Abstract
We analyzed Lewy body (LB) pathology in 18 autosomal dominant Alzheimer’s disease (ADAD) brains via immunohistochemistry. Real-time quaking induced conversion was used to detect misfolded α-synuclein (α-syn) in 18 living ADAD cerebrospinal fluid (CSF) samples. Concomitant LB pathology was present in 44% ADAD brains. Only 6% CSF samples were positive for misfolded α-syn. In an additional AD sample, all patients with confirmed LB presented misfolded α-syn in postmortem CSF regardless of the LB staging. In conclusion, misfolded α-syn in CSF was scarce in symptomatic living ADAD individuals, in contrast to postmortem brain tissue. These results suggest late appearance of LB pathology in ADAD.
Keywords
INTRODUCTION
Autosomal dominant Alzheimer’s disease (ADAD) is a rare form of Alzheimer’s disease (AD) caused by mutations in PSEN1, PSEN2, or APP genes. Both sporadic AD (sAD) and ADAD are characterized by extracellular amyloid-β (Aβ) plaque accumulation and intracellular aggregation of hyperphosphorylated tau proteins ending up in synaptic and neuronal loss [1]. AD brains can also contain other pathological aggregates, such as Lewy bodies (LBs), whose main component is misfolded α-synuclein (α-syn) [2]. LBs are observed in 25% to 50% of both sAD and ADAD brains [3, 4]. However, the pathological mechanisms linking α-syn aggregation and AD and the timing of α-syn accumulation in these subjects are not well understood.
Aggregated abnormal α-syn might be used as a biomarker for α-synucleinopathies [5]. The real-time quaking-induced conversion (RT-QuIC) technique has proven to be highly specific and sensitive to detect pathological α-syn seeds in the cerebrospinal fluid (CSF) of patients with LB diseases, even during the premotor stages of the disease [6, 7]. It has yet to be evaluated whether this technique can detect misfolded α-syn when presented as concomitant pathology with other primary proteinopathies, including AD.
The objective of our study was to gain a deeper understanding of the presence of abnormal α-syn in ADAD. We first describe the presence of LB pathology in our ADAD postmortem cohort and determine the seeding activity of misfolded α-syn in CSF from living symptomatic ADAD patients. Finally, we evaluate the correlation between the detection of α-syn seeding activity with the RT-QuIC method in postmortem CSF and the presence and staging of LB in AD brains.
METHODS
ADAD cohort
The study included a cohort of 18 neuropathologically-confirmed ADAD patients (14 males and 4 females aged 36–78 years, mean 54.06±7.52 years; 14 ADAD patients carried a PSEN1 mutation and 4 carried an APP mutation) (Table 1) and 18 living symptomatic ADAD individuals: 17 carrying a PSEN1 mutation and 1 an APP mutation (8 males and 10 females aged 26–59 years, mean 44.89±8.46 years) (Table 2).
The Hospital Clínic de Barcelona Ethics committee approved the study (HCB/2020/1410) and all the participants signed written informed consent for brain or CSF donation for research.
Neuropathology examination
Brain samples were obtained from the Neurological Tissue Bank (NTB), Biobank-Hospital Clínic-FRCB-IDIBAPS, Barcelona, Spain. Neuropathological examination was performed according to standardized protocols [8]. Main primary antibodies in the immunohistochemistry (IHC) protocol for this study were: anti-βA4 (6F/3D, Dako, Glostrup, Denmark), anti-tau (AT8; Thermo Scientific, USA), anti-α-synuclein (5G4; Analytik Jena, Germany), anti-TDP-43 phospho Ser409/410 (11-9; Cosmo Bio, Japan). Disease evaluation was performed according to international consensus criteria [9, 10].
For α-syn aggregates evaluation, the regions of the brainstem, amygdala, and olfactory bulb were first examined. If α-syn pathology was observed, then the study was expanded to the rest of the recommended areas. With this, AD patients were classified as AD patients without α-syn pathology or with LB pathology (olfactory only, amygdala predominant, brainstem, limbic, and neocortical). In only two subjects with ADAD without α-syn pathology in other regions, an olfactory-only pathology could not be ruled out due to lack of tissue (Table 1).
All brain donors with AD died in a stage of severe dementia.
CSF sampling
CSF samples from living individuals were obtained and processed as previously described [11].
