SV2A PET shows hippocampal synaptic loss in cognitively unimpaired APOE ε4/ε4 homozygotes.

INTRODUCTION: We investigated hippocampal synaptic density using synaptic vesicle 2A positron emission tomography (PET), and its association with amyloid beta (Aß) and cognitive performance in healthy apolipoprotein E (APOE) ε4 carriers. METHODS: Synaptic density was assessed in 46 individuals (APOE ε4/ε4 n = 14; APOE ε3/ε4 n = 16; APOE ε3/ε3 n = 16) with [11C]UCB-J-PET standardized uptake value ratios (SUVRs), by using the centrum semiovale as a reference region. Differences in hippocampal [11C]UCB-J SUVRs were analyzed with analysis of variance (ANOVA) and linear models. Associations among [11C]UCB-J SUVR, Aß, hippocampal volume, and cognitive variables were analyzed with Spearman correlation. RESULTS: Hippocampal synaptic density was different among the APOE groups (PANOVA = 0.016): APOE ε4/ε4 carriers had lower [11C]UCB-J SUVRs compared to APOE ε3/ε3 (p = 0.013). Hippocampal synaptic density did not correlate with Consortium to Establish a Registry for Alzheimer's Disease (CERAD) total score (rho = -0.052, p = 0.74), Alzheimer's Prevention Initiative Preclinical Cognitive Composite (APCC) score (rho = 0.17, p = 0.28), or [11C]PiB uptake (rho = -0.10, p = 0.50). DISCUSSION: Hippocampal synaptic loss emerges early in the AD continuum and is measurable in vivo in cognitively unimpaired high-risk individuals. HIGHLIGHTS: Synaptic density was studied in vivo in healthy older adults using [11C]UCB-J positron emission tomography. Apolipoprotein E (APOE) ε4/ε4 carriers had lower hippocampal synaptic density compared to APOE ε3/ε3. Synaptic density was not associated with cognitive performance in this population. Hippocampal synaptic alterations occur before clinical symptoms in APOE ε4/ε4 carriers.

[11C]carfentanil PET imaging for studying the peripheral opioid system in vivo: effect of photoperiod on mu-opioid receptor availability in brown adipose tissue.

PURPOSE: Photoperiod determines the metabolic activity of brown adipose tissue (BAT) and affects the food intake and body mass of mammals. Sympathetic innervation of the BAT controls thermogenesis and facilitates physiological adaption to seasonal changes, but the exact mechanism remains elusive. Previous studies have shown that central opioid signaling regulates BAT thermogenesis, and that the expression of the brain mu-opioid receptor (MOR) varies seasonally. Therefore, it is important to know whether MOR expression in BAT shows seasonal variation. METHODS: We determined the effect of photoperiod on BAT MOR availability using [11C]carfentanil positron emission tomography (PET). Adult rats (n = 9) were repeatedly imaged under various photoperiods in order to simulate seasonal changes. RESULTS: Long photoperiod was associated with low MOR expression in BAT (ß = - 0.04, 95% confidence interval: - 0.07, - 0.01), but not in muscles. We confirmed the expression of MOR in BAT and muscle using immunofluorescence staining. CONCLUSION: Photoperiod affects MOR availability in BAT. Sympathetic innervation of BAT may influence thermogenesis via the peripheral MOR system. The present study supports the utility of [11C]carfentanil PET to study the peripheral MOR system.

Obesity risk is associated with altered cerebral glucose metabolism and decreased µ-opioid and CB1 receptor availability.

BACKGROUND: Obesity is a pressing public health concern worldwide. Novel pharmacological means are urgently needed to combat the increase of obesity and accompanying type 2 diabetes (T2D). Although fully established obesity is associated with neuromolecular alterations and insulin resistance in the brain, potential obesity-promoting mechanisms in the central nervous system have remained elusive. In this triple-tracer positron emission tomography study, we investigated whether brain insulin signaling, µ-opioid receptors (MORs) and cannabinoid CB1 receptors (CB1Rs) are associated with risk for developing obesity. METHODS: Subjects were 41 young non-obese males with variable obesity risk profiles. Obesity risk was assessed by subjects' physical exercise habits, body mass index and familial risk factors, including parental obesity and T2D. Brain glucose uptake was quantified with [18F]FDG during hyperinsulinemic euglycemic clamp, MORs were quantified with [11C]carfentanil and CB1Rs with [18F]FMPEP-d2. RESULTS: Subjects with higher obesity risk had globally increased insulin-stimulated brain glucose uptake (19 high-risk subjects versus 19 low-risk subjects), and familial obesity risk factors were associated with increased brain glucose uptake (38 subjects) but decreased availability of MORs (41 subjects) and CB1Rs (36 subjects). CONCLUSIONS: These results suggest that the hereditary mechanisms promoting obesity may be partly mediated via insulin, opioid and endocannabinoid messaging systems in the brain.

