Survey for CAG repeat polymorphisms in the human MAP-2 gene.

Microtubule-associated protein-2 (MAP-2) expression is altered in response to a number of physiological insults such as Alzheimer's disease, schizophrenia, stroke and AIDS-dementia. Changes include alteration in MAP-2 transcription, translation, and state of phosphorylation. Multiple MAP-2 transcripts exist within the nervous system and, as noted for a number of genes expressed in the central nervous system, MAP-2 contains a region of trinucleotide repeats located in exon 1 of the 5' untranslated region (5' UTR). Since expansion of CAG repeats are found in several neurodegenerative disorders, we analysed the CAG repeats in MAP-2 for polymorphisms in 31 controls, 35 chronic schizophrenics, and 20 with other neuropsychiatric illnesses. Genomic DNA samples from 86 individuals were used as templates in PCR amplifications with primers within exon 1. Sequencing of the PCR products, or short tandem repeat polymorphism (STRP) analysis, demonstrated consistency in the size of the CAG repeats. This study demonstrates that the seven copies of the CAG repeat located in the 5' UTR of the MAP-2 gene are highly conserved in the general population, and that there is no evidence for expansion of the CAG repeat.

Molecular and functional characteristics of MAP-2a: ability of MAP-2a versus MAP-2b to induce stable microtubules in COS cells.

Microtubule-associated protein-2 (MAP-2) is a prominent cytoskeletal protein in the mammalian nervous system. Two high-molecular-weight (HMW) MAP-2 isoforms, MAP-2a and MAP-2b, are developmentally regulated. MAP-2b is expressed through the life of the neuron, while MAP-2a expression coincides with the time of synaptic formation. MAP-2a and MAP-2b differ in size by approximately 10 kD. Attempts to differentiate MAP-2a from MAP-2b led to the identification of additional exons; exons 7A, 8, 13, and 16. The focus of the present study was to define the complete molecular composition of MAP-2a that was prerequisite for investigating the functional characteristic of the MAP-2a protein. Detailed examination of rat brain mRNA by Northern blot analysis and RT-PCR showed that MAP-2a contains only exon 8 in addition to the exons found in the MAP-2b transcript. Exons 7A, 13, and 16 are not present in the MAP-2a transcript. Antibody generated to exon 8 expressed protein, immunoprecipitated a HMW protein from adult rat brain that co-migrated with MAP-2a and was immunopositive with other MAP-2 antibodies. Comparative transfections of full-length MAP-2a and MAP-2b cDNA into COS-7 cells demonstrated that MAP-2a influenced the microtubule network differently than MAP-2b by inducing rapid and stable microtubule bundle formation even in the presence of nocodazole.

Making sense of the multiple MAP-2 transcripts and their role in the neuron.

Microtubule-associated protein-2 (MAP-2) is a family of heat-stable, phosphoproteins expressed predominantly in the cell body and dendrites of neurons. Three major MAP-2 isoforms, (MAP-2a, MAP-2b, MAP-2c) are differentially expressed during the development of the nervous system and have an important role in microtubule dynamics. Several MAP-2 cDNA clones that correspond to the major MAP-2 transcripts and additional, novel MAP-2 transcripts expressed in the CNS and PNS have been characterized. The transcripts result from the alternative splicing of a single MAP-2 gene consisting of 20 exons. Studies are now being directed toward understanding the role of the multiple MAP-2 forms that contain novel exons in the nervous system. The expression, localization, and possible functions of the newly identified spliced forms are the focus of this review.

Distribution and subcellular localization of high-molecular-weight microtubule-associated protein-2 expressing exon 8 in brain and spinal cord.

The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2 + 8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2 + 8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2 + 8 comigrated with MAP-2a. In human adult brain, HMW MAP-2 + 8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultrastructural level, HMW MAP-2 + 8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2 + 8. The presence of HMW MAP-2 + 8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.

Expression of microtubule-associated protein-2a and other novel microtubule-associated protein-2 transcripts in human fetal spinal cord.

