Transcriptional changes in the rat brain induced by repetitive transcranial magnetic stimulation.

Introduction: Transcranial Magnetic Stimulation (TMS) is a noninvasive technique that uses pulsed magnetic fields to affect the physiology of the brain and central nervous system. Repetitive TMS (rTMS) has been used to study and treat several neurological conditions, but its complex molecular basis is largely unexplored. Methods: Utilizing three experimental rat models (in vitro, ex vivo, and in vivo) and employing genome-wide microarray analysis, our study reveals the extensive impact of rTMS treatment on gene expression patterns. Results: These effects are observed across various stimulation protocols, in diverse tissues, and are influenced by time and age. Notably, rTMS-induced alterations in gene expression span a wide range of biological pathways, such as glutamatergic, GABAergic, and anti-inflammatory pathways, ion channels, myelination, mitochondrial energetics, multiple neuron-and synapse-specific genes. Discussion: This comprehensive transcriptional analysis induced by rTMS stimulation serves as a foundational characterization for subsequent experimental investigations and the exploration of potential clinical applications.

AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences.

The choroid plexus (CP) is a source of trophic factors for the developing and mature brain. Insulin is produced in epithelial cells of the CP (EChPs), and its secretion is stimulated by Htr2c-mediated signaling. We modulated insulin expression in EChPs with intracerebroventricular injections of AAV5. Insulin overexpression in CP decelerates food intake, whereas its knockdown has the opposite effect. Insulin overexpression also results in reduced anxious behavior. Transcriptomic changes in the hypothalamus, especially in synapse-related processes, are also seen in mice overexpressing insulin in CP. Last, activation of Gq signaling in CP leads to acute Akt phosphorylation in neurons of the arcuate nucleus, indicating a direct action of CP-derived insulin on the hypothalamus. Taken together, our findings signify that CP is a relevant source of insulin in the central nervous system and that CP-derived insulin should be taken into consideration in future work pertaining to insulin actions in the brain.

Prolonged fasting times reap greater geroprotective effects when combined with caloric restriction in adult female mice.

Emerging new evidence highlights the importance of prolonged daily fasting periods for the health and survival benefits of calorie restriction (CR) and time-restricted feeding (TRF) in male mice; however, little is known about the impact of these feeding regimens in females. We placed 14-month-old female mice on five different dietary regimens, either CR or TRF with different feeding windows, and determined the effects of these regimens on physiological responses, progression of neoplasms and inflammatory diseases, serum metabolite levels, and lifespan. Compared with TRF feeding, CR elicited a robust systemic response, as it relates to energetics and healthspan metrics, a unique serum metabolomics signature in overnight fasted animals, and was associated with an increase in lifespan. These results indicate that daytime (rest-phase) feeding with prolonged fasting periods initiated late in life confer greater benefits when combined with imposed lower energy intake.

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates ß-amyloid aggregation.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-ß peptide (Aß1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aß1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aß1-42 peptide, the aggregation kinetics of Aß1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aß1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aß1-42. Aß1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aß1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aß1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aß1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Fasting blood glucose as a predictor of mortality: Lost in translation.

Aging leads to profound changes in glucose homeostasis, weight, and adiposity, which are considered good predictors of health and survival in humans. Direct evidence that these age-associated metabolic alterations are recapitulated in animal models is lacking, impeding progress to develop and test interventions that delay the onset of metabolic dysfunction and promote healthy aging and longevity. We compared longitudinal trajectories, rates of change, and mortality risks of fasting blood glucose, body weight, and fat mass in mice, nonhuman primates, and humans throughout their lifespans and found similar trajectories of body weight and fat in the three species. In contrast, fasting blood glucose decreased late in life in mice but increased over the lifespan of nonhuman primates and humans. Higher glucose was associated with lower mortality in mice but higher mortality in nonhuman primates and humans, providing a cautionary tale for translating age-associated metabolic changes from mice to humans.

Study of Longitudinal Aging in Mice: Presentation of Experimental Techniques.

