Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol.

Alzheimer's disease (AD) is characterized by the presence of amyloid ß (Aß) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic risk factor for sporadic AD. In vitro evidence suggests that apoE links to Aß production through nanoscale lipid compartments (lipid clusters), but its regulation in vivo is unclear. Here, we use superresolution imaging in the mouse brain to show that apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) in and out of lipid clusters, where it interacts with ß- and γ-secretases to generate Aß-peptide. We find that the targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid clusters, where it interacts with α-secretase and gives rise to soluble APP-α (sAPP-α), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, ß-, and γ-secretase trafficking, suggesting that the ratio of Aß to sAPP-α is regulated by the trafficking of the substrate, not the enzymes. We conclude that cholesterol is kept low in neurons, which inhibits Aß accumulation and enables the astrocyte regulation of Aß accumulation by cholesterol signaling.

SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-ß oligomers.

Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimer's disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We previously discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-ß oligomers (AßOs), a primary factor in AD pathogenesis (Norambuena et al., 2018). Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. AßOs inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from tuberous sclerosis (TS) patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.

Neutrophil Enzyme Myeloperoxidase Modulates Neuronal Response in a Model of Subarachnoid Hemorrhage by Venous Injury.

Background and Purpose: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delayed cognitive deficits. Neutrophil infiltration into the central nervous system is linked to the development of these deficits after SAH. It is however unclear how neutrophil activity influences central nervous system function in SAH. The present project aims to elucidate which neutrophil factors mediate central nervous system injury and cognitive deficits after SAH. Methods: Using a murine model of SAH and mice deficient in neutrophil effector functions, we determined which neutrophil effector function is critical to the development of deficits after SAH. In vivo and in vitro techniques were used to investigate possible pathways of neutrophils effect after SAH. Results: Our results show that mice lacking functional MPO (myeloperoxidase), a neutrophil enzyme, lack both the meningeal neutrophil infiltration (wild type, sham 872 cells/meninges versus SAH 3047, P=0.023; myeloperoxidase knockout [MPOKO], sham 1677 versus SAH 1636, P=NS) and erase the cognitive deficits on Barnes maze associated with SAH (MPOKO sham versus SAH, P=NS). The reintroduction of biologically active MPO, and its substrate hydrogen peroxide (H2O2), to the cerebrospinal fluid of MPOKO mice at the time of hemorrhage restores the spatial memory deficit observed after SAH (time to goal box MPOKO sham versus MPOKO+MPO/H2O2, P=0.001). We find evidence of changes in neurons, astrocytes, and microglia with MPO/H2O2 suggesting the effect of MPO may have complex interactions with many cell types. Neurons exposed to MPO/H2O2 show decreased calcium activity at baseline and after stimulation with potassium chloride. Although astrocytes and microglia are affected, changes seen in astrocytes are most consistent with inflammatory changes that likely affect neurons. Conclusions: These results implicate MPO as a mediator of neuronal dysfunction in SAH through its effect on both neurons and glia. These results show that, in SAH, the activity of innate immune cells in the meninges modulates the activity and function of the underlying brain tissue.

Loss of astrocyte cholesterol synthesis disrupts neuronal function and alters whole-body metabolism.

Cholesterol is important for normal brain function. The brain synthesizes its own cholesterol, presumably in astrocytes. We have previously shown that diabetes results in decreased brain cholesterol synthesis by a reduction in sterol regulatory element-binding protein 2 (SREBP2)-regulated transcription. Here we show that coculture of control astrocytes with neurons enhances neurite outgrowth, and this is reduced with SREBP2 knockdown astrocytes. In vivo, mice with knockout of SREBP2 in astrocytes have impaired brain development and behavioral and motor defects. These mice also have altered energy balance, altered body composition, and a shift in metabolism toward carbohydrate oxidation driven by increased glucose oxidation by the brain. Thus, SREBP2-mediated cholesterol synthesis in astrocytes plays an important role in brain and neuronal development and function, and altered brain cholesterol synthesis may contribute to the interaction between metabolic diseases, such as diabetes and altered brain function.

Insulin resistance and impaired lipid metabolism as a potential link between diabetes and Alzheimer's disease.

