Abstract
We aimed to study the expression of circulating heat-shock protein HSP70 and exosomes in plasma of a cohort of patients with Alzheimer’s disease (AD) and frontotemporal dementia (FTD) at different stages. We performed correlations with clinical scales and FDG-PET. HSP70 levels were higher within exosomes than free in plasma. Moderate correlations were found between exosomal HSP70 and CDR, FTLD-CDR, and extension of hypometabolism. Our results suggest modifications in the level of exosomal HSP70 during the course of neurodegeneration, regardless of AD or FTD, and therefore HSP70 could have a potential role in the follow-up of these disorders.
INTRODUCTION
Heat-shock proteins (HSP) constitute a group of highly conserved ubiquitous chaperones, which are expressed in response to several conditions. Among the different HSPs, inducible HSP70 (HSPA1) is the most universally stress-inducible chaperone. Expression of HSP70 has been often associated with bad prognosis in cancer, and it has been shown to play a role in infectious diseases, atherosclerosis, or diabetes, among others [1]. Interestingly, HSP70 is key regulator of proteostasis and it interacts with misfolded proteins present in neurodegenerative disorders regulating their aggregation or refolding and amending those that are incorrectly folded [1]. Like other HSPs, HSP70 can be found in the extracellular compartment, and notably in exosomes [2]. Exosomes are nanovesicles with a key role in intercellular communication and many physiological and pathological states. Exosomes contain several proteins and genetic material, and they are released through exocytosis from several cells including neurons and other central nervous system cells. In cancer research, investigation of exosomes is providing interesting opportunities from a theranostic perspective [2]. In this regard, we have recently shown that exosomes expressing HSP70 are potential circulating biomarkers of cancer dissemination and are targets in cancer immunotherapy because of their effect activating myeloid-derived suppressive cells [3].
In the setting of neurodegenerative disorders, experimental studies have shown that exosomes could contribute to propagation of proteins associated to neurodegeneration such as tau, alpha-synuclein, or amyloid-beta [4–6]. In fact, multiple roles for exosomes have been suggested in neurodegenerative diseases, and have gained greater interest with the prion-like hypothesis [7]. For instance, recent studies have shown changes in the levels of several exosomal proteins (e.g., some lysosomal and synaptic proteins, in plasma neuron-derived exosomes) in preclinical Alzheimer’s disease (AD), during the course of AD, or frontotemporal dementia (FTD), among other neurodegenerative disorders [8–10].
However, to our knowledge, there are very few studies analyzing HSP within exosomes in clinical samples of patients with dementia, and specifically in peripheral blood [11]. HSPs are major constituents of the chaperome, and their role in the pathogenesis of AD and other neurodegenerative disorders have been emphasized in recent years [12]. In this regard, increased levels of several HSPs and other associated proteins have been found in AD brains and animal models. Moreover, experimental studies have also shown a significant influence of HSP70 and HSP90 in amyloid-beta oligomers toxicity and protein tau, although detailed mechanisms explaining the interaction between HSP and neurodegenerative disorders’ pathophysiology seem to be complex [13]. Because both exosomes and HSP70 have emerged as important regulators in neurodegeneration, we aimed to study the expression of circulating HSP70 and exosomes in plasma of a cohort of patients with AD and FTD at different stages.
METHODS
Study population, diagnostic classification, and staging
We included 63 participants, who were classified as follows: 21 patients with AD (10 patients with typical amnestic AD and 11 patients with logopenic variant of primary progressive aphasia), 29 patients within the FTD spectrum (11 behavioral variant FTD, 10 non-fluent variant primary progressive aphasia, and 8 semantic dementia), and 13 healthy controls (Table 1). All patients with logopenic aphasia had positive amyloid biomarkers and were therefore included within the AD group.
Main characteristics of the sample
Diagnosis was performed at the onset of disease using a comprehensive clinical and neuropsychological protocol that has been specified elsewhere [14]. All patients met current diagnostic criteria for each disorder [15–17] and diagnosis was confirmed with 18F-FDG-PET and clinical follow-up. Patients with a previous history of cancer were excluded from the study.
All cases were assessed with Clinical Dementia Scale (CDR) (global score and sum of boxes) and the FTDL-CDR (sum of boxes), which adds 2 domains (behavior and language) to the classic 6 domains of CDR (memory, orientation, judgement and problem solving, community affairs, home and hobbies, and personal care) [18, 19]. According to CDR scale, 14 (22.2%), 11 (17.5%), 16 (25.4%), 12 (19.0%), and 10 (15.9%) participants scored 0, 0.5, 1, 2, and 3, respectively.
Isolation, characterization, and quantification of exosomes
Stored plasma was thawed and centrifuged at 3000 g for 15 min at 4°C. Exosomes were isolated from plasma samples using ExoQuick precipitation solution kit (System Biosciences) according to the manufacturer’ protocol. The starting volume for isolation of exosomes was 250μL. Briefly, ExoQuick solution was added to the supernatant and incubated at 4°C for 30 min after mixing. Then, the mixed solution was centrifuged at 1500 g for 30 min and the supernatant removed. The exosome-containing pellets were then subsequently resuspended in phosphate-buffered saline (PBS) or 1X Cell Lysis Buffer (Cell Signalling Technologies), and stored at – 80°C until further use.