Aging is associated with functional and metabolic decline and is a risk factor for all noncommunicable diseases. Even though mice are routinely used for modeling human aging and aging-related conditions, no comprehensive assessment to date has been conducted on normative mouse aging. To address this gap, the Study of Longitudinal Aging in Mice (SLAM) was designed and implemented by the National Institute on Aging (NIA/NIH) as the mouse counterpart to the Baltimore Longitudinal Study of Aging (BLSA). In this manuscript, we describe the premise, study design, methodologies, and technologies currently employed in SLAM. We also discuss current and future study directions. In this large population mouse study, inbred C57BL/6J and outbred UM-HET3 mice of both sexes are longitudinally evaluated for functional, phenotypic, and biological health, and collection of biospecimens is conducted throughout their life span. Within the longitudinal cohorts, a cross-sectional arm of the study has also been implemented for the well-controlled collection of tissues to generate a biorepository. SLAM and studies stemming from SLAM seek to identify and characterize phenotypic and biological predictors of mouse aging and age-associated conditions, examine the degrees of functional and biomolecular variability that occur within inbred and genetically heterogeneous mouse populations with age, and assess whether these changes are consistent with alterations observed in human aging in BLSA. The findings from these studies will be critical for evaluating the utility of mouse models for studying different aspects of aging, both in terms of interpreting prior findings and designing and implementing future studies.

Spontaneous chordoma: a case report on a female UM-HET3 mouse from the SLAM study.

A female UM-HET3 mouse from the Study of Longitudinal Aging in Mice (SLAM) was euthanized at 164 weeks of age due to hind limb weakness. Necropsy and histological analysis revealed that the most probable cause of the clinical finding was the compression of the thoracolumbar segment of the spinal cord by herniated intervertebral disks. In addition, a spontaneous chordoma was incidentally found in the coccygeal bones. Given the rarity of this type of tumor, bio-clinical annotations acquired throughout lifespan, detailed histopathological assessment, and comparative clinical-pathological correlations for this mouse are presented and discussed.

Release of insulin produced by the choroid plexis is regulated by serotonergic signaling.

The choroid plexus (ChP) is a highly vascularized tissue found in the brain ventricles, with an apical epithelial cell layer surrounding fenestrated capillaries. It is responsible for the production of most of the cerebrospinal fluid (CSF) in the ventricular system, subarachnoid space, and central canal of the spinal cord, while also constituting the blood-CSF barrier (BCSFB). In addition, epithelial cells of the ChP (EChP) synthesize neurotrophic factors and other signaling molecules that are released into the CSF. Here, we show that insulin is produced in EChP of mice and humans, and its expression and release are regulated by serotonin. Insulin mRNA and immune-reactive protein, including C-peptide, are present in EChP, as detected by several experimental approaches, and appear in much higher levels than any other brain region. Moreover, insulin is produced in primary cultured mouse EChP, and its release, albeit Ca2+ sensitive, is not regulated by glucose. Instead, activation of the 5HT2C receptor by serotonin treatment led to activation of IP3-sensitive channels and Ca2+ mobilization from intracellular storage, leading to insulin secretion. In vivo depletion of brain serotonin in the dorsal raphe nucleus negatively affected insulin expression in the ChP, suggesting an endogenous modulation of ChP insulin by serotonin. Here, we show for the first time to our knowledge that insulin is produced by EChP in the brain, and its release is modulated at least by serotonin but not glucose.

Activity-dependent neuronal Klotho enhances astrocytic aerobic glycolysis.

Mutations of the ß-glucuronidase protein α-Klotho have been associated with premature aging, and altered cognitive function. Although highly expressed in specific areas of the brain, Klotho functions in the central nervous system remain unknown. Here, we show that cultured hippocampal neurons respond to insulin and glutamate stimulation by elevating Klotho protein levels. Conversely, AMPA and NMDA antagonism suppress neuronal Klotho expression. We also provide evidence that soluble Klotho enhances astrocytic aerobic glycolysis by hindering pyruvate metabolism through the mitochondria, and stimulating its processing by lactate dehydrogenase. Pharmacological inhibition of FGFR1, Erk phosphorylation, and monocarboxylic acid transporters prevents Klotho-induced lactate release from astrocytes. Taken together, these data suggest Klotho is a potential new player in the metabolic coupling between neurons and astrocytes. Neuronal glutamatergic activity and insulin modulation elicit Klotho release, which in turn stimulates astrocytic lactate formation and release. Lactate can then be used by neurons and other cells types as a metabolic substrate.

