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1.  Heteromers of amyloid precursor protein in cerebrospinal fluid 
Background
Soluble fragments of the amyloid precursor protein (APP) generated by α- and β-secretases, sAPPα and sAPPβ, have been postulated as promising new cerebrospinal fluid (CSF) biomarkers for the clinical diagnosis of Alzheimer’s disease (AD). However, the capacity of these soluble proteins to assemble has not been explored and could be relevant. Our aim is to characterize possible sAPP oligomers that could contribute to the quantification of sAPPα and sAPPβ in CSF by ELISA, as well as to characterize the possible presence of soluble full-length APP (sAPPf).
Results
We employed co-immunoprecipitation, native polyacrylamide gel electrophoresis and ultracentrifugation in sucrose density gradients to characterize sAPP oligomers in CSF. We have characterized the presence of sAPPf in CSF from NDC and AD subjects and demonstrated that all forms, including sAPPα and sAPPβ, are capable of assembling into heteromers, which differ from brain APP membrane-dimers. We measured sAPPf, sAPPα and sAPPβ by ELISA in CSF samples from AD (n = 13) and non-disease subjects (NDC, n = 13) before and after immunoprecipitation with antibodies against the C-terminal APP or against sAPPβ. We demonstrated that these sAPP heteromers participate in the quantification of sAPPα and sAPPβ by ELISA. Immunoprecipitation with a C-terminal antibody to remove sAPPf reduced by ~30% the determinations of sAPPα and sAPPβ by ELISA, whereas immunoprecipitation with an APPβ antibody reduced by ~80% the determination of sAPPf and sAPPα.
Conclusions
The presence of sAPPf and sAPP heteromers should be taken into consideration when exploring the levels of sAPPα and sAPPβ as potential CSF biomarkers.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-10-2) contains supplementary material, which is available to authorized users.
doi:10.1186/1750-1326-10-2
PMCID: PMC4298044  PMID: 25573162
sAPPα; sAPPβ; Heteromers; Cerebrospinal fluid; Alzheimer’s disease; ELISA
2.  Amyloid and tau cerebrospinal fluid biomarkers in HIV infection 
BMC Neurology  2009;9:63.
Background
Because of the emerging intersections of HIV infection and Alzheimer's disease, we examined cerebrospinal fluid (CSF) biomarkers related of amyloid and tau metabolism in HIV-infected patients.
Methods
In this cross-sectional study we measured soluble amyloid precursor proteins alpha and beta (sAPPα and sAPPβ), amyloid beta fragment 1-42 (Aβ1-42), and total and hyperphosphorylated tau (t-tau and p-tau) in CSF of 86 HIV-infected (HIV+) subjects, including 21 with AIDS dementia complex (ADC), 25 with central nervous system (CNS) opportunistic infections and 40 without neurological symptoms and signs. We also measured these CSF biomarkers in 64 uninfected (HIV-) subjects, including 21 with Alzheimer's disease, and both younger and older controls without neurological disease.
Results
CSF sAPPα and sAPPβ concentrations were highly correlated and reduced in patients with ADC and opportunistic infections compared to the other groups. The opportunistic infection group but not the ADC patients had lower CSF Aβ1-42 in comparison to the other HIV+ subjects. CSF t-tau levels were high in some ADC patients, but did not differ significantly from the HIV+ neuroasymptomatic group, while CSF p-tau was not increased in any of the HIV+ groups. Together, CSF amyloid and tau markers segregated the ADC patients from both HIV+ and HIV- neuroasymptomatics and from Alzheimer's disease patients, but not from those with opportunistic infections.
Conclusions
Parallel reductions of CSF sAPPα and sAPPβ in ADC and CNS opportunistic infections suggest an effect of CNS immune activation or inflammation on neuronal amyloid synthesis or processing. Elevation of CSF t-tau in some ADC and CNS infection patients without concomitant increase in p-tau indicates neural injury without preferential accumulation of hyperphosphorylated tau as found in Alzheimer's disease. These biomarker changes define pathogenetic pathways to brain injury in ADC that differ from those of Alzheimer's disease.
doi:10.1186/1471-2377-9-63
PMCID: PMC2807422  PMID: 20028512
3.  CNS Amyloid-β, Soluble APP-α and -β Kinetics during BACE Inhibition 
The Journal of Neuroscience  2014;34(24):8336-8346.
BACE, a β-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the β-secretase pathway and a lowering of CNS amyloid-β (Aβ) levels. The interaction of the β-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aβ and soluble APPβ (sAPPβ), with increased newly generated sAPPα.
A stable isotope labeling kinetics experiment in NHPs was performed with a 13C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPβ, and Aβ in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated.
sAPPβ and sAPPα kinetics were similar, but both significantly slower than Aβ. BACE inhibition resulted in decreased labeled sAPPβ and Aβ in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPβ and Aβ both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the β-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.
doi:10.1523/JNEUROSCI.0540-14.2014
PMCID: PMC4051982  PMID: 24920637
amyloid beta; amyloid precursor protein; BACE inhibitor; sAPPα; sAPPβ; SILK
4.  Diurnal Patterns of Soluble Amyloid Precursor Protein Metabolites in the Human Central Nervous System 
PLoS ONE  2014;9(3):e89998.
