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Transthyretin (TTR) is secreted by hepatocytes, retinal pigment epithelial
cells, pancreatic
Transthyretin (TTR) was first identified in the 1940s as a human serum and
cerebrospinal fluid (CSF) protein [1]. Initially classified based on its
electrophoretic mobility (prealbumin), it was more precisely described as
thyroxine binding prealbumin (TBPA) after it was shown to bind T4 in plasma [2].
The molecule was formally named transthyretin after being found to bind plasma
retinol binding protein charged with retinol, hence its official name,
Transporter of Thyroxine and Retinol binding protein
(RBP) [3]. Two laboratories independently identified functional TTR mRNA and
protein in both the liver and the choroid plexus in rats and humans, suggesting
that CSF TTR was likely to be synthesized locally [4, 5]. The observed
TTR transcriptional responses to inflammation differed between
hepatocytes and choroidal plexus epithelial (CPE) cells, indicating that the gene
was probably regulated differently in the two organs [6]. In the liver,
transcription was regulated by a series of transcription factors (Hepatocyte nuclear factors 1 [HNF1], 3, 4)
that were suppressed by the pro-inflammatory cytokines IL-1 and tumor necrosis factor alpha (TNF
A possible association between Alzheimer’s disease (AD) and CSF TTR was first addressed in a survey of CSF proteins in three groups of Finnish subjects: patients hospitalized for dementia (n = 10), patients ambulatory but with evidence of cognitive impairment (n = 22), and an age-matched group of individuals without defined intellectual deficits (n = 22). In this relatively crude analysis, immunoglobulin classes, haptoglobin, transferrin, albumin, and TTR (as prealbumin) in serum and CSF were measured nephelometrically. The only significant finding with respect to TTR was a lower mean serum level in the institutionalized AD patients, which appeared to be a function of their nutritional status, since the serum albumin concentration also trended lower in this group than in the controls or the ambulatory AD cohort [9]. CSF TTR concentrations did not differ significantly among the three clinical groups. In a subsequent publication, the same investigators compared the values in the ambulatory AD group with similar measurements in 29 patients with multi-infarct dementia and did not report any significant differences in CSF or serum prealbumin or albumin levels [10].
In a contemporaneous Norwegian study, CSF TTR concentrations in 24 subjects with
dementia of the Alzheimer type, seven with multi-infarct dementia, 14 age- and
sex-matched, non-demented individuals with a variety of medical conditions, a
younger group with multiple sclerosis (n = 17), a group with amyotrophic lateral
sclerosis (n = 6), and a group with post subarachnoid hemorrhage patients (n =
10) were measured. The degree of dementia was determined using the no longer
utilized Roth dementia scale and the concentration of TTR in serum and CSF was
measured by rocket immunoelectrophoresis [11]. The only patient group that had a
significantly different mean CSF TTR concentration was the sub-arachnoid
hemorrhage cohort in which it was lower. The authors assumed this to be a
function of reduced choroid plexus function. However, they did find a significant
inverse correlation between the level of dementia as measured by the Roth score
and the total CSF TTR concentration (p
At the time of these analyses, A
The demonstration that human CSF could inhibit A
The first published systematic examination of CSF TTR concentrations in AD
performed after the in vitro demonstration of inhibition of A
A later report describing CSF TTR concentrations (as determined by radial
immunodiffusion) in 49 individuals ranging in age from 27 to 82 years with a
variety of neurologic disorders, but none with a history of dementia, stroke, or
recent head trauma, found no relationship between ApoE genotypes and TTR
levels nor with A
An examination of CSF obtained by lumbar puncture from 26 patients with AD, eight with vascular dementia and 18 age- and sex-matched controls, was designed to enhance the quantitative sensitivity of the determination of proteins present in low concentrations by removing high abundance serum proteins (Blue Sepharose for albumin, protein G Sepharose for immunoglobulins, and an immunosorbent column with a multi-specific anti human serum protein antibodies). The “cleared” CSF was then separated by micro-reverse high-performance liquid chromatography (HPLC) and the fractions analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Mass spectrometry was performed on some fractions and some samples were analyzed by isoelectric focusing. The results of the isoelectric point and mass spectrometry analyses were not conclusive. When the TTR quantitation was performed by nephelometry with a commercially available TTR-specific antibody, total CSF TTR in AD patients was lower than in controls or patients with vascular dementia. Serum TTR in the three dementia groups was higher than controls, but not significantly so [34].
