Synaptic vesicle protein 2A (SV2A) is a universal marker of synaptic density. Recent advances in SV2A-targeted radiotracers have opened new windows into synaptic imaging. In focal epilepsy, synaptic dysfunction is a central pathologic feature. Unlike [18F]FDG PET, which reflects neuronal metabolism only indirectly, SV2A PET allows for direct quantification of synaptic density. We therefore evaluated [18F]UCB-H SV2A PET in comparison to [18F]FDG PET in patients with pharmacoresistant, unilateral focal epilepsy, aiming to assess its complementary value to established metabolic imaging. Methods: In total, 29 patients with unilateral focal epilepsy underwent both dynamic [18F]UCB-H PET (0-60 min) and static [18F]FDG PET (30-50 min) imaging. Eight patients were treated with the SV2A-binding medications levetiracetam or brivaracetam. [18F]UCB-H PET time-activity curves were extracted from 35 frames across cortical and subcortical regions, and Pearson correlation coefficients with [18F]FDG uptake were calculated for each frame to identify the most suitable imaging windows. Voxelwise percentage differences between epileptogenic and contralateral healthy hemispheres were computed to determine lesion severity and volume. Finally, we evaluated gaussian smoothing kernels for minimizing background noise while preserving contrast during lesion detection. Results: Treatment with SV2A-binding medications reduced late-phase [18F]UCB-H binding up to 75% compared with untreated individuals, demonstrating high target specificity. Framewise correlation analysis in unaffected contralateral hemispheres revealed significant associations between [18F]UCB-H and [18F]FDG uptake within the 0-10-min and 30-60-min postinjection intervals. These time windows were therefore selected for early- and late-phase analyses, respectively. Within epileptogenic foci, SV2A PET lesion severity correlated with [18F]FDG uptake for both early-phase (r = 0.61, P = 0.0042) and late-phase (r = 0.63, P = 0.0027) imaging. However, only early-phase SV2A PET lesion volume correlated with [18F]FDG lesion volume (r = 0.70, P = 0.0004), whereas late-phase SV2A PET volume did not. In line, [18F]FDG and early-phase [18F]UCB-H PET visually showed broad hypometabolic and hypoperfused areas around the epileptogenic zone, whereas late-phase [18F]UCB-H PET yielded sharper, high-contrast delineation of synaptic abnormalities. Conclusion: Dual-phase [18F]UCB-H PET provides complementary perfusion-like and synaptic information in focal epilepsy and shows spatial correspondence with [18F]FDG PET while offering more spatially confined synaptic signal changes.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and loss-of-function coding variants are major risk factors for late onset Alzheimer’s disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling in microglia, we generated a TREM2 reporter mouse. In APP transgenic animals, bulk RNA-sequencing of isolated microglia sorted based on reporter expression highlighted TREM2 level-related changes in major immunometabolic pathways, and enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. Metabolic and lipidomic profiling of sorted microglia showed that, independent of Aβ pathology, TREM2 expression correlated with signatures consistent with increased cellular redox, energetics, and cholesterol homoeostasis. In accordance, metabolic activity correlated with phagocytic capacity. Finally, we performed chronic treatment with a TREM2 agonist antibody and identified a window of TREM2 expression where microglia are most responsive, thereby informing clinical applications of TREM2 agonists. TREM2 is an important AD risk factor playing essential roles in the microglial response to amyloid pathology. Here, authors show using a TREM2 reporter mouse that TREM2 levels are critical for efficacy of TREM2 agonism informing current clinical efforts.

Strong expression of the 18 kDa translocator protein (TSPO) is observed in glioma and tumor-associated myeloid cells within the tumor microenvironment. However, TSPO expression also extends beyond the tumor and may reflect systemic immune regulation. We therefore assessed inter-organ associations of TSPO expression in mice at different stages of glioblastoma using whole-body TSPO-PET imaging. Whole-body TSPO-PET images were acquired using [18F]GE-180 in sham-inoculated mice (n = 18) and glioblastoma-bearing mice at early (5–13 days, n = 20) and late (18–19 days, n = 29) stages. The tumor and organs (brain, heart, lungs, skull, and various bones) were segmented to compare TSPO-PET signals between mouse cohorts. Pearson correlation served to analyze cohort-specific organ-organ interaction, and deviations were quantified using correlation abnormality scores. [18F]DPA-714 TSPO-PET scans (n = 53) of a validation cohort served for longitudinal assessment of TSPO organ-organ interaction, which was analyzed relative to tumor stage and survival. Late-stage glioblastoma mice showed increased TSPO expression in the tumor region, but no significant TSPO alterations in other organs. In contrast, organ-organ interactions, including lungs and non-tumor brain regions, were disrupted in early- and late-stage glioblastoma mice compared to sham mice. Late-stage glioblastoma mice had high correlation abnormality scores across several organ systems, as was also observed in data obtained with the second TSPO tracer. Longitudinal TSPO organ-organ interactions, but not TSPO-PET signals in single organs, were associated with tumor stage and survival. Glioblastoma induces stage-dependent systemic TSPO alterations and organ–organ interaction changes, suggesting that whole-body TSPO-PET network analysis may track tumor-associated immune dynamics.

The role of microglia has emerged as a critical driver of disease progression in multiple sclerosis (MS), but we lack broadly applicable monitoring tools. Here, we investigated whether hyperreflective foci (HRF), as detected by optical coherence tomography (OCT) within the inner nuclear layer (INL) of the retina, can be used as a marker for microglial pathology. We demonstrate that HRF counts are increased in persons with relapsing and progressive MS and correlate with global white and gray matter, as well as deep gray matter [18F]GE-180 uptake. [Color figure can be viewed at www.annalsofneurology.org] ANN NEUROL 2026;99:1480-1485.

The triggering receptor expressed on myeloid cells 2 (TREM2) plays a pivotal role in the activation of myeloid cells and is currently being investigated as a potential therapeutic target in several diseases. In this study, we established enhanced quantification of PET images of a 64Cu-labeled antibody-based PET radiotracer as a noninvasive tool for the assessment of TREM2 expression in the brain and peripheral organs of mice. We used TREM2 knockout mice that lack target expression to investigate data-driven blood normalization of PET images against percentage of injected dose normalization. Methods: TREM2 knockout and wild-type mice (n = 11 each) were injected with the radiotracer [64Cu]Cu-NODAGA-ATV:4D9 (ATV is antibody transport vehicle). Twenty hours after injection, TREM2 PET was conducted and blood samples were collected. A voxelwise analysis with statistical parametric mapping served to determine voxels that correlate with ex vivo blood radioactivity levels. Furthermore, TREM2 PET signals were compared between mice with and those without TREM2 expression using image-derived blood normalization. Correlation with TREM2 protein expression levels in the lung, liver, spleen, and bone marrow was used to validate organ-specific PET results. Disease models of brain amyloidosis and myocardial infarction were investigated to test for the value of image-derived normalization in mice. Results: Blood radioactivity levels derived from a statistical parametric mapping-derived region of interest demonstrated a robust correlation with radioactivity measurements obtained from ex vivo blood samples. Voxelwise clusters of TREM2 PET signals were more robustly detected after blood normalization of the PET images. Significant voxelwise clusters of TREM2 PET signals in peripheral organs correlated with TREM2 protein expression levels. Furthermore, image-derived normalization enhanced the significance of voxelwise clusters of TREM2 in the brains of App SAA;TfRmu/hu mice, as well as the TREM2 signal in the myocardial infarct region. Both strongly correlated with ex vivo autoradiography. Conclusion: Normalization of PET images to account for blood levels enhanced the detection of TREM2. This improved methodology for TREM2 PET analysis provides a promising basis for future assessments of TREM2 imaging.

Rationale: Radiopharmaceutical therapy (RPT) has become an effective treatment option for neuroendocrine tumors (NETs) and castration-resistant prostate cancer and is in clinical development for many indications. One of the major advantages of theranostic RPT is that the distribution of radiopharmaceuticals in the human body can be imaged, and radiation doses to the patient's organs can be calculated. However, accurate dosimetry may be fundamentally limited by microscopic heterogeneity of radiopharmaceutical distribution. Methods: We developed fluorescent analogs of somatostatin-receptor-subtype 2 (SSTR2) targeting Lutetium-177 labeled radiopharmaceuticals that are clinically used in patients with NETs and studied their uptake by hematopoietic stem and progenitor cells (HSPC) using flow cytometry and microscopy. Results: Hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) showed high and specific SSTR2-ligand uptake, which was at similar levels as NET cells. Furthermore, they displayed a several-fold higher uptake of SSTR2-antagonists than of SSTR2-agonists. HSPC treatment with a 177Lu-labeled antagonist and agonist showed a stronger reduction of HSC proliferation by the antagonist. Due to the scarcity of HSCs and MPPs, their contribution to total bone marrow uptake of SSTR2-radiopharmaceuticals is negligible in imaging-based dosimetry. This likely explains why SSTR2-antagonists caused pancytopenia in clinical trials despite safe dosimetry estimates. Conclusion: Target expression heterogeneity can lead to underestimation of radiopharmaceutical toxicity and should be considered when designing clinical trials for new radiopharmaceuticals. The implications of our findings go beyond SSTR2-targeted radiopharmaceuticals and suggest more generally that first-in-human studies should not only be guided by radiation dosimetry but should also include careful escalation of the administered therapeutic activity. Our multimodal ligand design is modular and can be applied to other peptide or protein-based radiopharmaceuticals to study cellular distribution and potential bone marrow uptake prior to clinical testing.

