Validity and value of metabolic connectivity in mouse models of ß-amyloid and tauopathy.

Among functional imaging methods, metabolic connectivity (MC) is increasingly used for investigation of regional network changes to examine the pathophysiology of neurodegenerative diseases such as Alzheimer's disease (AD) or movement disorders. Hitherto, MC was mostly used in clinical studies, but only a few studies demonstrated the usefulness of MC in the rodent brain. The goal of the current work was to analyze and validate metabolic regional network alterations in three different mouse models of neurodegenerative diseases (ß-amyloid and tau) by use of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) imaging. We compared the results of FDG-µPET MC with conventional VOI-based analysis and behavioral assessment in the Morris water maze (MWM). The impact of awake versus anesthesia conditions on MC read-outs was studied and the robustness of MC data deriving from different scanners was tested. MC proved to be an accurate and robust indicator of functional connectivity loss when sample sizes ≥12 were considered. MC readouts were robust across scanners and in awake/ anesthesia conditions. MC loss was observed throughout all brain regions in tauopathy mice, whereas ß-amyloid indicated MC loss mainly in spatial learning areas and subcortical networks. This study established a methodological basis for the utilization of MC in different ß-amyloid and tau mouse models. MC has the potential to serve as a read-out of pathological changes within neuronal networks in these models.

[18F]F-DED PET imaging of reactive astrogliosis in neurodegenerative diseases: preclinical proof of concept and first-in-human data.

OBJECTIVES: Reactive gliosis is a common pathological hallmark of CNS pathology resulting from neurodegeneration and neuroinflammation. In this study we investigate the capability of a novel monoamine oxidase B (MAO-B) PET ligand to monitor reactive astrogliosis in a transgenic mouse model of Alzheimer`s disease (AD). Furthermore, we performed a pilot study in patients with a range of neurodegenerative and neuroinflammatory conditions. METHODS: A cross-sectional cohort of 24 transgenic (PS2APP) and 25 wild-type mice (age range: 4.3-21.0 months) underwent 60 min dynamic [18F]fluorodeprenyl-D2 ([18F]F-DED), static 18 kDa translocator protein (TSPO, [18F]GE-180) and ß-amyloid ([18F]florbetaben) PET imaging. Quantification was performed via image derived input function (IDIF, cardiac input), simplified non-invasive reference tissue modelling (SRTM2, DVR) and late-phase standardized uptake value ratios (SUVr). Immunohistochemical (IHC) analyses of glial fibrillary acidic protein (GFAP) and MAO-B were performed to validate PET imaging by gold standard assessments. Patients belonging to the Alzheimer's disease continuum (AD, n = 2), Parkinson's disease (PD, n = 2), multiple system atrophy (MSA, n = 2), autoimmune encephalitis (n = 1), oligodendroglioma (n = 1) and one healthy control underwent 60 min dynamic [18F]F-DED PET and the data were analyzed using equivalent quantification strategies. RESULTS: We selected the cerebellum as a pseudo-reference region based on the immunohistochemical comparison of age-matched PS2APP and WT mice. Subsequent PET imaging revealed that PS2APP mice showed elevated hippocampal and thalamic [18F]F-DED DVR when compared to age-matched WT mice at 5 months (thalamus: + 4.3%; p = 0.048), 13 months (hippocampus: + 7.6%, p = 0.022) and 19 months (hippocampus: + 12.3%, p < 0.0001; thalamus: + 15.2%, p < 0.0001). Specific [18F]F-DED DVR increases of PS2APP mice occurred earlier when compared to signal alterations in TSPO and ß-amyloid PET and [18F]F-DED DVR correlated with quantitative immunohistochemistry (hippocampus: R = 0.720, p < 0.001; thalamus: R = 0.727, p = 0.002). Preliminary experience in patients showed [18F]F-DED VT and SUVr patterns, matching the expected topology of reactive astrogliosis in neurodegenerative (MSA) and neuroinflammatory conditions, whereas the patient with oligodendroglioma and the healthy control indicated [18F]F-DED binding following the known physiological MAO-B expression in brain. CONCLUSIONS: [18F]F-DED PET imaging is a promising approach to assess reactive astrogliosis in AD mouse models and patients with neurological diseases.

