NMR-based comparative metabolomics of quiescent muscle cells.

Adult muscle tissue largely comprised of differentiated myofibers also harbors quiescent muscle-resident stem cells (MuSCs) that are responsible for its maintenance, repair and regeneration. Emerging evidence suggests that quiescent MuSCs exhibit a specific metabolic state, which is regulated during physiological and pathological alterations. However, a detailed understanding of the metabolic state of quiescent MuSCs and its alteration during activation and repair is lacking. Direct profiling of MuSCs in vivo is challenging because the cells are rare and dispersed, while isolation and enrichment leads to their activation and loss of quiescence. In this study, we employed 1H-nuclear magnetic resonance (NMR) spectroscopy to profile metabolites in an established culture model of quiescent MuSC-derived myoblasts and compared with activated, proliferative and differentiated muscle cells to determine the state-specific metabolome. We report that the proliferating and differentiated cells are highly enriched in metabolites involved in energy generation, the quiescent state is enriched in metabolites related to phospholipid catabolism (glycerophosphocholine and choline) and depleted for phosphocholine which is enriched in proliferating cells. We propose that the ratio of these metabolites may be useful as a biomarker of MuSC quiescence.

Chronic lead exposure disrupts neurometabolic activity in mouse brain: An ex vivo1H-[13C]-NMR study.

Lead poisoning has been identified as a problem in adults as well as in children. Chronic exposure to lead has been implicated in neurological disorders such as amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. In the present study, we evaluated the impact of chronic lead exposure on cerebral glutamatergic and GABAergic metabolic activity in mice. C57BL6 mice were provided lead acetate in drinking water for two months. The regional cerebral metabolic activity was measured using 1H-[13C]-NMR spectroscopy in conjunction with infusion of [1,6-13C2]glucose. The blood Pb2+ increased significantly in lead acetate treated mice. Concomitantly, there was a significant reduction in the forelimb strength. The level of myo-inositol was elevated in the cerebral cortex of mice chronically exposed to lead. The glutamatergic neurometabolic activity was found to be reduced following chronic lead exposure in the cerebral cortex, hippocampus, and striatum. In contrast, the GABAergic fluxes were impaired in the hippocampus and thalamus only. The metabolic fluxes in the cerebellum were unperturbed to Pb2+ toxicity. In conclusion, we report that chronic lead exposure in mice leads to an impairment in forelimb strength, and a perturbation in neurometabolism in brain regions involving cognition and movement.

Riluzole restores memory and brain energy metabolism in AßPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to memory loss and impaired cognition. Despite several decades of research, AD therapeutic is not available. In this study, we have investigated the impact of a chronic intervention of riluzole on memory and neurometabolism in the AßPP-PS1 mouse model of AD. The 10-month-old AßPP-PS1 mice were administered 30 doses of riluzole (6 mg/kg, intragastrically) on an alternate day for two months. The memory was assessed using Morris Water Maze, while neurometabolism was evaluated by 1H-[13C]-NMR spectroscopy together with an intravenous infusion of [1,6-13C2]glucose. The normal saline-treated AßPP-PS1 mice exhibited a decrease in learning and memory that were restored to the control level following riluzole treatment. Most interestingly, the reduced 13C labeling of GluC4 and AspC3 from [1,6-13C]glucose in the AßPP-PS1 mice was restored to the control level following riluzole intervention. As a consequence, chronic riluzole treatment improved metabolic activity of glutamatergic neurons in AßPP-PS1 mice. Together these data suggest that riluzole may be useful for improving cognition in AD.

A comparative study of antibody response, virus neutralization efficiency & metabolites in SARS-CoV-2-infected adults & children.

