Extracellular vesicle biomarkers for cognitive impairment in Parkinson's disease.

Besides motor symptoms, many individuals with Parkinson's disease develop cognitive impairment perhaps due to coexisting α-synuclein and Alzheimer's disease pathologies and impaired brain insulin signalling. Discovering biomarkers for cognitive impairment in Parkinson's disease could help clarify the underlying pathogenic processes and improve Parkinson's disease diagnosis and prognosis. This study used plasma samples from 273 participants: 103 Parkinson's disease individuals with normal cognition, 121 Parkinson's disease individuals with cognitive impairment (81 with mild cognitive impairment, 40 with dementia) and 49 age- and sex-matched controls. Plasma extracellular vesicles enriched for neuronal origin were immunocaptured by targeting the L1 cell adhesion molecule, then biomarkers were quantified using immunoassays. α-Synuclein was lower in Parkinson's disease compared to control individuals (P = 0.004) and in cognitively impaired Parkinson's disease individuals compared to Parkinson's disease with normal cognition (P < 0.001) and control (P < 0.001) individuals. Amyloid-ß42 did not differ between groups. Phosphorylated tau (T181) was higher in Parkinson's disease than control individuals (P = 0.003) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and controls (P < 0.001). Total tau was not different between groups. Tyrosine-phosphorylated insulin receptor substrate-1 was lower in Parkinson's disease compared to control individuals (P = 0.03) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and controls (P = 0.01), and also decreased with increasing motor symptom severity (P = 0.005); serine312-phosphorylated insulin receptor substrate-1 was not different between groups. Mechanistic target of rapamycin was not different between groups, whereas phosphorylated mechanistic target of rapamycin trended lower in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.05). The ratio of α-synuclein to phosphorylated tau181 was lower in Parkinson's disease compared to controls (P = 0.001), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and decreased with increasing motor symptom severity (P < 0.001). The ratio of insulin receptor substrate-1 phosphorylated serine312 to insulin receptor substrate-1 phosphorylated tyrosine was higher in Parkinson's disease compared to control individuals (P = 0.01), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and increased with increasing motor symptom severity (P = 0.003). α-Synuclein, phosphorylated tau181 and insulin receptor substrate-1 phosphorylated tyrosine contributed in diagnostic classification between groups. These findings suggest that both α-synuclein and tau pathologies and impaired insulin signalling underlie Parkinson's disease with cognitive impairment. Plasma neuronal extracellular vesicles biomarkers may inform cognitive prognosis in Parkinson's disease.

SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19.

OBJECTIVE: As SARS-CoV-2 is known to invade neural cell mitochondria, a plasma system for quantifying central nervous system proteins in living humans was used to investigate neuropathogenic mechanisms of long-COVID-19. METHODS: SARS-CoV-2 proteins and mitochondrial proteins (MPs) in enriched plasma neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) were quantified in resolved acute COVID-19 without post-acute sequelae of SARS-CoV-2 (PASC), PASC without neuropsychiatric manifestations (NP), PASC with NP and healthy controls. RESULTS: NDEV and ADEV mean levels of SARS-CoV-2 S1 and nucleocapsid (N) proteins were higher in all PASC sub-groups than controls, but only N levels were higher in PASC with than without NP. Exosome marker CD81-normalized NDEV mean levels of subunit 6 of MP respiratory chain complex I and subunit 10 of complex III, and neuroprotective MPs Humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were decreased significantly in PASC with NP but not in PASC without NP relative to controls. NDEV levels of MPs voltage-dependent anion-selective channel protein 1 (VDAC1) and N-methyl-D-aspartate receptor 1 (NMDAR1) were decreased in PASC without and with NP, whereas those of calcium channel MPs mitochondrial calcium uniporter (MCU), sodium/calcium exchanger (NCLX) and leucine zipper EF-hand containing transmembrane 1 protein (LETM1) were decreased only in PASC with NP. ADEV levels of MCU and NCLX only were increased in PASC without and with NP. INTERPRETATION: Abnormal NDEV and ADEV levels of SARS-CoV-2 N and S1 protein and MPs correlate with NP and may be biomarkers for long-COVID prognostics and therapeutic trials. ANN NEUROL 2022;91:772-781.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n = 10) than Cs (n = 10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

Endothelial-derived plasma exosome proteins in Alzheimer's disease angiopathy.

