Low neural exosomal levels of cellular survival factors in Alzheimer’s disease

Transcription factors that mediate neuronal defenses against diverse stresses were quantified in plasma neural-derived exosomes of Alzheimer’s disease or frontotemporal dementia patients and matched controls. Exosomal levels of low-density lipoprotein receptor-related protein 6, heat-shock factor-1, and repressor element 1-silencing transcription factor all were significantly lower in Alzheimer’s disease patients than controls (P < 0.0001). In frontotemporal dementia, the only significant difference was higher levels of repressor element 1-silencing transcription factor than in controls. Exosomal transcription factors were diminished 2–10 years before clinical diagnosis of Alzheimer’s disease. Low exosomal levels of survival proteins may explain decreased neuronal resistance to Alzheimer’s disease neurotoxic proteins.


Introduction
Deficiencies of transcription factors that protect neurons from diverse stresses have been implicated in progression and severity of Alzheimer's disease (AD). 1,2 Repressor element 1-silencing transcription factor (REST) is expressed at high levels in aging human brain, where it maintains factors that enhance neuronal resistance to stresses and apoptosis. 1,3 Nuclear levels of REST and its recognition motif, termed RE1, are lower in autopsy brain tissues of patients with AD than cognitively normal controls. 1 A complex of low-density lipoprotein receptor-related protein 6 (LRP6) and Frizzled receptors bind Wnt ligands and thereby evoke nuclear translocation of b-catenin that activates genes encoding survival factors such as REST. [4][5][6] As for REST, LRP6-Wnt signaling is decreased in autopsy brain tissue from AD patients and deletion of the Lrp6 gene selectively in mouse forebrain neurons enhances AD-like pathology. 2 The heat-shock factor-1 (HSF1) maintains neuronal defenses by increasing expression of heat-shock family chaperones and cytoskeletal-protective calcium-sensing proteins, but has not been adequately studied in human neurodegenerative diseases. [7][8][9] Recent analyses of proteins extracted from immunochemically isolated neurally derived plasma exosomes have shown significantly higher levels of pathogenic P-T181-tau, P-S396-tau and Ab1-42, as well as altered levels of phosphorylated forms of the insulin receptor proximal signaling protein, termed insulin receptor substrate (IRS) in AD as contrasted with controls. 10,11 Plasma neurally derived exosomal levels of P-T181-tau, P-S396-tau, Ab1-42, and phosphorylated forms of IRS were significantly This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. different in preclinical AD (AP) than for controls up to 10 years before appearance of neurological signs. 10 Here, we demonstrate significant differences between AD patients and controls in neural exosomal contents of transcription factors mediating neuronal resistance to diverse stresses.

Experimental design and patient evaluation
We identified 24 patients for cross-sectional studies (one blood sample at the National Institute on Aging [NIA]) and 16 patients for longitudinal studies (one blood sample at diagnosis [longitudinal AD set] and another 2-10 years earlier [longitudinal AP set] at the Mayo Clinic or the University of Kentucky) ( Table 1). Samples from the longitudinal study were analyzed without knowledge of any clinical data. Amnestic mild cognitive impairment (MCI) and dementia from AD were diagnosed as described, using established criteria. [10][11][12][13][14][15][16][17] Ten patients with the behavioral variant of frontotemporal dementia (FTD) were from the Memory and Aging Center of the Department of Neurology of the University of California, San Francisco. Their diagnosis and assignment to mild or moderate dementia groups (Table 1) were based on standard criteria. 18,19 Cognitively normal control subjects for FTD (FTC) and both AD sets (AC) were from the NIA (28/50) or the Jewish Home of San Francisco (22/50) and were matched by age, gender, and race to FTD and AD patients in cross-sectional and longitudinal studies.
Each subject and some patient designates signed consent forms approved with the protocol at each institution. Thirty milliliter of venous blood were drawn into 100 U/mL of heparin and centrifuged for 15 min at 2000g. Plasmas were stored in 0.5 mL aliquots at À80°C.
The mean value for all determinations of CD81 in each assay group was set at 1.00 and the relative values for each sample used to normalize their exosomal content. A neural origin and enrichment of neural exosomes by anti-L1CAM antibody absorption were supported by enhanced content of NCAM-1 and NS-enolase (Table S1). 20

