InteractiveFly: GeneBrief

Ankyrin 2: Biological Overview | References

BIOLOGICAL OVERVIEW

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein G_o functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the G_o-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the G_o-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane (Bennett, 2001). Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2] (Dubreuil, 1994; Bouley, 2000). Ank2 is expressed exclusively in neurons and exists in several splicing variants (Koch, 2008; Pielage, 2008). The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function (Hortsche, 2002; Koch, 2008; Pielage, 2008). The C-terminal part of Ank2L can bind to microtubules (Pielage, 2008). Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric G_o protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ (Koch, 2008; Pielage, 2008), is a target of Gαo in this signaling (Luchtenborg, 2014).

This study found that the α subunit of G_o is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that G_o is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, G_o acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than G_o might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R (Koon, 2012). This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octopaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers (Koon, 2012). It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

Upon dissociation of the heterotrimeric G_o protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

Hox function is required for the development and maintenance of the Drosophila feeding motor unit

Feeding is an evolutionarily conserved and integral behavior that depends on the rhythmic activity of feeding muscles stimulated by specific motoneurons. However, critical molecular determinants underlying the development of the neuromuscular feeding unit are largely unknown. This study identified the Hox transcription factor Deformed (Dfd) as essential for feeding unit formation, from initial specification to the establishment of active synapses, by controlling stage-specific sets of target genes. Importantly, Dfd was found to control the expression of functional components of synapses, such as Ankyrin2-XL, a protein known to be critical for synaptic stability and connectivity. Furthermore, Dfd was uncovered as a potential regulator of synaptic specificity, as it represses expression of the synaptic cell adhesion molecule Connectin (Con). These results demonstrate that Dfd is critical for the establishment and maintenance of the neuromuscular unit required for feeding behavior, which might be shared by other group 4 Hox genes (Friedrich, 2016).

Stereotypical motor behaviors are the primary means by which animals interact with their environment, forming the final output of most CNS activity. One such behavior is feeding, a crucial and highly conserved activity in all animals. The motor output consists of coordinated contractions of distinct head muscles in a rhythmic pattern required for chewing, sucking, and swallowing of food. Food uptake in adult flies has recently been shown to be controlled by a single pair of interneurons emanating from the subesophageal ganglion (SEG), an insect brain region primarily associated with taste and feeding. Although a substantial number of neurons are linked to different aspects of feeding behavior in flies, molecular factors critical for the establishment and development of feeding motor patterns have not been identified so far (Friedrich, 2016).

The fruit fly Drosophila melanogaster is an excellent model to study the developmental aspect of feeding behavior for several reasons. First, Drosophila takes up food extensively during its larval stage, when the organism almost exclusively feeds to increase its body weight and size. Additionally, the anatomical framework and motor patterns critical for larval food uptake are well described. Feeding requires the rhythmic extension and retraction of the head skeleton, the cephalopharyngeal skeleton (CPS), coupled with coordinated elevation and depression of the mouth hooks (MHs), mandible-derived structures required for chopping up solid food, and subsequent food ingestion. The repetitive larval feeding movements are controlled by head muscles innervated by CNS nerves emerging from the SEG. CPS protraction and tilting are mediated by protractor muscles receiving input from the prothoracic nerve, while MH motor patterns are controlled by the mouth hook elevator (MHE) and depressor (MHD), which are innervated by the maxillary nerve. Food ingestion is achieved by the cibarial dilator muscle (CDM), which is connected to the CNS via the antennal nerve. The cellular framework of Drosophila larval feeding is established during embryogenesis. Thus, molecular and genetic approaches can be used to identify and analyze factors controlling specification and communication of cell types critical for larval motor patterns. In contrast, neuromuscular units required for motor activities in adult flies develop from stem cell systems during larval and pupal stages in a process called metamorphosis. Due to the limited accessibility of this transitional phase, embryonic stages are better suited to study the development of neuromuscular units required for regional movements (Friedrich, 2016).

The Hox family of transcription factors (TFs) have emerged as key regulators of motor behaviors. One such behavior is locomotion, which Drosophila larvae perform by region-specific contractions of abdominal segments allowing them to crawl on substrate. Segment-specific changes of peristaltic movements in animals carrying mutations in the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A) led to the assumption that Hox genes orchestrate the development of regional motor activities. Recent studies have now revealed that Hox proteins perform their task in a very refined manner and seem to have a direct transcriptional input on successive steps of motoneuronal development. As one such example, Hox5 function was shown to be required in motoneurons that control the contraction of breathing muscles in vertebrates: Hox5 deletion in mice leads to progressive death of phrenic motor column (PMC) neurons as well as to the inability of surviving PMC neurons to innervate the diaphragm muscle. However, despite the fact that blocking motoneuron apoptosis did rescue the decline in PMC neuron number, branching and innervation defects were still unchanged under these conditions. These findings imply that Hox5 proteins directly regulate early and late processes in the course of PMC neuron differentiation, a hypothesis still awaiting confirmation (Friedrich, 2016).

Hox genes are segmentally expressed along the anterior-posterior body axis of animals, suggesting that members of this gene family expressed in the head region should control food uptake. Intriguingly, the Drosophila group 4 Hox gene Deformed (Dfd), which is known to specify the SEG, has already been associated with feeding behavior before: animals carrying a hypomorphic Dfd allele starve to death as adult flies due to the inability to move their proboscis, an action crucial for food ingestion. This work reveals that Dfd, which is expressed in many cell types, including a large number of SEG neurons, is functional in motoneurons and muscles that drive the movements critical for hatching and feeding. Most interestingly, it was shown that Dfd exerts its function via direct and stage-specific regulation of target genes. Importantly, Ank2-XL, a microtubule organizing protein required for synaptic stability, was shown to be under direct Dfd control throughout different stages in the animal's life. Furthermore, synchronous expression of Dfd targets with critical function in synaptic target specificity, in particular, the cell adhesion molecule (CAM) Connectin (Con), was found in feeding neurons and muscles. This suggests that Dfd positively and/or negatively regulates different CAMs providing a specificity code required for the establishment of regional motor units (Friedrich, 2016).

Consistent with a temporal requirement of Dfd for motoneuronal development, an over-representation of neuronal genes was found among those genes associated with ChIP-seq identified Dfd binding regions, which were classified as Dfd target genes. Importantly, grouping of these genes based on similar GO annotations showed that they operate at different time points in neuronal development: during neurogenesis and neuronal specification (32/182), when axon outgrowth and guidance decisions occur (86/182), and during synapse-related processes (85/182). To test the temporal control of these genes by Dfd, their expression was analyzed when Dfd function was abolished at two different developmental stages. For early interference, ^Dfd16 loss-of-function embryos was used, while late interference was achieved using animals that carry the temperature-sensitive ^Dfd3 allele and were shifted to the restrictive temperature only during larval stages. Early neurogenesis target genes, including prospero, a gene involved in asymmetric neuroblast division, was found to be mis-localized in ^Dfd16 null mutant embryos. Consequently the expression of genes required for subsequent processes in motoneuronal development, like the axon guidance genes capricious (caps), roundabout 2 (robo2), roundabout 3 (robo3), and Neural Lazarillo (NLaz), were also affected. Thus, ^Dfd16 null mutants are unable to form the neuromuscular unit required for MH movements due to the inability to activate the proper developmental program. In contrast, the MH-associated motor unit of ^Dfd3 animals shifted to the restrictive temperature during larval stages was intact, with respect to outgrowth of maxillary nerve projecting motoneurons and MHE innervation. Accordingly, the expression of early Dfd neuronal targets was unchanged (data not shown). However, compared to the control group the expression of Dfd target genes critical for synapse-related processes was substantially altered in these late-shifted ^Dfd3 third-instar larvae. This includes Ankyrin2 extra large (Ank2-XL), which is encoded in the ank2 locus. Ank2-XL, which is part of a membrane-associated microtubule-organizing complex, is known to be required for the establishment of appropriate synaptic dimensions and release properties (Stephan, 2015). This study found that not only was Ank2 mRNA levels reduced in SEG neurons in late-shifted ^Dfd3 third-instar larvae, but also observed decreased Ank2-XL protein expression in synaptic boutons, axons and their terminals on the MHE. Similar to a recent report (Stephan, 2015), Futsch/MAP1B, a microtubule-associated protein known to form a membrane-associated complex with Ank2-XL, was also found to be reduced in synaptic boutons of late-shifted ^Dfd3 third-instar larvae. Concomitantly, the morphology of synaptic boutons on this muscle was also changed in late-shifted ^Dfd3 third-instar larvae: they were not of uniform size but appeared often dramatically increased compared to boutons of control animals. This is in line with the described phenotype of ank2-XL mutant animals (Stephan, 2015), which was suggested to reflect the failed separation of neighboring boutons. The effects observed are due to Dfd's action in (moto)neurons, as tissue-specific knockdown of Dfd activity in neuronal cells only using the elav-GAL4 driver in combination with two independent UAS-DfdRNAi lines resulted in severe bouton phenotypes and Ank2-XL expression changes, while the muscle architecture was completely normal. Similar results on Ank2-XL expression and synapse morphology were obtained in Dfd13/Df(3R)Scr third-instar larvae that survived to this stage. The effect of Dfd on synapses on the MHE is specific, since neuromuscular junctions on control muscles, like the CDM, were completely normal with respect to their morphology and Ank2-XL expression in late-shifted ^Dfd3 third-instar animals. These results show that Dfd activity is continuously required during the formation of the feeding motor unit, from its specification to the establishment of synaptic connections, and that Dfd executes this function by directly regulating the transcription of phase-specific components. Intriguingly, these findings demonstrate that the Hox TF Dfd is one of the upstream regulators coordinating Ankyrin-dependent microtubule organization and synapse stability and provides evidence that Dfd function is required even after the initial establishment of the motor unit to control synapse-related processes via its synaptic targets, like Ank2-XL. Finally, the results indicate that synaptic stability and plasticity is not only determined by the half-life of synaptic proteins, but is dependent on a robust transcriptional program that provides a continuous supply of essential synaptic components that maintain the system (Friedrich, 2016).

This analysis has shown that Dfd is expressed in SEG motoneurons. In addition, Dfd was found to be present in embryonic muscles, which later form the feeding/hatching motor unit. Defects were observed in the structure and number of the MH-associated muscles in the embryo when Dfd function was abolished. As was the case in the CNS, Dfd seems to execute its muscle-specific function in an immediate manner, since a substantial fraction (7.4%) of the genome-wide identified Dfd target genes is associated with mesoderm-related functions. The innervation of the Dfd-expressing MHE by Dfd-positive motoneurons raised the intriguing possibility that the activity of the Hox protein Dfd provides a code on the functionally connected neurons and muscles crucial for the recognition and matching of the synaptic partners and, thus, the execution of rhythmic motor patterns. Consistent with this hypothesis, 27 of the ChiP-seq identified Dfd target genes encode factors with described functions in muscles and the nervous system, and importantly nine of these genes play an important role in synaptic target recognition, like tartan (trn), Connectin (Con), or capricious (caps). Therefore, the expression of the homophilic cell adhesion molecule Con was analyzed, and it was found to be exclusively expressed in motoneurons and muscles devoid of Dfd protein in wild-type embryos, suggesting that Dfd might function as a suppressor of Con expression. In order to provide vigorous proof for this hypothesis, cells were specifically labeled that were devoid of Dfd function in Dfd mutants, and their ability to now express Con was analyzed. Use was made of the fact that ^Dfd16 mutants that do not produce any functional protein (protein-null mutants) still express Dfd mRNA. Consistent with this hypothesis, de-repression of Con mRNA expression was found in many Dfd mutant neuronal cells that were labeled by the presence of Dfd mRNA. Due to the inability of Dfd mutant embryos to involute their heads (which reorganizes the order of the head muscles) and due to the high variance of the muscle phenotype, Con expression in this tissue was not shown in the Dfd loss-of-function situation. Taken together, these results demonstrate that Dfd is one of the critical upstream regulators, which coordinates the interdependent events of neuromuscular development and connectivity by positively or negatively regulating the expression of synaptic target selection molecules on the interacting motoneurons and muscles. Furthermore, it shows that the expression of synaptic cues is tightly regulated even in neurons located in close or direct proximity, allowing these cells to express different sets of synaptic recognition molecules thereby ensuring that they make the proper connections with their synaptic partners (Friedrich, 2016).

