Perspective Strategies for Interventions in Parkinsonism: Remedying the Neglected Role of TPPP.

Neurological disorders such as Parkinsonism cause serious socio-economic problems as there are, at present, only therapies that treat their symptoms. The well-established hallmark alpha-synuclein (SYN) is enriched in the inclusion bodies characteristic of Parkinsonism. We discovered a prominent partner of SYN, termed Tubulin Polymerization Promoting Protein (TPPP), which has important physiological and pathological activities such as the regulation of the microtubule network and the promotion of SYN aggregation. The role of TPPP in Parkinsonism is often neglected in research, which we here attempt to remedy. In the normal brain, SYN and TPPP are expressed endogenously in neurons and oligodendrocytes, respectively, whilst, at an early stage of Parkinsonism, soluble hetero-associations of these proteins are found in both cell types. The cell-to-cell transmission of these proteins, which is central to disease progression, provides a unique situation for specific drug targeting. Different strategies for intervention and for the discovery of biomarkers include (i) interface targeting of the SYN-TPPP hetero-complex; (ii) proteolytic degradation of SYN and/or TPPP using the PROTAC technology; and (iii) depletion of the proteins by miRNA technology. We also discuss the potential roles of SYN and TPPP in the phenotype stabilization of neurons and oligodendrocytes.

Modulatory Role of TPPP3 in Microtubule Organization and Its Impact on Alpha-Synuclein Pathology.

Parkinson's disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting Protein (TPPP/p25 or TPPP1) is also implicated in these processes. The moonlighting and chameleon TPPP1 modulates the dynamics/stability of the multifunctional microtubule network by promoting its acetylation and bundling. Previously, we identified the microtubule-associated TPPP3, a homologue of TPPP1 lacking its N-terminus; however, its involvement in physiological or pathological processes was not elucidated. In this work, we have shown the modulatory role of TPPP3, similarly to TPPP1, in microtubule organization, as well as its homo- and hetero-associations with TPPP1. TPPP3, in contrast to TPPP1, virtually does not bind to SYN; consequently, it does not promote SYN aggregation. Its anti-aggregative potency is achieved by counteracting the formation of the TPPP1-SYN pathological complex/aggregation leading to Parkinsonism. The interactions of TPPP3 have been determined and quantified in vitro with recombinant human proteins, cell extracts and in living human cells using different methods including bifunctional fluorescence complementation. The tight association of TPPP3 with TPPP1, but not with SYN, may ensure a unique mechanism for its inhibitory effect. TPPP3 or its selected fragments may become a leading agent for developing anti-Parkinson agents.

Challenges in Discovering Drugs That Target the Protein-Protein Interactions of Disordered Proteins.

Protein-protein interactions (PPIs) outnumber proteins and are crucial to many fundamental processes; in consequence, PPIs are associated with several pathological conditions including neurodegeneration and modulating them by drugs constitutes a potentially major class of therapy. Classically, however, the discovery of small molecules for use as drugs entails targeting individual proteins rather than targeting PPIs. This is largely because discovering small molecules to modulate PPIs has been seen as extremely challenging. Here, we review the difficulties and limitations of strategies to discover drugs that target PPIs directly or indirectly, taking as examples the disordered proteins involved in neurodegenerative diseases.

Anti-Aggregative Effect of the Antioxidant DJ-1 on the TPPP/p25-Derived Pathological Associations of Alpha-Synuclein.