Impact of Coffee and Cacao Purine Metabolites on Neuroplasticity and Neurodegenerative Disease.

Increasing evidence suggests that regular consumption of coffee, tea and dark chocolate (cacao) can promote brain health and may reduce the risk of age-related neurodegenerative disorders. However, the complex array of phytochemicals in coffee and cacao beans and tea leaves has hindered a clear understanding of the component(s) that affect neuronal plasticity and resilience. One class of phytochemicals present in relatively high amounts in coffee, tea and cacao are methylxanthines. Among such methylxanthines, caffeine has been the most widely studied and has clear effects on neuronal network activity, promotes sustained cognitive performance and can protect neurons against dysfunction and death in animal models of stroke, Alzheimer's disease and Parkinson's disease. Caffeine's mechanism of action relies on antagonism of various subclasses of adenosine receptors. Downstream xanthine metabolites, such as theobromine and theophylline, may also contribute to the beneficial effects of coffee, tea and cacao on brain health.

Uric acid enhances longevity and endurance and protects the brain against ischemia.

Among mammals, there is a positive correlation between serum uric acid (UA) levels and life span. Humans have high levels of UA because they lack a functional urate oxidase (UOX) enzyme that is present in shorter lived mammals. Here, we show that male and female mice with UOX haploinsufficiency exhibit an age-related elevation of UA levels, and that the life span of female but not male UOX+/- mice is significantly increased compared to wild-type mice. Serum UA levels are elevated in response to treadmill exercise in UOX+/- mice, but not wild-type mice, and the endurance of the UOX+/- mice is significantly greater than wild-type mice. UOX+/- mice exhibit elevated levels of brain-derived neurotrophic factor, reduced brain damage and improved functional outcome in a model of focal ischemic stroke. Levels of oxidative protein nitration and lipid peroxidation are reduced in muscle and brain tissues of UOX+/- mice under conditions of metabolic and oxidative stress (running in the case of muscle and ischemia in the case of the brain), consistent with prior evidence that UA can scavenge peroxynitrite and hydroxyl radical. Our findings reveal roles for UA in life span determination, endurance and adaptive responses to brain injury, and suggest novel approaches for protecting cells against injury and for optimizing physical performance.

Astrocytes, emerging stars of energy homeostasis.

Astrocytes have historically been considered structural supporting cells for neurons. Thanks to new molecular tools, allowing specific cell ablation or over-expression of genes, new unexpected astrocytic functions have recently been unveiled. This review focus on emerging groundbreaking findings showing that hypothalamic astrocytes are pivotal for the regulation of whole body energy homeostasis. Hypothalamic astrocytes sense glucose and fatty acids, and express receptors for several peripheral hormones such as leptin and insulin. Furthermore, they display striking sexual dimorphism which may account, at least partially, for gender specific differences in energy homeostasis. Metabolic alterations have been shown to influence the initiation and progression of many neurodegenerative disorders. A better understanding of the roles and interplay between the different brain cells in regulating energy homeostasis could help develop new therapeutic strategies to prevent or cure neurodegenerative disorders.

Mice lacking the transcriptional regulator Bhlhe40 have enhanced neuronal excitability and impaired synaptic plasticity in the hippocampus.

