genes associated with Spastic paraplegia
of the disease
Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative disorders marked by lower-limb spasticity (stiffness) and weakness, leading to progressive difficulty walking. Supportive treatments exist, but are of mixed efficacy and do not restore mobility. The most common form, pure autosomal dominant HSP (AD-HSP), accounts for 70–80% of HSP-afflicted families. AD-HSP pathology is characterized primarily by degeneration of the longest descending axons of the central nervous system (CNS). These originate from the upper motor neurons in the cortex and terminate in the lumbar spine, innervating the α1 motor neurons that control leg movement. In regards to providing a model for AD-HSP, Drosophila Spastin, like its vertebrate orthologs, severs purified microtubules and those in Drosophila S2 cells. Knocking down fly Spastin using a RNA-interference (RNAi) transgene or deletion of the endogenous gene both cause synaptic defects at the Drosophila larval neuromuscular junction (NMJ), supporting a role for spastin in regulating synaptic morphology and function. Orthologs of several other HSP causative genes studied in Drosophila also exhibit progressive neurodegeneration, supporting the relevance of flies in providing insights into mechanisms underlying this disease (Baxter, 2014 and references therein).
Relevant studies of Spastic paraplegia
Julien, C., Lissouba, A., Madabattula, S., Fardghassemi, Y., Rosenfelt, C., Androschuk, A., Strautman, J., Wong, C., Bysice, A., O'Sullivan, J., Rouleau, G.A., Drapeau, P., Parker, J.A. and Bolduc, F.V. (2016). Conserved pharmacological rescue of hereditary spastic paraplegia-related phenotypes across model organisms. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26744324
Srivastava, S., Shaked, H. M., Gable, K., Gupta, S. D., Pan, X., Somashekarappa, N., Han, G., Mohassel, P., Gotkine, M., Doney, E., Goldenberg, P., Tan, Q. K. G., Gong, Y., Kleinstiver, B., Wishart, B., Cope, H., Pires, C. B., Stutzman, H., Spillmann, R. C., Sadjadi, R., Elpeleg, O., Lee, C. H., Bellen, H. J., Edvardson, S., Eichler, F. and Dunn, T. M. (2023). SPTSSA variants alter sphingolipid synthesis and cause a complex hereditary spastic paraplegia. Brain. PubMed ID: 36718090
Melentev, P. A., Ryabova, E. V., Surina, N. V., Zhmujdina, D. R., Komissarov, A. E., Ivanova, E. A., Boltneva, N. P., Makhaeva, G. F., Sliusarenko, M. I., Yatsenko, A. S., Mohylyak, II, Matiytsiv, N. P., Shcherbata, H. R. and Sarantseva, S. V. (2021). Loss of swiss cheese in Neurons Contributes to Neurodegeneration with Mitochondria Abnormalities, Reactive Oxygen Species Acceleration and Accumulation of Lipid Droplets in Drosophila Brain. Int J Mol Sci 22(15). PubMed ID: 34361042
Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. This study used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)-the evolutionarily conserved ortholog of human NTE/PNPLA6-in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. These results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. It is proposed that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay (Melentev, 2021).
Allison, R., Edgar, J. R., Pearson, G., Rizo, T., Newton, T., Gunther, S., Berner, F., Hague, J., Connell, J. W., Winkler, J., Lippincott-Schwartz, J., Beetz, C., Winner, B. and Reid, E. (2017). Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia. J Cell Biol 216(5): 1337-1355. PubMed ID: 28389476
Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. This study found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. These results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration (Allison, 2017).
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
Mutations in over 50 genes including spastin and atlastin lead to Hereditary Spastic Paraplegia (HSP). It was previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin and spichthyin were reduced. Impaired regeneration was dependent on genetic background, and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. Other markers were examined to identify additional changes associated with regeneration. While mitochondria, endosomes and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was only observed during axon regeneration, and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. It is proposed that the HSP proteins Spastin and Atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip (Rao, 2016).
Previous work has shown that axon regeneration is impaired when one copy of spastin is mutant. This study now shows that atlastin is also haploinsufficient for axon regeneration and that reduction of several other HSP proteins using RNAi impairs regeneration. Thus axon regeneration seems to be a postdevelopmental process that involves at minimum a subset of HSP protein (Rao, 2016).
