InteractiveFly: GeneBrief

ringmaker: Biological Overview | References


Gene name - ringmaker

Synonyms -

Cytological map position - 72E2-72E2

Function - cytoskeleton

Keywords - PNS, maintains microtubule stability/dynamics with the microtubule-associated protein Futsch, splice factor Rtca suppresses Xbp1 via nonconventional mRNA splicing, which in turn reduces ringer expression to inhibit axon regeneration, lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6

Symbol - ringer

FlyBase ID: FBgn0266417

Genetic map position - chr3L:16,354,746-16,359,428

Classification - p25-alpha

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and anti-regeneration factors are being identified. Previous work has shown that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Through transcriptome profiling this study has shown that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein Futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, these findings suggest that Ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration (Monahan Vargas, 2020).

In recent years, several strategies have shown efficacy augmenting nerve regeneration in various experimental models. Unfortunately, therapeutic interventions to promote nerve regeneration and functional recovery still do not exist. Previous work has also helped shape the approach researchers have taken toward better understanding regeneration and drawing connections between successful paradigms. This study reports a link between two cellular mechanisms that are essential for regeneration: RNA processing and microtubule dynamics (Monahan Vargas, 2020).

In Drosophila, sensory dendritic arborization (da) neurons show differential regenerative potentials between the periphery and the central nervous system (CNS), resembling that of mammalian neurons. Moreover, distinct subclasses of da neurons also regenerate differently. A previous study developed a two-photon-based axon injury model that assays class III (C3da) and class IV (C4da) da neurons to identify and analyze targets that enhance regeneration (Li, 2018). Using this model, Rtca (RNA 3'-terminal phosphate cyclase), an RNA-binding protein (RBP), was identified as an inhibitor of axon regeneration (Song, 2015). Rtca is involved in stress induced Xbp1 mRNA splicing, and its knockout or neuronal knockdown promotes axon regeneration both in the peripheral nervous system (PNS) and CNS. However, its downstream effectors and signaling mechanisms remain unexplored. RBPs are increasingly shown to regulate complex cellular processes associated with neurodegenerative diseases and regeneration. This study reports the results from transcriptome profiling revealing that a microtubule associated protein, Ringer (also known as Ringmaker, which is the fly homolog of the mammalian tubulin polymerization-promoting proteins [TPPPs]), is strongly increased following Rtca removal (Monahan Vargas, 2020).

Microtubules and the cytoskeletal network are essential for neuronal function and are paramount to an axon's ability to respond to guidance cues, transport proteins and organelles, grow, survive, and regenerate. Microtubule-binding small molecules and microtubule-associated proteins (MAPs) that regulate microtubule dynamics are attractive therapeutic targets to augment axon regeneration. Ringer belongs to the brain-specific protein, p25α, also known as the TPPP protein family. TPPPs regulate tubulin polymerization and are implicated in neurodegenerative disorders such as α-synucleinopathies and Multiple System Atrophy. Drosophila has only one TPPP ortholog, Ringer, and it directly binds tubulin, promotes microtubule bundling and polymerization in vitro, and is critical for microtubule stabilization and developmental axon growth (Mino, 2016). This study shows that transcription of ringer is negatively regulated by Rtca via Xbp1. ringer was found to function as a neuronal intrinsic promoter of axon regeneration, working in concert with other MAPs, specifically Futsch/MAP1B and HDAC6, which have been previously shown to be integral for axonal health and integrity. The results reveal MAPs as important arbiters of axon regeneration, and ringer (TPPP homologs) is proposed as an attractive therapeutic target for promoting axon regeneration (Monahan Vargas, 2020).

RBPs have been shown to be crucial in regulating complex cellular processes such as mRNA editing, transport and local translation. Aberrant processing of RNA is present in neuronal diseases and injury. How these processes are affected after nervous system trauma and their regulation during neural repair are poorly understood. Previous work has identified Rtca, an RNA-binding protein regulating RNA repair and splicing, as a potential damage sensor that inhibits axon regeneration. Rtca LOF enhances axon regeneration in both fly and mammalian neurons. To better understand its underlying mechanism, RNA-seq was performed to assess the transcriptome of Rtca mutant neurons; ringer transcripts were found to be highly expressed. Ringer is a MAP homologous to the mammalian tubulin polymerization-promoting proteins (TPPPs), in particular TPPP3 or TPPP1, which has been shown to be a regulator of axonal microtubule organization by promoting microtubule polymerization, assembly, and stability both in vitro and in vivo (Mino, 2016). This study has revealed a connection between the injury-evoked RNA repair/splicing system and the MAP ringer; it is proposed that Rtca suppresses Xbp1 via nonconventional mRNA splicing, which in turn reduces ringer expression to inhibit axon regeneration. Furthermore, evidence is provided for an association between Futsch and HDAC6, additional MAPs capable of regulating microtubule stability and posttranslational modifications. Ringer is also inhibited by HDAC6, and it cooperates with Futsch to relay a cellular stress signal to the microtubule network. In addition, these data suggest that Rtca and Xbp1 likely have additional downstream effectors independent of ringer, and that Futsch likely receives additional inputs, in parallel to Ringer, to support axonal regeneration. Future studies to directly monitor microtubule dynamics in Rtca LOF mutants will help further validate this model and offer clues to the identity of additional players in this pathway (Monahan Vargas, 2020).

