Amyloid imaging by small-animal PET in models of Alzheimer disease (AD) offers the possibility to track amyloidogenesis and brain energy metabolism. Because microglial activation is thought to ...contribute to AD pathology, we undertook a triple-tracer small-animal PET study to assess microglial activation and glucose metabolism in association with amyloid plaque load in a transgenic AD mouse model.
Groups of PS2APP and C57BL/6 wild-type mice of various ages were examined by small-animal PET. We acquired 90-min dynamic emission data with (18)F-GE180 for imaging activated microglia (18-kD translocator protein ligand TSPO) and static 30- to 60-min recordings with (18)F-FDG for energy metabolism and (18)F-florbetaben for amyloidosis. Optimal fusion of PET data was obtained through automatic nonlinear spatial normalization, and SUVRs were calculated. For the novel TSPO tracer (18)F-GE180, we then calculated distribution volume ratios after establishing a suitable reference region. Immunohistochemical analyses with TSPO antisera, methoxy-X04 staining for fibrillary β-amyloid, and ex vivo autoradiography served as terminal gold standard assessments.
SUVR at 60-90 min after injection gave robust quantitation of (18)F-GE180, which correlated well with distribution volume ratios calculated from the entire recording and using a white matter reference region. Relative to age-matched wild-type, (18)F-GE180 SUVR was slightly elevated in PS2APP mice at 5 mo (+9%; P < 0.01) and distinctly increased at 16 mo (+25%; P < 0.001). Over this age range, there was a high positive correlation between small-animal PET findings of microglial activation with amyloid load (R = 0.85; P < 0.001) and likewise with metabolism (R = 0.61; P < 0.005). Immunohistochemical and autoradiographic findings confirmed the in vivo small-animal PET data.
In this first triple-tracer small-animal PET in a well-established AD mouse model, we found evidence for age-dependent microglial activation. This activation, correlating positively with the amyloid load, implies a relationship between amyloidosis and inflammation in the PS2APP AD mouse model.
Synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal α-synuclein in intraneuronal inclusions, named Lewy bodies. ...Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase, have been identified as the most common genetic risk factor for PD and DLB. However, despite extensive research, the mechanism by which glucocerebrosidase dysfunction increases the risk for PD or DLB still remains elusive.
In our study we expand the toolbox for PD-DLB post-mortem studies by introducing new quantitative biochemical assays for glucocerebrosidase and α-synuclein. Applying causal modelling, we determine how these parameters are interrelated and ultimately impact disease manifestation.
We developed quantitative immuno-based assays for glucocerebrosidase and α-synuclein (total and phosphorylated at Serine 129) protein levels, as well as a liquid chromatography–mass spectrometry method for the detection of the glucocerebrosidase lipid substrate glucosylsphingosine. These assays were applied on tissue samples from frontal cortex, putamen and substantia nigra of PD (n = 15) and DLB (n = 15) patients and age-matched non-demented controls (n = 15).
Our results confirm elevated p-129 over total α-synuclein levels in the insoluble fraction of PD and DLB post-mortem brain tissue and we found significantly increased α-synuclein levels in the soluble fractions in PD and DLB. Furthermore, we identified an inverse correlation between reduced glucocerebrosidase enzyme activity and protein levels with increased glucosylsphingosine levels. In the substantia nigra, a brain region particularly vulnerable in Parkinson's disease, we found a significant correlation between glucocerebrosidase protein reduction and increased p129/total α-synuclein ratios.
We assessed the direction and strength of the interrelation between all measured parameters by confirmatory path analysis. Interestingly, we found that glucocerebrosidase dysfunction impacts the PD-DLB status by increasing α-synuclein ratios in the substantia nigra, which was partly mediated by increasing glucosylsphingosine levels.
In conclusion, we show that the introduced immuno-based assays enable the quantitative assessment of glucocerebrosidase and α-synuclein parameters in post-mortem brain. In the substantia nigra, reduced glucocerebrosidase levels contribute to the increase in α-synuclein levels and to PD-DLB disease manifestation partly by increasing its glycolipid substrate glucosylsphingosine. This interrelation between glucocerebrosidase, glucosylsphingosine and α-synuclein parameters supports the hypothesis that glucocerebrosidase acts as a modulator of PD-DLB.
•Path analysis - a new tool to understand the GBA/ a-syn axis•Ser129 phosphorylated a-syn levels are increased in LBD•In the SN reduced glucocerebrosidase protein correlates with increased α-synuclein•Glucocerebrosidase contributes to LBD partly by increasing glucosylsphingosine
Causal associations between microglia activation and β-amyloid (Aβ) accumulation during the progression of Alzheimer's disease (AD) remain a matter of controversy. Therefore, we used longitudinal ...dual tracer in vivo small animal positron emission tomography (μPET) imaging to resolve the progression of the association between Aβ deposition and microglial responses during aging of an Aβ mouse model.
APP-SL70 mice (N = 17; baseline age 3.2-8.5 months) and age-matched C57Bl/6 controls (wildtype (wt)) were investigated longitudinally for 6 months using Aβ (18F-florbetaben) and 18 kDa translocator protein (TSPO) μPET (18F-GE180). Changes in cortical binding were transformed to Z-scores relative to wt mice, and microglial activation relative to amyloidosis was defined as the Z-score difference (TSPO-Aβ). Using 3D immunohistochemistry for activated microglia (Iba-1) and histology for fibrillary Aβ (methoxy-X04), we measure microglial brain fraction relative to plaque size and the distance from plaque margins.
Aβ-PET binding increased exponentially as a function of age in APP-SL70 mice, whereas TSPO binding had an inverse U-shape growth function. Longitudinal Z-score differences declined with aging, suggesting that microglial response declined relative to increasing amyloidosis in aging APP-SL70 mice. Microglial brain volume fraction was inversely related to adjacent plaque size, while the proximity to Aβ plaques increased with age.