Background & objectives: COVID-19 has been a global pandemic since early 2020. It has diverse clinical manifestations, but consistent immunological and metabolic correlates of disease severity and protection are not clear. This study was undertaken to compare seropositivity rate, antibody levels against nucleocapsid and spike proteins, virus neutralization and metabolites between adult and child COVID-19 patients. Methods: Plasma samples from naïve control (n=14) and reverse transcription (RT)-PCR positive COVID-19 participants (n=132) were tested for reactivity with nucleocapsid and spike proteins by ELISA, neutralization of SARS-CoV-2 infectivity in Vero cells and metabolites by [1]H nuclear magnetic resonance (NMR) spectroscopy. Results: An ELISA platform was developed using nucleocapsid and spike proteins for COVID-19 serosurvey. The participants showed greater seropositivity for nucleocapsid (72%) than spike (55.3%), and males showed higher seropositivity than females for both the proteins. Antibody levels to both the proteins were higher in intensive care unit (ICU) than ward patients. Children showed lower seropositivity and antibody levels than adults. In contrast to ICU adults (81.3%), ICU children (33.3%) showed lower seropositivity for spike. Notably, the neutralization efficiency correlated with levels of anti-nucleocapsid antibodies. The levels of plasma metabolites were perturbed differentially in COVID-19 patients as compared with the naive controls. Interpretation & conclusions: Our results reflect the complexity of human immune response and metabolome to SARS-CoV-2 infection. While innate and cellular immune responses are likely to be a major determinant of disease severity and protection, antibodies to multiple viral proteins likely affect COVID-19 pathogenesis. In children, not adults, lower seropositivity rate for spike was associated with disease severity.

Brain energy metabolism in intracerebroventricularly administered streptozotocin mouse model of Alzheimer's disease: A 1H-[13C]-NMR study.

Alzheimer's disease (AD) is a very common neurodegenerative disorder. Although a majority of the AD cases are sporadic, most of the studies are conducted using transgenic models. Intracerebroventricular (ICV) administered streptozotocin (STZ) animals have been used to explore mechanisms in sporadic AD. In this study, we have investigated memory and neurometabolism of ICV-STZ-administered C57BL6/J mice. The neuronal and astroglial metabolic activity was measured in 1H-[13C]-NMR spectrum of cortical and hippocampal tissue extracts of mice infused with [1,6-13C2]glucose and [2-13C]acetate, respectively. STZ-administered mice exhibited reduced (p = 0.00002) recognition index for memory. The levels of creatine, GABA, glutamate and NAA were reduced (p ≤ 0.04), while that of myo-inositol was increased (p < 0.05) in STZ-treated mice. There was a significant (p ≤ 0.014) reduction in aspartate-C3, glutamate-C4/C3, GABA-C2 and glutamine-C4 labeling from [1,6-13C2]glucose. This resulted in decreased rate of glucose oxidation in the cerebral cortex (0.64 ± 0.05 vs. 0.77 ± 0.05 µmol/g/min, p = 0.0008) and hippocampus (0.60 ± 0.04 vs. 0.73 ± 0.07 µmol/g/min, p = 0.001) of STZ-treated mice, due to similar reductions of glucose oxidation in glutamatergic and GABAergic neurons. Additionally, reduced glutamine-C4 labeling points towards compromised synaptic neurotransmission in STZ-treated mice. These data suggest that the ICV-STZ model exhibits neurometabolic deficits typically observed in AD, and its utility in understanding the mechanism of sporadic AD.

Chronic postnatal chemogenetic activation of forebrain excitatory neurons evokes persistent changes in mood behavior.

Early adversity is a risk factor for the development of adult psychopathology. Common across multiple rodent models of early adversity is increased signaling via forebrain Gq-coupled neurotransmitter receptors. We addressed whether enhanced Gq-mediated signaling in forebrain excitatory neurons during postnatal life can evoke persistent mood-related behavioral changes. Excitatory hM3Dq DREADD-mediated chemogenetic activation of forebrain excitatory neurons during postnatal life (P2-14), but not in juvenile or adult windows, increased anxiety-, despair-, and schizophrenia-like behavior in adulthood. This was accompanied by an enhanced metabolic rate of cortical and hippocampal glutamatergic and GABAergic neurons. Furthermore, we observed reduced activity and plasticity-associated marker expression, and perturbed excitatory/inhibitory currents in the hippocampus. These results indicate that Gq-signaling-mediated activation of forebrain excitatory neurons during the critical postnatal window is sufficient to program altered mood-related behavior, as well as functional changes in forebrain glutamate and GABA systems, recapitulating aspects of the consequences of early adversity.