Small cerebral vascular disease (SCeVD) demonstrated by white matter hyperintensity (WMH) on MRI contributes to the development of dementia in Alzheimer's disease (AD), but it has not been possible to correlate onset, severity, or protein components of SCeVD with characteristics of WMH in living patients. Plasma endothelial-derived exosomes (EDEs) were enriched by two-step immunoabsorption from four groups of participants with no clinical evidence of cerebrovascular disease: cognitively normal (CN) without WMH (CN without SCeVD, n = 20), CN with SCeVD (n = 22), preclinical AD (pAD) + mild cognitive impairment (MCI) without SCeVD (pAD/MCI without SCeVD, n = 22), and pAD/MCI with SCeVD (n = 16) for ELISA quantification of cargo proteins. Exosome marker CD81-normalized EDE levels of the cerebrovascular-selective biomarkers large neutral amino acid transporter 1 (LAT-1), glucose transporter type 1 (Glut-1), and permeability-glycoprotein (p-GP, ABCB1) were similarly significantly higher in the CN with SCeVD and pAD/MCI with SCeVD groups than their corresponding control groups without SCeVD. CD81-normalized EDE levels of Aß40 and Aß42 were significantly higher in the pAD/MCI with SCeVD group but not in the CN with SCeVD group relative to controls without SCeVD. Levels of normal cellular prion protein (PrPc), a receptor for amyloid peptides, and phospho-181T-tau were higher in both CN and pAD/MCI with SCeVD groups than in the corresponding controls. High EDE levels of Aß40, Aß42, and phospho-181T-tau in patients with WMH suggesting SCeVD appear at the pre-clinical or MCI stage of AD and therapeutic lowering of neurotoxic peptide levels may delay progression of AD angiopathy.

Traumatic brain injury increases plasma astrocyte-derived exosome levels of neurotoxic complement proteins.

Possible involvement of complement (C) systems in the pathogenesis of traumatic brain injury (TBI) was investigated by quantifying Cproteins in plasma astrocyte-derived exosomes (ADEs) of subjects with sports-related TBI (sTBI) and TBI in military veterans (mtTBI) without cognitive impairment. All sTBI subjects (n = 24) had mild injuries, whereas eight of the mtTBI subjects had moderate, and 17 had mild injuries. Plasma levels of ADEs were decreased after acute sTBI and returned to normal within months. Cprotein levels in ADEs were from 12- to 35-fold higher than the corresponding levels in neuron-derived exosomes. CD81 exosome marker-normalized ADE levels of classical pathway C4b, alternative pathway factor D and Bb, lectin pathway mannose-binding lectin (MBL), and shared neurotoxic effectors C3b and C5b-9 terminal C complex were significantly higher and those of C regulatory proteins CR1 and CD59 were lower in the first week of acute sTBI (n = 12) than in controls (n = 12). Most C abnormalities were no longer detected in chronic sTBI at 3-12 months after acute sTBI, except for elevated levels of factor D, Bb, and MBL. In contrast, significant elevations of ADE levels of C4b, factor D, Bb, MBL, C3b and C5b-9 terminal C complex, and depressions of CR1 and CD59 relative to those of controls were observed after 1-4 years in early chronic mtTBI (n = 10) and persisted for decades except for normalization of Bb, MBL, and CD59 in late chronic mtTBI (n = 15). Complement inhibitors may be useful therapeutically in acute TBI and post-concussion syndrome.

Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

Exosomes derived from chondroitin sulfate proteoglycan (CSPG) 4 type neural precursor cells (CSPG4Es) were purified from human plasma by sequential immunoabsorption with anti-CSPG4 and anti-platelet growth factor receptor α mAb to characterize the potential in vivo roles of CSPG4 cells in neuronal repair. Hepatocyte growth factor, fibroblast growth factors (FGFs)-2 and -13, and type 1 insulin-like growth factor (IGF-1), which enhance neuronal survival and functions, were quantified in CSPG4E extracts. For CSPG4Es of 24 healthy control subjects, mean levels of hepatocyte growth factor, FGF-13, and IGF-1, but not FGF-2, were significantly higher by up to 7-fold than in their neuronal-derived exosomes, and mean levels of all 4 growth factors were significantly higher by up to 8-fold than in their astrocyte-derived exosomes. Mean CSPG4E levels of all growth factors were significantly lower in patients with mild Alzheimer disease (AD) ( n = 24) than in age- and sex-matched cognitively normal control subjects ( n = 24). Mean CSPG4E levels of all growth factors were also significantly lower in 15 patients at the stage of moderate dementia from AD (AD2) and at their preclinical stage 3 to 8 yr earlier (AD1), with no differences between values at stages AD1 and AD2. Current findings suggest that CSPG4 cells export in exosomes higher levels of neurotrophic factors than neurons or astrocytes and that CSPG4E neurotrophic factors are diminished early in AD, with no significant progression of decreases later in the course.-Goetzl, E. J., Nogueras-Ortiz, C., Mustapic, M., Mullins, R. J., Abner, E. L., Schwartz, J. B., Kapogiannis, D. Deficient neurotrophic factors of CSPG4-type neural cell exosomes in Alzheimer disease.

