Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.
We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.
We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.
Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.
Electronic supplementary material
The online version of this article (doi:10.1186/s13024-016-0138-8) contains supplementary material, which is available to authorized users.
Glymphatic pathways; AQP4; Alzheimer’s disease; Lymphatic system; Brain clearance; Sleep/wake
Wang et al. report that TREM2 protects mice from Alzheimer's disease by enabling resident microglia to insulate and alter Aβ plaque structure, thereby limiting neuritic damage.
Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor that recognizes changes in the lipid microenvironment, which may occur during amyloid β (Aβ) accumulation and neuronal degeneration in Alzheimer’s disease (AD). Rare TREM2 variants that affect TREM2 function lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents microglial clustering around Aβ deposits. However, the origin of myeloid cells surrounding amyloid and the impact of TREM2 on Aβ accumulation are a matter of debate. Using parabiosis, we found that amyloid-associated myeloid cells derive from brain-resident microglia rather than from recruitment of peripheral blood monocytes. To determine the impact of TREM2 deficiency on Aβ accumulation, we examined Aβ plaques in the 5XFAD model of AD at the onset of Aβ-related pathology. At this early time point, Aβ accumulation was similar in TREM2-deficient and -sufficient 5XFAD mice. However, in the absence of TREM2, Aβ plaques were not fully enclosed by microglia; they were more diffuse, less dense, and were associated with significantly greater neuritic damage. Thus, TREM2 protects from AD by enabling microglia to surround and alter Aβ plaque structure, thereby limiting neuritic damage.
Alzheimer’s disease (AD) pathology appears several years before clinical symptoms, so identifying ways to detect individuals in the preclinical stage is imperative. The cerebrospinal fluid (CSF) Tau/Aβ42 ratio is currently the best known predictor of AD status and cognitive decline, and the ratio of CSF levels of chitinase-3-like 1 protein (CHI3L1, YKL-40) and amyloid beta (Aβ42) were reported as predictive, but individual variability and group overlap inhibits their utility for individual diagnosis making it necessary to find ways to improve sensitivity of these biomarkers.
We used linear regression to identify genetic loci associated with CSF YKL-40 levels in 379 individuals (80 cognitively impaired and 299 cognitively normal) from the Charles F and Joanne Knight Alzheimer’s Disease Research Center. We tested correlations between YKL-40 and CSF Tau/Aβ42 ratio, Aβ42, tau, and phosphorylated tau (ptau181). We used studentized residuals from a linear regression model of the log-transformed, standardized protein levels and the additive reference allele counts from the most significant locus to adjust YKL-40 values and tested the differences in correlations with CSF Tau/Aβ42 ratio, Aβ42, tau, and ptau181.
We found that genetic variants on the CH13L1 locus were significantly associated with CSF YKL-40 levels, but not AD risk, age at onset, or disease progression. The most significant variant is a reported expression quantitative trait locus for CHI3L1, the gene which encodes YKL-40, and explained 12.74 % of the variance in CSF YKL-40 in our study. YKL-40 was positively correlated with ptau181 (r = 0.521) and the strength of the correlation significantly increased with the addition of genetic information (r = 0.573, p = 0.006).
CSF YKL-40 levels are likely a biomarker for AD, but we found no evidence that they are an AD endophenotype. YKL-40 levels are highly regulated by genetic variation, and by including genetic information the strength of the correlation between YKL-40 and ptau181 levels is significantly improved. Our results suggest that studies of potential biomarkers may benefit from including genetic information.
Electronic supplementary material
The online version of this article (doi:10.1186/s12883-016-0742-9) contains supplementary material, which is available to authorized users.
CHI3L1; YKL-40; Cerebrospinal fluid; Alzheimer disease
The influence of reserve variables and Alzheimer’s disease (AD) biomarkers on cognitive test performance has been fairly well-characterized. However, less is known about the influence of these factors on “non-cognitive” outcomes, including functional abilities and mood.
We examined whether cognitive and brain reserve variables mediate how AD biomarker levels in cognitively normal persons predict future changes in function, mood, and neuropsychiatric behavior.
Non-cognitive outcomes were examined in 328 individuals 50 years and older enrolled in ongoing studies of aging and dementia at the Knight Alzheimer Disease Research Center (ADRC). All participants were cognitively normal at baseline (Clinical Dementia Rating [CDR] 0), completed cerebrospinal fluid (CSF) and structural neuroimaging studies within one year of baseline, and were followed for an average of 4.6 annual visits. Linear mixed effects models explored how cognitive reserve and brain reserve variables mediate the relationships between AD biomarker levels and changes in function, mood, and neuropsychiatric behavior in cognitively normal participants.