Hox genes have been shown to control several motor activities along the anterior-posterior axis of animals; however, critical determinants regulating feeding movements have not been previously identified. This study has shown that the Drosophila group 4 Hox gene Dfd controls multiple aspects in both the establishment and maintenance of the neural network controlling feeding behavior (Friedrich, 2016).

A crucial finding from this study is that Hox TFs are required throughout the formation of regional motor units and mediate their effect not only through the induction of downstream TFs. In fact, it was shown that Hox factors control distinct effector target genes, which realize stage-specific processes in a very immediate manner. This is true for Ankyrin2-XL, which, along with the MAP1B homolog Futsch, forms a membrane-associated microtubule-organizing complex that determines axonal diameter, supports axonal transport, and controls synaptic dimensions and stability (Stephan, 2015). Interestingly, it was found that Dfd is required for the maintenance of Ank2-XL expression, not only when the motor system is established but also when it is fully operational. In the light of recent findings showing that mis-regulation of Ankyrin 1 (ANK1) has an important role in the neurodegenerative Alzheimer disease, these results raise the intriguing possibility that Hox genes have a neuro-protective function (Friedrich, 2016).

An important question arising from this study is whether the establishment of feeding-related motor patterns is one of the basic functions of group 4 Hox genes and thus conserved in the animal kingdom. Promisingly, it is known that tongue muscles critical for rhythmic feeding movements in mammals are innervated by the hypoglossal nerve. This nerve has its origin in rhombomere 8, which expresses several group 4 Hox genes, including Hoxb4. Preliminary analysis using a previously identified fish Hoxb4 promoter as a reporter in the teleost fish medaka (Oryzias latipes) shows that GFP is expressed in distinct neuronal subpopulations of the post-otic hindbrain and the spinal cord in stable Hoxb4-GFP medaka embryos. Intriguingly, a subset of neurons co-expressing GFP and Hoxb4 project their axons ventrally toward Hoxb4-positive cells within the pharyngeal region. Both the branchial muscles and the pharyngeal jaw specialized for feeding in teleost fish develop from this area. When medaka embryos have developed into hatchlings, axons emerging from GFP-labeled neurons innervate branchial muscles and the sternohyodeus, muscle groups required for mouth opening and food swallowing. Thus, the regulatory and transcriptional network dictating the formation of the respective feeding units in flies and fish could be conserved despite the fact that muscles and bones responsible for the execution of feeding movements are of different origin. In future, more functional studies are needed to validate the potentially conserved role of homology group 4 Hox genes in regulating rhythmic feeding movements throughout the animal kingdom (Friedrich, 2016).

The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. This study addresses the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. A P[acman] based rescue approach was used to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. Specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, these data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance (Weber, 2019).

Giant ankyrins serve as central organizing adaptor molecules in both invertebrate and vertebrate neurons. This study provides first insights into the control of synaptic localization and function of giant Ankyrins by the N-terminal ankyrin repeat domain in vivo. By selectively deleting subdomains of the ARD of ank2 this study unravel critical requirements of specific regions of the ARD for the synaptic localization of the neuronal specific giant isoforms Ank2-L and Ank2-XL. The data demonstrate that the N-terminal domain controls not only synaptic targeting of individual isoforms but also contributes to synaptic localization of the alternative isoform. The functional requirements of Ank2-L and Ank2-XL for synaptic stability and microtubule organization clearly correlate with ARD-dependent regulation of protein abundance at the presynaptic terminal, with individual subdomains providing unique functional features (Weber, 2019).

The N-terminal ARD mediates membrane-association of Ankyrins and is essential for the subcellular localization and organization of transmembrane binding partners. Prior work largely focused on the organizational roles of giant Ankyrins at the AIS and nodes of Ranvier in vertebrate neurons. In vivo analysis of ARD deletions now revealed a critical importance of this domain for the localization of Ank2-L at the presynaptic terminal of motoneurons. The synaptic localization of Ank2-L depends on AnkD2-4 (repeats 7-24) while the first domain (repeats 1-6) only had a minor impact on protein abundance. Interestingly, axonal targeting of Ank2-L was only partially affected by these deletions. These results indicate that separate and distinctive mechanisms exist in vivo that enable selective localization of giant Ankyrins in axons and within the presynaptic motoneuron terminal. As similar localization defects were observed already at earlier stages of development it argues that AnkD2-4 are essential for initial synaptic localization. Previous work demonstrated that a fusion protein comprising the specific C-terminal tail domain of Ank2-L efficiently localizes to both axonal and synaptic areas. The demonstration that synaptic localization of full length Ank2-L requires an intact ARD indicates that subcellular distribution is precisely regulated and not achieved by passive distribution within the neuron. Of particular interest is the observation that the first six ankyrin repeats are dispensable for synaptic localization of Ank2-L in the ank2^ΔL mutant background. The recent characterization of the structure of the human ARD demonstrated distinct and independent binding sites for voltage-gated sodium channels (Nav1.2) and for L1-family CAMs (Neurofascin) in vitro and in cultured neurons. Interestingly, in cultured hippocampal neurons the L1-family CAM binding sites within AnkD2 (ankyrin repeats 8-9 and 11-13) are essential for clustering of the 270 kDa AnkG isoform at the AIS. In contrast, AnkG lacking the Nav1.2 binding site within AnkD1 (ankyrin repeats 2-6) efficiently localized to the AIS but failed to cluster Nav channels at the AIS. Thus, similar to the situation at the vertebrate AIS, the synaptic localization of Drosophila Ank2-L largely depends on interactions with L1-family CAMs or alternative transmembrane proteins that occupy binding sites within the central and C-terminal part of the ARD. This localized L1-CAM-Ankyrin complex can then serve as an assembly platform for additional molecules like voltage-gated sodium channels that acquired Ankyrin-binding capacities during chordate evolution to determine the physiological properties of specific subcellular neuronal compartments. While the AnkD1 of Drosophila ank2 had the least impact on synaptic stability phenotypes it will be interesting to identify putative binding proteins that may provide specific functional properties as absence of this domain resulted in decreased survival and locomotion of adult flies (Weber, 2019).

In contrast to Ank2-L, the deletion of individual parts of the ARD had smaller effects on synaptic targeting of Ank2-XL in the ank2^ΔXL mutant background. In this study, only the deletion of AnkD3 (repeats 13-18) resulted in a significant reduction of axonal and synaptic localization. However, the analysis revealed a striking dependence of Ank2-XL on the presence of the intact ARD of Ank2-L. Impairments of Ank2-L localization, including the minor effects caused by deletion of AnkD1, resulted in a severe reduction of synaptic Ank2-XL levels. This shows that Ank2-XL localizes via Ank2-L to the presynaptic terminal consistent with prior observations. Interestingly, deletions of parts of the ARD of Ank2-XL also significantly reduced synaptic Ank2-L levels indicating a critical co-dependence of Ank2-L and Ank2-XL at the NMJ. This interaction may depend on direct interactions between the two Ankyrin isoforms, potentially mediated within the ARD. However, it is equally possible that incorporation of mutated Ankyrins with altered binding properties resulted in a disruption of the larger Ankyrin-Spectrin scaffold that in turn leads to reduced targeting of the other isoform. Indeed, in vertebrate axons it has been demonstrated that different affinities of specific giant Ankyrins control the isoform-specific incorporation into selective axonal compartments like the nodes of Ranvier (Weber, 2019).

At a functional level, a strong correlation was observed between synaptic Ank2-L level and the control of synaptic stability. At all muscle groups analyzed it was observed that deletion of AnkD1 domain had the mildest impact on synaptic stability consistent with the least impairments of synaptic Ank2-L localization. Importantly, the analysis of synaptic stability at muscle 4 also revealed that AnkD3 is most critical for synaptic maintenance. Despite an almost identical reduction in Ank2-L level compared to AnkD2 and 4 the rescue construct lacking the third domain was unable to restore any synaptic stability of ank2^ΔL mutant animals. Interestingly, this assay also highlighted the specificity of the individual ankyrin repeats within the ARD. The exchange of second domain sequences with analogous sequences of the first domain failed to restore both Ank2-L localization and the synaptic stability phenotype of ank2ΔL mutants. The failure to support synaptic stability is at least in part due to a failure to organize and stabilize synaptic cell adhesion molecules. This analysis demonstrated that AnkD2 and 3 are critical for normal clustering of the NCAM homolog Fas II and of the L1 CAM homolog Nrg. This is consistent with prior observations that Ank2-L supports synaptic organization of both Fas II and Nrg and the co-dependence of Ank2-L and these CAMs at the synapse. This function is evolutionary conserved as mutations in the second and third binding site of vertebrate AnkG failed to restore clustering of the L1 CAM family members Neurofascin or Nr-CAM at the AIS of hippocampal neurons (Weber, 2019).

In contrast to the clear requirements of the ARD for presynaptic targeting of Ank2-L the same mutations only partially affected localization of Ank2-XL. Consistently, only mild disruptions were observed of the presynaptic microtubule cytoskeleton and of the synaptic bouton organization compared to the ank2^ΔXL mutation. The effects were most severe when deleting AnkD2 despite the fact that Ank2-XL protein levels were more compromised in AnkD3 mutants. As the large C-terminal repeat region of Ank2-XL is essential for the interaction with microtubules these results indicate that inappropriate ARD-dependent complex assembly may influence the functional properties of Ank2-XL at the NMJ (Weber, 2019).

Together, this in vivo analysis of the ARD of Drosophila giant Ankyrins uncovered a structural basis for presynaptic localization and provided a genetic basis for the identification of regulatory mechanisms controlling structural synaptic plasticity via the selective Ankyrin complex assembly (Weber, 2019).

Expression of a fragment of Ankyrin 2 disrupts the structure of the axon initial segment and causes axonal degeneration in Drosophila

Neurodegenerative stimuli are often associated with perturbation of the axon initial segment (AIS), but it remains unclear whether AIS disruption is causative for neurodegeneration or is a downstream step in disease progression. This study demonstrates that either of two separate, genetically parallel pathways that disrupt the AIS induce axonal degeneration and loss of neurons in the central brain of Drosophila. Expression of a portion of the C-terminal tail of the Ank2-L isoform of Ankyrin severely shortens the AIS in Drosophila mushroom body (MB) neurons, and this shortening occurs through a mechanism that is genetically separate from the previously described Cdk5alpha-dependent pathway of AIS regulation. Further, either manipulation triggers morphological degeneration of MB axons and is accompanied by neuron loss. Taken together, these results are consistent with the hypothesis that disruption of the AIS is causally related to degeneration of fly central brain neurons, and it is suggested that similar mechanisms may contribute to neurodegeneration in mammals (Spurrier, 2019).

The axon initial segment (AIS) is a key neuronal domain that lies between the somatodendritic and axonal compartments. The AIS is a gatekeeper for intracellular transport that maintains neuronal polarity, and it is essential for proper neuronal excitability as it is the site of action potential (AP) initiation and a critical target for AP modulation. The AIS performs these tasks by virtue of a unique composition of membrane proteins, including a distinctive set of voltage-gated ion channels and submembranous cytoskeletal proteins. The structure of the AIS in vertebrate neurons depends on the presence of a 'master organizer,' Ankyrin G (ankG), a giant ankyrin isoform that recruits and anchors the specialized set of proteins that performs its distinctive functions (Spurrier, 2019).

Aberrant AIS regulation and function have been linked to a variety of diseases. Changes in sequence or expression levels of proteins localized to the AIS are involved in epilepsy, bipolar disorder, schizophrenia [, and autism spectrum disorder. Perturbation of the AIS has also been linked to neurodegeneration under a variety of contexts. The AIS contributes to proper tau localization, and breakdown of its barrier function leads to tau missorting, while expression of Alzheimer's disease (AD)-related tau mutants was found to decrease AIS-associated proteins and shorten the overall length of the AIS, SPTBN4 (encoding non-erythrocyte β-spectrin 4) was identified in a DNA methylation screen of genes related to AD; β-spectrin works in coordination with ankG to localize proteins to the AIS. Further, in a mouse model of AD, it was found that AIS length was reduced near Aβ plaques. Aβ plaques are also associated with microglia recruitment, which is itself associated with AIS structural plasticity. While recent literature suggests that various provocations, including pathology, can trigger structural plasticity of the AIS, the underlying mechanisms remain enigmatic (Spurrier, 2019).

