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1.  Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment 
Clinical proteomics  2014;11(1):12.
Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD) it is of interest to characterize CSF-TTR isoform distribution in AD patients and controls. Here, TTR isoforms are profiled directly from CSF by an optimized immunoaffinity-mass spectrometry method in 76 samples from patients with AD (n = 37), mild cognitive impairment (MCI, n = 17)), and normal pressure hydrocephalus (NPH, n = 15), as well as healthy controls (HC, n = 7). Fractions of three specific oxidative modifications (S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation) were quantitated relative to the total TTR protein. Results were correlated with diagnostic information and with levels of CSF AD biomarkers tau, phosphorylated tau, and amyloid β1-42 peptide.
Preliminary data highlighted the high risk of artifactual TTR modification due to ex vivo oxidation and thus the samples for this study were all collected using strict and uniform guidelines. The results show that TTR is significantly more modified on Cys(10) in the AD and MCI groups than in controls (NPH and HC) (p ≤ 0.0012). Furthermore, the NPH group, while having normal TTR isoform distribution, had significantly decreased amyloid β peptide but normal tau values. No obvious correlations between levels of routine CSF biomarkers for AD and the degree of TTR modification were found.
AD and MCI patients display a significantly higher fraction of oxidatively modified TTR in CSF than the control groups of NPH patients and HC. Quantitation of CSF-TTR isoforms thus may provide diagnostic information in patients with dementia symptoms but this should be explored in larger studies including prospective studies of MCI patients. The development of methods for simple, robust, and reproducible inhibition of in vitro oxidation during CSF sampling and sample handling is highly warranted. In addition to the diagnostic information the possibility of using TTR as a CSF oxymeter is of potential value in studies monitoring disease activity and developing new drugs for neurodegenerative diseases.
PMCID: PMC3973606  PMID: 24678637
Immunoaffinity; Mass spectrometry; Isoform profiling; Oxidation; Alzheimer’s disease; Transthyretin; Cerebrospinal fluid; Normal pressure hydrocephalus
2.  Cerebrospinal Fluid Levels of sAPPα and sAPPβ in Lewy Body and Alzheimer's Disease: Clinical and Neurochemical Correlates 
We measured cerebrospinal fluid (CSF) levels of the soluble isoforms of amyloid precursor protein (APP; sAPPα sAPPβ) and other CSF biomarkers in 107 patients with Alzheimer's disease (AD), dementia with Lewy body dementia (DLB), Parkinson's disease dementia (PDD), and normal controls (NC) using commercial kits. DLB and PDD were combined in a Lewy body dementia group (LBD). No differences were observed in sAPPα and sAPPβ levels between the groups. Significant correlations were observed between sAPPα and sAPPβ and between sAPPβ and Mini-Mental State Examination scores in the total group analysis as well as when LBD and AD groups were analyzed separately. sAPPα and sAPPβ levels correlated with Aβ38, Aβ40, Aβ42, and Tau in the LBD group. In AD, sAPPα correlated with p-Tau and sAPPβ with Aβ40. The differential association between sAPPα and sAPPβ with Aβ and Tau species between LBD and AD groups suggests a possible relationship with the underlying pathologies in LBD and AD.
PMCID: PMC3182340  PMID: 21966597
3.  Transthyretin, Thyroxine, and Retinol-Binding Protein in Human Cerebrospinal Fluid: Effect of Lead Exposure 
Transthyretin (TTR), synthesized by the choroid plexus, is proposed to have a role in transport of thyroid hormones in the brain. Our previous studies in animals suggest that sequestration of lead (Pb) in the choroid plexus may lead to a marked decrease in TTR levels in the cerebrospinal fluid (CSF). The objectives of this study were to establish in humans whether TTR and thyroxine (T4) are correlated in the CSF, and whether CSF levels of Pb are associated with those of TTR, T4, and/or retinol-binding protein (RBP). Eighty-two paired CSF and blood/serum samples were collected from patients undergoing clinical diagnosis of CSF chemistry. Results showed that the mean value of CSF concentrations for TTR was 3.33 ± 1.60 μg/mg of CSF proteins (mean ± SD, n = 82), for total T4 (TT4) was 1.56 ± 1.68 ng/mg (n = 82), for RBP was 0.34 ± 0.19 μg/mg (n = 82), and for Pb was 0.53 ± 0.69 μg/dl (n = 61 for those above the detection limit). Linear regression analyses revealed that CSF TTR levels were positively associated with those of CSF TT4 (r = 0.33, p < 0.005). CSF TTR concentrations, however, were inversely associated with CSF Pb concentrations (r = −0.29, p < 0.05). There was an inverse, albeit weak, correlation between CSF TT4 and CSF Pb concentrations (r = −0.22, p = 0.09). The concentrations of TTR, TT4, and Pb in the CSF did not vary as the function of their levels in blood or serum, but RBP concentrations in the CSF did correlate to those of serum (r = 0.39, p < 0.0005). Unlike TTR, CSF RBP concentrations were not influenced by Pb. These human data are consistent with our earlier observations in animals, which suggest that TTR is required for thyroxine transport in the CSF and that Pb exposure is likely associated with diminished TTR levels in the CSF.
PMCID: PMC4126162  PMID: 11294981
transthyretin; thyroxine; retinol-binding protein; lead; cerebrospinal fluid; choroid plexus
4.  α-Synuclein improves diagnostic and prognostic performance of tau and Aβ in Alzheimer's disease 
Acta neuropathologica  2013;126(5):10.1007/s00401-013-1148-z.
Alzheimer's disease (AD) and Lewy body diseases (LBD), e.g. Parkinson's disease (PD) dementia and dementia with Lewy bodies (DLB), are common causes of geriatric cognitive impairments. In addition, AD and LBD are often found in the same patients at autopsy; therefore, biomarkers that can detect the presence of both pathologies in living subjects are needed. In this investigation, we report the assessment of α-synuclein (α-syn) in cerebrospinal fluid (CSF) and its association with CSF total tau (t-tau), phosphorylated tau181 (p-tau181), and amyloid beta1-42 (Aβ1-42) in subjects of the Alzheimer's Disease Neuroimaging Initiative (ADNI; n=389), with longitudinal clinical assessments. A strong correlation was noted between α-syn and t-tau in controls, as well as in patients with AD and mild cognitive impairment (MCI). However, the correlation is not specific to subjects in the ADNI cohort, as it was also seen in PD patients and controls enrolled in the Parkinson's Progression Markers Initiative (PPMI; n=102). A bimodal distribution of CSF α-syn levels was observed in the ADNI cohort, with high levels of α-syn in the subjects with abnormally increased t-tau values. Although a correlation was also noted between α-syn and p-tau181, there was a mismatch (α-syn-p-tau181-Mis), i.e. higher p-tau181 levels accompanied by lower α-syn levels in a subset of ADNI patients. We hypothesize that this α-syn-p-tau181-Mis is a CSF signature of concomitant LBD pathology in AD patients. Hence, we suggest that inclusion of measures of CSF α-syn and calculation of α-syn-p-tau181-Mis improves the diagnostic sensitivity/specificity of classic CSF AD biomarkers and better predicts longitudinal cognitive changes.
PMCID: PMC3812407  PMID: 23812319
Alzheimer's disease; Parkinson's disease; dementia with Lewy body; Cerebrospinal fluid; Amyloid β; tau; α-synuclein
5.  Low CSF Levels of Both α-Synuclein and the α-Synuclein Cleaving Enzyme Neurosin in Patients with Synucleinopathy 
PLoS ONE  2013;8(1):e53250.
Neurosin is a protease that in vitro degrades α-synuclein, the main constituent of Lewy bodies found in brains of patients with synucleinopathy including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Several studies have reported reduced cerebrospinal fluid (CSF) levels of α-synuclein in synucleinopathy patients and recent data also proposes a significant role of α-synuclein in the pathophysiology of Alzheimer's disease (AD). To investigate potential links between neurosin and its substrate α-synuclein in vivo we used a commercially available sandwich ELISA and an in-house developed direct ELISA to quantify CSF levels of α-synuclein and neurosin in patients diagnosed with DLB, PD and PD dementia (PDD) versus AD patients and non-demented controls. We found that patients with synucleinopathy displayed lower CSF levels of neurosin and α-synuclein compared to controls and AD patients. In contrast, AD patients demonstrated significantly increased CSF α-synuclein but similar neurosin levels compared to non-demented controls. Further, CSF neurosin and α-synuclein concentrations were positively associated in controls, PD and PDD patients and both proteins were highly correlated to CSF levels of phosphorylated tau in all investigated groups. We observed no effect of gender or presence of the apolipoprotein Eε4 allele on neither neurosin or α-synuclein CSF levels. In concordance with the current literature our study demonstrates decreased CSF levels of α-synuclein in synucleinopathy patients versus AD patients and controls. Importantly, decreased α-synuclein levels in patients with synucleinopathy appear linked to low levels of the α-synuclein cleaving enzyme neurosin. In contrast, elevated levels of α-synuclein in AD patients were not related to any altered CSF neurosin levels. Thus, altered CSF levels of α-synuclein and neurosin in patients with synucleinopathy versus AD may not only mirror disease-specific neuropathological mechanisms but may also serve as fit candidates for future biomarker studies aiming at identifying specific markers of synucleinopathy.
PMCID: PMC3540093  PMID: 23308173
6.  Characterization of the interaction of β-amyloid with transthyretin monomers and tetramers# 
Biochemistry  2010;49(38):8276-8289.
β-amyloid (Aβ) is the main protein component of the amyloid plaques associated with Alzheimer’s disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type transthyretin (TTR) amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from toxic effects of Aβ deposition [Stein, T.D. and Johnson, J.A. (2002) J. Neurosci. 22: 7380–7388]. We examined the interaction of Aβ with wt TTR as well as two mutants: F87M/L110M, engineered to be a stable monomer, and T119M, a naturally occurring mutant with higher tetrameric stability than wildtype. Based on enzyme-linked immunoassays as well as crosslinking experiments, we conclude that Aβ monomers bind more strongly to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aβ aggregates rather than Aβ monomers. Through tandem mass spectrometry analysis of crosslinked TTR-Aβ fragments, we identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Aβ aggregate growth. These data provide important new insights into the nature of TTR-Aβ interactions. Such interactions may regulate TTR-mediated protection against Aβ toxicity.
PMCID: PMC2943652  PMID: 20795734
7.  Protein Clearance Mechanisms of Alpha-Synuclein and Amyloid-Beta in Lewy Body Disorders 
Protein clearance is critical for the maintenance of the integrity of neuronal cells, and there is accumulating evidence that in most—if not all—neurodegenerative disorders, impaired protein clearance fundamentally contributes to functional and structural alterations eventually leading to clinical symptoms. Dysfunction of protein clearance leads to intra- and extraneuronal accumulation of misfolded proteins and aggregates. The pathological hallmark of Lewy body disorders (LBDs) is the abnormal accumulation of misfolded proteins such as alpha-synuclein (Asyn) and amyloid-beta (Abeta) in a specific subset of neurons, which in turn has been related to deficits in protein clearance. In this paper we will highlight common intraneuronal (including autophagy and unfolded protein stress response) and extraneuronal (including interaction of neurons with astrocytes and microglia, phagocytic clearance, autoimmunity, cerebrospinal fluid transport, and transport across the blood-brain barrier) protein clearance mechanisms, which may be altered across the spectrum of LBDs. A better understanding of the pathways underlying protein clearance—in particular of Asyn and Abeta—in LBDs may result in the identification of novel biomarkers for disease onset and progression and of new therapeutic targets.
PMCID: PMC3485523  PMID: 23133788
8.  Microheterogeneity of transthyretin in serum and ascitic fluid of ovarian cancer patients 
BMC Cancer  2005;5:133.
Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR.
Subjects were 48 women with primary invasive epithelial ovarian cancer or recurrent ovarian carcinoma. The control group consisted of 20 postmenopausal women. TTR and retinol-binding protein (RBP) levels were measured by enzyme-linked immunoassay (ELISA) and C-reactive protein (CRP) levels by a high-sensitivity latex particle turbidimetric assay. The molecular heterogeneity of TTR was analysed using immunoprecipitation and matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Presence of TTR in tumor tissue was determined with indirect peroxidase immunostaining.
TTR and RBP (μg/ml) levels in serum were 148.5 ± 96.7 and 22.5 ± 14.8 in affected women compared to 363.3 ± 105.5 and 55.8 ± 9.3 in healthy postmenopausal women (p < 0.01). In ascitic fluid, levels were 1.02 ± 0.24 and 4.63 ± 1.57 μg/ml, respectively. The mean levels of TTR and RBP in serum showed a tendency to decrease with the severity of the disease and were lower in affected women whose CRP levels were > 40 mg/ml (p = 0.08 for TTR; p < 0.05 for RBP). No differences in TTR microheterogeneity were observed between TTR isolated from serum of affected and healthy women or from ascitic fluid. TTR occurred rather consistently in four variants. Mass signals were at 13758 ± 7, 13876 ± 13 (greatest intensity), 13924 ± 21 and 14062 ± 24 Da, representing native, S-cysteinylated, S-cysteinglycinylated and glutathionylated TTR, respectively. Serum of healthy and affected women as well as ascitic fluid contained the truncated fragment of TTR (12828 ± 11 Da). No immunoreactive TTR was observed in the tumor sites.
The severity of the cancer associated catabolism as well as the inflammation status affect serum TTR and RBP levels. Neither TTR nor its truncated form originates from tumor tissue and its occurrence in ascites may well reflect the filtration from blood into ascitic fluid.
PMCID: PMC1274304  PMID: 16225703
9.  APOE ε4 Increases Risk for Dementia in Pure Synucleinopathies 
JAMA neurology  2013;70(2):223-228.