Diabetes disrupts organs throughout the body including the brain. Evidence suggests diabetes is a risk factor for Alzheimer's disease (AD) and neurodegeneration. In this review, we focus on understanding how diabetes contributes to the progression of neurodegeneration by influencing several aspects of the disease process. We emphasize the potential roles of brain insulin resistance, as well as cholesterol and lipid disruption, as factors which worsen AD.

Effect of cholesterol reduction on receptor signaling in neurons.

Diabetes mellitus is associated with a variety of complications, including alterations in the central nervous system (CNS). We have recently shown that diabetes results in a reduction of cholesterol synthesis in the brain due to decreased insulin stimulation of SREBP2-mediated cholesterol synthesis in neuronal and glial cells. In the present study, we explored the effects of the decrease in cholesterol on neuronal cell function using GT1-7 hypothalamic cells subjected to cholesterol depletion in vitro using three independent methods: 1) exposure to methyl-ß-cyclodextrin, 2) treatment with the HMG-CoA reductase inhibitor simvastatin, and 3) shRNA-mediated knockdown of SREBP2. All three methods produced 20-31% reductions in cellular cholesterol content, similar to the decrease in cholesterol synthesis observed in diabetes. All cholesterol-depleted neuron-derived cells, independent of the method of reduction, exhibited decreased phosphorylation/activation of IRS-1 and AKT following stimulation by insulin, insulin-like growth factor-1, or the neurotrophins (NGF and BDNF). ERK phosphorylation/activation was also decreased after methyl-ß-cyclodextrin and statin treatment but increased in cells following SREBP2 knockdown. In addition, apoptosis in the presence of amyloid-ß was increased. Reduction in cellular cholesterol also resulted in increased basal autophagy and impairment of induction of autophagy by glucose deprivation. Together, these data indicate that a reduction in neuron-derived cholesterol content, similar to that observed in diabetic brain, creates a state of insulin and growth factor resistance that could contribute to CNS-related complications of diabetes, including increased risk of neurodegenerative diseases, such as Alzheimer disease.

Reduction of the cholesterol sensor SCAP in the brains of mice causes impaired synaptic transmission and altered cognitive function.

The sterol sensor SCAP is a key regulator of SREBP-2, the major transcription factor controlling cholesterol synthesis. Recently, we showed that there is a global down-regulation of cholesterol synthetic genes, as well as SREBP-2, in the brains of diabetic mice, leading to a reduction of cholesterol synthesis. We now show that in mouse models of type 1 and type 2 diabetes, this is, in part, the result of a decrease of SCAP. Homozygous disruption of the Scap gene in the brains of mice causes perinatal lethality associated with microcephaly and gliosis. Mice with haploinsufficiency of Scap in the brain show a 60% reduction of SCAP protein and ~30% reduction in brain cholesterol synthesis, similar to what is observed in diabetic mice. This results in impaired synaptic transmission, as measured by decreased paired pulse facilitation and long-term potentiation, and is associated with behavioral and cognitive changes. Thus, reduction of SCAP and the consequent suppression of cholesterol synthesis in the brain may play an important role in the increased rates of cognitive decline and Alzheimer disease observed in diabetic states.

Incidental Diagnosis of Williams Syndrome in an Adult With Recurrent Hypercalcemia.

Williams syndrome (WS) is a rare genetic disorder with multisystem involvement associated with hypercalcemia. The cause of this hypercalcemia is poorly understood and while primarily associated with WS children, it is also observed in adults. A 51-year-old woman with intellectual disability, renal insufficiency, recurrent pancreatitis, and intermittent hypercalcemia despite partial parathyroidectomy presented with hypercalcemia to 14 mg/dL (3.49 mmol/L; normal 8.6-10.5 mg/dL [2.12-2.62 mmol/L]) at routine follow-up. Laboratory testing was notable for acute-on-chronic renal failure with unremarkable vitamin D, urine calcium, and parathyroid hormone. She presented to the emergency department and was admitted. Treatment with bisphosphonates, calcitonin, and intravenous fluids decreased calcium to 9.4 mg/dL (2.35 mmol/L) and improved kidney function. She was discharged with recommendations for increased oral hydration, a low-calcium diet, and outpatient follow-up. Her phenotype was suspicious for WS, later confirmed with genetic testing. This case exemplifies both the increased risk of hypercalcemia in WS adults and the need to consider WS in hypercalcemic adults with intellectual disability. It also serves to illustrate the importance of recognizing WS features in potentially undiagnosed adults and reviews guidelines for hypercalcemia surveillance and management in WS adults.