Altered levels of plasma neuron-derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury.

Neuron-derived exosomes (NDEs) were enriched by anti-L1CAM antibody immunoabsorption from plasmas of subjects ages 18-26 yr within 1 wk after a sports-related mild traumatic brain injury (acute mTBI) ( n = 18), 3 mo or longer after the last of 2-4 mTBIs (chronic mTBI) ( n = 14) and with no recent history of TBI (controls) ( n = 21). Plasma concentrations of NDEs, assessed by counts and levels of extracted exosome marker CD81, were significantly depressed by a mean of 45% in acute mTBI ( P < 0.0001), but not chronic mTBI, compared with controls. Mean CD81-normalized NDE levels of a range of functional brain proteins were significantly abnormal relative to those of controls in acute but not chronic mTBI, including ras-related small GTPase 10, 73% decrease; annexin VII, 8.8-fold increase; ubiquitin C-terminal hydrolase L1, 2.5-fold increase; AII spectrin fragments, 1.9-fold increase; claudin-5, 2.7-fold increase; sodium-potassium-chloride cotransporter-1, 2.8-fold increase; aquaporin 4, 8.9-fold increase (3.6-fold increase in chronic mTBI); and synaptogyrin-3, 3.1-fold increase (1.3-fold increase in chronic mTBI) (all acute mTBI proteins P < 0.0001). In chronic mTBI, there were elevated CD81-normalized NDE levels of usually pathologic ß-amyloid peptide 1-42 (1.6-fold, P < 0.0001), P-T181-tau (2.2-fold, P < 0.0001), P-S396-tau (1.6-fold, P < 0.01), IL-6 (16-fold, P < 0.0001), and prion cellular protein (PRPc) (5.1-fold, P < 0.0001) with lesser or greater (IL-6, PRPc) increases in acute mTBI. Increases in NDE levels of most neurofunctional proteins in acute, but not chronic, mTBI, and elevations of most NDE neuropathological proteins in chronic and acute mTBI delineated phase-specificity. Longitudinal studies of more mTBI subjects may identify biomarkers predictive of and etiologically involved in mTBI-induced neurodegeneration.-Goetzl, E. J., Elahi, F. M., Mustapic, M., Kapogiannis, D., Pryhoda, M., Gilmore, A., Gorgens, K. A., Davidson, B., Granholm, A.-C., Ledreux, A. Altered levels of plasma neuron-derived exosomes and their cargo proteins characterize acute and chronic mild traumatic brain injury.

Plasma neuronal exosomes serve as biomarkers of cognitive impairment in HIV infection and Alzheimer's disease.