A post-mortem analysis, in which brain (middle temporal gyrus, middle frontal
gyrus, inferior parietal lobule, hippocampal CA1) and ventricular CSF samples
were obtained within 24 hours of death, was performed with material obtained from
20 patients with confirmed AD and 10 sex- and age-matched controls without AD or
any other neurologic diseases (mean ages 84.4 years and 83.1 years respectively)
although eight had cerebrovascular disease (three with multi-infarct dementia).
There was no significant difference in CSF ApoE concentrations between the AD and
control cohort, but the TTR levels were significantly lower in the AD subjects
(p = 0.0094). There appeared to be an inverse relationship between TTR
concentration and senile plaque number as determined immunohistochemically (using
antihuman
A dementia-focused analysis of CSF samples from 106 elderly (ages 66–74) German
individuals with a variety of conditions including AD (n = 23), Creutzfeldt Jakob disease (CJD) (n = 18),
dementia with Lewy bodies (DLB) (n = 23), frontotemporal dementia (FTD) (n = 10), normal pressure
hydrocephalus (NPH) (n = 13), and 19 non-demented controls used enzyme-linked
immunosorbent assays (ELISAs) for Tau, A
CSF concentrations of TTR, Cystatin C,
The first serious attempt to broadly examine CSF proteomics in AD utilized micro
2D gel analysis with SYPRO staining and mass spectrometric analysis of eluted
proteins in samples obtained from 15 AD patients (diagnosed clinically according
to NINCDS-ADRDA criteria with a mean age of 77.2 years) and 12 controls (mean age
67.3 years) participating in a longitudinal geriatric population study in
Northern Sweden. The investigators found increases in RBP, TTR (not significant),
In a subsequent study, a modification of this approach was used, in which
iso-electric focusing was incorporated into the preparation for 2 dimensional gel
electrophoresis (2DGE) in CSF samples to enhance the detection of changes in
proteins present in low concentrations, from a small number (n = 7) of AD patients
and controls (n = 7). The pre-clearing procedure resulted in the identification of
nine proteins that differed significantly in the AD patients from the controls.
Only
In a technically more sophisticated manner, CSF samples, obtained within 4 hours
of death from patients in whom brain pathology confirmed the presence or absence
of AD, were analyzed. The samples were pooled from 43 AD and 43 non-demented
subjects and analyzed by 2D gel electrophoresis. Protein spots exhibiting
differences in the two pools were recovered and analyzed by matrix-assisted laser
desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Hemopexin and
two pigment epithelium-derived isoforms (PEDFs) were shown to be higher in the AD
pool, while ApoA1, Cathepsin D, and TTR were significantly reduced [40]. In two
follow-up studies, the pools from the same AD and elderly non-demented patients
were compared with a non-AD dementia group (17 subjects) using an improved 2DGE
technique. Twenty-one different proteins showed differences in the comparisons
among the pools. TTR was significantly lower in both the non-AD demented and the
AD subject pools than in the normal pool, without significant differences between
the two dementia groups. An increase in hemopexin was confirmed, but no increase
in the PEDFs, ApoA1, or cathepsin D was seen [41]. In a parallel analysis, the
same investigators used commercially available ELISAs for ApoA1,
In a similar 2DGE analysis of CSF from 30 Korean subjects with either mild cognitive impairment (n = 3) or overt AD (n = 27) evaluated using the clinical dementia rating (CDR) to group the subjects, 350 spots were detected. The investigators found that retinol binding protein decreased with an increase in the CDR. While they also found a decrease in haptoglobin precursor 1 they did not report any results for CSF TTR that could have been responsible for the decrease in RBP, as it is the main carrier of RBP charged with retinol in the plasma and the CSF, thus diminishing the informative value of the study with respect to TTR [43].
