach will provide more convincing clues concerning the microtubule-based processes in photoreceptor morphogenesis (Chen, 2010).
Thus, analysis of the microtubule-modulating Spastin in Drosophila photoreceptors may provide important insights into the understanding of the functional basis of the microtubule-based structure and the microtubule-related genes involved the formation and development of photoreceptors. Evolutionary conservation in the structure and function of eye morphogenesis genes makes the Drosophila eye an excellent model for studying the genetic and molecular basis of retinal cell organization (Chen, 2010).
Future work will help to uncover other genes that might affect the microtubule cytoskeleton and cell polarity targeting during the extensive membrane growth phase of the pupal eye. Determining the role of Spastin in photoreceptor development will help in understanding retinitis pigmentosa, spastic paraplegia and other retinal degenerative diseases that involve mutations in crb and spastin. The finding that human mutations in CRB1 lead to retinitis pigmentosa emphasizes the importance of deciphering the molecular networks associated with Crb in the apical membrane domain of the Drosophila photoreceptor (Chen, 2010).
In summary, this study has examined the role of Spastin in the regulation of the apical Crb domain in developing photoreceptors. The data strongly suggests that Spastin plays important functions in the modulation of cell membrane domains including the apical domains of photoreceptors during pupal eye development. Because proper maintenance of the apical Crb domain is important for the massive growth observed in rhabdomeres at the apical region of photoreceptor cells, malfunction of spastin results in severe defects in the formation of functional photoreceptors (Chen, 2010).
Dendrite shape is considered a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their function depends on these morphologies, remain unclear. This study demonstrates a requirement for the microtubule-severing protein katanin p60-like 1 (Kat-60L1) in regulating the elaborate dendrite morphology and nocifensive functions of Drosophila larval class IV dendritic arborization neurons. Kat-60L1 mutants exhibit diminished responsiveness to noxious mechanical and thermal stimuli. Class IV dendrite branch number and length are also reduced, supporting a correspondence between neuronal function and the full extent of the dendritic arbor. These arborization defects occur particularly in late larval development, and live imaging reveals that Kat-60L1 is required for dynamic, filopodia-like nascent branches to stabilize during this stage. Mutant dendrites exhibit fewer EB1-GFP-labeled microtubules, suggesting that Kat-60L1 increases polymerizing microtubules to establish terminal branch stability and full arbor complexity. Although loss of the related microtubule-severing protein Spastin also reduces the class IV dendrite arbor, microtubule polymerization within dendrites is unaffected. Conversely, Spastin overexpression destroys stable microtubules within these neurons, while Kat-60L1 has no effect. Kat-60L1 thus sculpts the class IV dendritic arbor through microtubule regulatory mechanisms distinct from Spastin. The data support differential roles of microtubule-severing proteins in regulating neuronal morphology and function, and provide evidence that dendritic arbor development is the product of multiple pathways functioning at distinct developmental stages (Stewart, 2012).
Autosomal-dominant hereditary spastic paraplegia (AD-HSP) is a crippling neurodegenerative disease for which effective treatment or cure remains unknown. Victims experience progressive mobility loss due to degeneration of the longest axons in the spinal cord. Over half of AD-HSP cases arise from loss-of-function mutations in spastin, which encodes a microtubule-severing AAA ATPase. In Drosophila models of AD-HSP, larvae lacking Spastin exhibit abnormal motor neuron morphology and function, and most die as pupae. Adult survivors display impaired mobility, reminiscent of the human disease. This study shows that rearing pupae or adults at reduced temperature (18°C), compared with the standard temperature of 24°C, improves the survival and mobility of adult spastin mutants but leaves wild-type flies unaffected. Flies expressing human spastin with pathogenic mutations are similarly rescued. Additionally, larval cooling partially rescues the larval synaptic phenotype. Cooling thus alleviates known spastin phenotypes for each developmental stage at which it is administered and, notably, is effective even in mature adults. Further, cold treatment rescues larval synaptic defects in flies with mutations in Flower (a protein with no known relation to Spastin) and mobility defects in flies lacking Kat60-L1, another microtubule-severing protein enriched in the CNS. Together, these data support the hypothesis that the beneficial effects of cold extend beyond specific alleviation of Spastin dysfunction, to at least a subset of cellular and behavioral neuronal defects. Mild hypothermia, a common neuroprotective technique in clinical treatment of acute anoxia, might thus hold additional promise as a therapeutic approach for AD-HSP and, potentially, for other neurodegenerative diseases (Baxter, 2014).
This study demonstrates that cold temperature alleviates reduced mobility and survival caused by loss of Spastin function in Drosophila. This is the case for flies lacking endogenous spastin, as well as those expressing pathogenic human Spastin. Cold treatment during the pupal stage of development is sufficient to enhance the eclosion rate, climbing ability and lifespan of spastin mutant adults. Furthermore, cold administered only after pupal development, to fully developed adults, also improves mutant mobility. The timing of these two effective periods is consistent with the idea that cold alleviates spastin mutant phenotypes by acting on the developing adult nervous system during pupal metamorphosis, but is also potent after the nervous system has matured. This is extremely promising from a clinical viewpoint, suggesting that the therapeutic window in AD-HSP includes both developing and mature nervous systems (Baxter, 2014).
Although wild-type levels of mobility and survival were not often achieved, the temperature shift to 18°C confers considerable improvement. Some cold-treated flies are able to jump and even fly briefly, behaviors not observed in untreated mutants. Cooling can match or exceed the efficacy of rescue by the microtubule destabilizing drug vinblastine, which has been proposed as a therapeutic approach for AD-HSP. It has been shown that vinblastine doubles the ~12% eclosion rate of spastin5.75 null mutants; however, this study found the drug to be ineffective for null and HL44,HR388 eclosion, but improved eclosion by 65% for HWT,HR388, which is a more common, representative AD-HSP genotype associated with milder pathogenesis. In comparison, pupal cooling of spastin5.75 null mutants increases eclosion by 70% (Baxter, 2014).
Importantly, cooling during the pupal and adult stages does not affect eclosion or motor behavior in wild-type flies. This suggests that cooling not only compensates for defects in neuronal function caused by lack of Spastin (or other mutations), but is also innocuous to properly functioning neurons. Although cooling administered at the larval stage is ultimately deleterious to both control and spastin mutant adults, mutant larval synapses are effectively restored to wild-type morphologies. This suggests that cold is beneficial for some spastin-mediated defects at this stage, but also has nonspecific, toxic effects on a cell population required later, in adults (Baxter, 2014).
What is the mechanism(s) underlying the rescuing effect of cold? The demonstration that cold alleviates not just spastin mutant phenotypes, but also mutant phenotypes in fwe and kat-60L1, indicates that that rescuing effects of cold on nervous system function might be quite broad. All three genes are important in synapse formation, although kat-60L1 has been shown to act post-rather than pre-synaptically at larval and pupal stages. Reduced temperature could thus be generally beneficial to synaptic dysfunction, perhaps by reducing activity or metabolic load. Alternatively, fwe, spastin and kat-60L1 might share a common pathway component(s), as yet undiscovered, that is directly affected by cold. For example, cold itself is well known to destabilize microtubules, particularly at temperatures below 20°C, and is often used in experiments to depolymerize microtubules. Cold could thus substitute directly for the microtubule-severing function of Spastin by promoting microtubule destabilization. Cold-mediated rescue of Kat-60L1 mutants support this idea; however, obvious differences in stable microtubule distribution at cold-treated spastin5.75 synapses or in Drosophila S2R+ cells were not observed; fwe mutants, which have not been implicated in microtubule dysregulation, were also rescued by cooling (Baxter, 2014).
In humans, cooling has been shown to be generally neuroprotective, and mild or moderate therapeutic hypothermia (e.g. 33–35°C) has long had clinical applications, including reducing neurological injury in patients following cardiac arrest, traumatic brain injury, epilepsy and stroke. Furthermore, exposure to even near-freezing temperatures results in minimal neuropathology in rat and cat neocortex and hippocampus. Although commonly administered in situations involving acute brain injury, the mechanism by which cooling confers neuroprotection or therapeutic improvement is unknown, multifactorial and context-dependent (Baxter, 2014).
It will be important to characterize the in vivo effects of cold in mouse models of AD-HSP. The specificity of the effect of cold on mutant and not wild-type animals, together with the spatially localized neurodegeneration in AD-HSP, suggest that moderate hypothermia could be applied in a highly targeted manner in this disease context, with minimal negative effects. Future studies should furthermore elucidate the underlying cellular mechanisms and potentially broader applications of cold in alleviating neuronal dysfunction in neurodegeneration. Because Drosophila are ectothermic, with body temperatures that vary with their environment, they provide a straightforward system in which the cell biological effects of temperature change can be studied in vivo (Baxter, 2014).
Baxter, S.L., Allard, D.E., Crowl, C. and Sherwood, N.T. (2014). Cold temperature improves mobility and survival in Drosophila models of autosomal-dominant hereditary spastic paraplegia (AD-HSP). Dis Model Mech 7: 1005-1012. PubMed ID: 24906373
Casari, G., De Fusco, M., Ciarmatori, S., Zeviani, M., Mora, M., Fernandez, P., De Michele, G., Filla, A., Cocozza, S. and Marconi, R. et al. (1998). Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a neuclear-encoded mitochondrial metalloprotease. Cell 93: 973-983. 9635427
Charvin, D., Cifuentes-Diaz, C., Fonknechten, N., Joshi, V., Hazan, J., Melki, J. and Betuing, S. (2003). Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus. Hum. Mol. Genet. 12: 71-78. 12490534
Chen, G., League, G. P. and Nam, S. C. (2010). Role of spastin in apical domain control along the rhabdomere elongation in Drosophila photoreceptor. PLoS One 5(3): e9480. PubMed Citation: 20209135
Ciccarelli, F. D., Proukakis, C., Patel, H., Cross, H., Azam, S., Patton, M. A., Bork, P. and Crosby, A. H. (2003). The identification of a conserved domain in both spartin and spastin, mutated in hereditary spastic paraplegia. Genomics 81: 437-441. 12676568
De Michele, G., De Fusco, M., Cvalvani, F., Filla, A., Marconi, R., Volpe, G., Monticelli, A., Ballabio, A., Casari, G. and Cocozza, S. (1998). A new locus for autosomal recessive hereditary spastic paraplegia maps to chromosome 16q24.3. Am. J. Hum. Genet. 63: 135-139. 9634528
Errico, A., Ballabio, A. and Rugarli, E. I. (2002). Spastin, the protein mutated in autosomal dominant hereditary spastic paraplegia, is involved in microtubule dynamics. Hum. Mol. Genet. 11: 153-163. 11809724
Errico, A., Claudiani, P., D'Addio, M. and Rugarli, E. I. (2004). Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon. Hum. Mol. Genet. 13(18): 2121-32. 1526918
Fonknechten, N., Mavel, D., Byrne, P., Davpome, C.S., Cruaud, C., Boentsch, D., Samson, D., Coutinho, P., Hutchinson, M. and McMonagle, P. et al. (2000). Spectrum of SPG4 mutations in autosomal dominant spastic paraplegia. Hum. Mol. Genet. 9: 637-644. 10699187
Hartman, J. J., Mahr, J., McNally, K., Okawa, K., Iwamatsu, A., Thomas, S., Cheesman, S., Heuser, J., Vale, R. D. and McNally, F. J. (1998). Katanin, a microtubule-severing protein is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell 93: 277-287. 9568719
Hazan, J., Fonknechten, N., Mavel, D., Paternotte, C., Samson, D., Artiguenave, F., Davoine, C. S., Cruaud, C., Durr, A. and Wincker, P. et al. (1999). Spastin, a new AAA protein, is latered in the most frequent form of autosomal dominant spastic paraplegia. Nat. Genet. 23: 296-303. 10610178
Jin, S., Pan, L., Liu, Z., Wang, Q., Xu, Z. and Zhang, Y. Q. (2009). Drosophila Tubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation. Development 136(9): 1571-81. PubMed Citation: 19297412
Jinushi-Nakao, S., et al. (2007). Knot/Collier and Cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape. Neuron 56: 963-978. PubMed citation: 18093520
Jouet, M., Rsoenthal, A., Armstrong, G., MacFarlane, J., Stenson, R., Paterson, J. and Metxenberg, A. (1994). X-linked hydrocephalus result from mutations in the L1 gene. Nat. Genet. 7: 402-407. 7920659
Kammermeier, L., Spring, J., Stierwald, M., Burgunder, J. M. and Reichert, H. (2003). Identification of the Drosophila homolog of the human spastin gene. Dev. Genes Evol. 213: 412-415. 12908108
Karabay, A., Yu, W., Solowska, J. M., Baird, D. H. and Baas, P. W. (2004). Axonal growth is sensitive to the levels of katanin, a protein that severs microtubules. J. Neurosci. 24: 5778-5788. PubMed citation: 15215300
Laub F., et al. (2005) Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system. Mol. Cell Biol. 25: 5699-5711. PubMed Citation: 15964824
Lee, M., et al. (2009). Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development. Dev. Biol. 330(2): 250-62. PubMed Citation: 19341724
Lei L., et al. (2005). The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons. Genes Dev. 19: 1354-1364. PubMed Citation: 15937222
Lei L., Zhou, J., Lin, L. and Parada, L. F. (2006). Brn3a and Klf7 cooperate to control TrkA expression in sensory neurons. Dev. Biol. 300: 758-769. PubMed Citation: 17011544
Molon, A., et al. (2004). Large-scale disruption of microtubule pathways in morphologically normal human spastin muscle. Neurology 62(7): 1097-104. 15079007
Moore, D. L., et al. (2009). KLF family members regulate intrinsic axon regeneration ability. Science 326: 298-301. PubMed Citation: 19815778
Orso, G. et al. (2005). Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine. J. Clin. Invest. 115: 3026-3034. PubMed Citation: 16276413
Patel, H., Cross, H., Proukakis, C., Ershberver, R., Bork, P., Ciccarelli, F. D., Patton, M. A., McKusick, V. A. and Crosby, A. H. (2002). SPG20 is mutated in Troyer syndrome, an hereditary spastic paraplegia. Nat. Genet. 31: 347-348. 12134148
Papadopoulos, C., Orso, G., Mancuso, G., Herholz, M., Gumeni, S., Tadepalle, N., Jüngst, C., Tzschichholz, A., Schauss, A., Höning, S., Trifunovic, A., Daga, A. and Rugarli, E.I. (2015). Spastin binds to lipid droplets and affects lipid metabolism. PLoS Genet 11: e1005149. PubMed ID: 25875445
Rao, K., Stone, M. C., Weiner, A. T., Gheres, K. W., Zhou, C., Deitcher, D. L., Levitan, E. S. and Rolls, M. M. (2016). Spastin, atlastin and ER relocalization are involved in axon, but not dendrite, regeneration. Mol Biol Cell 27(21):3245-3256. PubMed ID: 27605706
Reid, E., Kloos, M., Ahley-Koch, A., Hughes, L., Bevan, S., Svenson, I. K., Lennon, G., Graham, F., Gaskell, P. C. and Dearlove, A. et al. (2002). A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10). Am. J. Hum. Genet. 71: 1189-1194. 12355402
Reid, E. (2003). Science in motion: common molecular pathological themes emerge in the hereditary spastic paraplegias. J. Med. Genet. 40: 81-86. 12566514
Riano, E., et al. (2009). Pleiotropic effects of spastin on neurite growth depending on expression levels. J. Neurochem. 108: 1277-1288. PubMed Citation: 19141076
Roll-Mecak, A. and Vale, R. D. (2005). The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules. Curr. Biol. 15: 650-655. PubMed citation: 15823537
Roll-Mecak, A. and Vale, R. D. (2008). Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451: 363-367. PubMed citation: 18202664
Roll-Mecak, A. and McNally, F. J. (2010). Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22: 96-103. PubMed Citation: 19963362
Sherwood, N. T., Sun, Q., Xue, M., Zhang, B. and Zinn, K. (2004). Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol. 2: e429. PubMed citation: 15562320
Stegmuller, J., et al. (2006). Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN. Neuron 50(3): 389-400. PubMed Citation: 16675394
Stewart, A., Tsubouchi, A., Rolls, M. M., Tracey, W. D. and Sherwood, N. T. (2012). Katanin p60-like1 promotes microtubule growth and terminal dendrite stability in the larval class IV sensory neurons of Drosophila. J Neurosci 32: 11631-11642. Pubmed: 22915107
Suagier-Veber, P., Munnich, A., Bonneau, D., Rozet, J. M., LeMerrer, M., Gil, R. and Boespflug-Tanguy, O. (1994). X-linked spastic paraplegia and Pelizaeus-Merzbacher disease are allelic disorders at the proteolipid protein locus. Nat. Genet. 6: 257-262. 8012387
Tahirovic, S. and Bradke, F. (2009). Neuronal polarity. Cold Spring Harb. Perspect. Biol. 1: a001644. PubMed Citation: 20066106
Trotta, N., Orso, G., Rossetto, M. G., Daga, A. and Broadie, K. (2004). The Hereditary Spastic Paraplegia Gene, spastin, regulates microtubule stability to modulate synaptic structure and function. Curr. Biol. 14: 1135-1147. 15242610
Wharton, S. B., et al. (2003). The cellular and molecular pathology of the motor system in hereditary spastic paraparesis due to mutation of the spastin gene. J. Neuropathol. Exp. Neurol. 62: 1166-1177. PubMed citation: 14656074
Wiggin, G. R., Fawcett, J. P. and Pawson, T. (2005) Polarity proteins in axon specification and synaptogenesis. Dev. Cell 8: 803-816. PubMed Citation: 15935771
Wood, J. D. et al. (2006) The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo, Hum. Mol. Genet. 15: 2763-2771. PubMed citation: 16893913
Ye, B., et al. (2007). Growing dendrites and axons differ in their reliance on the secretory pathway. Cell 130: 717-729. PubMed Citation: 17719548
Ye, B., et al. (2011). Differential regulation of dendritic and axonal development by the novel Kruppel-like factor dar1. J. Neurosci. 31(9): 3309-19. PubMed Citation: 21368042
Yeaman, C., et al. (2004). Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network. Nat. Cell Biol. 6: 106-112. PubMed Citation: 14743217
Zhang, D., Rogers, G. C., Buster, D. W. and Sharp, D. J. (2007). Three microtubule severing enzymes contribute to the 'Pacman-flux' machinery that moves chromosomes. J. Cell Biol. 177: 231-242. PubMed Citation: 17452528
Zhao, X., Alvarado, D., Rainier, S., Lemons, R., Hedera, P., Weber, C. H., Tukel, T., Apak, M., Heiman-Patterson, T. and Ming, L. et al. (2001). Mutations in a newly identified GTPase cause autosomal dominant hereditary spastic paraparesis. Nat. Genet. 29: 326-331. 11685207
date revised: 10 February 2016 <!References updated>
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.