Neurofibrillary degeneration in progressive supranuclear palsy and corticobasal degeneration: tau pathologies with exclusively "exon 10" isoforms

Pathological tau proteins that constitute the basic matrix of neuronal inclusions observed in numerous neurodegenerative disorders are disease specific. This is mainly the consequence of the aggregation of specific sets of tau isoforms according to the diseases, i.e., six isoforms in Alzheimer's disease (AD) and exclusively the three tau isoforms lacking the corresponding sequence of exon 10 (E10-) in Pick's disease (PiD). By using antibodies specific to the different tau isoforms and one- and two-dimensional gel electrophoresis followed by western blots, we demonstrate herein a third group of neurodegenerative disorders characterized by intraneuronal inclusions exclusively constituted of tau isoforms containing the sequence corresponding to exon 10, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Together, tau isoforms with exon 10 clearly differentiate three groups of neurodegenerative diseases: AD, PiD, and PSP/CBD. For each group, the neuropathological and clinical phenotypes are most likely related to specific sets of tau isoforms expressed by the vulnerable neuronal populations. The recently described mutations of the tau gene responsible for familial frontotemporal dementias also support this hypothesis.     

Mitochondrial dysfunction in progressive supranuclear palsy

A progressive impairment of mitochondrial function has been suggested to play a critical role in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease and Huntington's disease. Mitochondrial dysfunction can lead to number of deleterious consequences including impaired calcium buffering, generation of free radicals, activation of the mitochondrial permeability transition pore and secondary excitotoxicity. Progressive supranuclear palsy (PSP) is a rare neurological disorder characterized by the appearance of supranuclear gaze palsy and extrapyramidal symptoms [Arch. Neurol. 10 (1964) 333]. Although the etiological basis of PSP is unknown, compelling evidence from spectroscopy studies in PSP patients, biochemical studies in post-mortem PSP brain tissue and PSP cybrids has emerged that supports a contributory role of bio-energetic defects in the pathogenesis of PSP.     

Frontal lobe dysfunction in progressive supranuclear palsy: evidence for oxidative stress and mitochondrial impairment

Recent data from our laboratory have shown a regionally specific increase in lipid peroxidation in postmortem progressive supranuclear palsy (PSP) brain. To extend this finding, we measured activities of mitochondrial enzymes as well as tissue malondialdehyde (MDA) levels in postmortem superior frontal cortex (Brodmann's area 9; SFC) from 14 pathologically confirmed cases of PSP and 13 age-matched control brains. Significant decreases (-39%) in alpha-ketoglutarate dehydrogenase complex/glutamate dehydrogenase ratio and significant increases (+36%) in tissue MDA levels were observed in the SFC in PSP; no differences in complex I or complex IV activities were detected. Together, these results suggest that mitochondrial dysfunction and lipid peroxidation may underlie the frontal metabolic and functional deficits observed in PSP.     

Mitochondrial dysfunction in cybrid lines expressing mitochondrial genes from patients with progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement disorder of unknown etiology. We hypothesized that mitochondrial DNA (mtDNA) aberration could occur in this disease and contribute to its pathogenesis. To address this we created transmitochondrial cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from persons with PSP. The presence of cybrid mtDNA aberration was screened for by biochemical assay of mitochondrial gene products. Relative to a control cybrid set, complex I activity was reduced in PSP cybrid lines (p<0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.     

Disease severity and progression in progressive supranuclear palsy and multiple system atrophy: validation of the NNIPPS--Parkinson Plus Scale

Background

The Natural History and Neuroprotection in Parkinson Plus Syndromes (NNIPPS) study was a large phase III randomized placebo-controlled trial of riluzole in Progressive Supranuclear Palsy (PSP, n = 362) and Multiple System Atrophy (MSA, n = 398). To assess disease severity and progression, we constructed and validated a new clinical rating scale as an ancillary study.

