High Molecular Weight Tau Research Articles

Heterogeneity of the high molecular weight tau proteins in N115 neuroblastoma cells.

The sequence of a high molecular weight (HMW) tau cDNA cloned from a neuroblastoma N115 library contains, in addition to the C- and N-terminal and middle regions present in the low molecular weight mouse brain tau proteins, a 711-bp nonhomologous domain (exon 4a) and a region of 198 bp corresponding to exon 6 of the tau gene. Protein immunoblot analysis, performed with antibodies specific either for a sequence present in the N-terminal region of all the tau variants or for exon 4a revealed several bands suggesting that more than one tau form is expressed in this cell line. Northern blot experiments performed with a number of cDNA probes spanning domains common and uncommon to low molecular weight and HMW tau allowed the identification of four tau transcripts differing in the size of their coding and noncoding regions. All these transcripts contain the sequence encoded by exon 6, but two of them lack exon 4a. As shown by RNase protection assays, the N-terminal region of these transcripts is also variable and contains either exon 1, or exons 1 and 2, or exons 1-3. Yet all these HMW tau forms contain four homologous repeats in their C-terminal domain both in the differentiated and nondifferentiated cells, i.e., have adult characteristics. In conclusion, the data reported in this article demonstrate that several HMW tau variants are expressed in neuroblastoma N115 cells and that the transition between immature to mature tau forms occurring during brain development is not required for neurite outgrowth during morphological differentiation of this cell line.

High and low molecular weight tau proteins are differentially expressed from a single gene.

Both high and low molecular weight (HMW and LMW) tau proteins are expressed in the immature and adult mouse spinal cord. Northern blot analysis, performed with probes complementary to domains common and uncommon to the LMW and HMW entities, suggested that HMW tau proteins found in the immature mouse spinal cord are not translated from the single transcript of 6 kb expressed at these stages, but are transported within this nervous structure by axons arising in the periphery. In contrast, another minor transcript of 8 kb was detected in the adult mouse spinal cord by a HMW tau specific probe, suggesting that a small fraction of the HMW tau forms present in adulthood are translated within mouse spinal cord neurons. LMW spinal cord tau forms are encoded by mRNAs of 6 kb that contain three and four homologous repeats at immature and mature stages, respectively, whereas adult HMW entities contain four repeats. PCR analysis performed with mouse genomic DNA also showed that the nonhomologous region specific for HMW tau is a single exon. Southern blot and gene mapping showed that the same gene, located on the murine chromosome 11, encodes all the LMW and HMW tau variants. All these tau forms, therefore, are produced by an alternative splicing mechanism that is neuron-specific and developmentally regulated.

Differential regulation of beta III and other tubulin genes during peripheral and central neuron development.

Mammalian peripheral and central neurons differ considerably in the composition and properties of their axonal cytoskeletons. Recent reports of the selective expression of a high molecular weight (HMW) tau protein in neurons with peripherally projecting axons have furthered the idea that the microtubules in central and peripheral neurons are disparate. In the present study, we examined the possibility that the various tubulin genes are differentially expressed in central versus peripheral neurons. To examine this, we compared the expression of four of the beta-tubulin mRNAs (classes beta I, beta II, beta III, beta IV) and the alpha 1-tubulin mRNA in rat dorsal root ganglion (DRG) neurons with their expression in cerebral cortex during postnatal development (P5-90), using northern blots and in situ hybridization. We document both similarities and differences in tubulin gene expression in these two regions of the neuraxis during postnatal development. In both DRG and cortex, the expression of the class beta I- and beta II-tubulin mRNAs and the alpha 1-tubulin mRNA was higher at earlier stages of postnatal development than in the adult. However, class beta IV-tubulin mRNA levels increased during cortical development but decreased during DRG postnatal development. The opposite pattern was found for the neuron-specific class beta III-tubulin gene, the mRNA levels of which were high in cortex, at birth and then decreased with increasing postnatal development. In DRG, the beta III-tubulin mRNA levels generally increased during postnatal development. Beta III-tubulin protein levels were examined qualitatively at different developmental stages (5-90 days) by immunoblotting and immunocytochemistry.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional distribution and biochemical characteristics of high molecular weight tau in the nervous system.