Education levels did not have a significant effect on predicting non-cognitive decline. However, participants with smaller brain volumes exhibited the worst outcomes on measures of mood, functional abilities, and behavioral disturbance. This effect was most pronounced in individuals who also had abnormal CSF biomarkers.
The findings suggest that brain reserve plays a stronger, or earlier, role than cognitive reserve in protecting against non-cognitive impairment in AD.
Alzheimer’s disease; biomarkers; cognitive reserve; dementia
Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by impairment of cognitive function, extracellular amyloid plaques, intracellular neurofibrillary tangles, and synaptic and neuronal loss. There is substantial evidence that the aggregation of amyloid β (Aβ) in the brain plays a key role in the pathogenesis of AD and that Aβ aggregation is a concentration dependent process. Recently, it was found that Aβ levels in the brain interstitial fluid (ISF) are regulated by the sleep-wake cycle in both humans and mice; ISF Aβ is higher during wakefulness and lower during sleep. Intracerebroventricular infusion of orexin increased wakefulness and ISF Aβ levels, and chronic sleep deprivation significantly increased Aβ plaque formation in amyloid precursor protein transgenic (APP) mice. Growth hormone-releasing hormone (GHRH) is a well-documented sleep regulatory substance which promotes non-rapid eye movement sleep. GHRHRlit/lit mice that lack functional GHRH receptor have shorter sleep duration and longer wakefulness during light periods. The current study was undertaken to determine whether manipulating sleep by interfering with GHRH signaling affects brain ISF Aβ levels in APPswe/PS1ΔE9 (PS1APP) transgenic mice that overexpress mutant forms of APP and PSEN1 that cause autosomal dominant AD. We found that intraperitoneal injection of GHRH at dark onset increased sleep and decreased ISF Aβ and that delivery of a GHRH antagonist via reverse-microdialysis suppressed sleep and increased ISF Aβ. The diurnal fluctuation of ISF Aβ in PS1APP/GHRHRlit/lit mice was significantly smaller than that in PS1APP/GHRHRlit/+ mice. However despite decreased sleep in GHRHR deficient mice, this was not associated with an increase in Aβ accumulation later in life. One of several possibilities for the finding is the fact that GHRHR deficient mice have GHRH-dependent but sleep-independent factors which protect against Aβ deposition.
Alzheimer’s disease; Amyloid-β; growth hormone-releasing hormone; sleep
The dynamic range of cerebrospinal fluid (CSF) amyloid β (Aβ1–42) measurement does not parallel to cognitive changes in Alzheimer’s disease (AD) and cognitively normal (CN) subjects across different studies. Therefore, identifying novel proteins to characterize symptomatic AD samples is important.
Proteins were profiled using a multianalyte platform by Rules Based Medicine (MAP-RBM). Due to underlying heterogeneity and unbalanced sample size, we combined subjects (344 AD and 325 CN) from three cohorts: Alzheimer’s Disease Neuroimaging Initiative, Penn Center for Neurodegenerative Disease Research of the University of Pennsylvania, and Knight Alzheimer’s Disease Research Center at Washington University in St. Louis. We focused on samples whose cognitive and amyloid status was consistent. We performed linear regression (accounted for age, gender, number of APOE e4 alleles, and cohort variable) to identify amyloid-related proteins for symptomatic AD subjects in this largest ever CSF–based MAP-RBM study. ANOVA and Tukey’s test were used to evaluate if these proteins were related to cognitive impairment changes as measured by mini-mental state examination (MMSE).
Seven proteins were significantly associated with Aβ1–42 levels in the combined cohort (false discovery rate adjusted P < .05), of which lipoprotein a (Lp(a)), prolactin (PRL), resistin, and vascular endothelial growth factor (VEGF) have consistent direction of associations across every individual cohort. VEGF was strongly associated with MMSE scores, followed by pancreatic polypeptide and immunoglobulin A (IgA), suggesting they may be related to staging of AD.
Lp(a), PRL, IgA, and tissue factor/thromboplastin have never been reported for AD diagnosis in previous individual CSF–based MAP-RBM studies. Although some of our reported analytes are related to AD pathophysiology, others’ roles in symptomatic AD samples worth further explorations.