The sensitivity of axon regeneration to partial reduction of HSP proteins, however, depends on the genetic background. In a previous study, EB1-GFP was used as a dual-purpose marker of cell shape and microtubule dynamics. This fusion protein is not, however, completely neutral. GFP fused to the C-terminus of EB1 can interfere with binding of partner proteins to EB1. Because EB1 acts as a dynamic platform at growing microtubule ends that recruits other proteins, the presence of large amounts of EB1-GFP could reduce recruitment of other plus end-binding proteins. Indeed, in Drosophila, neurons EB1 binds Apc, which in turn brings kinesin-2 to growing dendritic microtubules to help maintain minus-end-out polarity, and high levels of EB1-GFP result in mixed polarity. Because of this, this study expressed EB1-GFP at low levels, but it is still possible that there is a subtle defect in microtubule growth or organization. Under normal circumstances, this does not result in any defects in regeneration, which is indistinguishable in control neurons expressing EB1-GFP, mCD8-RFP, or Rtnl1-GFP. Only when combined with partial reduction of HSP proteins was a difference seen among neurons expressing different markers. This difference was most clearly demonstrated in spastin heterozygotes, which had a very strong reduction in regenerative growth in neurons labeled with EB1-GFP but not with mCD8-RFP. The synthetic interaction between EB1-GFP and spastin suggests that even though the early microtubule changes triggered by axon injury were normal, with reduced levels of HSP proteins, microtubules were somehow involved in the phenotype. This conclusion was supported by a similar effect of EB1-CT, a dominant-negative form of EB1, and the fact that introduction of tdEOS-αtubulin suppressed the spastin phenotype (Rao, 2016).
To probe in more depth how HSP protein function related to regenerative axon growth, several other approaches were taken. First, dendrite regeneration was examined. Complete regeneration of dendrites after removal of the entire arbor involves very rapid outgrowth, and so it was reasoned that if HSP proteins were generally involved in facilitating growth of neuronal processes, they should be required for dendrite regeneration. No defects in dendrite regeneration were observed, and this suggested that the cells were healthy and that a process specific to axon regeneration was sensitive to HSP protein reduction. Second, a catalogue of intracellular markers was examined to look for rearrangements associated with regenerative axon growth. The ER was most dramatically different in regenerating axons and accumulated near the growing tip. Moreover, this was specific to axon regeneration and not observed in dendrite regeneration (Rao, 2016).
All of these observations were assembled into a model for HSP protein function during regeneration. It is proposed that a subset of HSP proteins is involved in concentrating the ER, together with underlying microtubules, at tips of axons undergoing regenerative growth. This model makes particular sense for spastin and atlastin. Because spastin is a microtubule regulator that in flies and mammals also has a transmembrane domain and binds the ER regulatory protein atlastin, their combined action could facilitate concentration of the ER at growing axon tips. In support of this idea, reduction of either protein disrupted ER accumulation at single growing axon tips (Rao, 2016).
Although it is believed that atlastin and spastin help to concentrate ER at growing axon tips by linking the ER to microtubules, which also accumulate at growing tips, it is not known what mediates the microtubule redistribution. It is suspected, however, that microtubule polarity is involved in setting up tip accumulation of tubulin and ER. Tip accumulation is seen only during regenerative axon growth, which requires microtubules in the growing process to be plus-end-out, and not during regenerative dendrite growth, when microtubules are largely minus-end-out. In initiation of regenerative axon outgrowth in cultured Drosophila neurons, kinesin-mediated microtubule sliding is important, and so one possibility is that motors slide short pieces of microtubules out to the new tip (Rao, 2016).
It is intriguing that ER accumulates at regenerating axon but not dendrite tips. This suggests that increased amounts of local ER are specifically important for promoting maximal axon growth. Local Smooth Endoplasmic Reticulum (SER) could promote axon growth by increasing local lipid production or increasing availability of intracellular calcium. A recent study in Caenorhabditis elegans suggests that it is the calcium storage function of SER that is important in this context. In this system, release of ER calcium through ryanodine receptors is required for maximal axon outgrowth, and high levels of calcium were seen at axon tips up to 5 h (the latest time point examined) after axon injury. Thus perhaps atlastin and spastin help concentrate ER at growing axon tips to provide a local source of intracellular calcium stores, which in turn facilitate regenerative growth (Rao, 2016).
It is difficult to know how a function for atlastin and spastin, and potentially other HSP proteins, in ER localization during regenerative axon growth relates to the axon degeneration that occurs in the disease. All HSP proteins seem to have important basic cellular functions that are quite universal. For example, atlastin is biochemically an ER fusion protein, but only a subset of disease-causing atlastin mutations affect ER fusion. Perhaps the function of atlastin, spastin, and other HSP proteins important for disease is not the core function but a subtler role these proteins play in very long neurons. ER relocalization during regenerative growth of axons is worth considering as a disease-relevant function for several reasons: 1) it is important for mature neurons, 2) at least two different HSP proteins contribute to it, 3) repeated small failures of regeneration could lead to accumulated axon loss over a long time period, and 4) at least in some genetic backgrounds, the capacity for regenerative growth is reduced when only one allele of the gene is mutant. An interesting further speculation is how this function might relate to cell-type susceptibility to regeneration. The hallmark of HSP is degeneration of upper motor neurons. As in many neurodegenerative diseases, it is unclear why these cells might be more sensitive than others. One possibility raised by the data is that the suite of microtubule regulators expressed in different neurons could influence sensitivity of regeneration to reduction of HSP proteins. For example, slightly higher levels of a microtubule-stabilizing protein might eliminate the need for full HSP protein function during regeneration in the same way that tdEOS-αtubulin bypassed the requirement for spastin and atlastin for regeneration. Similarly, a different set or ration of microtubule plus end-binding proteins could make a particular neuron type more sensitive to partial reduction of HSP proteins in the same way that EB1-GFP and EB1-CT did in this study (Rao, 2016).