Fluid biomarkers for cognitive impairment have the advantage of being relatively noninvasive and capable of monitoring neuronal and other brain cell health in real time. Biomarkers can predict the onset of dementing illness, but also correlate with cognition in a dynamic way allowing us to follow treatment responses and determine brain recovery. Chronic HIV infection causes cognitive impairment in a subset of individuals suggesting "premature aging." Exosomes are small extracellular vesicles that are shed from all cells. They are important in normal cell-to-cell communication as they contain cellular proteins, mRNA transcripts, and miRNAs. Exosome cargo varies depending on the health of the cell and pathological state; specific proteins/mRNAs and/or miRNAs are present and may serve as biomarkers. Exosomes of variable cellular origin can be isolated from peripheral blood by various methods. Neuron-derived exosomes (NDEs) can be isolated using a precipitation/immunoaffinity approach using antibodies against neuronal cell adhesion molecule L1CAM and the contents queried for central nervous system (CNS) disorders including HIV-associated neurological disorders (HAND) and Alzheimer's disease (AD). As these studies are recent, numerous questions arise including which neuronal proteins are in NDEs and whether their contents differ in different CNS pathologies or with age. In addition, can the NDE cargo predict as well as diagnose cognitive impairment and could exosomal contents be used as therapeutic biomarkers, or theramarkers, of neuronal recovery from effective treatment? This mini-review will show some new data and review recent studies on NDE from individuals with HIV infection and AD. HIV-associated neurocognitive disorders (HAND) are pathologies seen in a subset of individuals with chronic HIV infection. They belong to the spectrum of neurodegenerative diseases that result in death or dysfunction of neurons with similarities to Alzheimer disease (AD) but also distinctive differences (reviewed (Canet et al., Front Cell Neurosci 12: 307, 2018)). Both disorders are difficult to diagnose without neuropsychological testing and both need new biomarkers to judge progression as well as recovery with treatment. Both disorders involve neuroinflammation and several common targets. AD is associated with aging and HIV is thought to initiate premature aging. In HIV infection, amyloid beta (Aß), which is deposited in "plaques" in AD, is soluble and its relevance to HIV-associated cognitive impairment is controversial (Achim et al., J Neuroimmune Pharmacol 4: 190-199, 2009; Rempel and Pulliam, AIDS 19: 127-135, 2005). Aß deposition is required for AD pathological diagnosis, but is not necessarily causative (Barage and Sonawane, Neuropeptides 52: 1-18, 2015; Hardy and Selkoe, Science 297: 353-356, 2002; Morris et al., Acta Neuropathol Commun 2: 135, 2014). Neurofilament light (NF-L) is a surrogate marker in plasma and cerebrospinal fluid (CSF) for neurodegeneration (Abu-Rumeileh et al., Alzheimers Res Ther 10: 3, 2018; Mattsson et al., JAMA Neurol 74: 557-566, 2017) but continues to be a controversial biomarker for both HAND and AD (Gisslen et al., EBioMedicine 3: 135-140, 2016; Kovacs et al., Eur J Neurol 24:1326-e77, 2017; Norgren et al., Brain Res 987: 25-31, 2003; Rolstad et al., J Alzheimers Dis 45: 873-881, 2015; Yilmaz et al., Expert Rev Mol Diagn 17: 761-770, 2017). Blood biomarkers are needed to advance both HAND and AD fields, as blood draws are less costly than neuroimaging and are minimally invasive compared to lumbar punctures required for CSF acquisition. Extracellular vesicles (EVs) are nanoscale membranous vesicles shed from all cells including those of the central nervous system (CNS) and found in all biofluids; they are divided into exosomes (30-150 nm) originating from late endosomes/multivesicular bodies and microvesicles (150-1000 nm) produced through budding of the plasma membrane. Both types of vesicles are implicated in the pathogenesis of neurodegenerative diseases and may provide biomarkers (Bellingham et al., Front Physiol 3: 124, 2012). In this report, we call the vesicles exosomes, since they are the predominant vesicles in our preparations. They are involved in cell-to-cell communication in normal homeostasis and can be carriers of toxic proteins (Aß, tau) (Sardar Sinha et al., Acta Neuropathol 136: 41-56, 2018) shed by cells as waste or actively secreted in a degenerative process (review Gupta and Pulliam, J Neuroinflammation 11: 68, 2014). The idea that exosomes originating from a specific cell can be recovered in the plasma using cellular surface markers of interest is intriguing. Neuron derived exosomes (NDEs) were first described in 2015 and isolated using antibodies against neural cell adhesion molecules NCAM or L1CAM, after total plasma exosome isolation (Fiandaca et al., Alzheimers Dement 11: 600-607 e1, 2015). Characterization of NDEs follows guidelines endorsed by the International Society for Extracellular Vesicles and includes Nanoparticle Tracking Analysis (NTA) to determine EV concentration and average diameter; Western Blots for EV markers; ELISAs for neuronal proteins and transmission EM for visualization (Sun et al., AIDS 31: F9-F17, 2017; Tang et al., FASEB J 30: 3097-106, 2016). This innovative isolation of an exosome sub-population has generated interest in using NDE as biomarkers for neurodegenerative diseases like AD, HAND, traumatic brain injury, posttraumatic stress disorder and more (reviews Agoston et al., Brain Inj 31: 1195-1203, 2017; Gupta and Pulliam, J Neuroinflammation 11: 68, 2014; Hu et al., Cell Death Dis 7: e2481, 2016; Karnati et al., J Neurotrauma, 2018; Osier et al., Mol Neurobiol, 2018). Several biomarkers from plasma NDEs were recently reported by the Pulliam lab to be elevated in general cognitive impairment (Sun et al., AIDS 31: F9-F17, 2017). We review our collective data here on HAND and AD and add to the characterization of plasma NDEs as exciting biomarkers of neurodegeneration.

Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease.

Plasma endothelial cell-derived exosomes (EDEs) and platelet-derived exosomes (PDEs) were precipitated and enriched separately by immunospecific absorption procedures for analyses of cargo proteins relevant to atherosclerosis. EDEs had usual exosome size and marker protein content, and significantly higher levels than PDEs of the endothelial proteins vascular cell adhesion molecule-1 (VCAM-1) and endothelial nitric oxide synthase, whereas PDEs had significantly higher levels of platelet glycoprotein VI. EDE levels of VCAM-1, von Willebrand factor, platelet-derived growth factor (PDGF)-BB, angiopoietin-1, and lysyl oxidase-2 and the cerebrovascular-selective proteins glucose transporter 1, permeability-glycoprotein, and large neutral amino acid transporter 1 were significantly higher for 18 patients with cerebrovascular disease (CeVD) than for 18 age- and gender-matched control subjects. PDE levels of PDGF-AA, platelet glycoprotein VI, integrin-linked kinase-1, high mobility group box-1 protein, chemokine CXCL4, and thrombospondin-1 were significantly higher in patients with CeVD than in control subjects, but differences were less with greater overlaps than for EDE proteins. EDE levels of Yes-associated protein (YAP) were higher and of P(S127)-YAP lower in patients with CeVD than in control subjects, consistent with heightened activity of this mechanical force-sensitive system in atherosclerosis. Elevated EDE and PDE levels of atherosclerosis-promoting proteins in CeVD justify clinical studies of their potential value as biomarkers.-Goetzl, E. J., Schwartz, J. B., Mustapic, M., Lobach, I. V., Daneman, R., Abner, E. L., Jicha, G. A. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease.

Higher exosomal tau, amyloid-beta 42 and IL-10 are associated with mild TBIs and chronic symptoms in military personnel.

OBJECTIVE: Identify biomarkers in peripheral blood that relate to chronic post-concussive and behavioural symptoms following traumatic brain injuries (TBIs) to ultimately improve clinical management. RESEARCH DESIGN: We compared military personnel with mild TBIs (mTBIs) (n = 42) to those without TBIs (n = 22) in concentrations of tau, amyloid-beta (Aß42) and cytokines (tumour necrosis factor alpha (TNFα, interleukin (IL)-6 and -10) in neuronal-derived exosomes from the peripheral blood. We utilized nanosight technology coupled with ultra-sensitivity immunoassay methods. We also examined the impact of post-concussive and behavioural symptoms including depression and post-traumatic stress disorder (PTSD) on these neuronal-derived markers. RESULTS: We report that concentrations of exosomal tau (F1, 62 = 10.50), Aß42 (F1, 61 = 5.32) and IL-10 (F1, 59 = 4.32) were elevated in the mTBI group compared to the controls. Within the mTBI group, regression models show that post-concussive symptoms were most related to exosomal tau elevations, whereas exosomal IL-10 levels were related to PTSD symptoms. CONCLUSIONS: These findings suggest that chronic post-concussive symptoms following an mTBI relate to altered exosomal activity, and that greater tau pathology may underlie chronic post-concussive symptoms that develop following mTBIs. It also suggests that central inflammatory activity contributes to PTSD symptoms following an mTBI, providing necessary insights into the role of inflammation in chronic PTSD symptoms.

Detection of Aggregation-Competent Tau in Neuron-Derived Extracellular Vesicles.

Progressive cerebral accumulation of tau aggregates is a defining feature of Alzheimer's disease (AD). A popular theory that seeks to explain the apparent spread of neurofibrillary tangle pathology proposes that aggregated tau is passed from neuron to neuron. Such a templated seeding process requires that the transferred tau contains the microtubule binding repeat domains that are necessary for aggregation. While it is not clear how a protein such as tau can move from cell to cell, previous reports have suggested that this may involve extracellular vesicles (EVs). Thus, measurement of tau in EVs may both provide insights on the molecular pathology of AD and facilitate biomarker development. Here, we report the use of sensitive immunoassays specific for full-length (FL) tau and mid-region tau, which we applied to analyze EVs from human induced pluripotent stem cell (iPSC)-derived neuron (iN) conditioned media, cerebrospinal fluid (CSF), and plasma. In each case, most tau was free-floating with a small component inside EVs. The majority of free-floating tau detected by the mid-region assay was not detected by our FL assays, indicating that most free-floating tau is truncated. Inside EVs, the mid-region assay also detected more tau than the FL assay, but the ratio of FL-positive to mid-region-positive tau was higher inside exosomes than in free solution. These studies demonstrate the presence of minute amounts of free-floating and exosome-contained FL tau in human biofluids. Given the potential for FL tau to aggregate, we conclude that further investigation of these pools of extracellular tau and how they change during disease is merited.