Cerebrospinal fluid; Biomarkers; Alzheimer’s disease; Cognitive impairment; Amyloid beta; Dementia
Aerobic glycolysis and lactate production in the brain plays a key role in memory, yet the role of this metabolism in the cognitive decline associated with Alzheimer's disease (AD) remains poorly understood. Here we examined the relationship between cerebral lactate levels and memory performance in an APP/PS1 mouse model of AD, which progressively accumulates amyloid-β. In vivo
1H-magnetic resonance spectroscopy revealed an age-dependent decline in lactate levels within the frontal cortex of control mice, whereas lactate levels remained unaltered in APP/PS1 mice from 3 to 12 months of age. Analysis of hippocampal interstitial fluid by in vivo microdialysis revealed a significant elevation in lactate levels in APP/PS1 mice relative to control mice at 12 months of age. An age-dependent decline in the levels of key aerobic glycolysis enzymes and a concomitant increase in lactate transporter expression was detected in control mice. Increased expression of lactate-producing enzymes correlated with improved memory in control mice. Interestingly, in APP/PS1 mice the opposite effect was detected. In these mice, increased expression of lactate producing enzymes correlated with poorer memory performance. Immunofluorescent staining revealed localization of the aerobic glycolysis enzymes pyruvate dehydrogenase kinase and lactate dehydrogenase A within cortical and hippocampal neurons in control mice, as well as within astrocytes surrounding amyloid plaques in APP/PS1 mice. These observations collectively indicate that production of lactate, via aerobic glycolysis, is beneficial for memory function during normal aging. However, elevated lactate levels in APP/PS1 mice indicate perturbed lactate processing, a factor that may contribute to cognitive decline in AD.
SIGNIFICANCE STATEMENT Lactate has recently emerged as a key metabolite necessary for memory consolidation. Lactate is the end product of aerobic glycolysis, a unique form of metabolism that occurs within certain regions of the brain. Here we detected an age-dependent decline in the expression of aerobic glycolysis enzymes and a concomitant decrease in lactate levels within the frontal cortex of wild-type mice. Improved memory performance in wild-type mice correlated with elevated expression of aerobic glycolysis enzymes. Surprisingly, lactate levels remained elevated with age and increased aerobic glycolysis enzyme expression correlated with poorer memory performance in APP/PS1 mice. These findings suggest that while lactate production is beneficial for memory in the healthy aging brain, it might be detrimental in an Alzheimer's disease context.
aerobic glycolysis; Alzheimer's disease; amyloid; lactate; magnetic resonance spectroscopy; memory
Biomarkers for Alzheimer's disease (AD) have improved our understanding of the temporal sequence of biological events that lead to AD dementia (Jack et al, 2013). AD is characterized neuropathologically by amyloid plaques comprised of the amyloid‐β peptide and neurofibrillary tangles comprised of tau. Brain amyloid deposition, as evidenced by a decline in amyloid‐β peptide 42 (Aβ42) in the cerebrospinal fluid (CSF) or by binding of amyloid PET ligands, is thought to be a key initiating event in AD and begins many years prior to the onset of dementia. A rise in CSF tau and phosphorylated tau in the setting of Aβ deposition appears to reflect neurodegeneration and also begins years prior to the onset of dementia but after Aβ deposition has begun to accumulate. Individuals with “preclinical AD,” that is, normal cognition but abnormal AD biomarkers, have a much higher risk for developing AD dementia but may remain cognitively normal for years (Vos et al, 2013). While deposition of amyloid and formation of tau tangles are necessary for AD to occur, it is likely that additional events involving inflammation or other processes contribute to crossing the tipping point from preclinical AD to AD dementia. Current efforts are aimed at defining the biomarker(s) that best predict the transition from cognitive normality to abnormality. A biomarker that is closely associated with the onset of cognitive decline could help us to understand the biological events that connect amyloid deposition and tangle formation to cognitive decline and could have significant practical value in AD diagnosis and clinical trial design.
Biomarkers & Diagnostic Imaging; Neuroscience
Severe amyloidosis and plaque-localized neuro-inflammation are key pathological features of Alzheimer’s disease (AD). In addition to astrocyte and microglial reactivity, emerging evidence suggests a role of gut microbiota in regulating innate immunity and influencing brain function. Here, we examine the role of the host microbiome in regulating amyloidosis in the APPSWE/PS1ΔE9 mouse model of AD. We show that prolonged shifts in gut microbial composition and diversity induced by long-term broad-spectrum combinatorial antibiotic treatment regime decreases Aβ plaque deposition. We also show that levels of soluble Aβ are elevated and that levels of circulating cytokine and chemokine signatures are altered in this setting. Finally, we observe attenuated plaque-localised glial reactivity in these mice and significantly altered microglial morphology. These findings suggest the gut microbiota community diversity can regulate host innate immunity mechanisms that impact Aβ amyloidosis.
