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.

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.

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.

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.

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.

Microdissected Pyramidal Cell Proteomics of Alzheimer Brain Reveals Alterations in Creatine Kinase B-Type, 14-3-3-γ, and Heat Shock Cognate 71.

Novel insights on proteins involved in Alzheimer's disease (AD) are needed. Since multiple cell types and matrix components are altered in AD, bulk analysis of brain tissue maybe difficult to interpret. In the current study, we isolated pyramidal cells from the cornu ammonis 1 (CA1) region of the hippocampus from five AD and five neurologically healthy donors using laser capture microdissection (LCM). The samples were analyzed by proteomics using 18O-labeled internal standard and nano-high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for relative quantification. Fold change between AD and control was calculated for the proteins that were identified in at least two individual proteomes from each group. From the 10 cases analyzed, 62 proteins were identified in at least two AD cases and two control cases. Creatine kinase B-type (CKB), 14-3-3-γ, and heat shock cognate 71 (Hsc71), which have not been extensively studied in the context of the human AD brain previously, were selected for further studies by immunohistochemistry (IHC). In hippocampus, semi-quantitative measures of IHC staining of the three proteins confirmed the findings from our proteomic analysis. Studies of the same proteins in the frontal cortex revealed that the alterations remained for CKB and 14-3-3-γ but not for Hsc71. Protein upregulation in CA1 neurons of final stage AD is either a result of detrimental, pathological effects, or from cell-specific protective response mechanisms in surviving neurons. Based on previous findings from experimental studies, CKB and Hsc71 likely exhibit protective effects, whereas 14-3-3-γ may represent a detrimental pathway. These new players could reflect pathways of importance for the development of new therapeutic strategies.

Serum proBDNF Is Associated With Changes in the Ketone Body ß-Hydroxybutyrate and Shows Superior Repeatability Over Mature BDNF: Secondary Outcomes From a Cross-Over Trial in Healthy Older Adults.

Background: ß-hydroxybutyrate (BHB) can upregulate brain-derived neurotrophic factor (BDNF) in mice, but little is known about the associations between BHB and BDNF in humans. The primary aim here was to investigate whether ketosis (i.e., raised BHB levels), induced by a ketogenic supplement, influences serum levels of mature BDNF (mBDNF) and its precursor proBDNF in healthy older adults. A secondary aim was to determine the intra-individual stability (repeatability) of those biomarkers, measured as intra-class correlation coefficients (ICC). Method: Three of the arms in a 6-arm randomized cross-over trial were used for the current sub-study. Fifteen healthy volunteers, 65-75 y, 53% women, were tested once a week. Test oils, mixed in coffee and cream, were ingested after a 12-h fast. Labeled by their level of ketosis, the arms provided: sunflower oil (lowK); coconut oil (midK); caprylic acid + coconut oil (highK). Repeated blood samples were collected for 4 h after ingestion. Serum BDNF levels were analyzed for changes from baseline to 1, 2 and 4 h to compare the arms. Individual associations between BHB and BDNF were analyzed cross-sectionally and for a delayed response (changes in BHB 0-2 h to changes in BDNF at 0-4 h). ICC estimates were calculated from baseline levels from the three study days. Results: proBDNF increased more in highK vs. lowK between 0 and 4 h (z-score: ß = 0.25, 95% CI 0.07-0.44; p = 0.007). Individual change in BHB 0-2 h, predicted change in proBDNF 0-4 h, (ß = 0.40, CI 0.12-0.67; p = 0.006). Change in mBDNF was lower in highK vs. lowK at 0-2 h (ß = -0.88, CI -1.37 to -0.40; p < 0.001) and cumulatively 0-4 h (ß = -1.01, CI -1.75 to -0.27; p = 0.01), but this could not be predicted by BHB levels. ICC was 0.96 (95% CI 0.92-0.99) for proBDNF, and 0.72 (CI 0.47-0.89) for mBDNF. Conclusions: The findings support a link between changes in peripheral BHB and proBDNF in healthy older adults. For mBDNF, changes differed between arms but independent to BHB levels. Replication is warranted due to the small sample. Excellent repeatability encourages future investigations on proBDNF as a predictor of brain health. Clinical Trial Registration:ClinicalTrials.gov, NCT03904433.

27-Hydroxycholesterol, cognition, and brain imaging markers in the FINGER randomized controlled trial.