By 2011 it appeared likely that lower mean levels of CSF A
To define longitudinal changes in CSF biomarkers defined in a discovery analysis
of CSF pools from controls (n = 10), mild cognitive impairment (MCI) (n = 5) subjects and
patients with fully expressed AD dementia (n = 45) a 1.2-fold increase in TTR was
observed in both MCI and AD pools relative to controls. However, when samples
collected from individual members of the pools were longitudinally examined by a
multiplex proteomic assay over time and compared with changes in
A
In a very small study, CSF was obtained from four age- and sex-matched cognitively normal controls, four subjects defined as having MCI and four with mild AD. The samples were treated with protease inhibitors and the endogenous peptides filtered, concentrated, desalted in formic acid, dried down, resuspended, and examined by LC-MS/MS analysis. 645 peptides, representing 93 protein precursors were identified. In parallel, aliquots of the same samples were passed through a wheat germ agglutinin (WGA) column with the flow through discarded (and not analyzed) and the adherent proteins eluted, tryptic digested and subject to mass spectrometric analysis. In this study it appeared that the TTR concentrations in the MCI subjects were higher than the controls and those from the AD subjects lower. Surprising in this analysis was the identification of TTR peptides in the WGA eluted fraction, since WGA should selectively bind glycoproteins and TTR is not generally glycosylated, although there have been some reports indicating that under some circumstances this might be true. If these findings, obtained in a unique analytic mode, were correct, they would be consistent with the notion that CSF TTR is increased early in AD pathogenesis and diminished in the severely affected. Alternatively, the results may be an artifact of the preparative methodology [46].
A combined study including clinically diagnosed AD (n = 59) and DLB (n = 13) patients and age matched controls from institutions in Denmark, and Sweden explored CSF TTR levels and their relationship to the presence of depression. Mini-mental state examination showed that the AD and DLB patients were clearly more compromised than the controls. There were no differences in mean CSF TTR concentrations as determined immunochemically (enhanced Mancini method). However, TTR was significantly lower in AD patients treated with cholinesterase inhibitors, a phenomenon not previously reported. It was not stated whether these patients received the anti-cholinesterase drugs because they had more severe disease than those who did not. The investigators concluded from their work and previously published studies that “CSF TTR does not appear to be a robust biomarker for differentiating AD from DLB and controls in all cohorts”. They also could not reproduce the results from prior published studies indicating that CSF TTR levels were low in depression [47].
In a study focusing on Lewy body dementia (LBD) with AD defined clinically (and
with decreased CSF A
Two studies examined the oxidation status of TTR Cys10 in CSF from AD subjects to determine if it had any relationship to pathogenesis. In the first, CSF samples from 39 patients identified clinically as having probable AD (CDR 1–1.5) were compared with those from 27 cognitively normal individuals matched for sex, although not necessarily age. Using two different mass spectrometry protocols to quantitate -Cys-Cys and -Cys-Gly, they found the conjugated forms to be significantly less abundant in the AD cohort with individual AUCs of 0.893 and 0.866, making them reasonably efficient in sorting out CSF from AD and non-AD subjects. Unfortunately, the study did not report total TTR concentrations immunologically in order to determine if they were higher or lower than normal; the modifications detected reflected changes in total CSF TTR [50].
In a later study, using rocket immunoelectrophoresis to measure total TTR and
immunoaffinity isolation and mass spectrometry to specifically examine
S-cysteinylation, S-cysteinylglycinylation, and S-glutathionylation in clinically
defined age- and sex-matched AD patients (n = 37), patients with mild cognitive
impairment (MCI) (n = 17), patients with normal pressure hydrocephalus (n = 15), and
healthy controls (n = 7), the AD and MCI patients had a significantly higher fraction
of oxidatively modified CSF TTR, a clear difference from the findings in the
earlier analysis. Mean total TTR was about the same in the AD and NPH patients,
being somewhat higher than in the MCI and normal control cohorts. Parallel
studies of the same samples using standard ELISAs for A
In an analysis of a different post-translational modification of TTR, carbonylated CSF proteins in a small number of AD subjects by 2D gel (oxy-blotting) and mass spectrometry, TTR was among the most abundant carbonylated proteins, but it was significantly decreased in patients with probable AD relative to controls, suggesting that reduced epitope exposure was not responsible. However, the study did not measure total TTR concentration immunologically [52].