In human fetal spinal cord (HFSC), six additional microtubule-associated protein-2 (MAP-2) transcripts are generated by alternative splicing of two recently described exons, exon 8 and exon 13. The following three translated proteins are detected by western blot analysis: MAP-2b expressing exon 8 (MAP-2b + 8; MAP-2a), MAP-2b expressing exon 13 (MAP-2b + 13), and MAP-2c expressing exon 8 and exon 13 (MAP-2c + 8 + 13). The finding that MAP-2b + 8 is expressed in HFSC demonstrates for the first time the presence of MAP-2a in human fetal CNS. Immunocytochemical studies show that exon 8-specific antibody and exon 13-specific antibody stain independent and overlapping populations of neurons in the lumbar region of the HFSC. Antibody 13-immunopositive neurons have predominantly cytosolic staining, whereas in the antibody 8-immunoreactive neurons staining was observed in the cytosol, dendrites, and some synapses. The prenatal expression of MAP-2a, which has been used as a marker of synaptogenesis, not only demonstrates the presence of a mature MAP-2 isoform in HFSC, but suggests that MAP-2a is important during human fetal as well as postnatal synaptogenesis.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.

Three unique 5' untranslated regions are spliced to common coding exons of high- and low-molecular-weight microtubule-associated protein-2.

Three unique 5' untranslated regions (UTRs) have been characterized for human microtubule-associated protein-2 (MAP-2) transcripts. All three UTRs shared a common 171-bp sequence adjacent to the MAP-2 coding region and then diverged upstream. The size of the unique upstream sequence was 281, 146, or 104 bp. PCR of genomic DNA demonstrated that the 5' UTRs span multiple exons. The unique region of the UTRs recognizes a 9.5- and a 6-kb MAP-2 transcript in poly(A)+ mRNA isolated from human MSN cells, and PCR analysis demonstrated that each unique UTR is contained in multiple high- and low-molecular-weight MAP-2 transcripts. Reverse transcription-PCR (RT-PCR) performed on MSN mRNA isolated from polysomes demonstrated that all three of the UTRs contained within multiple MAP-2 transcripts were associated with polysomes and hence translated. RT-PCR from human fetal spinal cord and adult brain mRNA demonstrated that all of the UTRs are expressed at these developmental time points.

Localization of specific epitopes on human microtubule-associated protein 2.

Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169-219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.

Characterization of the transcripts encoding two isoforms of human microtubule-associated protein-2 (MAP-2).

Through the isolation of a series of overlapping clones from human fetal and adult cDNA libraries, we have generated the complete cDNA sequences encoding human high- and low-molecular-weight microtubule-associated protein-2 (MAP-2) which have strong sequence homology with rodent MAP-2.

Geographic correlation between the occurrence of endemic nephropathy and urinary tract tumours in vratza district, Bulgaria.

Data on the occurrence of endemic nephropathy (EN) and urinary tract and other cancers in an endemic region of Vratza district, Bulgaria, for the years 1965-1974, are presented. In endemic villages a high incidence of urinary tract tumours, affecting in particular the renal pelvis and ureter, closely correlated with the EN incidence and mortality rates. In the villages with high and moderate EN incidences urinary tract tumours are the most common neoplasms. They account for 25% of all tumour sites in males and 30% in females. In hyperendemic villages age-adjusted incidences in EN and urinary tract tumours were 506/10(5) and 104/10(5) in females, and 315/10(5) and 89/10(5) in males respectively. EN mortality in these villages accounted for over 40% of all deaths in females and about 30% in males. Both diseases displayed peculiar geographic clustering. Females and middle-aged persons were most often affected. Urinary tract neoplasms were often multiple and nearly 90% of them originated in the uro-epithelium. In endemic and non-endemic villages of the region studied, the frequency and pattern of non-urinary tract cancers were rather similar, with statistical values close to those of the rural population of Vratza District and Bulgaria as a whole.