Previous work has shown that some Drosophila neurons include a domain that exhibits the characteristic hallmarks of the mammalian AIS, providing a simpler model in which to study the molecular pathways contributing to AIS biogenesis and regulation (Trunova, 2011). First, there is selective accumulation of an anchoring protein within this subcellular domain. Drosophila only has two ankyrin genes: Ank1, which is expressed ubiquitously, and the neuron-specific Ank2. While Ank1 is expressed in all cells, it is present at elevated levels in the AIS of MB neurons (Trunova, 2011). Second, the Drosophila AIS contains a unique combination of voltage-gated potassium channels. Specifically, Elk, Shaw (Kv3), and Shal (Kv4) channels were enriched in the AIS, while dORK-C2 (Ork1), Shaker (Kv1.3), and EKO (Kv1.3) were selectively excluded. Lastly, there is an altered F-actin cytoskeleton within the AIS. In the somatodendritic and axonal regions, actin is highly expressed and ubiquitous; within the AIS, actin levels are reduced significantly and appear to have a distinctively patterned distribution. An AIS-like domain has also been observed in multipolar dendritic arborization neurons of flies. Jegla (2016) identified a diffusion barrier localized to the proximal axon of ddaE neurons that coincided with the enrichment of Shal and Elk potassium channels. Furthermore, they found that a fragment of Ank2-L was targeted to the proximal axon in the same area. As Ank2-L is one of the two Ank2 isoforms that have giant exons that share structural similarities with AnkG, this suggests that Ank2-L is an analog, and possibly an ortholog, of mammalian AnkG (Spurrier, 2019).

Similar to the mammalian AIS, the AIS in flies is also linked to neurodegenerative stimuli. Deletion of Cdk5α (also called D-p35), encoding the activating subunit for cyclin dependent kinase 5 (Cdk5), has been shown to induce multiple degenerative phenotypes in Drosophila central brain neurons, including impaired autophagy, swelling of proximal axons, histologically-evident tissue loss (i.e., formation of 'vacuoles' in the brain), and age-dependent loss of neurons. Cdk5α-null flies also exhibit an AIS that is severely shortened and indeed nearly absent: though a barrier remains that separates the somatodendritic and axonal compartments, the domain is so short that the characteristic pattern of accumulation of molecular markers cannot be detected. Notably, the position of the missing AIS correlates with the location where axonal swellings form in affected neurons and where histological tissue loss is observed. However, it remains unclear if disruption of the AIS merely correlates with neurodegeneration or plays a causative role (Spurrier, 2019).

This study shows that overexpression of a portion of the carboxyl domain of the Ank2-L isoform results in a severely shortened AIS in Drosophila central brain neurons, reminiscent of that observed in Cdk5a-null. Ank2-L4-mediated modulation of the AIS, however, occurs through a pathway that is genetically parallel to the previously identified Cdk5α-dependent mechanism. Strikingly, dysregulation of either pathway results in morphological degeneration of axons, and is associated with neuron loss. Therefore, an orthogonal molecular manipulation that shortens the AIS, acting by a genetically independent pathway, also causes axonal degeneration, just as was observed with altered Cdk5α. This provides evidence supporting the hypothesis that disruption of the AIS is apt to be causal for degeneration in fly MB neurons, and may contribute to neurodegeneration in mammalian disease (Spurrier, 2019).

Previous experiments have shown that defects in the axon initial segment are sometimes observed in neurons that have been subjected to neurodegenerative insults, but whether the AIS defects are themselves causal for degeneration has remained unknown. This study has identified a novel reagent to manipulate the AIS in Drosophila central brain neurons. Overexpression of a C-terminal portion of the Ank2-L isoform drastically shortens the AIS in MB neurons, as measured with multiple markers, and that this modulation of the AIS occurs through a genetic pathway that is parallel to Cdk5α-mediated regulation of the AIS. Further, this study demonstrated that dysregulation of either mechanism triggers axonal degeneration, and in some cases, age-dependent loss of MB neurons (Spurrier, 2019).

Based on structural similarities and the presence of a giant exon, Drosophila Ank2-L has been proposed to be the ortholog of the mammalian AnkG and serves as a master organizer of the AIS. The current results confirm an important role of Ank2-L for AIS formation and maintenance but suggest that other components can compensate for its function in regulating the size of the AIS, at least in part. In mammals, deletion of AnkG results in the complete loss of the AIS. Consistent with this, a partial shortening of the AIS was observed when Ank2-L levels were reduced, either genetically, with a null mutant, or with RNAi. However, complete absence of the AIS from reduced Ank2-L levels was not seen, even when those levels are decreased enough to cause lethality. This discrepancy could stem from the differing sets of ankyrin genes between flies and mammals. Drosophila only has two identified ankyrins while mammals carry three ankyrin genes. It may be that as mammals have an expanded ankyrin repertoire with more specialized functions for each gene, the loss of one completely ablates the associated function. In Drosophila, the neuron-specific Ank2 is accompanied by Ank1, which is also enriched at the AIS. It is possible that deletion of Ank2 disrupts the AIS to a certain degree, but there is compensation by Ank1, or by another Ank2 isoform, such as Ank2-XL, preventing the complete dissolution of the AIS. Interplay between multiple ankyrins at the AIS has been shown in mammals, as overexpression of AnkB in cultured hippocampal neurons resulted in a more restricted distribution of AnkG at the AIS. Another possibility is that Ank1 plays a significant role in the organization of the Drosophila AIS. However, Ank1 lacks the giant exons common to mammalian AnkG, so if it were to be capable of acting as the sole organizer of the AIS, it would likely do so through somewhat different mechanisms. Experiments reducing Ank1 levels selectively in the MB will be informative to reveal the specific contributions of Ank1 in establishment and maintenance of the AIS. Furthermore, it will be interesting to see if Ank1 is altered in MB neurons when Ank2-L levels are perturbed or Ank2-L4 is overexpressed (Spurrier, 2019).

It was shown recently that Ank2-L localizes to the AIS in multipolar ddaE neurons, and Ank2-null mutants exhibit loss of a diffusion barrier and loss of potassium channel localization in these neurons (Jegla, 2016). One potential explanation for the apparent difference in AIS phenotype in MB vs ddaE neurons upon Ank2-deletion could be cell type differences. At the neuromuscular junction (NMJ) of Drosophila motoneurons, Ank2-L was localized to the axon and pre-synaptic terminal. In ddaE neurons, in contrast, Ank2-L4 accumulated in the proximal axon and regulated the diffusion barrier, while Ank2-S appeared ubiquitously throughout the entire neuron. In the current study, neither Ank2-S, Ank2-L4, nor Ank2-L8 showed any specific localization within MB gamma neurons, as accumulation of all three was observed in the cell bodies, dendrites, and axonal lobes. Thus, it seems that different neuronal subtypes may use different targeting paradigms. A second possible explanation for the differences between these results could be the sensitivity of the assays used to measure AIS defects. Measuring changes in the boundary of antibody staining is largely qualitative, whereas intermediate effects might be easier to recognize when assaying a quantitative measure, such as diffusion rates. Regardless, both the current dzta and the study by Jegla (2016). support the conclusion that Ank2 contributes to the proper maintenance of the axon initial segment (Spurrier, 2019).

Overexpression of the C-terminal portion of Ank2-L drastically shortened the AIS, as did genetic reduction or removal of Ank2-L. This was initially unexpected, as overexpression of AnkG in cultured hippocampal neurons nearly tripled the length of the AIS, and suggests that the isolated C-terminal domain of Ank2-L acts as a dominant negative and not as a gain of function. Indeed, expression of the Ank2-L C-terminus had a significantly stronger effect on the AIS than even the Ank2-L null mutant, a property known genetically as an 'antimorph'. It may be, for example, that Ank2-L4 acts by mislocalizing the normal binding partners of Ank2-L, either directly or by changing the localization of the endogenous Ank2-L, or that it mislocalizes or inactivates components that would normally be able to compensate for the absence of Ank2. Formally, it cannot be rule out that Ank2-L4 acts as a neomorph with regard to AIS regulation, as the total absence of Ank2-L in homozygous mutants failed to cause the severely shortened phenotype seen with Ank2-L4 overexpression. It has been demonstrated previously that the complete C-terminal domain of Ank2 exhibits neomorphic activity during neuromuscular junction development, as expression of Ank2-L8 in presynaptic neurons at the NMJ results in the formation of small, highly ramified satellite boutons at the synapse. It is also striking that the antimorphic effect of Ank2-L is assay dependent; the AIS phenotype is stronger than that of the null mutant, but unlike the genetic mutant, the Ank2-L C-terminus does not induce lethality even when expressed pan-neuronally at high level. Such assay-specific differences in phenotypic strength are not uncommon, and may arise, for example, from differences in the spectrum of proteins that bind a particular domain in different tissues (Spurrier, 2019).

The cell loss observed from expression of Ank2-L was far less extensive than the axonal morphological phenotypes. This finding raises questions about the relationship of axonal degeneration to loss of the cell soma. Deletion of Cdk5α results in a shortened AIS, as well as causing degenerative phenotypes and neuronal loss that stem, in part, from an acceleration of physiological aging and hyperactivation of the immune system. However, the level of neuron loss in Cdk5α-null flies was greater than what was observed in this study. This suggests that some aging-associated degenerative pathways are more strongly productive of soma loss, while axonal degeneration, which one would expect to be equally effective at disrupting neuronal function, may not always lead to death of the soma, at least in Drosophila (Spurrier, 2019).

Disruption of the AIS has been observed in association with multiple neurodegenerative stimuli, including altered expression of Cdk5α in Drosophila and tau mutation or acetylation in mic]. The data in this study show that a manipulation that directly targets a structural component of the AIS leads to axonal degeneration, as Ank2-L4 overexpression resulted in a significant increase in the prevalence of axonal swelling within the proximal axon, and blebbing of the axon in aged flies, in concert with shortening the AIS. Additionally, Ank2-L4 overexpression resulted in a significant increase in cell loss relative to control. The observed degenerative phenotypes and cell loss do not appear to be due to non-specific detrimental effects on neuronal morphology or physiology during development. Expression of neither Ank2-L4 nor Ank2-L8, which had a more severe effect on the AIS, significantly altered the gross morphological structure of the MB neurons of 3rd instar larva, indicating that any phenotypes are largely restricted to subcellular organization. Moreover, it is worth noting that even though the AIS was shortened to the point that it could not be detected with molecular markers when Ank2-L4 was overexpressed, Futsch remained restricted to the somatodendritic region suggesting maintenance of a diffusion barrier, just as was observed in Cdk5α-null mutant flies. Thus, it is unlikely that the observed neurodegeneration stems from gross disruption of neuronal polarity or morphology (Spurrier, 2019).

The data reported in this study show that the structure of the AIS is regulated by at least two parallel pathways, one defined by Cdk5α and one by Ank2-L, and that perturbation of either pathway leads to axonal degeneration and neuron loss. As such, the most parsimonious interpretation is that a shared feature, such as the altered AIS, is responsible for the observed degenerative phenotypes. However, it cannot be excluded that overexpression of the dominant-negative Ank2-L4 disrupts some other aspect(s) of cell structure or physiology that it also shares with Cdk5α. Modest structural plasticity of the AIS does not always lead to degeneration; indeed, modulation of AIS length is a vital part of fine-tuning neuronal excitability. Thus, shortening of the AIS by overexpression of Ank2-L4 may be only one aspect of disturbed AIS function and neuronal physiology from this manipulation. For example, the data also demonstrate alteration of actin organization in the AIS, and the localized swelling of the axon is likely associated with defects in axonal transport. Moreover, the drastic reduction observed in the extent of the AIS is expected to have severe consequences for neuronal excitability. Finally, Ank2-L4 expression may cause unrecognized changes in other parts of the cell. The key point, however, is that the data seems to exclude the hypothesis that AIS loss is simply a secondary, late-stage marker of neurodegeneration, but rather suggests that it is associated with the earliest stages of the process, as targeting either of two independent regulators of the AIS, one a core structural component, is sufficient to induce degeneration (Spurrier, 2019).

