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Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis in vivo and require glycolysis for normal function.

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2'-deoxy-2'-18F-fluoro-9-β-d-arabinofuranosylguanine (18F-FAraG) PET imaging to noninvasively assess infiltrating T cells and to provide, in combination with MRI, a novel tool to determine lesion types. Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis to reproducibly induce 2 brain inflammatory lesion types, differentiated by their T cell content. 18F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed before disease induction, during demyelination with high levels of innate immune cells, and after T cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T cells, microglia/macrophages, myelin, and blood-brain barrier (BBB) integrity were performed to validate the in vivo findings. Results: 18F-FAraG signal was significantly increased in the brain and spinal cord at the time point of T cell infiltration. 18F-FAraG signal from white matter (corpus callosum) and gray matter (cortex, hippocampus) further correlated with T cell density. T2-weighted MRI detected white matter lesions independently of T cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T cell infiltration. Fingolimod treatment prevented motor deficits and decreased T cell and microglia/macrophage levels. In agreement, 18F-FAraG signal was decreased in the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and 18F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses.

Glutamine synthetase (GS) is a key enzyme that metabolizes glutamate into glutamine. While GS is highly enriched in astrocytes, expression in other glial lineages has been noted. Using a combination of reporter mice and cell type-specific markers, we show that GS is expressed in myelinating oligodendrocytes (OL) but not oligodendrocyte progenitor cells of the mouse and human ventral spinal cord. To investigate the role of GS in mature OL, we used a conditional knockout (cKO) approach to selectively delete GS-encoding gene (Glul) in OL, which caused a significant decrease in glutamine levels on mouse spinal cord extracts. GS cKO mice (CNP-cre+ :Glulfl/fl ) showed no differences in motor neuron numbers, size or axon density; OL differentiation and myelination in the ventral spinal cord was normal up to 6 months of age. Interestingly, GS cKO mice showed a transient and specific decrease in peak force while locomotion and motor coordination remained unaffected. Last, GS expression in OL was increased in chronic pathological conditions in both mouse and humans. We found a disease-stage dependent increase of OL expressing GS in the ventral spinal cord of SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Moreover, we showed that GLUL transcripts levels were increased in OL in leukocortical tissue from multiple sclerosis but not control patients. These findings provide evidence towards OL-encoded GS function in spinal cord sensorimotor axis, which is dysregulated in chronic neurological diseases.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13 C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-13 C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-13 C] pyruvate and HP 13 C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-13 C] pyruvate and HP 13 C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-13 C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP 13 C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP 13 C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13 C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13 C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-13 C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-13 C] pyruvate and HP 13 C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-13 C] pyruvate and HP 13 C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-13 C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP 13 C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP 13 C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13 C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

Traumatic brain injury (TBI) is of particular concern for the aging community since there is both increased incidence of TBI and decreased functional recovery in this population. In addition, TBI is the strongest environmental risk factor for development of Alzheimer's disease and other dementia-related neurodegenerative disorders. Critical changes that affect cognition take place over time following the initial insult. Our previous work identified immune system activation as a key contributor to cognitive deficits observed in aged animals. Using a focal contusion model in the current study, we demonstrate a brain lesion and cavitation formation, as well as prolonged blood⁻brain barrier breakdown. These changes were associated with a prolonged inflammatory response, characterized by increased microglial cell number and phagocytic activity 30 days post injury, corresponding to significant memory deficits. We next aimed to identify the injury-induced cellular and molecular changes that lead to chronic cognitive deficits in aged animals, and measured increases in complement initiation components C1q, C3, and CR3, which are known to regulate microglial⁻synapse interactions. Specifically, we found significant accumulation of C1q on synapses within the hippocampus, which was paralleled by synapse loss 30 days post injury. We used genetic and pharmacological approaches to determine the mechanistic role of complement initiation on cognitive loss in aging animals after TBI. Notably, both genetic and pharmacological blockade of the complement pathway prevented memory deficits in aged injured animals. Thus, therapeutically targeting early components of the complement cascade represents a significant avenue for possible clinical intervention following TBI in the aging population.

Abstract In primary central nervous system lymphoma (PCNSL), L265P mutation of the myeloid differentiation 88 (MYD88) protein is the most common, and induces a constitutive activation of IRAK4 kinase, IRAK1 phosphorylation and increased NF-kB signaling, offering a unique window for therapeutic development. We previously show that AZ1495, a potent inhibitor of IRAK4, significantly delayed progression and improved OS in L265P-mutated PCNSL models [ASH 2016], highlighting the clinical potential of IRAK inhibition. In the clinic however, early monitoring of such targeted therapies remains challenging using conventional imaging, as treatment often results in non-readily detectable anatomical changes. Here, we questioned the potential of a new neuroimaging method, hyperpolarized 13C magnetic resonance spectroscopic imaging (HP-13C MRSI), to non-invasively monitor IRAK inhibition in PCNSL for the first time. Two PCNSL patient-derived xenografts models were studied: one harboring the L265P MYD88 mutation (MYD88mut), and the other, wild type for this protein (MYD88wt). Using HP-13C MRSI, a high production of HP [1-13C] lactate was detected in both MYD88wt and MYD88mut (0.5 ± 0.04, n=2 MYD88wt; 0.61 ± 0.2, n=4 MYD88mut), indicating PCNSL presence throughout the brain. At day 2 and 4 of AZ1495 treatment, the HP [1-13C] lactate-to-pyruvate ratio remains unchanged in MYD88wt animals at all treatment time points (p=0.29 and p=0.29 at day 2 and 4 when compared to baseline, respectively) On the other hand, this ratio significantly decreased in MYD88mut animals at all time points (36.6% at day 2, p=0.07 and 56.3% at day 4, p=0.0006), in line with the previously reported effect of AZ1495 treatment on MYD88mut PCNSL only. To conlude, we showed that HP-13C MRSI can detect MYD88-mutation-specific modulations of HP lactate production following IRAK inhibition. Because HP-13C MRSI is clinically translatable and expanding rapidly, this study is of high significance for future clinical trials, to enhance monitoring of PCNSL progression and response of targeted therapies.

