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Aberrant crosstalk between insulin signaling and mTOR in young Down syndrome individuals revealed by neuronal‐derived extracellular vesicles

Intellectual disability, accelerated aging, and early‐onset Alzheimer‐like neurodegeneration are key brain pathological features of Down syndrome (DS). Although growing research aims at the identification of molecular pathways underlying the aging trajectory of DS population, data on infants and adolescents with DS are missing.


HIGHLIGHTS
• Brain insulin resistance develops early in Down syndrome (DS) independent of peripheral alterations. • Brain insulin resistance is associated with mTOR hyperactivation in DS.
• Neuronal-derived extracellular vesicles (nEVs) allow detection of alterations of the insulin/mTOR pathway in DS brain.
• PTEN inhibition drives insulin/mTOR alterations in DS brain.
• Alterations of the insulin/mTOR pathway allow discrimination of DS versus healthy individuals. excessive numbers of astrocytes, dendritic atrophy, and impaired connectivity. 1,2 Over the last decades, the lifespan of people with DS has significantly extended. However, the increased life expectancy brings about a higher risk of developing Alzheimer's disease (AD)-like dementia. 3,4 Due to the triplication of the amyloid precursor protein gene (APP) on Hsa21, DS may potentially be envisioned as a genetic form of AD, similar to its autosomal dominant form. 3,4 Therefore, persons with DS represent a population to explore the molecular mechanisms underlying intellectual disability as well as neurodegeneration. Several studies have focused on the identification of neuronal pathways involved in synaptic plasticity, neurogenesis, and neurotransmission, which are altered early in life in people with DS. 3 However, no single gene or region of Hsa21 has been found to be responsible for all of the common features of DS. 5 This suggests that the coordination of multiple genes and other factors may be responsible for the development of major DS phenotypes. 3,6 Previous studies support the idea that altered insulin signaling in the brain, referred to as brain insulin resistance, may affect the molecular pathways involved in synaptic plasticity and adult neurogenesis. 7 Indeed, the development of brain insulin resistance may lead to a reduction of "mindspan"-the ability of the brain to preserve mental capabilities throughout life-and increase the risk of neurodegeneration. 8 The mammalian target of rapamycin (mTOR) is a "master switch" between anabolic and catabolic cellular processes 9 through regulating glucose metabolism, bioenergetics, mitochondrial function, and autophagy, also in response to insulin. 10,11 mTOR has also been involved in long-lasting synaptic adaptations at the basis of higher-order brain function (ie, synaptic plasticity, memory preservation, and neuronal recovery). 10 Previous studies by our group and by others indicate that both insulin and mTOR signaling pathways are impaired early in life in the brain of people with DS 12,13 and DS animal models. [14][15][16] Moreover, crosstalk between insulin signaling and mTOR pathway has been indicated to play a key role in the maintenance of mindspan. Therefore, understanding at which time point and through which trajectory the disturbance of the insulin/mTOR pathway occurs In this regard, plasma-resident neuronal-derived extracellular vesicles (nEVs) show great potential as a diagnostic tool. 17 EVs are a heterogeneous population of vesicles released into the extracellular space by all cell types, including brain cells for cell-to-cell communication. 18 EVs carry surface markers and biologically active cargo molecules that are specific to their originating tissue/cell and that may reflect the tissue/cell's physiological state. 19,20 Thus the isolation of nEVs from the blood may provide a minimally invasive approach for sampling neuronal components in DS and may, therefore, be considered a form of "liquid biopsy." 20 Recently, the characterization of nEVs cargo provided valuable information on the molecular alterations underlying AD neuropathology. 17,21 Increased levels of AD biomarkers, such as amyloid beta (Aβ) and phosphorylated tau (p-tau), were observed in nEVs from persons with AD relative to controls. 22,23 Furthermore, the analysis of nEVs collected from participants of the Baltimore Longitudinal Study of Aging revealed that markers of brain insulin resistance were among the strongest individual predictors of AD development, thereby highlighting a role for altered insulin signaling in AD onset and progression. 17 In addition, high levels of Aβ1-42, pTau181, and p-s396-Tau were identified in the nEVs of persons with DS compared with age-matched controls, already in childhood. 24 Although a role for insulin/mTOR pathway during brain development and in the maintenance of brain functions has been proposed, no evidence is available in children with DS. In the present study, we applied a multiplex immunoassay for the simultaneous evaluation of all the mediators of the insulin/mTOR pathway in circulating nEVs isolated from infants and adolescents with DS and age-matched controls.

