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1.  In Vivo Microdialysis Reveals Age-Dependent Decrease of Brain Interstitial Fluid Tau Levels in P301S Human Tau Transgenic Mice 
Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.
PMCID: PMC4299126  PMID: 21917794
2.  Anti-ApoE Antibody Given after Plaque Onset Decreases Aβ Accumulation and Improves Brain Function in a Mouse Model of Aβ Amyloidosis 
The Journal of Neuroscience  2014;34(21):7281-7292.
Apolipoprotein E (apoE) is the strongest known genetic risk factor for late onset Alzheimer's disease (AD). It influences amyloid-β (Aβ) clearance and aggregation, which likely contributes in large part to its role in AD pathogenesis. We recently found that HJ6.3, a monoclonal antibody against apoE, significantly reduced Aβ plaque load when given to APPswe/PS1ΔE9 (APP/PS1) mice starting before the onset of plaque deposition. To determine whether the anti-apoE antibody HJ6.3 affects Aβ plaques, neuronal network function, and behavior in APP/PS1 mice after plaque onset, we administered HJ6.3 (10 mg/kg/week) or PBS intraperitoneally to 7-month-old APP/PS1 mice for 21 weeks. HJ6.3 mildly improved spatial learning performance in the water maze, restored resting-state functional connectivity, and modestly reduced brain Aβ plaque load. There was no effect of HJ6.3 on total plasma cholesterol or cerebral amyloid angiopathy. To investigate the underlying mechanisms of anti-apoE immunotherapy, HJ6.3 was applied to the brain cortical surface and amyloid deposition was followed over 2 weeks using in vivo imaging. Acute exposure to HJ6.3 affected the course of amyloid deposition in that it prevented the formation of new amyloid deposits, limited their growth, and was associated with occasional clearance of plaques, a process likely associated with direct binding to amyloid aggregates. Topical application of HJ6.3 for only 14 d also decreased the density of amyloid plaques assessed postmortem. Collectively, these studies suggest that anti-apoE antibodies have therapeutic potential when given before or after the onset of Aβ pathology.
PMCID: PMC4028501  PMID: 24849360
Alzheimer's; amyloid; antibody; apolipoprotein E
3.  Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo 
Neuron  2013;80(2):402-414.
Tau aggregation occurs in neurodegenerative diseases including Alzheimer's disease and many other disorders collectively termed tauopathies. Trans-cellular propagation of tau pathology, mediated by extracellular tau aggregates, may underlie pathogenesis of these conditions. P301S tau transgenic mice express mutant human tau protein, and develop progressive tau pathology. Using a cell-based biosensor assay, we screened anti-tau monoclonal antibodies for their ability to block seeding activity present in P301S brain lysates. We infused 3 effective antibodies or controls into the lateral ventricle of P301S mice for 3 months. The antibodies markedly reduced hyperphosphorylated, aggregated, and insoluble tau. They also blocked development of tau seeding activity detected in brain lysates using the biosensor assay, reduced microglial activation, and improved cognitive deficits. These data imply a central role for extracellular tau aggregates in the development of pathology. They also suggest immunotherapy specifically designed to block trans-cellular aggregate propagation will be a productive treatment strategy.
PMCID: PMC3924573  PMID: 24075978
4.  Neuronal activity regulates extracellular tau in vivo 
Neuronal activity promotes the release of extracellular tau in vivo.
Tau is primarily a cytoplasmic protein that stabilizes microtubules. However, it is also found in the extracellular space of the brain at appreciable concentrations. Although its presence there may be relevant to the intercellular spread of tau pathology, the cellular mechanisms regulating tau release into the extracellular space are not well understood. To test this in the context of neuronal networks in vivo, we used in vivo microdialysis. Increasing neuronal activity rapidly increased the steady-state levels of extracellular tau in vivo. Importantly, presynaptic glutamate release is sufficient to drive tau release. Although tau release occurred within hours in response to neuronal activity, the elimination rate of tau from the extracellular compartment and the brain is slow (half-life of ∼11 d). The in vivo results provide one mechanism underlying neuronal tau release and may link trans-synaptic spread of tau pathology with synaptic activity itself.