Postmortem CSF samples were obtained from the NTB. Only 2 postmortem CSF samples were available from ADAD individuals. We increased the sample with postmortem CSF from 9 neuropathologically confirmed sAD, to reach a total number of 11 samples (7 males and 4 females aged 44–86 years, mean 68.18±11.79 years; PM19-27). Ventricular postmortem CSF was collected at the time of brain extraction into 15 ml siliconized polypropilene tubes. CSF was centrifuged at 4°C, 4000xg for 10 min, then transferred to 1.5 mL siliconized polypropilene tubes and immediately frozen at –80°C.
Real-time quaking induced conversion (RT-QuIC) analysis
RT-QuIC buffer composition was as follows: 10 mM phosphate buffer (pH 8), 10μM Thioflavin-T (ThT), and 0.1 mg/mL human recombinant full-length (1–140 aa) α-syn (Sigma-Aldrich, Cambridge, UK). Reactions were prepared in a black 96-well, optical bottomed plate (Nalgene Nunc International, #265301) with 85μL of RT-QuIC buffer and 15μL undiluted CSF for a final reaction volume of 100μL. Each sample was run in triplicate. The plates were sealed and incubated in a BMG OPTIMA FluoSTAR plate reader at 35°C for 120 h with intermittent shaking cycles: double orbital with 1 min shake (400 rpm), 14 min rest. ThT fluorescence measurements (450 nm excitation and 480 nm emission) were taken every 15 min. A positive response was defined as a relative fluorescence unit (rfu) value of 60.000. A positive response in two or more of the replicates was considered positive.
RESULTS
Neuropathological diagnosis in postmortem ADAD brains
AD staging revealed an A3B3C3 score in 17 subjects (94.4%) and an A3B2C3 score due to Braak IV in 1 subject (5.6%; PM4, Table 1). α-syn aggregates in the form of LB were present in 8 subjects (44.4%). In 4 positive subjects the deposits were present predominantly in amygdala (50%), in 2 deposits were limbic (25%), in 1 neocortical (12.5%) and in 1 were present in olfactory bulb only (12.5%) (Table 1). In addition, TDP-43 concomitant pathology was observed in 1 patient (5.6%, PM9) (LATE-NC stage 2).
Neuropathological data from the 18 ADAD postmortem (PM) subjects
PM, postmortem; PSEN, Presenilin; APP, Amyloid-beta precursor protein. *olfactory bulb not available.
RT-QuIC in CSF from living ADAD individuals
Analysis of CSF samples via RT-QuIC from living ADAD showed that only one PSEN1 mutation carrier was positive for misfolded α-syn (5.6%) (Table 2).
Misfolded α-syn CSF RT-QuIC results from the 18 AD living (L) subjects and patient characteristics per diagnosis
L, Living; PSEN, Presenilin; APP, Amyloid-beta precursor protein.
RT-QuIC in postmortem CSF and IHC results comparison
Seven (63.6%) postmortem CSF samples from AD patients (1 ADAD and 6 sAD) were positive for misfolded α-syn using the RT-QuIC technique. All of them showed the presence of LB at the IHC at different stages (1 amygdalar, 3 limbic, 3 neocortical). In the same way, all the patients with positive IHC presented positive CSF results. None of the AD cases without LB pathology or the negative control used were RT-QuIC positive even after 120 h (Fig. 1).
RT-QuIC detection of pathological α-synuclein (α-syn) seeding activity in CSF from 11 AD postmortem samples. RT-QuIC kinetics measured by relative ThT fluorescence (rfu) from CSF of each individual cases with ADAD (PM6 and PM8) and with sAD (PM19–27) at 120 h were positive (+) for one ADAD individual (PM6) and 6 sAD subjects (PM22–27). Each curve consists of 3 duplicates. PM, postmortem; PBS+BSA1%, negative control; C+ 1/1000, positive control.
DISCUSSION
In the present study, we observed that 44% of ADAD brains showed LB pathology, the majority with predominant amygdala deposits. In contrast, only one ADAD patient carrying one PSEN1 mutation, 6% of samples tested from symptomatic living ADAD individuals, presented in CSF detectable abnormal α-syn using RT-QuIC technology.
The presence of LB pathology in ADAD in our cohort was similar to previous cohorts, where they found LB presence in 50% of ADAD postmortem cases [3, 4]. The low level of misfolded α-syn RT-QuIC positivity in CSF from living symptomatic ADAD individuals was unexpected based on previous and own data regarding the postmortem IHC. This discrepancy could suggest a low sensitivity of the technique to detect abnormal α-syn in these individuals. Another possibility might be that the α-syn pathology appears late in the course of the disease in ADAD, as all the individuals tested were in mild-moderate phases of the disease.