Epidemiological studies show that patients with type 2 diabetes (T2DM) and individuals with a diabetes-independent elevation in blood glucose have an increased risk for developing dementia, specifically dementia due to Alzheimer’s disease (AD). These observations suggest that abnormal glucose metabolism likely plays a role in some aspects of AD pathogenesis, leading us to investigate the link between aberrant glucose metabolism, T2DM, and AD in murine models. Here, we combined two techniques — glucose clamps and in vivo microdialysis — as a means to dynamically modulate blood glucose levels in awake, freely moving mice while measuring real-time changes in amyloid-β (Aβ), glucose, and lactate within the hippocampal interstitial fluid (ISF). In a murine model of AD, induction of acute hyperglycemia in young animals increased ISF Aβ production and ISF lactate, which serves as a marker of neuronal activity. These effects were exacerbated in aged AD mice with marked Aβ plaque pathology. Inward rectifying, ATP-sensitive potassium (KATP) channels mediated the response to elevated glucose levels, as pharmacological manipulation of KATP channels in the hippocampus altered both ISF Aβ levels and neuronal activity. Taken together, these results suggest that KATP channel activation mediates the response of hippocampal neurons to hyperglycemia by coupling metabolism with neuronal activity and ISF Aβ levels.
Endocrinology; Metabolism; Neuroscience
The Amyloid Hypothesis, which has been the predominant framework for research in Alzheimer’s Disease (AD) over the past two decades, has also been the source of considerable controversy within the field. The Amyloid Hypothesis postulates that amyloid-beta peptide (Aβ) is the causative agent in AD, and is strongly supported by data from rare autosomal dominant forms of AD. However, the evidence that Aβ causes age-associated sporadic AD is more complex and less clear, prompting criticism of the hypothesis. Herein, we provide an overview of the major arguments for and against the Amyloid Hypothesis, and review key data supporting or refuting these arguments. We conclude that Aβ likely is the key initiator of a complex pathogenic cascade which causes AD, thus supporting the Amyloid Hypothesis in general. However, we argue that Aβ acts primarily as a trigger of other downstream processes, in particular tau aggregation, which mediate neurodegeneration. Thus, Aβ appears to be necessary but not sufficient to causes AD, and its major pathogenic effects may occur very early in the disease process. We discuss implications for therapeutic development and future research.
amyloid hypothesis; Aβ; tau; neurodegeneration; Alzheimer Disease
The nicotinamide‐nucleotide adenylyltransferase protein Nmnat1 is a potent inhibitor of axonal degeneration in models of acute axonal injury. Hyperphosphorylation and aggregation of the microtubule‐associated protein Tau are associated with neurodegeneration in Alzheimer's Disease and other disorders. Previous studies have demonstrated that other Nmnat isoforms can act both as axonoprotective agents and have protein chaperone function, exerting protective effects in drosophila and mouse models of tauopathy. Nmnat1 targeted to the cytoplasm (cytNmnat1) is neuroprotective in a mouse model of neonatal hypoxia‐ischemia, but the effect of cytNmnat1 on tauopathy remains unknown.
We examined the impact of overexpression of cytNmnat1 on tau pathology, neurodegeneration, and brain functional connectivity in the P301S mouse model of chronic tauopathy.
Overexpression of cytNmnat1 preserved cortical neuron functional connectivity in P301S mice in vivo. However, whereas Nmnat1 overexpression decreased the accumulation of detergent‐insoluble tau aggregates in the cerebral cortex, it exerted no effect on immunohistochemical evidence of pathologic tau phosphorylation and misfolding, hippocampal atrophy, or inflammatory markers in P301S mice.
Our results demonstrate that cytNmnat1 partially preserves neuronal function and decreases biochemically insoluble tau in a mouse model of chronic tauopathy without preventing tau phosphorylation, formation of soluble aggregates, or tau‐induced inflammation and atrophy. Nmnat1 might thus represent a therapeutic target for tauopathies.
Dysregulation of amyloid-β (Aβ) metabolism is critical for Alzheimer's disease (AD) pathogenesis. Mounting evidence suggests that apolipoprotein E (ApoE) is involved in Aβ metabolism. ATP-binding cassette transporter A1 (ABCA1) is a key regulator of ApoE lipidation, which affects Aβ levels. Therefore, identifying regulatory mechanisms of ABCA1 expression in the brain may provide new therapeutic targets for AD. Here, we demonstrate that microRNA-33 (miR-33) regulates ABCA1 and Aβ levels in the brain. Overexpression of miR-33 impaired cellular cholesterol efflux and dramatically increased extracellular Aβ levels by promoting Aβ secretion and impairing Aβ clearance in neural cells. In contrast, genetic deletion of mir-33 in mice dramatically increased ABCA1 levels and ApoE lipidation, but it decreased endogenous Aβ levels in cortex. Most importantly, pharmacological inhibition of miR-33 via antisense oligonucleotide specifically in the brain markedly decreased Aβ levels in cortex of APP/PS1 mice, representing a potential therapeutic strategy for AD.