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition.

We undertook longitudinal ß-amyloid positron emission tomography (Aß-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone in Aß model mice. We thus tested the hypothesis this treatment would rescue from increases of the Aß-PET signal while promoting spatial learning and preservation of synaptic density. Here, we investigated longitudinally for 5 months PS2APP mice (N = 23; baseline age: 8 months) and App NL-G-F mice (N = 37; baseline age: 5 months) using Aß-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, Aß-PET and immunohistochemistry revealed a shift toward higher fibrillary composition of Aß-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 Aß-PET signal upon immunomodulatory treatments targeting Aß aggregation can thus be protective.

Glitter in the Darkness? Nonfibrillar ß-Amyloid Plaque Components Significantly Impact the ß-Amyloid PET Signal in Mouse Models of Alzheimer Disease.

ß-amyloid (Aß) PET is an important tool for quantification of amyloidosis in the brain of suspected Alzheimer disease (AD) patients and transgenic AD mouse models. Despite the excellent correlation of Aß PET with gold standard immunohistochemical assessments, the relative contributions of fibrillar and nonfibrillar Aß components to the in vivo Aß PET signal remain unclear. Thus, we obtained 2 murine cerebral amyloidosis models that present with distinct Aß plaque compositions and performed regression analysis between immunohistochemistry and Aß PET to determine the biochemical contributions to Aß PET signal in vivo. Methods: We investigated groups of AppNL-G-F and APPPS1 mice at 3, 6, and 12 mo of age by longitudinal 18F-florbetaben Aß PET and with immunohistochemical analysis of the fibrillar and total Aß burdens. We then applied group-level intermodality regression models using age- and genotype-matched sets of fibrillar and nonfibrillar Aß data (predictors) and Aß PET results (outcome) for both Aß mouse models. An independent group of double-hit APPPS1 mice with dysfunctional microglia due to knockout of triggering receptor expression on myeloid cells 2 (Trem2-/-) served for validation and evaluation of translational impact. Results: Neither fibrillar nor nonfibrillar Aß content alone sufficed to explain the Aß PET findings in either AD model. However, a regression model compiling fibrillar and nonfibrillar Aß together with the estimate of individual heterogeneity and age at scanning could explain a 93% of variance of the Aß PET signal (P < 0.001). Fibrillar Aß burden had a 16-fold higher contribution to the Aß PET signal than nonfibrillar Aß. However, given the relatively greater abundance of nonfibrillar Aß, we estimate that nonfibrillar Aß produced 79% ± 25% of the net in vivo Aß PET signal in AppNL-G-F mice and 25% ± 12% in APPPS1 mice. Corresponding results in separate groups of APPPS1/Trem2-/- and APPPS1/Trem2+/+ mice validated the calculated regression factors and revealed that the altered fibrillarity due to Trem2 knockout impacts the Aß PET signal. Conclusion: Taken together, the in vivo Aß PET signal derives from the composite of fibrillar and nonfibrillar Aß plaque components. Although fibrillar Aß has inherently higher PET tracer binding, the greater abundance of nonfibrillar Aß plaque in AD-model mice contributes importantly to the PET signal.

Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases.

2-Deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) is widely used to study cerebral glucose metabolism. Here, we investigated whether the FDG-PET signal is directly influenced by microglial glucose uptake in mouse models and patients with neurodegenerative diseases. Using a recently developed approach for cell sorting after FDG injection, we found that, at cellular resolution, microglia displayed higher glucose uptake than neurons and astrocytes. Alterations in microglial glucose uptake were responsible for both the FDG-PET signal decrease in Trem2-deficient mice and the FDG-PET signal increase in mouse models for amyloidosis. Thus, opposite microglial activation states determine the differential FDG uptake. Consistently, 12 patients with Alzheimer's disease and 21 patients with four-repeat tauopathies also exhibited a positive association between glucose uptake and microglial activity as determined by 18F-GE-180 18-kDa translocator protein PET (TSPO-PET) in preserved brain regions, indicating that the cerebral glucose uptake in humans is also strongly influenced by microglial activity. Our findings suggest that microglia activation states are responsible for FDG-PET signal alterations in patients with neurodegenerative diseases and mouse models for amyloidosis. Microglial activation states should therefore be considered when performing FDG-PET.

