A comparative study on pathological features of transgenic rat lines expressing either three or four repeat misfolded tau

Human tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by distinct clinical features, typical histopathological structures, and defined ratio(s) of three‐repeat and four‐repeat tau isoforms within pathological aggregates. How the optional microtubule‐binding repeat of tau influences this differentiation of pathologies is understudied. We have previously generated and characterized transgenic rodent models expressing human truncated tau aa151–391 with either three (SHR24) or four microtubule‐binding repeats (SHR72). Here, we compare the behavioral and neuropathological hallmarks of these two transgenic lines using a battery of tests for sensorimotor, cognitive, and neurological functions over the age range of 3.5–15 months. Progression of sensorimotor and neurological deficits was similar in both transgenic lines; however, the lifespan of transgenic line SHR72 expressing truncated four‐repeat tau was markedly shorter than SHR24. Moreover, the expression of three or four‐repeat tau induced distinct neurofibrillary pathology in these lines. Transgenic lines displayed different distribution of tau pathology and different type of neurofibrillary tangles. Our results suggest that three‐ and four‐repeat isoforms of tau may display different modes of action in the diseased brain.     

Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in the tauopathies, which include Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration and Pick's disease. Tau isoform composition and cellular and regional distribution as well as morphology of these inclusions vary in each disorder. Recently, several pathological missense and exon 10 splice-donor site mutations of the tau gene were identified in FTDP-17. Exon 10 codes for the second of four microtubule-binding repeat domains. The splice-site mutations result in increased inclusion of exon 10 which causes a relative increase in tau isoforms containing four microtubule-binding repeat domains over those containing three repeat domains. This could be a central aetiological mechanism in FTDP-17 and, perhaps, other related tauopathies. We have investigated changes in the ratio and distribution of three-repeat and four-repeat tau in the different tauopathies as a basis of the phenotypic range of these disorders and the selective vulnerability of different subsets of neurones. In this study, we have developed two monoclonal antibodies, RD3 and RD4 that effectively distinguish these closely related tau isoforms. These new isoform-specific antibodies are useful tools for analysing tau isoform expression and distribution as well as pathological changes in the human brain.     

Differential expression of exons 10 and 11 in normal tau and tau associated with paired helical filaments

Antibodies were raised to two synthetic peptides with amino acid sequences encoded by a variable region of exons 10 and 11 of the tau gene. The affinity-purified antibodies, designated E-10 and E-11, were used to determine whether PHF-tau and normal tau differ in variants containing three or four repeats in the microtubule-binding domain, respectively. Normal adult human brain was shown by gel electrophoresis to contain six isoforms of tau. All of the isoforms reacted with E-11, whereas only four of them with slower electrophoretic mobility were recognized by E-10. Fetal brain tau was readily recognized by E-11 but reacted poorly with E-10. In PHF preparations, E-11 bound to all three polypeptides of PHF-tau of 68 kD, 64 kD, and 60 kD and reacted intensely with a material smearing from the top of the gel to about the 50-kD region. In contrast, E-10 only weakly recognized the two higher molecular weight PHF-tau polypeptides of 68 kD and 64 kD, as well as smeared material, and the binding was not affected by phosphatase treatment. Using recombinant tau with four repeats as a reference, the immunoreactivity of E-10 with PHF-tau was estimated to be approximately 5% of that of E-11. By comparison, the immunoreactivity of E-10 with four isoforms of normal tau was comparable to that of E-11. These results indicate that the ratio of three vs. four repeat variants in PHF-tau is higher than in normal tau and suggest that Alzheimer disease may be associated with the disproportional expression of fetal (or juvenile) forms of tau. Alternatively, the weak reactivity of PHF-tau with E-10 antibody could be due to post-translational modifications other than phosphorylation. © 1995 Wiley-Liss, Inc.     

Hyperphosphorylation-induced self assembly of murine tau: a comparison with human tau

Summary. Alzheimer’s disease-like neurofibrillary pathology is neither seen in rodents nor in transgenic animals expressing the disease causing mutant human APP or mutant human presenilins. Whether the absence of this pathology is due to inability of the murine tau to self assemble into filaments or due to some other factors is not understood. In this study, we compared recombinant murine and human taus in their ability to form filaments by AD-like hyperphosphorylation in vitro. Human and murine taus, 0N4R, were generated as recombinant proteins and phosphorylated with rat brain extract as a source of protein kinases. We found that murine tau could be hyperphosphorylated to similar stoichiometry and manner as human tau. Upon hyperphosphorylation, murine tau was able to self polymerize into bundles of paired helical filament- and straight filament-like morphology. The filaments obtained from self assembly of murine tau closely resembled those formed from identically treated human tau. Moreover, like human tau, 60–70% of murine tau aggregated on hyperphosphorylation.     

Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration

Microtubule associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and related tauopathies; in this form it is the major protein subunit of paired helical filaments (PHF)/neurofibrillary tangles. However, the nature of protein kinases and phosphatases and tau sites involved in this lesion has been elusive. We investigated self-assembly and microtubule assembly promoting activities of hyperphosphorylated tau isolated from Alzheimer disease brain cytosol, the AD abnormally hyperphosphorylated tau (AD P-tau) before and after dephosphorylation by phosphoseryl/phosphothreonyl protein phosphatase-2A (PP-2A), and then rephosphorylation by cyclic AMP-dependent protein kinase (PKA), calcium, calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent protein kinase 5 (cdk5) in different kinase combinations. We found that (i) dephosphorylation of AD P-tau by PP-2A inhibits its polymerization into PHF/straight filaments (SF) and restores its binding and ability to promote assembly of tubulin into microtubules; (ii) rephosphorylation of PP-2A-dephosphorylated AD P-tau by sequential phosphorylation by PKA, CaMKII and GSK-3beta or cdk5, and as well as by cdk5 and GSK-3beta, promotes its self-assembly into tangles of PHF similar to those seen in Alzheimer brain, and (iii) phosphorylation of tau sites required for this pathology are Thr231 and Ser262, along with several sites flanking the microtubule binding repeat region. Phosphorylation of recombinant human brain tau(441) yielded similar results as the PP-2A dephosphorylated AD P-tau, except that mostly SF were formed. The conditions for the abnormal hyperphosphorylation of tau that promoted its self-assembly also induced the microtubule assembly inhibitory activity. These findings suggest that activation of PP-2A or inhibition of either both GSK-3beta and cdk5 or one of these two kinases plus PKA or CaMKII might be required to inhibit Alzheimer neurofibrillary degeneration.     

Increase in the relative expression of tau with four microtubule binding repeat regions in frontotemporal lobar degeneration and progressive supranuclear palsy brains

Some cases of familial frontotemporal dementia (FTD) leading to frontotemporal lobar degeneration (FTLD) are caused by mutations in tau on chromosome 17 (FTDP-17). Certain mutations alter the ratio between four (4R tau) and three (3R tau) repeat tau isoforms whereas cases with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) mainly have 4R tau brain pathology. We assessed tau mRNA and protein levels in frontal cortex from 15 sporadic FTLD, 21 PSP, 5 CBD, 15 Alzheimer’s disease (AD) and 16 control brains. Moreover, we investigated the disease association and possible tau splicing effects of the tau H1 haplotype. Cases with FTLD and PSP had lower tau mRNA levels than control brains. When analyzing 4R tau and 3R tau mRNA separately, control subjects displayed a 4R tau/3R tau ratio of 0.48. Surprisingly, FTLD brains displayed a more elevated ratio (1.32) than PSP brains (1.12). Also, several FTLD and PSP cases had higher 4R tau/3R tau mRNA than FTDP-17 cases, included as reference tissues, and the ratio increase was seen regardless of underlying histopathology, i.e. both for tau-positive and tau-negative FTLD cases. Furthermore, total tau protein levels were slightly decreased in both FTLD and AD as compared to control subjects. Finally, we confirmed the association of tau H1 with PSP, but could not find any haplotype-related effect on tau exon 10 splicing. In conclusion, we demonstrated increased but largely variable 4R tau/3R tau mRNA ratios in FTLD and PSP cases, suggesting heterogeneous pathophysiological processes within these disorders.     

Neuron-selective toxicity of tau peptide in a cell culture model of neurodegenerative tauopathy: essential role for aggregation in neurotoxicity

Intracellular aggregation of tau is a pathological hallmark in Alzheimer's disease and other tauopathies. The mechanisms underlying tau aggregation and the role that these aggregates play in neuronal death have remained controversial. To study these issues, we established a cell culture model of tauopathy using a hexameric peptide with the sequence (306)VQIVYK(311) located within the third microtubule-binding repeat of tau, rendered cell-permeable by a tag of nine arginine residues (R(9)). This peptide (VQIVYK-R(9)), designated as T-peptide, self-assembles in vitro into paired helical filament-like aggregates. Primary neuronal cells treated with T-peptide die within 24 hr. Neurodegeneration correlates with the ability of the peptide to aggregate. Two peptides with mutations in the hexameric core, K-peptide (VQIVKK) and VV-peptide (VQVVVK), that are incapable of aggregating are not toxic, whereas two other mutant peptides, V-peptide (VQVVYK) and F-peptide (VQIVFK), which aggregate, are also neurotoxic. Two other peptides that aggregate in vitro, but are not derived from tau, are not neurotoxic suggesting sequence dependence. Although localizing to the nucleus, T-peptide induces aggregation of cellular proteins in the cytoplasm. These aggregates are not caused by disruption of endogenous tau localization, although endogenous tau is reduced in neurons exposed to T-peptide. Interestingly, nonneuronal cells are less sensitive to T-peptide toxicity, recapitulating in part the selective loss of neurons in tauopathies. Moreover, T-peptide treatment leads to mitochondrial dysfunction, a common feature of neurodegenerative disorders. The model system described here represents a convenient paradigm for studying the mechanisms underlying tau aggregation and neurotoxicity and for identifying compounds that can prevent these effects.     