PMCID: PMC3949564  PMID: 24534188
5.  Sleep quality and preclinical Alzheimer Disease 
JAMA neurology  2013;70(5):587-593.
Sleep and circadian problems are very common in Alzheimer Disease (AD). Recent animal studies suggest a bidirectional relationship between sleep and amyloid-β (Aβ), a key molecule involved in AD pathogenesis. This study tested whether Aβ deposition in preclinical AD, prior to the appearance of cognitive impairment, is associated with changes in quality or quantity of sleep.
Cognitively normal, middle-aged individuals (n=142) had sleep objectively measured using actigraphy for 2 weeks. Concurrent sleep diaries provided nap information. Cerebrospinal fluid Aβ42 levels were used to determine whether amyloid deposition was present or absent. Sleep parameters were assessed with regard to amyloid deposition.
Amyloid deposition was associated with worse sleep quality, specifically worse sleep efficiency (% time in bed that was spent asleep), compared to those without amyloid deposition. In contrast, quantity of sleep was not different between groups, as measured by total sleep time. Frequent napping was associated with amyloid deposition.
Amyloid deposition in the preclinical stage of AD appears to be associated with worse sleep quality, but not with changes in sleep quantity.
PMCID: PMC3676720  PMID: 23479184
6.  Cerebral amyloid angiopathy increases susceptibility to infarction following focal cerebral ischemia in Tg2576 mice 
We and others have shown that soluble amyloid-β peptide (Aβ) and cerebral amyloid angiopathy (CAA) cause significant cerebrovascular dysfunction in mutant amyloid precursor protein (APP) mice, and that these deficits are greater in aged APP mice having CAA compared to young APP mice lacking CAA. Aβ in young APP mice also increases infarction following focal cerebral ischemia, but the impact of CAA on ischemic brain injury is unknown. To determine this, we assessed cerebrovascular reactivity, cerebral blood flow (CBF), and extent of infarction and neurological deficits following transient middle cerebral artery occlusion (MCAO) in aged APP mice having extensive CAA versus young APP mice lacking CAA (and aged-matched littermate controls). We found that aged APP mice have more severe cerebrovascular dysfunction that is CAA-dependent, have greater CBF compromise during and immediately following MCAO, and develop larger infarctions after MCAO. These data indicate CAA induces a more severe form of cerebrovascular dysfunction than Aβ alone, leading to intra- and post-ischemic CBF deficits that ultimately exacerbate cerebral infarction. Our results shed mechanistic light on human studies identifying CAA as an independent risk factor for ischemic brain injury.
PMCID: PMC4288816  PMID: 25190447
Alzheimer's disease; amyloid angiopathy; cerebral ischemia; transgenic mice; cerebrovascular; amyloid beta; cerebral blood flow
7.  Biomarker Modeling of Alzheimer's Disease 
Neuron  2013;80(6):1347-1358.
Alzheimer's disease (AD) is a slowly progressing disorder in which pathophysiological abnormalities, detectable in vivo by biomarkers, precede overt clinical symptoms by many years to decades. Five AD biomarkers are sufficiently validated to have been incorporated into clinical diagnostic criteria and commonly used in therapeutic trials. Current AD biomarkers fall into 2 categories: biomarkers of amyloid-β plaques and of tau-related neurodegeneration. Three of the 5 are imaging measures and two are cerebrospinal fluid analytes. AD biomarkers do not evolve in an identical manner but rather in a sequential but temporally overlapping manner. Models of the temporal evolution of AD biomarkers can take the form of plots of biomarker severity (degree of abnormality) vs. time. In this review we discuss several time-dependent models of AD which take into consideration varying age of onset (early vs. late) and the influence of aging and co-occurring brain pathologies that commonly arise in the elderly.
PMCID: PMC3928967  PMID: 24360540
Alzheimer's disease; Alzheimer's biomarkers; amyloid imaging; Alzheimer's imaging; Alzheimer's modeling; PET AND Alzheimer's; MRI AND Alzheimer's
8.  CSF biomarkers of Alzheimer disease 
Neurology  2013;81(23):2028-2031.