Although the idea that the function identified for HSP proteins during axon regeneration is appealing in many ways, it is not a perfect fit. It is not known whether axon regeneration is triggered during normal wear and tear of axons in the spinal cord, and this is a critical missing piece of information necessary to evaluate whether reduction in regeneration might lead to long-term degeneration (Rao, 2016).
Independently of potential relevance to disease, the differential effect of reduction of HSP proteins on axon and dendrite regeneration is intriguing. One might expect that proteins with core cellular functions like ER and microtubule regulation would be equally required for both types of outgrowth. Similarly, if the ER is concentrated at growing axon tips to provide an extra calcium reservoir, why is this not important for dendrite regenerative growth? It will be extremely interesting to learn what promotes dendrite regeneration in future studies (Rao, 2016).
Xu, S., Stern, M. and McNew, J. A. (2016). Beneficial effects of rapamycin in a Drosophila model for hereditary spastic paraplegia. J Cell Sci [Epub ahead of print]. PubMed ID: 27909242
The locomotor deficits in the hereditary spastic paraplegias (HSPs) reflect degeneration of upper motor neurons, but the mechanisms underlying this neurodegeneration are unknown. This study established a Drosophila model for the HSP atlastin (atl), which encodes an ER fusion protein. Neuronal atl loss causes degeneration of specific thoracic muscles that is preceded by other pathologies including accumulation of aggregates containing poly-ubiquitin (poly-UB), increased generation of reactive oxygen species, and activation of the JNK/Foxo stress response pathway. Inhibiting the Tor kinase, either genetically or by administering rapamycin, at least partially reversed many of these pathologies. atl loss from muscle also triggers muscle degeneration and rapamycin-sensitive locomotor deficits and poly-UB aggregate accumulation. These results indicate that atl loss triggers muscle degeneration both cell autonomously and nonautonomously (Xu, 2016).
Galikova, M., Klepsatel, P., Munch, J. and Kuhnlein, R. P. (2017). Spastic paraplegia-linked phospholipase PAPLA1 is necessary for development, reproduction, and energy metabolism in Drosophila. Sci Rep 7: 46516. PubMed ID: 28422159
The human PAPLA1 phospholipase family is associated with hereditary spastic paraplegia (HSP), a neurodegenerative syndrome characterized by progressive spasticity and weakness of the lower limbs. Taking advantage of a new Drosophila PAPLA1 mutant, this study describes novel functions of this phospholipase family in fly development, reproduction, and energy metabolism. Loss of Drosophila PAPLA1 reduces egg hatchability, pre-adult viability, developmental speed, and impairs reproductive functions of both males and females. In addition, this work describes novel metabolic roles of PAPLA1, manifested as decreased food intake, lower energy expenditure, and reduced ATP levels of the mutants. Moreover, PAPLA1 has an important role in the glycogen metabolism, being required for expression of several regulators of carbohydrate metabolism and for glycogen storage. In contrast, global loss of PAPLA1 does not affect fat reserves in adult flies. Interestingly, several of the PAPLA1 phenotypes in fly are reminiscent of symptoms described in some HSP patients, suggesting evolutionary conserved functions of PAPLA1 family in the affected processes. Altogether, this work reveals novel physiological functions of PAPLA1, which are likely evolutionary conserved from flies to humans (Galikova, 2017).
Ring, J., et al. (2017). Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin. Microb Cell 4(12): 411-422. PubMed ID: 29234670
Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. This study shows that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. Spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. It is concluded that mitochondria-related energy metabolism is crucial for spartin's vital function during aging; this study uncovers a network of specific interactors required for this function (Ring, 2017).
Yalcin, B., Zhao, L., Stofanko, M., O'Sullivan, N. C., Kang, Z. H., Roost, A., Thomas, M. R., Zaessinger, S., Blard, O., Patto, A. L., Sohail, A., Baena, V., Terasaki, M. and O'Kane, C. J.(2017). Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins. Elife 6. PubMed ID: 28742022
Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP (Drosophila Reep1) proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). This study shows that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function (Yalcin, 2017).
