Diurnal cortisol, neuroinflammation, and neuroimaging visual rating scales in memory clinic patients.

BACKGROUND: Neuroinflammation is a hallmark of the Alzheimer's disease (AD) pathogenic process. Cortisol dysregulation may increase AD risk and is related to brain atrophy. This cross-sectional study aims to examine interactions of cortisol patterns and neuroinflammation markers in their association with neuroimaging correlates. METHOD: 134 participants were recruited from the Karolinska University Hospital memory clinic (Stockholm, Sweden). Four visual rating scales were applied to magnetic resonance imaging or computed tomography scans: medial temporal lobe atrophy (MTA), global cortical atrophy (GCA), white matter lesions (WML), and posterior atrophy. Participants provided saliva samples for assessment of diurnal cortisol patterns, and underwent lumbar punctures for cerebrospinal fluid (CSF) sampling. Three cortisol measures were used: the cortisol awakening response, total daily output, and the ratio of awakening to bedtime levels. Nineteen CSF neuroinflammation markers were categorized into five composite scores: proinflammatory cytokines, other cytokines, angiogenesis markers, vascular injury markers, and glial activation markers. Ordinal logistic regressions were conducted to assess associations between cortisol patterns, neuroinflammation scores, and visual rating scales, and interactions between cortisol patterns and neuroinflammation scores in relation to visual rating scales. RESULT: Higher levels of angiogenesis markers were associated with more severe WML. Some evidence was found for interactions between dysregulated diurnal cortisol patterns and greater neuroinflammation-related biomarkers in relation to more severe GCA and WML. No associations were found between cortisol patterns and visual rating scales. CONCLUSION: This study suggests an interplay between diurnal cortisol patterns and neuroinflammation in relation to brain structure. While this cross-sectional study does not provide information on causality or temporality, these findings suggest that neuroinflammation may be involved in the relationship between HPA-axis functioning and AD.

Assessment of brain-derived extracellular vesicle enrichment for blood biomarker analysis in age-related neurodegenerative diseases: An international overview.

INTRODUCTION: Brain-derived extracellular vesicles (BEVs) in blood allows for minimally-invasive investigations of central nervous system (CNS) -specific markers of age-related neurodegenerative diseases (NDDs). Polymer-based EV- and immunoprecipitation (IP)-based BEV-enrichment protocols from blood have gained popularity. We systematically investigated protocol consistency across studies, and determined CNS-specificity of proteins associated with these protocols. METHODS: NDD articles investigating BEVs in blood using polymer-based and/or IP-based BEV enrichment protocols were systematically identified, and protocols compared. Proteins used for BEV-enrichment and/or post-enrichment were assessed for CNS- and brain-cell-type-specificity, extracellular domains (ECD+), and presence in EV-databases. RESULTS: A total of 82.1% of studies used polymer-based (ExoQuick) EV-enrichment, and 92.3% used L1CAM for IP-based BEV-enrichment. Centrifugation times differed across studies. A total of 26.8% of 82 proteins systematically identified were CNS-specific: 50% ECD+, 77.3% were listed in EV-databases. CONCLUSIONS: We identified protocol steps requiring standardization, and recommend additional CNS-specific proteins that can be used for BEV-enrichment or as BEV-biomarkers. HIGHLIGHTS: Across NDDs, we identified protocols commonly used for EV/BEV enrichment from blood. We identified protocol steps showing variability that require harmonization. We assessed CNS-specificity of proteins used for BEV-enrichment or found in BEV cargo. CNS-specific EV proteins with ECD+ or without were identified. We recommend evaluation of blood-BEV enrichment using these additional ECD+ proteins.

DEAD Box Helicase 24 Is Increased in the Brain in Alzheimer's Disease and AppN-LF Mice and Influences Presymptomatic Pathology.

