Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.     

Developmentally regulated expression of specific tau sequences

Tau protein undergoes a shift in its molecular mass and its electrophoretic complexity during early postnatal development. We have sequenced a tau cDNA from an adult rat brain expression library and have found two inserted sequences. One of these inserts predicts a fourth repeated sequence homologous to the other three in the carboxyl end of tau that have the property of microtubule binding. Oligonucleotide probes directed against the insert hybridized only to tau mRNA at postnatal time points, even though tau is first expressed as early as embryonic day 13. A probe directed against the junction revealed expression of non-insert-containing tau mRNA from embryonic day 14 until postnatal day 8, after which time there was an abrupt decline in the expression of this immature form. Comparison of the developmentally expressed tau sequences with those sequences obtained directly from Alzheimer paired helical filaments revealed the presence of both the mature and the immature tau mRNA sequences.     

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.     

Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies.

Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer's disease paired helical filaments. Previously, we reported that ABalphaC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABalphaC binds a domain on tau that is indistinguishable from its MT-binding domain. ABalphaC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABalphaC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABalphaC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer's disease.     

Identification, expression, and localization of β keratin gene products during development of avian scutate scales

Epidermal-dermal interactions influence morphogenesis and expression of the beta keratin gene family during development of scales in the embryonic chick. The underlying mechanisms by which these interactions control beta keratin expression are not understood. However, the present study of beta keratin gene expression during avian epidermal differentiation contributes new information with which to investigate the role of tissue interactions in this process. Using beta keratin-specific synthetic oligonucleotide probe, beta keratin mRNA was hybrid-selected from total poly A+ RNA of scutate scales. Seven beta keratin polypeptides were translated in vitro and could be identified by their positions in two-dimensional gels among the detergent-insoluble extracts of scutate scale epidermis. In vivo phosphorylation studies suggested that an additional three beta keratin polypeptides were present as phosphoproteins. The temporal appearance of beta keratin mRNA and the corresponding polypeptides was followed during scutate scale development. Polyclonal antiserum made against two of the beta keratin polypeptides was used for immunohistochemical and immunogold electron-microscopic analysis of beta keratin tissue distribution. Immunological reactivity was observed specifically along the outer scale surface in epidermal cells above the stratum germinativum. Immunogold beads were localized on 3-nm filament bundles. In situ hybridization with a beta keratin-specific RNA probe demonstrated that mRNA accumulated in the same regional manner as the polypeptides. This selective expression of beta keratin genes in specific regions of the developing scutate scale suggests that epidermal-dermal interactions provide not only for morphological events, but also for control of complex patterns of histogenesis and biochemical differentiation.     

Cell-free Synthesis of the Myelin Basic Proteins in a Wheat Germ System Programmed with Brain Messenger RNA

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3-week-old mice and translated in a cell-free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mM) and low mRNA concentration (1-10 microgram/ml). The translational system was dependent on an energy-generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co-chromatographed and co-electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.     

Copine1 regulates neural stem cell functions during brain development

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development.     

Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo

Truncated tau protein is the characteristic feature of human sporadic Alzheimer's disease. We have identified truncated tau proteins conformationally different from normal healthy tau. Subpopulations of these structurally different tau species promoted abnormal microtubule assembly in vitro suggesting toxic gain of function. To validate pathological activity in vivo we expressed active form of human truncated tau protein as transgene, in the rat brain. Its neuronal expression led to the development of the neurofibrillary degeneration of Alzheimer's type. Furthermore, biochemical analysis of neurofibrillary changes revealed that massive sarcosyl insoluble tau complexes consisted of human Alzheimer's tau and endogenous rat tau in ratio 1:1 including characteristic Alzheimer's disease (AD)-specific proteins (A68). This work represents first insight into the possible causative role of truncated tau in AD neurofibrillary degeneration in vivo.     

Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.     

Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer's disease

Hyperphosphorylated tau, which is the major protein of the neurofibrillary tangles in Alzheimer's disease brain, is most probably the result of an imbalance of tau kinase and phosphatase activities in the affected neurons. By using metabolically competent rat brain slices as a model, we found that selective inhibition of protein phosphatase 2A by okadaic acid induced an Alzheimer-like hyperphosphorylation and accumulation of tau. The hyperphosphorylated tau had a reduced ability to bind to microtubules and to promote microtubule assembly in vitro. Immunocytochemical staining revealed hyperphosphorylated tau accumulation in pyramidal neurons in cornu ammonis and in neocortical neurons. The topography of these changes recalls the distribution of neurofibrillary tangles in Alzheimer's disease brain. Selective inhibition of protein phosphatase 2B with cyclosporin A did not have any significant effect on tau phosphorylation, accumulation, or function. These studies suggest that protein phosphatase 2A participates in regulation of tau phosphorylation, processing, and function in vivo. A down-regulation of protein phosphatase 2A activity can lead to Alzheimer-like abnormal hyperphosphorylation of tau.     