Methods and Findings

Patients were assessed at entry and 6-montly for up to 3 years. Evaluation of the scale''s psychometric properties included reliability (n = 116), validity (n = 760), and responsiveness (n = 642). Among the 85 items of the initial scale, factor analysis revealed 83 items contributing to 15 clinically relevant dimensions, including Activity of daily Living/Mobility, Axial bradykinesia, Limb bradykinesia, Rigidity, Oculomotor, Cerebellar, Bulbar/Pseudo-bulbar, Mental, Orthostatic, Urinary, Limb dystonia, Axial dystonia, Pyramidal, Myoclonus and Tremor. All but the Pyramidal dimension demonstrated good internal consistency (Cronbach α≥0.70). Inter-rater reliability was high for the total score (Intra-class coefficient = 0.94) and 9 dimensions (Intra-class coefficient = 0.80–0.93), and moderate (Intra-class coefficient = 0.54–0.77) for 6. Correlations of the total score with other clinical measures of severity were good (rho≥0.70). The total score was significantly and linearly related to survival (p<0.0001). Responsiveness expressed as the Standardized Response Mean was high for the total score slope of change (SRM = 1.10), though higher in PSP (SRM = 1.25) than in MSA (SRM = 1.0), indicating a more rapid progression of PSP. The slope of change was constant with increasing disease severity demonstrating good linearity of the scale throughout disease stages. Although MSA and PSP differed quantitatively on the total score at entry and on rate of progression, the relative contribution of clinical dimensions to overall severity and progression was similar.

Conclusions

The NNIPPS-PPS has suitable validity, is reliable and sensitive, and therefore is appropriate for use in clinical studies with PSP or MSA.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00211224","term_id":"NCT00211224"}}NCT00211224     

The economic costs of progressive supranuclear palsy and multiple system atrophy in France, Germany and the United Kingdom

Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are progressive disabling neurological conditions usually fatal within 10 years of onset. Little is known about the economic costs of these conditions. This paper reports service use and costs from France, Germany and the UK and identifies patient characteristics that are associated with cost. 767 patients were recruited, and 760 included in the study, from 44 centres as part of the NNIPPS trial. Service use during the previous six months was measured at entry to the study and costs calculated. Mean six-month costs were calculated for 742 patients. Data on patient sociodemographic and clinical characteristics were recorded and used in regression models to identify predictors of service costs and unpaid care costs (i.e., care from family and friends). The mean six-month service costs of PSP were €24,491 in France, €30,643 in Germany and €25,655 in the UK. The costs for MSA were €28,924, €25,645 and €19,103 respectively. Unpaid care accounted for 68-76%. Formal and unpaid costs were significantly higher the more severe the illness, as indicated by the Parkinson's Plus Symptom scale. There was a significant inverse relationship between service and unpaid care costs.     

Tau gene mutation in familial progressive subcortical gliosis

Familial forms of frontotemporal dementias are associated with mutations in the tau gene. A kindred affected by progressive subcortical gliosis (PSG), a rare form of presenile dementia, has genetic linkage to chromosome 17q21-22. This kindred (PSG-1) is included in the 'frontotemporal dementias and Parkinsonism linked to chromosome 17' group along with kindreds affected by apparently different forms of atypical dementias. Some of these kindreds have mutations in the tau gene. We report here that PSG-1 has a tau mutation at position +16 of the intron after exon 10. The mutation destabilizes a predicted stem-loop structure and leads to an over-representation of the soluble four-repeat tau isoforms, which assemble into wide, twisted, ribbon-like filaments and ultimately result in abundant neuronal and glial tau pathology. The mutations associated with PSG and other atypical dementias can be subdivided into three groups according to their tau gene locations and effects on tau. The existence of tau mutations with distinct pathogenetic mechanisms may explain the phenotypic heterogeneity of atypical dementias that previously led to their classification into separate disease entities.     