Exosomes were evaluated for their size and concentration by nanoparticle tracking analysis (NTA) using a NS300 Instrument (Malvern Instruments, United Kingdom). Exosome preparations were homogenized by vortexing followed by dilution (1:500) in filtered PBS. Each sample analysis was conducted for 60 s. Data was analyzed by Nanosight NTA 3.2 Analytical Software (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom) with the detection threshold optimized for each sample and screen gain at 10 to track as many particles as possible with minimal background. A blank 0.2μm-filtered 1× PBS was also run as a negative control. HSPs were quantified using a High Sensitivity ELISA kits (Enzo Life science) according to the manufacturer protocol. Exosome isolation and analysis were performed in all the participants.
18F-FDG-PET acquisition, preprocessing, and analysis
PET imaging was acquired in a PET-CT Siemens Biograph True Point. Specific protocol and parameters of acquisition have been reported elsewhere [14]. In patients (n = 31) in which FDG-PET was performed <6 months since the plasma extraction, correlation between HSP70 and brain metabolism was examined. This subgroup of patients comprised 8 patients with non-fluent progressive aphasia, 7 with semantic dementia, 7 with behavioral variant FTD, and 9 patients with logopenic progressive aphasia. A single-subject brain metabolism analysis was conducted in order to assess extension of the hypometabolism in each particular case. Each patient was compared with a healthy control group of 40 participants matched for age and sex. A two-sample T test was applied, using age and sex as nuisance covariates. Scaling to global mean metabolism was used, as recommended for this type of analysis [20]. An uncorrected p < 0.001 and k = 20 was used as statistical threshold. This analysis provided the number of significant voxels displaying hypometabolism for each patient, which is a marker of the extension of the neurodegeneration.
Statistical analysis
Statistical analysis was conducted using IBM(R) SPSS Statistics 20.0. Correlations between HSPs levels and clinical measures, as well as the number of significant voxels of the single-subject brain metabolism analysis were calculated using Pearson’s coefficient. The Mann Whitney U and Kruskall-Wallis tests were used to compare means between two or more groups, respectively. A p-value <0.05 was considered statistically significant.
RESULTS
Circulating HSP70 and exosomes in patients with Alzheimer’s disease and frontotemporal dementia
Since exosomes are a source of potential biomarkers, which are stabilized by the nanovesicle lipid membrane, we first quantified HSP70 either free (soluble) or within exosomes (Fig. 1A) in plasma of patients with AD (n = 12). As shown in Fig. 2A, HSP70 levels were higher when quantified within exosomes than when soluble in plasma.
Characterization of exosomes in AD and FTD patients. A) After nanovesicles purification, exosomes markers were determined by western blot. B) Analysis of exosomes size by NanoSight. C) Quantification of the data obtained by NanoSight.
Analysis of circulating HSP70 in AD and FTD patients. A) HSP70 was quantified by ELISA in plasma from AD patients (n = 12) either free (soluble) or within exosomes (mean: 3.402 ng/mL±0.6916), ****p < 0.0001 (Mann Whitney test). B) Total amount of exosomes quantified by NanoSight in the plasma of patients (n = 63, mean: 2.865E + 11 particles/mL±1.463E + 11) and healthy controls (HC, n = 13, mean: 3.361E + 11 particles/mL±8.80E + 10), p = 0.0448 (Mann Whitney test). C) HSP70 levels (ELISA) in exosomes purified from the plasma of AD and FTD patients (n = 50, mean: 2.521 ng/mL±0.3675) or HC (n = 13, mean: 1.275 ng/mL±0.1166), *p < 0.05 (Mann Whitney test). D) Ratio of HSP70, HSP90, and HSP27 levels per nanoparticle in patients and HC. Two-way ANOVA, *p < 0.05.
We, therefore, focused our study in exosomal HSP70, which was determined in our whole cohort of patients with AD (n = 21) and FTD (n = 29), and compared the results to that obtained in healthy volunteers (n = 13). While there was no difference between patients and healthy controls in the amount of exosomes and their size (Figs. 1B, C, and 2B), there was a difference in the amount of HSP70 in exosomes (Fig. 2C). The same statistical significance was reached when analyzing the ratio of HSP70 per particle. In contrast to HSP70, no differences between patients and controls were found for other exosomal HSPs such as HSP90 and HSP27 (Fig. 2D).