At the time of diagnosis, Alzheimer's disease (AD) patients already suffer from significant neuronal loss. The identification of proteins that influence disease progression before the onset of symptoms is thus an essential part of the development of new effective drugs and biomarkers. Here, we used an unbiased 18O labelling proteomics approach to identify proteins showing altered levels in the AD brain. We studied the relationship between the protein with the highest increase in hippocampus, DEAD box Helicase 24 (DDX24), and AD pathology. We visualised DDX24 in the human brain and in a mouse model for Aß42-induced AD pathology-AppNL-F-and studied the interaction between Aß and DDX24 in primary neurons. Immunohistochemistry in the AD brain confirmed the increased levels and indicated an altered subcellular distribution of DDX24. Immunohistochemical studies in AppNL-F mice showed that the increase of DDX24 starts before amyloid pathology or memory impairment is observed. Immunocytochemistry in AppNL-F primary hippocampal neurons showed increased DDX24 intensity in the soma, nucleus and nucleolus. Furthermore, siRNA targeting of DDX24 in neurons decreased APP and Aß42 levels, and the addition of Aß42 to the medium reduced DDX24. In conclusion, we have identified DDX24 as a protein with a potential role in Aß-induced AD pathology.

27-hydroxycholesterol promotes oligodendrocyte maturation: Implications for hypercholesterolemia-associated brain white matter changes.

Oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) is a potential link between hypercholesterolemia and neurodegenerative diseases since unlike peripheral cholesterol, 27-OH is transported across the blood-brain barrier. However, the effects of high 27-OH levels on oligodendrocyte function remain unexplored. We hypothesize that during hypercholesterolemia 27-OH may impact oligodendrocytes and myelin and thus contribute to the disconnection of neural networks in neurodegenerative diseases. To test this idea, we first investigated the effects of 27-OH in cultured oligodendrocytes and found that it induces cell death of immature O4+ /GalC+ oligodendrocytes along with stimulating differentiation of PDGFR+ oligodendrocyte progenitors (OPCs). Next, transgenic mice with increased systemic 27-OH levels (Cyp27Tg) underwent behavioral testing and their brains were immunohistochemically stained and lysed for immunoblotting. Chronic exposure to 27-OH in mice resulted in increased myelin basic protein (MBP) but not 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) or myelin oligodendrocyte glycoprotein (MOG) levels in the corpus callosum and cerebral cortex. Intriguingly, we also found impairment of spatial learning suggesting that subtle changes in myelinated axons of vulnerable areas like the hippocampus caused by 27-OH may contribute to impaired cognition. Finally, we found that 27-OH levels in cerebrospinal fluid from memory clinic patients were associated with levels of the myelination regulating CNPase, independently of Alzheimer's disease markers. Thus, 27-OH promotes OPC differentiation and is toxic to immature oligodendrocytes as well as it subtly alters myelin by targeting oligodendroglia. Taken together, these data indicate that hypercholesterolemia-derived higher 27-OH levels change the oligodendrocytic capacity for appropriate myelin remodeling which is a crucial factor in neurodegeneration and aging.

APOE-Genotype and Insulin Modulate Estimated Effect of Dietary Macronutrients on Cognitive Performance: Panel Analyses in Nondiabetic Older Adults at Risk of Dementia.

BACKGROUND: The apolipoprotein E gene (APOE ε-2/3/4, combined as 6 different genotypes: ε-22/23/24/33/34/44) and insulin status modulate dementia risk and play a role in the metabolism of macronutrients. OBJECTIVES: We aimed to examine APOE-genotype and fasting insulin as effect modifiers of the slopes between dietary macronutrients and cognitive performance among older adults at risk of dementia. METHODS: Panel analyses-with diet and cognition measured at baseline and follow-up at years 1 and 2-were performed in a sub-sample from the FINGER (Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability) trial (n = 676, 60-77 y, 46% females, all nondiabetics). The associations between macronutrients (3-d food records, z-scores) and global cognition (modified Neuropsychological Test Battery, z-score) were analyzed in mixed regression models adjusted for confounders selected a priori. After a gradient was implied by the point estimates in categorical APOE analyses, we investigated a continuous APOE variable [APOE-gradient, coded -1 (for ε-23), -0.5 (ε-24), 0 (ε-33), 1 (ε-34), 2 (ε-44)] as an effect-modifier. RESULTS: At increasing levels of the APOE-gradient, a relatively more favorable slope between diet and cognition was observed for a lower carbohydrate/fat ratio [ß = -0.040, 95% confidence interval (CI): -0.074, -0.006; P = 0.020 for interaction diet × APOE-gradient), and higher protein (ß = 0.075, 95% CI: 0.042, 0.109; P = 9.4 × 10-6). Insulin concentration (log-linear) modulated the association between the carbohydrate/fat ratio and cognition by a quadratic interaction (ß = -0.016, P = 0.039). Coherent findings for exploratory predictors (fiber, fat subtypes, composite score, metabolic biomarkers) were compatible with published hypotheses of differential dietary adaptation by APOE, with cognition among ε-33 being relatively independent of dietary parameters-implying "metabolic flexibility." Antagonistic slopes to cognition for ε-23 (positive) compared with ε-34 and ε-44 (negative) were found for a Higher-carbohydrates-fiber-Lower-fat-protein composite score, even as within-subjects effects. CONCLUSIONS: APOE-based precision nutrition appears conceptually promising, but replications in wider samples are warranted, as well as support from trials. Both relative hyper- and hypoinsulinemia might modulate the effect of diet on cognition.