DJ-1, a multi-functional protein with antioxidant properties, protects dopaminergic neurons against Parkinson's disease (PD). The oligomerization/assembly of alpha-synuclein (SYN), promoted by Tubulin Polymerization Promoting Protein (TPPP/p25), is fatal in the early stage of PD. The pathological assembly of SYN with TPPP/p25 inhibits their proteolytic degradation. In this work, we identified DJ-1 as a new interactive partner of TPPP/p25, and revealed its influence on the association of TPPP/p25 with SYN. DJ-1 did not affect the TPPP/p25-derived tubulin polymerization; however, it did impede the toxic assembly of TPPP/p25 with SYN. The interaction of DJ-1 with TPPP/p25 was visualized in living human cells by fluorescence confocal microscopy coupled with Bifunctional Fluorescence Complementation (BiFC). While the transfected DJ-1 displayed homogeneous intracellular distribution, the TPPP/p25-DJ-1 complex was aligned along the microtubule network. The anti-aggregative effect of DJ-1 on the pathological TPPP/p25-SYN assemblies was established by the decrease in the intensity of their intracellular fluorescence (BiFC signal) and the increase in the proteolytic degradation of SYN complexed with TPPP/p25 due to the DJ-1-derived disassembly of SYN with TPPP/p25. These data obtained with HeLa and SH-SY5Y cells revealed the protective effect of DJ-1 against toxic SYN assemblies, which assigns a new function to the antioxidant sensor DJ-1.

Co-Transmission of Alpha-Synuclein and TPPP/p25 Inhibits Their Proteolytic Degradation in Human Cell Models.

The pathological association of alpha-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) is a key factor in the etiology of synucleinopathies. In normal brains, the intrinsically disordered SYN and TPPP/p25 are not found together but exist separately in neurons and oligodendrocytes, respectively; in pathological states, however, they are found in both cell types due to their cell-to-cell transmission. The autophagy degradation of the accumulated/assembled SYN has been considered as a potential therapeutic target. We have shown that the hetero-association of SYN with TPPP/p25 after their uptake from the medium by human cells (which mimics cell-to-cell transmission) inhibits both their autophagy- and the ubiquitin-proteasome system-derived elimination. These results were obtained by ELISA, Western blot, FACS and immunofluorescence confocal microscopy using human recombinant proteins and living human cells; ANOVA statistical analysis confirmed that TPPP/p25 counteracts SYN degradation by hindering the autophagy maturation at the stage of LC3B-SQSTM1/p62-derived autophagosome formation and its fusion with lysosome. Recently, fragments of TPPP/p25 that bind to the interface between the two hallmark proteins have been shown to inhibit their pathological assembly. In this work, we show that the proteolytic degradation of SYN on its own is more effective than when it is complexed with TPPP/p25. The combined strategy of TPPP/p25 fragments and proteolysis may ensure prevention and/or elimination of pathological SYN assemblies.

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)*.

We have discovered the sirtuin-rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+ -dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis-targeting chimera (PROTAC) enabled small-molecule-induced degradation of Sirt2. Herein, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small-molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

Microtubule-Associated Proteins with Regulatory Functions by Day and Pathological Potency at Night.

The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeleton, including the microtubule network. Microtubules play crucial roles achieved by their decoration with proteins/enzymes as well as by posttranslational modifications. This review focuses on the Tubulin Polymerization Promoting Protein (TPPP/p25), a new microtubule associated protein, on its "regulatory functions by day and pathological functions at night". Physiologically, the moonlighting TPPP/p25 modulates the dynamics and stability of the microtubule network by bundling microtubules and enhancing the tubulin acetylation due to the inhibition of tubulin deacetylases. The optimal endogenous TPPP/p25 level is crucial for its physiological functions, to the differentiation of oligodendrocytes, which are the major constituents of the myelin sheath. Pathologically, TPPP/p25 forms toxic oligomers/aggregates with α-synuclein in neurons and oligodendrocytes in Parkinson's disease and Multiple System Atrophy, respectively; and their complex is a potential therapeutic drug target. TPPP/p25-derived microtubule hyperacetylation counteracts uncontrolled cell division. All these issues reveal the anti-mitotic and α-synuclein aggregation-promoting potency of TPPP/p25, consistent with the finding that Parkinson's disease patients have reduced risk for certain cancers.

Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8.

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.

New chemical tools for probing activity and inhibition of the NAD+-dependent lysine deacylase sirtuin 2.

