Tau Splicing Research Articles

Detection of all adult Tau isoforms in a 3D culture model of iPSC-derived neurons.

Tauopathies are a class of neurodegenerative diseases characterized by the presence of pathological intracellular deposits of Tau proteins. Six isoforms of Tau are expressed in the adult human brain, resulting from alternative splicing of the MAPT gene. Tau splicing is developmentally regulated such that only the smallest Tau isoform is expressed in fetal brain, contrary to the adult brain showing the expression of all 6 isoforms. Induced Pluripotent Stem Cell (iPSC) technology has opened up new perspectives in human disease modeling, including tauopathies. However, a major challenge to in vitro recapitulation of Tau pathology in iPSC-derived neurons is their relative immaturity. In this study, we examined the switch in Tau splicing from fetal-only to all adult Tau isoforms during the differentiation of iPSC-derived neurons in a new 3D culture system. First, we showed that iPSC-induced neurons inside Matrigel-coated alginate capsules were able to differentiate into cortical neurons. Then, using a new assay that allowed both the qualitative and the quantitative analysis of all adult MAPT mRNA isoforms individually, we demonstrated that BrainPhys-maintained neurons expressed the 6 adult MAPT mRNA transcripts from 25 weeks of maturation, making this model highly suitable for modeling Tau pathology and therapeutic purposes.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

SummaryTauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

High-resolution temporal and regional mapping of MAPT expression and splicing in human brain development.

The microtubule associated protein tau plays a critical role in the pathogenesis of neurodegenerative disease. Recent studies suggest that tau also plays a role in disorders of neuronal connectivity, including epilepsy and post-traumatic stress disorder. Animal studies have shown that the MAPT gene, which codes for the tau protein, undergoes complex pre-mRNA alternative splicing to produce multiple isoforms during brain development. Human data, particularly on temporal and regional variation in tau splicing during development are however lacking. In this study, we present the first detailed examination of the temporal and regional sequence of MAPT alternative splicing in the developing human brain. We used a novel computational analysis of large transcriptomic datasets (total n = 502 patients), quantitative polymerase chain reaction (qPCR) and western blotting to examine tau expression and splicing in post-mortem human fetal, pediatric and adult brains. We found that MAPT exons 2 and 10 undergo abrupt shifts in expression during the perinatal period that are unique in the canonical human microtubule-associated protein family, while exon 3 showed small but significant temporal variation. Tau isoform expression may be a marker of neuronal maturation, temporally correlated with the onset of axonal growth. Immature brain regions such as the ganglionic eminence and rhombic lip had very low tau expression, but within more mature regions, there was little variation in tau expression or splicing. We thus demonstrate an abrupt, evolutionarily conserved shift in tau isoform expression during the human perinatal period that may be due to tau expression in maturing neurons. Alternative splicing of the MAPT pre-mRNA may play a vital role in normal brain development across multiple species and provides a basis for future investigations into the developmental and pathological functions of the tau protein.

Liquid\u2013liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau

The protein Tau aggregates into tangles in the brain of patients with Alzheimer’s disease. In solution, however, Tau is intrinsically disordered, highly soluble, and binds to microtubules. It is still unclear what initiates the conversion from an innocuous phase of high solubility and functionality to solid-like neurotoxic deposits. Here, we show that the microtubule-binding repeats of Tau, which are lysine-rich, undergo liquid–liquid phase separation in solution. Liquid–liquid demixing causes molecular crowding of amyloid-promoting elements of Tau and drives electrostatic coacervation. Furthermore, we demonstrate that three-repeat and four-repeat isoforms of Tau differ in their ability for demixing. Alternative splicing of Tau can thus regulate the formation of Tau-containing membrane-less compartments. In addition, phosphorylation of Tau repeats promotes liquid–liquid phase separation at cellular protein conditions. The combined data propose a mechanism in which liquid droplets formed by the positively charged microtubule-binding domain of Tau undergo coacervation with negatively charged molecules to promote amyloid formation.

Hallmarks of Alzheimer’s Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, invitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.

Is Huntington's disease a tauopathy?

Tauopathies are a subclass of neurodegenerative diseases typified by the deposition of abnormal microtubule-associated tau protein within the cerebral tissue. Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy and some fronto-temporal dementias are examples of the extended family of tauopathies. In the last decades, intermittent reports of cerebral tau pathology in individuals afflicted with Huntington's disease-an autosomal dominant neurodegenerative disorder that manifests by severe motor, cognitive and psychiatric problems in adulthood-have also begun to surface. These observations remained anecdotal until recently when a series of publications brought forward compelling evidence that this monogenic disorder may, too, be a tauopathy. Collectively, these studies reported that: (i) patients with Huntington's disease present aggregated tau inclusions within various structures of the brain; (ii) tau haplotype influences the cognitive function of Huntington's disease patients; and (iii) that the genetic product of the disease, the mutant huntingtin protein, could alter tau splicing, phosphorylation, oligomerization and subcellular localization. Here, we review the past and current evidence in favour of the postulate that Huntington's disease is a new member of the family of tauopathies.