The capacity of an axon to regenerate depends on both the external environment and cell-intrinsic mechanisms, which ultimately converge onto axonal microtubules. MAPs have become popular targets for augmenting nerve regeneration given the importance of microtubule stability and polymerization in both the nascent axon and the regenerating axon's growth cone. As an axon elongates, microtubules engorge the growth cone to fill it with microtubule mass. As the growth cone advances, microtubules bundle and consolidate within the nascent axon to provide structure and support. Ringer has been shown to be essential for proper microtubule bundling (Mino, 2016). Microtubules are inherently polarized because newly added tubulin dimers only assemble and disassemble at the 'plus' end of the lattice, whereas the minus end of a microtubule is highly stabilized with special tubulin variants, abundant post translational modifications (e.g., acetylation of α-tubulin), and minus-end associating proteins. Therefore, a single microtubule can be thought of as having two general domains; a plus-end that is labile (i.e., where dynamic instability occurs) and a minus end that is stable and resists depolymerization. Microtubule stabilization prevents depolymerization and favors microtubule growth, which is beneficial for the axon's growth cone to advance. Inducing microtubule stabilization using extremely low doses of the drugs paclitaxel or epithilones has resulted in significant augmentation of nerve regeneration in vivo. The results of this study demonstrated a loss of microtubule acetylation in whole-cell lysate and specifically within the proximal axon of injured neurons in ringer mutants. This is in line with the function of Ringer, which has been associated with microtubule polymerization and stability. Future experiments to dynamically track Ringer proteins in accordance with microtubule polymerization during axon regeneration, and an extensive investigation of microtubule posttranslational modifications following axotomy are warranted (Monahan Vargas, 2020).

Futsch, a MAP1B homolog, was recently shown to associate with ringer (Shi, 2019). Together, Ringer and Futsch were found to regulate synapse formation at neuromuscular junctions via a microtubule-based mechanism. It can be inferred that Ringer and Futsch may help promote the formation of a growth cone rather than a retracting dystrophic end within injured axons, similar to its maintenance of synaptic integrity. Ringer mutation led to a decrease in futsch mRNAs and immunolabeling, suggesting a role in regulating futsch transcription, localization, and protein levels. Both ringer and futsch mutations impaired axon regeneration, albeit futsch had a more dramatic effect, suggesting that futsch may contribute to additional signaling independent of ringer. While heterozygous mutants for futsch and ringer did not have a reduction in regeneration, transheterozygotes of ringer and futsch mutations exhibited a similar reduction in regeneration as ringer mutants alone. Coimmunoprecipitation experiments showed that ringer, futsch, and tubulin physically interact and form a molecular complex, and that Ringer facilitates Futsch binding to tubulin. Epistasis analysis further demonstrated that overexpression of Futsch failed to rescue the reduced axon regeneration in ringer mutants, while overexpression of futsch is sufficient to promote axon regeneration despite the absence of futsch. Importantly, this study found that microtubule turnover is faster in injured versus uninjured axons, and that futsch LOF dysregulates microtubule dynamics, accelerating its turnover after injury. Taken together, sthe data suggest that Ringer and Futsch cooperate in the same complex with tubulin, to maintain microtubule dynamics/stability, and that both are critical to the ability of sensory neurons to regenerate. Futsch is phosphorylated by GSK3 and sustained GSK3 activity promotes axon regeneration and increases the pool of dynamic microtubule mass, which further leads to a speculation that futsch might be regulated by additional signaling pathways (Monahan Vargas, 2020).

Elucidating how microtubule stability properties are altered following an injury and the MAPs responsible for mediating those changes may identify novel therapeutic targets. This study found that acetylation properties were altered by ringer mutations and, therefore, attempts were made to explore the role HDAC6, the primary tubulin deacetylase, may play in instructing regeneration. HDAC6 knockout and pharmacological inhibition increased regeneration in C3da neurons, a subtype of sensory neurons incapable of regeneration in WT flies. Previous studies have shown that HDAC6 inhibition and deletion leads to the hyperacetylation of microtubules. Early studies found that HDAC6 was neuroprotective after a CNS injury and associated these findings with HDAC6's role in transcriptional regulation. However, more recent studies found that HDAC6 is neuroprotective in a manner that was associated with its deacetylation of microtubules. Other studies have shown that HDAC6 is essential for healthy axonal transport and influences MAP-microtubule interactions. This study showd that HDAC6 LOF leads to increased protein levels of ringer and futsch, likely through posttranscriptional mechanisms. It may also be possible that HDAC6 knockout affects microtubule-binding kinetics and the protein localization of Ringer and Futsch (i.e., concentrated versus diffuse). Augmented regeneration following HDAC6 knockout was lost with a ringer mutation. These results, along with the changes observed in Ac-Tub levels, suggest an interaction between HDAC6 and Ringer, where Ringer may function to either directly or indirectly restrict HDAC6 deacetylase activity with respect to α tubulin acetylation. This is likely, given that Ringer has been shown to regulate microtubule bundling and stability, which are associated with highly acetylated domains of microtubules. Ringer may be essential to protecting highly acetylated and stable microtubule domains from HDAC6 deacetylation by occluding its interaction with α tubulin or directly blocking deacetylase activity. This would be consistent with in vitro studies suggesting that mammalian TPPP modulates microtubule acetylation by binding to HDAC6 and inhibiting its activity. Alternatively, HDAC6 could inhibit TPPP nucleation by binding to TPPP and preventing its association to tubulin. Furthermore, HDAC6 can also physically modify kinases shown to negatively interrupt TPPP function such as ERK2. This network hypothesis could help explain an underlying positive feedback loop regulating microtubule stability: Increase of TPPP would inhibit HDAC6 leading to an enhancement of acetylated, potentially stable microtubule; in contrast, modification of kinases by HDAC6 could lead to kinase activation and downstream phosphorylation of TPPP, limiting its microtubule binding activity. It is believed that HDAC6 and ringer are involved in a pathway that ultimately affects the stability and dynamics of microtubules. Future studies will explore whether Ringer and HDAC6 expression, along with posttranslational modifications of tubulin, can predict the regenerative potential of da sensory neurons. C4da neurons show only ~75% regeneration and it is proposed that the other 25% will show differences in the expression of MAPs and microtubule posttranslational modifications, specifically acetylation of α-tubulin (Monahan Vargas, 2020).