The amyloid-β (Aβ) protein is diurnally regulated in both the cerebrospinal fluid and blood in healthy adults; circadian amplitudes decrease with aging and the presence of cerebral Aβ deposits. The cause of the Aβ diurnal pattern is poorly understood. One hypothesis is that the Amyloid Precursor Protein (APP) is diurnally regulated, leading to APP product diurnal patterns. APP in the central nervous system is processed either via the β-pathway (amyloidogenic), generating soluble APP-β (sAPPβ) and Aβ, or the α-pathway (non-amyloidogenic), releasing soluble APP-α (sAPPα). To elucidate the potential contributions of APP to the Aβ diurnal pattern and the balance of the α- and β- pathways in APP processing, we measured APP proteolytic products over 36 hours in human cerebrospinal fluid from cognitively normal and Alzheimer's disease participants. We found diurnal patterns in sAPPα, sAPPβ, Aβ40, and Aβ42, which diminish with increased age, that support the hypothesis that APP is diurnally regulated in the human central nervous system and thus results in Aβ diurnal patterns. We also found that the four APP metabolites were positively correlated in all participants without cerebral Aβ deposits. This positive correlation suggests that the α- and β- APP pathways are non-competitive under normal physiologic conditions where APP availability may be the limiting factor that determines sAPPα and sAPPβ production. However, in participants with cerebral Aβ deposits, there was no correlation of Aβ to sAPP metabolites, suggesting that normal physiologic regulation of cerebrospinal fluid Aβ is impaired in the presence of amyloidosis. Lastly, we found that the ratio of sAPPβ to sAPPα was significantly higher in participants with cerebral Aβ deposits versus those without deposits. Therefore, the sAPPβ to sAPPα ratio may be a useful biomarker for cerebral amyloidosis.
doi:10.1371/journal.pone.0089998
PMCID: PMC3960093  PMID: 24646516
5.  Impact of Cerebrospinal Fluid Shunting for Idiopathic Normal Pressure Hydrocephalus on the Amyloid Cascade 
PLoS ONE  2015;10(3):e0119973.
The aim of this study was to determine whether the improvement of cerebrospinal fluid (CSF) flow dynamics by CSF shunting, can suppress the oligomerization of amyloid β-peptide (Aβ), by measuring the levels of Alzheimer’s disease (AD)-related proteins in the CSF before and after lumboperitoneal shunting. Lumbar CSF from 32 patients with idiopathic normal pressure hydrocephalus (iNPH) (samples were obtained before and 1 year after shunting), 15 patients with AD, and 12 normal controls was analyzed for AD-related proteins and APLP1-derived Aβ-like peptides (APL1β) (a surrogate marker for Aβ). We found that before shunting, individuals with iNPH had significantly lower levels of soluble amyloid precursor proteins (sAPP) and Aβ38 compared to patients with AD and normal controls. We divided the patients with iNPH into patients with favorable (improvement ≥ 1 on the modified Rankin Scale) and unfavorable (no improvement on the modified Rankin Scale) outcomes. Compared to the unfavorable outcome group, the favorable outcome group showed significant increases in Aβ38, 40, 42, and phosphorylated-tau levels after shunting. In contrast, there were no significant changes in the levels of APL1β25, 27, and 28 after shunting. After shunting, we observed positive correlations between sAPPα and sAPPβ, Aβ38 and 42, and APL1β25 and 28, with shifts from sAPPβ to sAPPα, from APL1β28 to 25, and from Aβ42 to 38 in all patients with iNPH. Our results suggest that Aβ production remained unchanged by the shunt procedure because the levels of sAPP and APL1β were unchanged. Moreover, the shift of Aβ from oligomer to monomer due to the shift of Aβ42 (easy to aggregate) to Aβ38 (difficult to aggregate), and the improvement of interstitial-fluid flow, could lead to increased Aβ levels in the CSF. Our findings suggest that the shunting procedure can delay intracerebral deposition of Aβ in patients with iNPH.
doi:10.1371/journal.pone.0119973
PMCID: PMC4379026  PMID: 25821958
6.  Cerebrospinal Fluid Biomarker and Brain Biopsy Findings in Idiopathic Normal Pressure Hydrocephalus 
PLoS ONE  2014;9(3):e91974.
Background
The significance of amyloid precursor protein (APP) and neuroinflammation in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD) is unknown.
Objective
To investigate the role of soluble APP (sAPP) and amyloid beta (Aβ) isoforms, proinflammatory cytokines, and biomarkers of neuronal damage in the cerebrospinal fluid (CSF) in relation to brain biopsy Aβ and hyperphosphorylated tau (HPτ) findings.
Methods
The study population comprised 102 patients with possible NPH with cortical brain biopsies, ventricular and lumbar CSF samples, and DNA available. The final clinical diagnoses were: 53 iNPH (91% shunt-responders), 26 AD (10 mixed iNPH+AD), and 23 others. Biopsy samples were immunostained against Aβ and HPτ. CSF levels of AD-related biomarkers (Aβ42, p-tau, total tau), non-AD-related Aβ isoforms (Aβ38, Aβ40), sAPP isoforms (sAPPα, sAPPβ), proinflammatory cytokines (several interleukins (IL), interferon-gamma, monocyte chemoattractant protein-1, tumor necrosis factor-alpha) and biomarkers of neuronal damage (neurofilament light and myelin basic protein) were measured. All patients were genotyped for APOE.