To test whether CSF Alzheimer disease biomarkers (β-amyloid 42 [Aβ42], tau, phosphorylated tau at threonine 181 [ptau181], tau/Aβ42, and ptau181/Aβ42) predict future decline in noncognitive outcomes among individuals cognitively normal at baseline.
Longitudinal data from participants (N = 430) who donated CSF within 1 year of a clinical assessment indicating normal cognition and were aged 50 years or older were analyzed. Mixed linear models were used to test whether baseline biomarker values predicted future decline in function (instrumental activities of daily living), weight, behavior, and mood. Clinical Dementia Rating Sum of Boxes and Mini-Mental State Examination scores were also examined.
Abnormal levels of each biomarker were related to greater impairment with time in behavior (p < 0.035) and mood (p < 0.012) symptoms, and more difficulties with independent activities of daily living (p < 0.012). However, biomarker levels were unrelated to weight change with time (p > 0.115). As expected, abnormal biomarker values also predicted more rapidly changing Mini-Mental State Examination (p < 0.041) and Clinical Dementia Rating Sum of Boxes (p < 0.001) scores compared with normal values.
CSF biomarkers among cognitively normal individuals are associated with future decline in some, but not all, noncognitive Alzheimer disease symptoms studied. Additional work is needed to determine the extent to which these findings generalize to other samples.
PMCID: PMC3854826  PMID: 24212387
9.  Functional Connectivity in Autosomal Dominant and Late-Onset Alzheimer Disease 
JAMA neurology  2014;71(9):1111-1122.
Autosomal dominant Alzheimer disease (ADAD) is caused by rare genetic mutations in three specific genes, in contrast to late-onset Alzheimer Disease (LOAD), which has a more polygenetic risk profile.
Design, Setting, and Participants
We analyzed functional connectivity in multiple brain resting state networks (RSNs) in a cross-sectional cohort of ADAD (N=79) and LOAD (N=444) human participants using resting state functional connectivity MRI (rs-fcMRI) at multiple international academic sites.
Main Outcomes and Measures
For both types of AD, we quantified and compared functional connectivity changes in RSNs as a function of dementia severity as measured by clinical dementia rating (CDR). In ADAD, we qualitatively investigated functional connectivity changes with respect to estimated years from onset of symptoms within five RSNs.
Functional connectivity decreases with increasing CDR were similar for both LOAD and ADAD in multiple RSNs. Ordinal logistic regression models constructed in each type of AD accurately predicted CDR stage in the other, further demonstrating similarity of functional connectivity loss in each disease type. Among ADAD participants, functional connectivity in multiple RSNs appeared qualitatively lower in asymptomatic mutation carriers near their anticipated age of symptom onset compared to asymptomatic mutation non-carriers.
Conclusions and Relevance
rs-fcMRI changes with progressing AD severity are similar between ADAD and LOAD. Rs-fcMRI may be a useful endpoint for LOAD and ADAD therapy trials. ADAD disease process may be an effective model for LOAD disease process.
PMCID: PMC4240274  PMID: 25069482
Resting-state functional connectivity; autosomal dominant Alzheimer's disease; late-onset Alzheimer's disease; default mode network; apolipoprotein E (APOE)
10.  B-RAF unlocks axon regeneration 
PMCID: PMC4010911  PMID: 24799531
11.  Alzheimer Disease in 2020 
Remarkable advances in unraveling the biological underpinnings of Alzheimer disease (AD) have occurred during the last 25 years. Despite this, we have made only the smallest of dents in the development of truly disease-modifying treatments. What will change over the next 10 years? While the answer is not clear, we make several predictions on the state of the field in 2020, based on the rich knowledge described in the other contributions in this collection. As such, our predictions represent some of the principal unresolved questions that we believe deserve special investigative attention in the coming decade.
By 2020, thanks to a significantly advanced understanding of Alzheimer disease pathogenesis, we may be on the verge of treatments that delay the onset of the disease in many millions worldwide.