SIGNIFICANCE STATEMENT Brain lipid metabolism, in particular Apolipoprotein E (ApoE) lipidation, is critical to Aβ metabolism and Alzheimer's disease (AD). Brain lipid metabolism is largely separated from the periphery due to blood–brain barrier and different repertoire of lipoproteins. Therefore, identifying the novel regulatory mechanism of brain lipid metabolism may provide a new therapeutic strategy for AD. Although there have been studies on brain lipid metabolism, its regulation, in particular by microRNAs, is relatively unknown. Here, we demonstrate that inhibition of microRNA-33 increases lipidation of brain ApoE and reduces Aβ levels by inducing ABCA1. We provide a unique approach for AD therapeutics to increase ApoE lipidation and reduce Aβ levels via pharmacological inhibition of microRNA in vivo.
ABCA1; abeta; Alzheimer's disease; ApoE; miR-33
Past studies have shown that measures of attentional control and semantic memory are sensitive markers of Alzheimer disease (AD). The effects of established biomarkers of AD (cerebrospinal fluid tau and amyloid-beta42, PET-PIB, and APOE genotype) on concurrent cognitive performance in cognitively normal individuals have been mixed. The present study examined the utility of combining attentional control with semantic retrieval as a sensitive correlate of AD biomarkers and used mediation analyses to examine possible mechanisms by which the biomarkers influence cognition.
363 participants completed a category verification task (CVT) and 113 of them concurrently underwent biomarker assessments. On each trial, participants viewed a category (e.g. “unit of time”) and verified whether a subsequent target item was an exemplar of the category (“hour”) or not (“clock”). Importantly, the nonmembers of the category were associatively related to the category (e.g., “clock” is not “a unit of time”, but is highly related), and demanded attentional control to reject.
Accuracy to the foil items was the strongest discriminator between healthy aging and very mild symptomatic AD. CSF biomarkers had independent yet synergistic influence on CVT performance in cognitively healthy older adults. Furthermore, the influence of the biomarkers and APOE genotype was mediated primarily through increased levels of PIB.
The combined influence of attentional control with semantic retrieval is a marker of symptomatic AD and a sensitive correlate of established biomarkers for AD risk in cognitively healthy participants. The biomarkers influenced cognition primarily through increased levels of amyloid in the brain.
Alzheimer disease; biomarkers; attention; semantic retrieval
There is a growing emphasis on examining preclinical levels of Alzheimer Disease-related pathology in the absence of cognitive impairment. Prior work examining biomarkers has focused almost exclusively on memory, although there is mounting evidence that attention also declines early in disease progression. In the current experiment, two attentional control tasks were used to examine alterations in task-evoked functional magnetic resonance imaging (fMRI) data related to biomarkers of Alzheimer pathology. Seventy-one cognitively normal individuals (females=44, mean age=63.5) performed two attention-demanding cognitive tasks in a design that modeled both trial-level and task-level fMRI changes. Biomarkers included Aβ42, tau, and phosphorylated tau (ptau) measured from cerebrospinal fluid (CSF), and positron emission tomography (PET) measures of amyloid deposition. Both tasks elicited widespread patterns of activation and deactivation associated with large task-level manipulations of attention. Importantly, results from both tasks indicated that higher levels of tau and ptau pathology were associated with block level over-activations of attentional control areas. This suggests early alteration in attentional control with rising levels of Alzheimer Disease pathology.
Alzheimer; attention; biomarkers; fMRI; dementia; amyloid; Alzheimer’s; tau; ptau
Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia surface receptor that triggers intracellular protein tyrosine phosphorylation. Recent genome-wide association studies have shown that a rare R47H mutation of TREM2 correlates with a substantial increase in the risk of developing Alzheimer's disease (AD). To address the basis for this genetic association, we studied TREM2 deficiency in the 5XFAD mouse model of AD. We found that TREM2 deficiency and haploinsufficiency augment β-amyloid (Aβ) accumulation due to dysfunctional response of microglia, which become apoptotic and fail to cluster around Aβ plaques. We further demonstrate that TREM2 senses a broad array of anionic and zwitterionic lipids known to associate with fibrillar Aβ in lipid membranes and to be exposed on the surface of damaged neurons. Remarkably, the R47H mutation impairs TREM2 detection of lipid ligands. Thus, TREM2 detects damage-associated lipid patterns associated with neurodegeneration, sustaining microglia response to Aβ accumulation.
In Alzheimer’s disease, beta-amyloid peptides in the brain aggregate into toxic oligomers and plaques, a process which is associated with neuronal degeneration, memory loss, and cognitive decline. One therapeutic strategy is to decrease the production of potentially toxic beta-amyloid species by the use of inhibitors or modulators of the enzymes that produce beta-amyloid from amyloid precursor protein (APP). The failures of several such drug candidates by lack of effect or undesired side-effects underscore the importance to monitor the drug effects in the brain on a molecular level. Here we evaluate if peptidomic analysis in cerebrospinal fluid (CSF) can be used for this purpose.