Results
Lumbar CSF levels of sAPPα were lower (p<0.05) in patients with shunt-responsive iNPH compared to non-iNPH patients. sAPPβ showed a similar trend (p = 0.06). CSF sAPP isoform levels showed no association to Aβ or HPτ in the brain biopsy. Quantified Aβ load in the brain biopsy showed a negative correlation with CSF levels of Aβ42 in ventricular (r = −0.295, p = 0.003) and lumbar (r = −0.356, p = 0.01) samples, while the levels of Aβ38 and Aβ40 showed no correlation. CSF levels of proinflammatory cytokines and biomarkers of neuronal damage did not associate to the brain biopsy findings, diagnosis, or shunt response. Higher lumbar/ventricular CSF IL-8 ratios (p<0.001) were seen in lumbar samples collected after ventriculostomy compared to the samples collected before the procedure.
Conclusions
The role of sAPP isoforms in iNPH seems to be independent from the amyloid cascade. No neuroinflammatory background was observed in iNPH or AD.
doi:10.1371/journal.pone.0091974
PMCID: PMC3956805  PMID: 24638077
7.  Secreted Amyloid Precursor Protein β and Secreted Amyloid Precursor Protein α Induce Axon Outgrowth In Vitro through Egr1 Signaling Pathway 
PLoS ONE  2011;6(1):e16301.
Background
sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPβ released at the first step of amyloid peptide cleavage, raising the possibility that sAPPβ could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPβ on primary neurons and identified a key signaling event required for the changes observed.
Methodology/Principal Findings
Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPβ added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPβ induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPβ stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 −/− mice, sAPPs affected dendritic length but did not induce any increase of axon length.
Conclusion/Significance
sAPPα and sAPPβ decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPβ stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPβ is not deleterious per se. Since sAPPβ and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage.
doi:10.1371/journal.pone.0016301
PMCID: PMC3029320  PMID: 21298006
8.  Amyloid mis-metabolism in idiopathic normal pressure hydrocephalus 
Background
Patients with idiopathic normal pressure hydrocephalus (iNPH) have reduced cerebrospinal fluid (CSF) concentrations of amyloid-β (Aβ) and α- and β-cleaved soluble forms of amyloid precursor protein (sAPPα and sAPPβ). The aims of this study were to examine if changes could also be seen in the CSF for secreted metabolites of APP-like protein 1 (APLP1) and to explore the prognostic value of amyloid-related CSF biomarkers, as well as markers of neuronal injury and astroglial activation, as regards to clinical outcome after shunt surgery.
Methods
Twenty patients diagnosed with iNPH, 10 improved and 10 unchanged by shunt surgery, and 20 neurologically healthy controls were included. All patients were examined clinically prior to surgery and at 6-month follow-up after surgery using the iNPH scale. Lumbar puncture was performed pre-operatively. CSF samples were analyzed for neurofilament light (NFL), Aβ isoforms Aβ38, Aβ40 and Aβ42, sAPPα, sAPPβ, APLP1 β-derived peptides APL1β25, APL1β 27 and APL1β 28 and YKL40 by immunochemical methods.
Results
The concentrations of all soluble forms of APP, all Aβ isoforms and APL1β28 were lower, whilst APL1β25 and APL1β27 were higher in the CSF of iNPH patients compared to controls. There was no difference in biomarker concentrations between patients who improved after surgery and those who remained unchanged.
Conclusions
The reduced CSF concentrations of Aβ38, Aβ40, Aβ42, sAPPα and sAPPβ suggest that APP expression could be downregulated in iNPH. In contrast, APLP1 concentration in the CSF seems relatively unchanged. The increase of APL1β25 and APL1β27 in combination with a slight decreased APL1β28 could be caused by more available γ-secretase due to reduced availability of its primary substrate, APP. The data did not support the use of these markers as indicators of shunt responsiveness.
doi:10.1186/s12987-016-0037-y
PMCID: PMC4967298  PMID: 27472944
Neurodegeneration; Normal pressure hydrocephalus; Cerebrospinal fluid; Amyloid; NFL; YKL40; Prediction ; APLP1
9.  Increased Secreted Amyloid Precursor Protein-α (sAPPα) in Severe Autism: Proposal of a Specific, Anabolic Pathway and Putative Biomarker 
PLoS ONE  2011;6(6):e20405.
Autism is a neurodevelopmental disorder characterized by deficits in verbal communication, social interactions, and the presence of repetitive, stereotyped and compulsive behaviors. Excessive early brain growth is found commonly in some patients and may contribute to disease phenotype. Reports of increased levels of brain-derived neurotrophic factor (BDNF) and other neurotrophic-like factors in autistic neonates suggest that enhanced anabolic activity in CNS mediates this overgrowth effect. We have shown previously that in a subset of patients with severe autism and aggression, plasma levels of the secreted amyloid-β (Aβ) precursor protein-alpha form (sAPPα) were significantly elevated relative to controls and patients with mild-to-moderate autism. Here we further tested the hypothesis that levels of sAPPα and sAPPβ (proteolytic cleavage products of APP by α- and β-secretase, respectively) are deranged in autism and may contribute to an anabolic environment leading to brain overgrowth. We measured plasma levels of sAPPα, sAPPβ, Aβ peptides and BDNF by corresponding ELISA in a well characterized set of subjects. We included for analysis 18 control, 6 mild-to-moderate, and 15 severely autistic patient plasma samples. We have observed that sAPPα levels are increased and BDNF levels decreased in the plasma of patients with severe autism as compared to controls. Further, we show that Aβ1-40, Aβ1-42, and sAPPβ levels are significantly decreased in the plasma of patients with severe autism. These findings do not extend to patients with mild-to-moderate autism, providing a biochemical correlate of phenotypic severity. Taken together, this study provides evidence that sAPPα levels are generally elevated in severe autism and suggests that these patients may have aberrant non-amyloidogenic processing of APP.