The future treatments for nerve regeneration will most likely be combinatorial, with a need to address the extrinsic and intrinsic barriers to regeneration. This study has identified a link between RNA repair/splicing and microtubule organization via a damage-evoked mechanism involving Rtca and Ringer. Further evidence is presented that therapeutic targets capable of augmenting nerve regeneration ultimately converge on microtubules. Microtubules are a bottleneck to regeneration and identifying intrinsic signaling cascades that regulate microtubule dynamics using fly genetics will reveal pathways critical to microtubule-mediated nerve regeneration. Given the complexity of MAPs and the increasing number of candidate proteins, utilizing the fly injury model system allows screening for promising targets that warrant an investigation into their mammalian homologs with in vitro and in vivo mammalian nerve injury models. Excitingly, the zebrafish homolog of TPPP3 was recently shown to promote axon regeneration in Mauthner cells and is regulated at the transcript level by microRNA 133b. This corroborates the current findings, leading to the proposal that ringer/TPPP is tightly regulated and may function as a relay station at multiple levels. Moreover, HDAC6 was also recently shown to be inhibitory in a regeneration screen performed in C. elegans. In summary, this study has identified a RNA repair/splicing pathway that up-regulates the MAP Ringer, which interacts with other MAPs associated with microtubule stability/dynamics and tubulin posttranslational modifications, processes that are evolutionarily conserved and promising targets for regenerative therapies (Monahan Vargas, 2020).

Tubulin polymerization promoting protein, Ringmaker, and MAP1B homolog Futsch coordinate microtubule organization and synaptic growth

Drosophila Ringmaker (Ringer) is homologous to the human Tubulin Polymerization Promoting Proteins (TPPPs) that are implicated in the stabilization and bundling of microtubules (MTs) that are particularly important for neurons and are also implicated in synaptic organization and plasticity. No in vivo functional data exist that have addressed the role of TPPP in synapse organization in any system. This study presents the phenotypic and functional characterization of ringer mutants during Drosophila larval neuromuscular junction (NMJ) synaptic development. ringer mutants show reduced synaptic growth and transmission and display phenotypic similarities and genetic interactions with the Drosophila homolog of vertebrate Microtubule Associated Protein (MAP)1B, futsch. Immunohistochemical and biochemical analyses show that individual and combined loss of Ringer and Futsch cause a significant reduction in MT loops at the NMJs and reduced acetylated-tubulin levels. Presynaptic over-expression of Ringer and Futsch causes elevated levels of acetylated-tubulin and significant increase in NMJ MT loops. These results indicate that Ringer and Futsch regulate synaptic MT organization in addition to synaptic growth. Together these findings may inform studies on the close mammalian homolog, TPPP, and provide insights into the role of MTs and associated proteins in synapse growth and organization (Shi, 2019).

While regulation of synaptic MTs and the range of proteins that affect synaptic MT organization and function are not well-characterized, synaptic MTs have been implicated in regulating synaptic bouton growth. Thus, understanding the regulation of MT assembly, organization and dynamics in synaptic terminals is crucial for understanding synapse development and function. The current findings demonstrate that loss of Ringer affects synaptic bouton growth at the NMJ. The growth of NMJ synapses in Drosophila has been postulated to occur either through a process called intercalation where existing synaptic boutons space apart with new boutons inserted between them, or by end addition where new boutons are added at the ends of existing string of boutons. Synapse growth is also thought to occur from budding of existing boutons. While future studies will determine which of these processes may be compromised in ringer mutants leading to a reduction in the number of NMJ synaptic boutons, Ringer can be added to the increasing repertoire of proteins involved in the modulation of synaptic growth. It is likely that the regulation of the cytoskeleton by Ringer may have a profound impact on the balance between synaptic growth and stability. Since synaptic growth can be under both positive and negative regulations, one issue of interest would be to determine what genes are upstream and downstream of Ringer and define a signaling cascade that modulate synaptic growth (Shi, 2019).