Napoli, B., Gumeni, S., Forgiarini, A., Fantin, M., De Filippis, C., Panzeri, E., Vantaggiato, C. and Orso, G. (2019).Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes. Front Neurosci 13: 1202. PubMed ID: 31803000
Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. This study investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. The data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. These data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment (Napoli. 2019).
Summerville, J., Faust, J., Fan, E., Pendin, D., Daga, A., Formella, J., Stern, M. and McNew, J. A. (2016). The effects of ER morphology on synaptic structure and function in Drosophila melanogaster. J Cell Sci 129(8):1635-48. PubMed ID: 26906425
Hereditary Spastic Paraplegia (HSP) is a set of genetic diseases caused by mutations in one of 72 genes that results in age-dependent corticospinal axon degeneration accompanied by spasticity and paralysis. Two genes implicated in HSPs encode proteins that regulate ER morphology. Atlastin (SPG3A) encodes an ER membrane fusion GTPase and Reticulon 2 (SPG12) helps shape ER tube formation. This study used a new fluorescent ER marker to show that the ER within wildtype Drosophila motor nerve terminals forms a network of tubules that is fragmented and made diffuse by atl loss. atl or Rtnl1 loss decreases evoked transmitter release and increases arborization. Similarly to other HSP genes, atl inhibits bone morphogenetic protein (BMP) signaling, and loss of atl causes age-dependent locomotor deficits in adults. These results demonstrate a critical role for ER in neuronal function and identify mechanistic links between ER morphology, neuronal function, BMP signaling, and adult behavior (Summerville, 2016).
The function of intracellular organelles is tightly coordinated with location within the cytoplasm. The endoplasmic reticulum (ER) is an interconnected network of narrow tubes and flattened cisternae or sheets. In most cells, the ER is the most abundant subcellular organelle and extends elaborate processes throughout the cytoplasm. The ER membrane is formed into its tubular architecture by the action of structural proteins within the reticulon, REEP and DP1 family. The members of this diverse family of proteins share a common protein motif called the reticulon homology domain (RHD). The hydrophobic ~200-amino-acid RHD likely forms a helical hairpin structure that intercalates four hydrophobic helical segments into the outer leaflet of the ER membrane to induce curvature and maintain a tubular shape. Many members of the Reticulon, REEP and DP1 family also contain an extended N-terminal segment ranging from a few hundred to a thousand amino acids that likely provides additional functionality. The nature of most of these secondary functions remains to be revealed (Summerville, 2016).
The large ER network also maintains luminal and membrane continuity throughout the cytoplasm. This interconnected nature of the ER network is required for ER function and is maintained by the ER membrane fusion GTPase atlastin, which is a member of the fusion dynamin-related protein family (fusion DRP)(Summerville, 2016).
The ER is closely associated with and functionally connected to the plasma membrane. This connection is often associated with the management of Ca2+ stores in the ER lumen. The ER protein STIM1 diffuses through the ER membrane to find binding partners in the plasma membrane including the Orai channel. This set of protein-protein associations works to restore ER Ca2+ through the store-operated Ca2+ channel system. ER-plasma-membrane contact sites are also generated by the association of ER-integral extended synaptotagmins (E-Syt) with phospholipids in the plasma membrane as well as proteins like junctophillins in certain cell types (Summerville, 2016).
Most recently, the ER has been found to be stably associated with endosomal structures. In this circumstance, the specific proteins on each surface that interact remain to be precisely defined, but the consequence of the interaction is functional segregation of certain cargoes within the endosome that permits regulated sorting into membrane subdomains prior to an ER-directed membrane fission event (Summerville, 2016).
ER structure appears to be crucially important for cell function given that human disease results when components that control this structure are compromised by mutation. The hereditary spastic paraplegias (HSPs) are a group of related genetic disorders caused by mutations in any of more than 70 genes, denoted SPG1 to SPG72. Lower limb weakness and spasticity represent two prominent clinical features of these diseases, which occur as a consequence of dysfunction or degeneration of the upper motor neurons. The observation that atlastin 1 (ATL1) and reticulon 2 (RTN2) are HSP genes responsible for SPG3A and SPG12, respectively, implicates ER morphology in the neuronal dysfunction that causes HSPs (Summerville, 2016).
The properties of three additional HSP genes, spartin (SPG20), spastin (SPAST, also known as SPG4) and NIPA1 (also known as spichthyin or SPG6), implicate receptor trafficking through the endocytic system in HSP neuronal dysfunction. For example, loss of spartin attenuates both ligand-stimulated EGF receptor uptake as well as depolarization-stimulated FM1-43 uptake, whereas loss of spastin increases endosome tubule number and alters transferrin receptor sorting). NIPA1 is also located in endosomes and promotes the endocytosis of receptors for bone morphogenetic protein (BMP) (Summerville, 2016).