Stress and adversity in early childhood can have long-lasting effects, predisposing people to mental illness and mood disorders in adult life. The weeks immediately before and after birth are critical for establishing key networks of neurons in the brain. Therefore, any disruption to these neural circuits during this time can be detrimental to emotional development. However, it is still unclear which cellular mechanisms cause these lasting changes in behavior. Studies in animals suggest that these long-term effects could result from abnormalities in a few signaling pathways in the brain. For example, it has been proposed that overstimulating the cells that activate circuits in the forebrain ­ also known as excitatory neurons ­ may contribute to the behavioral changes that persist into adulthood. To test this theory, Pati et al. used genetic engineering to modulate a signaling pathway in male mice, which is known to stimulate excitatory neurons in the forebrain. The experiments showed that prolonged activation of excitatory neurons in the first two weeks after birth resulted in anxious and despair-like behaviors as the animals aged. The mice also displayed discrepancies in how they responded to certain external sensory information, which is a hallmark of schizophrenia-like behavior. However, engineering the same changes in adolescent and adult mice had no effect on their mood-related behaviors. This animal study reinforces just how critical the first few weeks of life are for optimal brain development. It provides an insight into a possible mechanism of how disruption during this time could alter emotional behavior. The findings are also relevant to psychiatrists interested in the underlying causes of mental illness after early childhood adversity.

Subanesthetic ketamine reverses neuronal and astroglial metabolic activity deficits in a social defeat model of depression.

Depression is one of the most debilitating neuropsychiatric disorders. Most of the current antidepressants have long remission time and low recovery rate. This study explores the impact of ketamine on neuronal and astroglial metabolic activity in prefrontal cortex in a social defeat (SD) model of depression. C57BL/6 mice were subjected to a social defeat paradigm for 5 min a day for 10 consecutive days. Ketamine (10 mg/kg, intraperitoneal) was administered to mice for two consecutive days following the last defeat stress. Mice were infused with [1,6-13 C2 ]glucose or [2-13 C]acetate to assess neuronal and astroglial metabolic activity, respectively, together with proton-observed carbon-edited nuclear magnetic resonance spectroscopy in prefrontal cortex tissue extract. The 13 C labeling of amino acids from glucose and acetate was decreased in SD mice. Ketamine treatment in SD mice restored sucrose preference, social interaction and immobility time to control values. Acute subanesthetic ketamine restored the 13 C labeling of brain amino acids from glucose as well as acetate in SD mice to the respective control values, suggesting that rates of neuronal and astroglial tricarboxylic acid (TCA) cycle and neurotransmitter cycling were re-established to normal levels. The finding of improved energy metabolism in SD mice suggests that fast anti-depressant action of ketamine is linked with improved neurotransmitter cycling.

Increased astroglial activity and reduced neuronal function across brain in AßPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with progressive loss of cognitive function, personality, and behavior. The present study evaluates neuronal and astroglial metabolic activity, and neurotransmitter cycle fluxes in AßPP-PS1 mouse model of AD by using 1H-[13C]-nuclear magnetic resonance (NMR) spectroscopy together with an infusion of either [1,6-13C2]glucose or [2-13C]acetate. The levels of N-acetyl-aspartate (NAA) and glutamate were found to be decreased in the cerebral cortex and hippocampus in AßPP-PS1 mice, when compared with wild type controls. The cerebral metabolic rate of acetate oxidation was increased in the hippocampus and cerebral cortex of AßPP-PS1 mice suggesting enhanced astroglial activity in AD. AßPP-PS1 mice exhibit severe reduction in glutamatergic and gamma-amino butyric acid (GABA)ergic neuronal metabolic activity and neurotransmitter cycling fluxes in the hippocampus, cerebral cortex, and striatum as compared with controls. These data suggest that metabolic activity of excitatory and inhibitory neurons is compromised across brain in AßPP-PS1 mouse model of AD.

Energetics of Excitatory and Inhibitory Neurotransmission in Aluminum Chloride Model of Alzheimer's Disease: Reversal of Behavioral and Metabolic Deficits by Rasa Sindoor.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder, characterized by progressive loss of cognitive functions and memory. Excessive intake of aluminum chloride in drinking water is associated with amyloid plaques and neurofibrillary tangles in the brain, which are the hallmark of AD. We have evaluated brain energy metabolism in aluminum chloride (AlCl3) mouse model of AD. In addition, effectiveness of Rasa Sindoor (RS), a formulation used in Indian Ayurvedic medicine, for alleviation of symptoms of AD was evaluated. Mice were administered AlCl3 (40 mg/kg) intraperitoneally once a day for 60 days. The memory of mice was measured using Morris Water Maze test. The 13C labeling of brain amino acids was measured ex vivo in tissue extracts using 1H-[13C]-NMR spectroscopy with timed infusion of [1,6-13C2]glucose. The 13C turnover of brain amino acids was analyzed using a three-compartment metabolic model to derive the neurotransmitter cycling and TCA cycle rates associated with glutamatergic and GABAergic pathways. Exposure of AlCl3 led to reduction in memory of mice. The glutamatergic and GABAergic neurotransmitter cycling and glucose oxidation were found to be reduced in the cerebral cortex, hippocampus, and striatum following chronic AlCl3 treatment. The perturbation in metabolic rates was highest in the cerebral cortex. However, reduction in metabolic fluxes was higher in hippocampus and striatum following one month post AlCl3 treatment. Most interestingly, oral administration of RS (2 g/kg) restored memory as well as the energetics of neurotransmission in mice exposed to AlCl3. These data suggest therapeutic potential of RS to manage cognitive functions and memory in preclinical AD.