Exosomal biomarkers of brain insulin resistance associated with regional atrophy in Alzheimer's disease.

Brain insulin resistance (IR), which depends on insulin-receptor-substrate-1 (IRS-1) phosphorylation, is characteristic of Alzheimer's disease (AD). Previously, we demonstrated higher pSer312-IRS-1 (ineffective insulin signaling) and lower p-panTyr-IRS-1 (effective insulin signaling) in neural origin-enriched plasma exosomes of AD patients vs. CONTROLS: Here, we hypothesized that these exosomal biomarkers associate with brain atrophy in AD. We studied 24 subjects with biomarker-supported probable AD (low CSF Aß42 ). Exosomes were isolated from plasma, enriched for neural origin using immunoprecipitation for L1CAM, and measured for pSer312 - and p-panTyr-IRS-1 phosphotypes. MPRAGE images were segmented by brain tissue type and voxel-based morphometry (VBM) analysis for gray matter against pSer312 - and p-panTyr-IRS-1 was conducted. Given the regionally variable brain expression of IRS-1, we used the Allen Brain Atlas to make spatial comparisons between VBM results and IRS-1 expression. Brain volume was positively associated with P-panTyr-IRS-1 and negatively associated with pSer312 -IRS-1 in a strikingly similar regional pattern (bilateral parietal-occipital junction, R middle temporal gyrus). This volumetric association pattern was spatially correlated with Allen Human Brain atlas normal brain IRS-1 expression. Exosomal biomarkers of brain IR are thus associated with atrophy in AD as could be expected by their pathophysiological roles and do so in a pattern that reflects regional IRS-1 expression. Furthermore, neural-origin plasma exosomes may recover molecular signals from specific brain regions. Hum Brain Mapp 38:1933-1940, 2017. © 2017 Wiley Periodicals, Inc.

Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

Efficient intercellular transfer of RNAs, proteins, and lipids as protected exosomal cargo has been demonstrated in the CNS, but distinct physiologic and pathologic roles have not been well defined for this pathway. The capacity to isolate immunochemically human plasma neuron-derived exosomes (NDEs), containing neuron-specific cargo, has permitted characterization of CNS-derived exosomes in living humans. Constituents of the amyloid ß-peptide (Aß)42-generating system now are examined in 2 distinct sets of human neural cells by quantification in astrocyte-derived exosomes (ADEs) and NDEs, enriched separately from plasmas of patients with Alzheimer's disease (AD) or frontotemporal dementia (FTD) and matched cognitively normal controls. ADE levels of ß-site amyloid precursor protein-cleaving enzyme 1 (BACE-1), γ-secretase, soluble Aß42, soluble amyloid precursor protein (sAPP)ß, sAPPα, glial-derived neurotrophic factor (GDNF), P-T181-tau, and P-S396-tau were significantly (3- to 20-fold) higher than levels in NDEs for patients and controls. BACE-1 levels also were a mean of 7-fold higher in ADEs than in NDEs from cultured rat type-specific neural cells. Levels of BACE-1 and sAPPß were significantly higher and of GDNF significantly lower in ADEs of patients with AD than in those of controls, but not significantly different in patients with FTD than in controls. Abundant proteins of the Aß42 peptide-generating system in ADEs may sustain levels in neurons. ADE cargo proteins may be useful for studies of mechanisms of cellular interactions and effects of BACE-1 inhibitors in AD.-Goetzl, E. J., Mustapic, M., Kapogiannis, D., Eitan, E., Lobach, I. V., Goetzl, L., Schwartz, J. B., Miller, B. L. Cargo proteins of plasma astrocyte-derived exosomes in Alzheimer's disease.

The catecholamine biosynthetic enzyme dopamine ß-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter.