Sirtuins are NAD+-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Chemically Induced Degradation of Sirtuin 2 (Sirt2) by a Proteolysis Targeting Chimera (PROTAC) Based on Sirtuin Rearranging Ligands (SirReals).

Here we report the development of a proteolysis targeting chimera (PROTAC) based on the combination of the unique features of the sirtuin rearranging ligands (SirReals) as highly potent and isotype-selective Sirt2 inhibitors with thalidomide, a bona fide cereblon ligand. For the first time, we report the formation of a PROTAC by Cu(I)-catalyzed cycloaddition of a thalidomide-derived azide to an alkynylated inhibitor. This thalidomide-derived azide as well as the highly versatile linking strategy can be readily adapted to alkynylated ligands of other targets. In HeLa cells, our SirReal-based PROTAC induced isotype-selective Sirt2 degradation that results in the hyperacetylation of the microtubule network coupled with enhanced process elongation. Thus, our SirReal-based PROTAC is the first example of a probe that is able to chemically induce the degradation of an epigenetic eraser protein.

Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency.

The microtubule network exerts multifarious functions controlled by its decoration with various proteins and post-translational modifications. The disordered microtubule associated Tubulin Polymerization Promoting Protein (TPPP/p25) and the NAD+-dependent tubulin deacetylase sirtuin-2 (SIRT2) play key roles in oligodendrocyte differentiation by acting as dominant factors in the organization of myelin proteome. Herein, we show that SIRT2 impedes the TPPP/p25-promoted microtubule assembly independently of NAD+; however, the TPPP/p25-assembled tubulin ultrastructures were resistant against SIRT2 activity. TPPP/p25 counteracts the SIRT2-derived tubulin deacetylation producing enhanced microtubule acetylation. The inhibition of the SIRT2 deacetylase activity by TPPP/p25 is evolved by the assembly of these tubulin binding proteins into a ternary complex, the concentration-dependent formation of which was quantified by experimental-based mathematical modelling. Co-localization of the SIRT2-TPPP/p25 complex on the microtubule network was visualized in HeLa cells by immunofluorescence microscopy using Bimolecular Fluorescence Complementation. We also revealed that a new potent SIRT2 inhibitor (MZ242) and its proteolysis targeting chimera (SH1) acting together with TPPP/p25 provoke microtubule hyperacetylation, which is coupled with process elongation only in the case of the degrader SH1. Both the structural and the functional effects manifesting themselves by this deacetylase proteome could lead to the fine-tuning of the regulation of microtubule dynamics and stability.

Challenging drug target for Parkinson's disease: Pathological complex of the chameleon TPPP/p25 and alpha-synuclein proteins.

The hallmarks of Parkinson's disease and other synucleinopathies, Tubulin Polymerization Promoting Protein (TPPP/p25) and α-synuclein (SYN) have two key features: they are disordered and co-enriched/co-localized in brain inclusions. These Neomorphic Moonlighting Proteins display both physiological and pathological functions due to their interactions with distinct partners. To achieve the selective targeting of the pathological TPPP/p25-SYN but not the physiological TPPP/p25-tubulin complex, their interfaces were identified as a specific innovative strategy for the development of anti-Parkinson drugs. Therefore, the interactions of TPPP/p25 with tubulin and SYN were characterized which suggested the involvements of the 178-187 aa and 147-156 aa segments in the complexation of TPPP/p25 with tubulin and SYN, respectively. However, various truncated and deletion mutants reduced but did not abolish the interactions except one mutant; in addition synthetized fragments corresponding to the potential binding segments of TPPP/p25 failed to interact with SYN. In fact, the studies of the multiple interactions at molecular and cellular levels revealed the high conformational plasticity, chameleon feature, of TPPP/p25 that ensures exceptional functional resilience; the lack of previously identified binding segments could be replaced by other segments. The experimental results are underlined by distinct bioinformatics tools. All these data revealed that although targeting chameleon proteins is a challenging task, nevertheless, the validation of a drug target can be achieved by identifying the interface of complexes of the partner proteins existing at the given pathological conditions.