doi:10.1371/journal.pone.0020405
PMCID: PMC3120811  PMID: 21731612
10.  Finding novel distinctions between the sAPPα-mediated anabolic biochemical pathways in Autism Spectrum Disorder and Fragile X Syndrome plasma and brain tissue 
Scientific Reports  2016;6:26052.
Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are developmental disorders. No validated blood-based biomarkers exist for either, which impedes bench-to-bedside approaches. Amyloid-β (Aβ) precursor protein (APP) and metabolites are usually associated with Alzheimer’s disease (AD). APP cleavage by α-secretase produces potentially neurotrophic secreted APPα (sAPPα) and the P3 peptide fragment. β-site APP cleaving enzyme (BACE1) cleavage produces secreted APPβ (sAPPβ) and intact Aβ. Excess Aβ is potentially neurotoxic and can lead to atrophy of brain regions such as amygdala in AD. By contrast, amygdala is enlarged in ASD but not FXS. We previously reported elevated levels of sAPPα in ASD and FXS vs. controls. We now report elevated plasma Aβ and total APP levels in FXS compared to both ASD and typically developing controls, and elevated levels of sAPPα in ASD and FXS vs. controls. By contrast, plasma and brain sAPPβ and Aβ were lower in ASD vs. controls but elevated in FXS plasma vs. controls. We also detected age-dependent increase in an α-secretase in ASD brains. We report a novel mechanistic difference in APP pathways between ASD (processing) and FXS (expression) leading to distinct APP metabolite profiles in these two disorders. These novel, distinctive biochemical differences between ASD and FXS pave the way for blood-based biomarkers for ASD and FXS.
doi:10.1038/srep26052
PMCID: PMC4876513  PMID: 27212113
11.  Cerebrospinal Fluid (CSF) Neuronal Biomarkers across the Spectrum of HIV Infection: Hierarchy of Injury and Detection 
PLoS ONE  2014;9(12):e116081.
The character of central nervous system (CNS) HIV infection and its effects on neuronal integrity vary with evolving systemic infection. Using a cross-sectional design and archived samples, we compared concentrations of cerebrospinal fluid (CSF) neuronal biomarkers in 143 samples from 8 HIV-infected subject groups representing a spectrum of untreated systemic HIV progression and viral suppression: primary infection; four groups of chronic HIV infection neuroasymptomatic (NA) subjects defined by blood CD4+ T cells of >350, 200–349, 50–199, and <50 cells/µL; HAD; treatment-induced viral suppression; and ‘elite’ controllers. Samples from 20 HIV-uninfected controls were also examined. The neuronal biomarkers included neurofilament light chain protein (NFL), total and phosphorylated tau (t-tau, p-tau), soluble amyloid precursor proteins alpha and beta (sAPPα, sAPPβ) and amyloid beta (Aβ) fragments 1–42, 1–40 and 1–38. Comparison of the biomarker changes showed a hierarchy of sensitivity in detection and suggested evolving mechanisms with progressive injury. NFL was the most sensitive neuronal biomarker. Its CSF concentration exceeded age-adjusted norms in all HAD patients, 75% of NA CD4<50, 40% of NA CD4 50–199, and 42% of primary infection, indicating common neuronal injury with untreated systemic HIV disease progression as well as transiently during early infection. By contrast, only 75% of HAD subjects had abnormal CSF t-tau levels, and there were no significant differences in t-tau levels among the remaining groups. sAPPα and β were also abnormal (decreased) in HAD, showed less marked change than NFL with CD4 decline in the absence of HAD, and were not decreased in PHI. The CSF Aβ peptides and p-tau concentrations did not differ among the groups, distinguishing the HIV CNS injury profile from Alzheimer's disease. These CSF biomarkers can serve as useful tools in selected research and clinical settings for patient classification, pathogenetic analysis, diagnosis and management.
doi:10.1371/journal.pone.0116081
PMCID: PMC4277428  PMID: 25541953
12.  Posiphen as a candidate drug to lower CSF amyloid precursor protein, amyloid-β peptide and τ levels: target engagement, tolerability and pharmacokinetics in humans 
Aim
A first in human study to evaluate tolerability and pharmacokinetics followed by an early proof of mechanism (POM) study to determine whether the small orally, available molecule, Posiphen tartrate (Posiphen), lowers secreted (s) amyloid-β precursor protein (APP) α and -β, amyloid-β peptide (Aβ), tau (τ) and inflammatory markers in CSF of patients with mild cognitive impairment (MCI).
Study design
Posiphen single and multiple ascending dose phase 1 randomised, double blind, placebo-controlled safety, tolerance, pharmacokinetic studies were undertaken in a total of 120 healthy volunteers to define a dose that was then used in a small non-randomised study of five MCI subjects, used as their own controls, to define target engagement.