Bhlhe40 is a transcription factor that is highly expressed in the hippocampus; however, its role in neuronal function is not well understood. Here, we used Bhlhe40 null mice on a congenic C57Bl6/J background (Bhlhe40 KO) to investigate the impact of Bhlhe40 on neuronal excitability and synaptic plasticity in the hippocampus. Bhlhe40 KO CA1 neurons had increased miniature excitatory post-synaptic current amplitude and decreased inhibitory post-synaptic current amplitude, indicating CA1 neuronal hyperexcitability. Increased CA1 neuronal excitability was not associated with increased seizure severity as Bhlhe40 KO relative to +/+ (WT) control mice injected with the convulsant kainic acid. However, significant reductions in long term potentiation and long term depression at CA1 synapses were observed in Bhlhe40 KO mice, indicating impaired hippocampal synaptic plasticity. Behavioral testing for spatial learning and memory on the Morris Water Maze (MWM) revealed that while Bhlhe40 KO mice performed similarly to WT controls initially, when the hidden platform was moved to the opposite quadrant Bhlhe40 KO mice showed impairments in relearning, consistent with decreased hippocampal synaptic plasticity. To investigate possible mechanisms for increased neuronal excitability and decreased synaptic plasticity, a whole genome mRNA expression profile of Bhlhe40 KO hippocampus was performed followed by a chromatin immunoprecipitation sequencing (ChIP-Seq) screen of the validated candidate genes for Bhlhe40 protein-DNA interactions consistent with transcriptional regulation. Of the validated genes identified from mRNA expression analysis, insulin degrading enzyme (Ide) had the most significantly altered expression in hippocampus and was significantly downregulated on the RNA and protein levels; although Bhlhe40 did not occupy the Ide gene by ChIP-Seq. Together, these findings support a role for Bhlhe40 in regulating neuronal excitability and synaptic plasticity in the hippocampus and that indirect regulation of Ide transcription may be involved in these phenotypes.

Toll-like receptor 4 mediates fat, sugar, and umami taste preference and food intake and body weight regulation.

OBJECTIVE: Immune and inflammatory pathways play important roles in the pathogenesis of metabolic disorders. This study investigated the role of toll-like receptor 4 (TLR4) in orosensory detection of dietary lipids and sugars. METHODS: Taste preferences of TLR4 knockout (KO) and wild-type (WT) male mice under a standard and a high-fat, high-sugar diet were assessed with two-bottle tests. Gene expression of taste signaling molecules was analyzed in the tongue epithelium. The role of TLR4 in food intake and weight gain was investigated in TLR4 KO and WT mice fed a high-fat and high-sugar diet for 12 weeks. RESULTS: Compared to WT mice, TLR4 KO mice showed reduced preference for lipids, sugars, and umami in a two-bottle preference test. The altered taste perception was associated with decreased levels of key taste regulatory molecules in the tongue epithelium. TLR4 KO mice on a high-fat and high-sugar diet consumed less food and drink, resulting in diminished weight gain. CONCLUSIONS: TLR4 signaling promotes ingestion of sugar and fat by a mechanism involving increased preference for such obesogenic foods.

skn-1 is required for interneuron sensory integration and foraging behavior in Caenorhabditis elegans.

Nrf2/skn-1, a transcription factor known to mediate adaptive responses of cells to stress, also regulates energy metabolism in response to changes in nutrient availability. The ability to locate food sources depends upon chemosensation. Here we show that Nrf2/skn-1 is expressed in olfactory interneurons, and is required for proper integration of multiple food-related sensory cues in Caenorhabditis elegans. Compared to wild type worms, skn-1 mutants fail to perceive that food density is limiting, and display altered chemo- and thermotactic responses. These behavioral deficits are associated with aberrant AIY interneuron morphology and migration in skn-1 mutants. Both skn-1-dependent AIY autonomous and non-autonomous mechanisms regulate the neural circuitry underlying multisensory integration of environmental cues related to energy acquisition.

Brain metabolism in health, aging, and neurodegeneration.

Brain cells normally respond adaptively to bioenergetic challenges resulting from ongoing activity in neuronal circuits, and from environmental energetic stressors such as food deprivation and physical exertion. At the cellular level, such adaptive responses include the "strengthening" of existing synapses, the formation of new synapses, and the production of new neurons from stem cells. At the molecular level, bioenergetic challenges result in the activation of transcription factors that induce the expression of proteins that bolster the resistance of neurons to the kinds of metabolic, oxidative, excitotoxic, and proteotoxic stresses involved in the pathogenesis of brain disorders including stroke, and Alzheimer's and Parkinson's diseases. Emerging findings suggest that lifestyles that include intermittent bioenergetic challenges, most notably exercise and dietary energy restriction, can increase the likelihood that the brain will function optimally and in the absence of disease throughout life. Here, we provide an overview of cellular and molecular mechanisms that regulate brain energy metabolism, how such mechanisms are altered during aging and in neurodegenerative disorders, and the potential applications to brain health and disease of interventions that engage pathways involved in neuronal adaptations to metabolic stress.