To test for an association between the apolipoprotein E (APOE) ε4 allele and dementias with synucleinopathy.
Genetic case-control association study.
Academic research.
Autopsied subjects were classified into 5 categories: dementia with high-level Alzheimer disease (AD) neuropathologic changes (NCs) but without Lewy body disease (LBD) NCs (AD group; n=244), dementia with LBDNCs and high-level ADNCs (LBD-AD group; n=224), dementia with LBDNCs and no or low levels of ADNCs (pure DLB [pDLB] group; n=91), Parkinson disease dementia (PDD) with no or low levels of ADNCs (n=81), and control group (n=269).
Main Outcome Measure
The APOE allele frequencies.
The APOE ε4 allele frequency was significantly higher in the AD (38.1%), LBD-AD (40.6%), pDLB (31.9%), and PDD (19.1%) groups compared with the control group (7.2%; overall χ42=185.25; P=5.56×10−39), and it was higher in the pDLB group than the PDD group (P=.01). In an age-adjusted and sex-adjusted dominant model, ε4 was strongly associated with AD (odds ratio, 9.9; 95% CI, 6.4–15.3), LBD-AD (odds ratio, 12.6; 95% CI, 8.1–19.8), pDLB (odds ratio, 6.1; 95% CI, 3.5–10.5), and PDD (odds ratio, 3.1; 95% CI, 1.7–5.6).
The APOE ε4 allele is a strong risk factor across the LBD spectrum and occurs at an increased frequency in pDLB relative to PDD. This suggests that ε4 increases the likelihood of presenting with dementia in the context of a pure synucleinopathy. The elevated ε4 frequency in the pDLB and PDD groups, in which the overall brain neuritic plaque burden was low, indicates that apoE might contribute to neurodegeneration through mechanisms unrelated to amyloid processing.
PMCID: PMC3580799  PMID: 23407718
10.  Detection of disease-associated α-synuclein in the cerebrospinal fluid: a feasibility study  
Clinical Neuropathology  2014;33(5):329-334.
With the aim to evaluate the significance and reliability of detecting disease-specific α-synuclein in the cerebrospinal fluid (CSF) we developed an ELISA and bead-assay. We used a commercial antibody (5G4) that does not bind to the physiological monomeric form of α-synuclein, but is highly specific for the disease-associated forms, including high molecular weight fraction of β-sheet rich oligomers. We applied both tests in CSF from a series of neuropathologically confirmed α-synucleinopathy cases, including Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB) (n = 7), as well as Alzheimer’s disease (n = 6), and control patients without neurodegenerative pathologies (n = 9). Disease-specific α-synuclein was detectable in the CSF in a subset of patients with α-synuclein pathology in the brain. When combined with the analysis of total α-synuclein, the bead-assay for disease-specific α-synuclein was highly specific for PDD/DLB. Detection of disease-associated α-synuclein combined with the total levels of α-synuclein is a promising tool for the in-vivo diagnosis of α-synucleinopathies, including PDD and LBD.
PMCID: PMC4151342  PMID: 25131945
α-synuclein; bead-assay; cerebrospinal fluid; Parkinson’s disease dementia; dementia with Lewy bodies; synucleinopathy
11.  Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice 
Serum retinol-binding protein (RBP4) is secreted by liver and adipocytes and is implicated in systemic insulin resistance in rodents and humans. RBP4 normally binds to the larger transthyretin (TTR) homotetramer, forming a protein complex that reduces renal clearance of RBP4. To determine whether alterations in RBP4-TTR binding contribute to elevated plasma RBP4 levels in insulin-resistant states, we investigated RBP4-TTR interactions in leptin-deficient ob/ob mice and high-fat-fed obese mice (HFD). Gel filtration chromatography of plasma showed that 88–94% of RBP4 is contained within the RBP4-TTR complex in ob/ob and lean mice. Coimmunoprecipitation with an RBP4 antibody brought down stoichiometrically equal amounts of TTR and RBP4, indicating that TTR was not more saturated with RBP4 in ob/ob mice than in controls. However, plasma TTR levels were elevated approximately fourfold in ob/ob mice vs. controls. RBP4 injected intravenously in lean mice cleared rapidly, whereas the t1/2 for disappearance was approximately twofold longer in ob/ob plasma. Urinary fractional excretion of RBP4 was reduced in ob/ob mice, consistent with increased retention. In HFD mice, plasma TTR levels and clearance of injected RBP4 were similar to chow-fed controls. Hepatic TTR mRNA levels were elevated approximately twofold in ob/ob but not in HFD mice. Since elevated circulating RBP4 causes insulin resistance and glucose intolerance in mice, these findings suggest that increased TTR or alterations in RBP4-TTR binding may contribute to insulin resistance by stabilizing RBP4 at higher steady-state concentrations in circulation. Lowering TTR levels or interfering with RBP4-TTR binding may enhance insulin sensitivity in obesity and type 2 diabetes.