Blood ß-synuclein is related to amyloid PET positivity in memory clinic patients.

INTRODUCTION: ß-synuclein is an emerging blood biomarker to study synaptic degeneration in Alzheimer´s disease (AD), but its relation to amyloid-ß (Αß) pathology is unclear. METHODS: We investigated the association of plasma ß-synuclein levels with [18F] flutemetamol positron emission tomography (PET) in patients with AD dementia (n = 51), mild cognitive impairment (MCI-Aß+ n = 18, MCI- Aß- n = 30), non-AD dementias (n = 22), and non-demented controls (n = 5). RESULTS: Plasma ß-synuclein levels were higher in Aß+ (AD dementia, MCI-Aß+) than in Aß- subjects (non-AD dementias, MCI-Aß-) with good discrimination of Aß+ from Aß- subjects and prediction of Aß status in MCI individuals. A positive correlation between plasma ß-synuclein and Aß PET was observed in multiple cortical regions across all lobes. DISCUSSION: Plasma ß-synuclein demonstrated discriminative properties for Aß PET positive and negative subjects. Our data underline that ß-synuclein is not a direct marker of Aß pathology and suggest different longitudinal dynamics of synaptic degeneration versus amyloid deposition across the AD continuum. HIGHLIGHTS: Blood and CSF ß-synuclein levels are higher in Aß+ than in Aß- subjects. Blood ß-synuclein level correlates with amyloid PET positivity in multiple regions. Blood ß-synuclein predicts Aß status in MCI individuals.

Capillary blood tests may overestimate ketosis: triangulation between three different measures of ß-hydroxybutyrate.

The ketone body ß-hydroxybutyrate (BHB), assessed by a point-of-care meter in venous whole blood (BHBv), was used as the main outcome in a study on nutritional ketosis in healthy older adults. Two other BHB measures were also used in the study for validation and exploratory purposes, and here we report findings on correlation and agreement between those three methods. Ketosis in the range of 0-1.5 mmol/L was induced in 15 healthy volunteers by intake of medium-chain fatty acids after a 12-h fast. BHBv was assessed at 12 time points for 4 h. The same point-of-care meter was also used to test capillary blood (BHBc) at three time points, and a laboratory test determined total ketones (TK) in plasma (BHBp + acetoacetate) at four time points. A total of 180 cases included simultaneous data on BHBv, BHBc, BHBp, and TK. TK correlated with BHBp (Pearson's r = 0.99), BHBv (r = 0.91), and BHBc (r = 0.91), all P < 0.0001. BHBv and BHBp had good agreement in absolute values. However, the slope between BHBc and BHBv, measured with the same device, was in the range of 0.64-0.78 in different regression models, indicating substantially higher BHB concentrations in capillary versus venous blood. We conclude that all three methods are valid to detect relative changes in ketosis, but our results highlight the importance of method considerations and the possible need to adjust cutoffs, e.g., in the management of ketoacidosis and in the evaluation and comparison of dietary interventions.

Proximity ligation assay reveals both pre- and postsynaptic localization of the APP-processing enzymes ADAM10 and BACE1 in rat and human adult brain.

BACKGROUND: Synaptic degeneration and accumulation of amyloid ß-peptides (Aß) are hallmarks of the Alzheimer diseased brain. Aß is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the ß-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aß will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. RESULTS: Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. CONCLUSIONS: We show that the α-secretase ADAM10 and the ß-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

Reduction of PINK1 or DJ-1 impair mitochondrial motility in neurites and alter ER-mitochondria contacts.

