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 (Gal4^2-21, 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 spastin^5.75 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; spastin^5.75/+ mutants had 43%, and kn¹/kn^KN2 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 Gal4^2-21, UAS-mCD8::GFP or UAS-kn; Gal4^2-21, 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).

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 spastinís 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).

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 Bolwigís 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).

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