Study participants
For the present study, we enrolled infants and adolescents with DS (n = 18) and age-matched healthy donors (HDs, n = 17) from the Down Syndrome and Pediatric Outpatient Clinic of the Bambino Gesù Children's Hospital in Rome (Italy). The study was approved by the ethics committee of the Bambino Gesù Children Hospital (protocol ID # 1771_OPBG_2019). We have received and archived written consent for participation/publication from every individual whose data are included. Consent for inclusion in the study was obtained during a visit to outpatient clinics from all participants.

nEV isolation
Blood samples were obtained from all participants after overnight fasting. All blood draws and processing followed established proto-

nEVs characterization by dynamic light scattering
Ten microliters of intact nEVs were used for the determination of the vesicle hydrodynamic diameter through dynamic light scattering (DLS).
The mean hydrodynamic diameter (Z-average size), size distribution, and correlation curve of nEVs in phosphate-buffered saline (PBS; pH 7.2, dilution factor 1:100) were measured at 25 • C using a Zetasizer Pro (Malvern Panalytical, UK) equipped with a solid state HeNe laser (λ = 632.8 nm) at the scattering angles of 173 • and 13 • . Size measurement data were analyzed by the general-purpose algorithm.

nEV characterization by transmission electron microscopy
Intact nEVs were used for morphological evaluation of nEVs. The purified suspension stored at 4 • C was deposited by drop casting on a Formvar copper grid and subsequently stained by Uranyless solution for 1 minute. Transmission electron microscopy (TEM) observations were performed using a Zeiss EM10 (Zeiss, Oberkochen, Germany) at 60 kV.

nEVs characterization for EVs and neuronal markers
The detection of both transmembrane and intravesicular EV markers is required to confirm the sequential enrichment of EVs from neat plasma using Exo-spin Blood kit EX-02 sedimentation of total EVs followed by immunoprecipitation of nEVs. EV enrichment was confirmed by showing the presence of transmembrane (cluster of differentiation 81, CD81) and intravesicular EV markers (Alix) as well as the relative depletion of lipoprotein (apolipoprotein A1, APOA1) in both total and nEVs compared with EV-depleted plasma. Moreover, enrichment of EVs of neuronal origin was confirmed by evaluating L1CAM and neuronal nuclear protein (NeuN) levels, as neuronal markers, in nEVs, total EVs, and EV-depleted plasma samples. The evaluation of the markers mentioned above was performed by Western blot analysis described below.

2.6
Western blot analysis Laboratories) and analyzed using Image Lab software (Bio-Rad Laboratories). Normalization was performed against total protein load signal.

Bioplex assay
A magnetic bead-based immunoassay was used to measure levels of eight phosphoproteins and total target proteins pertaining to the mTOR signaling pathway in nEVs. All mediators were assayed in multiplex using the Bio-Plex Pro Cell Signaling protein kinase B (Akt) Panel, Median fluorescence intensities (MFI) corrected for the blank background were obtained for all analytes and results were calculated as the ratio between the MFI of phosphorylated targets and total proteins.

Statistical analyses
Data were first tested for equal variance and normality (Shapiro-Wilk test i indicating the generic i th individual and the subscript norm explicitly referring to the above-mentioned normalization.

Demographics and clinical characteristics
Thirty-five participants ranging in age between 2 and 17 years, including 18 with DS and 17 controls, were enrolled and included in the analysis. Baseline characteristics of participants are listed in Table 1. Participants with and without DS did not differ for any demographic, clinical, or anthropometric characteristics.

Characterization of nEVs
nEV preparations were characterized for protein content, size, and morphology through DLS and TEM, along with immunoblotting analyses for positive and negative EV markers, according to established criteria 18 (Figure 1).
Quantification of the nEVs was performed by measuring the total protein content, which provides an estimation of nEV amount. 18,27 Higher nEV levels were observed in participants with DS relative to controls (P = .004) ( Figure 1A).
The size distribution of nEVs immunocaptured by L1CAM was studied with DLS ( Figure 1A and 1B). Correlation coefficients of nEVs from both participant groups were consistent with EVs guidelines 18 and so were mean hydrodynamic diameter values (204 ± 95 nm and 210 ± 96 nm, in controls and participants with DS, respectively) ( Figure 1C).
The size of nEVs was comparable between the groups (P = .77).
Bright-field (BF) image TEM showed well-separated nEVs (Figure 1D). Imaging analyses were performed to accurately measure nEV size at low magnification to conduct the appropriate statistics. 28,29 Quantitative morphometric measurements were performed by generating a 2D contour map of 419 vesicles distributed on a probed area of 6220 nm per 4587 nm ( Figure 1D). The analyzed size was plotted in a frequency profile of EVs with spherical shape. The bin distributions were processed by Gaussian fitting, and the frequency distribution of isolated nEVs was estimated to be centered around the mean value of 64 ± 0.71 nm, having a lowest polydispersity of about 1.10% ( Figure 1E). Furthermore, small populations of bins were fitted to estimate a mean size of 128 ± 0.51 nm of the nEVs ( Figure 1E). It should be noted that the mean hydrodynamic diameter of nEVs (detected by DLS measurements) was almost 3-fold greater than the size estimated with TEM, evidencing how the particle behaves in a fluid, thus taking into account the electric dipole layer that adheres to the vesicle surface.