PMCID: PMC2711779  PMID: 18285525
obesity; adipose; high-fat diet
12.  Biochemical marker in familial amyloidotic polyneuropathy, Portuguese type. Family studies on the transthyretin (prealbumin)-methionine-30 variant. 
Journal of Clinical Investigation  1985;76(6):2171-2177.
A transthyretin variant with a methionine for valine substitution at position 30 [TTR(Met30)] is found in Portuguese patients with familial amyloidotic polyneuropathy (FAP). Effective, rapid, small- and semimicro-scale (immunoblotting) procedures were developed to determine whether or not TTR(Met30) is present in the plasma of an individual subject. The immunoblotting procedure employs only 0.10 ml of serum and can serve as a reliable procedure for the screening of large numbers of persons for the presence of TTR(Met30). In family studies of seven FAP kindreds, TTR(Met30) was found in 21 out of 41 asymptomatic FAP offspring, and its presence was not related to either age or sex. Thus, the mutant TTR segregated in accordance with the known autosomal dominant mode of inheritance of FAP. Total plasma TTR levels were not reduced in asymptomatic FAP offspring who were carriers of TTR(Met30), and no difference was observed between carriers and noncarriers of the mutant TTR. The ratios of the variant to normal TTR in plasma were estimated in asymptomatic FAP offspring and were similar to those found in FAP patients. In contrast, TTR(Met30) was relatively enriched in cerebrospinal fluid samples from two FAP patients. The significance of this finding is not known, but might relate to the preferential deposition of amyloid in the nervous system in FAP. A limited study was conducted involving simultaneous analysis of both stored (collected in 1975) and fresh serum from 20 FAP offspring, all of whom had been asymptomatic in 1975. In every subject, the results obtained with the stored and the fresh serum samples were in agreement. Six of these subjects developed clinical FAP since 1975; TTR(Met30) was present in each of these subjects. These several studies strongly suggest that the presence of TTR(Met30) in plasma constitutes a predictive biochemical marker of FAP in the preclinical phase of the disease.
PMCID: PMC424338  PMID: 3908483
13.  Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites 
PLoS ONE  2012;7(9):e43522.
Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4) and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis.
Methodology and Principal Findings
We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site) than the second T4 site (BD site). Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy.
Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation.
PMCID: PMC3433419  PMID: 22973437
14.  Molecular Tweezers Targeting Transthyretin Amyloidosis 
Neurotherapeutics  2014;11(2):450-461.
Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the “molecular tweezers”, CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer’s disease and Parkinson’s disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0256-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3996111  PMID: 24459092
Molecular tweezers; Transthyretin; Amyloid; Familial amyloidotic polyneuropathy
15.  GBA mutations increase risk for Lewy body disease with and without Alzheimer disease pathology 
Neurology  2012;79(19):1944-1950.
Mutations in the GBA gene occur in 7% of patients with Parkinson disease (PD) and are a well-established susceptibility factor for PD, which is characterized by Lewy body disease (LBD) neuropathologic changes (LBDNCs). We sought to determine whether GBA influences risk of dementia with LBDNCs, Alzheimer disease (AD) neuropathologic changes (ADNCs), or both.
We screened the entire GBA coding region for mutations in controls and in subjects with dementia and LBDNCs and no or low levels of ADNCs (pure dementia with Lewy bodies [pDLB]), LBDNCs and high-level ADNCs (LBD-AD), and high-level ADNCs but without LBDNCs (AD).
Among white subjects, pathogenic GBA mutations were identified in 6 of 79 pDLB cases (7.6%), 8 of 222 LBD-AD cases (3.6%), 2 of 243 AD cases (0.8%), and 3 of 381 controls (0.8%). Subjects with pDLB and LBD-AD were more likely to carry mutations than controls (pDLB: odds ratio [OR] = 7.6; 95% confidence interval [CI] = 1.8–31.9; p = 0.006; LBD-AD: OR = 4.6; CI = 1.2–17.6; p = 0.025), but there was no significant difference in frequencies between the AD and control groups (OR = 1.1; CI = 0.2–6.6; p = 0.92). There was a highly significant trend test across groups (χ2(1) = 19.3; p = 1.1 × 10−5), with the likelihood of carrying a GBA mutation increasing in the following direction: control/AD < LBD-AD < pDLB.
GBA is a susceptibility gene across the LBD spectrum, but not in AD, and appears to convey a higher risk for PD and pDLB than for LBD-AD. PD and pDLB might be more similar to one another in genetic determinants and pathophysiology than either disease is to LBD-AD.