Subcellular distribution of mitochondria in neurons is crucial for meeting the energetic demands, as well as the necessity to buffer Ca2+ within the axon, dendrites and synapses. Mitochondrial impairment is an important feature of Parkinson disease (PD), in which both familial parkinsonism genes DJ-1 and PINK1 have a great impact on mitochondrial function. We used differentiated human dopaminergic neuroblastoma cell lines with stable PINK1 or DJ-1 knockdown to study live motility of mitochondria in neurites. The frequency of anterograde and retrograde mitochondrial motility was decreased in PINK1 knockdown cells and the frequency of total mitochondrial motility events was reduced in both cell lines. However, neither the distribution nor the size of mitochondria in the neurites differed from the control cells even after downregulation of the mitochondrial fission protein, Drp1. Furthermore, mitochondria from PINK1 knockdown cells, in which motility was most impaired, had increased levels of GSK3ßSer9 and higher release of mitochondrial Ca2+ when exposed to CCCP-induced mitochondrial uncoupling. Further analysis of the ER-mitochondria contacts involved in Ca2+ shuttling showed that PINK1 knockdown cells had reduced contacts between the two organelles. Our results give new insight on how PINK1 and DJ-1 influence mitochondria, thus providing clues to novel PD therapies.

Tau hyperphosphorylation induces oligomeric insulin accumulation and insulin resistance in neurons.

Insulin signalling deficiencies and insulin resistance have been directly linked to the progression of neurodegenerative disorders like Alzheimer's disease. However, to date little is known about the underlying molecular mechanisms or insulin state and distribution in the brain under pathological conditions. Here, we report that insulin is accumulated and retained as oligomers in hyperphosphorylated tau-bearing neurons in Alzheimer's disease and in several of the most prevalent human tauopathies. The intraneuronal accumulation of insulin is directly dependent on tau hyperphosphorylation, and follows the tauopathy progression. Furthermore, cells accumulating insulin show signs of insulin resistance and decreased insulin receptor levels. These results suggest that insulin retention in hyperphosphorylated tau-bearing neurons is a causative factor for the insulin resistance observed in tauopathies, and describe a novel neuropathological concept with important therapeutic implications.

Anthocyanins protect from complex I inhibition and APPswe mutation through modulation of the mitochondrial fission/fusion pathways.

Anthocyanins are a distinguished class of flavonoids with powerful free radical-scavenging activity that have been suggested as chemotherapeutic agents for the prevention of Alzheimer disease (AD). In this study, we examined the ability of nutraceutical Medox rich in purified cyanidin 3-O-glucoside (C3G), 3-O-b-glucosides and delphinidin 3-O-glucoside (D3G) to counteract mitochondrial deficiency induced by complex I inhibition and/or amyloid-ß peptide (Aß) induced toxicity. SH-SY5Y neuroblastoma cells were stably transfected with APP Swedish K670N/M671L double mutation (APPswe) or with the empty vector and treated with rotenone. We report that Medox treatment improves the metabolic activity and maintains cell integrity in both cell lines. At the mitochondrial level, APPswe and rotenone induced mitochondrial fragmentation, an effect that was counteracted by Medox through the modulation of fission and fusion proteins, resulting in a reshaped mitochondrial network. Although Medox was unable to fully neutralise the effects of rotenone on ATP levels and mitochondrial membrane potential, it was able to prevent rotenone-induced cytotoxicity. Our findings suggest that Medox anthocyanins, on top of their antioxidant capacity, ameliorate mitochondrial dysfunction generated by Aß overproduction or by chemical inhibition of mitochondrial complex I via stabilization of the fusion/fission processes. Modulation of the mitochondrial network has been suggested as a novel therapeutic approach in diseases involving mitochondrial dysfunction and oxidative stress. Hence, increasing the understanding of how anthocyanins influence mitochondrial dynamics in a neurodegenerative context, could be of future therapeutic value.

CSF protein ratios with enhanced potential to reflect Alzheimer's disease pathology and neurodegeneration.

