Glial activation and inflammation along the Alzheimer's disease continuum.

BACKGROUND: Neuronal and glial cell interaction is essential for synaptic homeostasis and may be affected in Alzheimer's disease (AD). We measured cerebrospinal fluid (CSF) neuronal and glia markers along the AD continuum, to reveal putative protective or harmful stage-dependent patterns of activation. METHODS: We included healthy controls (n = 36) and Aß-positive (Aß+) cases (as defined by pathological CSF amyloid beta 1-42 (Aß42)) with either subjective cognitive decline (SCD, n = 19), mild cognitive impairment (MCI, n = 39), or AD dementia (n = 27). The following CSF markers were measured: a microglial activation marker-soluble triggering receptor expressed on myeloid cells 2 (sTREM2), a marker of microglial inflammatory reaction-monocyte chemoattractant protein-1 (MCP-1), two astroglial activation markers-chitinase-3-like protein 1 (YKL-40) and clusterin, a neuron-microglia communication marker-fractalkine, and the CSF AD biomarkers (Aß42, phosphorylated tau (P-tau), total tau (T-tau)). Using ANOVA with planned comparisons, or Kruskal-Wallis tests with Dunn's pairwise comparisons, CSF levels were compared between clinical groups and between stages of biomarker severity using CSF biomarkers for classification based on amyloid pathology (A), tau pathology (T), and neurodegeneration (N) giving rise to the A/T/N score. RESULTS: Compared to healthy controls, sTREM2 was increased in SCD (p < .01), MCI (p < .05), and AD dementia cases (p < .001) and increased in AD dementia compared to MCI cases (p < .05). MCP-1 was increased in MCI (p < .05) and AD dementia compared to both healthy controls (p < .001) and SCD cases (p < .01). YKL-40 was increased in dementia compared to healthy controls (p < .01) and MCI (p < .05). All of the CSF activation markers were increased in subjects with pathological CSF T-tau (A+T-N+ and A+T+N+), compared to subjects without neurodegeneration (A-T-N- and A+T-N-). DISCUSSION: Microglial activation as indicated by increased sTREM2 is present already at the preclinical SCD stage; increased MCP-1 and astroglial activation markers (YKL-40 and clusterin) were noted only at the MCI and AD dementia stages, respectively, and in Aß+ cases (A+) with pathological T-tau (N+). Possible different effects of early and later glial activation need to be explored.

CSF sTREM2 in delirium-relation to Alzheimer's disease CSF biomarkers Aß42, t-tau and p-tau.

BACKGROUND: Delirium and dementia share symptoms of cognitive dysfunctions, and mechanisms of neuroinflammation appear involved in both conditions. Triggering receptor expressed on myeloid cells 2 (TREM2) is linked to dementia and neurodegenerative disease. It encodes expression of an innate immune receptor in the brain expressed by microglia. The level of the soluble fragment of TREM2 (sTREM2) is reported to increase in the cerebrospinal fluid (CSF) already in prodromal and asymptomatic Alzheimer's disease. METHODS: We analyzed the level of CSF sTREM2 in relation to delirium and dementia. The study included patients with or without pre-existing dementia who underwent acute hip fracture surgery (n = 120), and some of the patients developed delirium (n = 65). A medical delirium cohort (n = 26) was also examined. ELISA was used to determine the level of sTREM2 in CSF. RESULTS: Delirium was associated with a higher level of CSF sTREM2 only among those without pre-existing dementia (p = 0.046, n = 15, n = 44), particularly among patients developing delirium after CSF sampling (p = 0.02, n = 7, n = 44). Between patients with dementia, there was no group difference, but the CSF sTREM2 level increased with waiting time for surgery (rS = 0.39, p = 0.002, n = 60) and correlated well with the CSF Alzheimer's disease biomarkers, Aß42, and t-tau/p-tau (rS = 0.40, p = 0.002, rS = 0.46, p < 0.001/ rS = 0.49, p < 0.001, n = 60). Among patients with dementia, the level of Aß38 and Aß40 also correlated positively with sTREM2 in CSF (Aß38MSDrS = 0.44, p = 0.001; Aß40MSDrS = 0.48, p < 0.001; Aß42MSDrS = 0.43, p < 0.001, n = 60). CONCLUSION: The findings reinforce the involvement of neuroinflammation in delirium, yet with separate responses in patients with or without pre-existing dementia. Our findings support the concept of primed microglia in neurodegenerative disease and central immune activation after a peripheral trauma in such patients. A CSF biomarker panel of neuroinflammation might be valuable to prevent delirium by identifying patients at risk.