Gray matter pathology, including the formation of cortical lesions, predicts progression in people with multiple sclerosis (PwMS). Here, we investigated whether positron emission tomography (PET) imaging using the synaptic vesicle protein 2A (SV2A)-targeting radioligand [18F]UCB-H could help to detect and monitor synapse loss, an early feature of gray matter pathology in MS. First, we confirmed that SV2A is a suitable marker of synapse density in MS by analyzing SV2A mRNA and protein expression in cortical gray matter. We then used a mouse model of cortical MS pathology to demonstrate that SV2A-PET imaging can detect synapse loss in cortical lesions and that synapse densities measured by PET imaging correspond to the densities of genetically and immunohistochemically labeled synapses in the same lesions. Last, we performed SV2A-PET imaging in a total of 31 PwMS at different stages of the disease process, showing that PET imaging can detect synapse loss in cortical MS lesions in vivo. Moreover, we found that interhemispheric asymmetries in SV2A-PET tracer uptake can be leveraged to uncover further cortical alterations, the volume of which was more than 20-fold larger than the cortical lesion area detected by MRI. The extent of these PET-defined areas of cortical synapse pathology was larger in the progressive stage of the disease and correlated with the disability and cognitive performance of the same individuals. SV2A-PET imaging thus unmasked clinically relevant cortical pathology in MS thereby providing a promising tool to detect and monitor disease progression.

Ziel/Aim: [18F]D2-Deprenyl bindet an das Enzym MAO-B und ermöglicht so Rückschlüsse auf die Aktivität von Astrozyten, als Index der Neuroinflammation im ZNS. Bislang wurde [18F]D2-Deprenyl-PET vor allem zur Untersuchung von neurodegenerativen Erkrankungen wie Alzheimer und ALS eingesetzt. Glioblastome sind die häufigsten primären Hirntumore. Da sie von entarteten Astrozyten ausgehen könnte eine Bildgebung durch [18F]D2-Deprenyl-PET auch hier von Nutzen sein, jedoch auch zur Erfassung der Neuroinflammation dienen.

IntroductionMicroglial energy metabolism has gained attention for the treatment of neurodegenerative diseases. In vitro methods provide important insights; however, it remains unclear whether the metabolism of highly motile microglia is preserved outside their regular environment. Therefore, we directly compared the microglial glucose uptake in vivo and in vitro in mice.MethodsMicroglia and astrocytes were isolated from the brain using immunomagnetic cell sorting following [18F]FDG injection in living mice, followed by gamma and single-cell radiotracing (scRadiotracing). Enriched cell fractions were incubated with excess [18F]FDG (50,000-fold) in vivo, washed, and measured equivalently. For all fractions, radioactivity per cell was normalized to the injected or incubated radioactivity, and ratios of microglialuptake were calculated relative to astrocytes and the microglia/astrocyte-negative fraction. The experiment was repeated using a glucose-free buffer and validated by in vitro incubation without prior in vivo [18F]FDG injection to exclude the influence of fasting and glucose injection.ResultsscRadiotracing results were compared against cell culture [18F]-FDG incubation. The in vivo glucose uptake of microglia was higher when compared to astrocytes (50.4-fold, p < 0.0001) and non-microglia/ non-astrocyte cells (10.6-fold, p < 0.0001). Microglia still exhibited the highest glucose uptake in vitro, but with a distinct reduction in microglia-to-astrocyte (5.7-fold, p < 0.0015) and microglia-to-microglia/astrocyte-negative ratios (1.7 fold, p < 0.0001). Fasting and in vitro incubation were used to validate the results. Cell culture indicated low microglial uptake compared to that in neurons (1:100) or astrocytes (1:10).DiscussionCompared to astrocytes and other cells, microglia show a distinct reduction in uptake in vitro compared to in vivo uptake. Our results emphasize that in vitro experiments should be interpreted with caution when studying microglial energy metabolism.

Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues comparable to standard samples. We demonstrate its versatility by investigating tumor heterogeneity in a syngeneic glioblastoma mouse model, using 3D imaging to identify spatially distinct microenvironments and characterize their unique transcriptomic signatures. Moreover, DISCO-seq enabled unbiased, whole-body mapping of SARS-CoV-2 S1 protein deposition in mice, followed by transcriptomic profiling of spatially defined niches. By bridging mesoscale 3D imaging with single-cell transcriptomics, DISCO-seq establishes a paradigm for anatomically contextualized, hypothesis-free tissue interrogation. ### Competing Interest Statement A.E. is co-founder of Deep Piction, GmbH. M.B. is a member of the Neuroimaging Committee of the EANM. M.B. has received speaker honoraria from Roche, GE Healthcare, Iba, and Life Molecular Imaging; has advised Life Molecular Imaging, AC Immune, MIAC, and GE healthcare. ERC Consolidator Grant, GA 865323 Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), EXC 2145 SyNergy, ID 390857198 Nomis Foundation

Ziel/Aim Microglial energy demand and metabolism have gained high attention in neurodegenerative diseases. To this end, in vitro methods such as the seahorse assay are used, but it is unclear if energy metabolism of microglia highly motile in vivo is biased, when acting in vitro. Thus, we performed a direct comparison of in vivo and in vitro glucose uptake by scRadiotracing.

Elevated TSPO-PET signals in the non-lesional hemisphere of patients with glioblastoma. A, Patient selection process. Patients receiving TSPO-PET imaging at initial diagnosis of glioma were allocated. Patients with unilateral manifestation of glioblastoma (WHO IV; n = 41) or isocitrate dehydrogenase mutant astrocytoma WHO grade 2 (IDHmut astrocytoma WHO 2; n = 7) at the time of PET were selected for further analysis. B, Patients with newly diagnosed glioblastoma but not patients with IDHmut astrocytoma WHO 2 indicate higher TSPO-PET signal in the contralateral hemisphere compared to healthy controls. Images represent examples of TSPO-PET images of patients with newly diagnosed glioblastoma (left) and IDHmut astrocytoma WHO 2 (middle) in comparison to a healthy control (right). C, Surface projections and axial slices of the group average contralateral TSPO-PET signal of patients with glioblastoma, IDHmut astrocytoma WHO 2, and healthy controls. The non-lesional hemispheres were parcellated into 123 sub-regions using the Brainnetome Atlas. Axial slices show TSPO-PET signals of basal ganglia regions (masked cortical regions). D, Significant TSPO-PET signal elevation in brain regions of the contralateral hemisphere of patients with glioblastoma but not with IDHmut astrocytoma WHO 2 compared to healthy controls. E, Pronounced contralateral TSPO expression in orbito-frontal, superior-temporal, mid-temporal, and mesio-temporal regions of patients with glioblastoma. *, P < 0.01.

Contralateral TSPO-PET signal elevation is associated with persisting epileptic seizures and worse overall survival. A, Contralateral TSPO-PET shows no quantitative difference between patients presenting with (n = 20) and without (n = 20) epileptic seizures at inital diagnosis (top). Significant increase of contralateral TSPO expression in patients experiencing persisting epileptic seizures after treatment of the primary tumor site (n = 8) compared to patients with discontinued seizures (n = 12; bottom). B, Heatmap of contralateral TSPO-PET signal in anatomically and functionally predefined brain regions (n = 15) of all patients experiencing epileptic seizures at initial diagnosis highlights two patients (age: in their 70s) with strongest contralateral TSPO expression. Coronal slices of TSPO-PET of these two patients are illustrated in comparison to group average images of patients with discontinued and persisting epileptic seizures after tumor therapy. White arrows point toward the tumor. C, Patients with persisting epileptic seizures indicate significant signal elevation in several contralateral brain regions with predominance in motor cortex, mesial temporal lobe, and occipital lobe compared to patients with discontinued epileptic seizures. *, P < 0.01. D, Matrix of regional TSPO-PET inter-correlation coefficients in comparison of patients with discontinued and persisting seizures as well as controls. Single boxes indicate interregional Pearson’s R. E, Significant increase of the contralateral TSPO-PET signal in patients with short overall survival (≤10.7 months; median split). F, High contralateral TSPO-PET signal (>0.88 SUVr; median split) at initial diagnosis is associated with worse overall survival in patients with glioblastoma. Multivariate Cox regression was adjusted for age, glucocorticoid medication, subsequent radiotherapy, and TSPO-PET signal of the tumor. **, P < 0.01. G and H, Distinct predictive value of contralateral TSPO-PET signal in different sub-regions on overall survival. *, P < 0.05.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1 gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration. Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1 in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we report severe alterations in microglial lipidomic profiles, including the enrichment of bis(monoacylglycero)phosphate, increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hyperactivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a pathological cascade resembling NPC-like phenotypes, including a shortened life span, motor impairments, astrogliosis, neuroaxonal pathology, and increased neurofilament light chain (NF-L), a neuronal injury biomarker. As observed in the mouse model, patients with NPC showed increased NF-L in the blood and microglial hyperactivity, as visualized by TSPO-PET imaging. Reduced TSPO expression in blood-derived macrophages of patients with NPC was measured after N -acetyl- l -leucine treatment, which has been recently shown to have beneficial effects in patients with NPC, suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further investigated for therapeutic targeting and disease monitoring.