To investigate the cause of this discrepancy, we evaluated postmortem CSF and correlated the RT-QuIC results with the presence of LB pathology at postmortem brain, in samples from ADAD and sAD. CSF postmortem samples from patients with LB pathology were positive for abnormal α-syn using the RT-QuIC technique, unrelated to the stage of LB pathology (amygdalar, limbic, or neocortical) and vice versa. The CSF negative cases were also negative at IHC. Thus, postmortem CSF abnormal α-syn RT-QuIC results and the presence of LB pathology showed a complete concordance, supporting that the seeding evaluation of α-syn using RT-QuIC is a reliable technique for the study of the presence of LBs in AD. In a recent study, the sensibility of postmortem CSF using the α-syn RT-QuIC technique to detect LBs in 14 patients with LBs as concomitant pathology in neuropathological confirmed AD, was lower, showing that only 64% of the samples were positive [12]. In the same study, however, the sensitivity of the α-syn RT-QuIC ranged from 100% in those cases with cortical LBs to 37,5% in cases with olfactory bulb only or brainstem predominant LB (not included in our sample), suggesting that the positivity of the postmortem assay depends on the distribution of the LBs. In another study performed in cognitive impaired sporadic patients, 21% of participants with AD presented positive CSF α-syn RT-QuIC, a higher percentage of that observed in our study in ADAD, and much closer to the prevalence (33–42%) of LBs in neuropathological studies in sAD [13]. In this sense, these findings would then support the late appearance of relevant LB pathology in most ADAD individuals.
The presence of TDP-43 concomitant pathology was rare in this population (5.6%, LATE-NC stage 2), compared to late onset AD [3], which would suggest that even late, the concomitant presence of LB pathology is specific to AD [14], even in young genetic individuals.
We should, however, acknowledge we cannot exclude that the origin of the CSF (intraventricular in the postmortem in contrast to lumbar CSF in living individuals) might influence the results. Due to the limited number of postmortem CSF samples from ADAD (2), we included postmortem sAD samples in the analysis, although the performance of both subtypes of AD seemed quite similar. Because there were only 2 ADAD postmortem CSF samples, we can only speculate that the postmortem CSF α-syn RT-QuIC technique might perform in a similar way than in sAD.
In sAD, the positivity of syn RT-QuIC is associated to a faster cognitive decline [13]. At this point, there is not enough information to evaluate the clinical impact of the presence of LB pathology in ADAD. However, we can speculate that introducing α-syn RT-QuIC testing in the evaluation of ADAD individuals could help to stratify patients in terms of prognosis or response to therapeutic interventions.
Altogether, we provide new data about the presence and detection of α-syn aggregates in ADAD that suggest a late appearance of LB pathology in these individuals. The frequency and specificity of this concomitant pathology in postmortem species in ADAD suggest a direct result of the biological process. Additional studies with a broader sample are needed to confirm these results.
Footnotes
ACKNOWLEDGMENTS
We thank all the participants for their generosity as without them none of this research would have been possible. We are indebted to the Biobanc-Hospital Clinic-FRCB-IDIBAPS for samples and data procurement. We are also grateful to AGAUR SGR 2021/01126 Generalitat de Catalunya.
FUNDING
This work has been funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, “Una manera de hacer Europa” (PI20/00448 to Dr Sánchez-Valle and PI17/00670 to Dr Antonell and PFIS grant (FI18/00121) to Dr Ramos-Campoy); Dr Fort-Aznar is funded through the Marie Skłodowska-Curie Fellowship (PCI2021-122086-2B), MCIN/AEI/501100011033 and NextGenerationEU/PRTR. This study was partially funded by a generous donation from Grau-DeMiguel family.
CONFLICT OF INTEREST
RSV has served in Advisory Boards Meetings for Wave Life Sciences, Ionis, and Novo Nordisk and received personal fees for participating in educational activities from Janssen, Roche Diagnostics, and Neuroxpharma and funding to her institution for research projects from Biogen and Sage Pharmaceuticals.
AL is an Editorial Board Member of this journal but was not involved in the peer-review process nor had access to any information regarding its peer-review.
All other authors have no conflict of interest to report.
DATA AVAILABILITY
The data supporting the findings of this study are available within the article.