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

A novel, brain-specific mouse drebrin: cDNA cloning, chromosomal mapping, genomic structure, expression, and functional characterization

Drebrin A, a major neuronal actin-binding protein, regulates the dendritic spine shapes of neurons. Here, we have cloned and characterized a novel mouse cDNA clone encoding a truncated form of drebrin A, named s-drebrin A. Analysis of the genomic organization of the mouse drebrin gene (Dbn1), which mapped to the central portion of chromosome 13, revealed that isoforms including s-drebrin A are generated by alternative splicing from a single drebrin gene. The s-drebrin A mRNA was expressed in the brain, but not in non-neuronal tissues. The s-drebrin A expression was barely detected in the embryonic brain, but was upregulated during postnatal development of the brain. Overexpression of GFP-tagged s-drebrin A in fibroblasts showed it to be associated with actin filaments and with changes in actin cytoskeleton organization. These findings suggest that s-drebrin A has a role in spine morphogenesis, possibly by competing the actin-binding activity with drebrin A.     

MAP 4: occurrence in mouse tissues

A polyclonal antiserum to a microtubule-associated protein (MAP) from mouse neuroblastoma cells (MAP 4) was used to examine the distribution of this protein in mouse tissues. Immunoblots of neuroblastoma cell microtubule protein preparations demonstrated that the antiserum reacted with a triplet of proteins at 215,000-240,000 mol wt. Antibodies affinity purified from any of the bands showed cross- reaction with the other bands, indicating these polypeptides were all immunologically related. Antibodies specific to MAP 4 decorated microtubules in cultured murine cells fixed with glutaraldehyde, and diffuse staining was seen following treatment of cells with nocodazole. The antiserum reacted with MAP 4 in extracts of brain, heart, liver, and lung from adult mouse; the triplet in brain was more closely spaced than in the other tissues or neuroblastoma cells. In kidney, spleen, and stomach, only a single band (band 4) was labeled; this band was immunologically related to the triplet and was also present in all tissues positive for the triplet. Skeletal muscle, sperm, and peripheral blood contained no reactive polypeptides. After taxol- induced polymerization, the MAP 4 triplet was preferentially associated with the microtubule pellet whereas band 4 remained in the supernatant. These data indicate that there is tissue specificity in the distribution of MAP 4, and that some tissues contain a polypeptide related to MAP 4 (band 4) that does not bind to microtubules in vitro.     

Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa.

In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical.     

Expression profile of mouse BWF1, a protein with a BEACH domain, WD40 domain and FYVE domain

We isolated a mouse cDNA encoding a protein that contains a BEACH domain, 5 WD40 repeats and a FYVE domain, which we designated as BWF1. The mRNA is approximately 10 kb in size and encodes a protein consisting of 3508 amino acids with a predicted molecular weight of 385 kDa. BWF1 has 45% homology with the Drosophila protein, blue cheese (BCHS). The BWF1 gene consists of 67 exons, which span 270 kb of genomic sequence, and has been mapped to mouse chromosome 5. Northern blot analysis revealed that it was strongly expressed in the liver, moderately in the kidney and testis, and weakly in the brain of adult mice. During the development of the mouse brain, BWF1 mRNA was abundant on embryonic day (E) 14-16; after birth, the level of BWF1 mRNA expression decreased markedly to reach the adult level at postnatal day 3. In situ hybridization analysis revealed that the expressed BWF1 mRNA was restricted to the marginal region both in E14 and E16 embryonic brain, but became diffuse after birth. Confocal microscopy studies of the epitope-tagged BWF1 protein showed that the protein was a cytoplasmic one.     

Quantitation and characterization of tau factor in porcine tissues

Using a monospecific antibody against brain tau factor purified by affinity chromatography, we have studied the distribution of tau factor or related polypeptides in different cells. The presence of tau in all cell types tested was demonstrated by a radioimmunoassay. Tau factor-related proteins were found in liver, spleen, pancreas, kidney and lung, although at much lower levels than that found in neural cells. In all cases, they copolymerized with tubulin and were heat-resistant. When the distribution of tau factor-related proteins was studied by Western blotting, tau factor antiserum reacted against peptides with an electrophoretic mobility that was similar to those of brain tau factor peptides. Immunofluorescence studies have also been performed with the same antibody to determine the distribution of tau factor-related peptides in PK15 cells. Our results indicated that these peptides were associated to the microtubule network.     

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)-Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)-containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)-cellulose-purified poly(A)-containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L-[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9- to 20-fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two-dimensional polyacrylamide gel electrophoresis (PAGE); size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)-cellulose chromatography yielded 1.8 micrograms/g and 2.0 micrograms/g, respectively, of poly(A)-containing mRNA; this represents a two- to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two-dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.