Zn²+-induced rearrangement of the disordered TPPP/p25 affects its microtubule assembly and GTPase activity

Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn(2+) is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His(2)Cys(2)) (His(61)-Cys(83)) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions. The specific binding of the Zn(2+) to TPPP/p25 induced the formation of molten globule but not that of a well-defined tertiary structure. The Zn(2+)-induced partially folded structure accommodating the zinc binding motif is localized at the single Trp(76)-containing region as demonstrated by fluorescence resonance energy transfer and quenching experiments. We showed that the Zn(2+)-induced change in the TPPP/p25 structure modified its interaction with tubulin and GTP coupled with functional consequences: the TPPP/p25-promoted tubulin polymerization was increased while the TPPP/p25-catalyzed GTPase activity was decreased as detected by turbidimetry and by malachite green phosphate release/(31)P NMR assays, respectively. The finding that the Zn(2+) of the bivalent cations can uniquely influence physiological relavant interactions significantly contributes to our understanding of the role of Zn(2+)-related TPPP/p25 processes in the differentiation/myelination of oligodendrocytes possessing a high-affinity Zn(2+) uptake mechanism.     

An unstructured protein with destructive potential: TPPP/p25 in neurodegeneration

TPPP/p25 is a recently discovered, unstructured protein involved in brain function. It is found predominantly in oligodendrocytes in normal brain but is enriched in neuronal and glial inclusions of Parkinson's disease and other synucleinopathies. Its physiological function seems to be the dynamic stabilization of microtubular ultrastructures, as well as the projections of mature oligodendrocytes and ciliary structures. We reappraise the earlier belief that TPPP/p25 is a brain‐specific protein. We have identified and cloned two shorter (N‐terminal‐free) homologs of TPPP/p25 that behave differently from each other and from TPPP/p25. Two unique cell models have been established and used to study the effect of the unstructured protein on the energy metabolism and the formation of pathological aggregates. Our data suggest that the intracellular level of TPPP/p25 influences the cell differentiation, proliferation and the formation of protein aggregates, and consequently, the etiology of central nervous system diseases.     

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 α-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 α-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 μm 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.     

Disordered TPPP/p25 binds GTP and displays Mg2+-dependent GTPase activity

The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays a crucial role in differentiation of oligodendrocytes. Here we first demonstrated by multinuclear NMR that the extended disordered segments are localized at the N- and C-terminals straddling a flexible region. We showed by affinity chromatography, fluorescence spectroscopy and circular dichroism that GTP binds to TPPP/p25 likely within the flexible region; neither positions nor intensities of the peaks in the assigned terminals were affected by GTP. In addition, we demonstrated that TPPP/p25 specifically hydrolyses GTP in an Mg(2+)-dependent manner. The GTPase activity is comparable with the intrinsic activities of small G proteins suggesting its potential role in multiple physiological processes.     

Interaction of TPPP/p25 protein with glyceraldehyde-3-phosphate dehydrogenase and their co-localization in Lewy bodies

TPPP/p25, a flexible unstructured protein, binds to tubulin and induces aberrant microtubule assemblies. We identified hereby glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a new interacting partner of TPPP/p25. The immunoprecipitation and affinity chromatographic experiments with bovine brain cell-free extract revealed that the interaction was salt and NAD(+) sensitive while ELISA showed resistant and firm association of the two isolated proteins. In transfected HeLa cells at low expression level of EGFP-TPPP/p25, while the green fusion protein aligned at the microtubular network, GAPDH distributed uniformly in the cytosol. However, at high expression level, GAPDH co-localized with TPPP/p25 in the aggresome-like aggregate. Immunohistochemistry showed enrichment of TPPP/p25 and GAPDH within the alpha-synuclein positive Lewy body.     

A new myelin protein, TPPP/p25, reduced in demyelinated lesions is enriched in cerebrospinal fluid of multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease with variable extent of remyelination coupled with the differentiation of oligodendrocytes, in which Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) plays a crucial role. Previously we reported that the loss of TPPP/p25-positive oligodendrocytes in demyelinated lesions in the brain of MS patients could be a biomarker for MS [2]. In this work we tested the occurrence of TPPP/p25 in the cerebrospinal fluid (CSF) of MS patients, and by elaborating a sensitive assay for quantification of TPPP/p25 we showed that its level is significantly higher than in the case of non-MS patients. Patients with MS were diagnosed at the Department of Neurology, University of Szeged according to the clinical and laboratory diagnostic criteria of McDonald. In non-MS patients no significant pathological changes were detected on magnetic resonance imaging scans, while in MS patients multiple hyperintense T2 lesions in the white matter were detected. Kurtzke Expanded Disability Status Scale scores as well as IgG level and oligoclonal bands of MS patients were demonstrated. The sensitive assay elaborated in this study is based upon Western blot followed by chemiluminescent detection validated by human recombinant protein. The median TPPP/p25 contents in the CSF were 62.8 and 64.7 μg/L for patients with clinically isolated syndromes and relapsing remitting MS, respectively, while this value for non-MS patients was 27.9 μg/L. The enrichment of TPPP/p25 was independent of age, gender and the time period between lumbar puncture and relapse/shub. These data suggest that the TPPP/p25-based assay could be a powerful diagnostic test for MS patients.     