Using a novel reagent, this study has unlocked a new method for modulating the AIS independent of Cdk5/Cdk5α, the only known AIS regulator in Drosophila MB neurons. The regulation of the AIS presented in this study is likely to be direct as Ank2-L4 acts as an antimorph of the essential ankyrin isoform that helps construct the AIS. Remarkably, this study showa that a genetic treatment that disrupts the AIS by manipulation of one of its core components is also sufficient to cause axon degeneration and even neuron loss in flies, supporting the hypothesis that the AIS disruption observed in association with several neurodegenerative mechanisms contributes causally to the process of neurodegeneration (Spurrier, 2019).

The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. This study addresses the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. A P[acman] based rescue approach was used to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. Specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, these data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance (Weber, 2019).

Giant ankyrins serve as central organizing adaptor molecules in both invertebrate and vertebrate neurons. This study provides first insights into the control of synaptic localization and function of giant Ankyrins by the N-terminal ankyrin repeat domain in vivo. By selectively deleting subdomains of the ARD of ank2 this study has unravel critical requirements of specific regions of the ARD for the synaptic localization of the neuronal specific giant isoforms Ank2-L and Ank2-XL. The data demonstrate that the N-terminal domain controls not only synaptic targeting of individual isoforms but also contributes to synaptic localization of the alternative isoform. The functional requirements of Ank2-L and Ank2-XL for synaptic stability and microtubule organization clearly correlate with ARD-dependent regulation of protein abundance at the presynaptic terminal, with individual subdomains providing unique functional features (Weber, 2019).

The N-terminal ARD mediates membrane-association of Ankyrins and is essential for the subcellular localization and organization of transmembrane binding partners. Prior work largely focused on the organizational roles of giant Ankyrins at the AIS and nodes of Ranvier in vertebrate neurons. The current in vivo analysis of ARD deletions now revealed a critical importance of this domain for the localization of Ank2-L at the presynaptic terminal of motoneurons. The synaptic localization of Ank2-L depends on AnkD2-4 (repeats 7-24) while the first domain (repeats 1-6) only had a minor impact on protein abundance. Interestingly, axonal targeting of Ank2-L was only partially affected by these deletions. These results indicate that separate and distinctive mechanisms exist in vivo that enable selective localization of giant Ankyrins in axons and within the presynaptic motoneuron terminal. As similar localization defects are observed already at earlier stages of development argues that AnkD2-4 are essential for initial synaptic localization. It was previously demonstrated that a fusion protein comprising the specific C-terminal tail domain of Ank2-L efficiently localizes to both axonal and synaptic areas (Stephan, 2015). The demonstration that synaptic localization of full length Ank2-L requires an intact ARD indicates that subcellular distribution is precisely regulated and not achieved by passive distribution within the neuron. Of particular interest is the observation that the first six ankyrin repeats are dispensable for synaptic localization of Ank2-L in the ank2ΔL mutant background. The recent characterization of the structure of the human ARD demonstrated distinct and independent binding sites for voltage-gated sodium channels (Nav1.2) and for L1-family CAMs (Neurofascin) in vitro and in cultured neurons. Interestingly, in cultured hippocampal neurons the L1-family CAM binding sites within AnkD2 (ankyrin repeats 8-9 and 11-13) are essential for clustering of the 270 kDa AnkG isoform at the AIS. In contrast, AnkG lacking the Nav1.2 binding site within AnkD1 (ankyrin repeats 2-6) efficiently localized to the AIS but failed to cluster Nav channels at the AIS. Thus, similar to the situation at the vertebrate AIS, the synaptic localization of Drosophila Ank2-L largely depends on interactions with L1-family CAMs or alternative transmembrane proteins that occupy binding sites within the central and C-terminal part of the ARD. This localized L1-CAM-Ankyrin complex can then serve as an assembly platform for additional molecules like voltage-gated sodium channels that acquired Ankyrin-binding capacities during chordate evolution to determine the physiological properties of specific subcellular neuronal compartments. While the AnkD1 of Drosophila ank2 had the least impact on synaptic stability phenotypes it will be interesting to identify putative binding proteins that may provide specific functional properties as absence of this domain resulted in decreased survival and locomotion of adult flies (Weber, 2019).

In contrast to Ank2-L, the deletion of individual parts of the ARD had smaller effects on synaptic targeting of Ank2-XL in the ank2ΔXL mutant background. Here, only the deletion of AnkD3 (repeats 13-18) resulted in a significant reduction of axonal and synaptic localization. However, this analysis revealed a striking dependence of Ank2-XL on the presence of the intact ARD of Ank2-L. Impairments of Ank2-L localization, including the minor effects caused by deletion of AnkD1, resulted in a severe reduction of synaptic Ank2-XL levels. This shows that Ank2-XL localizes via Ank2-L to the presynaptic terminal consistent with prior observations (Stephan, 2015). Interestingly, deletions of parts of the ARD of Ank2-XL also significantly reduced synaptic Ank2-L levels indicating a critical co-dependence of Ank2-L and Ank2-XL at the NMJ. This interaction may depend on direct interactions between the two Ankyrin isoforms, potentially mediated within the ARD. However, it is equally possible that incorporation of mutated Ankyrins with altered binding properties resulted in a disruption of the larger Ankyrin-Spectrin scaffold that in turn leads to reduced targeting of the other isoform. Indeed, in vertebrate axons it has been demonstrated that different affinities of specific giant Ankyrins control the isoform-specific incorporation into selective axonal compartments like the nodes of Ranvier (Weber, 2019).

At a functional level a strong correlation was observed between synaptic Ank2-L level and the control of synaptic stability. At all muscle groups analyzed deletion of AnkD1 domain had the mildest impact on synaptic stability consistent with the least impairments of synaptic Ank2-L localization. Importantly, the analysis of synaptic stability at muscle 4 also revealed that AnkD3 is most critical for synaptic maintenance. Despite an almost identical reduction in Ank2-L level compared to AnkD2 and 4 the rescue construct lacking the third domain was unable to restore any synaptic stability of ank2ΔL mutant animals. Interestingly, this assay also highlighted the specificity of the individual ankyrin repeats within the ARD. The exchange of second domain sequences with analogous sequences of the first domain failed to restore both Ank2-L localization and the synaptic stability phenotype of ank2ΔL mutants. The failure to support synaptic stability is at least in part due to a failure to organize and stabilize synaptic cell adhesion molecules. This analysis demonstrated that AnkD2 and 3 are critical for normal clustering of the NCAM homolog Fas II and of the L1 CAM homolog Nrg. This is consistent with a prior observations that Ank2-L supports synaptic organization of both Fas II and Nrg and the co-dependence of Ank2-L and these CAMs at the synapse (Pielage, 2008; Enneking, 2013). This function is evolutionary conserved as mutations in the second and third binding site of vertebrate AnkG failed to restore clustering of the L1 CAM family members Neurofascin or Nr-CAM at the AIS of hippocampal neurons (Weber, 2019).

In contrast to the clear requirements of the ARD for presynaptic targeting of Ank2-L the same mutations only partially affected localization of Ank2-XL. Consistently, only mild disruptions of the presynaptic microtubule cytoskeleton and of the synaptic bouton organization compared to the ank2ΔXL mutation were observed. The effects were most severe when deleting AnkD2 despite the fact that Ank2-XL protein levels were more compromised in AnkD3 mutants. As the large C-terminal repeat region of Ank2-XL is essential for the interaction with microtubules these results indicate that inappropriate ARD-dependent complex assembly may influence the functional properties of Ank2-XL at the NMJ (Weber, 2019).

Together, this in vivo analysis of the ARD of Drosophila giant Ankyrins uncovered a structural basis for presynaptic localization and provided a genetic basis for the identification of regulatory mechanisms controlling structural synaptic plasticity via the selective Ankyrin complex assembly (Weber, 2019).

A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, this study has identified a novel long isoform of Drosophila ankyrin2 (ank2-L). Loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. Genetic evidence is provided that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that can directly bind and organize synaptic microtubules. It is proposed that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal (Pielage, 2008).

The pathology of many neurodegenerative diseases involves the dying back of neuronal processes and the eventual elimination of synaptic connections. It has become apparent that synapse loss and axon retraction often precede the onset of neurodegenerative disease symptoms and generally precede neuronal death. These observations implicate synapse disassembly and axon retraction as early events in neurodegenerative disease. However, the molecular mechanisms underlying the inappropriate disassembly and retraction of synaptic connections remain poorly understood. A common observation is that synapse destabilization and axon retraction are correlated with a disruption of the underlying microtubule cytoskeleton. Recently it has been proposed that disruption of the neuronal microtubule cytoskeleton could even be a causal or an inductive event in disease. However, it remains unclear how disruption of the synaptic microtubule cytoskeleton could lead to synapse disassembly, a process that would seem to require the disruption of strong trans-synaptic protein interactions that normally stabilize synaptic connections (Pielage, 2008).

Using a forward genetic approach in Drosophila this study has identified mutations in a novel giant isoform of Ankyrin2 (Ank2-L) that forms a sub-membranous lattice within the presynaptic nerve terminal at the neuromuscular junction (NMJ). Loss of presynaptic Ank2-L results in the inappropriate destabilization and retraction of the presynaptic nerve terminal at the NMJ. Retraction of the NMJ is correlated with the disorganization of synaptic cell adhesion molecules and the disruption of the underlying microtubule cytoskeleton. The C-terminal tail of Ank2-L can directly bind and organize microtubules and is necessary for synapse stability. Based upon these and additional data, a model is presented for synapse stability in which a presynaptic giant Ankryin provides a molecular link between intercellular adhesion and cytoskeletal stabilization that is necessary to maintain the integrity of the NMJ (Pielage, 2008).

Ankyrins are a family of adaptor proteins that have the potential to recruit the Spectrin-based membrane skeleton to specific transmembrane proteins. Ankyrins are organized into three conserved domains: an amino terminal membrane association domain that contains 24 Ankyrin repeats organized into 4 subdomains, a Spectrin-binding domain and a C-terminal tail region that varies significantly between Ankyrins (Bennett, 2001). While canonical Ankyrins do not exceed 220 kDa, both vertebrate AnkyrinB (AnkB) and AnkyrinG (AnkG) encode giant isoforms of 440 kDa and 270/480 kDa, respectively. Interestingly, these giant isoforms are specifically targeted to the axon and their C-terminal tail is predicted to be up to 220 nm long and to extend deep into the axoplasm. In contrast, AnkB isoforms that lack this C-terminal domain are restricted to the neuronal cell body. It is hypothesized that this long C-terminal extension might be required for axonal targeting and could mediate interactions with the underlying cytoskeleton and thereby co-ordinate Ankyrin-dependent protein interactions at the cell membrane with the cell interior (Bennett, 2001). However, there is no direct evidence demonstrating that Ankyrins control the organization of the underlying cytoskeleton (Pielage, 2008).

Within the vertebrate nervous system, giant Ankyrins are required for the organization and maintenance of specific membrane domains including the axon initial segment and nodes of Ranvier. AnkyrinG has been shown to bind cell adhesions molecules (e.g. L1 CAM, Nr CAM and Neurofascin), sodium channels (NaV1.6), potassium channels (KCNQ) and βIV-Spectrin and is required to organize and stabilize these proteins at the axon initial segment. A similar mechanism of cell adhesion molecule and ion channel localization has been observed at the nodes of Ranvier (Pielage, 2008).

The Drosophila genome encodes two ankyrin genes, ankyrin and ankyrin2. The ankyrin gene is ubiquitously expressed and is enriched within the postsynaptic muscle membranes of the Drosophila NMJ (Dubreuil, 1994; Pielage, 2006). RNAi-mediated depletion of Ankyrin from the muscle does not alter the stability or function of the NMJ (Pielage, 2006). In contrast, expression of ankyrin2 is restricted to the nervous system (Bouley, 2000; Hortsch, 2002). Ankyrin2 encodes two previously annotated isoform types, a short isoform (Ank2a referred to as Ank2-S; 1159 amino acids) and long isoforms (Ank2b-e; between 2386 and 2465 amino acids). Both previously annotated isoform types share the first 1126 amino acids (aa) that contain the characteristic Ankyrin repeat domains as well as the Spectrin binding domain. These proteins differ in their unique C-terminal tail regions (Hortsch, 2002). Using antibodies targeted against the unique C-terminal tails it has been demonstrated that the short isoform is restricted to neuronal cell bodies while the long isoforms (Ank2b-e) are present in axons (Hortsch, 2002). Initial studies provided evidence that the long isoforms (Ank2b-e) of Ank2 are required for animal viability (Hortsch, 2002) and that Ank2, together with the cell adhesion molecule Neuroglian, is required to suppress axonal sprouting during dendrite development (Yamamoto, 2006). This study presents the first formal genetic analysis of ank2 in Drosophila and, in so doing, identify a novel giant isoform of Ank2 (Ank2-L; 4083 aa) that is present within the presynaptic nerve terminal. Based upon these data a model is presented in which Ank2-L represents an important link between the membrane, the Spectrin skeleton and the underlying microtubules that is necessary for the long-term stabilization of the NMJ. It is hypothesized that this function of Ank2-L could be relevant for synapse stability in vertebrate systems where Ankyrins have already been implicated in the cause of neurodegeneration (Pielage, 2008).