BackgroundMicroglial activation is one hallmark of Alzheimer disease (AD) neuropathology but the impact of the regional interplay of microglia cells in the brain is poorly understood. We hypothesized that microglial activation is regionally synchronized in the healthy brain but experiences regional desynchronization with ongoing neurodegenerative disease. We addressed the existence of a microglia connectome and investigated microglial desynchronization as an AD biomarker.MethodsTo validate the concept, we performed microglia depletion in mice to test whether interregional correlation coefficients (ICCs) of 18 kDa translocator protein (TSPO)-PET change when microglia are cleared. Next, we evaluated the influence of dysfunctional microglia and AD pathophysiology on TSPO-PET ICCs in the mouse brain, followed by translation to a human AD-continuum dataset. We correlated a personalized microglia desynchronization index with cognitive performance. Finally, we performed single-cell radiotracing (scRadiotracing) in mice to ensure the microglial source of the measured desynchronization.ResultsMicroglia-depleted mice showed a strong ICC reduction in all brain compartments, indicating microglia-specific desynchronization. AD mouse models demonstrated significant reductions of microglial synchronicity, associated with increasing variability of cellular radiotracer uptake in pathologically altered brain regions. Humans within the AD-continuum indicated a stage-depended reduction of microglia synchronicity associated with cognitive decline. scRadiotracing in mice showed that the increased TSPO signal was attributed to microglia.ConclusionUsing TSPO-PET imaging of mice with depleted microglia and scRadiotracing in an amyloid model, we provide first evidence that a microglia connectome can be assessed in the mouse brain. Microglia synchronicity is closely associated with cognitive decline in AD and could serve as an independent personalized biomarker for disease progression.

Background: The translocator protein (TSPO) has been proven to have great potential as a target for the positron emission tomography (PET) imaging of glioblastoma. However, there is an ongoing debate about the potential various sources of the TSPO PET signal. This work investigates the impact of the inoculation-driven immune response on the PET signal in experimental orthotopic glioblastoma. Methods: Serial [18F]GE-180 and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) PET scans were performed at day 7/8 and day 14/15 after the inoculation of GL261 mouse glioblastoma cells (n = 24) or saline (sham, n = 6) into the right striatum of immunocompetent C57BL/6 mice. An additional n = 25 sham mice underwent [18F]GE-180 PET and/or autoradiography (ARG) at days 7, 14, 21, 28, 35, 50 and 90 in order to monitor potential reactive processes that were solely related to the inoculation procedure. In vivo imaging results were directly compared to tissue-based analyses including ARG and immunohistochemistry. Results: We found that the inoculation process represents an immunogenic event, which significantly contributes to TSPO radioligand uptake. [18F]GE-180 uptake in GL261-bearing mice surpassed [18F]FET uptake both in the extent and the intensity, e.g., mean target-to-background ratio (TBRmean) in PET at day 7/8: 1.22 for [18F]GE-180 vs. 1.04 for [18F]FET, p < 0.001. Sham mice showed increased [18F]GE-180 uptake at the inoculation channel, which, however, continuously decreased over time (e.g., TBRmean in PET: 1.20 at day 7 vs. 1.09 at day 35, p = 0.04). At the inoculation channel, the percentage of TSPO/IBA1 co-staining decreased, whereas TSPO/GFAP (glial fibrillary acidic protein) co-staining increased over time (p < 0.001). Conclusion: We identify the inoculation-driven immune response to be a relevant contributor to the PET signal and add a new aspect to consider for planning PET imaging studies in orthotopic glioblastoma models.