Phenotypic analysis of mutations in the HSP orthologs of model systems has provided additional clues to the cellular function of these proteins. In Drosophila, loss of spastin, spartin and spichthyin confers a similar, but not identical, set of phenotypes including stabilized microtubules, increased synaptic bouton number and decreased evoked transmitter release at the larval neuromuscular junction (NMJ), age-dependent locomotor deficits and increased BMP signaling at the larval NMJ. These shared phenotypes might reflect disruption of a common pathway in endocytic receptor trafficking in these mutants. Some of these phenotypes, such as stabilized microtubules, age-dependent locomotor deficits and increased synaptic bouton number, are also observed in flies lacking atlastin (atl) (Summerville, 2016).
This study extends this phenotypic analysis of altered atl and reticulon-like 1 (Rtnl1) activities in Drosophila. The ER in motor nerve terminals from wild-type larvae forms a network of tubules that resembles a 'basket', but is diffuse in larvae lacking atl. Neuronal RNA interference (RNAi)-mediated knockdown of either atl or Rtnl1 increases arborization at the larval neuromuscular junction and decreases evoked transmitter release from larval motor nerve terminals, and elevated bath [Ca2+] fully rescues these transmitter release phenotypes. This study also showed that atl is required only in motor neurons to affect transmitter release, whereas Rtnl1 is required additionally in the target muscle and peripheral glia. This study shows that loss of atl increases BMP signaling in larval motor neurons and causes age-dependent locomotor deficits in adults. Thus, loss of atl and Rtnl1 confers phenotypes similar, but not identical, to each other as well as to mutants defective in spartin, spastin and spichthyin. These results demonstrate specific mechanistic links between ER morphology and several aspects of neuronal anatomy and function (Summerville, 2016).
Mutations in two genes that affect ER morphology, atlastin (ATL1) and reticulon 2 (RTN2), cause two forms of hereditary spastic paraplegia (HSP), which result in progressive limb weakness, spasticity and degeneration of the longest motor axons. These observations suggest that altered ER morphology is causal for motor axon dysfunction, but the mechanisms underlying these dysfunctions are unclear. This study used Drosophila to evaluate the nervous system deficits caused by altered atl and Rtnl1 activity. Using a new fluorescent ER imaging reagent, it was shown that the ER in wild-type motor nerve terminals is present as a network of tubules, termed 'baskets', underlying the plasma membrane, and that these baskets are eliminated in larvae lacking or overexpressing atl. This study also shows that loss of either atl or Rtnl1 increases arborization and decreases evoked transmitter release, and that evoked release is restored to normal by elevated bath [Ca2+]. Finally, atl loss was shown to increase signaling through the bone morphogenetic protein (BMP) pathway and causes age-dependent declines in adult locomotion. This set of phenotypes is also exhibited by Drosophila mutant for the HSP orthologs of spartin, spastin and spichthyin, as well as for spinster and nervous wreck, which encode regulators of receptor trafficking through endosomes (Summerville, 2016).
Two adjustments were made to improve visualization of the ER. First, a transgene was introduced into flies that encoded an ER-localized superfolder GFP, which was optimized for efficient folding in the ER. Second, based on previous results indicating that the ER as well as the lysosomal tubule network is labile to fixation, ER was imaged in live tissues. Using these approaches, the ER was shown to be present within the axon initial segments of motor neurons as a polygonal structure with numerous crossbridges (three-way junctions), and in motor nerve terminals as a network of tubules that were termed 'baskets', which underlie the plasma membrane. It was also shown that these structures are disrupted by either loss of or overexpression of atl. In particular, atl overexpression causes the aberrant appearance of large punctae in motor neuron cell bodies or axon initial segments. In contrast, atl loss decreases the number of crossbridges in the axon initial segment, leading to excessively long tubules. A similar appearance was noted previously and attributed to deficits in fusion of orthogonal ER membranes. Loss of atl also disrupts nerve terminal baskets and appears to cause ER fragmentation. It is possible that the transition from tubules to baskets as the ER moves from the interbouton region to boutons occurs through ER fragmentation followed by Atl-dependent reassembly. In this view, loss of atl would prevent this reassembly, thus causing the fragmented ER that was observed (Summerville, 2016).