Although the ank2 gene encodes both short and long isoforms, several lines of evidence indicate that the stabilizing function of Ank2 is contributed primarily by the giant, presynaptic ank2-L isoform. First, the ank2²⁰⁰¹ transposon insertion specifically disrupts the ank2-L open reading frame and causes synapse retraction. Second, RNAi-mediated knock down of the ank2-L isoform causes synapse retraction. Third, the short isoform of ank2 is not sufficient to rescue synapse stability in the ank2 mutant background. Thus, while participation of the Ank2 short isoform at some level cannot be ruled out, the data support the conclusion that loss of Ank2-L is the primary cause of presynaptic retraction and degeneration (Pielage, 2008).

In the vertebrate nervous system, mutations in giant Ankyrins have been associated with neurodegeneration. For example, the cerebellar specific knockout of ankG causes progressive ataxia and loss of Purkinje cells. In ankB knockout mice, hypoplasia of corpus collosum and pyramidal tracts and a degeneration of the optic nerve have been observed (Scotland, 1998). While AnkB is not required for the formation of optic nerve connections, significant neurodegeneration is observed by postnatal day 9 in ankB mutant animals and by postnatal day 20 nearly complete degeneration of the optic nerve can be observed. AnkB is required for the maintenance of the axonal localization of the cell adhesion molecule L1-CAM, and the loss of L1-CAM precedes axon degeneration in the knockout mice. Although these important studies establish a causal link between giant Ankyrins and neurodegeneration, it is unknown whether synapse loss precedes neurodegeneration in ankB or ankG knockout mice. Mechanistically, it also remains unknown which essential cellular functions of ankB or ankG are particularly relevant to the initiation or progression of neurodegeneration (Pielage, 2008).

It is well established that the disruption of the axonal or synaptic microtubule cytoskeleton is an early event that is correlated with the induction and initial phases of neurodegeneration. This study has demonstrate that synapse retraction following loss of Ank2-L is correlated with a severe disruption and eventual elimination of the synaptic microtubule cytoskeleton, documented at both the light and EM levels. Several experiments suggest that Ank2-L directly controls synaptic microtubule organization and stability. First, presynaptic Ank2-L resides in close proximity to synaptic microtubules, as visualized by extended high-resolution structured illumination microscopy. Second, a region within the C-terminal tail of Ank2-L was found to bind microtubules in vitro. Third, the C-terminal microtubule-binding region of Ank2-L is sufficient to re-organize the microtubule cytoskeleton in S2 cells, resulting in aberrant microtubule bundling and increased Tubulin levels. However, expression of only the C-terminus of Ank2-L is not sufficient to rescue microtubule organization or synapse stability in ank2 mutant animals. Therefore, although Ank2-L is able to bind microtubules, it is hypothesized that the organization and stabilization of synaptic microtubules depends equally upon the association of Ank2-L with either the sub-membranous Spectrin-actin skeleton and/or with synaptic cell adhesion molecules. Evidence in favor of the involvement of synaptic cell adhesion molecules is the observation that Fas II (and Nrg) staining becomes severely disorganized and unstable in ank2-L mutant NMJs. In addition, it was demonstrate that Ank2-L resides in close proximity to Fas II within synaptic boutons. Importantly, it has been previously demonstrated that, in fasII null mutant animals, the NMJ can form normally but then retracts over time. Interestingly, the phenotype of complete NMJ elimination observed in these prior studies is quantitatively similar to what is observed in this study (e.g. complete eliminations at 5% of the NMJ on muscle 3 in fasII null mutant mosaic animals. Since Fas II is necessary for synapse stability, it seems reasonable to propose that the loss of Fas II protein in the ank2 mutants contributes to the loss of NMJ stability and subsequent NMJ retraction. It has previously been speculated that the large intracellular tail of giant Ankyrins could extend significant distances (220 nm) within the cell cytoplasm (Bennett, 2001). Taking this into account, Ank2-L is ideally suited to function as a stabilizing bridge between cell adhesion molecules at the synaptic plasma membrane and the underlying synaptic microtubule cytoskeleton. It is hypothesized, therefore, that the loss of this molecular link between microtubules and cell adhesion is the primary cause of synapse destabilization in Drosophila ank2 mutations (Pielage, 2008).

This study has visualized the organization of a giant Ankyrin within the axon and presynaptic nerve terminal by taking advantage of the increased resolution of structured illumination microscopy. The data are consistent with the presence of a highly organized Ank2-L lattice throughout the axon. Remarkably, the lattice-like organization of Ank2-L is modified within specific subdomains of the presynaptic nerve terminal. Ank2-L has a tight, lattice-like organization between synaptic boutons, but a less regular, sparsely connected organization within synaptic boutons. The modulation of Ank2-L organization may have functional significance for NMJ development and stabilization. Synaptic bouton organization is severely perturbed in ank2-L mutant animals including the loss of narrow-diameter, inter-bouton regions. The majority of the NMJ appears as a presynaptic ribbon of uniform width rather than the characteristic 'beads on a string' type organization. This phenotype suggest that the Ank2-L lattice may be involved in either the developmental partitioning the presynaptic nerve terminal into distinct synaptic bouton and inter-bouton regions, or the maintenance of these discrete domains over time. This phenotype is remarkable when one considers that the majority of identified Drosophila mutations affect either the number and size of synaptic boutons, or the branching pattern of boutons on the muscle surface, but do not prevent the neuron from generating the membrane expansions that will eventually become synaptic boutons (Pielage, 2008).

The mechanisms that differentiate presynaptic boutons from axons, whether one considers en passent boutons or boutons that form at the end of an axon, are fundamental to the organization and function of the nervous system. One model, based upon the current data, is that the highly organized Ank2-L lattice provides the structural integrity necessary to maintain a tight tubular shape for the axon and inter-bouton regions. Bouton formation might then be associated with the regulated relaxation of the Ank2-L lattice, which could allow for the expansion of the presynaptic membrane into a synaptic bouton. It is likely that the Ank2-L lattice is coincident with a similar organization of the presynaptic Spectrin skeleton. Indeed β-Spectrin staining shows a similar organization in the axon like Ank2-L with an approximate ~200 nm repeat structure and evidence of pentameric and hexameric structures. Thus, the model remains consistent with the well-established function of the Spectrin skeleton in maintaining cell shape (Pielage, 2008).

In vertebrates, expression of giant Ankyrins is restricted to the nervous system and these proteins are targeted to the axon. Interestingly, short Ankyrin isoforms, encoded by the same genes, are restricted to the cell body suggesting that the C-terminal domain is required for axonal targeting. The mechanisms that control the trafficking and localization of giant Ankyrins to discrete locations within a neuron are fundamentally important for ion channel and cell adhesion molecule organization at the nodes of Ranvier and the axon initial segment and, as shown in this study, both synapse morphology and stabilization. However, the detailed mechanisms that regulate the trafficking and localization of giant Ankyrins within a neuron are generally unknown. A structure-function analysis of the 270 kDa giant AnkG demonstrated that multiple domains, including the membrane-binding, Spectrin-binding and a serine-rich region within the C-terminal tail, cooperate in targeting and restriction of AnkG to specific axonal domains within dorsal root ganglion neurons (Zhang, 1998). In Drosophila, the short and long isoforms of Ank2 localize to different neuronal compartments. Similar to vertebrate AnkB, the short isoform is restricted to the cell body while the long isoform is found in axons and the presynaptic nerve terminal. This study has identified a domain within the novel C-terminal extension of Ank2-L that is necessary and sufficient for trafficking Ank2-L to the axon and presynaptic nerve terminal. Presumably, the mechanisms of microtubule binding and axonal trafficking will be separable functions that map to discrete sequences within the C-terminus of Ank2-L (Pielage, 2008).

Drosophila ankyrin 2 is required for synaptic stability

Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, mutations were uncovered in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability (Koch, 2008).

Mutations in ankyrin 2 affect the number, size, and spacing of synaptic boutons. Using immunohistochemical methods, transmission electron microscopy, and in vivo imaging, this study shows that these phenotypes arise through insufficient support of the synaptic cytoskeleton. Reduced stability of cytoskeletal elements leads to disassembly of transmitter-releasing active zones, withdrawal of microtubules, and retraction of entire boutons. Ank2 functions upstream of the microtubule-based cytoskeleton, suggesting that the synaptic core cytoskeleton is structurally supported by the membrane cytoskeleton. Because ankyrins have been shown to be important determinants of the subcellular organization of central synapses in vertebrates (Ango, 2004), they might play a general role in the stabilization of synapses (Koch, 2008).

Based on this molecular analysis of the ank2 locus, ank2 transcripts are alternatively spliced to nearby sequences, originally annotated as separate genes, SP2523 and CG32377. These new isoforms, Ank2-L and Ank2-XL, respectively, encode extremely large proteins. Ank2-L is highly expressed in axons and to a lesser extent in NMJs but was not found in the cell soma. Ank2-XL, in contrast, was consistently detected in neuronal cell bodies. In synaptic branches of NMJs, Ank2-L is expressed in the plasma membrane compartment, whereas Ank2-XL is tightly associated with the microtubule-based cytoskeleton (Koch, 2008).

However, the expression of Ank2-XL in terminal-most boutons lacking microtubules indicates that it also associates with membranes, at least partially. Smaller splice isoforms of Ank2 have been shown to localize to different neuronal compartments (Hortsch, 2002). While a short isoform (Ank2-S) was expressed in the cell soma, medium-sized isoforms (Ank2-M) were concentrated in axons. In various organisms, alternative splice isoforms of ankyrin genes are expressed in different tissues or subcellular regions. However, from worms to mice, the largest isoforms seem to be expressed always in the nervous system, indicating a functional requirement in neurons (Koch, 2008).

The identified point mutations in ank2 cause C-terminal truncation of both Ank2-L and Ank2-XL. However, the loss of a single giant ankyrin isoform already results in a synaptic phenotype. In the absence of only Ank2-L, Ank2-XL fails to extend into distal boutons, suggesting that Ank2-L is necessary for the synaptic localization of Ank2-XL (Koch, 2008).

The synaptic cytoskeleton is a major mediator of synaptic remodeling and stability in various organisms. During the period of synaptic growth in Drosophila larvae, new boutons are added to existing branches either by intercalation or by end addition. While these basic growth processes occur in ank2 mutants, live imaging of mutant NMJs revealed that a subset of synaptic branches is unable to expand or does retract. ank2 mutant NMJs have therefore overt defects in synaptic stabilization. This study found that this is most likely due to an unstable cytoskeleton. The destabilized cytoskeletal elements visibly resemble retraction bulbs, cone-shaped nerve endings undergoing synaptic elimination at vertebrate NMJs. Tubulin and the microtubule- associated protein Futsch are retracted from terminal boutons (Koch, 2008).

In this respect, it is interesting to note that Futsch is tightly associated with Ank2. Although coaligned in axons and interbouton regions, Ank2 and futsch do not completely overlap, and Ank2 consistently has a more peripheral distribution. The disparate location is most obvious in terminal boutons. While Futsch is detected only in a subset of terminal boutons, Ank2 is found in every terminal bouton, including very small (and probably very young) boutons at the tips of growing branches. These observations imply that Futsch is integrated only into relatively old and stable boutons, whereas Ank2 is immediately incorporated into nascent boutons. Although this scenario is only one possibility, it reveals a spatiotemporal succession of cytoskeletal components during synaptic expansion. Ank2 might therefore organize a synaptic scaffold for the stabilization of microtubules. This idea is supported by a recent study that implicated the ankyrin-binding protein neuroglian in the organization of synaptic microtubules during the formation of central synapses in Drosophila (Godenschwege, 2006). In addition, biochemical experiments showed that vertebrate ankyrins interact with microtubules assembled from purified tubulin. However, further experiments will be necessary to better characterize the linkage between synaptic microtubules and the membrane cytoskeleton (Koch, 2008).