PMCID: PMC3484986  PMID: 23035075
16.  Evaluating the relationship between amyloid-β and α-synuclein phosphorylated at Ser129 in dementia with Lewy bodies and Parkinson’s disease 
Lewy body and Alzheimer-type pathologies often co-exist. Several studies suggest a synergistic relationship between amyloid-β (Aβ) and α-synuclein (α-syn) accumulation. We have explored the relationship between Aβ accumulation and the phosphorylation of α-syn at serine-129 (pSer129 α-syn), in post-mortem human brain tissue and in SH-SY5Y neuroblastoma cells transfected to overexpress human α-syn.
We measured levels of Aβ40, Aβ42, α-syn and pSer129 α-syn by sandwich enzyme-linked immunosorbent assay, in soluble and insoluble fractions of midfrontal, cingulate and parahippocampal cortex and thalamus, from cases of Parkinson’s disease (PD) with (PDD; n = 12) and without dementia (PDND; n = 23), dementia with Lewy bodies (DLB; n = 10) and age-matched controls (n = 17). We also examined the relationship of these measurements to cognitive decline, as measured by time-to-dementia and the mini-mental state examination (MMSE) score in the PD patients, and to Braak tangle stage.
In most brain regions, the concentration of insoluble pSer129 α-syn correlated positively, and soluble pSer129 α-syn negatively, with the levels of soluble and insoluble Aβ. Insoluble pSer129 α-syn also correlated positively with Braak stage. In most regions, the levels of insoluble and soluble Aβ and the proportion of insoluble α-syn that was phosphorylated at Ser129 were significantly higher in the PD and DLB groups than the controls, and higher in the PDD and DLB groups than the PDND brains. In PD, the MMSE score correlated negatively with the level of insoluble pSer129 α-syn. Exposure of SH-SY5Y cells to aggregated Aβ42 significantly increased the proportion of α-syn that was phosphorylated at Ser129 (aggregated Aβ40 exposure had a smaller, non-significant effect).
Together, these data show that the concentration of pSer129 α-syn in brain tissue homogenates is directly related to the level of Aβ and Braak tangle stage, and predicts cognitive status in Lewy body diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13195-014-0077-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4248436  PMID: 25452767
17.  Differential modification of Cys10 alters transthyretin's effect on beta-amyloid aggregation and toxicity 
Tg2576 mice produce high levels of beta-amyloid (Aβ) and develop amyloid deposits, but lack neurofibrillary tangles and do not suffer the extensive neuronal cell loss characteristic of Alzheimer's disease. Protection from Aβ toxicity has been attributed to up-regulation of transthyretin (TTR), a normal component of plasma and cerebrospinal fluid. We compared the effect of TTR purified from human plasma (pTTR) with that produced recombinantly (rTTR) on Aβ aggregation and toxicity. pTTR slowed Aβ aggregation but failed to protect primary cortical neurons from Aβ toxicity. In contrast, rTTR accelerated aggregation, while effectively protecting neurons. This inverse correlation between Aβ aggregation kinetics and toxicity is consistent with the hypothesis that soluble intermediates rather than insoluble fibrils are the most toxic Aβ species. We carried out a detailed comparison of pTTR with rTTR to ascertain the probable cause of these different effects. No differences in secondary, tertiary or quaternary structure were detected. However, pTTR differed from rTTR in the extent and nature of modification at Cys10. We hypothesize that differential modification at Cys10 regulates TTR's effect on Aβ aggregation and toxicity.
PMCID: PMC2719498  PMID: 19549717
Alzheimer's disease; beta-amyloid; post-translational modification; transthyretin
18.  Presence of N-glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD 
Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N-glycosylation N-D-S at position 98, it is not secreted as a glycoprotein. The most common FAP-associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild-type and mutant V30M; SMT was undetectable upon N-glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M - FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.
PMCID: PMC3823024  PMID: 23387326
Transthyretin; N- glycosylation; ERAD
19.  Dementia in Parkinson's Disease Correlates with α-Synuclein Pathology but Not with Cortical Astrogliosis 
Parkinson's Disease  2012;2012:420957.