The observations that the apposition of the presynaptic AZ protein, BRP, with the GluR receptor fields were not severely disrupted in ringer mutants suggest that Ringer might not be crucial for proper placement of pre- and post-synaptic specializations at the synaptic boutons. Interestingly, number of BRP-positive puncta/bouton area was significantly increased in ringer mutants than wild type. The synaptic ultrastructure of ringer mutants also revealed an increase in AZ number as well as disrupted AZ morphology. Thus, one possibility is that Ringer may directly play a role in AZ organization by interacting with BRP or indirectly through other proteins. It is also possible that disorganized MTs due to loss of Ringer may simply impact the proper assembly of AZs in the synaptic boutons. While elucidating the role of Ringer in AZ organization is an interesting topic of future research, it is important to note that this role of Ringer may or may not be dependent on Futsch. Recent findings report that futsch mutants, contrary to ringer mutants, have a decrease in AZ number and density at the larval NMJs but normal AZ ultrastructural morphology) further underscoring the fact that these proteins may coordinate unique axonal cytoskeletal functions during synapse organization (Shi, 2019).

ringer mutants showed a decrease in bouton numbers but an increase in AZs/bouton area as revealed both by Brp immunostaining and EM analyses. This phenotype could result in unchanged spontaneous firing of the minis as is reflected from no significant changes in mEJP frequency. At the same time the evoked EJP amplitude was decreased in ringer mutants. The increase in AZ numbers did not translate directly into increased miniature frequency, as loss of Ringer may also affect synapse ultrastructure that could still be abnormal at the more molecular level. Analysis of the synaptic vesicles (clustered and docked) at the AZs in the presynaptic terminals of ringer mutants also did not reveal any significant differences compared to controls. It is quite likely that significant decrease in EJP amplitude and quantal content might reflect a lower release probability and possibly defects in the synaptic release machinery. Altogether, Ringer loss reflects a presynaptic defect in neurotransmission machinery (Shi, 2019).

Both present and previously published studies on Ringer (Mino, 2016) reveal an interesting spatio-temporal pattern and differential levels of Ringer localization in the Drosophila nervous system during development. Ringer displayed a temporally dynamic expression in neurons during early embryonic stages followed by an expression at the midline glia during later stages of embryonic ventral nerve cord development (Mino, 2016). Interestingly, in vertebrates, TPPP is predominantly expressed in the CNS oligodendrocytes and plays a critical role in myelin maturation (Skjoerringe, 2006; Lehotzky, 2010; Ota, 2014). Given Ringer's localization in both neuronal and glial cell types in the Drosophila embryonic CNS, it is possible that mammalian TPPP may also be expressed at lower/undetectable levels in neurons in physiological conditions. Under pathological conditions though, TPPPs are reported to be enriched and colocalize with α-Synuclein in neuronal and oligodendroglial inclusions that are characteristic of Synucleinopathies. Ringer also has differential levels of wild type localization in third instar larvae as it is expressed at higher levels in larval axons (Mino, 2016) but at much lower levels at the presynaptic NMJ terminals. The NMJ localization is mostly cytoplasmic but also seems to associate with Futsch, which localizes at higher levels to the core MT cytoskeleton (Shi, 2019).

MT assembly and dynamics are regulated by several factors and mechanisms, such as MT-assembly promoting factors, MT stabilizing/destabilizing factors, MT severing proteins and MT post-translational modifications that affect MT stability. As cells respond to physiological needs, they constantly adapt their MT arrays by modulating the balance between dynamic and stable MT subpopulations. This is also achieved through acetylation which occurs primarily on MTs and can be abundant on long lived stable MTs. These studies revealed that Ringer together with Futsch regulates levels of Ac-Tub at the NMJ with single and double mutants displaying significantly decreased levels of acetylation. These in vivo findings are in line with previously reported cell culture data showing down regulation of TPPP by specific si-RNA resulted in decrease of Ac-Tub levels. The control of acetylation level of MT network is an important factor for the regulation of MT architecture and maintenance of its integrity. The current data suggest that one of the aspects of Ringer functions would thus be to regulate the MT architecture possibly by regulating levels of MT acetylation (Shi, 2019).

The stabilization of MTs during neuronal maturation also underlies axonal specification and growth. Data from Drosophila have shown that the conversion of a motile growth cone into a presynaptic terminal is associated with the appearance of a hairpin MT loop in the growth cone. Homozygous mutations in both ringer and futsch alter MT loop formation, a process that has been implicated as a phenomenon reflective of MT stability and budding of new boutons. While individual and combined loss of ringer and futsch resulted in reduced levels of synaptic Ac-Tub and reduction in NMJ MT loops, overexpression of Ringer and Futsch showed the opposite. These findings are consistent with the in vitro cell culture experiments and biochemical Tubulin assays that showed that Ringer affects MT polymerization; with Ringer-expressing cells forming a circular ring instead of regularly distributed MTs (Mino, 2016). Vertebrate MAP1B may also be involved in MT loop formation as revealed by in vitro overexpression of MAP1B (Shi, 2019).