Amalaki Rasayana improved memory and neuronal metabolic activity in AbPP-PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive loss of memory and cognitive function. The cerebral metabolic rate of glucose oxidation has been shown to be reduced in AD. The present study evaluated efficacy of dietary Amalaki Rasayana (AR), an Ayurvedic formulation used in Indian traditional system, in AbPP-PS1 mouse model of AD in ameliorating memory and neurometabolism, and compared with donepezil, a standard FDA approved drug for AD. The memory of mice was measured using Morris Water Maze analysis. The cerebral metabolism was followed by 13C labelling of brain amino acids in tissue extracts ex vivo using 1H-[13C]-NMR spectroscopy together with a short time infusion of [1,6-13C2]glucose to mice. The intervention with Amalaki Rasayana showed improved learning and memory in AbPP-PS1 mice. The 13C labelings of GluC4, GABAC2 and GlnC4 were reduced in AbPP-PS1 mice when compared with wild-type controls. Intervention of AR increased the 13C labelling of amino acids suggesting a significant enhancement in glutamatergic and GABAergic metabolic activity in AbPP-PS1 mice similar to that observed with donepezil treatment. These data suggest that AR has potential to improve memory and cognitive function in AD.

Direct one-pot synthesis of glutathione capped hydrophilic FePt-CdS nanoprobe for efficient bimodal imaging application.

One-pot synthesis methods for development of hydrophilic imaging nanoprobes have advantages over multi-pot methods due to their simple procedures, less probability for degradation of efficiency, superior control over growth and morphology, cost effectiveness, improved scope for scale-up synthesis etc. Here, we present a novel one-pot facile synthesis of hydrophilic colloidal bimodal nanoprobe (FePt-CdS) prepared through a seed-mediated nucleation and growth technique. In this facile synthesis of complex nanostructure, glutathione (GSH) was used as the capping agent to render biocompatibility and dispersibility. The microstructure, surface, optical, magnetic, biocompatibility, relaxivity and imaging property of the developed nanoprobe have been studied. The microstructural characterizations reveal average size of the particle as ~9-11nm with bleb shaped morphology. Spectroscopic characterization depicts the development of GSH capped CdS QDs on FePt, surface functionalities and their stability. The magnetic measurements confirm the superparamagnetic property in the developed bimodal nanoprobe. In addition, the GSH capping imparts excellent biocompatibility, water dispersibility, and fluorescence property to the probe. In RAW 264.7 macrophage cells, the bimodal nanoprobes exhibit intense green and red fluorescence. The magnetic resonance imaging (MRI) and fluorescence imaging (FI) study depict high transverse relaxivity and visible range fluorescent property in the synthesized FePt-CdS nanoprobe. Hence, the developed bimodal nanoprobe can be used as a potential candidate in simultaneous FI and MR imaging.

Insights into the epigenetic mechanisms involving histone lysine methylation and demethylation in ischemia induced damage and repair has therapeutic implication.