BACKGROUND: Amyloid and tau aggregates are considered to cause neurodegeneration and consequently cognitive decline in individuals with Alzheimer's disease (AD). Here, we explore the potential of cerebrospinal fluid (CSF) proteins to reflect AD pathology and cognitive decline, aiming to identify potential biomarkers for monitoring outcomes of disease-modifying therapies targeting these aggregates. METHOD: We used a multiplex antibody-based suspension bead array to measure the levels of 49 proteins in CSF from the Swedish GEDOC memory clinic cohort at the Karolinska University Hospital. The cohort comprised 148 amyloid- and tau-negative individuals (A-T-) and 65 amyloid- and tau-positive individuals (A+T+). An independent sample set of 26 A-T- and 26 A+T+ individuals from the Amsterdam Dementia Cohort was used for validation. The measured proteins were clustered based on their correlation to CSF amyloid beta peptides, tau and NfL levels. Further, we used support vector machine modelling to identify protein pairs, matched based on their cluster origin, that reflect AD pathology and cognitive decline with improved performance compared to single proteins. RESULTS: The protein-clustering revealed 11 proteins strongly correlated to t-tau and p-tau (tau-associated group), including mainly synaptic proteins previously found elevated in AD such as NRGN, GAP43 and SNCB. Another 16 proteins showed predominant correlation with Aß42 (amyloid-associated group), including PTPRN2, NCAN and CHL1. Support vector machine modelling revealed that proteins from the two groups combined in pairs discriminated A-T- from A+T+ individuals with higher accuracy compared to single proteins, as well as compared to protein pairs composed of proteins originating from the same group. Moreover, combining the proteins from different groups in ratios (tau-associated protein/amyloid-associated protein) significantly increased their correlation to cognitive decline measured with cognitive scores. The results were validated in an independent cohort. CONCLUSIONS: Combining brain-derived proteins in pairs largely enhanced their capacity to discriminate between AD pathology-affected and unaffected individuals and increased their correlation to cognitive decline, potentially due to adjustment of inter-individual variability. With these results, we highlight the potential of protein pairs to monitor neurodegeneration and thereby possibly the efficacy of AD disease-modifying therapies.

CYP46A1-mediated cholesterol turnover induces sex-specific changes in cognition and counteracts memory loss in ovariectomized mice.

The brain-specific enzyme CYP46A1 controls cholesterol turnover by converting cholesterol into 24S-hydroxycholesterol (24OH). Dysregulation of brain cholesterol turnover and reduced CYP46A1 levels are observed in Alzheimer's disease (AD). In this study, we report that CYP46A1 overexpression in aged female mice leads to enhanced estrogen signaling in the hippocampus and improved cognitive functions. In contrast, age-matched CYP46A1 overexpressing males show anxiety-like behavior, worsened memory, and elevated levels of 5α-dihydrotestosterone in the hippocampus. We report that, in neurons, 24OH contributes to these divergent effects by activating sex hormone signaling, including estrogen receptors. CYP46A1 overexpression in female mice protects from memory impairments induced by ovariectomy while having no effects in gonadectomized males. Last, we measured cerebrospinal fluid levels of 24OH in a clinical cohort of patients with AD and found that 24OH negatively correlates with neurodegeneration markers only in women. We suggest that CYP46A1 activation is a valuable pharmacological target for enhancing estrogen signaling in women at risk of developing neurodegenerative diseases.

Profiling of plasma biomarkers in the context of memory assessment in a tertiary memory clinic.

Plasma biomarkers have shown promising performance in research cohorts in discriminating between different stages of Alzheimer's disease (AD). Studies in clinical populations are necessary to provide insights on the clinical utility of plasma biomarkers before their implementation in real-world settings. Here we investigated plasma biomarkers (glial fibrillary acidic protein (GFAP), tau phosphorylated at 181 and 231 (pTau181, pTau231), amyloid ß (Aß) 42/40 ratio, neurofilament light) in 126 patients (age = 65 ± 8) who were admitted to the Clinic for Cognitive Disorders, at Karolinska University Hospital. After extensive clinical assessment (including CSF analysis), patients were classified as: mild cognitive impairment (MCI) (n = 75), AD (n = 25), non-AD dementia (n = 16), no dementia (n = 9). To refine the diagnosis, patients were examined with [18F]flutemetamol PET (Aß-PET). Aß-PET images were visually rated for positivity/negativity and quantified in Centiloid. Accordingly, 68 Aß+ and 54 Aß- patients were identified. Plasma biomarkers were measured using single molecule arrays (SIMOA). Receiver-operated curve (ROC) analyses were performed to detect Aß-PET+ using the different biomarkers. In the whole cohort, the Aß-PET centiloid values correlated positively with plasma GFAP, pTau231, pTau181, and negatively with Aß42/40 ratio. While in the whole MCI group, only GFAP was associated with Aß PET centiloid. In ROC analyses, among the standalone biomarkers, GFAP showed the highest area under the curve discriminating Aß+ and Aß- compared to other plasma biomarkers. The combination of plasma biomarkers via regression was the most predictive of Aß-PET, especially in the MCI group (prior to PET, n = 75) (sensitivity = 100%, specificity = 82%, negative predictive value = 100%). In our cohort of memory clinic patients (mainly MCI), the combination of plasma biomarkers was sensitive in ruling out Aß-PET negative individuals, thus suggesting a potential role as rule-out tool in clinical practice.

