Neuronal exosomes reveal Alzheimer’s disease biomarkers in Down syndrome

1. Introduction

Down syndrome (DS) is the most common non-lethal aneuploidy in humans caused by complete or partial trisomy of chromosome 21 [1]. In addition to intellectual disability and atypical development often observed in DS, the prevalence of dementia is significantly higher, with onset at an earlier age than in the general population [2]. A strong candidate mechanism is the amyloid beta precursor protein (APP) gene which is located on chromosome 21 and triplicated in people with DS, giving rise to toxic amyloid peptides at an early age [3]. Amyloid pathology has been observed as early as 15 years of age in postmortem brain samples from individuals with DS [4]. By age 40, amyloid plaques and neurofibrillary tangles are present in sufficient numbers for a postmortem pathological diagnosis of Alzheimer’s disease (AD) [5]. Age-related increases in both soluble and insoluble amyloid beta (Aβ) peptides Aβ_1-40 and Aβ_1-42 within frontal cortex have been described in older individuals with DS, and are correlated with increased oxidative stress, suggesting that other pathological pathways are at play as well [6]. The presence of AD pathology in younger individuals with DS is relatively unknown, due in part to a paucity of brain tissue available for study at earlier age epochs. Biomarkers that reflect AD pathology at earlier ages are thus of considerable interest since neuroprotective therapies will eventually target younger individuals with DS.

Neuropathological biomarkers of AD, including Aβ_1-40 and Aβ_1-42, have been detected in brain tissue and cerebrospinal fluid (CSF) decades prior to onset of dementia in the general population [7], and in DS [8]. Performing lumbar punctures (LPs) in those with DS is challenging; thus there is a need for the development of reliable blood-based biomarkers for AD in this population. Most cell types in the body, including neurons, release small endosomally-derived vesicles, known as exosomes [9, 10]. Exosomes contain proteins, messenger RNA (mRNA) and microRNA (miRNA) that play a prominent role in cellular signaling, removal of unwanted proteins, and transfer of cellular pathogens to other cells [8]. Because of their small size, secreted exosomes diffuse into biological fluids (blood, CSF and urine) and circulate in the interstitial space, both in the brain and the periphery [11]. Neuronal exosomes have unique neuron-specific surface markers, which enable targeted examination from circulating biological fluids [10, 12, 13].

APP processing occurs in exosomes [14–17], and it has been suggested that exosomes play a role in Aβ clearance [18]. Exosomes receive APP from early endosomes after cleavage into Aβ peptides, which are secreted from the cells in exosomes [18]. Neuronal exosomes contain Aβ peptide products and Tau, and transmit these to neighboring cells, other brain regions and the circulatory system, suggesting that neuronal exosomes extracted from either plasma or CSF can specifically assess relevant neuropathological processes within CNS neurons [19, 20]. Other findings indicate that AD biomarkers in neuronal exosomes accurately predict onset of dementia as early as 10 years prior to symptom onset in patients with sporadic AD or frontotemporal dementia (FTD) [10, 21]. Exosomal biomarkers have not been analyzed in individuals with DS. In this study, we hypothesized that neuronal exosomes obtained from blood in DS would have elevated levels of amyloid-beta (Aβ) peptides and phosphorylated-Tau (P-Tau) that could document a preclinical AD phase in the DS population.

The results presented here are from an international collaboration, including investigators from Hospital de la Santa Creu i Sant Pau in Barcelona (Spain), University of California at Irvine (UCI), Barrow Neurological Institute and Banner Sun Health Research Institute (Arizona), the Linnaeus University in Sweden, and Medical University of South Carolina (MUSC). The aims were to examine alterations in exosome AD biomarkers in children, young adults, and older adults with DS in comparison to age-matched non-DS controls, and also whether AD biomarkers were altered in individuals with DS that display dementia.

2. Methods

3. Results

4. Discussion

We report Aβ_1-42 and P-Tau levels from neuronal exosomes isolated from individuals with DS, as a function of age and dementia status. We found a marked increase in AD biomarkers in individuals with DS as early as 8 years of age. Extrapolation of the linear regressions suggests that these abnormal neuronal exosome biomarker levels may be elevated already at birth in DS, but further studies are needed. Adults with DS diagnosed with dementia exhibited lower Aβ_1-42 and higher P-S396-Tau levels compared to non-cognitively impaired adults with DS, suggesting different biomarker trajectories in exosomes as a consequence of the AD process.