A broad review of proteomic analysis of CSF across the entire spectrum of intrinsic and extrinsic neurologic diseases failed to show any specific changes in TTR or its peptides in any of the studies analyzed. Hence, this exercise was not informative for our analysis [53]. A metanalysis, reviewing publications in PubMed from 2012 to 2017 found 28 papers reporting proteomic analyses of CSF that included AD as one of the diagnostic groups, emphasized the changes in technology that had been introduced during this period and cited several examples of potential biomarkers. Only one study of the 28 found significant changes in TTR [54]. A later metanalysis identified 14 studies that reported a difference between CSF TTR concentrations in AD patients and controls. The only proteins in which there were more studies showing differences were ApoE and nerve growth factor induced peptide VGF. TTR (and AponerE) were found to be increased and decreased in different proteomic studies, prompting the authors to comment, “suggesting that they cannot be considered as reliable CSF biomarkers of AD. While it is likely that heterogeneity in response direction may reflect irrelevant physiological or environmental factors, it is also interesting to speculate that the heterogeneity reflects unknown endophenotypes in AD or provides an indication of the profound general protein dysregulation that occurs during AD progression”. They also noted that RBP4 was reduced in four of five studies and that it circulates bound to TTR and it was unchanged in preclinical AD [55].
Reviewing AD biomarkers in 2021, the authors noted that “it is now established
that in AD, CSF A
Across a broad range of studies, it appears that CSF TTR determinations are not consistent within AD populations nor specific enough with respect to other neurodegenerative disorders to be clinically useful in determining the diagnosis or prognosis of AD or response to therapy. Nonetheless, given the frequency of the observations regarding TTR in AD patients, it is worth considering their relevance with respect to the pathogenesis of neurodegeneration and why they were considered in the first place.
There are three possible technical explanations as to why TTR has appeared to fail as an AD biomarker. The assay/measurement itself may have technical issues. TTR ELISAs have been around for a long time and have been reasonably reliable in looking at serum TTR concentrations, particularly of the wildtype molecule. Although it has been noted that the concentrations of some mutant TTRs are not accurately measured by all commercially available antibodies. It was suggested in some of the proteomic studies that post translational modifications of TTR, particularly at cys10, could have changed the reactivity with the antibodies, although the Poulsen study suggests this is not the case [51].
There are clearly differences when the same samples or sample pools are assayed
by both proteomic and immunochemical techniques. Several of the proteomic
analyses discuss the variation in the stoichiometry of the TTR peptides in 2DGE
or mass spectrometry; the former is perhaps related to post-translational change
while the latter may reflect variation in the cleavage of the protein either in
the preliminary proteolytic step or during the mass spectrometric analysis
itself. It is also possible that in some of the studies that used pre-clearing of
highly abundant proteins from the CSF samples, the TTR may have been
non-specifically bound to the presumably specifically cleared protein species. In
no instance was it stated that the CSF was assayed before and after
pre-clearance. There is no good reason why TTR should be specifically affected by
the pre-clearance; nonetheless, it would have been reassuring to see analyses of
the total and the pre-cleared CSF as well as the proteins included in the
clearance fraction. None of the studies examined the CSF for TTR–A
An additional potential source of the inconsistency may be related to the
clinical staging of the patient populations studied. Various clinical staging
systems have been used to assess the patients whose CSF was being assayed. It is
likely that the clinical classification systems are relatively crude measures of
the pathologic and biochemical changes taking place intra-cerebrally. Patients
within a given clinical class may also show considerable pathologic variation,
although this has not been examined in detail. The use of PET scan diagnostics
and the reasonably well correlated levels of CSF A
It is our conclusion, despite our reservations and the inconsistencies in the
studies summarized above, that CSF TTR may be increased early in the evolution of
AD but that in full blown, anatomically recognized AD, CSF TTR concentration is
lower than normal. In the absence of definitive data supporting that conclusion,
the potential dynamics of CSF TTR concentration related to the rate of
pathogenesis of AD makes TTR an unreliable marker for either prognosis,
diagnosis, or response to therapy. It would have been interesting if some of the
recent studies showing plaque clearance by PET scanning had included serial
determinations of CSF TTR to assess whether there was a systematic change related
to the reduction in A
Given the well documented observations that TTR inhibits A
JNB wrote the manuscript.
Not applicable.
Not applicable.
This research received no external funding.
The author is a member of the Scientific Advisory Board of Protego Biopharma, but has no conflict of interest with respect to the subject of this paper.
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