The Dietary Carbohydrate/Fat-Ratio and Cognitive Performance: Panel Analyses in Older Adults at Risk for Dementia.

Background: Roughly 80% of total energy intake (TEI) in most human diets originates from digestible carbohydrates (eCarb) and fat (eFat), but the impact of their proportions on cognitive performance is poorly understood. Objectives: Our primary aim was to investigate estimates of global cognition in relation to macronutrient intake, with the log-ratio eCarb/eFat (CFr) as the primary predictor variable of interest. Secondary predictors were protein and the saturated/total fat ratio. Exploratory comparisons of CFr with eCarb and eFat as separate predictors were an additional aim. Methods: The observations were made on panel data (years 0, 1, 2) from the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability, n = 1251; age 60-77 y; 47% females; selected by risk factors for dementia. Self-reported diet was assessed by 3-d food records. Global cognition was measured using a modified Neuropsychological Test Battery. A mixed linear regression model was used, adjusted for age, sex, education, body-mass index, cholesterol-lowering drugs, TEI, time, time × intervention/control group, with study site and subject as random factors. Estimates were standardized (mean = 0; SD = 1) with 95% CI. Results: CFr had a negative estimate to global cognition (ß = -0.022, CI: -0.039, -0.005; P = 0.011). The point estimate for protein was ß = 0.013 (P = 0.41), and for the saturated/total fat ratio, associations with cognition were nonlinear. CFr correlated highly with eCarb (Pearson's r = 0.92) and eFat (r = -0.94). The point estimate for CFr fell between eCarb (ß = -0.026, P < 0.001) and (inversely) eFat (ß = 0.017, P = 0.090). Conclusions: A lower CFr was associated with better global cognition among older adults at risk for dementia. Because this is an important target group for preventive interventions, clinical trials are warranted to further investigate the impact of macronutritional composition on cognitive health. The potential role of CFr as a predictor for cognitive health should be further studied.

Assessment of brain-derived extracellular vesicle enrichment for blood biomarker analysis in age-related neurodegenerative diseases: An international overview.

INTRODUCTION: Brain-derived extracellular vesicles (BEVs) in blood allows for minimally- invasive investigations of CNS-specific markers of age-related neurodegenerative diseases (NDDs). Polymer-based EV- and immunoprecipitation (IP)-based BEV-enrichment protocols from blood have gained popularity. We systematically investigated protocol consistency across studies, and determined CNS-specificity of proteins associated with these protocols. METHODS: NDD articles investigating BEVs in blood using polymer-based and/or IP-based BEV enrichment protocols were systematically identified, and protocols compared. Proteins used for BEV-enrichment and/or post-enrichment were assessed for CNS- and brain-cell-type- specificity; extracellular domains (ECD+); and presence in EV-databases. RESULTS: 82.1% of studies used polymer-based (ExoQuick) EV-enrichment, and 92.3% used L1CAM for IP-based BEV-enrichment. Centrifugation times differed across studies. 26.8% of 82 proteins systematically identified were CNS-specific: 50% ECD+, 77.3% were listed in EV- databases. DISCUSSION: We identified protocol steps requiring standardization, and recommend additional CNS-specific proteins that can be used for BEV-enrichment or as BEV-biomarkers.

SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer's Disease.