New improved radiometabolite analysis method for [18F]FTHA from human plasma: a test-retest study with postprandial and fasting state.

BACKGROUND: Fatty acid uptake can be measured using PET and 14-(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA). However, the relatively rapid rate of [18F]FTHA metabolism significantly affects kinetic modeling of tissue uptake. Thus, there is a need for accurate chromatographic methods to analyze the unmetabolized [18F]FTHA (parent fraction). Here we present a new radiometabolite analysis (RMA) method, with comparison to a previous method for parent fraction analysis, and its use in a test-retest clinical study under fasting and postprandial conditions. We developed a new thin-layer chromatography (TLC) RMA method for analysis of [18F]FTHA parent fraction and its radiometabolites from plasma, by testing stationary phases and eluent combinations. Next, we analyzed [18F]FTHA, its radiometabolites, and plasma radioactivity from subjects participating in a clinical study. A total of 17 obese or overweight participants were dosed with [18F]FTHA twice under fasting, and twice under postprandial conditions and plasma samples were obtained between 14 min (mean of first sample) and 72 min (mean of last sample) post-injection. Aliquots of 70 plasma samples were analyzed using both methods, enabling head-to-head comparisons. We performed test-retest and group comparisons of the parent fraction and plasma radioactivity. RESULTS: The new TLC method separated seven [18F]FTHA radiometabolite peaks, while the previous method separated three. The new method revealed at least one radiometabolite that was not previously separable from [18F]FTHA. From the plasma samples, the mean parent fraction value was on average 7.2 percentage points lower with the new method, compared to the previous method. Repeated [18F]FTHA investigations on the same subject revealed reproducible plasma SUV and parent fractions, with different kinetics between the fasted and postprandial conditions. CONCLUSIONS: The newly developed improved radio-TLC method for [18F]FTHA RMA enables accurate parent fraction correction, which is required to obtain quantitative data for modelling [18F]FTHA PET data. Our test-retest study of fasted and postprandial conditions showed robust reproducibility, and revealed clear differences in the [18F]FTHA metabolic rate under different study settings. TRIAL REGISTRATION: EudraCT No: 2020-005211-48, 04Feb2021; and Clinical Trials registry NCT05132335, 29Oct2021, URL: https://classic. CLINICALTRIALS: gov/ct2/show/NCT05132335 .

SGLT2 Inhibitor Dapagliflozin Increases Skeletal Muscle and Brain Fatty Acid Uptake in Individuals With Type 2 Diabetes: A Randomized Double-Blind Placebo-Controlled Positron Emission Tomography Study.

OBJECTIVE: The aim of this study was to investigate the impact of the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin on tissue fatty acid (FA) uptake in the skeletal muscle, brain, small intestine, and subcutaneous and visceral adipose tissue of individuals with type 2 diabetes by using positron emission tomography (PET). RESEARCH DESIGN AND METHODS: In a 6-week randomized double-blind placebo-controlled trial, 53 patients with type 2 diabetes treated with metformin received either 10 mg dapagliflozin or placebo daily. Tissue FA uptake was quantified at baseline and end of treatment with PET and the long-chain FA analog radiotracer 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid. Treatment effects were assessed using ANCOVA, and the results are reported as least square means and 95% CIs for the difference between groups. RESULTS: A total of 38 patients (dapagliflozin n = 21; placebo n = 17) completed the study. After 6 weeks, skeletal muscle FA uptake was increased by dapagliflozin compared with placebo (1.0 [0.07, 2.0] µmol ⋅ 100 g-1 ⋅ min-1; P = 0.032), whereas uptake was not significantly changed in the small intestine or visceral or subcutaneous adipose tissue. Dapagliflozin treatment significantly increased whole-brain FA uptake (0.10 [0.02, 0.17] µmol ⋅ 100 g-1 ⋅ min-1; P = 0.01), an effect observed in both gray and white matter regions. CONCLUSIONS: Six weeks of treatment with dapagliflozin increases skeletal muscle and brain FA uptake, partly driven by a rise in free FA availability. This finding is in accordance with previous indirect measurements showing enhanced FA metabolism in response to SGLT2 inhibition and extends the notion of a shift toward increased FA use to muscle and brain.

Glial reactivity in a mouse model of beta-amyloid deposition assessed by PET imaging of P2X7 receptor and TSPO using [11C]SMW139 and [18F]F-DPA.