Evoked transmitter release deficits in both atl2 and Rtnl11 mutants, and in pan-neuronal atl or Rtnl1 knockdown larvae, were rescued partially or completely by elevated bath [Ca2+]. These results indicate that loss of atl or Rtnl1 decreases evoked transmitter release at low bath [Ca2+] through causing deficits in evoked increases in cytoplasmic [Ca2+]. Insufficient Ca2+ influx could result from attenuated action potentials, which would decrease the opening of voltage-gated Ca2+ channels, decreases in number of plasma membrane Ca2+ channels or decreased Ca2+ release from the ER. Given the role of atlastin and the reticulons as ER-shaping molecules, effects on ER Ca2+ release would be the most direct explanation for this Ca2+ phenotype. ER-localized Ca2+ release channels such as the inositol 1,4,5-trisphosphate (IP3) receptor, the ryanodine receptor and the TRPV1 channel play key roles in evoked neurotransmitter release. In addition, dominant-negative mutations in the Drosophila ER-localized Ca2+ pump SERCA decrease evoked transmitter release by ~50%, which is consistent with the possibility that ER-derived Ca2+ contributes significantly to the Ca2+ required to trigger transmitter release (Summerville, 2016).
Unlike Atl, which appears to affect evoked transmitter release from neurons alone, Rtnl1 is required in neurons, muscles and peripheral glia for correct evoked transmitter release. This finding is consistent with previous data demonstrating that proper synaptic transmission requires intercellular signaling among these three cell types. In particular, loss of activity within the peripheral glia of the kinesin heavy chain gene or the inebriated-encoded neurotransmitter transporter alters evoked transmitter release. In addition, the peripheral glia secrete at least two proteins, the TGF-β ligand Maverick and Wingless/Wnt, that regulate synaptic function. The muscle, in turn, secretes the BMP ligand Gbb to regulate both evoked transmitter release and motor neuron arborization. It is possible that loss of Rtnl1 affects transmitter release from glia or muscle by perturbing secretion of these or other regulators (Summerville, 2016).
The most prominent clinical symptom in HSP patients is progressive, age-dependent locomotor difficulties. Drosophila mutant for any of several HSP orthologs, including spartin, spastin, atl and Rtnl1, as well as spinster, exhibit similar age-dependent locomotor deficits or lifespan deficits. This study shows locomotor impairment in adults with neuronal-specific atl knockdown. These results indicate a requirement for atl in neurons for proper locomotion but do not rule out crucial roles for atl in other tissues as well (Summerville, 2016).
Mutants in Drosophila orthologs of several HSP genes, including spartin, spastin and spichthyin, and the additional related genes spinster and nervous wreck share a common set of nervous system phenotypes, including increased arborization and BMP signaling at the larval NMJ, decreased evoked transmitter release and locomotor deficits (note that not all phenotypes have been reported for each mutant). The encoded proteins localize to various compartments within the endocytic receptor trafficking pathway. In fact, the increased BMP signaling in several of these mutants has been attributed to trafficking defects of the BMP receptor Wishful thinking (Wit). This study has shown that atl loss confers these same phenotypes, raising the possibility that atl acts in the endocytic receptor trafficking pathway as well. Although the ER is not known to play prominent roles in this pathway, a recent report has demonstrated that the ER is required for endosome fission in COS cells, and, in fact, the ER selects the location of fission. In addition, it has been found that this process is inhibited by overexpression of Rtn4a, which, similarly to atl loss, elongates ER tubules and inhibits formation of crossbridges. Thus, loss of atl could impact on the receptor trafficking pathway in Drosophila nerve terminals by similarly preventing endosome fission (Summerville, 2016).
The variety of phenotypes exhibited in common by the mutants described above raises the possibility that certain phenotypes might have causal relationships with others. The subcellular locations of these proteins suggest that they might directly affect receptor trafficking. If so, then the increased BMP signaling, as a consequence of altered Wit trafficking, might be the direct cause of the increased arborization and locomotor deficits. The phenotypes conferred by direct activation of the BMP pathway in neurons are consistent with this possibility. However, the increased BMP signaling is unlikely to cause the decreased transmitter release, as decreased BMP signaling, rather than increased BMP signaling, decreases evoked transmitter release. It is suggested that trafficking of receptors in addition to Wit are altered in the mutants described above, and it is the altered signaling of these additional receptors that is at least partly responsible for the transmitter release phenotype. Drosophila motor nerve terminals express a cholecystokinin-like receptor (CCKLR), a toll-like receptor, a metabotropic glutamate receptor (mGluRA) and likely the insulin receptor. Loss of mGluRA increases evoked transmitter release, raising the possibility that increased mGluRA signaling might decrease transmitter release, which could explain the decreased transmitter release observed in these receptor trafficking mutants (Summerville, 2016).