Dopamine beta-hydroxylase (DBH) is the biosynthetic enzyme catalyzing formation of norepinephrine. Changes in DBH expression or activity have been implicated in the pathogenesis of cardiovascular and neuropsychiatric disorders. Genetic determination of DBH enzymatic activity and its secretion are only incompletely understood. We began with a genome-wide association search for loci contributing to DBH activity in human plasma. Initially, in a population sample of European ancestry, we identified the proximal DBH promoter as a region harboring three common trait-determining variants (top hit rs1611115, P = 7.2 × 10(-51)). We confirmed their effects on transcription and showed that the three variants each acted additively on gene expression. Results were replicated in a population sample of Native American descent (top hit rs1611115, P = 4.1 × 10(-15)). Jointly, DBH variants accounted for 57% of DBH trait variation. We further identified a genome-wide significant SNP at the LOC338797 locus on chromosome 12 as trans-quantitative trait locus (QTL) (rs4255618, P = 4.62 × 10(-8)). Conditional analyses on DBH identified a third genomic region contributing to DBH variation: a likely cis-QTL adjacent to DBH in SARDH (rs7040170, P = 1.31 × 10(-14)) on chromosome 9q. We conclude that three common SNPs in the DBH promoter act additively to control phenotypic variation in DBH levels, and that two additional novel loci (SARDH and LOC338797) may also contribute to the expression of this catecholamine biosynthetic trait. Identification of DBH variants with strong effects makes it possible to take advantage of Mendelian randomization approaches to test causal effects of this intermediate trait on disease.

Serotonin risk factors for the development of hypertension in pregnancy.

PURPOSE: To determine platelet serotonin (5-HT) concentrations and genotype and allele frequencies of serotonergic type 2A receptor gene (HTR2A) and 5-HT transporter gene (SLC6A4) in women with pre-eclampsia, gestational hypertension without proteinuria (PIH) and in control normotensive pregnant women. METHODS: The study included 96 control women, 131 women with PIH and 84 women with pre-eclampsia (ICD-10 criteria) in the last trimester of pregnancy. Variants of the HTR2A T102C (rs6313) and SLC6A4 (5-HTTLPR and rs25531) were determined by the PCR and real-time PCR procedures. Platelet 5-HT concentrations were analyzed by the spectrofluorimetric method. RESULTS: The frequency of the 5HTTLPR and of HTR2A T102C genotypes or alleles was similar between three groups of pregnant women and was not associated with low platelet 5-HT concentrations observed in pregnant women with PIH or pre-eclampsia. CONCLUSIONS: The results confirm alterations in the peripheral 5-HT system in pregnancy-induced hypertension. Low platelet 5-HT concentration is a common feature of both PIH and pre-eclampsia. The results did not support the hypothesis that hypertension in pregnancy is a trait associated with polymorphic variants of the HTR2A and SLC6A4 or that they have a role in the predisposition to hypertensive disorders in pregnancy. The further studies are necessary to elucidate the role of 5-HT and genetic factors in the development of hypertensive disorders in pregnancy.

Chromogranin B: intra- and extra-cellular mechanisms to regulate catecholamine storage and release, in catecholaminergic cells and organisms.

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under-expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic-stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over-expression of CHGB was achieved by transduction of a CHGB-expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic-triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic-stimulated catecholamine release by ~ 72%. One conserved-region CHGB peptide inhibited nicotinic-triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi-directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra-cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

Two weeks of exercise alters neuronal extracellular vesicle insulin signaling proteins and pro-BDNF in older adults with prediabetes.

Adults with prediabetes are at risk for Alzheimer's Disease and Related Dementia (ADRD). While exercise may lower ADRD risk, the exact mechanism is unclear. We tested the hypothesis that short-term exercise would raise neuronal insulin signaling and pro-BDNF in neuronal extracellular vesicles (nEVs) in prediabetes. Twenty-one older adults (18F, 60.0 ± 8.6 yrs.; BMI: 33.5 ± 1.1 kg/m2) with prediabetes (ADA criteria; 75 g OGTT) were randomized to 12 supervised work-matched continuous (n = 13, 70% HRpeak) or interval (n = 8, 90% HRpeak and 50% HRpeak for 3 min each) sessions over 2-wks for 60 min/d. Aerobic fitness (VO2peak) and body weight were assessed. After an overnight fast, whole-body glucose tolerance (total area under the curve, tAUC) and insulin sensitivity (SIis) were determined from a 120 min 75 g OGTT. nEVs were acquired from 0 and 60 min time-points of the OGTT, and levels of insulin signaling proteins (i.e., p-IRS-1, total-/p-Akt, pERK1/2, pJNK1/2, and pp38) and pro-BNDF were measured. OGTT stimulatory effects were calculated from protein differences (i.e., OGTT 60-0 min). Adults were collapsed into a single group as exercise intensity did not affect nEV outcomes. Exercise raised VO2peak (+1.4 ± 2.0 mL/kg/min, p = 0.008) and insulin sensitivity (p = 0.01) as well as decreased weight (-0.4 ± 0.9 kg, p = 0.04) and whole-body glucose tAUC120min (p = 0.02). Training lowered 0-min pro-BDNF (704.1 ± 1019.0 vs. 414.5 ± 533.5, p = 0.04) and increased OGTT-stimulated tAkt (-51.8 ± 147.2 vs. 95 ± 204.5 a.u., p = 0.01), which was paralleled by reduced pAkt/tAkt at 60 min of the OGTT (1.3 ± 0.2 vs. 1.2 ± 0.1 a.u., p = 0.04). Thus, 2 weeks of exercise altered neuronal insulin signaling responses to glucose ingestion and lowered pro-BNDF among adults with prediabetes, thereby potentially lowering ADRD risk.