Role of the microtubule-associated TPPP/p25 in Parkinson’s and related diseases and its therapeutic potential

Introduction: The discovery and development of therapeutic strategies for the treatments of Parkinson’s disease (PD) and other synucleinopathies are limited by a lack of understanding of the pathomechanisms and their connection with different diseases such as cancers.

Areas covered: The hallmarks of these diseases are frequently multifunctional disordered proteins displaying moonlighting and/or chameleon features, which are challenging drug targets. A representative of these proteins is the disordered Tubulin Polymerization Promoting Protein (TPPP/p25) expressed specifically in oligodendrocytes (OLGs) in normal brain. Its non-physiological level is tightly related to the etiology of PD and Multiple System Atrophy (TPPP/p25 enrichment in inclusions of neurons and OLGs, respectively), multiple sclerosis (TPPP/p25-positive OLG destruction), as well as glioma (loss of TPPP/p25 expression). The established anti-proliferative potency of TPPP/p25 may raise its influence in cancer development. The recognition that whereas too much TPPP/p25 could kill neurons in PD, but its loss keeps cells alive in cancer could contribute to our understanding of the interrelationship of ‘TPPP/p25 diseases’.

Expert commentary: The knowledge accumulated so far underlines the key roles of the multifunctional TPPP/p25 in both physiological and diverse pathological processes, consequently its validation as drug target sorely needs a new innovative strategy that is briefly reviewed here.     

TPPP/p25: from unfolded protein to misfolding disease: prediction and experiments

TPPP/p25, the first representative of a new protein family, identified as a brain-specific unfolded protein induces aberrant microtubule assemblies in vitro, suppresses mitosis in Drosophila embryo and is accumulated in inclusion bodies of human pathological brain tissues. In this paper, we present prediction and additional experimental data that validate TPPP/p25 to be a new member of the "intrinsically unstructured" protein family. The comparison of these characteristics with that of alpha-synuclein and tau, involved also in neurodegenerative diseases, suggested that although the primary sequences of these proteins are entirely different, there are similarities in their well-defined unstructured segments interrupted by "stabilization centres", phosphorylation and tubulin binding motives. SK-N-MC neuroblastoma cells were transfected with pEGFP-TPPP/p25 construct and a stable clone denoted K4 was selected and used to establish the effect of this unstructured protein on the energy state/metabolism of the cells. Our data by analyzing the mitochondrial membrane polarization by fluorescence microscopy revealed that the high-energy phosphate production in K4 clone is not damaged by the TPPP/p25 expression. Biochemical analysis with cell homogenates provided quantitative data that the ATP level increased 1.5-fold and the activities of hexokinase, glucosephosphate isomerase, phosphofructokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were 1.2 to 2.0-fold higher in K4 as compared to the control. Our modelling using these data and rate equations of the individual enzymes suggests that the TPPP/p25 expression stimulates glucose metabolism. At pathological conditions TPPP/p25 is localized in inclusion bodies in multiple system atrophy, it tightly co-localizes with alpha-synuclein, partially with tubulin and not with vimentin. The previous and the present studies obtained with immunohistochemistry with pathological human brain tissues rendered it possible to classify among pathological inclusions on the basis of immunolabelling of TPPP/p25, and suggest this protein to be a potential linkage between Parkinson's and Alzheimer's diseases.     