Correlations between HSP70 levels and clinical stage and brain metabolism
Correlation between exosomal HSP70 and HSP70/particle with CDR scale was moderate (r = 0.354, p = 0.005; r = 0.401, p = 0.001, respectively). Exosomal HSP70 and HSP70/particle also correlated with CDR-SOB (r = 0.338, p = 0.008; r = 0.343, p = 0.007) and FTLD-CDR-SOB (r = 0.365, p = 0.004; r = 0.343, p = 0.007). After subclassification according to diagnosis, in the AD group CDR-SOB was correlated with HSP70 (r = 0.455, p = 0.015), and HSP70/particle (r = 0.399, p = 0.036). In FTD, on its side, there was a significant correlation with FTLD-CDR-SOB with HSP70 (r = 0.455, p = 0.015) and HSP70/particle (r = 0.399, p = 0.036).
As shown in Fig. 3, there was a decrease in mean values of HSP70 in patients with mild stages of disease, in comparison to healthy controls. In moderate-advanced stages, on the other hand, an increase of mean levels was detected.
Exosome HSP70 levels according to CDR staging. A weak decrease in HSP70 levels is observed in mild stages (CDR 0.5– 1), followed by a substantial increase in moderate and advance stages in both AD and FTD. Mean values±SD are shown across CDR stages. In CDR 0:1.77±1.12; CDR 0.5:1.20±0.72; CDR 1:1.27±0.42; CDR 2:2.78±1.20; CDR 3:3.26±1.33.
The number of hypometabolic voxels was correlated with exosomal HSP70 levels (r = 0.438, p = 0.015). Conversely, it was not correlated with the total number of exosomes or their size. FDG-PET scans of representative patients are shown in Fig. 4.
FDG-PET scans of four representative patients. A) Logopenic primary progressive aphasia in mild stage, left parieto-temporal hypometabolism; HSP70 = 0.92 ng/mL. B) Logopenic primary progressive aphasia in an advanced stage, showing bilateral parieto-temporal hypometabolism; HSP70 = 4.45 ng/mL. C) Mild FTD, with bilateral frontal hypometabolism, and HSP70 = 1.00 ng/mL. D) Advanced FTD, showing marked bilateral frontotemporal hypometabolism, and HSP70 = 8.02.
DISCUSSION
In this study, we evaluated exosomes and HSP70 associated to exosomes in a cohort of patients with different variants of FTD and AD. Exosome HSP70 levels showed differences among the various stages of neurodegeneration. A decrease in plasma levels was observed in the first stages of the diseases, which is in accordance with previous reports [21], followed by a marked increase in moderate and advanced stages. A plausible explanation is that HSP70 accumulates in the brain, in an attempt to unsuccessfully repair the protein aggregates, which increase as the disease progresses. Interestingly, other circulating HSPs have already been reported as markers (“danger signals”) of severity of a disease, such as HSP27 in myelofibrosis [22] or GP96 (HSP90B1) in graft versus host disease [23]. Furthermore, exosomal HSP70 levels correlate with the clinical staging scales, as well as the extent of hypometabolism. These are clinical or neuroimaging surrogates of the extension of the neurodegenerative process. Specifically, the correlation with FDG-PET suggests that exosomal HSP70 may be a marker of the degree of synaptic failure and/or neurodegeneration.
In contrast to the in-depth studies performed in the setting of cancer, where circulating exosomes have been already described as pharmacodynamic and/or prognosis biomarkers [2, 3], little is still known about this circulating nanovesicles in neurodegenerative disorders. Previous studies about exosomes in dementia have focused on several proteins with variable results: first, proteins directly associated to the pathophysiology, such as tau isoforms, alpha-synuclein or amyloid-beta; and second, proteins associated to other mechanisms, such as inflammation (cystatin-C, YKL-40, etc.), transcription factors (LRP6, REST, etc.) or synaptic damage (synaptoglobin, synaptopodin, etc.). Because each protein is associated to different roles in the pathophysiology of neurodegenerative disorders, different proteins could aid in responding to several clinical questions including early diagnosis, differential diagnosis, etc., if they are used as biomarkers [11]. Larger and longitudinal studies are needed to better understand the role of each exosomal protein in the neurodegenerative process, as well as their potential clinical use.
In conclusion, our study does not support the use of exosome HSP70 for diagnosis or differential diagnosis of AD and FTD. However, we found a moderate correlation with the severity of the neurodegeneration. Our results here suggest modifications in the level of exosomal HSP70 in plasma during the course of neurodegeneration, regardless of the specific disease, AD or FTD, and therefore a potential role in the monitoring of these diseases. Further studies deem necessary to disentail the clinical role of exosomal HSP70 in neurodegenerative disorders, and its potential use in terms of diagnostic or prognostic biomarker. In addition, further investigation is required to standardize the process of exosome analysis, especially regarding the search for more specific markers for exosomes derived from central nervous system cells [11], which could be associated to HSP70.
Footnotes
ACKNOWLEDGMENTS
This work was supported by grants from Conseil Régional de Bourgogne and a French Government grant managed by the French National Research Agency under the program “Investissements d’Avenir” with reference ANR-11-LABX-0021 (LabEX LipSTIC). MC had a fellowship from La Ligue Nationale contre le Cancer, and GC from the «Fondation pour la Recherche Médicale».