Statistical analyses
The statistical significance of differences between means for each patient group and their respective control group was determined with an unpaired t test including a Bonferroni correction (GraphPad Prism 6, La Jolla, CA). For longitudinal analyses, the significance of differences between AP and AD values was calculated with a paired t test (GraphPad). Discriminant classification modeling and receiver operating characteristic (ROC) analyses of proteins in the AD versus AC cross-sectional groups were conducted as described. 10,11 Results Transcription factor levels of plasma neural exosomes were significantly lower for AD patients than AC control subjects in the cross-sectional study (Fig. 1) (Fig. 1). The REST level was significantly higher for FTD patients (2065 AE 162 pg/mL) than FTC controls (691 AE 123 pg/ mL) (P < 0.0001), in contrast to earlier findings of lower values in brain tissues. 1 There were no differences between levels of the transcription factors in AD patients with MCI and those with dementia (Table S2). ROC analyses of all analytes correctly classified 79% of AC subjects and 92% of AD patients (Fig. S1), as had primary pathogenic proteins. 10 The results of longitudinal studies were confirmatory, as each of the plasma neural-derived exosomal protein levels for AD patients 2-10 years before (AP group) and at the time of diagnosis (AD group) were significantly lower than for AC control subjects (Fig. 2). The mean AE SEM levels of LRP6, HSF1, and REST for the AD group (568 AE 26.3, 140 AE 9.75, and 13.8 AE 1.87 pg/ mL, respectively) and for the AP group (689 AE 23.6, 183 AE 9.95, and 30.7 AE 5.37 pg/mL) were significantly lower than those for AC controls (1028 AE 64.7, 319 AE 32.0, and 667 AE 140 pg/mL) (all P < 0.0001). The respective levels for the AD group all were significantly lower than those for the AP group (P values of 0.0019, 0.0034, and 0.0066). Thus, levels of these transcription factors were significantly low years before appearance of clinical signs of AD and there was continued declination with time. There were no differences between levels of any of the transcription factors in AP samples obtained 2-5 years before and 6-10 years before diagnosis.

Discussion
It is difficult to assign distinct contributions to each of the pathogenic mechanisms and deficient neuronal defenses involved in AD. However, the low neural exosomal levels of critical survival factors in AD reported here (Figs. 1, 2) are of a degree similar to that of the increases in neural exosomal pathogenic proteins 10,11 and suggest a loss of resistance to neurotoxic proteins that is likely to increase susceptibility to development of AD. It also is possible to conclude that the extent of deficiency of these survival factors detected in neural plasma exosomes is similar to those found in human brain tissues from patients with AD at autopsy and analogous to those in mouse models with targeted deletions that result in earlier and more severe AD-like disease. 1,2 The Wnt signaling pathway regulates neurogenesis and diverse neurodevelopmental processes, as well as a variety of synaptic functions. 4 Of the transcriptional factors here found to be deficient in AD, LRP6 is a mediator of Wnt signaling and REST is a Wnt signal target. 1,2,5,6 Thus, a deficiency in LRP6 which diminishes Wnt signaling may contribute to reduced nuclear localization and activation of REST.
Major survival factor deficiencies 2-10 years before appearance of diagnostic clinical signs of AD (Fig. 2) suggest a possible early pathogenic contribution of increased neuronal susceptibility to neurotoxic proteins in AD beyond higher levels of the pathogenic proteins. 10,11 Validation of this platform will require larger studies analyzing profiles of these factors for individual patients and correlating results with those of cerebral bioimaging and pathology.

Supporting Information
Additional Supporting Information may be found in the online version of this article:  Table S1. Immunochemical enrichment of neuronal markers in plasma exosomes. Each pg/mL value is the mean AE SEM of determinations for five plasma samples from AD patients or matched AC controls after normalization for CD81 content. NCAM-1, type 1 neural cell adhesion molecule; L1CAM, L1 cell adhesion molecule; NS-enolase, neuron-specific enolase. As a mean of~8% of total precipitated plasma exosomes are neurally derived 10,20 this mean 10-fold enrichment suggests that up to 80% of the "neural set" studied are of neural origin. Table S2. Levels of serum exosome proteins in relation to severity of dementia in AD. All values are mean AE SEM, pg/mL. None of the differences between values for the MCI and dementia groups was significant.