Impact of TSPO Receptor Polymorphism on [18F]GE-180 Binding in Healthy Brain and Pseudo-Reference Regions of Neurooncological and Neurodegenerative Disorders.

TSPO-PET tracers are sensitive to a single-nucleotide polymorphism (rs6971-SNP), resulting in low-, medium- and high-affinity binders (LABs, MABs and HABS), but the clinical relevance of [18F]GE-180 is still unclear. We evaluated the impact of rs6971-SNP on in vivo [18F]GE-180 binding in a healthy brain and in pseudo-reference tissue in neuro-oncological and neurodegenerative diseases. Standardized uptake values (SUVs) of [18F]GE-180-PET were assessed using a manually drawn region of interest in the frontoparietal and cerebellar hemispheres. The SUVs were compared between the LABs, MABs and HABs in control, glioma, four-repeat tauopathy (4RT) and Alzheimer's disease (AD) subjects. Second, the SUVs were compared between the patients and controls within their rs6971-subgroups. After excluding patients with prior therapy, 24 LABs (7 control, 5 glioma, 6 4RT and 6 AD) were analyzed. Age- and sex-matched MABs (n = 38) and HABs (n = 50) were selected. The LABs had lower frontoparietal and cerebellar SUVs when compared with the MABs and HABs, but no significant difference was observed between the MABs and HABs. Within each rs6971 group, no SUV difference between the patients and controls was detected in the pseudo-reference tissues. The rs6971-SNP affects [18F]GE-180 quantification, revealing lower binding in the LABs when compared to the MABs and HABs. The frontoparietal and cerebellar ROIs were successfully validated as pseudo-reference regions.

Glial activation is moderated by sex in response to amyloidosis but not to tau pathology in mouse models of neurodegenerative diseases.

BACKGROUND: In vivo assessment of neuroinflammation by 18-kDa translocator protein positron-emission-tomography (TSPO-PET) ligands receives growing interest in preclinical and clinical research of neurodegenerative disorders. Higher TSPO-PET binding as a surrogate for microglial activation in females has been reported for cognitively normal humans, but such effects have not yet been evaluated in rodent models of neurodegeneration and their controls. Thus, we aimed to investigate the impact of sex on microglial activation in amyloid and tau mouse models and wild-type controls. METHODS: TSPO-PET (18F-GE-180) data of C57Bl/6 (wild-type), AppNL-G-F (ß-amyloid model), and P301S (tau model) mice was assessed longitudinally between 2 and 12 months of age. The AppNL-G-F group also underwent longitudinal ß-amyloid-PET imaging (Aß-PET; 18F-florbetaben). PET results were confirmed and validated by immunohistochemical investigation of microglial (Iba-1, CD68), astrocytic (GFAP), and tau (AT8) markers. Findings in cerebral cortex were compared by sex using linear mixed models for PET data and analysis of variance for immunohistochemistry. RESULTS: Wild-type mice showed an increased TSPO-PET signal over time (female +23%, male +4%), with a significant sex × age interaction (T = - 4.171, p < 0.001). The Aß model AppNL-G-F mice also showed a significant sex × age interaction (T = - 2.953, p = 0.0048), where cortical TSPO-PET values increased by 31% in female AppNL-G-F mice, versus only 6% in the male mice group from 2.5 to 10 months of age. Immunohistochemistry for the microglial markers Iba-1 and CD68 confirmed the TSPO-PET findings in male and female mice aged 10 months. Aß-PET in the same AppNL-G-F mice indicated no significant sex × age interaction (T = 0.425, p = 0.673). The P301S tau model showed strong cortical increases of TSPO-PET from 2 to 8.5 months of age (female + 32%, male + 36%), without any significant sex × age interaction (T = - 0.671, p = 0.504), and no sex differences in Iba-1, CD68, or AT8 immunohistochemistry. CONCLUSION: Female mice indicate sex-dependent microglia activation in aging and in response to amyloidosis but not in response to tau pathology. This calls for consideration of sex difference in TSPO-PET studies of microglial activation in mouse models of neurodegeneration and by extension in human studies.

