Regulation of alternative splicing of tau exon 10

The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer’s disease and related neurodegenerative disorders. The adult human brain expresses six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of its pre-mRNA. Exon 10 encodes the second microtubule-binding repeat of tau. Its alternative splicing produces tau isoforms with either three or four microtubule-binding repeats, termed 3R-tau and 4Rtau. In the normal adult human brain, the level of 3R-tau is approximately equal to that of 4R-tau. Several silent and intronic mutations of the tau gene associated with FTDP-17T (frontotemporal dementia with Parkinsonism linked to chromosome 17 and specifically characterized by tau pathology) only disrupt exon 10 splicing, but do not influence the primary sequence of the tau protein. Thus, abnormal exon 10 splicing is sufficient to cause neurodegeneration and dementia. Here, we review the regulation of tau exon 10 splicing by cis-elements and trans-factors and summarize all the mutations associated with FTDP-17T and related tauopathies. The findings suggest that correction of exon 10 splicing may be a potential target for tau exon 10 splicing-related tauopathies.     

Alternative splicing in protein associated with Myc (Pam) influences its binding to c-Myc

We recently identified Pam (for protein associated with c-Myc), as a binding partner for the tuberous sclerosis complex (TSC) protein tuberin in brain. The highly conserved Pam homologs in Drosophila and C. elegans are neuron-specific proteins that regulate synaptic growth. The Pam gene contains 83 exons and encodes a 4,641-amino-acid polypeptide with a predicted molecular weight of approximately 510 kDa. In a previous study, we demonstrated that Pam is expressed as two forms, approximately 450 kDa in rat embryonic and a approximately 350 kDa in rat adult brain. Here we have extended that work to show the approximately 450 kDa form is expressed in rat embryonic kidney, heart, and lung and in rat cell lines, and the approximately 350 kDa form is expressed in adult rat tissues as well as in human and mouse brain and human and mouse cell lines. To understand the size difference, we investigated alternative splicing of Pam in brain and detected six isoforms in the Myc-binding region resulting from splicing of exon 53, and three new exons, 52A, 56, and 56A. We also demonstrate that the presence of exon 52A in Pam significantly enhances binding to Myc, suggesting functional importance of this alternative splicing. The presence of Pam in many cellular compartments, its spliced variants, as well as its multiple binding partners, including tuberin, make it a complex, yet intriguing protein in the nervous system.     

PSF suppresses tau exon 10 inclusion by interacting with a stem-loop structure downstream of exon 10

Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA–protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5′ splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.     

Alternative splicing of amino-terminal Tau mRNA in rat spinal cord during development and following axonal injury

Tau is a family of microtubule-associated phosphoproteins in which isoform variation is produced by alternative splicing of a single gene and posttranslational modifications. Tau isoforms that include exon 10 are overexpressed in frontotemporal dementia and progressive supranuclear palsy. Therefore, we examined the expression of tau mRNA splice variants during axonal regeneration and abortive regeneration. Previous work in our laboratory demonstrated that expression of exon 10 tau isoforms during regeneration and abortive regeneration was altered and partially recapitulated the developmental patterns of tau isoform expression. Using RT-PCR, we examined the alternative splicing of exons 2 and 3 in tau during early postnatal development and regeneration in the rat spinal cord. The levels of tau lacking exons 2 and 3 were high on the day of birth and rapidly declined. Conversely, tau isoforms containing exon 2 or exons 2 and 3 first appeared at low levels and steadily increased. During axonal regeneration, the levels of all three tau mRNA isoforms were significantly lower 7 days after injury. In a model of abortive regeneration, all of the tau isoforms were elevated 14 and 42 days postinjury. The relative levels of exon 2 and 3 tau splice variants were not altered during regeneration or abortive regeneration as occurred during development. These results suggest that tau isoform expression following neuronal injury does not recapitulate the developmental pattern and is not independently regulated as in development. Our previous results together with these data suggest that alterations in tau mRNA isoform expression that occur in neurodegeneration are not secondary to axonal injury but may be a more primary event underlying cytoskeletal derangement.     

Tau isoform regulation is region- and cell-specific in mouse brain

Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, which are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R- and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wildtype mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R-tau was more "synaptic like," with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression.     

Alternative Splicing of HumanNrCAMin Neural and Nonneural Tissues

Neural cell adhesion molecule NrCAM exists in a variety of isoforms as a result of alternative splicing of individual exons during RNA processing. In this report we demonstrate that many of the alternative splicing events described for chick are conserved in man and describe a novel variant of NrCAM cDNA. Furthermore, we show that NrCAM is expressed at significant levels outside the nervous system; in particular in pancreas, adrenal glands, and placenta and that expression in both brain and other tissues is accompanied by a very variable pattern of exon utilization in fetal and adult cells.