The Alzheimer's disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) are genetically linked to Alzheimer's disease. Here, we investigated whether human ApoE mediates signal transduction through human and murine TREM2 and sought to identify a TREM2-binding domain in human ApoE. METHODS: To investigate cell signaling through TREM2, a cell line was used which expressed an NFAT-inducible ß-galactosidase reporter and human or murine TREM2, fused to CD8 transmembrane and CD3ζ intracellular signaling domains. ELISA-based binding assays were used to determine binding affinities of human ApoE isoforms to human TREM2 and to identify a TREM2-binding domain in ApoE. RESULTS: ApoE was found to be an agonist to human TREM2 with EC50 in the low nM range, and to murine TREM2 with reduced potency. In the reporter cells, TREM2 expression was lower than in nontransgenic mouse brain. Human ApoE isoforms ε2, ε3, and ε4 bound to human TREM2 with K d in the low nM range. The binding was displaced by an ApoE-mimetic peptide (amino acids 130-149). CONCLUSIONS: An ApoE-mediated dose-dependent signal transduction through TREM2 in reporter cells was demonstrated, and a TREM2-binding region in ApoE was identified. The relevance of an ApoE-TREM2 receptor signaling pathway to Alzheimer's disease is discussed.

Overexpression of heparanase lowers the amyloid burden in amyloid-ß precursor protein transgenic mice.

Heparan sulfate (HS) and HS proteoglycans (HSPGs) colocalize with amyloid-ß (Aß) deposits in Alzheimer disease brain and in Aß precursor protein (AßPP) transgenic mouse models. Heparanase is an endoglycosidase that specifically degrades the unbranched glycosaminoglycan side chains of HSPGs. The aim of this study was to test the hypothesis that HS and HSPGs are active participators of Aß pathogenesis in vivo. We therefore generated a double-transgenic mouse model overexpressing both human heparanase and human AßPP harboring the Swedish mutation (tgHpa*Swe). Overexpression of heparanase did not affect AßPP processing because the steady-state levels of Aß1-40, Aß1-42, and soluble AßPP ß were the same in 2- to 3-month-old double-transgenic tgHpa*Swe and single-transgenic tgSwe mice. In contrast, the Congo red-positive amyloid burden was significantly lower in 15-month-old tgHpa*Swe brain than in tgSwe brain. Likewise, the Aß burden, measured by Aßx-40 and Aßx-42 immunohistochemistry, was reduced significantly in tgHpa*Swe brain. The intensity of HS-stained plaques correlated with the Aßx-42 burden and was reduced in tgHpa*Swe mice. Moreover, the HS-like molecule heparin facilitated Aß1-42-aggregation in an in vitro Thioflavin T assay. The findings suggest that HSPGs contribute to amyloid deposition in tgSwe mice by increasing Aß fibril formation because heparanase-induced fragmentation of HS led to a reduced amyloid burden. Therefore, drugs interfering with Aß-HSPG interactions might be a potential strategy for Alzheimer disease treatment.

Isobaric Quantification of Cerebrospinal Fluid Amyloid-ß Peptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

The amyloid beta (Aß) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aß1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aß1-38 and Aß1-40 in addition to Aß1-42 has increased, quantitative assays of other Aß peptides remain rarely explored. We recently discovered novel Aß peptides in CSF using antibodies recognizing the Aß mid-domain region. Here we have developed a method using both Aß N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aß peptides in CSF. The developed method was used in a pilot study to produce Aß peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aß peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aß1-38 or Aß1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aß production or degradation.

Altered amyloid-ß structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion.

Deposition of amyloid beta (Aß) into plaques is a major hallmark of Alzheimer's disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aß. We recently identified the Uppsala APP mutation (APPUpp), which causes Aß pathology by a triple mechanism: increased ß-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aß conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aß pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aß pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased ß-secretase cleavage and suppressed α-secretase cleavage, resulting in AßUpp42 dominated diffuse plaque pathology appearing from the age of 5-6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [11C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aß pathology in all models, whereas the Aß protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aß pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AßUpp42 aggregates were found to affect their interaction with anti-Aß antibodies and profoundly modify the Aß-mediated glial response, which may be important aspects to consider for further development of AD therapies.

Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging.

BACKGROUND: Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease. RESULTS: Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10-/-) to study its functional role. Moreover, mice devoid of exon 10 (E10+/-) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10-/- mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/- and wild-type mice, and their muscular grip strength was less than that of E10+/- mice. The performance of E10+/- mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found. CONCLUSION: Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions.

Contrast Enhanced Magnetic Resonance Imaging of Amyloid-ß Plaques in a Murine Alzheimer's Disease Model.

BACKGROUND: Early detection of amyloid-ß (Aß) aggregates is a critical step to improve the treatment of Alzheimer's disease (AD) because neuronal damage by the Aß aggregates occurs before clinical symptoms are apparent. We have previously shown that luminescent conjugated oligothiophenes (LCOs), which are highly specific towards protein aggregates of Aß, can be used to fluorescently label amyloid plaque in living rodents. OBJECTIVE: We hypothesize that the LCO can be used to target gadolinium to the amyloid plaque and hence make the plaque detectable by T1-weighted magnetic resonance imaging (MRI). METHODS: A novel LCO-gadolinium construct was synthesized to selectively bind to Aß plaques and give contrast in conventional T1-weighted MR images after intravenous injection in Tg-APPSwe mice. RESULTS: We found that mice with high plaque-burden could be identified using the LCO-Gd constructs by conventional MRI. CONCLUSION: Our study shows that MR imaging of amyloid plaques is challenging but feasible, and hence contrast-mediated MR imaging could be a valuable tool for early AD detection.

Cryo-EM of Aß fibrils from mouse models find tg-APPArcSwe fibrils resemble those found in patients with sporadic Alzheimer's disease.

The use of transgenic mice displaying amyloid-ß (Aß) brain pathology has been essential for the preclinical assessment of new treatment strategies for Alzheimer's disease. However, the properties of Aß in such mice have not been systematically compared to Aß in the brains of patients with Alzheimer's disease. Here, we determined the structures of nine ex vivo Aß fibrils from six different mouse models by cryogenic-electron microscopy. We found novel Aß fibril structures in the APP/PS1, ARTE10 and tg-SwDI models, whereas the human type II filament fold was found in the ARTE10, tg-APPSwe and APP23 models. The tg-APPArcSwe mice showed an Aß fibril whose structure resembles the human type I filament found in patients with sporadic Alzheimer's disease. A detailed assessment of the Aß fibril structure is key to the selection of adequate mouse models for the preclinical development of novel plaque-targeting therapeutics and positron emission tomography imaging tracers in Alzheimer's disease.

Observations in APP bitransgenic mice suggest that diffuse and compact plaques form via independent processes in Alzheimer's disease.

Studies of familial Alzheimer's disease suggest that misfolding and aggregation of amyloid-ß (Aß) peptides initiate the pathogenesis. The Arctic mutation of Aß precursor protein (APP) results in AD, and Arctic Aß is more prone to form Aß protofibrils and extracellular deposits. Herein is demonstrated that the burden of diffuse Aß deposits but not compact plaques is increased when tg-Swe mice are crossed with tg-ArcSwe mice synthesizing low levels of Arctic Aß. The diffuse deposits in bitransgenic mice, which contain primarily wild-type Aß42, accumulate in regions both with and without transgene expression. However, APP processing, when compared with tg-Swe, remains unchanged in young bitransgenic mice, whereas wild-type Aß42 aggregation is accelerated and fibril architecture is altered in vitro and in vivo when a low level of Arctic Aß42 is introduced. Thus, the increased number of diffuse deposits is likely due to physical interactions between Arctic Aß and wild-type Aß42. The selective increase of a single type of parenchymal Aß deposit suggests that different pathways lead to formation of diffuse and compact plaques. These findings could have general implications for Alzheimer's disease pathogenesis and particular relevance to patients heterozygous for the Arctic APP mutation. Moreover, it further illustrates how Aß neuropathologic features can be manipulated in vivo by mechanisms similar to those originally conceptualized in prion research.

Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-ß in murine brain.

Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-ß peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-ß elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.

Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer's disease transgenic mice.

