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It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals.

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.

Background: 4-Aminopyridine (4AP) is a medication for the symptomatic treatment of multiple sclerosis. Several 4AP-based PET tracers have been developed for imaging demyelination. In preclinical studies, [11C]3MeO4AP has shown promise due to its high brain permeability, high metabolic stability, high plasma availability, and high in vivo binding affinity. To prepare for the translation to human studies, we developed a cGMP-compatible automated radiosynthesis protocol and evaluated the whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP in non-human primates (NHPs). Methods: Automated radiosynthesis was carried out using a GE TRACERlab FX-C Pro synthesis module. One male and one female adult rhesus macaques were used in the study. A high-resolution CT from cranial vertex to knee was acquired. PET data were collected using a dynamic acquisition protocol with four bed positions and 13 passes over a total scan time of ~ 150 min. Based on the CT and PET images, volumes of interest (VOIs) were manually drawn for selected organs. Non-decay corrected time-activity curves (TACs) were extracted for each VOI. Radiation dosimetry and effective dose were calculated from the integrated TACs using OLINDA software. Results: Fully automated radiosynthesis of [11C]3MeO4AP was achieved with 7.3 ± 1.2% (n = 4) of non-decay corrected radiochemical yield within 38 min of synthesis and purification time. [11C]3MeO4AP distributed quickly throughout the body and into the brain. The organs with highest dose were the kidneys. The average effective dose of [11C]3MeO4AP was 4.0 ± 0.6 μSv/MBq. No significant changes in vital signs were observed during the scan. Conclusion: A cGMP-compatible automated radiosynthesis of [11C]3MeO4AP was developed. The whole-body biodistribution and radiation dosimetry of [11C]3MeO4AP was successfully evaluated in NHPs. [11C]3MeO4AP shows lower average effective dose than [18F]3F4AP and similar average effective dose as other carbon-11 tracers. [ABSTRACT FROM AUTHOR]

Purpose: [18F]3F4AP is a novel PET radiotracer that targets voltage-gated potassium (K+) channels and has shown promise for imaging demyelinated lesions in animal models of neurological diseases. This study aimed to evaluate the biodistribution, safety, and radiation dosimetry of [18F]3F4AP in healthy human volunteers. Methods: Four healthy volunteers (2 females) underwent a 4-h dynamic PET scan from the cranial vertex to mid-thigh using multiple bed positions after administration of 368 ± 17.9 MBq (9.94 ± 0.48 mCi) of [18F]3F4AP. Volumes of interest for relevant organs were manually drawn guided by the CT, and PET images and time-activity curves (TACs) were extracted. Radiation dosimetry was estimated from the integrated TACs using OLINDA software. Safety assessments included measuring vital signs immediately before and after the scan, monitoring for adverse events, and obtaining a comprehensive metabolic panel and electrocardiogram within 30 days before and after the scan. Results: [18F]3F4AP distributed throughout the body with the highest levels of activity in the kidneys, urinary bladder, stomach, liver, spleen, and brain and with low accumulation in muscle and fat. The tracer cleared quickly from circulation and from most organs. The clearance of the tracer was noticeably faster than previously reported in nonhuman primates (NHPs). The average effective dose (ED) across all subjects was 12.1 ± 2.2 μSv/MBq, which is lower than the estimated ED from the NHP studies (21.6 ± 0.6 μSv/MBq) as well as the ED of other fluorine-18 radiotracers such as [18F]FDG (~ 20 μSv/MBq). No differences in ED between males and females were observed. No substantial changes in safety assessments or adverse events were recorded. Conclusion: The biodistribution and radiation dosimetry of [18F]3F4AP in humans are reported for the first time. The average total ED across four subjects was lower than most 18F-labeled PET tracers. The tracer and study procedures were well tolerated, and no adverse events occurred. [ABSTRACT FROM AUTHOR]

Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of molecular changes involved in demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K+ channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K+ channel PET tracer [ 18 F]3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging. [ 18 F]3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics. [ 18 F]3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of [ 18 F]3F4AP for the focal brain injury was higher than [ 18 F]FDG, [ 11 C]PiB, and [ 11 C]PBR28, and compared favorably to currently used MRI methods. [ABSTRACT FROM AUTHOR]