Sirtuin 2 (SIRT2) has been proposed to have a central role on aging, inflammation, cancer and neurodegenerative diseases; however, its specific function remains controversial. Recent studies propose SIRT2 pharmacological inhibition as a therapeutic strategy for several neurodegenerative diseases including Alzheimer's disease (AD). Surprisingly, none of these published studies regarding the potential interest of SIRT2 inhibition has assessed the peripheral adverse side consequences of this treatment. In this study, we demonstrate that the specific SIRT2 inhibitor, the compound 33i, does not exhibit genotoxic or mutagenic properties. Moreover, pharmacological treatment with 33i, improved cognitive dysfunction and long-term potentiation, reducing amyloid pathology and neuroinflammation in the APP/PS1 AD mouse model. However, this treatment increased peripheral levels of the inflammatory cytokines IL-1ß, TNF, IL-6 and MCP-1. Accordingly, peripheral SIRT2 inhibition with the blood brain barrier impermeable compound AGK-2, worsened the cognitive capacities and increased systemic inflammation. The analysis of human samples revealed that SIRT2 is increased in the brain but not in the serum of AD patients. These results suggest that, although SIRT2 pharmacological inhibition may have beneficial consequences in neurodegenerative diseases, its pharmacological inhibition at the periphery would not be recommended and the systemic adverse side effects should be considered. This information is essential to maximize the therapeutic potential of SIRT2 inhibition not only for AD but also for other neurodegenerative diseases.

Alzheimer's disease biomarker profiling in a memory clinic cohort without common comorbidities.

Alzheimer's disease is a multifactorial disorder with large heterogeneity. Comorbidities such as hypertension, hypercholesterolaemia and diabetes are known contributors to disease progression. However, less is known about their mechanistic contribution to Alzheimer's pathology and neurodegeneration. The aim of this study was to investigate the relationship of several biomarkers related to risk mechanisms in Alzheimer's disease with the well-established Alzheimer's disease markers in a memory clinic population without common comorbidities. We investigated 13 molecular markers representing key mechanisms underlying Alzheimer's disease pathogenesis in CSF from memory clinic patients without diagnosed hypertension, hypercholesterolaemia or diabetes nor other neurodegenerative disorders. An analysis of covariance was used to compare biomarker levels between clinical groups. Associations were analysed by linear regression. Two-step cluster analysis was used to determine patient clusters. Two key markers were analysed by immunofluorescence staining in the hippocampus of non-demented control and Alzheimer's disease individuals. CSF samples from a total of 90 participants were included in this study: 30 from patients with subjective cognitive decline (age 62.4 ± 4.38, female 60%), 30 with mild cognitive impairment (age 65.6 ± 7.48, female 50%) and 30 with Alzheimer's disease (age 68.2 ± 7.86, female 50%). Angiotensinogen, thioredoxin-1 and interleukin-15 had the most prominent associations with Alzheimer's disease pathology, synaptic and axonal damage markers. Synaptosomal-associated protein 25 kDa and neurofilament light chain were increased in mild cognitive impairment and Alzheimer's disease patients. Grouping biomarkers by biological function showed that inflammatory and survival components were associated with Alzheimer's disease pathology, synaptic dysfunction and axonal damage. Moreover, a vascular/metabolic component was associated with synaptic dysfunction. In the data-driven analysis, two patient clusters were identified: Cluster 1 had increased CSF markers of oxidative stress, vascular pathology and neuroinflammation and was characterized by elevated synaptic and axonal damage, compared with Cluster 2. Clinical groups were evenly distributed between the clusters. An analysis of post-mortem hippocampal tissue showed that compared with non-demented controls, angiotensinogen staining was higher in Alzheimer's disease and co-localized with phosphorylated-tau. The identification of biomarker-driven endophenotypes in cognitive disorder patients further highlights the biological heterogeneity of Alzheimer's disease and the importance of tailored prevention and treatment strategies.

Serum Thioredoxin-80 is associated with age, ApoE4, and neuropathological biomarkers in Alzheimer's disease: a potential early sign of AD.