PMCID: PMC3543098  PMID: 23125202
12.  Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation 
PLoS Genetics  2014;10(10):e1004758.
Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.
Author Summary
The use of quantitative endophenotypes from cerebrospinal fluid has led to the identification of several genetic variants that alter risk or rate of progression of Alzheimer's disease. Here we have analyzed the levels of 58 disease-related proteins in the cerebrospinal fluid for association with millions of variants across the human genome. We have identified significant, replicable associations with 5 analytes, Angiotensin-converting enzyme, Chemokine (C-C motif) ligand 2, Chemokine (C-C motif) ligand 4, Interleukin 6 receptor and Matrix metalloproteinase-3. Our results suggest that these variants play a regulatory role in the respective protein levels and are relevant to the inflammatory and amyloid processing pathways. Variants in associated with ACE and those associated with MMP3 levels also show association with risk for Alzheimer's disease in the expected directions. These associations are consistent in cerebrospinal fluid and plasma and in samples with only cognitively normal individuals suggesting that they are relevant in the regulation of these protein levels beyond the context of Alzheimer's disease.
PMCID: PMC4207667  PMID: 25340798
13.  Sleep-wake cycle and diurnal fluctuation of amyloid-β as biomarkers of brain amyloid pathology 
Science translational medicine  2012;4(150):150ra122.
Aggregation of amyloid-β (Aβ) in the brain begins to occur years prior to the clinical onset of Alzheimer’s disease (AD). Prior to Aβ aggregation, levels of extracellular, soluble interstitial fluid (ISF) Aβ, which are regulated by neuronal activity and the sleep-wake cycle, correlate with the amount of Aβ deposition in the brain seen later. The amount and quality of sleep declines with aging and to a greater extent in AD. How sleep quality amount as well as the diurnal fluctuation in Aβ change with age and Aβ aggregation are not well understood. We report that a normal sleep-wake cycle and diurnal fluctuation of ISF Aβ is present in the brain of APPswe/PS1δE9 mice before Aβ plaque formation. Following plaque formation, the sleep-wake cycle markedly deteriorated and diurnal fluctuation of ISF Aβ dissipated. As in mice, diurnal fluctuation of cerebrospinal fluid (CSF) Aβ in young adult humans with presenilin mutations was also markedly attenuated with Aβ plaque formation. Virtual elimination of Aβ deposits in the mouse brain by active immunization with Aβ42 normalized the sleep-wake cycle and the diurnal fluctuation of ISF Aβ. These data suggest that Aβ aggregation disrupts the sleep-wake cycle and diurnal fluctuation of Aβ. Sleep-wake behavior and diurnal fluctuation of Aβ in the central nervous system appear to be functional and biochemical markers respectively of Aβ-associated pathology that should be explored in humans diagnostically prior to and following symptom onset and in response to treatment.
PMCID: PMC3654377  PMID: 22956200
14.  Preclinical Alzheimer’s disease and its outcome: a longitudinal cohort study 
Lancet neurology  2013;12(10):957-965.
New research criteria for preclinical Alzheimer’s disease (AD)have been proposed by the National Institute on Aging and Alzheimer’s Association. They include stages for cognitively normal individuals with abnormal amyloid markers (stage 1), abnormal amyloid and injury markers (stage 2) and abnormal amyloid and injury markers and subtle cognitive changes (stage 3). We investigated the occurrence and long-term outcome of these stages.
Cerebrospinal fluidamyloid-β1–42 and tau levels and a memory composite score were used to classify 311 cognitively normal(Clinical Dementia Rating [CDR]=0) research participants ≥65 years as normal (both markers normal), preclinical AD stage 1–3, or Suspected Non-Alzheimer Pathophysiology (SNAP, abnormal injury marker without abnormal amyloid marker). Outcome measures were progression to CDR≥0·5 symptomatic AD and mortality up to 15 years after baseline (average=4 years).