Identification of a novel risk locus for progressive supranuclear palsy by a pooled genomewide scan of 500,288 single-nucleotide polymorphisms

To date, only the H1 MAPT haplotype has been consistently associated with risk of developing the neurodegenerative disease progressive supranuclear palsy (PSP). We hypothesized that additional genetic loci may be involved in conferring risk of PSP that could be identified through a pooling-based genomewide association study of >500,000 SNPs. Candidate SNPs with large differences in allelic frequency were identified by ranking all SNPs by their probe-intensity difference between cohorts. The MAPT H1 haplotype was strongly detected by this methodology, as was a second major locus on chromosome 11p12-p11 that showed evidence of association at allelic (P<.001), genotypic (P<.001), and haplotypic (P<.001) levels and was narrowed to a single haplotype block containing the DNA damage-binding protein 2 (DDB2) and lysosomal acid phosphatase 2 (ACP2) genes. Since DNA damage and lysosomal dysfunction have been implicated in aging and neurodegenerative processes, both genes are viable candidates for conferring risk of disease.     

Tau alteration and neuronal degeneration in tauopathies: mechanisms and models

Tau becomes characteristically altered both functionally and structurally in several neurodegenerative diseases now collectively called tauopathies. Although increasing evidence supports that alterations of tau may directly cause neuronal degeneration and cell death, the mechanisms, which render tau to become a toxic agent are still unclear. In addition, it is obscure, whether neurodegeneration in tauopathies occurs via a common mechanism or specific differences exist. The aim of this review is to provide an overview about the different experimental models that currently exist, how they are used to determine the role of tau during degeneration and what has been learnt from them concerning the mechanistic role of tau in the disease process. The review begins with a discussion about similarities and differences in tau alteration in paradigmatic tauopathies such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease (AD). The second part concentrates on major experimental models that have been used to address the mechanistic role of tau during degeneration. This will include a discussion of cell-free assays, culture models using cell lines or dissociated neurons, and animal models. How these models aid to understand (i) alterations in the function of tau as a microtubule-associated protein (MAP), (ii) direct cytotoxicity of altered tau protein, and (iii) the potential role of tau aggregation in neurodegenerative processes will be the central theme of this part. The review ends with concluding remarks about a general mechanistic model of the role of tau alteration and neuronal degeneration in tauopathies and future perspectives.     

Induction of Alzheimer-specific Tau epitope AT100 in apoptotic human fetal astrocytes

In Alzheimer's and other neurodegenerative diseases, hyperphosphorylated tau accumulates in affected neuronal and glial cells in the form of paired helical filaments (PHFs). This tau binds antibody AT100, which recognizes the double phosphorylation site (Thr212/Ser214) that is not present in normal biopsy tau. In primary cultures, highly enriched (>98%) in astrocytes of human fetal brain, three polypeptides of 52, 64, and 70 kD showed immunoreactivity with tau antibodies against non-phosphorylated epitopes, accounting for 88, 12, and <1%, respectively, of the total reactivity. All three polypeptides were phosphorylated at the PHF-1 epitope but not at the epitopes Tau-1, 12E8, AT8, and AT100. Treatment of cultures with okadaic acid resulted in apoptosis characterized by the blebbing of the plasma membrane, condensation of nuclear chromatin, and fragmentation of the nucleus. This treatment also resulted in a 3- to 5-fold increase in the content of both tau protein and phosphorylation. The increases were observed in all phosphorylation sites examined, and included the AT100 site. The AT100 site has been proposed to be generated by protein kinase B/Akt and Cdc2. Since okadaic acid can induce an AD-like hyperphosphorylated state of normal tau in primary cultures of human brain cells, a simple cellular model is available permitting study of self-aggregation of tau and phosphorylation events characteristic of neurodegeneration.     

Progressive supranuclear palsy as differential diagnosis of Parkinson's disease in the elderly

IntroductionProgressive supranuclear palsy (PSP) is a syndrome characterized by progressive parkinsonism with early falls due to postural instability, typically vertical gaze supranuclear ophthalmoplegia, pseudobulbar dysfunction, neck dystonia and upper trunk rigidity as well as mild cognitive dysfunction. Progressive supranuclear palsy must be differentiated from Parkinson's disease taking into account several so-called red flags.Materials and methodsWe report a case series hallmarked by gait abnormalities, falls and bradykinesia in which Parkinson's disease was the initial diagnosis.ResultsDue to a torpid clinical course, magnetic resonance imaging (MRI) was performed demonstrating midbrain atrophy, highly suggestive of progressive supranuclear palsy.ConclusionThe neuroradiological exams (magnetic resonance imaging, single photon emission computer tomography, and positron emission tomography) can be useful for diagnosis of PSP. Treatment with levodopa should be considered, especially in patients with a more parkinsonian phenotype.     