BACKGROUND: Thioredoxin-80 (Trx80) is a cleavage product from the redox-active protein Thioredoxin-1 and has been previously described as a pro-inflammatory cytokine secreted by immune cells. Previous studies in our group reported that Trx80 levels are depleted in Alzheimer's disease (AD) brains. However, no studies so far have investigated peripheral Trx80 levels in the context of AD pathology and whether could be associated with the main known AD risk factors and biomarkers. METHODS: Trx80 was measured in serum samples from participants from two different cohorts: the observational memory clinic biobank (GEDOC) (N = 99) with AD CSF biomarker data was available and the population-based lifestyle multidomain intervention trial Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) (N = 47), with neuroimaging data and blood markers of inflammation available. The GEDOC cohort consists of participants diagnosed with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), and AD, whereas the FINGER participants are older adults at-risk of dementia, but without substantial cognitive impairment. One-way ANOVA and multiple comparison tests were used to assess the levels of Trx80 between groups. Linear regression models were used to explore associations of Trx80 with cognition, AD CSF biomarkers (Aß42, t-tau, p-tau and p-tau/t-tau ratio), inflammatory cytokines, and neuroimaging markers. RESULTS: In the GEDOC cohort, Trx80 was associated to p-tau/t-tau ratio in the MCI group. In the FINGER cohort, serum Trx80 levels correlated with lower hippocampal volume and higher pro-inflammatory cytokine levels. In both GEDOC and FINGER cohorts, ApoE4 carriers had significantly higher serum Trx80 levels compared to non-ApoE4 carriers. However, Trx80 levels in the brain were further decreased in AD patients with ApoE4 genotype. CONCLUSION: We report that serum Trx80 levels are associated to AD disease stage as well as to several risk factors for AD such as age and ApoE4 genotype, which suggests that Trx80 could have potential as serum AD biomarker. Increased serum Trx80 and decreased brain Trx80 levels was particularly seen in ApoE4 carriers. Whether this could contribute to the mechanism by which ApoE4 show increased vulnerability to develop AD would need to be further investigated. TRIAL REGISTRATION: ClinicalTrials.gov NCT01041989 . Registered on 4 January 2010-retrospectively registered.

ß-Amyloid, Tau, Neurodegeneration Classification and Eligibility for Anti-amyloid Treatment in a Memory Clinic Population.

BACKGROUND AND OBJECTIVES: ATN (ß-amyloid [Aß], tau, neurodegeneration) system categorizes individuals based on their core Alzheimer disease (AD) biomarkers. An important potential future use for ATN is therapeutic decision-making in clinical practice once disease-modifying treatments (e.g., anti-amyloid), become widely available. In this cross-sectional study, we applied ATN and estimated potential eligibility for anti-amyloid treatment in a real-life memory clinic with biomarker assessments integrated into the routine diagnostic procedure and all specialized resources available for the implementation of novel treatments. METHODS: We included all consecutive patients at the Karolinska University Hospital Memory clinic in Solna, Stockholm, Sweden, who had their first diagnostic visit in April 2018-February 2021, informed consent for the clinic research database, and available clinical and biomarker (CSF and imaging) data. ATN classification was based on CSF Aß42 (or Aß42/40; A), CSF phosphorylated tau (T), and medial temporal lobe atrophy (N). For CSF markers, we applied laboratory cutoffs and data-driven cutoffs for comparison (determined with Gaussian mixture modeling). Eligibility for anti-amyloid treatment was assessed following the published recommendations for aducanumab (AD dementia or mild cognitive impairment [MCI] with no evidence of non-AD etiology, appropriate level of cognition, and AD-consistent CSF profile). RESULTS: The study population consisted of 410 patients (52% subjective cognitive impairment, 23% MCI, and 25% any dementia; age 59 ± 7 years, 56% women). Regardless of biomarker cutoffs, most patients were A-T-N- (54%-57%). A+ prevalence was 17%-30% (higher with data-driven cutoffs). Up to 13% of all patients (27% of those with MCI and 28% of those with dementia) were potentially eligible for anti-amyloid treatment when AD-consistent CSF was defined as any A+ profile. When A+T+ profile was required, treatment was targeted more to the dementia than MCI stage (eligibility up to 14% in MCI and 22% in dementia). The opposite applied to earlier-stage intervention (A+T-N-; eligibility up to 12% in MCI and 2% in dementia). DISCUSSION: In a memory clinic setting with all necessary infrastructure and national guidelines in place for dementia diagnostic examination (best-case scenario), most of the patients did not meet the eligibility criteria for anti-amyloid treatment. Continuing the development of disease-modifying treatments with different mechanisms of action is a priority.