129 (41·5%) of participants were normal, 47 (15%)were in stage 1, 36 (12%) in stage 2, 13 (4%)in stage 3, 72 (23%) had SNAP, and 14 (4·5%) remained unclassified. The proportion of preclinical AD (stage 1–3) in our cohort was higher in individuals older than 72 years and in APOE-ε4 carriers. The 5-year progression rate to CDR≥0·5 symptomatic AD was 2% for normal participants, 11% for stage 1, 26% for stage 2, 56% for stage 3, and 5% for SNAP. Compared with normal individuals, participants with preclinical AD had an increased risk of death (HR=6·2, p=0·0396).
Preclinical AD is common in cognitively normal elderly and strongly associated with future cognitive decline and mortality. Preclinical AD thus should be an important target for therapeutic interventions.
PMCID: PMC3904678  PMID: 24012374
15.  Relationship Between Stroop Performance and Resting State Functional Connectivity in Cognitively Normal Older Adults 
Neuropsychology  2013;27(5):10.1037/a0033402.
Early biomarkers of Alzheimer’s disease (AD) are needed for developing therapeutic interventions. Measures of attentional control in Stroop-type tasks discriminate healthy aging from early stage AD (Hutchison et al., 2010) and predict future development of AD (Balota et al., 2010) in cognitively normal individuals. Disruption in resting state functional connectivity magnetic resonance imaging (rs-fcMRI) has been reported in AD (Greicius et al., 2004), and in healthy controls at risk for AD (Sheline et al, 2010a). We explored the relationship among Stroop performance, rs-fcMRI, and CSF Aβ42 levels in cognitively normal older adults.
A computerized Stroop task (along with standard neuropsychological measures), rs-fcMRI, and CSF were obtained in 237 cognitively normal older adults. We compared the relationship between Stroop performance, including measures from reaction distributional analyses, and composite scores from four resting state networks (RSNs) [default mode (DMN), salience (SAL), dorsal attention (DAN), and sensory motor (SMN)], and the modulatory influence of CSF Aβ42 levels.
A larger Stroop effect in errors was associated with reduced rs-fcMRI within the DMN and SAL. Reaction time distributional analyses indicated the slow tail of the reaction time distribution was related to reduced rs-fcMRI functional connectivity within the SAL. Standard psychometric measures were not related to RSN composite scores. A relationship between Stroop performance and DMN (but not SAL) functional connectivity was stronger in CSF Aβ42 positive individuals.
A link exists between RSN composite scores and specific attentional performance measures. Both measures may be sensitive biomarkers for AD.
PMCID: PMC3837537  PMID: 24040929
resting state functional connectivity; Stroop; Alzheimer’s disease
16.  Quantitative Label-Free Proteomics for Discovery of Biomarkers in Cerebrospinal Fluid: Assessment of Technical and Inter-Individual Variation 
PLoS ONE  2013;8(5):e64314.
Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF) is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance) among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF) and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) for CSF biomarker discovery by (1) identifying reparable sources of technical variability, (2) assessing subject variance and residual technical variability for numerous CSF proteins, and (3) testing its ability to segregate samples on the basis of desired biomarker characteristics.
Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i) incomplete MAF and keratins; ii) globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii) oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5%) whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and individual samples.
Quantitative label-free LC-MS/MS can measure scores of CSF proteins with low technical variability and can segregate samples according to desired criteria. Thus, this technique shows potential for biomarker discovery for neurological diseases.
PMCID: PMC3659127  PMID: 23700471
17.  Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aβ amyloidosis 
The Journal of Experimental Medicine  2012;209(12):2149-2156.
Anti-ApoE antibody reduces amyloid deposition and enhances the microglial response to Aβ plaques in an Alzheimer’s disease mouse model.
The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer’s disease (AD). The influence of apoE on amyloid β (Aβ) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aβ and facilitates Aβ fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aβ antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60–80% and significantly reduced insoluble Aβ40 and Aβ42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aβ plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aβ binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases.