There is also a group of MT-severing proteins that regulate synaptic MT stability and growth at the NMJ. These are Spastin and Katanin 60. Spastin is enriched in axons and is highly abundant in presynaptic terminals. Knockdown of Spastin causes a severe reduction in synaptic arbor and an increase in stable and looped MTs at synaptic terminals. Similarly, loss of Katanin 60 also resulted in increased MT loops and levels of Ac-Tub suggesting that these protein functions are contrasting to that of Ringer and Futsch. Vertebrate Spastin is critically required for axonal outgrowth during zebrafish embryonic development. Also, axon branch loss at the developing mouse NMJ is mediated by branch-specific MT severing by Spastin, which results in local disassembly of the MT cytoskeleton with subsequent dismantling of branches. Mutations in Spastin have also been associated with increased stabilization of MT network. Recently, it has also been shown that in HeLa cells, the two isoforms of Spastin harboring a missense mutation increases the levels of Ac-Tub. Thus, the broader implications from all of these findings could be that a fine balance of acetylation/de-acetylation kinetics may underlie proper MT organization and synaptogenesis (Shi, 2019).

The primary intracellular target of TPPP is tubulin/MT under both in vitro and in vivo conditions and displays extensive MT bundling activity (Hlavanda, 2002; Mino, 2016). One of the crucial factors affecting the function of MT network is its acetylation by the action of acetyltransferase complex as well as histone deacetylase 6 (HDAC6) and Sirtuin-2 (SIRT2). In vitro studies suggest that mammalian TPPP modulates MT acetylation by binding to HDAC6 and inhibits its activity, resulting in a reciprocal increase in MT acetylation (Tokesi, 2010). HDAC6 is commonly considered to be a tubulin-deacetylase because chemical inhibition of this enzyme significantly increases MT acetylation in neurons. Similar to HDAC6, a more recent study showed the tubulin deacetylase (SIRT2) to play a role with TPPP in regulating MT dynamics and stability. Thus, TPPP-directed deacetylase inhibition can be speculated as one of the mechanisms for the fine control of the dynamics and stability of the MT network. It will be interesting to further investigate whether Drosophila Ringer and/or Futsch may form a larger molecular complex that involves aspects of HDAC6 and SIRT2 in regulating MT dynamics and potentially synaptic growth at the NMJs. In vitro studies have also demonstrated that TPPP influences MT dynamics by decreasing the growth velocity of MT plus ends. While future studies will investigate how Drosophila Ringer modulates the dynamics and stability of the MT network, one can speculate based on the findings from the vertebrate TPPP, that these mechanisms could involve its MT assembly promoting, cross-linking and/or acetylation enhancing activities (Shi, 2019).

The biochemical analyses of Ringer reported in this study provide important insights into its role in regulating the MT cytoskeleton. It is interesting that the overall levels of Ringer did not change in futsch mutants compared to the control. This finding was consistent whether the total Ringer levels were assayed from larval tissues or adult head lysates. However, while the total Ringer levels were unchanged, the synaptic Ringer localization displayed a significant alteration compared to control raising the possibilities that, in the absence of Futsch, either Ringer levels significantly decreases in the presynaptic terminals or Ringer just fails to localize in its proper place and instead gets diffuse. However, total Tub levels and that of Ac-Tub were consistent with what was observed at the synapses. Irrespective of tissue type, these findings reveal a remarkable consistency in demonstrating that Ringer and Futsch regulate synaptic and overall MT stability: (1) Ac-Tub levels in synapses, (2) synaptic MT loops and (3) total Ac-Tub levels, each of these parameters were found to be affected similarly with a reduction in individual and combined loss of ringer and futsch and an elevation in their respective overexpression (Shi, 2019).

Although not in the context of intercellular protein-protein interactions in the synapses, there are reports of some TPPP interacting proteins. Consistent with published reports, Ringer being a Tub-binding protein was further reiterated by their presence in the IP complex. As expected, the MAP1B/Futsch also existed in a complex with Tub. Interestingly, while endogenous Ringer and Futsch could not be detected in the same IP complex, m-Cherry tagged Ringer was detected from an overexpression experimental paradigm. These datasets are reflective of an inability of the endogenous proteins to be detected either due to: (1) a huge difference in their molecular weights (Ringer being ~25 kDa and Futsch over 550 kDa); (2) the relative abundance of the endogenous proteins; and (3) the binding affinity or the stoichiometry of the complex. However, the GST pull-down assays further established Futsch as an interacting partner of Ringer. Having established Ringer and Futsch as a complex, it will be interesting to investigate what other known as well as yet to be identified proteins will likely be recruited to this complex. Moreover, the large size and multiple domains of Futsch alone may allow it to complex with several others in the presynaptic terminals. An issue of interest, then, will be to determine how these complexes are assembled together with the variety of interactions with the post-synaptic targets. Also interesting will be to see if these protein-protein interactions are conserved across species, particularly in vertebrates and what role they will play in regulating MT dynamics. Together these results reveal that changes in MT organization are an essential aspect of synapse development and function and Ringer, a member of the unique and highly conserved TPPP family of proteins, plays a role in regulating MT stability and synaptic organization (Shi, 2019).

Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development

Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. Studies have suggested that members of the Tubulin Polymerization Promoting Protein (P25alpha/TPPP) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. This study reports the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25alpha proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic CNS. ringer null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together these studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization (Mino, 2016).