Cerebral ischemic stroke is one of the leading causes of death and disability worldwide. Therapeutic interventions to minimize ischemia-induced neural damage are limited due to poor understanding of molecular mechanisms mediating complex pathophysiology in stroke. Recently, epigenetic mechanisms mostly histone lysine (K) acetylation and deacetylation have been implicated in ischemic brain damage and have expanded the dimensions of potential therapeutic intervention to the systemic/local administration of histone deacetylase inhibitors. However, the role of other epigenetic mechanisms such as histone lysine methylation and demethylation in stroke-induced damage and subsequent recovery process is elusive. Here, we established an Internal Carotid Artery Occlusion (ICAO) model in CD1 mouse that resulted in mild to moderate level of ischemic damage to the striatum, as suggested by magnetic resonance imaging (MRI), TUNEL and histopathological staining along with an evaluation of neurological deficit score (NDS), grip strength and rotarod performance. The molecular investigations show dysregulation of a number of histone lysine methylases (KMTs) and few of histone lysine demethylases (KDMs) post-ICAO with significant global attenuation in the transcriptionally repressive epigenetic mark H3K9me2 in the striatum. Administration of Dimethyloxalylglycine (DMOG), an inhibitor of KDM4 or JMJD2 class of histone lysine demethylases, significantly ameliorated stroke-induced NDS by restoring perturbed H3K9me2 levels in the ischemia-affected striatum. Overall, these results highlight the novel role of epigenetic regulatory mechanisms controlling the epigenetic mark H3K9me2 in mediating the stroke-induced striatal damage and subsequent repair following mild to moderate cerebral ischemia.

Comparison of Glutamate Turnover in Nerve Terminals and Brain Tissue During [1,6-13C2]Glucose Metabolism in Anesthetized Rats.

The 13C turnover of neurotransmitter amino acids (glutamate, GABA and aspartate) were determined from extracts of forebrain nerve terminals and brain homogenate, and fronto-parietal cortex from anesthetized rats undergoing timed infusions of [1,6-13C2]glucose or [2-13C]acetate. Nerve terminal 13C fractional labeling of glutamate and aspartate was lower than those in whole cortical tissue at all times measured (up to 120 min), suggesting either the presence of a constant dilution flux from an unlabeled substrate or an unlabeled (effectively non-communicating on the measurement timescale) glutamate pool in the nerve terminals. Half times of 13C labeling from [1,6-13C2]glucose, as estimated by least squares exponential fitting to the time course data, were longer for nerve terminals (GluC4, 21.8 min; GABAC2 21.0 min) compared to cortical tissue (GluC4, 12.4 min; GABAC2, 14.5 min), except for AspC3, which was similar (26.5 vs. 27.0 min). The slower turnover of glutamate in the nerve terminals (but not GABA) compared to the cortex may reflect selective effects of anesthesia on activity-dependent glucose use, which might be more pronounced in the terminals. The 13C labeling ratio for glutamate-C4 from [2-13C]acetate over that of 13C-glucose was twice as large in nerve terminals compared to cortex, suggesting that astroglial glutamine under the 13C glucose infusion was the likely source of much of the nerve terminal dilution. The net replenishment of most of the nerve terminal amino acid pools occurs directly via trafficking of astroglial glutamine.

Neuroprotective effects of caffeine in MPTP model of Parkinson's disease: A (13)C NMR study.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of nigrostriatal dopaminergic neurons with an accompanying neuroinflammation leading to loss of dopamine in the basal ganglia. Caffeine, a well-known A2A receptor antagonist is reported to slow down the neuroinflammation caused by activated microglia and reduce the extracellular glutamate in the brain. In this study, we have evaluated the neuroprotective effect of caffeine in the MPTP model of PD by monitoring the region specific cerebral energy metabolism. Adult C57BL6 mice were treated with caffeine (30 mg/kg, i.p.) 30 min prior to MPTP (25 mg/kg, i.p.) administration for 8 days. The paw grip strength of mice was assessed in order to evaluate the motor function after various treatments. For metabolic studies, mice were infused with [1,6-(13)C2]glucose, and (13)C labeling of amino acids was monitored using ex vivo(1)H-[(13)C]-NMR spectroscopy. The paw grip strength was found to be reduced following the MPTP treatment. The caffeine pretreatment showed significant protection against the reduction of paw grip strength in MPTP treated mice. The levels of GABA and myo-inositol were found to be elevated in the striatum of MPTP treated mice. The (13)C labeling of GluC4, GABAC2 and GlnC4 from [1,6-(13)C2]glucose was decreased in the cerebral cortex, striatum, olfactory bulb, thalamus and cerebellum suggesting impaired glutamatergic and GABAergic neuronal activity and neurotransmission of the MPTP treated mice. Most interestingly, the pretreatment of caffeine maintained the (13)C labeling of amino acids to the control values in cortical, olfactory bulb and cerebellum regions while it partially retained in striatal and thalamic regions in MPTP treated mice. The pretreatment of caffeine provides a partial neuro-protection against severe striatal degeneration in the MPTP model of PD.