Dementia is a common feature in Parkinson's disease (PD) and is considered to be the result of limbic and cortical Lewy bodies and/or Alzheimer changes. Astrogliosis may also affect the development of dementia, since it correlates well with declining cognition in Alzheimer patients. Thus, we determined whether cortical astrogliosis occurs in PD, whether it is related to dementia, and whether this is reflected by the presence of glial fibrillary acidic protein (GFAP) and vimentin in cerebrospinal fluid (CSF). We have examined these proteins by immunohistochemistry in the frontal cortex and by Western blot in CSF of cases with PD, PD with dementia (PDD), dementia with Lewy bodies (DLB) and nondemented controls. We were neither able to detect an increase in cortical astrogliosis in PD, PDD, or DLB nor could we observe a correlation between the extent of astrogliosis and the degree of dementia. The levels of GFAP and vimentin in CSF did not correlate to the extent of astrogliosis or dementia. We did confirm the previously identified positive correlation between the presence of cortical Lewy bodies and dementia in PD. In conclusion, we have shown that cortical astrogliosis is not associated with the cognitive decline in Lewy body-related dementia.
PMCID: PMC3347756  PMID: 22577599
20.  Levels of cerebrospinal fluid α-synuclein oligomers are increased in Parkinson’s disease with dementia and dementia with Lewy bodies compared to Alzheimer’s disease 
The objective was to study whether α-synuclein oligomers are altered in the cerebrospinal fluid (CSF) of patients with dementia, including Parkinson disease with dementia (PDD), dementia with Lewy bodies (DLB), and Alzheimer disease (AD), compared with age-matched controls.
In total, 247 CSF samples were assessed in this study, including 71 patients with DLB, 30 patients with PDD, 48 patients with AD, and 98 healthy age-matched controls. Both total and oligomeric α-synuclein levels were evaluated by using well-established immunoassays.
The levels of α-synuclein oligomers in the CSF were increased in patients with PDD compared with the controls (P < 0.05), but not in patients with DLB compared with controls. Interestingly, the levels of α-synuclein oligomers in the CSF were also significantly higher in patients with PDD (P < 0.01) and DLB (P < 0.05) compared with patients with AD. The levels of CSF α-synuclein oligomers and the ratio of oligomeric/total-α-synuclein could distinguish DLB or PDD patients from AD patients, with areas under the curves (AUCs) of 0.64 and 0.75, respectively. In addition, total-α-synuclein alone could distinguish DLB or PDD patients from AD patients, with an AUC of 0.80.
The levels of α-synuclein oligomers were increased in the CSF from α-synucleinopathy patients with dementia compared with AD cases.
PMCID: PMC4075410  PMID: 24987465
21.  Neuronal Production of Transthyretin in Human and Murine Alzheimer’s Disease: is it Protective? 
Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with β-amyloid (Aβ) in vitro, inhibits Aβ fibril formation and suppresses the Alzheimer’s disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product up-regulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. QPCR for TTR mRNA and western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aβ pulled down TTR/Aβ complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments recombinant human TTR suppressed the cytotoxicity of soluble Aβ aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aβ, its precursor or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aβ/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aβ and/or its related peptides may play a role in natural resistance to human AD.
PMCID: PMC3172869  PMID: 21880910
Transthyretin; Alzheimer’s Disease; Beta Amyloid (Aβ); APP23 mice; Amyloidosis
22.  Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer’s disease 
Compromised secretory function of choroid plexus (CP) and defective cerebrospinal fluid (CSF) production, along with accumulation of beta-amyloid (Aβ) peptides at the blood-CSF barrier (BCSFB), contribute to complications of Alzheimer’s disease (AD). The AD triple transgenic mouse model (3xTg-AD) at 16 month-old mimics critical hallmarks of the human disease: β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) with a temporal- and regional- specific profile. Currently, little is known about transport and metabolic responses by CP to the disrupted homeostasis of CNS Aβ in AD. This study analyzed the effects of highly-expressed AD-linked human transgenes (APP, PS1 and tau) on lateral ventricle CP function. Confocal imaging and immunohistochemistry revealed an increase only of Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries; this buildup may reflect insufficient clearance transport from CSF to blood. Still, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: the low density lipoprotein receptor-related protein 1 (LRP1) and the receptor for advanced glycation end product (RAGE). A thickening of the epithelial basal membrane and greater collagen-IV deposition occurred around capillaries in CP, probably curtailing solute exchanges. Moreover, there was attenuated expression of epithelial aquaporin-1 and transthyretin (TTR) protein compared to Non-Tg mice. Collectively these findings indicate CP dysfunction hypothetically linked to increasing Aβ burden resulting in less efficient ion transport, concurrently with reduced production of CSF (less sink action on brain Aβ) and diminished secretion of TTR (less neuroprotection against cortical Aβ toxicity). The putative effects of a disabled CP-CSF system on CNS functions are discussed in the context of AD.