Individual boutons of a synaptic branch are normally connected by narrow cytoplasmic bridges termed 'bottlenecks'. Ank2 as well as Futsch showed a continuous distribution in interbouton regions but a rather reticular pattern within synaptic boutons. Ank2 could be important for the stabilization of bottleneck regions. These membrane constrictions dissolved into bouton-like structures in the absence of Ank2, fusing neighboring boutons to larger units. Ultrastructural analysis confirmed the existence of exceptionally large presynaptic cavities lacking bottlenecks in ank2 mutants. Similar vesicle-filled enlargements are not found in wild-type NMJs. Ank2 might therefore regulate the spatial separation of boutons by organizing compartmentalized membrane domains between individual boutons, potentially similar to the paranodal cytoskeleton established at the nodes of Ranvier by Ankyrin G in vertebrates (Koch, 2008).

The synaptic stability defects in ank2 mutants are most obvious in NMJs located on posterior muscles. NMJs are subjected to an anterior-posterior gradient of synaptic size and structure. A gradual decay of NMJs along the anterior-posterior axis is indicative of axonal transport problems, as transport distances increase toward NMJs on posterior muscles. The further away a synaptic terminal is from its supplying motor neuron the more transport defects would have an impact on its size and structure. This is exemplified by mutations in the kinesin heavy chain (Khc), an anterograde motor protein. khc mutants have axonal organelle jams and clearly less synaptic boutons in NMJs of anterior segments, but the reduction in bouton numbers is even more severe in posterior segments. A constant supply of adhesion proteins and other synaptic molecules appears therefore to be required for the maintenance of NMJs. However, the organelle jams in ank2 mutant axons could also arise due to an increased rate of membrane turnover during ongoing retraction processes, or even due to an excess supply of cargo proteins that cannot be incorporated into synaptic terminals. The vesicle accumulations, however, do suggest that loss of Ank2 not only affects the integrity of the neuronal cytoskeleton but also the dynamics of synaptic vesicles (Koch, 2008).

Because ankyrins interact with spectrins (Bennett, 2001), loss of spectrins is expected to cause similar phenotypes. α- and β-spectrins have been shown to play an important role in the stabilization of NMJs in Drosophila). Downregulation of spectrins by transgenic expression of interfering RNAs leads to branch-selective dismantling of NMJs and axonal transport defects. Many of the spectrin mutant phenotypes resemble the phenotypes in ank2 mutants, suggesting that spectrin and Ank2 function together to control the stability of synapses. This study found that β-spectrin is required to maintain the synaptic localization of Ank2, as loss of β-spectrin leads to retraction of Ank2 from synaptic branches. In contrast, lack of Ank2 had no effect on the localization of β-spectrin, suggesting that β-spectrin functions upstream of Ank2 in the assembly pathway (see also Das, 2006). The targeting of spectrin to the plasmamembrane seems therefore to be independent of ankyrins in Drosophila. Because mutations in both ankyrin and spectrin destabilize the microtubule cytoskeleton, this implies that ankyrin functions downstream of spectrin in the synaptic stabilization of microtubules (Koch, 2008).

Spectrin functions upstream of ankyrin in a spectrin cytoskeleton assembly pathway

Prevailing models place spectrin downstream of ankyrin in a pathway of assembly and function in polarized cells. A transgene rescue strategy was used in Drosophila to test contributions of four specific functional sites in beta spectrin to its assembly and function. (1) Removal of the pleckstrin homology domain blocked polarized spectrin assembly in midgut epithelial cells and was usually lethal. (2) A point mutation in the tetramer formation site, modeled after a hereditary elliptocytosis mutation in human erythrocyte spectrin, had no detectable effect on function. (3) Replacement of repetitive segments 4-11 of beta spectrin with repeats 2-9 of alpha spectrin abolished function but did not prevent polarized assembly. (4) Removal of the putative ankyrin-binding site had an unexpectedly mild phenotype with no detectable effect on spectrin targeting to the plasma membrane. The results suggest an alternate pathway in which spectrin directs ankyrin assembly and in which some important functions of spectrin are independent of ankyrin (Das, 2006; full text of article).

The results presented here provide several novel insights into the assembly and function of spectrin. There are two general models to explain the assembly of the spectrin cytoskeleton in polarized cells. Both models incorporate ankyrin as an adaptor that couples integral membrane proteins to the spectrin cytoskeleton. In the first case, assembly begins with a protein receptor that recruits ankyrin to a specific region of the plasma membrane (Das, 2006).

In this model, ankyrin serves two distinct roles: (1) as an adaptor that couples spectrin to a cue for assembly and (2) as an adaptor that links interacting proteins such as the Na,K ATPase and voltage-dependent sodium channels to the preassembled spectrin cytoskeleton. In the 'spectrin first' model, ankyrin functions as an adaptor that couples interacting membrane proteins to a preassembled spectrin cytoskeleton. In this model, the site of assembly is determined directly by spectrin and the role of ankyrin is to couple the diverse membrane proteins that interact with ankyrin to that site (Das, 2006).

The results of the present study provide the first direct evidence supporting the spectrin-first model. Ankyrin assembly at the basolateral membrane domain of copper cells was dependent on spectrin. Spectrin in turn was dependent on the PH domain of the ß subunit in copper cells and on an as-yet-unidentified signal in salivary gland cells. There are examples of ankyrin-independent assembly of spectrin in other systems: (1) During erythrocyte differentiation, ankyrin assembly occurs after the stable assembly of spectrin. (2) A related observation is that spectrin assembly appears remarkably normal in erythrocytes that lack band 3, the major membrane receptor for ankyrin in the erythrocyte. Thus, even in the best-characterized membrane model, it has been difficult to ascertain the sequence of events that leads to spectrin assembly. (3) Targeting of the alphaß_H isoforms of spectrin is thought to occur by an ankyrin-independent mechanism. These spectrins have unusually large and divergent ß subunits and are targeted to the apical membrane domain of polarized epithelia in D. melanogaster. Together with the current results, it appears that targeting to the plasma membrane is a shared property of spectrins, whether or not they interact with ankyrin (Das, 2006).

PH domains have been detected in hundreds of different proteins and in many cases they have physiological roles in binding to phosphoinositides. Structures have been determined for spectrin PH domains from mammals and Drosophila, and binding to phosphoinositides has been demonstrated. The PH domain of spectrins does not have the expected lipid specificity of a protein that mediates phosphatidylinositol (PtdIns)-3-kinase signaling. Although the structure of the spectrin PH domain appears to be compatible with binding to PtdIns(3,4,5)P_3, a more likely binding partner in vivo is PtdIns(4,5)P₂. The six residues that contact PtdIns(4,5)P₂ are conserved in the Drosophila PH domain and out of 37 amino acid identities among PH domains from mammalian ßI, ßII, ßIII, and ßIV spectrins, 31 are conserved in Drosophila. Overall, there was 44% identity between the fly PH domain and mammalian PH domains. For comparison, there was a mean of 62% identity between the PH domains of the four mammalian ß spectrin isoforms. Interestingly, in comparisons of full-length sequences, there was greater identity between Drosophila ß spectrin and human ßII spectrin (57%) than between human ßII and ßIV (53%) or between ßI and ßIV (49%). Therefore, it appears likely that the functions of ß spectrin, including the lipid-binding function of the PH domain, are conserved between Drosophila and mammals (Das, 2006).

The PH domain of spectrin may also mediate membrane targeting through interactions with protein receptors. For example, the membrane-binding activity originally described for brain ß spectrin was protease sensitive. Interactions between PH domains and protein ligands such as protein kinase C and G protein ßgamma subunits have been reported. However, the interaction between the PH domain of a mammalian ß spectrin and ßgamma subunits was tested and found to be comparatively weak. The reason for differential targeting of the ßspec^DeltaPH product in a mutant versus a wild-type background is not known. Further experiments will be necessary to determine whether mixed tetramers form and whether other sites in the molecule affect their targeting (Das, 2006).

Although the PH domain was required for the targeting of spectrin in copper cells, neither the PH domain nor ankyrin binding were required for targeting in the salivary gland. One obvious candidate to explain the recruitment observed in salivary gland is the ankyrin-independent membrane-binding site identified near the N terminus of mammalian ß spectrin. It is also possible that multiple membrane-binding sites contribute to targeting in some cells, even though the PH domain alone appears to be critical in copper cells. To help resolve these questions, it will be important in future studies to identify sites that are sufficient for membrane targeting in different cell types and to produce mutant transgenes in which multiple candidate targeting activities have been knocked out simultaneously (Das, 2006).

It was expected that loss of ankyrin binding would severely compromise the function of ß spectrin. Although there was a relatively low viability of flies rescued by the ßspec^alpha13 transgene, and they often had wing phenotypes and appeared less healthy than their wild-type siblings, a surprising number of these flies survived as fertile adults. In contrast, the four loss-of-function mutations that have been characterized all exhibited embryonic lethality. The rescue result reinforces the conclusion that spectrin targeting is independent of its interaction with ankyrin and further suggests that some important aspects of spectrin function are independent of its association with ankyrin (Das, 2006).

There are several possible interpretations of ßspec^alpha13 rescue that will require further experiments to resolve. (1) There may be redundant cellular mechanisms that can partially compensate for loss of the adaptor function of ankyrin. (2) The modification of the ankyrin-binding domain of ßspec^alpha13 may have selectively blocked its association with DAnk1 but left Dank2 binding intact. Once appropriate tools become available it will be important to test whether DAnk2 has an essential function and whether that function depends on the ankyrin-binding site defined here. (3) There may be residual ankyrin-binding activity in ßspec^alpha13 that was below the threshold of detection in these experiments. One reason to consider this possibility emerged from sequence comparisons between fly and human ß spectrins. Current structural models indicate that part of the putative ankyrin-binding site may include part of segment 15 (repeat 14), where there is striking sequence conservation. Future experiments will address whether this conservation represents part of the ankyrin-binding site or whether it is an as-yet-unidentified functional site in the ß spectrin molecule. In any case, it's apparent that most of the ankyrin 1-binding activity of spectrin was removed in ßspec^alpha13. (4) Finally, it is also formally possible, although unlikely given the degree of sequence conservation, that spectrins and ankyrins in vertebrates and invertebrates have fundamentally different roles in plasma membrane organization and function (Das, 2006).

Another surprising result in the present study was the finding that the behavior of the Na,K ATPase was more closely linked to the behavior of spectrin than to ankyrin. Based on biochemical evidence showing that purified mammalian ankyrin and Na,K ATPase directly interact with one another in vitro, it has been assumed that any effects of spectrin mutations on the behavior of the Na,K ATPase in vivo were likely to be mediated through ankyrin. Current evidence raises the possibility that the Na,K ATPase may be linked to spectrin either directly or perhaps by some other indirect mechanism. It is possible that vertebrates and invertebrates evolved independent mechanisms to link the Na,K ATPase to the spectrin cytoskeleton. It was recently suggested that mammalian KCNQ potassium channels and voltage-dependent sodium channels acquired their functional interaction with ankyrin through a process of convergent molecular evolution, after the split between vertebrates and invertebrates. The conserved ankyrin-binding sequence found in these mammalian proteins is not present in their Drosophila homologues. That does not appear to be the case with the Na,K ATPase, as the amino acid sequence that mediates interaction with ankyrins in vertebrates is remarkably conserved in the Drosophila Na,K ATPase. Future studies should address the possibility that, even though there is a direct interaction between ankyrin and the Na,K ATPase in vitro, there may be an important functional interaction with mammalian spectrin in vivo that is ankyrin independent (Das, 2006).