Fifteen human healthy volunteers, divided into three groups, received a single dose of placebo or either 140 mg or 280 mg of the γ-secretase inhibitor semagacestat (LY450139). Endogenous peptides in CSF, sampled prior to administration of the drug and at six subsequent time points, were analyzed by liquid chromatography coupled to mass spectrometry, using isobaric labeling based on the tandem mass tag approach for relative quantification.
Out of 302 reproducibly detected peptides, 11 were affected by the treatment. Among these, one was derived from APP and one from amyloid precursor-like protein 1. Nine peptides were derived from proteins that may not be γ-secretase substrates per se, but that are regulated in a γ-secretase-dependent manner.
These results indicate that a CSF peptidomic approach may be a valuable tool both to verify target engagement and to identify other pharmacodynamic effects of the drug. Data are available via ProteomeXchange with identifier PXD003075.
NCT00765115, registered 30/09/2008.
Electronic supplementary material
The online version of this article (doi:10.1186/s13195-016-0178-x) contains supplementary material, which is available to authorized users.
Measures of neuronal loss are likely good surrogates for clinical and radiological disease progression in Alzheimer disease (AD). Cerebrospinal fluid (CSF) markers of neuronal injury or neurodegeneration may offer usefulness in predicting disease progression and guiding outcome assessments and prognostic decisions in clinical trials of disease-modifying therapies. Visinin-like protein 1 (VILIP-1) has demonstrated potential usefulness as a marker of neuronal injury in AD.
To investigate the usefulness of CSF VILIP-1, tau, p-tau181, and Aβ42 levels in predicting rates of whole-brain and regional atrophy in early AD and cognitively normal control subjects over time.
DESIGN, SETTING, AND PARTICIPANTS
Longitudinal observational study of brain atrophy in participants with early AD and cognitively normal controls. Study participants had baseline CSF biomarker measurements and longitudinal magnetic resonance imaging assessments for a mean follow-up period of 2 to 3 years. Mixed linear models assessed the ability of standardized baseline CSF biomarker measures to predict rates of whole-brain and regional atrophy over the follow-up period. The setting was The Charles F. and Joanne Knight Alzheimer’s Disease Research Center, Washington University School of Medicine in St Louis. Participants (mean age, 72.6 years) were individuals with a clinical diagnosis of very mild AD (n = 23) and cognitively normal controls (n = 64) who were enrolled in longitudinal studies of healthy aging and dementia. The study dates were 2000 to 2010.
MAIN OUTCOMES AND MEASURES
Correlations between baseline CSF biomarker measures and rates of whole-brain or regional atrophy in the AD and control cohorts over the follow-up period.
Baseline CSF VILIP-1, tau, and p-tau181 levels (but not Aβ42 levels) predicted rates of whole-brain and regional atrophy in AD over the follow-up period. Baseline CSF VILIP-1 levels predicted whole-brain (P = .006), hippocampal (P = .01), and entorhinal (P = .001) atrophy rates at least as well as tau and p-tau181 in early AD. Cognitively normal controls whose CSF VILIP-1, tau, or p-tau181 levels were in the upper tercile had higher rates of whole-brain (P = .02, P = .003, and P = .02, respectively), hippocampal (P = .001, P = .01, and P = .02, respectively), and entorhinal (P = .007, P = .01, and P = .01, respectively) atrophy compared with those whose levels were in the lower 2 terciles.
CONCLUSIONS AND RELEVANCE
Cerebrospinal fluid VILIP-1 levels predict rates of whole-brain and regional atrophy similarly to tau and p-tau181 and may provide a useful CSF biomarker surrogate for neurodegeneration in early symptomatic and preclinical AD.
Neurogranin (NGRN) seems to be a promising novel cerebrospinal fluid (CSF) biomarker for synaptic loss; however, clinical, and especially longitudinal, data are sparse.
To examine the utility of NGRN, with repeated CSF sampling, for diagnosis, prognosis, and monitoring of Alzheimer disease (AD).
DESIGN, SETTING, AND PARTICIPANTS
Longitudinal study of consecutive patients who underwent 2 lumbar punctures between the beginning of 1995 and the end of 2010 within the memory clinic–based Amsterdam Dementia Cohort. The study included 163 patients: 37 cognitively normal participants (mean [SE] age, 64  years; 38% female; and mean [SE] Mini-Mental State Examination [MMSE] score, 28 [0.3]), 61 patients with mild cognitive impairment (MCI) (mean [SE] age, 68  years; 38% female; and mean [SE] MMSE score, 27 [0.3]), and 65 patients with AD (mean [SE] age, 65  years; 45% female; and mean [SE] MMSE score, 22 [0.7]). The mean (SE) interval between lumbar punctures was 2.0 (0.1) years, and the mean (SE) duration of cognitive follow-up was 3.8 (0.2) years. Measurements of CSF NGRN levels were obtained in January and February 2014.