Increased astrocytic Ca2+ signaling has been shown in Alzheimer's disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signaling in such mice. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signaling in 15-month-old plaque-bearing mice. Using the genetically encoded fluorescent norepinephrine sensor GRABNE, we demonstrate a reduced norepinephrine signaling during spontaneous running and startle responses in the transgenic mice, providing a possible mechanistic underpinning of the observed reduced astrocytic Ca2+ responses. Our data points to a dysfunction in the norepinephrine-astrocyte Ca2+ activity axis, which may account for some of the cognitive deficits observed in Alzheimer's disease.

Neurodegenerative conditions such as Parkinson's or Alzheimer's disease are characterized by neurons dying and being damaged. Yet neurons are only one type of brain actors; astrocytes, for example, are star-shaped 'companion' cells that have recently emerged as being able to fine-tune neuronal communication. In particular, they can respond to norepinephrine, a signaling molecule that acts to prepare the brain and body for action. This activation results, for instance, in astrocytes releasing chemicals that can act on neurons. Certain cognitive symptoms associated with Alzheimer's disease could be due to a lack of norepinephrine. In parallel, studies in anaesthetized mice have shown perturbed astrocyte signaling in a model of the condition. Disrupted norepinephrine-triggered astrocyte signaling could therefore be implicated in the symptoms of the disease. Experiments in awake mice are needed to investigate this link, especially as anesthesia is known to disrupt the activity of astrocytes. To explore this question, Åbjørsbråten, Skaaraas et al. conducted experiments in naturally behaving mice expressing mutations found in patients with early-onset Alzheimer's disease. These mice develop hallmarks of the disorder. Compared to their healthy counterparts, these animals had reduced astrocyte signaling when running or being startled. Similarly, a fluorescent molecular marker for norepinephrine demonstrated less signaling in the modified mice compared to healthy ones. Over 55 million individuals currently live with Alzheimer's disease. The results by Åbjørsbråten, Skaaraas et al. suggest that astrocyte­norepinephrine communication may be implicated in the condition, an avenue of research that could potentially lead to developing new treatments.

The CCAAT/enhancer binding protein (C/EBP) δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-ß peptide.

BACKGROUND: The transcription factors CCAAT/enhancer binding proteins (C/EBP) α, ß and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB). In general, C/EBPα is down-regulated, whereas both C/EBPß and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-ß (Aß) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aß deposits with the aim of defining new therapeutic targets. METHODS: Here we have investigated the effects of Aß on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aß deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay. RESULTS: We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aß inhibit up-regulation of C/EBPδ expression in interleukin-1ß-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPß was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aß deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1ß-induced C/EBPδ DNA binding activity to both C/EBP and κB sites is abolished after exposure to Aß. CONCLUSIONS: These data suggest that both expression and function of C/EBPδ are dysregulated in Alzheimer's disease. C/EBPδ seems to be differently regulated in response to different conformations of Aß. We propose that Aß induces an imbalance between NF-κB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.

The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid ß fibril formation.

Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid ß (Aß). Here, we describe the Uppsala APP mutation (Δ690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) Aß42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing ß-secretase cleavage and affecting α-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated Aß, AßUpp1-42Δ19-24, accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain.

Appearance of Cxcl10-expressing cell clusters is common for traumatic brain injury and neurodegenerative disorders.

Traumatic brain injury (TBI) in the mouse results in the rapid appearance of scattered clusters of cells expressing the chemokine Cxcl10 in cortical and subcortical areas. To extend the observation of this unique pattern, we used neuropathological mouse models using quantitative reverse transcriptase-polymerase chain reaction, gene array analysis, in-situ hybridization and flow cytometry. As for TBI, cell clusters of 150-200 mum expressing Cxcl10 characterize the cerebral cortex of mice carrying a transgene encoding the Swedish mutation of amyloid precursor protein, a model of amyloid Alzheimer pathology. The same pattern was found in experimental autoimmune encephalomyelitis in mice modelling multiple sclerosis. In contrast, mice carrying a SOD1(G93A) mutant mimicking amyotrophic lateral sclerosis pathology lacked such cell clusters in the cerebral cortex, whereas clusters appeared in the brainstem and spinal cord. Mice homozygous for a null mutation of the Cxcl10 gene did not show detectable levels of Cxcl10 transcript after TBI, confirming the quantitative reverse transcriptase-polymerase chain reaction and in-situ hybridization signals. Moreover, unbiased microarray expression analysis showed that Cxcl10 was among 112 transcripts in the neocortex upregulated at least threefold in both TBI and ageing TgSwe mice, many of them involved in inflammation. The identity of the Cxcl10(+) cells remains unclear but flow cytometry showed increased numbers of activated microglia/macrophages as well as myeloid dendritic cells in the TBI and experimental autoimmune encephalomyelitis models. It is concluded that the Cxcl10(+) cells appear in the inflamed central nervous system and may represent a novel population of cells that it may be possible to target pharmacologically in a broad range of neurodegenerative conditions.