Main outcome measures
Pharmacodynamic: sAPPα, sAPPβ, Aβ42, τ (total (t) and phosphorylated (p)) and inflammatory marker levels were time-dependently measured over 12 h and compared prior to and following 10 days of oral Posiphen treatment in four MCI subjects who completed the study. Pharmacokinetic: plasma and CSF drug and primary metabolite concentrations with estimated brain levels extrapolated from steady-state drug administration in rats.
Results
Posiphen proved well tolerated and significantly lowered CSF levels of sAPPα, sAPPβ, t-τ, p-τ and specific inflammatory markers, and demonstrated a trend to lower CSF Aβ42.
Conclusions
These results confirm preclinical POM studies, demonstrate that pharmacologically relevant drug/metabolite levels reach brain and support the continued clinical optimisation and evaluation of Posiphen for MCI and Alzheimer's disease.
doi:10.1136/jnnp-2012-302589
PMCID: PMC3415310  PMID: 22791904
Posiphen; amyloid precursor protein; amyloid-β peptide; inflammatory markers; mild cognitive impairment; genetics; B12 deficiency; neurochemistry; Alzheimer's disease; amyloid; head injury; Parkinson's disease
13.  Soluble β-amyloid Precursor Protein Alpha Binds to p75 Neurotrophin Receptor to Promote Neurite Outgrowth 
PLoS ONE  2013;8(12):e82321.
Background
The cleavage of β-amyloid precursor protein (APP) generates multiple proteins: Soluble β-amyloid Precursor Protein Alpha (sAPPα), sAPPβ, and amyloid β (Aβ). Previous studies have shown that sAPPα and sAPPβ possess neurotrophic properties, whereas Aβ is neurotoxic. However, the underlying mechanism of the opposing effects of APP fragments remains poorly understood. In this study, we have investigated the mechanism of sAPPα-mediated neurotrophic effects. sAPPα and sAPPβ interact with p75 neurotrophin receptor (p75NTR), and sAPPα promotes neurite outgrowth.
Methods and Findings
First, we investigated whether APP fragments interact with p75NTR, because full-length APP and Aβ have been shown to interact with p75NTR in vitro. Both sAPPα and sAPPβ were co-immunoprecipitated with p75NTR and co-localized with p75NTR on COS-7 cells. The binding affinity of sAPPα and sAPPβ for p75NTR was confirmed by enzyme-linked immunosorbent assay (ELISA). Next, we investigated the effect of sAPPα on neurite outgrowth in mouse cortical neurons. Neurite outgrowth was promoted by sAPPα, but sAPPα was uneffective in a knockdown of p75NTR.
Conclusion
We conclude that p75NTR is the receptor for sAPPα to mediate neurotrophic effects.
doi:10.1371/journal.pone.0082321
PMCID: PMC3864954  PMID: 24358169
14.  A panel of nine cerebrospinal fluid biomarkers may identify patients with atypical parkinsonian syndromes 
Background
Patients presenting with parkinsonian syndromes share many clinical features, which can make diagnosis difficult. This is important as atypical parkinsonian syndromes (APSs) such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA) and corticobasal syndrome (CBS) carry a poor prognosis, compared with patients with Parkinson’s disease (PD). In addition, there is overlap between APS and dementia diseases, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD).
Objective
To use a panel of cerebrospinal fluid (CSF) biomarkers to differentiate patients with APS from PD and dementia.
Methods
A prospective cohort of 160 patients and 30 control participants were recruited from a single specialist centre. Patients were clinically diagnosed according to current consensus criteria. CSF samples were obtained from patients with clinical diagnoses of PD (n=31), PSP (n=33), CBS (n=14), MSA (n=31), AD (n=26) and FTD (n=16). Healthy, elderly participants (n=30) were included as controls. Total τ (t-τ), phosphorylated τ (p-τ), β-amyloid 1–42 (Aβ42), neurofilament light chain (NFL), α-synuclein (α-syn), amyloid precursor protein soluble metabolites α and β (soluble amyloid precursor protein (sAPP)α, sAPPβ) and two neuroinflammatory markers (monocyte chemoattractant protein-1 and YKL-40) were measured in CSF. A reverse stepwise regression analysis and the false discovery rate procedure were used.
Results
CSF NFL (p<0.001), sAPPα (p<0.001) and a-syn (p=0.003) independently predicted diagnosis of PD versus APS. Together, these nine biomarkers could differentiate patients with PD from APS with an area under the curve of 0.95 and subtypes of APS from one another. There was good discriminatory power between parkinsonian groups, dementia disorders and healthy controls.
Conclusions
A panel of nine CSF biomarkers was able to differentiate APS from patients with PD and dementia. This may have important clinical utility in improving diagnostic accuracy, allowing better prognostication and earlier access to potential disease-modifying therapies.
doi:10.1136/jnnp-2014-309562
PMCID: PMC4564944  PMID: 25589779
15.  New highly sensitive rodent and human tests for soluble amyloid precursor protein alpha quantification: preclinical and clinical applications in Alzheimer’s disease 
BMC Neuroscience  2012;13:84.