MAIN OUTCOME AND MEASURE
Levels of NGRN in CSF samples.
Baseline CSF levels of NGRN in patients with AD (median level, 2381 pg/mL [interquartile range, 1651-3416 pg/mL]) were higher than in cognitively normal participants (median level, 1712 pg/mL [interquartile range, 1206-2724 pg/mL]) (P = .04). Baseline NGRN levels were highly correlated with total tau and tau phosphorylated at threonine 181 in all patient groups (all P < .001), but not with Aβ42. Baseline CSF levels of NGRN were also higher in patients with MCI who progressed to AD (median level, 2842 pg/mL [interquartile range, 1882-3950 pg/mL]) compared with those with stable MCI (median level, 1752 pg/mL [interquartile range, 1024-2438 pg/mL]) (P = .004), and they were predictive of progression from MCI to AD (hazard ratio, 1.8 [95% CI, 1.1-2.9]; stratified by tertiles). Linear mixed-model analyses demonstrated that within-person levels of NGRN increased over time in cognitively normal participants (mean [SE] level, 90  pg/mL per year; P < .05) but not in patients with MCI or AD.
CONCLUSIONS AND RELEVANCE
Neurogranin is a promising biomarker for AD because levels were elevated in patients with AD compared with cognitively normal participants and predicted progression from MCI to AD. Within-person levels of NGRN increased in cognitively normal participants but not in patients with later stage MCI or AD, which suggests that NGRN may reflect presymptomatic synaptic dysfunction or loss.
Modulation of orexin and its effects on sleep/wakefulness affect amyloid-β pathology in the brain of mouse models for Alzheimer’s disease.
Age-related aggregation of amyloid-β (Aβ) is an upstream pathological event in Alzheimer’s disease (AD) pathogenesis, and it disrupts the sleep–wake cycle. The amount of sleep declines with aging and to a greater extent in AD. Poor sleep quality and insufficient amounts of sleep have been noted in humans with preclinical evidence of AD. However, how the amount and quality of sleep affects Aβ aggregation is not yet well understood. Orexins (hypocretins) initiate and maintain wakefulness, and loss of orexin-producing neurons causes narcolepsy. We tried to determine whether orexin release or secondary changes in sleep via orexin modulation affect Aβ pathology. Amyloid precursor protein (APP)/Presenilin 1 (PS1) transgenic mice, in which the orexin gene is knocked out, showed a marked decrease in the amount of Aβ pathology in the brain with an increase in sleep time. Focal overexpression of orexin in the hippocampus in APP/PS1 mice did not alter the total amount of sleep/wakefulness and the amount of Aβ pathology. In contrast, sleep deprivation or increasing wakefulness by rescue of orexinergic neurons in APP/PS1 mice lacking orexin increased the amount of Aβ pathology in the brain. Collectively, modulation of orexin and its effects on sleep appear to modulate Aβ pathology in the brain.
Amyloid β (Aβ) accumulates in the extracellular space as diffuse and neuritic plaques in Alzheimer’s disease (AD). Aβ also deposits on the walls of arterioles as cerebral amyloid angiopathy (CAA) in most cases of AD and sometimes independently of AD. Apolipoprotein E (apoE) ɛ4 is associated with increases in both Aβ plaques and CAA in humans. Studies in mouse models that develop Aβ deposition have shown that murine apoE and human apoE4 have different abilities to facilitate plaque or CAA formation when studied independently. To better understand and compare the effects of murine apoE and human apoE4, we bred 5XFAD (line 7031) transgenic mice so that they expressed one copy of murine apoE and one copy of human apoE4 under the control of the normal murine apoE regulatory elements (5XFAD/apoEm/4).
The 5XFAD/apoEm/4 mice contained levels of parenchymal CAA that were intermediate between 5XFAD/apoEm/m and 5XFAD/apoE4/4 mice. In 5XFAD/apoEm/4 mice, we found that Aβ parenchymal plaques co-localized with much more apoE than did parenchymal CAA, suggesting differential co-aggregation of apoE with Aβ in plaques versus CAA. More importantly, within the brain parenchyma of the 5XFAD/apoEm/4 mice, plaques contained more murine apoE, which on its own results in more pronounced and earlier plaque formation, while CAA contained more human apoE4 which on its own results in more pronounced CAA formation. We further confirmed the co-aggregation of mouse apoE with Aβ in plaques by showing a strong correlation between insoluble mouse apoE and insoluble Aβ in PS1APP-21/apoEm/4 mice which develop plaques without CAA.
These studies suggest that both murine apoE and human apoE4 facilitate differential opposing effects in influencing Aβ plaques versus CAA via different co-aggregation with these two amyloid lesions and set the stage for understanding these effects at a molecular level.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0250-y) contains supplementary material, which is available to authorized users.