BACKGROUND: P2X7 receptor has emerged as a potentially superior PET imaging marker to TSPO, the gold standard for imaging glial reactivity. [11C]SMW139 is the most recently developed radiotracer to image P2X7 receptor. The aim of this study was to image reactive glia in the APP/PS1-21 transgenic (TG) mouse model of Aß deposition longitudinally using [11C]SMW139 targeting P2X7 receptor and to compare tracer uptake to that of [18F]F-DPA targeting TSPO at the final imaging time point. TG and wild type (WT) mice underwent longitudinal in vivo PET imaging using [11C]SMW139 at 5, 8, 11, and 14 months, followed by [18F]F-DPA PET scan only at 14 months. In vivo imaging results were verified by ex vivo brain autoradiography, immunohistochemical staining, and analysis of [11C]SMW139 unmetabolized fraction in TG and WT mice. RESULTS: Longitudinal change in [11C]SMW139 standardized uptake values (SUVs) showed no statistically significant increase in the neocortex and hippocampus of TG or WT mice, which was consistent with findings from ex vivo brain autoradiography. Significantly higher [18F]F-DPA SUVs were observed in brain regions of TG compared to WT mice. Quantified P2X7-positive staining in the cortex and thalamus of TG mice showed a minor increase in receptor expression with ageing, while TSPO-positive staining in the same regions showed a more robust increase in expression in TG mice as they aged. [11C]SMW139 was rapidly metabolized in mice, with 33% of unmetabolized fraction in plasma and 29% in brain homogenates 30 min after injection. CONCLUSIONS: [11C]SMW139, which has a lower affinity for the rodent P2X7 receptor than the human version of the receptor, was unable to image the low expression of P2X7 receptor in the APP/PS1-21 mouse model. Additionally, the rapid metabolism of [11C]SMW139 in mice and the presence of several brain-penetrating radiometabolites significantly impacted the analysis of in vivo PET signal of the tracer. Finally, [18F]F-DPA targeting TSPO was more suitable for imaging reactive glia and neuroinflammatory processes in the APP/PS1-21 mouse model, based on the findings presented in this study and previous studies with this mouse model.

Quantification of the purinergic P2X7 receptor with [11C]SMW139 improves through correction for brain-penetrating radiometabolites.

The membrane-based purinergic 7 receptor (P2X7R) is expressed on activated microglia and the target of the radioligand [11C]SMW139 for in vivo assessment of neuroinflammation. This study investigated the contribution of radiolabelled metabolites which potentially affect its quantification. Ex vivo high-performance liquid chromatography with a radio detector (radioHPLC) was used to evaluate the parent and radiometabolite fractions of [11C]SMW139 in the brain and plasma of eleven mice. Twelve healthy humans underwent 90-min [11C]SMW139 brain PET with arterial blood sampling and radiometabolite analysis. The volume of distribution was estimated by using one- and two- tissue compartment (TCM) modeling with single (VT) and dual (VTp) input functions. RadioHPLC showed three major groups of radiometabolite peaks with increasing concentrations in the plasma of all mice and humans. Two radiometabolite peaks were also visible in mice brain homogenates and therefore considered for dual input modeling in humans. 2TCM with single input function provided VT estimates with a wide range (0.10-10.74) and high coefficient of variation (COV: 159.9%), whereas dual input function model showed a narrow range of VTp estimates (0.04-0.24; COV: 33.3%). In conclusion, compartment modeling with correction for brain-penetrant radiometabolites improves the in vivo quantification of [11C]SMW139 binding to P2X7R in the human brain.

Lower abdominal adipose tissue cannabinoid type 1 receptor availability in young men with overweight.

OBJECTIVE: Cannabinoid type 1 receptors (CB1R) modulate feeding behavior and energy homeostasis, and the CB1R tone is dysgulated in obesity. This study aimed to investigate CB1R availability in peripheral tissue and brain in young men with overweight versus lean men. METHODS: Healthy males with high (HR, n = 16) or low (LR, n = 20) obesity risk were studied with fluoride 18-labeled FMPEP-d2 positron emission tomography to quantify CB1R availability in abdominal adipose tissue, brown adipose tissue, muscle, and brain. Obesity risk was assessed by BMI, physical exercise habits, and familial obesity risk, including parental overweight, obesity, and type 2 diabetes. To assess insulin sensitivity, fluoro-[18 F]-deoxy-2-D-glucose positron emission tomography during hyperinsulinemic-euglycemic clamp was performed. Serum endocannabinoids were analyzed. RESULTS: CB1R availability in abdominal adipose tissue was lower in the HR than in the LR group, whereas no difference was found in other tissues. CB1R availability of abdominal adipose tissue and brain correlated positively with insulin sensitivity and negatively with unfavorable lipid profile, BMI, body adiposity, and inflammatory markers. Serum arachidonoyl glycerol concentration was associated with lower CB1R availability of the whole brain, unfavorable lipid profile, and higher serum inflammatory markers. CONCLUSIONS: The results suggest endocannabinoid dysregulation already in the preobesity state.