Background
Amyloid precursor protein (APP), a key molecule in Alzheimer’s disease (AD), is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in AD pathology or the soluble form of APP (sAPPα). The level of amyloidogenic peptides in human cerebrospinal fluid (CSF) is considered to be a biomarker of AD, whereas the level of sAPPα in CSF as a biomarker has not been clearly established. sAPPα has neurotrophic and neuroprotective properties. Stimulating its formation and secretion is a promising therapeutic target in AD research. To this end, very sensitive tests for preclinical and clinical research are required.
Methods
The tests are based on homogenous time-resolved fluorescence and require no washing steps.
Results
We describe two new rapid and sensitive tests for quantifying mouse and human sAPPα. These 20 μl-volume tests quantify the levels of: i) endogenous mouse sAPPα in the conditioned medium of mouse neuron primary cultures, as well as in the CSF of wild-type mice, ii) human sAPPα in the CSF of AD mouse models, and iii) human sAPPα in the CSF of AD and non-AD patients. These tests require only 5 μl of conditioned medium from 5 × 104 mouse primary neurons, 1 μl of CSF from wild-type and transgenic mice, and 0.5 μl of human CSF.
Conclusions
The high sensitivity of the mouse sAPPα test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sAPPα in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sAPPα in the CSF of treated wild-type mice. Active molecules could then be tested in the AD mouse models by quantifying human sAPPα in the CSF through the progression of the disease. Finally, the human sAPPα test could strengthen the biological diagnosis of AD in large clinical investigations. Taken together, these new tests have a wide field of applications in preclinical and clinical studies.
doi:10.1186/1471-2202-13-84
PMCID: PMC3418197  PMID: 22824057
Alzheimer’s disease; Soluble amyloid precursor protein alpha; Homogeneous time-resolved fluorescence; Rodent; Human; Cerebrospinal fluid; Primary neurons; Sensitivity
16.  Soluble amyloid precursor protein β as blood-based biomarker of Alzheimer's disease 
Translational Psychiatry  2013;3(2):e227-.
The aim of this study was to explore concentrations differences of soluble amyloid precursor protein (sAPP) α and β in blood plasma in patients with probable Alzheimer's disease (AD) and cognitively healthy age-matched control subjects, as well as patients with behavioural variant frontotemporal dementia (bvFTD). Concentrations of sAPPα and β were measured using enzyme-linked immunosorbent assay technology in 80 patients with probable AD, 37 age-matched control subjects and 14 patients with bvFTD. Concentration differences were explored using parametric tests. Significantly decreased plasma concentrations in the AD group compared with both the control group and the bvFTD group were detected for sAPPβ (P=0.03 for both group comparisons), but not for sAPPα. The study provides a further piece of evidence in support of sAPPβ as a promising new biomarker of AD, which may potentially improve the diagnostic accuracy of existing markers and also enable a less invasive diagnostic workup. Further research is required to establish normal ranges and to replicate the results in independent cohorts including larger numbers of participants covering a wider spectrum of cognitive impairment.
doi:10.1038/tp.2013.11
PMCID: PMC3591004  PMID: 23423136
Alzheimer's disease; biomarker; early diagnosis; ELISA; plasma
17.  The cerebrospinal fluid biomarker profile in an HIV-infected subject with Alzheimer’s disease 
It is a challenge to differentiate between HIV-associated neurocognitive disorders (HAND) and other types of neurocognitive disease in the ageing HIV-infected population. Here we describe a 63 year old HIV-infected woman who had a history, neuropsychological test result, and PET examination consistent with characteristic Alzheimer’s disease (AD). The cerebrospinal fluid (CSF) biomarker profile was analogous to the profile typically found in AD in HIV-negative patients with increased t-tau and p-tau, a decreased level of Aβ42 and normal levels of CSF neurofilament light protein and sAPPα and sAPPβ, distinctly different from findings in HIV-associated dementia (HAD). Assessment of CSF biomarkers may be a valuable tool for clinicians to distinguish between HAD and AD.
doi:10.1186/s12981-015-0063-x
PMCID: PMC4501274  PMID: 26175795
Alzheimer’s disease; HIV; CSF biomarkers
18.  Interleukin-1 mediates Alzheimer and Lewy body pathologies 
Background
Clinical and neuropathological overlap between Alzheimer's (AD) and Parkinson's disease (PD) is now well recognized. Such cases of concurrent AD and Lewy body disease (AD/LBD) show neuropathological changes that include Lewy bodies (α-synuclein aggregates), neuritic amyloid plaques, and neurofibrillary tangles (hyperphosphorylated tau aggregates). The co-occurrence of these clinical and neuropathological changes suggests shared pathogenic mechanisms in these diseases, previously assumed to be distinct. Glial activation, with overexpression of interleukin-1 (IL-1) and other proinflammatory cytokines, has been increasingly implicated in the pathogenesis of both AD and PD.
Methods
Rat primary cultures of microglia and cortical neurons were cultured either separately or as mixed cultures. Microglia or cocultures were treated with a secreted fragment (sAPPα) of the β-amyloid precursor protein (βAPP). Neurons were treated with IL-1β or conditioned medium from sAPPα-activated microglia, with or without IL-1 receptor antagonist. Slow-release pellets containing either IL-1β or bovine serum albumin (control) were implanted in cortex of rats, and mRNA for various neuropathological markers was analyzed by RT-PCR. Many of the same markers were assessed in tissue sections from human cases of AD/LBD.