Alzheimer’s disease; Apolipoprotein E; Amyloid plaques; Cerebral amyloid angiopathy
Alzheimer’s disease (AD) is a growing health crisis around the world. While significant progress has been made in our understanding of AD pathogenesis, there is currently no effective treatment to delay the onset of or prevent the disease. The focus has now shifted to the identification and treatment of AD in the early clinical stages as well as before cognitive symptoms emerge – during the long preclinical stage. With this shift in focus, diagnosis of individuals with AD can be more accurate when clinical symptoms and signs are combined with biomarkers. Biomarkers can improve both the diagnostic and prognostic accuracy of AD and its differentiation from other neurodegenerative diseases. This review will discuss fluid and imaging biomarkers that have shown promise in such areas, as well as some of the current challenges that face the field.
Alzheimer disease; biomarker; cerebrospinal fluid; neuroimaging; diagnosis; prognosis
Intracellular accumulation of tau as neurofibrillary tangles (NFTs) is the hallmark of Alzheimer’s disease (AD) as well as in other tauopathies. Tau is present not only in the cytoplasm but also in the extracellular space such as cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Although clearance is one critical parameter leading to such intracellular/extracellular tau accumulation, in vivo turnover of tau has not been well characterized. The current study has attempted to precisely determine in vivo turnover rates of tau utilizing tet-off regulatable mice. In particular, we assessed intracellular tau and extracellular tau, soluble tau, insoluble tau and phosphorylated tau at certain sites utilizing a combination of in vivo microdialysis, biochemical analysis and specific ELISAs recognizing each species. To examine the effect of a tauopathy-associated mutation on tau clearance, half-lives of various tau species were compared between the mice with a FTDP-17 mutation that induces β-sheet formation, ΔK280 mutation (pro-aggregant mice) and control mice with additional β-sheet breaking mutations (anti-aggregant mice).
Here we report that tau is metabolized at much slower turnover rates in vivo than in cell culture. We found that insoluble tau in pro-aggregant mice had a significantly slower half-life (t1/2 = ~34.2 days) than soluble tau (t1/2 = ~9.7 days). In contrast, soluble tau phosphorylated in the proline rich region was cleared faster than total soluble tau. When comparing pro-aggregant mice to anti-agregant mice, turnover rates of soluble tau species were not significantly different.
The current study provides a comprehensive description of in vivo turnover of various tau species present in mice that express human tau. The turnover rate of soluble tau was not significantly altered between pro-aggregant mice and anti-aggregant mice. This suggests that altered conformation by ΔK280 does not have a major impact on clearance pathways for soluble tau. In contrast, different tau species displayed different half-lives. Turnover was significantly delayed for insoluble tau whereas it was accelerated for soluble tau phosphorylated in the proline rich region. These differences in susceptibilities to clearance suggest that aggregation and phosphorylation influences tau clearance which may be important in tau pathogenesis.
Alzheimer’s disease; Clearance; Half-life; Extracellular tau; Tauopathy model
We previously found a strong reduction in tau pathology and insoluble tau in P301S tau transgenic mice following intracerebroventricular infusion of the anti-tau antibody HJ8.5. We sought to determine the effects of HJ8.5 in the same model following peripheral administration.
The primary objective was to determine if HJ8.5 administered at a dose of 50 mg kg−1 week−1 by intraperitoneal (IP) injection to 6-month-old P301S mice for 3 months would influence phospho-tau (p-tau) accumulation, tau insolubility, and neurodegeneration.
Treatment with HJ8.5 at 50 mg/kg showed a very strong decrease in detergent-insoluble tau. Importantly, HJ8.5 significantly reduced the loss of cortical and hippocampal tissue volumes compared to control treated mice. HJ8.5 treatment reduced hippocampal CA1 cellular layer staining with the p-tau antibody AT8 and thio-S-positive tau aggregates in piriform cortex and amygdala. Moreover, mice treated with HJ8.5 at 50 mg/kg showed a decrease in motor/sensorimotor deficits compared to vehicle-treated mice. Some effects of HJ8.5, including reduction in brain atrophy, and p-tau immunostaining were also seen with a dose of 10 mg kg−1 week−1. In BV2-microglial cells, we observed significantly higher uptake of P301S tau aggregates in the presence of HJ8.5. HJ8.5 treatment also resulted in a large dose-dependent increase of tau in the plasma.
Our results indicate that systemically administered anti-tau antibody HJ8.5 significantly decreases insoluble tau, decreases brain atrophy, and improves motor/sensorimotor function in a mouse model of tauopathy. These data further support the idea that anti-tau antibodies should be further assessed as a potential treatment for tauopathies.