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

"Ouch!": you have just stabbed your little toe on the sharp corner of a coffee table. That painful sensation stems from nerve cells converting information about external forces into electric signals the brain can interpret. Increasingly, new evidence is suggesting that this process may be starting at fat-based structures within the membrane of these cells. The cell membrane is formed of two interconnected, flexible sheets of lipids in which embedded structures or molecules are free to move. This organisation allows the membrane to physically respond to external forces and, in turn, to set in motion chains of molecular events that help fine-tune how cells relay such information to the brain. For instance, an enzyme known as PLD2 is bound to lipid rafts ­ precisely arranged, rigid fatty 'clumps' in the membrane that are partly formed of cholesterol. PLD2 has also been shown to physically interact with and then activate the ion channel TREK-1, a membrane-based protein that helps to prevent nerve cells from relaying pain signals. However, the exact mechanism underpinning these interactions is difficult to study due to the nature and size of the molecules involved. To address this question, Petersen et al. combined a technology called super-resolution imaging with a new approach that allowed them to observe how membrane lipids respond to pressure and fluid shear. The experiments showed that mechanical forces disrupt the careful arrangement of lipid rafts, causing PLD2 and TREK-1 to be released. They can then move through the surrounding membrane where they reach a switch that turns on TREK-1. Further work revealed that the levels of cholesterol available to mouse cells directly influenced how the clumps could form and bind to PLD2, and in turn, dialled up and down the protective signal mediated by TREK-1. Overall, the study by Petersen et al. shows that the membrane of nerve cells can contain cholesterol-based 'fat sensors' that help to detect external forces and participate in pain regulation. By dissecting these processes, it may be possible to better understand and treat conditions such as diabetes and lupus, which are associated with both pain sensitivity and elevated levels of cholesterol in tissues.

Altered circadian behavior and light sensing in mouse models of Alzheimer's disease.

Circadian symptoms have long been observed in Alzheimer's disease (AD) and often appear before cognitive symptoms, but the mechanisms underlying circadian alterations in AD are poorly understood. We studied circadian re-entrainment in AD model mice using a "jet lag" paradigm, observing their behavior on a running wheel after a six hour advance in the light:dark cycle. Female 3xTg mice, which carry mutations producing progressive amyloid beta and tau pathology, re-entrained following jet lag more rapidly than age-matched wild type controls at both 8 and 13 months of age. This re-entrainment phenotype has not been previously reported in a murine AD model. Because microglia are activated in AD and in AD models, and inflammation can affect circadian rhythms, we hypothesized that microglia contribute to this re-entrainment phenotype. To test this, we used the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX3397, which rapidly depletes microglia from the brain. Microglia depletion did not alter re-entrainment in either wild type or 3xTg mice, demonstrating that microglia activation is not acutely responsible for the re-entrainment phenotype. To test whether mutant tau pathology is necessary for this behavioral phenotype, we repeated the jet lag behavioral test with the 5xFAD mouse model, which develops amyloid plaques, but not neurofibrillary tangles. As with 3xTg mice, 7-month-old female 5xFAD mice re-entrained more rapidly than controls, demonstrating that mutant tau is not necessary for the re-entrainment phenotype. Because AD pathology affects the retina, we tested whether differences in light sensing may contribute to altered entrainment behavior. 3xTg mice demonstrated heightened negative masking, an SCN-independent circadian behavior measuring responses to different levels of light, and re-entrained dramatically faster than WT mice in a jet lag experiment performed in dim light. 3xTg mice show a heightened sensitivity to light as a circadian cue that may contribute to accelerated photic re-entrainment. Together, these experiments demonstrate novel circadian behavioral phenotypes with heightened responses to photic cues in AD model mice which are not dependent on tauopathy or microglia.

Altered circadian behavior and light sensing in mouse models of Alzheimer's disease.