BACKGROUND: 27-Hydroxycholesterol (27-OH), the main circulating oxysterol in humans and the potential missing link between peripheral hypercholesterolemia and Alzheimer's disease (AD), has not been investigated previously in relation to cognition and neuroimaging markers in the context of preventive interventions. METHODS: The 2-year Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) included older individuals (60-77 years) at increased risk for dementia but without dementia or substantial cognitive impairment from the general population. Participants were randomized to a multidomain intervention (diet, exercise, cognitive training, and vascular risk management) or control group (general health advice) in a 1:1 ratio. Outcome assessors were masked to group allocation. This FINGER exploratory sub-study included 47 participants with measures of 27-OH, cognition, brain MRI, brain FDG-PET, and PiB-PET. Linear regression models were used to assess the cross-sectional and longitudinal associations between 27-OH, cognition, and neuroimaging markers, considering several potential confounders/intervention effect modifiers. RESULTS: 27-OH reduction during the intervention was associated with improvement in cognition (especially memory). This was not observed in the control group. The intervention reduced 27-OH particularly in individuals with the highest 27-OH levels and younger age. No associations were found between changes in 27-OH levels and neuroimaging markers. However, at baseline, a higher 27-OH was associated with lower total gray matter and hippocampal volume, and lower cognitive scores. These associations were unaffected by total cholesterol levels. While sex seemed to influence associations at baseline, it did not affect longitudinal associations. CONCLUSION: 27-OH appears to be a marker not only for dementia/AD risk, but also for monitoring the effects of preventive interventions on cholesterol metabolism. TRIAL REGISTRATION: ClinicalTrials.gov , NCT01041989 . Registered on 4 January 2010.

Sex difference in flux of 27-hydroxycholesterol into the brain.

BACKGROUND AND PURPOSE: The cerebrospinal fluid (CSF)/plasma albumin ratio (QAlb) is believed to reflect the integrity of the blood-brain barrier (BBB). Recently, we reported that QAlb is lower in females. This may be important for uptake of neurotoxic 27-hydroxycholesterol (27OH) by the brain in particular because plasma levels of 27OH are higher in males. We studied sex differences in the relation between CSF and plasma levels of 27OH and its major metabolite 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) with QAlb. We tested the possibility of sex differences in the brain metabolism of 27OH and if its flux into the brain disrupted integrity of the BBB. EXPERIMENTAL APPROACH: We have examined our earlier studies looking for sex differences in CSF levels of oxysterols and their relation to QAlb. We utilized an in vitro model for the BBB with primary cultured brain endothelial cells to test if 27OH has a disruptive effect on this barrier. We measured mRNA and protein levels of CYP7B1 in autopsy brain samples. KEY RESULTS: The correlation between CSF levels of 27OH and QAlb was higher in males whereas, with 7HOCA, the correlation was higher in females. No significant sex difference in the expression of CYP7B1 mRNA in brain autopsy samples. A correlation was found between plasma levels of 27OH and QAlb. No support was obtained for the hypothesis that plasma levels of 27OH have a disruptive effect on the BBB. CONCLUSIONS AND IMPLICATIONS: The sex differences are discussed in relation to negative effects of 27OH on different brain functions. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Ketosis After Intake of Coconut Oil and Caprylic Acid-With and Without Glucose: A Cross-Over Study in Healthy Older Adults.

Introduction: Medium-chain-triglycerides (MCT), formed by fatty acids with a length of 6-12 carbon atoms (C6-C12), constitute about two thirds of coconut oil (Coc). MCT have specific metabolic properties which has led them to be described as ketogenic even in the absence of carbohydrate restriction. This effect has mainly been demonstrated for caprylic acid (C8), which constitutes about 6-8% of coconut oil. Our aim was to quantify ketosis and blood glucose after intake of Coc and C8, with and without glucose intake. Sunflower oil (Suf) was used as control, expected to not break fasting ketosis, nor induce supply-driven ketosis. Method: In a 6-arm cross-over design, 15 healthy volunteers-age 65-73, 53% women-were tested once a week. After a 12-h fast, ketones were measured during 4 h after intake of coffee with cream, in combination with each of the intervention arms in a randomized order: 1. Suf (30 g); 2. C8 (20 g) + Suf (10 g); 3. C8 (20 g) + Suf (10 g) + Glucose (50 g); 4. Coc (30 g); 5. Coc (30 g) + Glucose (50 g); 6. C8 (20 g) + Coc (30 g). The primary outcome was absolute blood levels of the ketone ß-hydroxybutyrate, area under the curve (AUC). ANOVA for repeated measures was performed to compare arms. Results: ß-hydroxybutyrate, AUC/time (mean ± SD), for arms were 1: 0.18 ± 0.11; 2: 0.45 ± 0.19; 3: 0.28 ± 0.12; 4: 0.22 ± 0.12; 5: 0.08 ± 0.04; 6: 0.45 ± 0.20 (mmol/L). Differences were significant (all p ≤ 0.02), except for arm 2 vs. 6, and 4 vs. 1 & 3. Blood glucose was stable in arm 1, 2, 4, & 6, at levels slightly below baseline (p ≤ 0.05) at all timepoints hours 1-4 after intake. Conclusions: C8 had a higher ketogenic effect than the other components. Coc was not significantly different from Suf, or C8 with glucose. In addition, we report that a 16-h non-carbohydrate window contributed to a mild ketosis, while blood glucose remained stable. Our results suggest that time-restricted feeding regarding carbohydrates may optimize ketosis from intake of MCT. Clinical Trial Registration: The study was registered as a clinical trial on ClinicalTrials.gov, NCT03904433.