Structure-Based Development of an Affinity Probe for Sirtuin†2.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2-selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment-based screening, and affinity pull-down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.

Cross metathesis with hydroxamate and benzamide BOC-protected alkenes to access HDAC inhibitors and their biological evaluation highlighted intrinsic activity of BOC-protected dihydroxamates.

Conditions for the metathesis of alkenes in the convergent synthesis of HDAC inhibitors have been improved by continuous catalyst flow injection in the reaction media. Intermediate and target compounds obtained were tested for their ability to induce HDAC inhibition and tubulin acetylation, revealing the key role of the tert-butyloxycarbonyl (BOC) group for more HDAC6 selectivity. Molecular modelling added rationale for this BOC effect.

Aminothiazoles as Potent and Selective Sirt2 Inhibitors: A Structure-Activity Relationship Study.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

Zinc-induced structural changes of the disordered tppp/p25 inhibits its degradation by the proteasome.

Tubulin Polymerization Promoting Protein/p25 (TPPP/p25), a neomorphic moonlighting protein displaying both physiological and pathological functions, plays a crucial role in the differentiation of the zinc-rich oligodendrocytes, the major constituent of myelin sheath; and it is enriched and co-localizes with α-synuclein in brain inclusions hallmarking Parkinson's disease and other synucleinopathies. In this work we showed that the binding of Zn(2+) to TPPP/p25 promotes its dimerization resulting in increased tubulin polymerization promoting activity. We also demonstrated that the Zn(2+) increases the intracellular TPPP/p25 level resulting in a more decorated microtubule network in CHO10 and CG-4 cells expressing TPPP/p25 ectopically and endogenously, respectively. This stabilization effect is crucial for the differentiation and aggresome formation under physiological and pathological conditions, respectively. The Zn(2+)-mediated effect was similar to that produced by treatment of the cells with MG132, a proteasome inhibitor or Zn(2+) plus MG132 as quantified by cellular ELISA. The enhancing effect of zinc ion on the level of TPPP/p25 was independent of the expression level of the protein produced by doxycycline induction at different levels or inhibition of the protein synthesis by cycloheximide. Thus, we suggest that the zinc as a specific divalent cation could be involved in the fine-tuning of the physiological TPPP/p25 level counteracting both the enrichment and the lack of this protein leading to distinct central nervous system diseases.

Identification of motives mediating alternative functions of the neomorphic moonlighting TPPP/p25.

The disordered Tubulin Polymerization Promoting Protein (TPPP/p25), a prototype of neomorphic moonlighting proteins, displays physiological and pathological functions by interacting with distinct partners. Here the role of the disordered N- and C-termini straddling a middle flexible segment in the distinct functions of TPPP/p25 was established, and the binding motives responsible for its heteroassociations with tubulin and α-synuclein, its physiological and pathological interacting partner, respectively, were identified. We showed that the truncation of the disordered termini altered the folding state of the middle segment and has functional consequences concerning its physiological function. Double truncation diminished its binding to tubulin/microtubules, consequently the tubulin polymerization/microtubule bundling activities of TPPP/p25 were lost highlighting the role of the disordered termini in its physiological function. In contrast, interaction of TPPP/p25 with α-synuclein was not affected by the truncations and its α-synuclein aggregation promoting activity was preserved, showing that the α-synuclein binding motif is localized within the middle segment. The distinct tubulin and α-synuclein binding motives of TPPP/p25 were also demonstrated at the cellular level: the double truncated TPPP/p25 did not align along the microtubules in contrast to the full length form, while it induced α-synuclein aggregation. The localization of the binding motives on TPPP/p25 were established by specific ELISA experiments performed with designed and synthesized peptides: motives at the 178-187 and 147-156 segments are involved in the binding of tubulin and α-synuclein, respectively. The dissimilarity of these binding motives responsible for the neomorphic moonlighting feature of TPPP/p25 has significant innovative impact in anti-Parkinson drug research.

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. Here we 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. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we 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, we 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, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.

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. Here, we show 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.