A New Protein Superfamily: TPPP-Like Proteins

The introduction of the term ‘Tubulin Polymerization Promoting Protein (TPPP)-like proteins’ is suggested. They constitute a eukaryotic protein superfamily, characterized by the presence of the p25alpha domain (Pfam05517, IPR008907), and named after the first identified member, TPPP/p25, exhibiting microtubule stabilizing function. TPPP-like proteins can be grouped on the basis of two characteristics: the length of their p25alpha domain, which can be long, short, truncated or partial, and the presence or absence of additional domain(s). TPPPs, in the strict sense, contain no other domains but one long or short p25alpha one (long- and short-type TPPPs, respectively). Proteins possessing truncated p25alpha domain are first described in this paper. They evolved from the long-type TPPPs and can be considered as arthropod-specific paralogs of long-type TPPPs. Phylogenetic analysis shows that the two groups (long-type and truncated TPPPs) split in the common ancestor of arthropods. Incomplete p25alpha domains can be found in multidomain TPPP-like proteins as well. The various subfamilies occur with a characteristic phyletic distribution: e. g., animal genomes/proteomes contain almost without exception long-type TPPPs; the multidomain apicortins occur almost exclusively in apicomplexan parasites. There are no data about the physiological function of these proteins except two human long-type TPPP paralogs which are involved in developmental processes of the brain and the musculoskeletal system, respectively. I predict that the superfamily members containing long or partial p25alpha domain are often intrinsically disordered proteins, while those with short or truncated domain(s) are structurally ordered. Interestingly, members of this superfamily connected or maybe connected to diseases are intrinsically disordered proteins.     

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.     

On the TPPP-like proteins of flagellated fungi

TPPP-like proteins, exhibiting microtubule stabilizing function, constitute a eukaryotic superfamily, characterized by the presence of the p25alpha domain. TPPPs in the strict sense are present in animals except Trichoplax adhaerens, which instead contains apicortin where a part of the p25alpha domain is combined with a DCX domain. Apicortin is absent in other animals and occurs mostly in the protozoan phylum, Apicomplexa. A strong correlation between the occurrence of p25alpha domain and that of the eukaryotic cilium/flagellum was suggested. Species of the deeper branching clades of Fungi possess flagellum but others lost it thus investigation of fungal genomes can help testing of this suggestion. Indeed, these proteins are present in early branching Fungi. Both TPPP and apicortin are present in Rozellomycota (Cryptomycota) and Chytridiomycota, TPPP in Blastocladiomycota, apicortin in Neocallimastigomycota, Monoblepharomycota and the non-flagellated Mucoromycota. Beside the “normal” TPPP occurring in animals, a special, fungal-type TPPP is also present in Fungi, in which a part of the p25alpha domain is duplicated. Dikarya, the most developed subkingdom of Fungi, lacks both flagellum and TPPPs. Thus it is strengthened that each ciliated/flagellated organism contains p25alpha domain-containing proteins while there are very few non-flagellated ones where p25alpha domain can be found.     

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.     

TPPP orthologs are ciliary proteins

Tubulin polymerization promoting protein, (TPPP/p25), was identified as a brain-specific protein. The potential function of this protein resembled that of MAPs. It is mainly expressed in oligodendrocytes; however, immunopositivity was also detected in glial and neuronal inclusions in synucleinopathies. Here, we show that TPPP gene(s) are conserved in the genomes of ciliated organisms, but are lacking from the nonciliated ones. This recognition is based upon homologous gene sequence analysis, in silico comparative genomic studies, bioinformatic search and experimental evidence. Cilia (flagella) are microtubule-based cellular extensions of sensory and/or motile function. TPPP orthologs are among the only 16 genes that can be found in all ciliated organisms, suggesting that TPPP orthologs may be associated with a basic function of cilia.     

The relationship between developing oligodendrocyte units and maturing axons during myelinogenesis in the anterior medullary velum of neonatal rats

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between ～0.2 and 0.4 μm, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.     

Metabotropic Glutamate Receptors Protect Oligodendrocytes from Acute Ischemia in the Mouse Optic Nerve

Studies by Bruce Ransom and colleagues have made a major contribution to show that white matter is susceptible to ischemia/hypoxia. White matter contains axons and the glia that support them, notably myelinating oligodendrocytes, which are highly vulnerable to ischemic-hypoxic damage. Previous studies have shown that metabotropic GluRs (mGluRs) are cytoprotective for oligodendrocyte precursor cells and immature oligodendrocytes, but their potential role in adult white matter was unresolved. Here, we report that group 1 mGluR1/5 and group 2 mGluR3 subunits are expressed in optic nerves from mice aged postnatal day (P)8–12 and P30–35. We demonstrate that activation of group 1 mGluR protects oligodendrocytes against oxygen-glucose deprivation (OGD) in developing and young adult optic nerves. In contrast, group 2 mGluR are shown to be protective for oligodendrocytes against OGD in postnatal but not young adult optic nerves. The cytoprotective effect of group 1 mGluR requires activation of PKC, whilst group 2 mGluR are dependent on negatively regulating adenylyl cyclase and cAMP. Our results identify a role for mGluR in limiting injury of oligodendrocytes in developing and young adult white matter, which may be useful for protecting oligodendrocytes in neuropathologies involving excitoxicity and ischemia/hypoxia.     

