Myelin dysfunction drives amyloid-ß deposition in models of Alzheimer's disease.

The incidence of Alzheimer's disease (AD), the leading cause of dementia, increases rapidly with age, but why age constitutes the main risk factor is still poorly understood. Brain ageing affects oligodendrocytes and the structural integrity of myelin sheaths1, the latter of which is associated with secondary neuroinflammation2,3. As oligodendrocytes support axonal energy metabolism and neuronal health4-7, we hypothesized that loss of myelin integrity could be an upstream risk factor for neuronal amyloid-ß (Aß) deposition, the central neuropathological hallmark of AD. Here we identify genetic pathways of myelin dysfunction and demyelinating injuries as potent drivers of amyloid deposition in mouse models of AD. Mechanistically, myelin dysfunction causes the accumulation of the Aß-producing machinery within axonal swellings and increases the cleavage of cortical amyloid precursor protein. Suprisingly, AD mice with dysfunctional myelin lack plaque-corralling microglia despite an overall increase in their numbers. Bulk and single-cell transcriptomics of AD mouse models with myelin defects show that there is a concomitant induction of highly similar but distinct disease-associated microglia signatures specific to myelin damage and amyloid plaques, respectively. Despite successful induction, amyloid disease-associated microglia (DAM) that usually clear amyloid plaques are apparently distracted to nearby myelin damage. Our data suggest a working model whereby age-dependent structural defects of myelin promote Aß plaque formation directly and indirectly and are therefore an upstream AD risk factor. Improving oligodendrocyte health and myelin integrity could be a promising target to delay development and slow progression of AD.

Amino-terminally elongated Aß peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

AIMS: The aggregation and deposition of amyloid-ß (Aß) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aß peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aß peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aß4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aß peptides. METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aß peptide variants. Antibodies against these Aß variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aß peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aß-6-x and Aß-3-x peptides, of which the latter serve as a component in a promising Aß-based plasma biomarker. Aß-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aß-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aß-6/-3-x peptides. DISCUSSION: The current findings implicate ADAMTS4 in both the pathological process of Aß peptide aggregation and in the early detection of amyloid pathology in AD.

Assessment of immunoprecipitation with subsequent immunoassays for the blood-based diagnosis of Alzheimer's disease.

The Aß42/40 ratio and the concentration of phosphorylated Tau181 in blood plasma represent attractive biomarkers for Alzheimer's disease. As a means for reducing potential matrix effects, which may interfere with plasma immunoassays, we have previously developed a pre-analytical sample workup by semi-automated immunoprecipitation. Here we test the compatibility of pre-analytical immunoprecipitations with automated Aß1-40, Aß1-42 and phosphorylated Tau181 immunoassays on the Lumipulse platform and compare the diagnostic performance of the respective immunoprecipitation immunoassay approaches with direct plasma measurements. 71 participants were dichotomized according to their Aß42/40 ratios in cerebrospinal fluid into the diagnostic groups amyloid-positive (n = 32) and amyloid-negative (n = 39). The plasma Aß1-42/1-40 ratio and phosphorylated Tau181 levels were determined on the Lumipulse G600II platform (Fujirebio) by direct measurements in EDTA-plasma or after Aß- or Tau-immunoprecipitation, respectively. Pre-analytical immunoprecipitation of Aß turned out to be compatible with the Lumipulse Aß assays and resulted in a numerical, yet statistically not significant increase in the area under the ROC curve for plasma Aß1-42/1-40. Additionally, we observed a significant increase in the standardised effect size (Cohen's D). Pre-analytical immunoprecipitation of Tau resulted in increased differences between the diagnostic groups in terms of median and mean phosphorylated Tau 181 levels. Furthermore, we observed a greater Cohen's d (p < 0.001) and a larger area under the ROC curve (p = 0.038) after Tau-IP. Our preliminary findings in a small, preselected sample indicate that pre-analytical immunoprecipitation may have the potential to improve the diagnostic performance of plasma biomarker immunoassays for Aß1-42/1-40 and phosphorylated Tau181 to predict brain amyloid deposition.