PMCID: PMC3501350  PMID: 23129750
18.  Blocking the apoE/Aβ interaction ameliorates Aβ-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice 
Accumulation of β-amyloid (Aβ) in the brain is essential to Alzheimer’s disease (AD) pathogenesis. Carriers of the apolipoprotein E (APOE) ε4 allele demonstrate greatly increased AD risk and enhanced brain Aβ deposition. In contrast, APOE ε2 allele carries show reduced AD risk, later age of disease onset, and lesser Aβ accumulation. However, it remains elusive whether the apoE2 isoform exerts truly protective effect against Aβ pathology or apoE2 plays deleterious role albeit less pronounced than the apoE4 isoform. Here, we characterized APPSW/PS1dE9/APOE ε2-TR (APP/E2) and APPSW/PS1dE9/APOE ε4-TR (APP/E4) mice, with targeted replacement (TR) of the murine Apoe for human ε2 or ε4 alleles, and used these models to investigate effects of pharmacological inhibition of the apoE/Aβ interaction on Aβ deposition and neuritic degeneration. APP/E2 and APP/E4 mice replicate differential effect of human apoE isoforms on Aβ pathology with APP/E4 mice showing a several-fold greater load of Aβ plaques, insoluble brain Aβ levels, Aβ oligomers, and density of neuritic plaques than APP/E2 mice. Furthermore, APP/E4 mice, but not APP/E2 mice, exhibit memory impairment on object recognition and radial arm maze tests. Between the age of 6 and 10 months APP/E2 and APP/E4 mice received treatment with Aβ12-28P, a non-toxic, synthetic peptide homologous to the apoE binding motif within the Aβ sequence, which competitively blocks the apoE/Aβ interaction. In both lines, the treatment significantly reduced brain Aβ accumulation, co-accumulation of apoE within Aβ plaques, and neuritic degeneration, and prevented memory deficit in APP/E4 mice. These results indicate that both apoE2 and apoE4 isoforms contribute to Aβ deposition and future therapies targeting the apoE/Aβ interaction could produce favorable outcome in APOE ε2 and ε4 allele carriers.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-014-0075-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4174325  PMID: 24972680
Apolipoprotein E; Alzheimer’s disease; β-amyloid; Neurodegeneration; Therapy
19.  CSF biomarkers for Alzheimer’s disease: Current utility and potential future use 
Neurobiology of aging  2011;32(Suppl 1):S4-S9.
Over the last 15 years, cerebrospinal fluid (CSF) biomarkers have been shown to be useful for both the diagnosis as well as the prognosis in Alzheimer’s disease. It has been shown the CSF levels of Aβ42 are a very good marker for the presence of amyloid deposition in the brain regardless of clinical status and that total tau and phosphorylated forms of tau are useful in detection of neurodegeneration. When combined together, these CSF markers are useful not only in differential diagnosis but also in predicting conversion and rate of progression from mild cognitive impairment/very mild dementia to more severe impairment. The markers are also useful in predicting conversion from cognitive normalcy to very mild dementia. This field is briefly reviewed and recommendations for future studies in this area is provided.
PMCID: PMC3233690  PMID: 22078172
20.  Neuronal Clearance of Amyloid-β by Endocytic Receptor LRP1 
The Journal of Neuroscience  2013;33(49):19276-19283.
Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly population. Accumulation, aggregation, and deposition of amyloid-β (Aβ) peptides generated through proteolytic cleavage of amyloid precursor protein (APP) are likely initiating events in the pathogenesis of AD. While Aβ production is accelerated in familial AD, increasing evidence indicates that impaired clearance of Aβ is responsible for late-onset AD. Because Aβ is mainly generated in neurons, these cells are predicted to have the highest risk of encountering Aβ among all cell types in the brain. However, it is still unclear whether they are also involved in Aβ clearance. Here we show that receptor-mediated endocytosis in neurons by the low-density lipoprotein receptor-related protein 1 (LRP1) plays a critical role in brain Aβ clearance. LRP1 is known to be an endocytic receptor for multiple ligands including Aβ. Conditional knock-out of Lrp1 in mouse forebrain neurons leads to increased brain Aβ levels and exacerbated amyloid plaque deposition selectively in the cortex of amyloid model APP/PS1 mice without affecting Aβ production. In vivo microdialysis studies demonstrated that Aβ clearance in brain interstitial fluid is impaired in neuronal Lrp1 knock-out mice. Because the neuronal LRP1-deletion did not affect the mRNA levels of major Aβ degrading enzymes, neprilysin and insulin-degrading enzyme, the disturbed Aβ clearance is likely due to the suppression of LRP1-mediated neuronal Aβ uptake and degradation. Together, our results demonstrate that LRP1 plays an important role in receptor-mediated clearance of Aβ and indicate that neurons not only produce but also clear Aβ.