Results
Activation of microglia with sAPPα resulted in a dose-dependent increase in secreted IL-1β. Cortical neurons treated with IL-1β showed a dose-dependent increase in sAPPα release, an effect that was enhanced in the presence of microglia. IL-1β also elevated the levels of α-synuclein, activated MAPK-p38, and phosphorylated tau; a concomitant decrease in levels of synaptophysin occurred. Delivery of IL-1β by slow-release pellets elevated mRNAs encoding α-synuclein, βAPP, tau, and MAPK-p38 compared to controls. Finally, human cases of AD/LBD showed colocalization of IL-1-expressing microglia with neurons that simultaneously overexpressed βAPP and contained both Lewy bodies and neurofibrillary tangles.
Conclusion
Our findings suggest that IL-1 drives production of substrates necessary for formation of the major neuropathological changes characteristic of AD/LBD.
doi:10.1186/1742-2094-3-5
PMCID: PMC1435743  PMID: 16542445
19.  No diurnal variation of classical and candidate biomarkers of Alzheimer’s disease in CSF 
Background
Cerebrospinal fluid (CSF) biomarkers have gained increasing importance in the diagnostic work-up of Alzheimer’s disease (AD). The core CSF biomarkers related to AD pathology (Aβ42, t-tau and p-tau) are currently used in CSF diagnostics, while candidate markers of amyloid metabolism (Aβ38, Aβ40, sAPPα, sAPPβ), synaptic loss (neurogranin), neuroinflammation (YKL-40), neuronal damage (VILIP-1) and genetic risk (apolipoprotein E) are undergoing evaluation. Diurnal fluctuation in the concentration of CSF biomarkers has been reported and may represent a preanalytical confounding factor in the laboratory diagnosis of AD. The aim of the present study was to investigate the diurnal variability of classical and candidate CSF biomarkers in a cohort of neurosurgical patients carrying a CSF drainage.
Method
Samples were collected from a cohort of 13 neurosurgical patients from either ventricular (n = 6) or lumbar (n = 7) CSF drainage at six time points during the day, 1–7 days following the neurosurgical intervention. Concentrations of the core biomarkers were determined by immunoassays.
Results
Although absolute values largely varied among subjects, none of the biomarkers showed significant diurnal variation. Site of drainage (lumbar vs. ventricular) did not influence this result. The different immunoassays used for tau and Aβ markers provided similar results.
Conclusion
Time of day at CSF collection does not ultimately affect the concentration levels of classical and candidate AD biomarkers. Similar trends were found when using different immunoassays, thus corroborating the consistency of the data.
Electronic supplementary material
The online version of this article (doi:10.1186/s13024-016-0130-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13024-016-0130-3
PMCID: PMC5013624  PMID: 27605218
20.  Effects of TACE inhibition on Amyloid β production and APP processing in vitro and in vivo 
TNFα is a pro-inflammatory cytokine that is elevated in Alzheimer’s disease (AD) brains. Since TNFα is released from cell membranes by the TNFα converting enzyme (TACE), inhibition of TACE has the potential to mitigate TNFα effects in AD brain. TACE also cleaves amyloid precursor protein (APP) and generates sAPPα, precluding the formation of potentially harmful Aβ peptides by β-site APP cleaving enzymes (BACE). Hence, the anti-inflammatory benefits of TACE inhibition might be offset by an increase in Aβ. We have examined the effects of the highly selective TACE inhibitor, BMS-561392, on APP processing in vitro and in vivo. In CHO cells expressing APP, BMS-561392 significantly reduced secretion of sAPPα without a corresponding increase in Aβ production. Conversely, a BACE inhibitor decreased sAPPβ and Aβ peptides with no change in the secretion of sAPPα. These data indicate an absence of TACE and BACE competition for the APP substrate. Despite this, we observed competition for APP when TACE activity was enhanced via phorbol ester treatment or if APP was modified such that it was retained within the trans Golgi network (TGN). These results suggest that BACE and TACE share a common TGN localization, but under normal conditions do not compete for APP. To confirm this finding in vivo, BMS-561392 was infused into the brains of Tg2576 and wild-type mice. While decreased brain sAPPα levels were observed, steady-state Aβ levels were not significantly changed. Accordingly, it is possible that TACE inhibitors could reduce TNFα levels without increasing Aβ levels within the AD brain.
doi:10.1523/JNEUROSCI.2913-08.2008
PMCID: PMC2646261  PMID: 19005070
Alzheimer’s disease; amyloid-beta; inflammation; neuroinflammation; tumor necrosis factor; TNFα
21.  Physiological Mouse Brain Aβ Levels Are Not Related to the Phosphorylation State of Threonine-668 of Alzheimer's APP 
PLoS ONE  2006;1(1):e51.
Background
Amyloid-β peptide species ending at positions 40 and 42 (Aβ40, Αβ42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Aβ peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Aβ42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFα, CTFβ). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Aβ levels, prompting the current study.
Methodology
In order to evaluate whether the phosphorylation state of Thr668 controlled brain Aβ levels, we studied the levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in brains from “knock-in” mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein.
Conclusions
The levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Aβ40 or Αβ42 in vivo.
doi:10.1371/journal.pone.0000051
PMCID: PMC1762327  PMID: 17183681
22.  Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers 
PLoS ONE  2013;8(11):e81249.
Introduction
Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI.
Materials and Methods
Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed.