A novel interface between the N-terminal and coiled-coil domain of STAT1 functions in an auto-inhibitory manner.

BACKGROUND: STAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation. RESULTS: In this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner. CONCLUSION: Based on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process. Video Abstract.

Combined long-term enriched environment and caffeine supplementation improve memory function in C57Bl6 mice.

Regular physical activity has been associated with healthy brain aging, reflected by beneficial effects on cognition and learning and memory. Nutritional supplements such as caffeine have been shown to act as cognitive enhancers and may possess neuroprotective properties. Interestingly, caffeine also improves athletic capabilities and is widely used by athletes because of its performance-enhancing effect, while information on potential additive beneficial effects of physical activity and caffeine on cognitive performance is scarce. In the present study, the effects of caffeine supplementation in combination with prolonged physical and cognitive stimulation in the form of the enriched environment (EE) housing for a duration of 4 months were analyzed. We demonstrate that caffeine supplementation together with prolonged environmental enrichment led to enhanced memory function, resulting in improved recognition and spatial working memory in behavioral paradigms such as the novel object recognition task or the Morris water maze in C57Bl6 wild-type mice. Mice housed under EE conditions showed increased gene expression levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. The present findings underscore the potential impact of continuous physical activity in the prevention of age-related cognitive decline and may offer new options for combinatorial approaches.

Meprin ß knockout reduces brain Aß levels and rescues learning and memory impairments in the APP/lon mouse model for Alzheimer's disease.

ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.

A Combination of Caffeine Supplementation and Enriched Environment in an Alzheimer's Disease Mouse Model.

A variety of factors has been associated with healthy brain aging, and epidemiological studies suggest that physical activity and nutritional supplements such as caffeine may reduce the risk of developing dementia and, in particular, Alzheimer's disease (AD) in later life. Caffeine is known to act as a cognitive enhancer but has been also shown to positively affect exercise performance in endurance activities. We have previously observed that chronic oral caffeine supplementation and a treatment paradigm encompassing physical and cognitive stimulation by enriched environment (EE) housing can improve learning and memory performance and ameliorate hippocampal neuron loss in the Tg4-42 mouse model of AD. Here, we investigated whether these effects were synergistic. To that end, previous findings on individual treatments were complemented with unpublished, additional data and analyzed in depth by ANOVA followed by Bonferroni multiple comparison post tests. We further evaluated whether plasma neurofilament light chain levels reflect neuropathological and behavioral changes observed in the experimental groups. While a treatment combining physical activity and caffeine supplementation significantly improved learning and memory function compared to standard-housed vehicle-treated Tg4-42 in tasks such as the Morris water maze, no major additive effect outperforming the effects of the single interventions was observed.

Detection and quantification of Aß-3-40 (APP669-711) in cerebrospinal fluid.

Neurochemical biomarkers can support the diagnosis of Alzheimer's disease and may facilitate clinical trials. In blood plasma, the ratio of the amyloid-ß (Aß) peptides Aß-3-40/Aß1-42 can predict cerebral amyloid-ß pathology with high accuracy (Nakamura et al., 2018). Whether or not Aß-3-40 (aka. amyloid precursor protein (APP) 669-711) is also present in cerebrospinal fluid (CSF) is not clear. Here, we investigated whether Aß-3-40 can be detected in CSF and to what extent the CSF Aß-3-40/Aß42 ratio is able to differentiate between individuals with or without amyloid-ß positron emission tomography (PET) evidence of brain amyloid. The occurrence of Aß-3-40 in human CSF was assessed by immunoprecipitation followed by mass spectrometry. For quantifying the CSF concentrations of Aß-3-40 in 23 amyloid PET-negative and 17 amyloid PET-positive subjects, we applied a sandwich-type immunoassay. Our findings provide clear evidence of the presence of Aß-3-40 and Aß-3-38 in human CSF. While there was no statistically significant difference in the CSF concentration of Aß-3-40 between the two diagnostic groups, the CSF Aß-3-40/Aß42 ratio was increased in the amyloid PET-positive individuals. We conclude that Aß-3-40 appears to be a regular constituent of CSF and may potentially serve to accentuate the selective decrease in CSF Aß42 in Alzheimer's disease.