Sensitive detection of Aß protofibrils by proximity ligation--relevance for Alzheimer's disease.

BACKGROUND: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aß peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aß protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. RESULTS: For specific detection of Aß protofibrils we have used a monoclonal antibody, mAb158, selective for Aß protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aß aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aß protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aß protofibril detection by up to 25-fold. The assay was used to measure soluble Aß aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aß aggregation. CONCLUSIONS: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aß aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

Analyzing microglial-associated Aß in Alzheimer's disease transgenic mice with a novel mid-domain Aß-antibody.

The mechanisms of amyloid-ß (Aß)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aß-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aß-antibody raised against an Aß mid-domain and used it to investigate microglial Aß-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aß-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (Aß21-34), was characterized with biochemical and histological techniques. To identify the epitope in Aß recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10-10 M) and showed preference for C-terminal truncated Aß-peptides ending at amino acid 34 and Aß-mid domain peptides with high scores of ß-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aß-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aß-clearance in vivo. The ab338 antibody might be a valuable tool to study Aß-clearance by microglial uptake and Aß-mid-domain peptides generated by enzymatic degradation and alternate production.

A high cerebrospinal fluid soluble TREM2 level is associated with slow clinical progression of Alzheimer's disease.

INTRODUCTION: The progression rate of Alzheimer's disease (AD) varies and might be affected by the triggering receptor expressed on myeloid cells (TREM2) activity. We explored if cerebrospinal fluid (CSF) soluble TREM2 (sTREM2), a proxy of microglial activity, is associated with clinical progression rate. METHODS: Patients with clinical AD (N = 231) were followed for up to 3 years after diagnosis. Cognitively healthy controls (N = 42) were followed for 5 years. CSF sTREM2 was analyzed by enzyme-linked immunosorbent assay. Group-based trajectory modeling revealed distinct clinical progression groups. RESULTS: Higher CSF sTREM2 was associated with slow clinical progression. The slow- and medium-progressing groups had higher CSF sTREM2 than the cognitively healthy, who had a similar level to patients with rapid clinical progression. DISCUSSION: CSF sTREM2 levels were associated with clinical progression in AD, regardless of core biomarkers. This could be useful in assessing disease development in relation to patient care and clinical trial recruitment.

Cerebrospinal fluid sTREM2 in Alzheimer's disease: comparisons between clinical presentation and AT classification.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor expressed by microglia. Its cleaved fragments, soluble TREM2 (sTREM2), can be measured in the cerebrospinal fluid (CSF). Previous studies indicate higher CSF sTREM2 in symptomatic AD; however most of these studies have included biomarker positive AD cases and biomarker negative controls. The aim of the study was to explore potential differences in the CSF level of sTREM2 and factors associated with an increased sTREM2 level in patients diagnosed with mild cognitive impairment (MCI) or dementia due to AD compared with cognitively unimpaired controls as judged by clinical symptoms and biomarker category (AT). We included 299 memory clinic patients, 62 (20.7%) with AD-MCI and 237 (79.3%) with AD dementia, and 113 cognitively unimpaired controls. CSF measures of the core biomarkers were applied to determine AT status. CSF sTREM2 was analyzed by ELISA. Patients presented with comparable CSF sTREM2 levels as the cognitively unimpaired (9.6 ng/ml [SD 4.7] versus 8.8 ng/ml [SD 3.6], p = 0.27). We found that CSF sTREM2 associated with age-related neuroinflammation and tauopathy irrespectively of amyloid ß, APOE ε4 status or gender. The findings were similar in both symptomatic and non-symptomatic individuals.

An amyloid-beta protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease.

Human genetics link Alzheimer's disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimer's disease brain.