Beta-defensin overexpression induces progressive muscle degeneration in mice

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse -defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IB accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases. muscular dystrophy; innate immunity; NF-B     

Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies

The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.     

Initial studies with high resolution TEM and electron energy loss spectroscopy studies of ferritin cores extracted from brains of patients with progressive supranuclear palsy and Alzheimer disease.

Studies of crystallographic structure and composition of core nanocrystals of ferritin bound to aberrant tau filaments extracted from progressive supranuclear Palsy (PSP) and Alzheimer disease (AD) brain tissues were performed using high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). The results were compared with those obtained from synthetic Fe3O4 crystal (magnetite) and horse spleen ferritin cores. Core dimensions of ferritin molecules from PSP and AD were similar to those found in normal brain. Ferritin cores nanocrystals in AD seems to have less ordered structure than in PSP. Some nanocrystals did not have the hexagonal ferrihydrite structure generally found in healthy ferritin but rather a cubic structure similar to magnetite, a crystalline form in which both Fe2+ and Fe3+ are present. The presence of ferrous ion, Fe2+, may indicate some dysfunction in these pathological ferritins that might contribute to production of free radicals via the Fenton reaction involved in neurodegeneration.     

Mimotopes and proteome analyses using human genomic and cDNA epitope phage display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 x 106 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as 'mimotopes' (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences.ABBREVIATIONS USED: IL, interleukin; ELISA, enzyme linked immunoabsorbant assay; PBS, phospho-buffered saline; cfu, colony forming units.     

Phage display of a CTL epitope elicits a long-term in vivo cytotoxic response

The Ovalbumin(257-264) CTL epitope on the major coat protein of the filamentous bacteriophage in different antigen formulations was displayed and the immune response in C57BL6/J mice studied. The display of single cytotoxic epitope on the surface of the virion is sufficient to induce priming and sustain long-term major histocompatibility complex class I restricted cytotoxic T lymphocytes response in vivo. The filamentous bacteriophage is a versatile carrier able to display simultaneously either single or multiple epitopes and can elicit a cellular response carrying very little peptide (<1.5 microg).     

Baculovirus surface display: construction and screening of a eukaryotic epitope library.

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.     

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.     

Loss of tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila

Accumulations of Tau, a microtubule‐associated protein (MAP), into neurofibrillary tangles is a hallmark of Alzheimer's disease and other tauopathies. However, the mechanisms leading to this pathology are still unclear: the aggregates themselves could be toxic or the sequestration of Tau into tangles might prevent Tau from fulfilling its normal functions, thereby inducing a loss of function defect. Surprisingly, the consequences of losing normal Tau expression in vivo are still not well understood, in part due to the fact that Tau knockout mice show only subtle phenotypes, presumably due to the fact that mammals express several MAPs with partially overlapping functions. In contrast, flies express fewer MAP, with Tau being the only member of the Tau/MAP2/MAP4 family. Therefore, we used Drosophila to address the physiological consequences caused by the loss of Tau. Reducing the levels of fly Tau (dTau) ubiquitously resulted in developmental lethality, whereas deleting Tau specifically in neurons or the eye caused progressive neurodegeneration. Similarly, chromosomal mutations affecting dTau also caused progressive degeneration in both the eye and brain. Although photoreceptor cells initially developed normally in dTau knockdown animals, they subsequently degenerated during late pupal stages whereas weaker dTau alleles caused an age‐dependent defect in rhabdomere structure. Expression of wild type human Tau partially rescued the neurodegenerative phenotype caused by the loss of endogenous dTau, suggesting that the functions of Tau proteins are functionally conserved from flies to humans. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1210–1225, 2014