3-Hydroxybutyrate regulates energy metabolism and induces BDNF expression in cerebral cortical neurons.

During fasting and vigorous exercise, a shift of brain cell energy substrate utilization from glucose to the ketone 3-hydroxybutyrate (3OHB) occurs. Studies have shown that 3OHB can protect neurons against excitotoxicity and oxidative stress, but the underlying mechanisms remain unclear. Neurons maintained in the presence of 3OHB exhibited increased oxygen consumption and ATP production, and an elevated NAD+ /NADH ratio. We found that 3OHB metabolism increases mitochondrial respiration which drives changes in expression of brain-derived neurotrophic factor (BDNF) in cultured cerebral cortical neurons. The mechanism by which 3OHB induces Bdnf gene expression involves generation of reactive oxygen species, activation of the transcription factor NF-κB, and activity of the histone acetyltransferase p300/EP300. Because BDNF plays important roles in synaptic plasticity and neuronal stress resistance, our findings suggest cellular signaling mechanisms by which 3OHB may mediate adaptive responses of neurons to fasting, exercise, and ketogenic diets.

Enantioselective inhibition of d-serine transport by (S)-ketamine.

BACKGROUND AND PURPOSE: Patients with major depressive disorder receiving racemic ketamine, (R,S)-ketamine, experience transient increases in Clinician-Administered Dissociative States Scale scores and a coincident drop in plasma d-serine levels. The results suggest that (R,S)-ketamine produces an immediate, concentration-dependent pharmacological effect on d-serine plasma concentrations. One potential source of this effect is (R,S)-ketamine-induced inhibition of the transporter ASCT2, which regulates intracellular d-serine concentrations. In this study, we tested this hypothesis by examining the effect of (S)- and (R)-ketamine on ASCT2-mediated transport of d-serine in PC-12 and 1321N1 cells and primary neuronal cells in culture. EXPERIMENTAL APPROACH: Intracellular and extracellular d-serine levels were determined using capillary electrophoresis-laser-induced fluorescence and liquid chromatography-mass spectrometry respectively. Expression of ASCT2, Asc-1 and serine racemase was determined utilizing Western blotting. KEY RESULTS: (S)-Ketamine produced a concentration-dependent increase in intracellular d-serine and reduced extracellular d-serine accumulation. In contrast, (R)-ketamine decreased both intracellular and extracellular d-serine levels. The ASCT2 inhibitor, benzyl-d-serine (BDS), and ASCT2 gene knockdown mimicked the action of (S)-ketamine on d-serine in PC-12 cells, while the Asc-1 agonist d-isoleucine reduced intracellular d-serine and increased extracellular d-serine accumulation. This response to d-isoleucine was not affected by BDS or (S)-ketamine. Primary cultures of rat neuronal cells expressed ASCT2 and were responsive to (S)-ketamine and BDS. (S)- and (R)-ketamine increased the expression of monomeric serine racemase in all the cells studied, with (S)-ketamine having the greatest effect. CONCLUSIONS AND IMPLICATIONS: (S)-Ketamine decreased cellular export of d-serine via selective inhibition of ASCT2, and this could represent a possible source of dissociative effects observed with (R,S)-ketamine.

The Role of Uric Acid and Methyl Derivatives in the Prevention of Age-Related Neurodegenerative Disorders.

High uric acid (UA levels have been correlated with a reduced risk of many neurodegenerative diseases through mechanisms involving chelating Fenton reaction transitional metals, antioxidant quenching of superoxide and hydroxyl free radicals, and as an electron donor that increases antioxidant enzyme activity (e.g. SOD. However, the clinical usefulness of UA is limited by its' low water solubility and propensity to form inflammatory crystals at hyperuricemic levels. This review focuses on the role of UA in neuroprotection, as well as potential strategies aimed at increasing UA levels in the soluble range, and the potential therapeutic use of more water-soluble methyl-UA derivatives from the natural catabolic end-products of dietary caffeine, theophylline, and theobromine.

Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans.

Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1-C24:1), gangliosides (e.g., GM1-C24:1), and sphingomyelins (e.g., dC18:1-C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides.