Ian D'Souza

Determinants of 4-repeat tau expression. Coordination between enhancing and inhibitory splicing sequences for exon 10 inclusion.

Mutations in the tau gene are pathogenic causing autosomal dominant frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17). Some mutations intau exon 10 (E10) and immediately adjacent sequences cause disease by altering E10 splicing. To determine the mechanism of normal E10 splicing regulation and how FTDP-17 mutations alter splicing, mutational analysis of E10 was performed. The results show that E10 contains a complex array of both enhancer and inhibitorcis-acting elements that modulate usage of a weak 5′ splice site. The 5′ end of E10 contains a previously unrecognized multipartite exon splicing enhancer (ESE) composed of an SC35-like binding sequence, a purine-rich sequence, and an AC-rich element. Downstream of this ESE is a purine-rich exon splicing inhibitor. Intronic sequences immediately downstream of E10 also are inhibitory. The results support an alternative model in which I10 inhibitory sequences appear to function as a linear sequence. The cis-elements described are not redundant, and all appear required for normal E10 splicing. Results with double mutations demonstrate that the ESE and the intronic inhibitory element collaborate to regulate splicing. Thus splicing oftau E10 is regulated by a complex set ofcis-acting elements that span nearly the entire exon and also include intronic sequences.

A novel mutation at position +12 in the intron following Exon 10 of the tau gene in familial frontotemporal dementia (FTD‐Kumamoto)

Exonic and intronic mutations in the tau gene cause familial frontotemporal dementia and parkinsonism linked to chromosome 17. Here, we describe a new mutation, consisting of a C‐to‐T transition at position +12 of the intron following exon 10 of the tau gene in the Kumamoto pedigree, showing frontotemporal dementia. The mutation caused a marked reduction in melting temperature of the tau exon 10–splicing regulatory element RNA and a large increase in exon 10–containing transcripts. Brain tissue from affected individuals showed an abnormal preponderance of exon 10–containing transcripts that was reflected at the protein level by an overproduction of tau isoforms with four microtubule‐binding repeats. Immunostaining revealed the presence of tau aggregates in degenerating neurons and glial cells. Isolated tau filaments had a twisted ribbon‐like morphology and were made of hyperphosphorylated four‐repeat tau isoforms. The additional mutation located close to the splice‐donor site of the intron following exon 10 of the tau gene supports the view that intronic mutations exercize their pathogenic effect by destabilizing RNA secondary structure. Ann Neurol 2000;47:422–429.

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. J. Comp. Neurol. 511:788–803, 2008.

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.

A genomic sequence analysis of the mouse and human microtubule-associated protein tau

Abstract. Microtubule associated protein tau (MAPT) encodes the microtubule associated protein tau, the primary component of neurofibrillary tangles found in Alzheimers disease and other neurodegenerative disorders. Mutations in the coding and intronic sequences of MAPT cause autosomal dominant frontotemporal dementia (FTDP-17). MAPT is also a candidate gene for progressive supranuclear palsy and hereditary dysphagic dementia. A human PAC (201 kb) and a mouse BAC (161 kb) containing the entire MAPT and Mtapt genes, respectively, were identified and sequenced. Comparative DNA sequence analysis revealed over 100 conserved non-repeat potential cis-acting regulatory sequences in or close to MAPT. Those islands with greater than 67% nucleotide identity range in size from 20 to greater than 1700 nucleotides. Over 90 single nucleotide polymorphisms were identified in MAPT that are candidate susceptibility alleles for neurodegenerative disease. The 5′ and 3′ flanking genes for MAPT are the corticotrophin-releasing factor receptor (CRFR) gene and KIAA1267, a gene of unknown function expressed in brain.

Arginine/Serine-rich Protein Interaction Domain-dependent Modulation of a Tau Exon 10 Splicing Enhancer ALTERED INTERACTIONS AND MECHANISMS FOR FUNCTIONALLY ANTAGONISTIC FTDP-17 MUTATIONS Δ280K AND N279K

Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2β, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5′ end of exon 10. Disease splicing mutations N279K and Δ280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2β compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Δ280K targets are nearly undetectable, explaining why Δ280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2β significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Δ280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2β are required for normal exon 10 splicing in vivo. In contrast to Tra2β, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2β has a supporting role in exon 10 splicing.

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation

Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimers disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17). We show here that an FTDP‐17 tau mutation (V337M) in SH‐SY5Y cells reduces microtubule polymerization, increases voltage‐dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L‐type Ca channel blocker), whereas ω‐conotoxin GVIA (N‐type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L‐type calcium channel activity. The depolarization‐induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH‐SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule‐binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.

Pro-apoptotic effects of tau mutations in chromosome 17 frontotemporal dementia and parkinsonism.

It was recently discovered that mutations of tau cause hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we report that cultured SH-SY5Y human neuroblastoma cells transfected with mutated tau genes are more vulnerable to apoptotic stimulus. Two kinds of mutations of tau causing FTDP-17 were examined in the present study: one was in exon 10 (N279K) and the other was in exon 12 (V337M). SH-SY5Y cells transfected with either mutated tau were more vulnerable to serum withdrawal, whereas cells transfected with the wild-type tau or vector alone showed no significant change in apoptotic vulnerability. The increase in the intracellular calcium concentration by the serum withdrawal was significantly greater in the SH-SY5Y cells transfected with mutated tau genes than in cells transfected with the wild-type tau or vector alone. These results suggest that mutations of tau might cause FTDP-17 by these proapoptotic functions by disrupting the intracellular calcium homeostasis.