Novel plasma protein binding analysis method for a PET tracer and its radiometabolites: A case study with [11C]SMW139 to explain the high uptake of radiometabolites in mouse brain.

Radiometabolites of PET tracers interfere with imaging and need to be taken into account when modeling PET data. Various tracer and radiometabolite characteristics affect the uptake rate into tissue. In this study, we investigated two such factors, lipophilicity and protein-free fraction. A novel rapid method was developed using thin-layer chromatography with digital autoradiography (radioTLC) and ultrafiltration for analyzing the protein-free fractions of an exemplar PET tracer, [11C]SMW139 (fP, free parent tracer over all radioactivity), and its radiometabolites (fM, free radiometabolites over all radioactivity). Detailed understanding of the uptake of radiometabolites into extravascular cells requires analyzing fM, which has not previously been performed for PET tracers. Mice were injected with [11C]SMW139, and time-activity curves from plasma and brain coupled with the parent fraction and free fraction data were analyzed to demonstrate the true levels of protein-free and protein-bound [11C]SMW139 and its radiometabolites in plasma. The ultrafiltration method included separate membrane correction factors for the parent tracer and its radiometabolites for analysis of unbiased fP and fM. Metabolism of [11C]SMW139 was rapid, and after 45 min, the parent fraction was 0.33 in plasma and 0.28 in brain. Ultrafiltration membrane correction had a significant effect on the fP but not the fM. From 10-45 min, the fP decreased from 0.032 to 0.007, while fM remained between 0.52 and 0.35. The much higher fM in plasma could explain why the less lipophilic radiometabolites enter the brain efficiently. This detailed understanding of fP and fM from rodents can be used in translational studies to explain the behavior of the tracer in humans. Similar parent fraction and plasma protein binding methods can be used for human in vivo analysis.

ASIC-E4: Interplay of Beta-Amyloid, Synaptic Density and Neuroinflammation in Cognitively Normal Volunteers With Three Levels of Genetic Risk for Late-Onset Alzheimer's Disease - Study Protocol and Baseline Characteristics.

BACKGROUND: Detailed characterization of early pathophysiological changes in preclinical Alzheimer's disease (AD) is necessary to enable development of correctly targeted and timed disease-modifying treatments. ASIC-E4 study ("Beta-Amyloid, Synaptic loss, Inflammation and Cognition in healthy APOE ε4 carriers") combines state-of-the-art neuroimaging and fluid-based biomarker measurements to study the early interplay of three key pathological features of AD, i.e., beta-amyloid (Aß) deposition, neuroinflammation and synaptic dysfunction and loss in cognitively normal volunteers with three different levels of genetic (APOE-related) risk for late-onset AD. OBJECTIVE: Here, our objective is to describe the study design, used protocols and baseline demographics of the ASIC-E4 study. METHODS/DESIGN: ASIC-E4 is a prospective observational multimodal imaging study performed in Turku PET Centre in collaboration with University of Gothenburg. Cognitively normal 60-75-year-old-individuals with known APOE ε4/ε4 genotype were recruited via local Auria Biobank (Turku, Finland). Recruitment of the project has been completed in July 2020 and 63 individuals were enrolled to three study groups (Group 1: APOE ε4/ε4, N = 19; Group 2: APOE ε4/ε3, N = 22; Group 3: APOE ε3/ε3, N = 22). At baseline, all participants will undergo positron emission tomography imaging with tracers targeted against Aß deposition (11C-PIB), activated glia (11C-PK11195) and synaptic vesicle glycoprotein 2A (11C-UCB-J), two brain magnetic resonance imaging scans, and extensive cognitive testing. In addition, blood samples are collected for various laboratory measurements and blood biomarker analysis and cerebrospinal fluid samples are collected from a subset of participants based on additional voluntary informed consent. To evaluate the predictive value of the early neuroimaging findings, neuropsychological evaluation and blood biomarker measurements will be repeated after a 4-year follow-up period. DISCUSSION: Results of the ASIC-E4 project will bridge the gap related to limited knowledge of the synaptic and inflammatory changes and their association with each other and Aß in "at-risk" individuals. Thorough in vivo characterization of the biomarker profiles in this population will produce valuable information for diagnostic purposes and future drug development, where the field has already started to look beyond Aß.