PMCID: PMC3850043  PMID: 24305823
21.  Altered microglial response to Aβ plaques in APPPS1-21 mice heterozygous for TREM2 
Recent genome-wide association studies linked variants in TREM2 to a strong increase in the odds of developing Alzheimer’s disease. The mechanism by which TREM2 influences the susceptibility to Alzheimer’s disease is currently unknown. TREM2 is expressed by microglia and is thought to regulate phagocytic and inflammatory microglial responses to brain pathology. Given that a single allele of variant TREM2, likely resulting in a loss of function, conferred an increased risk of developing Alzheimer’s disease, we tested whether loss of one functional trem2 allele would affect Aβ plaque deposition or the microglial response to Aβ pathology in APPPS1-21 mice.
There was no significant difference in Aβ deposition in 3-month old or 7-month old APPPS1-21 mice expressing one or two copies of trem2. However, 3-month old mice with one copy of trem2 exhibited a marked decrease in the number and size of plaque-associated microglia. While there were no statistically significant differences in cytokine levels or markers of microglial activation in 3- or 7-month old animals, there were trends towards decreased expression of NOS2, C1qa, and IL1a in 3-month old TREM2+/− vs. TREM2+/+ mice.
Loss of a single copy of trem2 had no effect on Aβ pathology, but altered the morphological phenotype of plaque-associated microglia. These data suggest that TREM2 is important for the microglial response to Aβ deposition but that a 50% decrease inTREM2 expression does not affect Aβ plaque burden.
PMCID: PMC4049806  PMID: 24893973
Alzheimer’s disease; TREM2; Microglia; Amyloid β
22.  Bidirectional relationship between functional connectivity and amyloid-β deposition in mouse brain 
The Journal of Neuroscience  2012;32(13):4334-4340.
Brain region-specific deposition of extracellular amyloid plaques principally composed of aggregated amyloid-β (Aβ) peptide is a pathological signature of Alzheimer’s disease (AD). Recent human neuroimaging data suggest that resting-state functional connectivity strength is reduced in patients with AD, cognitively normal elderly harboring elevated amyloid burden, and in advanced aging. Interestingly, there exists a striking spatial correlation between functional connectivity strength in cognitively normal adults and the location of Aβ plaque deposition in AD. However, technical limitations have heretofore precluded examination of the relationship between functional connectivity, Aβ deposition, and normal aging in mouse models. Using a novel functional connectivity optical intrinsic signal (fcOIS) imaging technique, we demonstrate that Aβ deposition is associated with significantly reduced bilateral functional connectivity in multiple brain regions of older APP/PS1 transgenic mice. The amount of Aβ deposition in each brain region was associated with the degree of local, age-related bilateral functional connectivity decline. Normal aging was associated with reduced bilateral functional connectivity specifically in retrosplenial cortex. Furthermore, we found that the magnitude of regional bilateral functional correlation in young APP/PS1 mice prior to Aβ plaque formation was proportional to the amount of region-specific plaque deposition seen later in older APP/PS1 mice. Together, these findings suggest that Aβ deposition and normal aging are associated with region-specific disruption of functional connectivity and that the magnitude of local bilateral functional connectivity predicts regional vulnerability to subsequent Aβ deposition in mouse brain.
PMCID: PMC3326343  PMID: 22457485
23.  The Choroid Plexus and Cerebrospinal Fluid: Emerging Roles in Development, Disease, and Therapy 
The Journal of Neuroscience  2013;33(45):17553-17559.