An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form.

BACKGROUND: Unphosphorylated signal transducer and activator of transcription 1 (U-STAT1) has been reported to elicit a distinct gene expression profile as compared to tyrosine-phosphorylated STAT1 (P-STAT1) homodimers. However, the impact of U-STAT1 on the IFNγ-induced immune response mediated by P-STAT1 is unknown. By generating a double mutant of STAT1 with mutation R602L in the Src-homology 2 (SH2) domain and Y701F in the carboxy-terminal transactivation domain mimicking U-STAT1, we investigated the effects of U-STAT1 on P-STAT1-mediated signal transduction. RESULTS: In this study, we discovered a novel activity of U-STAT1 that alters the nucleo-cytoplasmic distribution of cytokine-stimulated P-STAT1. While the dimerization-deficient mutant R602L/Y701F was not able to display cytokine-induced nuclear accumulation, it inhibited the nuclear accumulation of co-expressed IFNγ-stimulated wild-type P-STAT1. Disruption of the anti-parallel dimer interface in the R602L/Y701F mutant via additional R274W and T385A mutations did not rescue the impaired nuclear accumulation of co-expressed P-STAT1. The mutant U-STAT1 affected neither the binding of co-expressed P-STAT1 to gamma-activated sites in vitro, nor the transcription of reporter constructs and the activation of STAT1 target genes. However, the nuclear accumulation of P-STAT1 was diminished in the presence of mutant U-STAT1, which was not restored by mutations reducing the DNA affinity of mutant U-STAT1. Whereas single mutations in the amino-terminus of dimerization-deficient U-STAT1 similarly inhibited the nuclear accumulation of co-expressed P-STAT1, a complete deletion of the amino-terminus restored cytokine-stimulated nuclear accumulation of P-STAT1. Likewise, the disruption of a dimer-specific nuclear localization signal also rescued the U-STAT1-mediated inhibition of P-STAT1 nuclear accumulation. CONCLUSION: Our data demonstrate a novel role of U-STAT1 in affecting nuclear accumulation of P-STAT1, such that a high intracellular concentration of U-STAT1 inhibits the detection of nuclear P-STAT1 in immunofluorescence assays. These observations hint at a possible physiological function of U-STAT1 in buffering the nuclear import of P-STAT1, while preserving IFNγ-induced gene expression. Based on these results, we propose a model of a hypothetical import structure, the assembly of which is impaired under high concentrations of U-STAT1. This mechanism maintains high levels of cytoplasmic STAT1, while simultaneously retaining signal transduction by IFNγ. Video Abstract.

Long-term caffeine treatment of Alzheimer mouse models ameliorates behavioural deficits and neuron loss and promotes cellular and molecular markers of neurogenesis.

Epidemiological studies indicate that the consumption of caffeine, the most commonly ingested psychoactive substance found in coffee, tea or soft drinks, reduces the risk of developing Alzheimer's disease (AD). Previous treatment studies with transgenic AD mouse models reported a reduced amyloid plaque load and an amelioration of behavioral deficits. It has been further shown that moderate doses of caffeine have the potential to attenuate the health burden in preclinical mouse models of a variety of brain disorders (reviewed in Cunha in J Neurochem 139:1019-1055, 2016). In the current study, we assessed whether long-term caffeine consumption affected hippocampal neuron loss and associated behavioral deficits in the Tg4-42 mouse model of AD. Treatment over a 4-month period reduced hippocampal neuron loss, rescued learning and memory deficits, and ameliorated impaired neurogenesis. Neuron-specific RNA sequencing analysis in the hippocampus revealed an altered expression profile distinguished by the up-regulation of genes linked to synaptic function and processes, and to neural progenitor proliferation. Treatment of 5xFAD mice, which develop prominent amyloid pathology, with the same paradigm also rescued behavioral deficits but did not affect extracellular amyloid-ß (Aß) levels or amyloid precursor protein (APP) processing. These findings challenge previous assumptions that caffeine is anti-amyloidogenic and indicate that the promotion of neurogenesis might play a role in its beneficial effects.