It is not surprising that Aβ_1-42 levels were elevated early in DS because the APP gene is located on chromosome 21 [24,25], and elevated toxic amyloid peptides in the plasma of young adults with DS have been reported [26]. A positron emission tomography (PET) imaging study, using the beta-amyloid Pittsburgh compound-B (PIB), showed increased load of fibrillar Aß in DS, in those >35 years of age [27]. In contrast, others have shown lower Aβ_1-42 levels in plasma in individuals with DS diagnosed with dementia compared to subjects with DS but no dementia [28]. Diffuse amyloid plaques have been described as early as age 15 in brain samples from people with DS [29, 30]. Increased levels of Aβ_1-42 have also been reported in the CSF of adults [1,31,32] and children [8] with DS. The Dominantly Inherited Alzheimer Network (DIAN) showed reduced concentrations of CSF Aβ_1-42 and elevated concentrations of CSF P-Tau, in asymptomatic mutation carriers 10 to 20 years before their estimated age at symptom onset [32], data that may lend support to the current findings [33]. The sensitivity discrepancy between PET imaging versus plasma and CSF studies may reflect PIB binding affinity preferences to protein-lipid components [34] or PIB tracer signal strength. Longitudinal studies need to be performed in the DS population to address potential differences in sensitivity of different diagnostic methods.

Since the Tau gene is not known to be affected by trisomy 21, it is more surprising that P-Tau levels are elevated early in DS. Phosphorylation of Tau protein at the carboxyl terminus has been shown to be one of the earliest pathological events in both AD and in DS-AD [32], and may occur prior to accumulation of brain fibrillary P-Tau. Here, we found that neuronal exosome levels of both P-T181-Tau and P-S396-Tau were significantly increased in younger individuals with DS compared to age-matched controls and that hyper-phosphorylation persisted into late adulthood when DS co-occurred with AD dementia. The highest levels of exosomal P-S396-Tau were found in cases diagnosed with dementia (Fig. 4C), suggesting that P-Tau continues to accumulate in exosomes as AD pathology progresses. Our data support previous P-Tau observations regarding AD-related progression in adults with DS but also suggest that significant elevations occur, at least in neuronal exosomes, as early as 8 years of age in DS. Future studies targeting earlier age ranges in DS should be undertaken to gain insight into the early neuropathological processes associated with DS. The data reported here may also bolster new in vivo imaging studies, using novel P-Tau radioligands [35]. The biological mechanisms driving the elevated P-Tau levels early in those with DS remain unknown.

Potential mechanisms for Tau dysregulation can be found in several genes involved in the trisomy. For example, superoxide dismutase 1 (SOD1) is located on the triplicated segment of Chr. 21, and is involved in oxidative stress which exacerbates AD pathology in the DS brain [36]. Dysregulation of alternative splicing of Tau has been reported in DS as well as in AD [37] and may also underlie the abnormal hyper-phosphorylation of Tau [38]. Furthermore, two genes located on Chromosome 21, the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene, and regulator of calcineurin 1 (RCAN1), are highly expressed in the DS brain [39–41], and have been implicated in the dysregulation of Tau phosphorylation associated with early onset AD in DS [42, 43, 44]. Taken together, dysregulation of these genes suggest several alternative molecular mechanisms underlying the increase in Aβ_1-42, P-S396-Tau and P-T181-Tau observed in neuronal exosomes isolated from children with DS, which will be further explored in ongoing expanded studies.

The capacity to quantify Aβ_1-42 and P-Tau in neuronal exosomes from easily acquirable blood samples has permitted us to show significant abnormalities in these proteins at an early age in individuals with DS. As expected, due to the increase in APP gene dosage, Aβ_1-42 levels were increased throughout the lifespan in participants with DS, but paradoxically were significantly reduced in those with a clinical diagnosis of dementia. The decrease in neuronal exosome Aβ_1-42 levels paralleled Aβ_1-42 decreases in plasma observed with conversion to AD in the DS population [28]. If exosomes express intra-neuronal signatures that are unique compared to the extracellular environment reflected by CSF or serum biomarkers, one could hypothesize that neuronal exosome Aβ_1-42 has a distinct interface with extracellular plaques in the brain compared to serum and CSF Aβ_1-42. Measurements of serum and CSF Aβ_1-42 occurring concurrently with Aβ plaque formation and cognitive impairment in those with DS-AD [25, 26, 28] may give different and complementary results compared to neuronal exosome Aβ_1-42 and longitudinal studies will have to be performed to test this hypothesis. Also, future studies will be needed to clarify important questions about both inter- and intra-exosomal amyloid peptide production, effects on different populations of amyloid deposits and correlations with clinical dementia status in the DS population. While amyloid-beta is a central concern, elevated Tau signatures are paramount as well. P-S396-Tau and P-T181-Tau levels were increased early in life, and P-S396-Tau showed a significant further increase in dementia suggesting that Tau phosphorylation plays a role in dementia onset and progression in DS.

We suggest that the neuronal exosome approach offers several benefits for the interrogation of early events in AD pathogenesis. Another significant benefit is that outcome measures can be reliably obtained either from serum or plasma samples, since data are normalized against the same neuronal marker. Finally, a neuronal exosome approach may prove useful in evaluating potential drug targets for early intervention of AD, while enabling reliable measures of therapeutic efficacy. Future studies will include comparison studies between whole plasma and neuronal exosome amyloid levels, correlation studies between exosome markers and cognitive measures, and an expanded search for other biomarkers in neuronal exosomes that may predict dementia and/or other conditions in adults with DS.

Supplementary Material