Spinal cord injuries (SCIs) often lead to lifelong disability. Among the various types of injuries, incomplete and discomplete injuries, where some axons remain intact, offer potential for recovery. However, demyelination of these spared axons can worsen disability. Demyelination is a reversible phenomenon, and drugs such as 4-aminopyridine (4AP), which target K + channels in demyelinated axons, show that conduction can be restored. Yet, accurately assessing and monitoring demyelination after SCI remains challenging because of the lack of suitable imaging methods. In this study, we introduce a novel approach using the PET tracer, 3-[ 18 F]fluoro-4-aminopyridine ([ 18 F]3F4AP), specifically targeting K + channels in demyelinated axons for SCI imaging. Methods: Rats with incomplete contusion injuries were imaged with [ 18 F]3F4AP PET up to 1 mo after injury, followed by further validation of PET imaging results with autoradiography and immunohistochemistry of postmortem spinal cord tissue. A proof-of-concept study in 2 human subjects with incomplete injuries of different severities and etiologies was also performed. Results: [ 18 F]3F4AP PET of SCI rats revealed a more than 2-fold increase in tracer binding highly localized to the injured segment of the cord at 7 d after injury relative to baseline (SUV ratio = 2.49 ± 0.09 for 7 d after injury vs. 1.14 ± 0.10 for baseline), revealing [ 18 F]3F4AP's exceptional sensitivity to injury and its ability to detect temporal changes. Autoradiography, histology, and immunohistochemistry confirmed [ 18 F]3F4AP's targeting of demyelinated axons. In humans, [ 18 F]3F4AP differentiated between a severe and a largely recovered incomplete injury, indicating axonal loss and demyelination, respectively. Moreover, alterations in tracer delivery were evident on dynamic PET images, suggestive of differences in spinal cord blood flow between the injuries. Conclusion: [ 18 F]3F4AP demonstrates efficacy in detecting incomplete SCI in both animal models and humans. The potential for monitoring post-SCI demyelination changes and response to therapy underscores the utility of [ 18 F]3F4AP in advancing our understanding and management of SCI., (© 2025 by the Society of Nuclear Medicine and Molecular Imaging.)

Carfentanil, a highly potent synthetic opioid, paradoxically serves as a crucial positron emission tomography (PET) imaging tool in neurobiological studies of the mu-opioid receptor (MOR) system when labeled with carbon-11 ([ 11 C]CFN). However, its clinical research use is hindered by extreme potency and the limited availability of short-lived carbon-11 ( t 1/2 = 20.4 min). We present fluorine-18-labeled fluorocarfentanils ([ 18 F]FCFNs), which can be produced at higher molar activity, allowing for lower mass doses and benefiting from the longer half-life of fluorine-18 ( t 1/2 = 109.8 min), facilitating broader accessibility. Using copper-mediated radiofluorination, we synthesized a small [ 18 F]FCFN library and conducted preclinical imaging evaluations. Two candidates, o - 18 F-1 and p - 18 F-2 , showed optimal brain uptake, favorable pharmacokinetics, and high MOR-specific binding. Selectivity was confirmed through in vitro binding assays and in vivo PET scans. These [ 18 F]FCFNs are promising for accessible human brain MOR imaging.

Spinal cord injuries (SCI) often lead to lifelong disability. Among the various types of injuries, incomplete and discomplete injuries, where some axons remain intact, offer potential for recovery. However, demyelination of these spared axons can worsen disability. Demyelination is a reversible phenomenon, and drugs like 4-aminopyridine (4AP), which target K+ channels in demyelinated axons, show that conduction can be restored. Yet, accurately assessing and monitoring demyelination post-SCI remains challenging due to the lack of suitable imaging methods. In this study, we introduce a novel approach utilizing the positron emission tomography (PET) tracer, [ 18 F]3F4AP, specifically targeting K+ channels in demyelinated axons for SCI imaging. Rats with incomplete contusion injuries were imaged up to one month post-injury, revealing [ 18 F]3F4AP's exceptional sensitivity to injury and its ability to detect temporal changes. Further validation through autoradiography and immunohistochemistry confirmed [ 18 F]3F4AP's targeting of demyelinated axons. In a proof-of-concept study involving human subjects, [ 18 F]3F4AP differentiated between a severe and a largely recovered incomplete injury, indicating axonal loss and demyelination, respectively. Moreover, alterations in tracer delivery were evident on dynamic PET images, suggestive of differences in spinal cord blood flow between the injuries. In conclusion, [ 18 F]3F4AP demonstrates efficacy in detecting incomplete SCI in both animal models and humans. The potential for monitoring post-SCI demyelination changes and response to therapy underscores the utility of [ 18 F]3F4AP in advancing our understanding and management of spinal cord injuries.