Du, F., Ozdowski, E.F., Kotowski, I.K., Marchuk, D.A. and Sherwood, N.T. (2010). Functional conservation of human Spastin in a Drosophila model of autosomal dominant-hereditary spastic paraplegia. Hum Mol Genet 19: 1883-1896. PubMed ID: 20154342
Consistent with their functional conservation, both the fly and human spastin proteins are diffusely distributed in the cytoplasm and can permeate throughout long neuronal processes. Overexpression does reveal some differences between the two proteins, however. Human Spastin forms more aggregates than does fly Spastin; this is observed independent of the transgene insertion, and whether a genomic or cDNA fly transgene is used. Additionally, two copies of the fly wild-type transgene become deleterious to rescue, although no significant differences are observed between one and two copies of the human transgene. Processing of these two proteins is therefore not identical in this overexpression context, although their ability to substitute for the loss of endogenous spastin is indistinguishable (Du, 2010).
Although differences in transgene expression levels are one caveat of the GAL4-UAS expression system, analysis of the various insertion lines used in these experiments reveals that the observed mutant phenotypes are relatively insensitive to expression levels, correlating instead with the different recombinant genotypes. Pathogenic AD-HSP genotypes resemble spastin loss of function phenotypes at behavioral, cellular and subcellular levels. The ability of the human pathogenic mutations to mimic the cellular and subcellular spastin null phenotypes is even more remarkable given that this phenotype of numerous small, clustered boutons devoid of microtubules has not been observed in other mutants affecting the larval NMJ, including mutations in other closely related microtubule severing proteins. This underscores the specificity of these phenotypes and their direct relation to Spastin function (Du, 2010).
Further data support the validity of these flies as a model for the human disease, and furthermore as the only model demonstrating robust behavioral phenotypes in the heterozygous mutant condition that typifies most AD-HSP patients. Using this model, human pedigree analyses, correlating disease augmentation with trans-heterozygous expression of L44 or Q45 and mutations interfering with Spastin's catalytic domain, were confirmed, substantiating the significance of this amino-terminal region in normal Spastin function. Although humans heterozygous for the S44L mutation are typically asymptomatic, the mutation may have some effect on protein function at the cellular and subcellular levels. Consistent with this observation, abnormal electromyogram measurements have been found in L44 heterozygous individuals despite their outward lack of disease symptoms (Du, 2010).
Several hypotheses have been proposed for the mechanism underlying exacerbated disease severity in the compound heterozygous condition. Modifier effects could occur at the level of the spastin protein; for instance, S44/P45 may be a phosphorylation target important in the regulation of Spastin function. S44 is also predicted to comprise part of a PEST sequence, such that mutations in it would interfere with degradation of full-length (but not truncated) Spastin isoforms. Alternatively, the base pair mutations underlying these amino acid substitutions fall within a predicted cryptic promoter in the first exon, and could affect its activity and inhibit the transcription of the shorter spastin isoform. These data argue against a transcriptional mechanism, given that transgene expression in the AD-HSP model flies is under the control of an exogenous promoter. However, the precise mechanism of the exacerbating effect remains to be resolved, and can be directly tested in this model (Du, 2010).
A growing list of Drosophila models of HSP is contributing key insights to the function of the human spastic gait (SPG) genes: in addition to SPG4/spastin, these include SPG3A/atlastin, a novel GTPase shown in flies to be required for ER morphogenesis, SPG6/NIPA1, elucidated as a regulator of BMP signaling through studies of its Drosophila ortholog spicthyn and SPG39/NTE, Drosophila Swiss-cheese, mutations in which disrupt the kinase activity and localization of PKA-C3 and cause progressive neurodegeneration (Du, 2010).
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
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).
Sujkowski, A., Rainier, S., Fink, J.K. and Wessells, R.J. (2015). Delayed induction of human NTE (PNPLA6) rescues neurodegeneration and mobility defects of Drosophila swiss cheese (sws) mutants. PLoS One 10: e0145356. PubMed ID: 26671664
Whereas previous studies also observe reduced vacuole formation in sws1 flies overexpressing Drosophila, mouse, or human PLNPA6, this study correlates reduced vacuole formation with longitudinal measures of mobility performance using a recently developed protocol for measuring Drosophila endurance. Measuring endurance with an automated system for inducing negative geotaxis behavior offers a non-invasive assessment of both coordination and strength, suitable for longitudinal assessment of performance decline. This system effectively measures performance decline as a result of either muscle-specific interventions. This system can be useful for future assessments of degenerative disease models or as an assessment of functional aging (Sujkowski, 2015).
Because hNTE expression is not induced in mutants until after development is complete, some developmental mobility defects could persist in adult hNTE rescue flies. However, hNTE rescue flies exhibit consistently faster climbing speed and higher endurance in comparison to both sws5 mutants and transgenic controls. Furthermore, induction of rescue even after substantial decline has taken place in adults can not only slow decline, but actually reverse the decline in endurance of a longitudinally measured cohort (Sujkowski, 2015).