A water-soluble and water-coordinated Mn(II) complex: synthesis, characterization and phantom MRI image study.

Ligand H4bedik was reacted with MnCl2·4H2O at pH ∼ 6.5 to give a highly water-soluble and water-coordinated Mn(ii) complex (). The complex was found to show r1 = 3.11 mM(-1) s(-1) per Mn(ii) at 1.4 T and 6.26 mM(-1) s(-1) per Mn(ii) at 14.1 T at 25 °C, pH = 7.4. In addition to r1, the r2 at 14.1 T was found to be 132.78 mM(-1) s(-1) per Mn(ii) at 25 °C, pH = 7.4.

Effects of γ-Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and γ-Aminobutyric acid metabolism in the anesthetized rat In vivo.

γ-Aminobutyric acid (GABA) clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters (GATs). The relative flows through these two pathways for GABA released from neurons remains unclear. This study determines the effect of tiagabine, a selective inhibitor of neuronal GAT-1, on the rates of glutamate (Glu) and GABA metabolism and GABA resynthesis via the GABA-glutamine (Gln) cycle. Halothane-anesthetized rats were administered tiagabine (30 mg/kg, i.p.) and 45 min later received an intravenous infusion of either [1,6-(13)C2]glucose (in vivo) or [2-(13)C]acetate (ex vivo). Nontreated rats served as controls. Metabolites and (13)C enrichments were measured with (1)H-[(13)C]-nuclear magnetic resonance spectroscopy and referenced to their corresponding endpoint values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the (13)C turnover data measured in vivo during [1,6-(13)C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P > 0.05) from controls in tissue amino acid levels and in (13)C enrichments from [2-(13)C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMR(glc(ox)Glu): control, 0.27 ± 0.05 vs. tiagabine, 0.22 ± 0.04 µmol/g/min; ↓11%, V(cyc(Glu-Gln)): control 0.23 ± 0.05 vs. tiagabine 0.21 ± 0.04 µmol/g/min and GABAergic neurons (↓18-25%, CMR(glc(ox)GABA): control 0.09 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min; V(cyc(GABA-Gln)): control 0.08 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P > 0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects.

RNA Interference Using c-Myc-Conjugated Nanoparticles Suppresses Breast and Colorectal Cancer Models.

In this article, we report the development and preclinical validation of combinatorial therapy for treatment of cancers using RNA interference (RNAi). RNAi technology is an attractive approach to silence genes responsible for disease onset and progression. Currently, the critical challenge facing the clinical success of RNAi technology is in the difficulty of delivery of RNAi inducers, due to low transfection efficiency, difficulties of integration into host DNA and unstable expression. Using the macromolecule polyglycidal methacrylate (PGMA) as a platform to graft multiple polyethyleneimine (PEI) chains, we demonstrate effective delivery of small oligos (anti-miRs and mimics) and larger DNAs (encoding shRNAs) in a wide variety of cancer cell lines by successful silencing/activation of their respective target genes. Furthermore, the effectiveness of this therapy was validated for in vivo tumor suppression using two transgenic mouse models; first, tumor growth arrest and increased animal survival was seen in mice bearing Brca2/p53-mutant mammary tumors following daily intratumoral treatment with nanoparticles conjugated to c-Myc shRNA. Second, oral delivery of the conjugate to an Apc-deficient crypt progenitor colon cancer model increased animal survival and returned intestinal tissue to a non-wnt-deregulated state. This study demonstrates, through careful design of nonviral nanoparticles and appropriate selection of therapeutic gene targets, that RNAi technology can be made an affordable and amenable therapy for cancer.

Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data.

(13)C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-(13)C]/[1,6-(13)C2]glucose as labeled substrate have consistently found a lower enrichment (∼25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using (1)H-[(13)C]-NMR spectroscopy together with intravenous infusion of [U-(13)C5]glutamine for 3, 15, 30, and 60 minutes in mice. (13)C labeling of brain glutamine was found to be saturated at plasma glutamine levels >1.0 mmol/L. Fitting a blood-astrocyte-neuron metabolic model to the (13)C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036±0.002 µmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (∼0.6 mmol/L), VGln(in) would be ∼0.010 µmol/g per minute, which corresponds to ∼6% of the glutamine synthesis rate and rises to ∼11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ∼20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-(13)C]glucose.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.