The proteolipid protein promoter drives expression outside of the oligodendrocyte lineage during embryonic and early postnatal development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin--PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26(LacZ) and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreER(t) line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology.     

Developmental expression of the myelin gene MOBP in the rat nervous system

The myelin-associated/oligodendrocyte basic proteins (MOBPs) are recently discovered constituents of myelin and are small, cytoplasmic, and highly basic proteins exclusively expressed postnatally by oligodendrocytes. Due to a clustering of positively charged amino acids observed in the most abundant MOBP isoform similar to myelin basic protein (MBP) and P0, it was speculated that MOBP could function in myelin sheath compaction. The present report strongly supports this view. A direct comparison of MBP and proteolipid protein (PLP) gene expression with that of MOBP by in situ hybridization revealed a very similar regional distribution. It was found that MOBP expression was abundant in the rat CNS at postnatal day 15 (P 15) but is restricted to densely myelinated regions. In contrast to MBP and PLP, expression of MOBP was undetectable in the peripheral nervous system during the entire development. Interestingly, MOBP mRNA was localized in oligodendrocyte processes even at early postnatal stages and throughout development. MOBP showed a very specific timing of expression: in spinal cord and brain, MOBP gene expression occurred significantly later (2–3 days) than that of MBP and PLP, but slightly earlier than myelin oligodendrocyte glycoprotein gene expression. MOBP proteins appeared in spinal cord and brain stem also after MBP protein, suggesting that the MOBPs functionally act after the structural myelin proteins MBP and PLP. Our findings imply a function of MOBP during the late steps of myelin formation, presumably at the initiation of sheath compaction.     

Myelination of prospective large fibres in chicken ventral funiculus

In mammals, the oligodendrocyte population includes morphological and molecular varieties. We reported previously that an antiserum against the T4-O molecule labels a subgroup of oligodendrocytes related to large myelinated axons in adult chicken white matter. We also reported that T4-O immunoreactive cells first appear in the developing ventral funiculus (VF) at embryonic day (E)15, subsequently increasing rapidly in number. Relevant fine structural data for comparison are not available in the literature. This prompted the present morphological analysis of developing and mature VF white matter in the chicken. The first axon-oligodendrocyte connections were seen at E10 and formation of compact myelin had started at E12. Between E12 and E15 the first myelinating oligodendrocytes attained a Schwann cell-like morphology. At hatching (E21) 60% of all VF axons were myelinated and in the adult this proportion had increased to 85%. The semilunar or polygonal oligodendrocytes associated with adult myelinated axons contained many organelles indicating a vivid metabolic activity. Domeshaped outbulgings with gap junction-like connections to astrocytic profiles were frequent. Oligodendrocytes surrounded by large myelinated axons and those surrounded by small myelinated axons were cytologically similar. But, thick and thin myelin sheaths had dissimilar periodicities and Marchi-positive myelinoid bodies occurred preferentially in relation to large myelinated axons. We conclude that early oligodendrocytes contact axons and form myelin well before the first expression of T4-O and that emergence of a T4-O immunoreactivity coincides in time with development of a Type IV phenotype. Our data also show that oligodendrocytes associated with thick axons are cytologically similar to cells related to thin axons. In addition, the development of chicken VF white matter was found to be similar to the development of mammalian white matter, except for the rapid time course.     

The distribution of heat shock proteins in the nervous system of the unstressed mouse embryo suggests a role in neuronal and non-neuronal differentiation

Heat shock proteins (Hsps) act as molecular chaperones and are generally constitutively expressed in the absence of stress. Hsps are also inducible by a variety of stressors whose effects could be disastrous on the brain. It has been shown previously that Hsps are differentially expressed in glial and neuronal cells, as well as in the different structures of the brain. This differential expression has been related to specific functions distinct from their general chaperone function, such as intracellular transport. We investigated here the constitutive expression of 5 Hsps (the small Hsp, Hsp25, the constitutive Hsc70 and Hsp90beta, the mainly inducible Hsp70 and Hsp90alpha), and of a molecular chaperone, TCP-1alpha during mouse nervous system development. We analyzed, by immunohistochemistry, their distribution in the central nervous system and in the ganglia of the peripheral nervous system from day 9.5 (E9.5) to day 17.5 (E17.5) of gestation. Hsps are expressed in different cell classes (neuronal, glial, and vascular). The different proteins display different but often overlapping patterns of expression in different regions of the developing nervous system, suggesting unique roles at different stages of neural maturation. Their putative function in cell remodeling during migration or differentiation and in protein transport is discussed. Moreover we consider Hsp90 function in cell signaling and the role of Hsp25 in apoptosis protection.