Variable alternative spliced exon (VASE)-containing and VASE-lacking neural cell adhesion molecule in the dorsal and ventral hippocampus of SAMP8 mice

The neural cell adhesion molecule (NCAM) is involved in the development and synaptic plasticity of the brain. Differential splicing of the variable alternative spliced exon (VASE) in the fourth immunoglobulin domain can dramatically change the functional properties of NCAM. This paper discusses our analysis of the levels of different expression of VASE-containing NCAM (NCAM-VASE(+)) and VASE-lacking NCAM (NCAM-VASE(-)) mRNAs in the dorsal and ventral hippocampus of senescence-accelerated mice (SAM). We further investigated the individual level of NCAM-VASE(+) and NCAM-VASE(-) in relation to the capacity for spatial learning and memory as assessed by a Morris water maze task. The results showed that the levels of both NCAM-VASE(+) and NCAM-VASE(-) were increased significantly in dorsal but not ventral hippocampus in aged SAMP8 mice. The mean latencies to find the hidden platform of the learning task on the last day were positively correlated with the levels of NCAM-VASE(+) in the dorsal hippocampus of SAMP8, which reveals that the mice with high levels of NCAM-VASE(+) have poor learning performances. These results suggest that the up-regulation of NCAM-VASE(+) could be involved in the impairments of spatial learning and memory.     

Human choline acetyltransferase gene: Localization of alternative first exons

The avian embryonic retina is widely used as a model system for cellular and molecular studies on central nervous system neurons. We aimed at the generation of cell lines from the early embryonic quail retina by retroviral oncogene transduction. For this, we made use of the retina organ culture system which exhibits both proliferation, necessary for stable oncogene transduction, and initial neuronal differentiation, a prerequisite for the generation of cell lines with mature neuronal properties. The oncogene myc was chosen ac it is both proliferation-inducing and differentiation-compatible. A chimeric gene, mycER, containing v-myc and the hormone-binding domain of the estrogen receptor, was used for transduction in order to allow for hormone regulation of myc activity. Transduced organ-cultured cells from temporal and nasal retina were passaged into sparse single cell cultures. From these, colonies of rapidly dividing cells were isolated and the progeny expanded as cell lines. The lines contained cells with features of neuroepithelial cells, showing vimentin and A2B5. They also contained spontaneously differentiated neuronal cells showing neurofilament L and N-CAM18O. A subpopulation of the neuronal cells exhibited the morphological characteristics of retinal ganglion cells, i.e., large pear-shaped somata each emitting one long process with a distinct growth cone. In addition, they showed the marker profile of retinal ganglion cells, i.e., expression of Thy-1, G4, DMGRASP, Nr-CAM, neurofilament H, and tau. Neuronal differentiation could be induced by the addition of db cAMP and retinoic acid. The mature neuronal features of the lines open new possibilities to study properties of retinal neurons, including ganglion cells, in a defined and manipulable experimental system. © Wiley-Liss, Inc.     

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.     

与应力调控相关的基因选择性剪接*****☆◆

背景：适当的力学环境是生物体正常生长发育、结构重建以及功能维持的重要因素,也是损伤组织功能性修复的关键因素之一。应力调控基因表达不仅体现在基因的开关或表达水平的调节,还可以体现在转录后的选择性剪接。 目的：结合力生长因子介绍选择性剪接这种新的应力调控方式,并推测可能的调控机制。 方法：检索PubMed 数据库(1964/2010)、CNKI数据库(2000/2009)有关力信号转导和基因选择性剪接方面的综述文章和研究报告,分析二者的联系和可能的调控机制。 结果与结论：应力刺激可以导致肌细胞、成骨细胞中的胰岛素样生长因子1基因发生选择性剪接,产生一种新的应力敏感的生长因子力生长因子,这种新型调控方式的机制还不明确,推测与应力导致的剪接小体的位置(位移运动)以及改变了剪接酶(如RNP酶)的位置和空间结构(变形)有关。     

Toxic tau: structural origins of tau aggregation in Alzheimer's disease

Alzheimer’s disease is characterized by the extracellular accumulation of the amyloidβin the form of amyloid plaques and the intracellular deposition of the microtubule-associated protein tau in the form of neurofibrillary tangles.Most of the Alzheimer’s drugs targeting amyloidβhave been failed in clinical trials.Particularly,tau pathology connects greatly in the pathogenesis of Alzheimer’s disease.Tau protein enhances the stabilization of microtubules that leads to the appropriate function of the neuron.Changes in the quantity or the conformation of tau protein could affect its function as a microtubules stabilizer and some of the processes wherein it is involved.The molecular mechanisms leading to the accumulation of tau are principally signified by numerous posttranslational modifications that change its conformation and structural state.Therefore,aberrant phosphorylation,as well as truncation of tau protein,has come into focus as significant mechanisms that make tau protein in a pathological entity.Furthermore,the shape-shifting nature of tau advocates to comprehend the progression of Alzheimer’s disease precisely.In this review,we emphasize the recent studies about the toxic and shape-shifting nature of tau in the pathogenesis of Alzheimer’s disease.