Circadian symptoms have long been observed in Alzheimer's disease (AD) and often appear before cognitive symptoms, but the mechanisms underlying circadian alterations in AD are poorly understood. We studied circadian re-entrainment in AD model mice using a "jet lag" paradigm, observing their behavior on a running wheel after a 6 h advance in the light:dark cycle. Female 3xTg mice, which carry mutations producing progressive amyloid beta and tau pathology, re-entrained following jet lag more rapidly than age-matched wild type controls at both 8 and 13 months of age. This re-entrainment phenotype has not been previously reported in a murine AD model. Because microglia are activated in AD and in AD models, and inflammation can affect circadian rhythms, we hypothesized that microglia contribute to this re-entrainment phenotype. To test this, we used the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX3397, which rapidly depletes microglia from the brain. Microglia depletion did not alter re-entrainment in either wild type or 3xTg mice, demonstrating that microglia activation is not acutely responsible for the re-entrainment phenotype. To test whether mutant tau pathology is necessary for this behavioral phenotype, we repeated the jet lag behavioral test with the 5xFAD mouse model, which develops amyloid plaques, but not neurofibrillary tangles. As with 3xTg mice, 7-month-old female 5xFAD mice re-entrained more rapidly than controls, demonstrating that mutant tau is not necessary for the re-entrainment phenotype. Because AD pathology affects the retina, we tested whether differences in light sensing may contribute to altered entrainment behavior. 3xTg mice demonstrated heightened negative masking, a circadian behavior measuring responses to different levels of light, and re-entrained dramatically faster than WT mice in a jet lag experiment performed in dim light. 3xTg mice show a heightened sensitivity to light as a circadian cue that may contribute to accelerated photic re-entrainment. Together, these experiments demonstrate novel circadian behavioral phenotypes with heightened responses to photic cues in AD model mice which are not dependent on tauopathy or microglia.

Inceptor as a regulator of brain insulin sensitivity.

While historically viewed as an insulin insensitive organ, it is now accepted that insulin has a role in brain physiology. Changes in brain insulin and IGF1 signaling have been associated with neurological diseases, however the molecular factors regulating brain insulin sensitivity remain uncertain. In this study, we proposed that a recently described protein, termed Inceptor, may play a role in brain insulin and IGF1 resistance. We studied Inceptor in healthy and diseased nervous tissue to understand the distribution of the protein and examine how it may change in states of insulin resistance. We found that Inceptor is in fact present in cerebellum, hippocampus, hypothalamus, and cortex of the brain in neurons, with higher levels in cortex of female compared to male mice. We also confirmed that Inceptor colocalized with IR and IGF1R in brain. We saw little difference in insulin receptor signaling following Inceptor knockdown in neuron cultures, or in Inceptor levels with high-fat diet in mouse or Alzheimer's disease in mouse or human tissue. These results all provide significant advancements to our understanding of Inceptor in the brain. PROTOCOL REGISTRATION: The Stage 1 registered report manuscript was accepted-in-principle on 9 August 2022. This manuscript was registered through Open Science Forum (OSF) on 24 August 2022 and is available here: https://osf.io/9q8sw .

Global profiling identifies a stress-responsive tyrosine site on EDC3 regulating biomolecular condensate formation.

RNA granules are cytoplasmic condensates that organize biochemical and signaling complexes in response to cellular stress. Functional proteomic investigations under RNA-granule-inducing conditions are needed to identify protein sites involved in coupling stress response with ribonucleoprotein regulation. Here, we apply chemical proteomics using sulfonyl-triazole (SuTEx) probes to capture cellular responses to oxidative and nutrient stress. The stress-responsive tyrosine and lysine sites detected mapped to known proteins involved in processing body (PB) and stress granule (SG) pathways, including LSM14A, FUS, and Enhancer of mRNA-decapping protein 3 (EDC3). Notably, disruption of EDC3 tyrosine 475 (Y475) resulted in hypo-phosphorylation at S161 and S131 and altered protein-protein interactions (PPIs) with decapping complex components (DDX6, DCP1A/B) and 14-3-3 proteins. This resulting mutant form of EDC3 was capable of rescuing the PB-deficient phenotype of EDC3 knockout cells. Taken together, our findings identify Y475 as an arsenic-responsive site that regulates RNA granule formation by coupling EDC3 post-translational modification and PPI states.

The brain as an insulin-sensitive metabolic organ.

BACKGROUND: The brain was once thought of as an insulin-insensitive organ. We now know that the insulin receptor is present throughout the brain and serves important functions in whole-body metabolism and brain function. Brain insulin signaling is involved not only in brain homeostatic processes but also neuropathological processes such as cognitive decline and Alzheimer's disease. SCOPE OF REVIEW: In this review, we provide an overview of insulin signaling within the brain and the metabolic impact of brain insulin resistance and discuss Alzheimer's disease, one of the neurologic diseases most closely associated with brain insulin resistance. MAJOR CONCLUSIONS: While brain insulin signaling plays only a small role in central nervous system glucose regulation, it has a significant impact on the brain's metabolic health. Normal insulin signaling is important for mitochondrial functioning and normal food intake. Brain insulin resistance contributes to obesity and may also play an important role in neurodegeneration.

Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes.

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.

Oxidized cholesterol species as signaling molecules in the brain: diabetes and Alzheimer's disease.

Type 2 diabetes is associated with adverse central nervous system effects, including a doubled risk for Alzheimer's disease (AD) and increased risk of cognitive impairment, but the mechanisms connecting diabetes to cognitive decline and dementia are unknown. One possible link between these diseases may be the associated alterations to cholesterol oxidation and metabolism in the brain. We will survey evidence demonstrating alterations to oxysterols in the brain in AD and diabetes and how these oxysterols could contribute to pathology, as well as identifying research questions that have not yet been addressed to allow for a fuller understanding of the role of oxysterols in AD and diabetes.

Insulin regulates astrocyte gliotransmission and modulates behavior.

Complications of diabetes affect tissues throughout the body, including the central nervous system. Epidemiological studies show that diabetic patients have an increased risk of depression, anxiety, age-related cognitive decline, and Alzheimer's disease. Mice lacking insulin receptor (IR) in the brain or on hypothalamic neurons display an array of metabolic abnormalities; however, the role of insulin action on astrocytes and neurobehaviors remains less well studied. Here, we demonstrate that astrocytes are a direct insulin target in the brain and that knockout of IR on astrocytes causes increased anxiety- and depressive-like behaviors in mice. This can be reproduced in part by deletion of IR on astrocytes in the nucleus accumbens. At a molecular level, loss of insulin signaling in astrocytes impaired tyrosine phosphorylation of Munc18c. This led to decreased exocytosis of ATP from astrocytes, resulting in decreased purinergic signaling on dopaminergic neurons. These reductions contributed to decreased dopamine release from brain slices. Central administration of ATP analogs could reverse depressive-like behaviors in mice with astrocyte IR knockout. Thus, astrocytic insulin signaling plays an important role in dopaminergic signaling, providing a potential mechanism by which astrocytic insulin action may contribute to increased rates of depression in people with diabetes, obesity, and other insulin-resistant states.

Regional differences in brain glucose metabolism determined by imaging mass spectrometry.

OBJECTIVE: Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). METHODS: To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 µm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. RESULTS: In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. CONCLUSION: These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution.

Small nuclear RING finger protein stimulates the rat luteinizing hormone-beta promoter by interacting with Sp1 and steroidogenic factor-1 and protects from androgen suppression.

GnRH controls expression of the LH subunit genes, alpha and LHbeta, with the LHbeta subunit regulated most dramatically. Two enhancer regions, distal and proximal, on the rat LHbeta gene promoter cooperate for full basal expression and GnRH stimulation. It has been hypothesized that the transcription factors binding to these regions, Sp1, Egr-1, and steroidogenic factor 1 (SF-1), may interact directly or indirectly via a coactivator. One such coactivator may be small nuclear RING finger protein (SNURF), which is expressed in pituitary tissue and the LbetaT2 gonadotrope cell line. In transfection experiments in LbetaT2 cells, SNURF stimulated basal expression of LHbeta and increased overall GnRH stimulation. SNURF specifically stimulated LHbeta, with no effect on the alpha-subunit promoter. SNURF interacts with Sp1 and SF-1, but not Egr-1, in pull-down experiments. Point mutations or deletions of SNURF functional domains demonstrated that Sp1 and SF-1 interactions with SNURF are required for SNURF stimulatory effects on the LHbeta promoter. Endogenous SNURF is associated with the LHbeta promoter on native chromatin, suggesting that it plays a physiological role in LHbeta gene expression. SNURF also binds the androgen receptor, and SNURF overexpression overcomes androgen suppression of GnRH-stimulated LHbeta but not alphasubunit promoter activity. SNURF mutations that disrupt Sp1 or SF-1 binding eliminate rescue by SNURF. We conclude that SNURF may mediate interactions between the distal and proximal GnRH response regions of the LHbeta promoter to stimulate transcription and can also protect the promoter from androgen suppression.