Asymmetry of Fibrillar Plaque Burden in Amyloid Mouse Models.

Asymmetries of amyloid-ß (Aß) burden are well known in Alzheimer disease (AD) but did not receive attention in Aß mouse models of Alzheimer disease. Therefore, we investigated Aß asymmetries in Aß mouse models examined by Aß small-animal PET and tested if such asymmetries have an association with microglial activation. Methods: We analyzed 523 cross-sectional Aß PET scans of 5 different Aß mouse models (APP/PS1, PS2APP, APP-SL70, AppNL-G-F , and APPswe) together with 136 18-kDa translocator protein (TSPO) PET scans for microglial activation. The asymmetry index (AI) was calculated between tracer uptake in both hemispheres. AIs of Aß PET were analyzed in correlation with TSPO PET AIs. Extrapolated required sample sizes were compared between analyses of single and combined hemispheres. Results: Relevant asymmetries of Aß deposition were identified in at least 30% of all investigated mice. There was a significant correlation between AIs of Aß PET and TSPO PET in 4 investigated Aß mouse models (APP/PS1: R = 0.593, P = 0.001; PS2APP: R = 0.485, P = 0.019; APP-SL70: R = 0.410, P = 0.037; AppNL-G-F : R = 0.385, P = 0.002). Asymmetry was associated with higher variance of tracer uptake in single hemispheres, leading to higher required sample sizes. Conclusion: Asymmetry of fibrillar plaque neuropathology occurs frequently in Aß mouse models and acts as a potential confounder in experimental designs. Concomitant asymmetry of microglial activation indicates a neuroinflammatory component to hemispheric predominance of fibrillary amyloidosis.

Longitudinal PET Monitoring of Amyloidosis and Microglial Activation in a Second-Generation Amyloid-ß Mouse Model.

Nonphysiologic overexpression of amyloid-ß (Aß) precursor protein in common transgenic Aß mouse models of Alzheimer disease likely hampers their translational potential. The novel AppNL-G-F mouse incorporates a mutated knock-in, potentially presenting an improved model of Alzheimer disease for Aß-targeting treatment trials. We aimed to establish serial small-animal PET of amyloidosis and neuroinflammation in AppNL-G-F mice as a tool for therapy monitoring. Methods:AppNL-G-F mice (20 homozygous and 21 heterogeneous) and 12 age-matched wild-type mice were investigated longitudinally from 2.5 to 10 mo of age with 18F-florbetaben Aß PET and 18F-GE-180 18-kDa translocator protein (TSPO) PET. Voxelwise analysis of SUV ratio images was performed using statistical parametric mapping. All mice underwent a Morris water maze test of spatial learning after their final scan. Quantification of fibrillar Aß and activated microglia by immunohistochemistry and biochemistry served for validation of the PET results. Results: The periaqueductal gray emerged as a suitable pseudo reference tissue for both tracers. Homozygous AppNL-G-F mice had a rising SUV ratio in cortex and hippocampus for Aß (+9.1%, +3.8%) and TSPO (+19.8%, +14.2%) PET from 2.5 to 10 mo of age (all P < 0.05), whereas heterozygous AppNL-G-F mice did not show significant changes with age. Significant voxelwise clusters of Aß deposition and microglial activation in homozygous mice appeared at 5 mo of age. Immunohistochemical and biochemical findings correlated strongly with the PET data. Water maze escape latency was significantly elevated in homozygous AppNL-G-F mice compared with wild-type at 10 mo of age and was associated with high TSPO binding. Conclusion: Longitudinal PET in AppNL-G-F knock-in mice enables monitoring of amyloidogenesis and neuroinflammation in homozygous mice but is insensitive to minor changes in heterozygous animals. The combination of PET with behavioral tasks in AppNL-G-F treatment trials is poised to provide important insights in preclinical drug development.