nEV biomarker comparison between participants with DS and controls
The interrogation of nEVs for markers of the insulin/mTOR pathway We also evaluated the levels of CD81 and L1CAM in nEVs and found no significant differences between participant groups (Supplementary Figure 3). To further confirm our results, we normalized insulin/mTOR pathway markers levels as well as those of synaptic proteins for both CD81 and L1CAM levels, as in previous reports. 18 Figures 3A-D). In addition, a strong negative association was found between pCamK IIα Thr286 and both pAkt Ser243 and pmTOR Ser2448 in controls but not in participants with DS ( Figures 3A-D).

nEV biomarker comparison between participants with DS and controls according to age
To verify whether the observed changes varied according to age, correlation analyses were performed. No significant associations with age were observed in either participant group (Figures 3A-D). Based on that, to unravel whether alterations in DS occur in childhood and remain consistent in older children and in adolescents independent of age, participants were categorized according to the age range reported in Table 1. Although differences were evident between DS and controls, the small sample size of each sub-group did not allow us to perform a reliable statistical analysis (data not shown). Hence, participant age was then categorized according to the median value (7 years for DS; 8 years for controls), and significantly higher levels of pIRS1 Ser636  Figures 4A and 4C). A similar pattern was observed in participants above the median age, although differences did not reach statistical significance ( Figure 4B). No significant differences were observed for any synaptic proteins (Supplementary Figure 6).

F I G U R E 2 nEV biomarker levels in healthy donors (HDs) and children with Down syndrome (DS). (A)
Schematic representation of the insulin/mTOR pathway. Arrows: activation; lines: inhibition; green circles: phosphorylation sites associated with protein activation; red circles: inhibitory phosphorylation sites. (B-I) Levels of biomarkers of the insulin/mTOR pathway evaluated in nEVs isolated from HD (n = 17) and DS (n = 18) children. Data are presented as means ± SD. *P < .05 and ** P < .01 versus HD (Mann-Whitney)

SIMCA analysis
The whole set of measured mediator signals was used to build a mul-