Lipidomic Analysis of Plasma Extracellular Vesicles Derived from Alzheimer's Disease Patients.

Analysis of blood-based indicators of brain health could provide an understanding of early disease mechanisms and pinpoint possible intervention strategies. By examining lipid profiles in extracellular vesicles (EVs), secreted particles from all cells, including astrocytes and neurons, and circulating in clinical samples, important insights regarding the brain's composition can be gained. Herein, a targeted lipidomic analysis was carried out in EVs derived from plasma samples after removal of lipoproteins from individuals with Alzheimer's disease (AD) and healthy controls. Differences were observed for selected lipid species of glycerolipids (GLs), glycerophospholipids (GPLs), lysophospholipids (LPLs) and sphingolipids (SLs) across three distinct EV subpopulations (all-cell origin, derived by immunocapture of CD9, CD81 and CD63; neuronal origin, derived by immunocapture of L1CAM; and astrocytic origin, derived by immunocapture of GLAST). The findings provide new insights into the lipid composition of EVs isolated from plasma samples regarding specific lipid families (MG, DG, Cer, PA, PC, PE, PI, LPI, LPE, LPC), as well as differences between AD and control individuals. This study emphasizes the crucial role of plasma EV lipidomics analysis as a comprehensive approach for identifying biomarkers and biological targets in AD and related disorders, facilitating early diagnosis and potentially informing novel interventions.

Single-extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron-derived EVs.

Isolation of neuron-derived extracellular vesicles (NDEVs) with L1 Cell Adhesion Molecule (L1CAM)-specific antibodies has been widely used to identify blood biomarkers of CNS disorders. However, full methodological validation requires demonstration of L1CAM in individual NDEVs and lower levels or absence of L1CAM in individual EVs from other cells. Here, we used multiple single-EV techniques to establish the neuronal origin and determine the abundance of L1CAM-positive EVs in human blood. L1CAM epitopes of the ectodomain are shown to be co-expressed on single-EVs with the neuronal proteins ß-III-tubulin, GAP43, and VAMP2, the levels of which increase in parallel with the enrichment of L1CAM-positive EVs. Levels of L1CAM-positive EVs carrying the neuronal proteins VAMP2 and ß-III-tubulin range from 30% to 63%, in contrast to 0.8%-3.9% of L1CAM-negative EVs. Plasma fluid-phase L1CAM does not bind to single-EVs. Our findings support the use of L1CAM as a target for isolating plasma NDEVs and leveraging their cargo to identify biomarkers reflecting neuronal function.

Brain responses to intermittent fasting and the healthy living diet in older adults.

Diet may promote brain health in metabolically impaired older individuals. In an 8-week randomized clinical trial involving 40 cognitively intact older adults with insulin resistance, we examined the effects of 5:2 intermittent fasting and the healthy living diet on brain health. Although intermittent fasting induced greater weight loss, the two diets had comparable effects in improving insulin signaling biomarkers in neuron-derived extracellular vesicles, decreasing the brain-age-gap estimate (reflecting the pace of biological aging of the brain) on magnetic resonance imaging, reducing brain glucose on magnetic resonance spectroscopy, and improving blood biomarkers of carbohydrate and lipid metabolism, with minimal changes in cerebrospinal fluid biomarkers for Alzheimer's disease. Intermittent fasting and healthy living improved executive function and memory, with intermittent fasting benefiting more certain cognitive measures. In exploratory analyses, sex, body mass index, and apolipoprotein E and SLC16A7 genotypes modulated diet effects. The study provides a blueprint for assessing brain effects of dietary interventions and motivates further research on intermittent fasting and continuous diets for brain health optimization. For further information, please see ClinicalTrials.gov registration: NCT02460783.