The present study examined the distribution of the high molecular weight (HMW) tau protein isoform in the nervous system by immunoblotting and immunohistochemistry. Some of the biochemical properties of this 110 kDa tau protein were explored, including its heat stability, phosphorylation and partitioning with cold/Ca2+ stable vs. soluble microtubules. Qualitative western blot analysis revealed that HMW tau is preferentially expressed in neurons with peripherally projecting axons. For example, this isotype was present in sciatic nerve, ventral and dorsal roots, trigeminal nerve, vagus nerve, dorsal root ganglia (DRG) and spinal cord, but was present in only trace amounts in CNS regions. Another tau isoform of slightly smaller size (90-100 kDa), termed mid-molecular weight (MMW) tau, was present in abundant quantity in optic nerve samples and detectable in several other CNS regions, including hippocampus and cerebellum. The 110 kDa HMW tau as well as MMW tau and the other tau isoforms were found to be heat stable proteins. The HMW and MMW tau isoforms preferentially partitioned with the cold and Ca+2 insoluble tubulin fraction, but the association of HMW tau with stable microtubules was very susceptible to proteolysis. Dephosphorylation of fresh tissue with alkaline phosphatase produced no apparent shift in the mobility of HMW tau on SDS-PAGE but did alter the mobility of other brain tau isoforms, including MMW tau. Immunocytochemical staining with tau-1 antibody in the DRG, which contains HMW tau but no other tau isotypes, showed localization to mainly small neurons and was not altered by dephosphorylation of the histological sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of high molecular weight tau present in the peripheral nervous system.

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.

Differentiation of neuroblastoma cells correlates with an altered splicing pattern of tau RNA

Morphological differentiation of N2A nouroblastoma cells is associated with an altered splicing of the gene of the microtubule-associated protein, tau. Two populations of RNA (coding for tau proteins containing three or four tubulin-binding motifs) are present in a similar proportion in undifferentiated neuroblastoma cells while in differentiated cells the proportion is changed in favour of that population coding for tau protein containing four tubulin-binding motifs. An increase in a high molecular weight tau isoforms correlates with the increase in the RNA population coding for four tubulin-binding motifs. A possible consequence of expressing a higher proportion of the tau protein containing four tubulin-binding motifs could be an increase in microtubule stability of differentiated neuroblastoma cells.

High molecular weight tau: Preferential localization in the peripheral nervous system

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 x 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.

Tau gene expression in rat sensory neurons during development and regeneration

In the mature rat dorsal root ganglion (DRG), only one tau isoform is expressed, and this protein (110 kDa in apparent molecular weight) is considerably larger in size than the predominant tau isoforms found in brain. The size of the mRNA encoding the "big" tau mRNA in DRG [approximately 8 kilobases (kb)] is also much larger than that of the major rat brain tau mRNA species (approximately 6 kb). In this study, we examined the pattern of normal developmental changes in expression of this high-molecular-weight (HMW) tau and its encoding mRNA and also determined how axonal injury of adult DRG neurons effected the expression of this gene. RNA blotting experiments revealed that higher levels of HMW tau mRNA were present in the DRG at early postnatal times than in the adult. Immunoblotting of total DRG protein using a monoclonal tau antibody revealed that the immature DRG (7 d postnatal) contained a 62-kDa tau isoform in addition to the HMW tau isoform that was expressed in the adult DRG. Neither of the tau isoforms expressed in the immature DRG was present to any significant extent in either immature or adult rat brain. To examine how tau expression changed in adult DRG neurons during regeneration, the sciatic nerves of rats were unilaterally crushed, and the L4 and L5 DRG were harvested 1, 7, and 14 d later.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal microtubule-associated proteins in the embryonic avian spinal cord.