Precise axon growth and guidance rely on microtubule polymerization, stabilization and bundling. These processes are central in establishing neuronal connectivity. Various proteins affecting microtubule dynamics have been characterized in the context of process extension. Proteins containing p25α domains are expressed in embryonic and postnatal brains, and are known to alter microtubule dynamics. However, the majority of studies do not address their relevance during early development. Using in vivo and in vitro studies, this study addressed the previously uncharacterized function of Drosophila TPPP (Ringer), the only long p25α-containing protein in Drosophila, and its importance in neuronal development (Mino, 2016).

Through mRNA and protein localization, this work uncovers that Ringer is present in the nervous system and that its expression is variable and tightly modulated in the embryonic CNS midline. Evidence is provided that Ringer is necessary for correct nervous system development. Loss of Ringer results in soma misplacement, and defects in axonal extension and guidance in agreement with neuron-specific knockdown experiments showing similar defects. That loss of Ringer results in axonal disruption is strengthened by the findings of knockdown studies in vitro and in zebrafish, which have shown TPPPs have an effect on process extension. Similarly, ringer has been identified as a neuronal outgrowth modifier candidate (Mino, 2016).

ringer mutants exhibit phenotypic variability. Initially, it was supposed that these differences were due to a contribution of maternal Ringer, a suspicion arising from experiments involving deficiency lines. However, all ringer-mutant embryos analyzed were from homozygous stocks, which rules out this possibility. Phenotypic variance could also arise owing to compensation by other proteins. For instance, TPPP has been suggested to bundle microtubules in manner similar to that of Tau. In Drosophila neurons, Tau knockdown only shows exacerbated neuronal degeneration when combined with futsch mutations. It is hypothesized that Ringer acts in a manner similar to Tau. Additionally, ringer-null mutants exhibit decreased organism viability. Lack of Ringer, as in the case of Tau, leads to reduced viability but not complete lethality (Mino, 2016).

These studies determined that Ringer, like mammalian TPPPs, is able to regulate microtubule dynamics. This is evidenced in vivo by microtubule disruption at segmental nerves in ringer mutants and supported by primary culture studies in which changes in Ringer translate into changes in acetylated tubulin. Ringer is likely to have a conserved stabilizing and bundling function similar to that of mammalian TPPPs. Cell culture experiments too suggest this, as they show Ringer can protect microtubules from depolymerization in addition to altering microtubule architecture, further underscoring a stabilizing function. Furthermore, purified Ringer data show that no other external factors are necessary to induce changes in microtubule dynamics. Thus, this work demonstrates that Ringer alone is sufficient to induce higher rates of microtubule polymerization as well as bundling and stabilization (Mino, 2016).

This work provides evidence that Ringer regulates microtubule changes necessary for axonal development. Ringer is expressed along the axon in primary neurons, and at cellular margins and membrane-ruffle areas in S2 cells, a location concomitant with process growth. Moreover, axon extension and growth cone advancement rely on microtubules. Consequently, in ringer mutants exhibiting axonal stalling and breaks, phenotypes might be representative of lower microtubule polymerization rates that result from lack of Ringer function. This is supported by evidence that Ringer is necessary endogenously for proper axonal extension, and by micrographs showing axonal microtubule disruption in ringer mutants. Surprisingly, both ringer-mutant and -overexpressing neurons exhibit delayed axonal extension. Although defects observed in Ringer overexpression in vivo could be explained by the contribution of Ringer from surrounding cells, overexpression in primary neurons and in vivo Eve-positive neurons also results in soma placement and axonal phenotypes, revealing that there is a cell autonomous Ringer function (Mino, 2016).

The similar phenotypes produced by Ringer loss and gain of function appear counterintuitive. However, in vitro data show that Ringer has the ability to promote microtubule polymerization and bundling. Studies have revealed that modest microtubule overstabilization leads to an overall decrease in dynamics. It is possible that Ringer overexpression stabilizes microtubules sufficiently to prevent axonal advancement, whereas in ringer mutants, axons delay advancement owing to lower tubulin polymerization. Additionally, Ringer loss might lead to depolymerization due to higher susceptibility to severing agents. Perhaps there are Ringer concentration thresholds, post-translational modifications or other factors that decide in favor of a specific function (Mino, 2016).

Conversely, the CNS axon mistargeting observed in ringer mutants might be an indirect result of delays in axonal extension. During embryogenesis, VNC midline neurons extend their axons as they migrate ventrally. In mutants, axons from misplaced neurons might not extend at a normal rate, causing them to miss cues resulting in guidance defects. Additionally, axon guidance defects are repeatedly accompanied by severe neuronal misplacement, suggesting these two phenotypes are linked to migration errors. Alternatively, guidance phenotypes might result from a function of Ringer in growth cone directional movement through differential microtubule stabilization. Thus, it is postulated that during axonal development, Ringer regulates microtubule stabilization that is necessary for correct spatial distribution and polymerization to direct growth (Mino, 2016).

Interestingly, none of the phenotypic rescue experiments yielded a full recovery. Besides FASII embryonic phenotypic rescue, other attempts proved modest at best. These differences are not due to changes in transgene expression but from diverging protein level requirements between systems. Moreover, FASII rescue measurements were performed relative to the integrity of the combined neuronal connections, whereas single-cell measurements were made unhindered by environmental cues. Another possibility is that Ringer is necessary in surrounding cells, such as lateral glia, and that the antibodies are not robust enough to detect Ringer expression in such cells. If this is the case, elav-GAL4-mediated rescue, which expresses Ringer in all neurons and lateral glia at early stages, would be the only driver able to rescue phenotypes. Although these observations do not discard the notion of a function for Ringer in other cells that could influence development, they support the idea of an endogenous cell autonomous Ringer function in neurons (Mino, 2016).

In summary, this work has demonstrated that Ringer contributes to development in the regulation of axonal extension. Ringer was shown to be sufficient to promote microtubule stabilization, bundling and polymerization and that its absence is likely to affect axonal microtubule dynamics, leading to extension delays, mistargeting and, consequently, abnormal neural development (Mino, 2016).


Functions of Ringer orthologs in other species

Further evidence for microtubule-independent dimerization of TPPP/p25

Tubulin Polymerization Promoting Protein (TPPP/p25) is a brain-specific disordered protein that modulates the dynamics and stability of the microtubule network by its assembly promoting, cross-linking and acetylation enhancing activities. In normal brain it is expressed primarily in differentiated oligodendrocytes; however, at pathological conditions it is enriched in inclusions of both neurons and oligodendrocytes characteristic for Parkinson's disease and multiple system atrophy, respectively. The objective of this paper is to highlight a critical point of a recently published paper (Skoufias, 2015) in which the crucial role of the microtubules in TPPP/p25 dimerization leading to microtubule bundling was suggested. However, previous and present data provide evidence for the microtubule-independent dimerization of TPPP/p25 and its stabilization by disulphide bridges. In addition, bimolecular fluorescence complementation experiments revealed the dimerization ability of both the full length and the terminal-free (CORE) TPPP/p25 forms, however, while TPPP/p25 aligned along the bundled microtubule network, the associated CORE segments distributed mostly homogeneously within the cytosol. This study has identified a molecular model from the possible ones suggested in the Skoufias's paper that could be responsible for stabilization of the microtubule network in the course of the oligodendrocyte differentiation, consequently in the constitution of the myelin sheath (Olah, 2017).

Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling

TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules (DeBonis, 2015).

Relocation of p25alpha/tubulin polymerization promoting protein from the nucleus to the perinuclear cytoplasm in the oligodendroglia of sporadic and COQ2 mutant multiple system atrophy

p25alpha/tubulin polymerization promoting protein (TPPP) is an oligodendroglial protein that plays crucial roles including myelination, and the stabilization of microtubules. In multiple system atrophy (MSA), TPPP is suggested to relocate from the myelin sheath to the oligodendroglial cell body, before the formation of glial cytoplasmic inclusions (GCIs), the pathologic hallmark of MSA. However, much is left unknown about the re-distribution of TPPP in MSA. This study generated new antibodies against the N- and C-terminus of TPPP, and analyzed control and MSA brains, including the brain of a familial MSA patient carrying homozygous mutations in the coenzyme Q2 gene (COQ2). In control brain tissues, TPPP was localized not only in the cytoplasmic component of the oligodendroglia including perinuclear cytoplasm and peripheral processes in the white matter, but also in the nucleus of a fraction (62.4%) of oligodendroglial cells. Immunoelectron microscopic analysis showed TPPP in the nucleus and mitochondrial membrane of normal oligodendroglia, while Western blot also supported its nuclear and mitochondrial existence. In MSA, the prevalence of nuclear TPPP was 48.6% in the oligodendroglia lacking GCIs, whereas it was further decreased to 19.6% in the oligodendroglia with phosphorylated alpha-synuclein (palpha-syn)-positive GCIs, both showing a significant decrease compared to controls (62.4%). In contrast, TPPP accumulated in the perinuclear cytoplasm where mitochondrial membrane (TOM20 and cytochrome C) and fission (DRP1) proteins were often immunoreactive. It is concluded that in MSA-oligodendroglia, TPPP is reduced, not only in the peripheral cytoplasm, but also in the nucleus and relocated to the perinuclear cytoplasm (Ota, 2014).

TPPP/p25 promotes tubulin acetylation by inhibiting histone deacetylase 6

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. This study demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of alpha-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of alpha-tubulin. These results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, this study demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 microM concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, it was demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, it is suggested that TPPP/p25 is a multiple effector of the microtubule organization (Tokesi, 2010).

Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation

TPPP/p25, a recently identified tubulin polymerization-promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. This study shows that TPPP/p25 is strongly upregulated during the differentiation of primary oligodendrocyte cells as well as the CG-4 cell line. The microRNA expression profile of CG-4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR-206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Overexpression of miR-206 led to downregulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination (Lehotzky, 2010).

P25alpha/Tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain

P25alpha/tubulin polymerization promoting protein (TPPP) is a brain specific phosphoprotein that displays microtubule bundling activity. In the mature brain, p25alpha/TPPP distributes to oligodendrocytes and choroid plexus epithelium. This study mapped the spatial and temporal distribution of p25alpha/TPPP in the developing rat brain. Having localized its expression to neuronal tissue by Western blot analyses, the distribution of p25alpha/TPPP to developing oligodendrocytes was confirmed using a specific antibody. In the pre-natal and post-natal brain, p25alpha/TPPP was localized to the perinuclear cytoplasm of myelinating oligodendrocytes from embryonic (E) day E20 as verified from cellular co-localization with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Oligodendrocyte progenitor cells and pre-myelinating oligodendrocytes identified by the expression of NG2 proteoglycan and CD9, respectively, both failed to contain p25alpha/TPPP. In contrast, P25alpha/TPPP co-localized with beta(IV)-tubulin from post-natal (p) day P10 suggesting that p25alpha/TPPP plays an important role for tubulin-related transport in developing, myelinating oligodendrocytes (Skjoerringe, 2006).

Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations

Previous work has demonstrated the presence of a protein factor [tubulin polymerization perturbing protein (TPPP)] in brain and neuroblastoma cell but not in muscle extract that uniquely influences the microtubule assembly. This study describes a procedure for isolation of this protein from the cytosolic fraction of bovine brain and presents evidence that this protein is a target of both tubulin and microtubules in vitro. The crucial step of the purification is the cationic exchange chromatography; the bound TPPP is eluted at high salt concentrations, indicating the basic character of the protein. By IDA-nanoLC-MS analysis of the peptides extracted from the gel-digested purified TPPP, this study shows the presence of a single protein in the purified fraction that corresponds to p25, a brain-specific protein the function of which has not been identified. Circular dichroism data have revealed that, on one hand, the alpha-helix content of p25 is very low (4%) with respect to the predicted values (30-43%), and its binding to tubulin induces remarkable alteration in the secondary structure of the protein(s). As shown by turbidimetry, pelleting experiments, and electron microscopy, p25 binds to paclitaxel-stabilized microtubules and bundles them. p25 induces formation of unusual (mainly double-walled) microtubules from tubulin in the absence of paclitaxel. The amount of aberrant tubules formed depends on the p25 concentration, and the process occurs at substoichiometric concentrations. These in vitro data suggest that p25 could act as a unique MAP in vivo (Hlavanda, 2002).


REFERENCES

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DeBonis, S., Neumann, E. and Skoufias, D. A. (2015). Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling. Sci Rep 5: 13242. PubMed ID: 26289831

Hlavanda, E., Kovacs, J., Olah, J., Orosz, F., Medzihradszky, K. F. and Ovadi, J. (2002). Brain-specific p25 protein binds to tubulin and microtubules and induces aberrant microtubule assemblies at substoichiometric concentrations. Biochemistry 41(27): 8657-8664. PubMed ID: 12093283

Lehotzky, A., Lau, P., Tokesi, N., Muja, N., Hudson, L. D. and Ovadi, J. (2010). Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation. Glia 58(2): 157-168. PubMed ID: 19606501

Li, D., Li, F., Guttipatti, P. and Song, Y. (2018). A Drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system. J Vis Exp(135). PubMed ID: 29781994

Mino, R. E., Rogers, S. L., Risinger, A. L., Rohena, C., Banerjee, S. and Bhat, M. A. (2016). Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development. J Cell Sci [Epub ahead of print]. PubMed ID: 27422099

Monahan Vargas, E. J., Matamoros, A. J., Qiu, J., Jan, C. H., Wang, Q., Gorczyca, D., Han, T. W., Weissman, J. S., Jan, Y. N., Banerjee, S. and Song, Y. (2020). The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration. Genes Dev 34(3-4): 194-208. PubMed ID: 31919191

Olah, J., Szenasi, T., Szunyogh, S., Szabo, A., Lehotzky, A. and Ovadi, J. (2017). Further evidence for microtubule-independent dimerization of TPPP/p25. Sci Rep 7: 40594. PubMed ID: 28074911

Ota, K., Obayashi, M., Ozaki, K., Ichinose, S., Kakita, A., Tada, M., Takahashi, H., Ando, N., Eishi, Y., Mizusawa, H. and Ishikawa, K. (2014). Relocation of p25alpha/tubulin polymerization promoting protein from the nucleus to the perinuclear cytoplasm in the oligodendroglia of sporadic and COQ2 mutant multiple system atrophy. Acta Neuropathol Commun 2: 136. PubMed ID: 25208467

Shi, Q., Lin, Y. Q., Saliba, A., Xie, J., Neely, G. G. and Banerjee, S. (2019). Tubulin polymerization promoting protein, Ringmaker, and MAP1B homolog Futsch coordinate microtubule organization and synaptic growth. Front Cell Neurosci 13: 192. PubMed ID: 31156389

Skjoerringe, T., Lundvig, D. M., Jensen, P. H. and Moos, T. (2006). P25alpha/Tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain. J Neurochem 99(1): 333-342. PubMed ID: 16879710

Song, Y., Sretavan, D., Salegio, E. A., Berg, J., Huang, X., Cheng, T., Xiong, X., Meltzer, S., Han, C., Nguyen, T. T., Bresnahan, J. C., Beattie, M. S., Jan, L. Y. and Jan, Y. N. (2015). Regulation of axon regeneration by the RNA repair and splicing pathway. Nat Neurosci 18(6): 817-825. PubMed ID: 25961792

Tokesi, N., Lehotzky, A., Horvath, I., Szabo, B., Olah, J., Lau, P. and Ovadi, J. (2010). TPPP/p25 promotes tubulin acetylation by inhibiting histone deacetylase 6. J Biol Chem 285(23): 17896-17906. PubMed ID: 20308065


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date revised: 15 February, 2020

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