Microglial activity decreases relative to ongoing amyloidosis with aging in APP-SL70 mice. The plaque-associated microglial brain fraction saturated and correlated negatively with increasing plaque size with aging.
We aimed to compare 18F-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer's disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). ...A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of β-amyloid with PET results, and undertook ex vivo autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95±0.04 at five months (N = 5) and 1.04±0.03 (p<0.05) at eight months (N = 7) to 1.07±0.04 (p<0.005) at ten months (N = 6), 1.28±0.06 (p<0.001) at 16 months (N = 6) and 1.39±0.09 (p<0.001) at 19 months (N = 6). SUVR was 0.95±0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to β-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from -22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p<0.001) and PVE-corrected (R = 0.95, p<0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We undertook longitudinal β-amyloid positron emission tomography (Aβ-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma ...(PPARγ) agonist pioglitazone in Aβ model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aβ-PET signal while promoting spatial learning and preservation of synaptic density. Here, we investigated longitudinally for 5 months PS2APP mice (
= 23; baseline age: 8 months) and
mice (
= 37; baseline age: 5 months) using Aβ-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. Surprisingly, Aβ-PET and immunohistochemistry revealed a shift toward higher fibrillary composition of Aβ-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. These translational data suggest that a shift toward higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the Aβ-PET signal upon immunomodulatory treatments targeting Aβ aggregation can thus be protective.
•18FUCB-H was evaluated as a preclinical biomarker of synapse density in Alzheimer’s disease (AD) models.•Decreased PET signals reflected synapse loss in transgenic mice with tau and Aβ ...pathology.•Multitracer comparison showed that 18FFDG but not 18FUCB-H PET is affected by glial metabolism.•Synaptic density may be a more reliable surrogate of neurodegeneration than glucose metabolism in AD models.
In preclinical research, the use of 18FFluorodesoxyglucose (FDG) as a biomarker for neurodegeneration may induce bias due to enhanced glucose uptake by immune cells. In this study, we sought to investigate synaptic vesicle glycoprotein 2A (SV2A) PET with 18FUCB-H as an alternative preclinical biomarker for neurodegenerative processes in two mouse models representing the pathological hallmarks of Alzheimer’s disease (AD).
A total of 29 PS2APP, 20 P301S and 12 wild-type mice aged 4.4 to 19.8 months received a dynamic 18FUCB-H SV2A-PET scan (14.7 ± 1.5 MBq) 0–60 min post injection. Quantification of tracer uptake in cortical, cerebellar and brainstem target regions was implemented by calculating relative volumes of distribution (VT) from an image-derived-input-function (IDIF). 18FUCB-H binding was compared across all target regions between transgenic and wild-type mice. Additional static scans were performed in a subset of mice to compare 18FFDG and 18FGE180 (18 kDa translocator protein tracer as a surrogate for microglial activation) standardized uptake values (SUV) with 18FUCB-H binding at different ages. Following the final scan, a subset of mouse brains was immunohistochemically stained with synaptic markers for gold standard validation of the PET results.
18FUCB-H binding in all target regions was significantly reduced in 8-months old P301S transgenic mice when compared to wild-type controls (temporal lobe: p = 0.014; cerebellum: p = 0.0018; brainstem: p = 0.0014). Significantly lower SV2A tracer uptake was also observed in 13-months (temporal lobe: p = 0.0080; cerebellum: p = 0.006) and 19-months old (temporal lobe: p = 0.0042; cerebellum: p = 0.011) PS2APP transgenic versus wild-type mice, whereas the brainstem revealed no significantly altered 18FUCB-H binding. Immunohistochemical analyses of post-mortem mouse brain tissue confirmed the SV2A PET findings. Correlational analyses of 18FUCB-H and 18FFDG using Pearson’s correlation coefficient revealed a significant negative association in the PS2APP mouse model (R = -0.26, p = 0.018). Exploratory analyses further stressed microglial activation as a potential reason for this inverse relationship, since 18FFDG and 18FGE180 quantification were positively correlated in this cohort (R = 0.36, p = 0.0076).
18FUCB-H reliably depicts progressive synaptic loss in PS2APP and P301S transgenic mice, potentially qualifying as a more reliable alternative to 18FFDG as a biomarker for assessment of neurodegeneration in preclinical research.
We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to ...a complete loss of glucocerebrosidase (GCase) protein expression.
Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and ...the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.
•Dual active and exosite designed BACE1 inhibitors with cell and in vivo activity.•X-ray structures explain the potency and selectivity of the BACE1 inhibitors.•The BACE1 peptide inhibitors become active within the brain mediated by Brain Shuttle transport across the blood brain barrier.
Alzheimer's disease (AD) is a devastating brain disorder that affects many people. This work describe how specifically designed inhibitors against the enzyme BACE1, which is known to produce toxic molecules that is claimed to be involved in causing AD. The BACE1 inhibitors are very effective and selective as they bind to two sites at the same time to this enzyme and accumulate at the cell membrane where the enzyme is located. As these inhibitors are too big to penetrate into the brain we use the Brain Shuttle technology. Potentially this approach could be used to treat people with the risk of getting AD.
In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing ...evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.
Data in this article show radioligand uptake (to gamma counter and positron-emission-tomography) as well as polymerase chain reaction analyses of 18 kDa translocator protein (TSPO) quantification. We ...confirmed specificity of 18FGE180 binding of rodent brain and myocardium by blocking experiments with prior application of non-radioactive GE180, using dynamic in vivo positron-emission-tomography and ex vivo gamma counter measurements. Expression of TSPO was compared between rodent brain and myocardium by quantitative polymerase chain reaction.
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