DISCUSSION
Our work reports for the first time a significant alteration of the insulin/mTOR pathway in infants and adolescents with DS through the analysis of nEVs. Furthermore, our data show that changes in the levels of protein phosphorylation are consistent across adjacent kinases of the insulin/mTOR pathway, indicating that the integrity of the signaling cascade is maintained in nEVs. Hence, nEVs are a valuable tool to investigate the neuronal insulin/mTOR pathway through a minimally invasive approach.
The first striking finding of our study is the significant IRS1 inhibition observed in nEVs from participants with DS. Inhibition of IRS1 is a marker of brain insulin resistance. 7 From a molecular point of view, IRS1 Ser636 phosphorylation is responsible for the uncoupling between insulin receptor (IR) and IRS1, which results in the inability of insulin (2) increased production and secretion of Aβ peptides 7,34 ; and (3) increased tau phosphorylation. 7,35,36 Of interest, all of the above-cited neuropathological alterations have been described in the brain of adult people with DS (reviewed in 3 ). Moreover, these observations are in keeping with clinical studies showing that the failure in brain energy metabolism responsible for cognitive decline during aging or AD could be driven by the development of brain insulin resistance, particularly at early stages. 7,22 In addition, IRS1 inhibition within the brain seems to occur independent of peripheral alterations. Fasting glycemia, insulinemia, and HOMA-IR-known to represent powerful risk factors for the development of brain insulin resistance and cognitive decline 7 -were within normal ranges and did not differ between participants with DS and controls. Similarly, metabolic disturbances/diseases associated with peripheral insulin resistance, such as overweight, obesity, type 2 diabetes, and non-alcoholic fatty liver disease, were not prevalent in our participant cohort.
Despite IRS1 inhibition, we observed that the activation of Akt, mTOR, and mTOR-associated targets, that is, P70S6K and S6, was consistently increased in nEVs isolated from participants with DS compared with controls, thus indicating an overactivated state. This observation confirms previous studies from ours and other groups show-ing aberrant mTOR signaling in post-mortem brain from people with DS, with and without AD, and in AD brain. 12,37 Hyperactivation of mTOR has been associated with defects of autophagosome formation and autophagy and with accumulation of AD pathology markers. 1.2,14 Accordingly, published data show defects in endo-lysosomal pathways in DS [38][39][40] and efficacy of mTOR inhibitors to rescue this abnormality in primary fibroblast from persons with DS. 41 A protective role for EVs was previously proposed in DS by showing that higher EV release may be a mechanism to alleviate endo-lysosomal dysfunctions both in mice and humans. 27,42 Enhanced EVs secretion, 27,31,42 as observed also in our work, might serve as a delayed cellular response to reduce the size and number of endosomal compartments in DS by shedding more endosomal content into the brain extracellular space. 27,42 However, it is important to highlight that, in addition to the mean of increased nEVs release in DS, the analysis of nEV content provides information about the molecular alterations occurring within the brain, as we observed for the insulin/mTOR pathway, thus representing a powerful tool of investigation.
mTOR is involved in neuronal development and proper functioning of mature neurons, 43 and changes in mTOR activity are often observed in neurological diseases, including genetic disorders (eg, tuberous sclerosis and fragile X syndrome), epilepsy, brain tumors, and neurodegenerative disorders (AD, Parkinson disease, and Huntington disease). 44 The analysis of nEVs in children with DS, although at a different age, recapitulates the main findings obtained from post-mortem brain of  Hyperactivation of the Akt/mTOR axis might also be responsible for increased BAD phosphorylation observed in nEVs isolated from participants with DS. Indeed, both Akt and P70S6K are known to phosphorylate BAD to promote anti-apoptotic signals and, thus, cell survival. 52,53 This is the first observation of increased BAD inhibition in DS and is in agreement with the concept that compensatory events promote survival mechanisms prior to the development of AD pathology both in persons with DS and transgenic mice. [54][55][56] Moreover, we previously reported that pro-apoptotic signals were significantly increased in persons with DS only after the development of AD pathology. 57 In the search for putative candidates responsible for the impairment of insulin/mTOR pathway crosstalk, we suggest that PTEN may play a role. PTEN is a widely known negative regulator of insulin/PI3K F I G U R E 5 SIMCA model: cross-validated projection of the samples onto the space of the healthy donor (HD) category. In the space spanned by the two variables T 2 norm and Q norm , the region associated with the class HD is represented by the area below the dashed black line identifying the classification threshold. The plot shows that almost all HD participants (blue circles), falling below the line, are correctly identified as members of the class. Likewise, the majority of participants with DS (red circles), falling above the decision line, are correctly identified as not being HD signaling. 58,59 We hypothesize that the significant PTEN inhibition (pPTEN Ser380 ) observed in nEVs from participants with DS would lead to an accumulation of phosphatidylinositol-3, 4, 5-triphosphate (PIP3), responsible for sustained overactivation of Akt and its downstream targets, that is, mTOR. A dysregulation of the axis is also supported by the lack of a significant association between PTEN and either mTOR Ser2448 or BAD Ser136 in participants with DS, but not in controls, as well as by SIMCA results, suggesting that PTEN inhibition may promote an unchecked Akt hyperactivation, which further contributes to aberrant mTOR activation and BAD inhibition. This proposed scenario is reinforced by recent observations showing that PTEN inhibition is coupled with Akt/mTOR hyperactivation in post-mortem brain samples obtained from people with DS and AD. 12,37,45 Observations from the current study also suggest that alterations of the insulin/mTOR pathway occur quite early in DS brain, already during the childhood. Because people with DS show intellectual disability ranging from mild to severe with cognitive functions below chronological age expectations 60 and because the insulin/mTOR pathway regulates processes associated with neuronal plasticity, 7,8 we propose that our results may add new insight about the molecular mechanisms responsible for worse cognitive performance in DS children.
We acknowledge that one limitation of the current study is the lack of correlations with cognitive measures, such as mental age or memory and learning, and further research is warranted to fully understand the pathophysiologic processes behind our observations.
In conclusion, our findings contribute to expand the knowledge on the neuropathological alterations in DS brain by analyzing a very young population still poorly characterized. As discussed earlier, defects of insulin/mTOR pathway, besides being involved in cognitive dysfunc-

CONFLICTS OF INTEREST
The authors disclose that this work was supported by Jerome-Lejeune