PMCID: PMC4319477
Alzheimer disease; 3xTg-AD mice; choroid plexus; dysfunction; amyloid-β; collagen-IV; transthyretin; aquaporin-1
23.  Transthyretin Cardiac Amyloidoses in Older North Americans 
The amyloidoses are a group of hereditary or acquired disorders caused by the extracellular deposition of insoluble protein fibrils that impair tissue structure and function. All amyloidoses result from protein misfolding, a common mechanism for disorders in older persons including Alzheimer's disease and Parkinson's disease. Cardiac amyloidoses in the elderly are most often caused by abnormalities in the protein transthyretin (TTR), a serum transporter of thyroxine and retinol. Mutations in TTR can result in familial amyloidotic cardiomyopathy, and wild-type TTR can result in senile cardiac amyloidosis. These underdiagnosed disorders are much more common than previously thought. The resulting restrictive cardiomyopathy can cause congestive heart failure, arrhythmias, and advanced conduction system disease. Although historically difficult to make, the diagnosis of TTR cardiac amyloidosis has become easier in recent years with advances in cardiac imaging and more widespread use of genetic analysis. While therapy to this point has largely involved supportive medical care, avoidance of potentially toxic agents, and rarely organ transplantation, the near future brings the possibility of targeted pharmacotherapies designed to prevent TTR misfolding and amyloid deposition. As these disease modifying agents are designed to prevent disease progression, it has become increasingly important that older persons with TTR amyloidosis be expeditiously identified and considered for enrollment in clinical registries and trials.
PMCID: PMC3325376  PMID: 22329529
amyloidosis; transthyretin; cardiomyopathy; heart failure; aging
24.  Chronic lithium administration down regulates transthyretin mRNA expression in rat choroid plexus 
Transthyretin (TTR) accounts for a quarter of the protein content of ventricular cerebrospinal fluid (CSF) yet its exact role in the brain remains unknown. Patients with a diagnosis of depression have reduced CSF levels of TTR and the locus encoding the TTR gene has been implicated in a Danish pedigree of bipolar patients. Lithium, the major treatment for bipolar disorder in the UK, was subcutaneously infused into rats for 28 days in the form of lithium chloride using osmotic minipumps. In situ hybridizations using oligonucleotide probes targeted against the TTR transcript were performed on coronal brain sections. Lithium significantly reduced the level of transthyretin mRNA in the rat choroid plexus within the lateral and third ventricle. The down-regulation was confirmed using semi-quantitative reverse transcription PCR on dissected brain tissue. Recent studies in mice suggest that the TTR gene is implicated in depression-like behavior therefore this effect of lithium may be relevant to its use as a mood stabilizer or an adjuvant to antidepressant drugs.
PMCID: PMC2671955  PMID: 19412503
bipolar disorder; in-situ hybridization; mood stabilizer; minipump; (SQ)-RT-PCR
25.  Validation of Serum Markers for Blood-Brain Barrier Disruption in Traumatic Brain Injury 
Journal of Neurotrauma  2009;26(9):1497-1507.
The blood-brain barrier (BBB), which prevents the entry into the central nervous system (CNS) of most water-soluble molecules over 500 Da, is often disrupted after trauma. Post-traumatic BBB disruption may have important implications for prognosis and therapy. Assessment of BBB status is not routine in clinical practice because available techniques are invasive. The gold-standard measure, the cerebrospinal fluide (CSF)-serum albumin quotient (QA), requires the measurement of albumin in CSF and serum collected contemporaneously. Accurate, less invasive techniques are necessary. The objective of this study was to evaluate the relationship between QA and serum concentrations of monomeric transthyretin (TTR) or S100B. Nine subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score ≤ 8) and 11 subjects with non-traumatic headache who had CSF collected by ventriculostomy or lumbar puncture (LP) were enrolled. Serum and CSF were collected at the time of LP for headache subjects and at 12, 24, and 48 h after ventriculostomy for TBI subjects. The QA was calculated for all time points at which paired CSF and serum samples were available. Serum S100B and TTR levels were also measured. Pearson's correlation coefficient and area under the receiver operating characteristic (ROC) curve were used to determine the relationship between the serum proteins and QA. Seven TBI subjects had abnormal QA's indicating BBB dysfunction. The remaining TBI and control subjects had normal BBB function. No significant relationship between TTR and QA was found. A statistically significant linear correlation between serum S100B and QA was present (r = 0.432, p = 0.02). ROC analysis demonstrated a significant relationship between QA and serum S100B concentrations at 12 h after TBI (AUC = 0.800; SE 0.147, 95% CI 0.511–1.089). Using an S100B concentration cutoff of 0.027 ng/ml, specificity for abnormal QA was 90% or higher at each time point. We conclude that serum S100B concentrations accurately indicate BBB dysfunction at 12 h after TBI.
PMCID: PMC2822805  PMID: 19257803
biomarkers; blood-brain barrier dysfunction; traumatic brain injury

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