Although universally recognized as the source of cerebrospinal fluid (CSF), the choroid plexus (ChP) has been one of the most understudied tissues in neuroscience. The reasons for this are multiple and varied, including historical perceptions about passive and permissive roles for the ChP, experimental issues, and lack of clinical salience. However, recent work on the ChP and instructive signals in the CSF have sparked new hypotheses about how the ChP and CSF provide unexpected means for regulating nervous system structure and function in health and disease, as well as new ChP-based therapeutic approaches using pluripotent stem cell technology. This minisymposium combines new and established investigators to capture some of the newfound excitement surrounding the ChP-CSF system.
PMCID: PMC3818536  PMID: 24198345
24.  GWAS of cerebrospinal fluid tau levels identifies novel risk variants for Alzheimer’s disease 
Neuron  2013;78(2):256-268.
Cerebrospinal fluid (CSF) tau, tau phosphorylated at threonine 181 (ptau) and Aβ42 are established biomarkers for Alzheimer’s Disease (AD), and have been used as quantitative traits for genetic analyses. We performed the largest genome-wide association study for cerebrospinal fluid (CSF) tau/ptau levels published to date (n=1,269), identifying three novel genome-wide significant loci for CSF tau and ptau: rs9877502 (P=4.89×10−9 for tau) located at 3q28 between GEMC1 and OSTN, rs514716 (P=1.07×10−8 and P=3.22×10−9 for tau and ptau respectively), located at 9p24.2 within GLIS3 and rs6922617 (P = 3.58×10−8 for CSF ptau) at 6p21.1 within the TREM gene cluster, a region recently reported to harbor rare variants that increase AD risk. In independent datasets rs9877502 showed a strong association with risk for AD, tangle pathology and global cognitive decline (P=2.67×10−4, 0.039, 4.86×10−5 respectively) illustrating how this endophenotype-based approach can be used to identify new AD risk loci.
PMCID: PMC3664945  PMID: 23562540
Annals of neurology  2011;70(2):274-285.
There is a growing need to identify cerebrospinal fluid (CSF) markers that can detect Alzheimer’s disease (AD) pathology in cognitively normal individuals since it is in this population that disease-modifying therapies may have the greatest chance of success. While AD pathology is estimated to begin ~10–15 years prior to the onset of cognitive decline, substantial neuronal loss is present by the time the earliest signs of cognitive impairment appear. Visinin-like protein −1 (VILIP-1) has demonstrated potential utility as a marker of neuronal injury. We here investigate CSF VILIP-1 and VILIP-1/amyloid-β42 (Aβ42) ratio as diagnostic and prognostic markers in early AD.
We assessed CSF levels of VILIP-1, tau, phosphorylated-tau181 (p-tau181), and Aβ42 in cognitively normal controls [CNC] (n=211), individuals with early symptomatic AD (n=98), and individuals with other dementias (n=19). Structural magnetic resonance imaging (n=192) and amyloid imaging with Pittsburgh Compound-B (n=156) were obtained in subsets of this cohort. Among the CNC cohort, 164 individuals had follow-up annual cognitive assessments for 2–3 years.
CSF VILIP-1 levels differentiated individuals with AD from CNC and individuals with other dementias. CSF VILIP-1 levels correlated with CSF tau, p-tau181, and brain volumes in AD. VILIP-1 and VILIP-1/Aβ42 predicted future cognitive impairment in CNC over the follow-up period. Importantly, CSF VILIP-1/Aβ42 predicted future cognitive impairment at least as well as tau/Aβ42 and p-tau181/Aβ42.
These findings suggest that CSF VILIP-1 and VILIP-1/Aβ42 offer diagnostic utility for early AD, and can predict future cognitive impairment in cognitively normal individuals similarly to tau and tau/Aβ42, respectively.
PMCID: PMC3154071  PMID: 21823155
Visinin-like protein-1; Alzheimer’s disease; biomarkers; cerebrospinal fluid; neuronal injury

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