In vivo carbon-13 ( 13 C) MRS opens unique insights into the metabolism of intact organisms, and has led to major advancements in the understanding of cellular metabolism under normal and pathological conditions in various organs such as skeletal muscles, the heart, the liver and the brain. However, the technique comes at the expense of significant experimental difficulties. In this review we focus on the experimental aspects of non-hyperpolarized 13 C MRS in vivo . Some of the enrichment strategies which have been proposed so far are described; the various MRS acquisition paradigms to measure 13 C labeling are then presented. Finally, practical aspects of 13 C spectral quantification are discussed. [ABSTRACT FROM AUTHOR]

Background: The correction of γ-photon attenuation in PET-MRI remains a critical issue, especially for bone attenuation. This problem is of great importance for brain studies due to the density of the skull. Current techniques for skull attenuation correction (AC) provide indirect estimates of cortical bone density, leading to inaccurate estimates of brain activity. The purpose of this study was to develop an alternate method for bone attenuation correction based on NMR. The proposed approach relies on the detection of hydroxyapatite crystals by zero echo time (ZTE) MRI of P, providing individual and quantitative assessment of bone density. This work presents a proof of concept of this approach. The first step of the method is a calibration experiment to determine the conversion relationship between the P signal and the linear attenuation coefficient μ. Then P-ZTE was performed in vivo in rodent to estimate the μ-map of the skull. F-FDG PET data were acquired in the same animal and reconstructed with three different AC methods: P-based AC, AC neglecting the bone and the gold standard, CT-based AC, used to comparison for the other two methods. Results: The calibration experiment provided a conversion factor of P signal into μ. In vivo P-ZTE made it possible to acquire 3D images of the rat skull. Brain PET images showed underestimation of F activity in peripheral regions close to the skull when AC neglected the bone (as compared with CT-based AC). The use of P-derived μ-map for AC leads to increased peripheral activity, and therefore a global overestimation of brain F activity. Conclusions: In vivo P-ZTE MRI of hydroxyapatite provides μ-map of the skull, which can be used for attenuation correction of F-FDG PET images. This study is limited by several intrinsic biases associated with the size of the rat brain, which are unlikely to affect human data on a clinical PET-MRI system. [ABSTRACT FROM AUTHOR]

Most current brain metabolic models are not capable of taking into account the dynamic isotopomer information available from fine structure multiplets in C spectra, due to the difficulty of implementing such models. Here we present a new approach that allows automatic implementation of multi-compartment metabolic models capable of fitting any number of C isotopomer curves in the brain. The new automated approach also makes it possible to quickly modify and test new models to best describe the experimental data. We demonstrate the power of the new approach by testing the effect of adding separate pyruvate pools in astrocytes and neurons, and adding a vesicular neuronal glutamate pool. Including both changes reduced the global fit residual by half and pointed to dilution of label prior to entry into the astrocytic TCA cycle as the main source of glutamine dilution. The glutamate-glutamine cycle rate was particularly sensitive to changes in the model. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1–13C] pyruvate has shown potential in assessing neuroinflammation‐linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS‐induced neuroinflammatory changes using HP [1–13C] pyruvate and HP 13C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1–13C] pyruvate and HP 13C urea was performed at baseline (day 0) and at days 3 and 7 post‐intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1–13C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS‐treated mouse brain, but not in either the contralateral side or saline‐injected animals. HP 13C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS‐injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post‐LPS injection. In conclusion, we can detect LPS‐induced changes in the mouse brain using HP 13C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation. [ABSTRACT FROM AUTHOR]

Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2'-deoxy-2'- 18 F-fluoro-9-β-d-arabinofuranosylguanine ( 18 F-FAraG) PET imaging to noninvasively assess infiltrating T cells and to provide, in combination with MRI, a novel tool to determine lesion types. Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis to reproducibly induce 2 brain inflammatory lesion types, differentiated by their T cell content. 18 F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed before disease induction, during demyelination with high levels of innate immune cells, and after T cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T cells, microglia/macrophages, myelin, and blood-brain barrier (BBB) integrity were performed to validate the in vivo findings. Results: 18 F-FAraG signal was significantly increased in the brain and spinal cord at the time point of T cell infiltration. 18 F-FAraG signal from white matter (corpus callosum) and gray matter (cortex, hippocampus) further correlated with T cell density. T2-weighted MRI detected white matter lesions independently of T cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T cell infiltration. Fingolimod treatment prevented motor deficits and decreased T cell and microglia/macrophage levels. In agreement, 18 F-FAraG signal was decreased in the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and 18 F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)