ADNP: in search for molecular mechanisms and innovative therapeutic strategies for frontotemporal degeneration.

Activity-dependent neuroprotective protein (ADNP) is deregulated in Alzheimer's disease (AD) and in schizophrenia and mutated in autism. In mice, ADNP is essential for brain formation and ADNP haploinsufficiency is associated with cognitive and social deficits and tauopathy. Tauopathy, a major pathology in AD, is also found in ~45% of frontotemporal dementias (FTDs). Tau transcript, a product of a single gene, undergoes alternative splicing. Tau splicing seems to be altered in FTD brain. In transgenic mice overexpressing a mutated tau in the cerebral cortex, significant increases in ADNP transcript expression were observed in the cerebral cortex of young transgenic mice (~disease onset) and a marked decrease with aging as compared to control littermates. ADNP is a member of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex also associated with alternative splicing, including tau transcript splicing. Further cellular interactions of ADNP include association with microtubules, with tau being a microtubule—associated protein. NAP (davundetide), a novel drug candidate derived from ADNP, increases ADNP-microtubule association and protects against tauopathy and cognitive deficiencies in mice. Although, NAP did not provide protection in progressive supranuclear palsy (PSP), a pure tauopathy, it increased cognitive scores in amnestic mild cognitively impaired patients and protected functional activity in schizophrenia patients. This mini-review focuses on ADNP in the context of FTD and tau/microtubules and proposes NAP as a novel drug target for future clinical evaluations.

Lack of association between TDP-43 pathology and tau mis-splicing in Alzheimer's disease

A proportion of Alzheimer's disease cases displays inclusions of the RNA-binding protein, TDP-43. Considering the pathogenic role of tau mis-splicing, we compared tau isoform expression between Alzheimer's disease cases with or without TDP-43 inclusions. The average ratio of tau isoforms containing or lacking exon 10 (4R/3R ratio) or the total level of tau mRNA was not significantly different between cases with or without TDP-43 pathology in any of the brain regions examined. Although TDP-43 functions may be affected, TDP-43 does not critically regulate expression or splicing of tau in Alzheimer's disease suggesting that TDP-43 contributes to Alzheimer's disease through mechanisms independent of tau.

Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

Tau splicing in vivo: Application in mouse models of tauopathy confers functional and pathological changes

Tau splicing in vivo: Application in mouse models of tauopathy confers functional and pathological changes

Tau splicing factor expression pattern in Alzheimer’s disease brain

Tau splicing factor expression pattern in Alzheimer’s disease brain

Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I: A MOLECULAR MECHANISM LINKED TO TAU PATHOLOGY IN ALZHEIMER DISEASE

Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca(2+)/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain.

Genome modification leads to phenotype reversal in human myotonic dystrophy type 1 induced pluripotent stem cell-derived neural stem cells.

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3'-untranslated region (3' UTR) of the DMPK gene. Correcting the mutation in DM1 stem cells would be an important step toward autologous stem cell therapy. The objective of this study is to demonstrate in vitro genome editing to prevent production of toxic mutant transcripts and reverse phenotypes in DM1 stem cells. Genome editing was performed in DM1 neural stem cells (NSCs) derived from human DM1 induced pluripotent stem (iPS) cells. An editing cassette containing SV40/bGH polyA signals was integrated upstream of the CTG repeats by TALEN-mediated homologous recombination (HR). The expression of mutant CUG repeats transcript was monitored by nuclear RNA foci, the molecular hallmarks of DM1, using RNA fluorescence in situ hybridization. Alternative splicing of microtubule-associated protein tau (MAPT) and muscleblind-like (MBNL) proteins were analyzed to further monitor the phenotype reversal after genome modification. The cassette was successfully inserted into DMPK intron 9 and this genomic modification led to complete disappearance of nuclear RNA foci. MAPT and MBNL 1, 2 aberrant splicing in DM1 NSCs were reversed to normal pattern in genome-modified NSCs. Genome modification by integration of exogenous polyA signals upstream of the DMPK CTG repeat expansion prevents the production of toxic RNA and leads to phenotype reversal in human DM1 iPS-cells derived stem cells. Our data provide proof-of-principle evidence that genome modification may be used to generate genetically modified progenitor cells as a first step toward autologous cell transfer therapy for DM1.

The cytoskeleton as a drug target for neuroprotection: the case of the autism- mutated ADNP.