Proteomics Time-Course Study of App Knock-In Mice Reveals Novel Presymptomatic Aß42-Induced Pathways to Alzheimer's Disease Pathology.

BACKGROUND: The 42 amino acids long amyloid-ß peptide, Aß42, may initiate a cascade of events leading to the severe neurodegeneration observed in Alzheimer's disease (AD) brain. However, the underlying molecular mechanisms remain to be established. OBJECTIVE: To find early Aß42-induced AD related mechanisms, we performed a brain proteomics time-course study on a novel App knock-in AD mouse model, AppNL-F, expressing high levels of Aß42 without AßPP overexpression artifacts. METHODS: Hippocampus and cortex were analyzed separately by using 18O-labelling mass spectrometry to reveal alterations in protein levels. Pathway analysis of proteomics data was used to identify altered biological functions. Immunohistochemistry was used to further investigate a significant key regulatory protein. RESULTS: Around 100 proteins were differently expressed in AppNL-F mice at each time point (3, 6, 9, and 18 months of age) as compared to wild type mice. Strikingly, already at 3 months of age-long before Aß plaque development and memory impairment-several pathways, including long-term potentiation and synaptic plasticity, were downregulated, and neuritogenesis was increased. Huntingtin (HTT) was identified as an upstream regulator, i.e., a key protein affecting the levels of several proteins. Increased levels of HTT in hippocampus of AppNL-F mice was supported by immunofluorescence microscopy. CONCLUSION: Notably, the proteome was significantly altered already at 3 months of age, 6 months before the development of plaques. Differentially expressed proteins varied over time, indicating that increased Aß42 levels initiate a cascade of events that eventually manifests in amyloid depositions, inflammation, and decline in memory.

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.

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.

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.

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid ß-peptide levels.

BACKGROUND: Increased levels of the pathogenic amyloid ß-peptide (Aß), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aß in neurons. METHODS: MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aß production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aß42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. RESULTS: Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aß42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aß42. Furthermore, overexpression of MAO-B enhanced Aß production. CONCLUSIONS: This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aß production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aß levels using protein-protein interaction breakers.

Apolipoprotein E4 Elicits Lysosomal Cathepsin D Release, Decreased Thioredoxin-1 Levels, and Apoptosis.

The major genetic risk factor for Alzheimer's disease (AD), apolipoprotein E4 (ApoE4), has been suggested to have detrimental effects on neurons, including direct toxicity via apoptosis. Thioredoxin-1 (Trx1) is an endogenous antioxidant protein important for redox regulation and participates in the regulation of apoptosis through the inhibition of apoptosis signal-regulating kinase-1 (Ask-1). In this study, we have investigated the effects of ApoE on Trx1 in the brain. Our results showed that the protein levels of Trx1 were reduced in the hippocampus of ApoE4 targeted replacement (TR) mice compared to ApoE3 TR mice. The reduction was also seen in vitro after treatment of both human primary cortical neurons and neuroblastoma cells with human recombinant ApoE4 (rApoE4). Furthermore, ApoE4 caused a disruption of lysosomal integrity and a shift in the localization of Cathepsin D, an enzyme known to degrade Trx1. ApoE4 treatment induced in addition apoptosis through translocation of Death-domain associated protein-6 (Daxx) from the nucleus to the cytosol, suggesting an activation of the Ask-1 pathway. This toxicity was prevented by overexpression of Trx1 and other endogenous Ask-1 inhibitors. Our data suggests that down-regulation of Trx1 is involved in the toxicity caused by ApoE4. An activated ASK-1 pathway might indeed make cells more vulnerable to other insults such as amyloid-ß, which could partially explain the mechanism behind the strongest genetic risk factor for AD.

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.