Results
CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers.
Conclusions
Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI.
doi:10.1371/journal.pone.0081249
PMCID: PMC3829937  PMID: 24260563
23.  Soluble amyloid precursor protein alpha (sAPPα) inhibits tau phosphorylation through modulation of GSK3β signaling pathway 
Journal of neurochemistry  2015;135(3):630-637.
We recently found that sAPPα decreases Aβ generation by directly associating with β-site amyloid precursor protein (APP) converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases GSK3β-mediated Alzheimer’s disease (AD)-like tau phosphorylation in AD patient-derived neurons, we determined whether sAPPα also reduces GSK3β-mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3β in primary neurons from sAPPα over-expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH-SY5Y cells. Further, in SH-SY5Y cells overexpressing BACE1, and HeLa cells overexpressing human tau, sAPPα reduced GSK3β activity and tau phosphorylation. Importantly, the reductions in GSK3β activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ-secretase inhibition. In accord, AD mice overexpressing human sAPPα had less GSK3β activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP β-processing and GSK3β-mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis.
doi:10.1111/jnc.13351
PMCID: PMC4624213  PMID: 26342176
Alzheimer’s disease; sAPPα; tau; BACE1; GSK3β
24.  Time-dependent changes in gene expression induced by secreted amyloid precursor protein-alpha in the rat hippocampus 
BMC Genomics  2013;14:376.
Background
Differential processing of the amyloid precursor protein liberates either amyloid-ß, a causative agent of Alzheimer’s disease, or secreted amyloid precursor protein-alpha (sAPPα), which promotes neuroprotection, neurotrophism, neurogenesis and synaptic plasticity. The underlying molecular mechanisms recruited by sAPPα that underpin these considerable cellular effects are not well elucidated. As these effects are enduring, we hypothesised that regulation of gene expression may be of importance and examined temporally specific gene networks and pathways induced by sAPPα in rat hippocampal organotypic slice cultures. Slices were exposed to 1 nM sAPPα or phosphate buffered saline for 15 min, 2 h or 24 h and sAPPα-associated gene expression profiles were produced for each time-point using Affymetrix Rat Gene 1.0 ST arrays (moderated t-test using Limma: p < 0.05, and fold change ± 1.15).
Results
Treatment of organotypic hippocampal slice cultures with 1 nM sAPPα induced temporally distinct gene expression profiles, including mRNA and microRNA associated with Alzheimer’s disease. Having demonstrated that treatment with human recombinant sAPPα was protective against N-methyl d-aspartate-induced toxicity, we next explored the sAPPα-induced gene expression profiles. Ingenuity Pathway Analysis predicted that short-term exposure to sAPPα elicited a multi-level transcriptional response, including upregulation of immediate early gene transcription factors (AP-1, Egr1), modulation of the chromatin environment, and apparent activation of the constitutive transcription factors CREB and NF-κB. Importantly, dynamic regulation of NF-κB appears to be integral to the transcriptional response across all time-points. In contrast, medium and long exposure to sAPPα resulted in an overall downregulation of gene expression. While these results suggest commonality between sAPPα and our previously reported analysis of plasticity-related gene expression, we found little crossover between these datasets. The gene networks formed following medium and long exposure to sAPPα were associated with inflammatory response, apoptosis, neurogenesis and cell survival; functions likely to be the basis of the neuroprotective effects of sAPPα.
Conclusions
Our results demonstrate that sAPPα rapidly and persistently regulates gene expression in rat hippocampus. This regulation is multi-level, temporally specific and is likely to underpin the neuroprotective effects of sAPPα.
doi:10.1186/1471-2164-14-376
PMCID: PMC3691674  PMID: 23742273
Secreted amyloid precursor protein alpha; Hippocampus; Organotypic slice cultures; Microarray; Ingenuity pathway analysis; Neuroprotection; Immediate early genes; MicroRNA; NF-κB
25.  Cerebrospinal fluid BACE1 activity and markers of amyloid precursor protein metabolism and axonal degeneration in Alzheimer’s disease 
Objective
To assess cerebrospinal fluid (CSF) β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) activity in relation to Alzheimer’s disease (AD) and to correlate the enzyme activity with protein markers of APP metabolism and axonal degeneration.
Methods
BACE1 activity and protein concentrations were measured and analysed in 342 participants of the AD Neuroimaging Initiative, including 99 normal controls, 75 stable mild cognitive impairment (MCI), 87 progressive MCI and 79 AD dementia cases. All statistical analyses were Bonferroni corrected for multiple comparisons.
Results
No significant differences between controls and any of the three patient groups were detected for BACE1 activity and soluble APP (sAPP)β concentrations in CSF. Significant correlations with BACE1 activity were found for CSF APPβ and total tau in all four groups; and for CSF phosphorylated tau181 in all groups but the progressive MCI group. There were no correlations for CSF amyloid β (Aβ)1-42 nor for plasma Aβ1-42 and Aβ1-40.
Conclusions
The consistent correlation between BACE1 activity and sAPPβ supports their role as biomarkers of target engagement in clinical trials on BACE1 inhibition.
doi:10.1016/j.jalz.2013.09.006
PMCID: PMC4038661  PMID: 24239250
Biomarker; dementia; mild cognitive impairment; diagnosis; prognosis; amyloid precursor protein; amyloid beta; tau; correlation

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