The greatest sequence identity between Drosophila and mammalian ß spectrins occurs in the first three segments of the protein, which includes the actin-binding site and tail-end subunit interaction sites in segments 2 and 3. Another site of striking sequence conservation among ß spectrins is the partial repeat 18, where alpha and ß spectrin interact to form tetramers. Spectrin is thought to have evolved from a large single-subunit ancestor by fracturing of the coding sequence at a site within an ancestral structural repeat. A point mutation in the N-terminal partial repeat of alpha spectrin (R22S) produced a temperature-sensitive defect in spectrin tetramer formation. This study tested the effect of a comparable mutation in the ß subunit that was also identified by its role in human anemia. The W2033R mutation corresponds to the W2024 position of human erythroid ß spectrin, and that residue is also conserved in human ßII, ßIII, and ßIV, but not in ßV spectrin. This tryptophan residue was dispensable for ß spectrin function in Drosophila, presumably because it does not affect tetramer formation. Polarized targeting of the transgene product was also unaffected by the mutation. Another tryptophan at position 2061 of human erythroid ß spectrin that has been implicated in hereditary elliptocytosis is conserved in human ßII, ßIII, and ßIV spectrin, but not in Drosophila ß or in human ßV spectrin. The importance of these tryptophan residues in nonerythroid spectrin tetramer formation and function has not been tested (Das, 2006).

Relatively little is known about the function of repetitive segments 4-14 in ß spectrin. This region has more limited sequence conservation than segments near the ends of the molecule. Further studies using smaller scale modifications will be required to identify the functional sites that explain the mutant phenotype of ßspec^alpha2-9. For now, this transgene helps to establish that not all functional defects in ß spectrin result in mislocalization. Thus, gene modifications that do affect protein targeting, such as ßspec^DeltaPH, are probably identifying functional sites that are responsible for targeting (Das, 2006).

The domain swap strategy described in this study takes advantage of the powerful genetic tools available in Drosophila to study protein function. This approach is especially well suited to studies of a modular protein such as spectrin and has provided valuable insights into both the function and the targeting of the protein in vivo. Combined with the fact that Drosophila is a simpler system, having only three spectrin genes and two ankyrin genes, this approach should continue to provide valuable information that will be more difficult to obtain in mammalian systems. One intriguing observation in this study was the effect of the DeltaPH mutation on the size of the rare flies that survived to adulthood. This phenotype was reminiscent of Drosophila mutations that affect PH domain-containing components of the insulin/insulin-like growth factor signaling pathway. Given the importance of a phosphoinositide-binding PH domain to ß spectrin function, it will be interesting to genetically test the possibility that spectrin has a functional interaction with growth factor signaling pathways (Das, 2006).

The axonal localization of large Drosophila ankyrin2 protein isoforms is essential for neuronal functionality

In polarized cells, such as neurons, ankyrin-type proteins are the major molecules that link the actin-spectrin-based membrane cytoskeleton to the plasma membrane. In Drosophila the second ankyrin gene, Dank2, is exclusively expressed in neuronal cells. Similar to ankyrin genes in other organisms, the Dank2 gene generates several ankyrin protein isoforms by differential splicing. This study reports that in Drosophila, the short Dank2 protein isoform is restricted to neuronal cell bodies and is excluded from axons, whereas the long Dank2 isoforms are localized specifically to axons. Thus the long and short Dank2 protein isoforms are localized to complementary neuronal subdomains, demonstrating that in vivo the composition of the neuronal cortical cytoskeleton is highly polarized. Once polarization is established, it persists during later stages of Drosophila development. Genetic evidence is presented that the absence of axonal Dank2 protein is lethal (Hortsch, 2002).

beta-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization

A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of β-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. This study shows in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, this study provides evidence that the mutation alters the mobility and recruitment of β-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, a Drosophila SCA5 model was developed in which an equivalent mutant Drosophila β-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased β-spectrin localization in distal dendrites. SCA5 β-spectrin dominantly mislocalizes α-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. The data suggest that high-affinity actin binding by SCA5 β-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth (Avery, 2017).

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that causes gait and limb ataxia, slurred speech, and abnormal eye movements. SCA5 stems from autosomal dominant mutations in the SPTBN2 gene that encodes β-III-spectrin, a cytoskeletal protein predominantly expressed in the brain and enriched in cerebellar Purkinje cells. A necessary function of β-III-spectrin in Purkinje cells was demonstrated by β-III-spectrin-null mice, which show ataxic phenotypes and decreased Purkinje cell dendritic arborization. β-III-spectrin consists of an N-terminal actin-binding domain (ABD) followed by 17 spectrin-repeat domains and a C-terminal pleckstrin homology domain. SCA5 mutations that result in single amino acid substitutions or small in-frame deletions have been identified in the ABD and neighboring spectrin-repeat domains. In a SCA5 mouse model, expression in Purkinje cells of a β-III-spectrin transgene containing a spectrin-repeat domain mutation, E532_M544del, causes ataxic phenotypes and thinning of the cerebellar molecular layer that contains Purkinje cell dendrites. This suggests that the cellular mechanism underlying SCA5 pathogenesis is a Purkinje cell deficit linked to the loss of dendritic arborization (Avery, 2017).

The functional unit of β-III-spectrin is considered to be a heterotetrameric complex containing two β-spectrin subunits and two α-spectrin subunits. Through the β-spectrin subunits the spectrin heterotetramer binds and cross-links actin filaments. Multiple β-spectrin protein isoforms have been shown to form a spectrin-actin cytoskeletal structure that lines the plasma membrane of axons and dendrites. The spectrin-actin lattice is a highly conserved neuronal structure identified in the axons of a broad range of neuron types in mammals and in invertebrates, including Drosophila. A spectrin-actin lattice containing β-III-spectrin, or the homolog β-II-spectrin, was identified in the dendrites of hippocampal neurons. Recent studies suggest that the dendritic spectrin-actin cytoskeleton is a ubiquitous feature of neurons, prominent in both dendritic shafts and spines. The widespread localization of β-III-spectrin within the Purkinje cell dendritic arbor suggests that similar spectrin-actin interactions are important for Purkinje cell dendritic function (Avery, 2017).

The spectrin-actin cytoskeleton functions to organize integral membrane proteins through the spectrin adaptor ankyrin and provides mechanical stability to neuronal processes. A form of erythrocyte ankyrin, ankyrin-R, is expressed in Purkinje cells and appears to be required for Purkinje cell health and normal motor function. A hypomorphic ankyrin-R mutation, termed 'normoblastosis', causes Purkinje cell degeneration and ataxia in mice. The subcellular localization of ankyrin-R in the Purkinje cell soma and dendrites mirrors the distribution of β-III-spectrin, and recently β-III-spectrin was shown to physically interact with ankyrin-R. In β-III-spectrin-null mice, ankyrin-R is present in the soma but absent in Purkinje cell dendrites, suggesting that Purkinje cell degeneration and ataxic phenotypes observed in the absence of β-III-spectrin may be linked to a loss of ankyrin-R function in dendrites. A SCA5 mutation that results in a leucine 253-to-proline (L253P) substitution in the ABD of β-III-spectrin causes ectopically expressed β-III-spectrin and ankyrin-R to colocalize internally in HEK293T cells, in contrast to control cells where wild-type β-III-spectrin colocalizes with ankyrin-R at the plasma membrane. This previous study suggests that neurotoxicity caused by the L253P mutation may be connected to spectrin mislocalization and the concomitant mislocalization of ankyrin-R. However, it has not been established whether the L253P mutation affects the dendritic localization of β-spectrin or ankyrin proteins in any neuronal system (Avery, 2017).

This report extends an analysis of the β-III-spectrin L253P mutation, which was recently demonstrated to cause an ∼1,000-fold increase in the binding affinity of the β-III-spectrin ABD for actin filaments in vitro. The mutation is also destabilizing in vitro, causing the ABD to begin to unfold near physiological temperature. Given these results, a key question with important implications for the SCA5 disease mechanism is whether the previously described mislocalization of L253P β-III-spectrin in mammalian cells is driven by a loss of ABD-binding activity, as originally proposed, or instead is the consequence of increased ABD-binding activity. To address the mechanistic basis of β-III-spectrin dysfunction, this study has characterized the L235P mutant protein behavior in mammalian cells. In addition, a Drosophila SCA5 model was generated in which a Drosophila β-spectrin transgene containing the equivalent mutation is conditionally expressed in dendritic arborization sensory neurons. This study used the Drosophila model to analyze the impact of the mutation on dendritic morphology, an aspect of Purkinje cell dysfunction that potentially underlies SCA5 pathology. In living, fully intact larvae, the consequence were examined of the ABD mutation on dendritic arborization, β-spectrin subcellular localization, and the functional interaction of β-spectrin and ankyrin in dendrites (Avery, 2017).

The morphology of dendritic arbors dictates the connectivity of neuronal networks, integrating inputs and propagating signals. The question of how neurons modulate dendritic morphology is of keen interest in the study of neuronal function and neurodegeneration. For example, the molecular and cell biological mechanisms that control branch stability and remodeling within a dendritic field remain largely elusive. This report describes the consequence of a SCA5 mutation on the binding of β-III-spectrin to actin in mammalian cells and leveraged the Drosophila model system to reveal the impact of the SCA5 disease mutation on the neuronal spectrin-actin cytoskeleton and dendritic arborization. This work identifies an important cytoskeletal mechanism in distal dendrites required for formation of large, complex arbors, critical to the function of Purkinje cells targeted in hereditary ataxias (Avery, 2017).

The data suggest that high-affinity actin binding acts dominantly as a driver of L253P β-III-spectrin neurotoxicity by impacting the dynamics of the spectrin-actin network. Drosophila SCA5 β-spectrin containing the equivalent L253P mutation accumulates in the da neuron soma and is absent in distal dendritic regions, in contrast to wild-type β-spectrin that localizes throughout the arbor. In the axons of mammalian neurons the spectrin-actin lattice initially forms near the soma and propagates distally, suggesting that the loss of Drosophila SCA5 β-spectrin in distal dendrites reflects a defect in expansion of the spectrin-actin cytoskeleton from the soma into dendrites. Such an expansion defect may be a consequence of a slow dissociation rate that is typical of high-affinity molecular interactions. Specifically, high-affinity actin binding caused by the mutation may limit the pool of free β-spectrin molecules available to be recruited to an expanding cytoskeleton. Like the loss of Drosophila SCA5 β-spectrin in da neuron dendritic extensions, a reduction was observed of human L253P β-III-spectrin in HEK293T cell plasma membrane protrusions. The absence of L253P β-III-spectrin in filopodium-like and lamellipodium-like extensions, despite abundant localization elsewhere at the plasma membrane, suggests a partitioning between structurally dynamic and stable membrane regions. This partitioning supports the idea that high-affinity actin binding reduces the availability of β-III-spectrin to be recruited to newly formed membrane structures. The data predict that high-affinity binding of L253P β-III-spectrin to actin filaments within the neuronal spectrin-actin lattice negatively impacts Purkinje cell arborization and function by impeding the expansion of the spectrin-actin cytoskeleton in dynamically growing or remodeling dendritic branches and spines (Avery, 2017).

In addition to increasing actin-binding affinity, the L253P mutation destabilizes β-III-spectrin, causing the ABD to begin to unfold near physiological temperature in vitro. This denaturation raised the possibility that cellular phenotypes in mammalian cells are the consequence of ABD protein unfolding and loss of ABD-binding activity rather than elevated ABD-binding activity. Experiments do not support this interpretation, showing instead through co-IP assays that the mutant ABD retains high-affinity actin binding in cells. Indeed, in cultured mammalian cells, protein unfolding reflected in minor degradation products was detected only when the mutant ABD was highly overexpressed. Significantly, the high-affinity actin binding observed for the L253P mutation is mimicked by the alternative substitution, L253A, which, like the L253P mutation, is predicted to disrupt the normal hydrophobic contacts of leucine 253 in the β-III-spectrin ABD. In this case, no degradation of the L253A mutant is detected, and the increased protein stability of the L253A mutation was confirmed in vitro. In light of these results, the observation that the L253A and L253P mutations cause the same β-III-spectrin subcellular localization phenotypes in HEK293T cells supports the conclusion that the behavior of L253P β-III-spectrin is driven by increased ABD-binding activity. These results support the hypothesis that high-affinity actin binding contributes to the L253P β-III-spectrin neurotoxicity that underlies SCA5 pathology (Avery, 2017).