Characterization of a Mouse Model of Alzheimer's Disease Expressing Aß4-42 and Human Mutant Tau.

The relationship between the two most prominent neuropathological hallmarks of Alzheimer's Disease (AD), extracellular amyloid-ß (Aß) deposits and intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFT), remains at present not fully understood. A large body of evidence places Aß upstream in the cascade of pathological events, triggering NFTs formation and the subsequent neuron loss. Extracellular Aß deposits were indeed causative of an increased tau phosphorylation and accumulation in several transgenic models but the contribution of soluble Aß peptides is still controversial. Among the different Aß variants, the N-terminally truncated peptide Aß4-42 is among the most abundant. To understand whether soluble Aß4-42 peptides impact the onset or extent of tau pathology, we have crossed the homozygous Tg4-42 mouse model of AD, exclusively expressing Aß4-42 peptides, with the PS19 (P301S) tau transgenic model. Behavioral assessment showed that the resulting double-transgenic line presented a partial worsening of motor performance and spatial memory deficits in the aged group. While an increased loss of distal CA1 pyramidal neurons was detected in young mice, no significant alterations in hippocampal tau phosphorylation were observed in immunohistochemical analyses.

Neuron Loss in Alzheimer's Disease: Translation in Transgenic Mouse Models.

Transgenic mouse models represent an essential tool for the exploration of Alzheimer's disease (AD) pathological mechanisms and the development of novel treatments, which at present provide only symptomatic and transient effects. While a variety of mouse models successfully reflects the main neuropathological hallmarks of AD, such as extracellular amyloid-ß (Aß) deposits, intracellular accumulation of Tau protein, the development of micro- and astrogliosis, as well as behavioral deficits, substantial neuron loss, as a key feature of the disease, seems to be more difficult to achieve. In this review, we summarize information on classic and more recent transgenic mouse models for AD, focusing in particular on loss of pyramidal, inter-, and cholinergic neurons. Although the cause of neuron loss in AD is still a matter of scientific debate, it seems to be linked to intraneuronal Aß accumulation in several transgenic mouse models, especially in pyramidal neurons.

Development and Technical Validation of an Immunoassay for the Detection of APP669-711 (Aß-3-40) in Biological Samples.

The ratio of amyloid precursor protein (APP)669-711 (Aß-3-40)/Aß1-42 in blood plasma was reported to represent a novel Alzheimer's disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aß-3-x and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of Aß-3-40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aß immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aß-3-40 with no appreciable cross reactivity with Aß1-40 or N-terminally truncated Aß variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aß-3-40 with a quantitative assay range of 22 pg/mL-7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aß42/Aß-3-40 and Aß42/Aß40 ratios were lower in patients with dementia of the Alzheimer's type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aß42/Aß-3-40 ratio as a novel biomarker candidate for Alzheimer's disease has been set.

The metalloprotease ADAMTS4 generates N-truncated Aß4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.

Phosphorylation of the amyloid ß-peptide at Ser26 stabilizes oligomeric assembly and increases neurotoxicity.

Aggregation and toxicity of the amyloid ß-peptide (Aß) are considered as critical events in the initiation and progression of Alzheimer's disease (AD). Recent evidence indicated that soluble oligomeric Aß assemblies exert pronounced toxicity, rather than larger fibrillar aggregates that deposit in the forms of extracellular plaques. While some rare mutations in the Aß sequence that cause early-onset AD promote the oligomerization, molecular mechanisms that induce the formation or stabilization of oligomers of the wild-type Aß remain unclear. Here, we identified an Aß variant phosphorylated at Ser26 residue (pSer26Aß) in transgenic mouse models of AD and in human brain that shows contrasting spatio-temporal distribution as compared to non-phosphorylated Aß (npAß) or other modified Aß species. pSer26Aß is particularly abundant in intraneuronal deposits at very early stages of AD, but much less in extracellular plaques. pSer26Aß assembles into a specific oligomeric form that does not proceed further into larger fibrillar aggregates, and accumulates in characteristic intracellular compartments of granulovacuolar degeneration together with TDP-43 and phosphorylated tau. Importantly, pSer26Aß oligomers exert increased toxicity in human neurons as compared to other known Aß species. Thus, pSer26Aß could represent a critical species in the neurodegeneration during AD pathogenesis.