Taken together, these observations imply that protection of mobility may not absolutely require reversal of vacuole formation. Blocking the formation of additional vacuoles, or blocking the expansion of existing vacuoles may be sufficient to allow some degree of functional recovery. These results support the idea that induction of NTE activity may be therapeutically valuable even after substantial degeneration has occurred. Extending these studies to vertebrate models of NTE mutation will be critical (Sujkowski, 2015).
Wang, X., Shaw, W.R., Tsang, H.T., Reid, E. and O'Kane, C.J. (2007). Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules. Nat Neurosci 10: 177-185. PubMed ID: 17220882
The posterior crossveinless phenotype in some spictmut adult wings is also typical of reduced BMP signaling in pupal wing discs. At first sight a crossveinless phenotype is inconsistent with Spict being an antagonist of BMP signaling. However, lowered BMP signaling in the posterior crossvein primordium could be due not only to direct downregulation of signaling, but also to upregulation of receptors that reduces diffusion of BMP ligands. Any changes in the level of BMP signaling about the time when the posterior crossvein primordium develops were not detected, but this could be due to either the partial penetrance of the phenotype, or the robustness of the regulatory and feedback mechanisms that translate smooth gradients of BMP ligands into more sharply defined developmental features (Wang, 2007).
How might an endosomal protein regulate BMP signaling? Membrane trafficking from the plasma membrane to lysosomes regulates many signaling pathways including BMP/TGF-β. For example, mutations that impair endosome to lysosome traffic cause an increase in BMP signaling, in at least some cases accompanied by increased levels of Tkv. However, the predominant localization of Spict on early endosomes, and its ability to internalize Wit to this compartment suggest that Spict functions at some step of plasma membrane to endosome traffic. First, Rab5 compartments fail to accumulate at spictmut NMJs, rather than enlarge as in Hrs mutants. Second, Spict overexpression in S2 cells redistributes Wit mainly to early endosomes, rather than to late endosomes or lysosomes. Third, there is no obvious degradation of Wit in Spict-overexpressing cells that internalize Wit, suggesting that Spict does not directly target Wit for degradation, at least in S2 cells. While levels of BMP receptors are elevated locally in NMJ boutons that lack Spict, this could be either to altered trafficking or degradation, and BMP signaling in S2 cells can be affected by Spict, without detectable changes in levels of BMP receptors. Therefore, Spict might inhibit BMP signaling by internalizing vacant receptors and thus preventing them from responding to ligand; since clathrin RNAi treatment redistributes Spict to the plasma membrane, Spict probably appears at least transiently at the plasma membrane. However, more complex models are possible. For example, Spict might sequester BMP receptors in a compartment from which they cannot signal; Notch receptors apparently have to reach a specific endosomal compartment before they can signal (Wang, 2007).
By studying Spict, the study identifies a role for BMP signaling in maintenance of axonal microtubules. Notably, local loss of presynaptic microtubules has also been seen in loss of BMP signaling at the NMJ, and apical microtubule arrays are eliminated in tkv mutant clones in wing imaginal discs. Since BMP signaling promotes synaptic growth and synaptic strength at the NMJ, it would be logical for it also to stimulate the additional transport of materials and organelles that a larger more active synapse requires (Wang, 2007).
If human SPG6 alleles are dominant gain-of-function, then the HSP that they cause would resemble the situation of Spict overexpression in Drosophila, and axonal degeneration in HSP could then be caused by inhibition of BMP signaling, loss of axonal microtubules, and impaired axonal transport. Given the effect of BMP signaling on axonal microtubules, other HSP gene products apart from SPG6 may affect BMP signaling and thus maintenance of axonal microtubules (Wang, 2007).
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
Solowska, J.M., D'Rozario, M., Jean, D.C., Davidson, M.W., Marenda, D.R. and Baas, P.W. (2014). Pathogenic mutation of spastin has gain-of-function effects on microtubule dynamics. J Neurosci 34: 1856-1867. PubMed ID: 24478365
Orso, G., Martinuzzi, A., Rossetto, M.G., Sartori, E., Feany, M. and Daga, A. (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 ID: 16276413
Dutta, S., Rieche, F., Eckl, N., Duch, C. and Kretzschmar, D. (2015). Glial expression of Swiss-cheese (SWS), the Drosophila orthologue of Neuropathy Target Esterase, is required for neuronal ensheathment and function. Dis Model Mech [Epub ahead of print]. PubMed ID: 26634819
O'Sullivan, N.C., Jahn, T.R., Reid, E. and O'Kane, C.J. (2012). Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons. Hum Mol Genet 21: 3356-3365. PubMed ID: 22543973
Deshpande, M. and Rodal, A.A. (2016). The crossroads of synaptic growth signaling, membrane traffic and neurological disease: Insights from Drosophila. Traffic 17: 87-101. PubMed ID: 26538429
Date revised:25 April 2019
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