How do the L253P and L253A substitutions account for elevated actin-binding affinity? The location of the mutations in the CH2 domain is consistent with a suspected regulatory role for the domain in mediating actin binding. Biochemical studies of the isolated CH domains of β-spectrin or of the related α-actinin ABD previously documented actin-binding activity for the CH1 domain but not for the CH2 domain. Confinement of binding activity to the CH1 domain is further supported by a structural model for α-actinin ABD-actin complexes in which only a single CH domain is bound to actin filaments. Consistent with the idea that the L253P mutation disrupts a CH2 domain regulatory function, leucine 253 in the CH2 domain is predicted to interface with the CH1 domain and physically bridge the two domains through hydrophobic contacts. The decrease in hydrophobicity introduced by the L253P or L253A substitution is thus predicted to disrupt inter-CH domain contacts and relieve CH2 inhibition. Significantly, disease-causing mutations located in the CH2 domain of α-actinin or filamin also increase actin-binding affinity (Avery, 2017).

In addition to binding filaments of conventional actin, the β-III-spectrin ABD also interacts with ARP1, a component of the dynactin complex that facilitates transport mediated by microtubule motors. Consistent with an ARP1 interaction, a previous study reported that expression of SCA5 β-spectrin in Drosophila motoneurons impairs axonal transport. Given the ~75% similarity in actin and ARP1 primary structures, it is predicted that the L253P mutation will similarly enhance the binding of β-III-spectrin to ARP1. Current studies have not directly addressed this prediction. However, in a previous study, a bimolecular fluorescence complementation assay conducted in HEK293T cells overexpressing ARP1 concluded that the L253P mutation reduces the interaction of β-III-spectrin ABD with ARP1. Nonetheless, a direct test of how L253P β-III-spectrin impacts ARP1 binding is lacking. Indeed, ARP1-binding studies are not straightforward; the native ARP1 filament is difficult to purify, ARP1-specific antibodies are not available, and there is a strong propensity for ARP1 to form nonnative structures when overexpressed in cells. Further experiments are needed to fully understand the impact of the L253P mutation on ARP1 binding and intracellular vesicular transport (Avery, 2017).

In Drosophila, the lone homolog of β-III-spectrin, β-spectrin, localizes to both dendrites and axons, where the function of the spectrin cytoskeleton has been extensively studied. The impact of SCA5/L246P β-spectrin expression on synaptic organization at the neuromuscular junction (NMJ) has been reported. Spectrin RNAi also disrupts synaptic bouton organization but further leads to NMJ retraction, a phenotype not observed in SCA5 motoneurons. The current work shows that SCA5 β-spectrin is absent not only in distal dendrites but also at the axon terminus of da neurons. Thus, SCA5 β-spectrin may dominantly mislocalize the spectrin cytoskeleton not only in dendrites but also in distal axons. In light of these findings, the reduced size of the Drosophila NMJ reported in motor neurons expressing SCA5 β-spectrin may in part reflect a disruption of the spectrin cytoskeleton and associated ankyrin-2 (Avery, 2017).

This work points to a molecular mechanism in the somatodendritic compartment of neurons that enables the formation of large, complex dendritic arbors. As the dendritic arbor grows, dynamic actin assembly in the distal dendrites drives the formation of new terminal branches. It is suggested that during arbor growth expansion of the spectrin-actin cytoskeleton is required to stabilize terminal branches and allow for continued expansion of the arbor. To explain the mutant SCA5 arbor phenotypes, it is proposed that high-affinity binding of the mutant β-spectrin decreases spectrin-actin dynamics and consequently constrains expansion of the spectrin-actin cytoskeleton and stabilization of growing dendrites. The spectrin cytoskeleton has similarly been implicated in axonal growth and stabilization of synaptic junctions. In support of this cytoskeletal mechanism regulating dendritic arbor stability and potentially underlying SCA5 pathology, this study shows that the loss of β-spectrin, as well as ankyrin-2, in the distal dendrites of Drosophila da neurons correlates with a proximal shift in dendritic branching. Importantly, expression of mutant SCA5 β-spectrin and ankyrin-2 RNAi resulted in similar dendritic arborization defects, and SCA5 β-spectrin causes a loss of ankyrin-2 XL in distal dendrites. This study characterizes a progressive elimination of distal dendrites at segmental boundaries in SCA5 arbors. Moreover, lacking expansion of the spectrin-actin cytoskeleton in terminal dendrites, dynamic actin-based assembly drives complex terminal branching at the periphery of SCA5 arbors. However, the stability of the SCA5 terminal branching is compromised, and the outward growth of the arbor field is defective. One possibility is that impaired expansion of the spectrin-actin cytoskeleton and loss of ankyrin-2 in dendrites impacts localization of neuroglian, a cell-adhesion molecule required for arborization and which may mediate stability of dendritic branching in Drosophila da neurons. In Purkinje cells, it is predicted that L253P β-III-spectrin will similarly impair expansion of the spectrin-actin lattice, disrupting dendritic localization of critical membrane proteins ankyrin-R, EAAT4, and mGluR1α, and in consequence promoting defects in arborization and postsynaptic signaling that characterize SCA5 pathology (Avery, 2017).

Significantly, this model for the impact of a SCA5 mutation on cytoskeletal dynamics and distal arborization is similar to a disease model proposed for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) in which a decrease in mitochondrial dynamics is suggested to disrupt distal Purkinje cell arborization. Like the mislocalization of SCA5 β-spectrin in da neurons, loss of function of the ARSACS disease protein sacsin, a mitochondrial protein, causes mitochondria to accumulate in the soma and proximal dendrites but fail to reach distal dendrites in mammalian neurons. Neurons such as Purkinje cells and da neurons that extend complex arbors appear to be especially vulnerable to disruptions to pathways in distal dendrites that support arborization, and this sensitivity possibly explains the cerebellar specificity of SCA5 pathology (Avery, 2017).

Tissue-specific regulation of alternative polyadenylation represses expression of neuronal ankyrin isoform in C. elegans epidermal development

Differential mRNA polyadenylation plays an important role in shaping the neuronal transcriptome. In C. elegans, several ankyrin isoforms are produced from the unc-44 locus through alternative polyadenylation. This study identify a key role for an intronic polyadenylation site (PAS) in temporal- and tissue-specific regulation of UNC-44/ankyrin isoforms. Removing an intronic PAS results in ectopic expression of the neuronal ankyrin isoform in non-neural tissues. This mis-expression underlies epidermal developmental defects in mutants of the conserved tumor suppressor death-associated protein kinase, dapk-1. Previous studies reported that the use of this intronic PAS depends on the nuclear polyadenylation factor SYDN-1, which inhibits the RNA polymerase II CTD phosphatase SSUP-72. Consistent with this, loss of sydn-1 blocks ectopic expression of neuronal ankyrin and suppresses epidermal morphology defects of dapk-1. These effects of sydn-1 are mediated by ssup-72 autonomously in the epidermis. A peptidyl-prolyl isomerase PINN-1 antagonizes SYDN-1 in the spatiotemporal control of neuronal ankyrin isoform. Moreover, the nuclear localization of PINN-1 is altered in dapk-1 mutants. These data reveal that tissue and stage-specific expression of ankyrin isoforms relies on differential activity of positive and negative regulators of alternative polyadenylation (Chen, 2017).

Search PubMed for articles about Drosophila Ankyrin2

Ango, F., di Cristo, G., Higashiyama, H., Bennett, V., Wu, P. and Huang, Z. J. (2004). Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at purkinje axon initial segment. Cell 119: 257-272. PubMed ID: 15479642

Avery, A. W., Thomas, D. D. and Hays, T. S. (2017). beta-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization. Proc Natl Acad Sci U S A 114(44): E9376-E9385. PubMed ID: 29078305

Bennett, V., A. J. (2001). Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol Rev 81: 1353-1392. PubMed ID: 11427698

Bouley, M., Tian, M. Z., Paisley, K., Shen, Y. C., Malhotra, J. D. and Hortsch, M. (2000). The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization. J Neurosci 20: 4515-4523. PubMed ID: 10844021

Chen, F., Chisholm, A. D. and Jin, Y. (2017). Tissue-specific regulation of alternative polyadenylation represses expression of neuronal ankyrin isoform in C. elegans epidermal development. Development [Epub ahead of print]. PubMed ID: 28087624

Das, A., Base, C., Dhulipala, S. and Dubreuil, R. R. (2006). Spectrin functions upstream of ankyrin in a spectrin cytoskeleton assembly pathway. J Cell Biol 175: 325-335. PubMed ID: 17060500

Dubreuil, R. R. and Yu, J. (1994). Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila. Proc Natl Acad Sci U S A 91: 10285-10289. PubMed ID: 7937942

Enneking, E. M., Kudumala, S. R., Moreno, E., Stephan, R., Boerner, J., Godenschwege, T. A. and Pielage, J. (2013). Transsynaptic coordination of synaptic growth, function, and stability by the L1-type CAM Neuroglian. PLoS Biol 11(4): e1001537. PubMed ID: 23610557

Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M. and Murphey, R. K. (2006). A conserved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation. Curr Biol 16: 12-23. PubMed ID: 16401420

Friedrich, J., Sorge, S., Bujupi, F., Eichenlaub, M. P., Schulz, N. G., Wittbrodt, J. and Lohmann, I. (2016). Hox function is required for the development and maintenance of the Drosophila feeding motor unit. Cell Rep 14: 850-860. PubMed ID: 26776518

Hortsch, M., Paisley, K. L., Tian, M. Z., Qian, M., Bouley, M. and Chandler, R. (2002). The axonal localization of large Drosophila ankyrin2 protein isoforms is essential for neuronal functionality. Mol Cell Neurosci 20: 43-55. PubMed ID: 12056839

Jegla, T., Nguyen, M. M., Feng, C., Goetschius, D. J., Luna, E., van Rossum, D. B., Kamel, B., Pisupati, A., Milner, E. S. and Rolls, M. M. (2016). Bilaterian giant Ankyrins have a common evolutionary origin and play a conserved role in patterning the axon initial segment. PLoS Genet 12(12): e1006457. PubMed ID: 27911898

Koch, I., Schwarz, H., Beuchle, D., Goellner, B., Langegger, M. and Aberle, H. (2008). Drosophila ankyrin 2 is required for synaptic stability. Neuron 58: 210-222. PubMed ID: 18439406

Koon, A. C. and Budnik, V. (2012). Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling. J Neurosci 32: 6312-6322. PubMed ID: 22553037

Luchtenborg, A. M., Solis, G. P., Egger-Adam, D., Koval, A., Lin, C., Blanchard, M. G., Kellenberger, S. and Katanaev, V. L. (2014). Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton. Development 141: 3399-3409. PubMed ID: 25139856

Pielage, J., Fetter, R. D. and Davis, G. W. (2006). A postsynaptic spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction. J Cell Biol 175: 491-503. PubMed ID: 17088429

Pielage, J., Cheng, L., Fetter, R. D., Carlton, P. M., Sedat, J. W. and Davis, G. W. (2008). A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion. Neuron 58: 195-209. PubMed ID: 18439405

Scotland, P., Zhou, D., Benveniste, H. and Bennett, V. (1998). Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons. J Cell Biol 143: 1305-1315. PubMed ID: 9832558

Spurrier, J., Shukla, A. K., Buckley, T., Smith-Trunova, S., Kuzina, I., Gu, Q. and Giniger, E. (2019). Expression of a fragment of Ankyrin 2 disrupts the structure of the axon initial segment and causes axonal degeneration in Drosophila. Mol Neurobiol. PubMed ID: 30666562

Stephan, R., Goellner, B., Moreno, E., Frank, C. A., Hugenschmidt, T., Genoud, C., Aberle, H. and Pielage, J. (2015). Hierarchical microtubule organization controls axon caliber and transport and determines synaptic structure and stability. Dev Cell 33: 5-21. PubMed ID: 25800091

Trunova, S., Baek, B. and Giniger, E. (2011). Cdk5 regulates the size of an axon initial segment-like compartment in mushroom body neurons of the Drosophila central brain. J Neurosci 31(29): 10451-10462. PubMed ID: 21775591

Weber, T., Stephan, R., Moreno, E. and Pielage, J. (2019). The ankyrin repeat domain controls presynaptic localization of Drosophila Ankyrin2 and is essential for synaptic stability. Front Cell Dev Biol 7: 148. PubMed ID: 31475145

Yamamoto, M., Ueda, R., Takahashi, K., Saigo, K. and Uemura, T. (2006). Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface. Curr Biol 16: 1678-1683. PubMed ID: 16920632

Zhang, X. and Bennett, V. (1998). Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains. J Cell Biol 142: 1571-1581. PubMed ID: 9744885

date revised: 11 January 2024

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