We have used monoclonal antibodies to study the distribution of three developmentally regulated microtubule-associated proteins-MAP2, MAP5, and tau-during the morphogenesis of the thoracic spinal cord and peripheral nervous system in the quail. MAP5 is the only one of the three that is present in growing motor neuron processes in the day 3 embryo. The low-molecular weight form of MAP2, MAP2c, is found in motor neuron cell bodies at embryonic day 3. At later stages MAP2c appears in axons and in glia; it decreases in abundance between embryonic days 5 and 7. High-molecular weight MAP2 appears in motor neuron cell bodies and spinal cord gray matter at embryonic day 4, and is never encountered in axons. Tau is found in axons, but only at embryonic day 3.5, after they have commenced active extension. The molecular form and patterns of intracellular compartmentalization of each of the microtubule-associated proteins studied is conserved in mammalian and avian neurons. We conclude that MAP5 may be involved in the active growth of neuronal processes, whereas MAP2 and tau are not, and that high-molecular weight MAP2 and tau may stabilize dendritic and axonal processes, respectively.

A monoclonal antibody that recognizes a phosphorylated epitope in Alzheimer neurofibrillary tangles, neurofilaments and tau proteins immunostains granulovacuolar degeneration

A monoclonal antibody, raised against extracts from Alzheimer brain, that recognizes a phosphorylated epitope in high molecular weight neurofilament proteins and tau proteins also immunostains Alzheimer neurofibrillary tangles, neurites in senile plaques and granulovacuolar degeneration. This result suggest that granulovacuolar degeneration may contain phosphorylated proteins, possibly due to autophagy of phosphorylated perikaryal proteins that appear to be increased in Alzheimer's disease.

Analysis of the relationship of bovine high molecular weight protein-2 and tau protein using radioimmunoassay

Microtubule-associated proteins isolated from cycle-purified bovine brain tubulin by boiling in high salt were fractionated into two samples by chromatography on A15M agarose. One sample is composed primarily of a 300 000 dalton polypeptide (HMW-2) and the other is composed of four polypeptides with apparent molecular weights between 53 000 and 68 000 (tau). Aliquots of the HMW-2 and tan samples were labelled with 125I and used in a radioimmunoassay to determine their crossreactivity. The antiserum for the radioimmunoassay was produced in a rabbit by injection of cycle-purified bovine brain tubulin and was found to contain antibodies against HMW-2 and tau when assayed by quantitative precipitation and radioimmunoassay. The data from the quantitative precipitation experiment demonstrate that the antiserum recognizes approximately 30 antigenic domains on the HMW-2 molecule. Several radioimmunoassays were done to determine the competition between either labelled HMW-2 as the indicator molecule and unlabelled tau as the competitor or labelled tau as the indicator molecule and unlabelled HMW-2 as the competitor. In all assays less than 5% of the competitor crossreacted with antibodies to the labelled protein. The results demonstrate that our antiserum recognizes HMW-2 and tau as distinct antigenic species.

39] Purification and assay of microtubule-associated proteins (MAPs)

Publisher Summary This chapter presents procedure for purification and assay of microtubule-associated proteins (MAPS). The procedures begin with microtubules assembled in vitro from either brain or tissue culture cells. The solution of microtubule proteins is usually carried through at least two to three complete assembly-disassembly cycles prior to beginning one of the following MAP isolation procedures. The strategies employed to isolate MAPs from the total microtubule protein preparation are based on several criteria: (1) net charge distribution affecting their behavior on ion exchange resins, (2) overall molecular size and axial ratio, which affect their elution from molecular sieve columns, and (3) varying stability to the effects of elevated temperatures (100°). One or more combinations of these properties can be used as the basis for the isolation procedures. Both the high molecular weight MAPs and Tau from brain and the MAPs from tissue culture cells have been assayed by their ability to promote the assembly of tubulin in vitro. Generally, these assays are carried out by determining whether the putative MAP will enhance the initial rate and total amount of assembly of a given concentration of PC or DEAE-purified brain tubulin.