Focusing the amyloid cascade hypothesis on N-truncated Abeta peptides as drug targets against Alzheimer's disease.

Although N-truncated Aß variants are known to be the main constituent of amyloid plaques in the brains of patients with Alzheimer's disease, their potential as targets for pharmacological intervention has only recently been investigated. In the last few years, the Alzheimer field has experienced a paradigm shift with the ever increasing understanding that targeting amyloid plaques has not led to a successful immunotherapy. On the other hand, there can be no doubt that the amyloid cascade hypothesis is central to the etiology of Alzheimer's disease, raising the question as to why it is apparently failing to translate into the clinic. In this review, we aim to refocus the amyloid hypothesis integrating N-truncated Aß peptides based on mounting evidence that they may represent better targets than full-length Aß. In addition to Aß peptides starting with an Asp at position 1, a variety of different N-truncated Aß peptides have been identified starting with amino residue Ala-2, pyroglutamylated Glu-3, Phe-4, Arg-5, His-6, Asp-7, Ser-8, Gly-9, Tyr-10 and pyroglutamylated Glu-11. Certain forms of N-truncated species are better correlates for early pathological changes found pre-symptomatically more often than others. There is also evidence that, together with full-length Aß, they might be physiologically detectable and are naturally secreted by neurons. Others are known to form soluble aggregates, which have neurotoxic properties in transgenic mouse models. It has been clearly demonstrated by several groups that some N-truncated Aßs dominate full-length Aß in the brains of Alzheimer's patients. We try to address which of the N-truncated variants may be promising therapeutic targets and which enzymes might be involved in the generation of these peptides.

Pyroglutamate amyloid ß (Aß) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease.

Pyroglutamate-modified Aß peptides at amino acid position three (Aß(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aß(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated Aß(pE3-42) levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces Aß(pE3-42). TBA42 mice showed age-dependent behavioral deficits and Aß(pE3-42) accumulation. The Aß profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-terminal truncated, and modified Aß peptides: Aß(1-42), Aß(1-40), Aß(pE3-40), Aß(pE3-42), Aß(3-42), Aß(4-42), and Aß(5-42). 5XFAD and TBA42 mice were then crossed to generate transgenic FAD42 mice. At 6 months of age, FAD42 mice showed an aggravated behavioral phenotype compared with single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that Aß(pE3) levels were elevated in FAD42 mice. No change in Aß(x)(-42) or other Aß isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of Aß(pE-42) in FAD42 mice.

Amyloid precursor protein is a biomarker for transformed human pluripotent stem cells.

Increasing evidence suggests an important function of the ß-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors.

Early intraneuronal accumulation and increased aggregation of phosphorylated Abeta in a mouse model of Alzheimer's disease.

The progressive accumulation of extracellular amyloid plaques in the brain is a common hallmark of Alzheimer's disease (AD). We recently identified a novel species of Aß phosphorylated at serine residue 8 with increased propensity to form toxic aggregates as compared to non-phosphorylated species. The age-dependent analysis of Aß depositions using novel monoclonal phosphorylation-state specific antibodies revealed that phosphorylated Aß variants accumulate first inside of neurons in a mouse model of AD already at 2 month of age. At higher ages, phosphorylated Aß is also abundantly detected in extracellular plaques. Besides a large overlap in the spatiotemporal deposition of phosphorylated and non-phosphorylated Aß species, fractionized extraction of Aß from brains revealed an increased accumulation of phosphorylated Aß in oligomeric assemblies as compared to non-phosphorylated Aß in vivo. Thus, phosphorylated Aß could represent an important species in the formation and stabilization of neurotoxic aggregates, and might be targeted for AD therapy and diagnosis.