Fifteen years ago we discovered activity-dependent neuroprotective protein (ADNP), and showed that it is essential for brain formation/function. Our protein interaction studies identified ADNP as a member of the chromatin remodeling complex, SWI/SNF also associated with alternative splicing of tau and prediction of tauopathy. Recently, we have identified cytoplasmic ADNP interactions with the autophagy regulating microtubule-associated protein 1 light chain 3 (LC3) and with microtubule end-binding (EB) proteins. The ADNP-EB-binding SIP domain is shared with the ADNP snippet drug candidate, NAPVSIPQ termed NAP (davunetide). Thus, we identified a precise target for ADNP/NAP (davunetide) neuroprotection toward improved drug development.

Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig.

ABSTRACTAnimal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

E-Pharmacophore Based Virtual Screening to Identify Agonist for PKA-Cα

Owing to PKA-Cα unique functions in regulating tau splicing alternatively in neurons results in aggregation of tau which contributes to neurofibrillary tangles and taupathies generation a hallmark of Alzheimer’s disease (AD). PKACα also inhibits GSK3β thus it has been a therapeutic target for AD intervention. In this study, e-pharmacophore and multiple docking strategies were followed to propose a novel PKA-Cα agonists. Nine e-pharmacophores were developed from nine co-crystal structures such that all the critical pharmacophoric features involved in their bioactivity of PKA-Cα were effectively mapped. Rigid receptor docking (RRD) was performed with the library of PKA-Cα activators having 3512 shape screened compounds towards PKA-Cα. To derive the best leads, dock complexes were further subjected to QPLD, IFD and MM-GBSA calculations. PKA-Cα-lead1 dock complex was subjected to 50 ns MD simulations run. Comparative analysis between obtained 25 leads and 9 co-crystal ligands revealed three the best leads. Among the three, lead1 has the least docking score with lowest binding free energy with better binding orientation towards PKA-Cα. The constancy of PKA-Cα-lead1 interactions was revealed by 50 ns MD simulations run. Thus ADME predictions and results from RRD, QPLD, IFD and MD simulations affirmed that the proposed three leads might be used as a potent agonists for PKA-Cα.

Mitochondrial complex 1 inhibition increases 4-repeat isoform tau by SRSF2 upregulation.

Progressive Supranuclear Palsy (PSP) is a neurodegenerative disorder characterised by intracellular aggregation of the microtubule-associated protein tau. The tau protein exists in 6 predominant isoforms. Depending on alternative splicing of exon 10, three of these isoforms have four microtubule-binding repeat domains (4R), whilst the others only have three (3R). In PSP there is an excess of the 4R tau isoforms, which are thought to contribute significantly to the pathological process. The cause of this 4R increase is so far unknown. Several lines of evidence link mitochondrial complex I inhibition to the pathogenesis of PSP. We demonstrate here for the first time that annonacin and MPP+, two prototypical mitochondrial complex I inhibitors, increase the 4R isoforms of tau in human neurons. We show that the splicing factor SRSF2 is necessary to increase 4R tau with complex I inhibition. We also found SRSF2, as well as another tau splicing factor, TRA2B, to be increased in brains of PSP patients. Thereby, we provide new evidence that mitochondrial complex I inhibition may contribute as an upstream event to the pathogenesis of PSP and suggest that splicing factors may represent an attractive therapeutic target to intervene in the disease process.

High-fat diet increases tau expression in the brain of T2DM and AD mice independently of peripheral metabolic status

Alzheimer’s disease and type 2 diabetes mellitus are risk factors for each other. To investigate the effects of both genetic and high-fat-induced diabetic phenotype on the expression and exon 10 splicing of tau, we used the Alzheimer’s disease mouse model (APdE9) cross-bred with the type 2 diabetes mouse model over-expressing insulin-like growth factor 2 in the pancreas. High-fat diet, regardless of the genotype, significantly induced the expression of four repeat tau mRNA and protein in the temporal cortex of female mice. The mRNA levels of three repeat tau were also significantly increased by high-fat diet in the temporal cortex, although three repeat tau expression was considerably lower as compared to four repeat tau. Moreover, high-fat diet significantly increased the mRNA ratio of four repeat tau vs. three repeat tau in the temporal cortex of these mice. All of these effects were independent of the peripheral hyperglycemia, hyperinsulinemia and insulin resistance. Increased four repeat tau and three repeat tau levels significantly associated with impaired memory and reduced rearing in the female mice. High-fat diet did not affect neuroinflammation, Akt/GSK3β signaling pathway or the expression of tau exon 10 splicing enhancers in the temporal cortex. Our study suggests that the high-fat diet independently of type 2 diabetes or Alzheimer’s disease background induces the expression and exon 10 inclusion of tau in the brain of female mice.

Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n>50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscleblind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.