Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency.

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain.

Depletion of COPI in cancer cells: the role of reactive oxygen species in the induction of lipid accumulation, noncanonical lipophagy and apoptosis.

The coatomer protein complex 1 (COPI) is a multisubunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells.

Pyridostigmine bromide elicits progressive and chronic impairments in the cholinergic anti-inflammatory pathway in the prefrontal cortex and hippocampus of male rats.

Gulf War Illness (GWI) is a multi-symptom illness that continues to affect over 250,000 American Gulf War veterans. The causes of GWI remain equivocal; however, prophylactic use of the acetylcholinesterase inhibitor pyridostigmine bromide (PB), and the stress of combat have been identified as two potential causative factors. Both PB and stress alter acetylcholine (ACh), which mediates both cognition and anti-inflammatory responses.

Hippocampal-specific insulin resistance elicits behavioral despair and hippocampal dendritic atrophy.

Insulin resistance is a major contributor to the neuroplasticity deficits observed in patients with metabolic disorders. However, the relative contribution of peripheral versus central insulin resistance in the development of neuroplasticity deficits remains equivocal. To distinguish between peripheral and central insulin resistance, we developed a lentiviral vector containing an antisense sequence selective for the insulin receptor (LV-IRAS).

High-fat diet induces neuroinflammation and reduces the serotonergic response to escitalopram in the hippocampus of obese rats.

Clinical studies indicate that obese individuals have an increased risk of developing co-morbid depressive illness and that these patients have reduced responses to antidepressant therapy, including selective serotonin reuptake inhibitors (SSRIs). Obesity, a condition of chronic mild inflammation including obesity-induced neuroinflammation, is proposed to contribute to decreases in synaptic concentrations of neurotransmitters like serotonin (5HT) by decreasing 5HT synthesis in the dorsal raphe nucleus (DRN) and/or affecting 5HT reuptake in DRN target regions like the hippocampus. In view of these observations, the goal of the current study was to examine inflammatory markers and serotonergic dynamics in co-morbid obesity and depression.

Fumarate and oxidative stress synergize to promote stability of C/EBP homologous protein in the adipocyte.

C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown.

A real time screening assay for cannabinoid CB1 receptor-mediated signaling.

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus.

Succination of Protein Disulfide Isomerase Links Mitochondrial Stress and Endoplasmic Reticulum Stress in the Adipocyte During Diabetes.

Aims: Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress.

Results: Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations.

Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis.

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth.

Tribbles 3 inhibits brown adipocyte differentiation and function by suppressing insulin signaling.

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function.

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome.

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus.

Hippocampal Insulin Resistance Impairs Spatial Learning and Synaptic Plasticity.

Insulin receptors (IRs) are expressed in discrete neuronal populations in the central nervous system, including the hippocampus. To elucidate the functional role of hippocampal IRs independent of metabolic function, we generated a model of hippocampal-specific insulin resistance using a lentiviral vector expressing an IR antisense sequence (LV-IRAS). LV-IRAS effectively downregulates IR expression in the rat hippocampus without affecting body weight, adiposity, or peripheral glucose homeostasis.

The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes.

While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions.

Repeated restraint stress-induced atrophy of glutamatergic pyramidal neurons and decreases in glutamatergic efflux in the rat amygdala are prevented by the antidepressant agomelatine.

Major depressive illness is among the most prevalent neuropsychiatric disorders and is associated with neuroplasticity deficits in limbic structures such as the amygdala. Since exposure to stressful life events is proposed to contribute to depressive illness, our recent studies examined the effects of stress on amygdalar neuroplasticity. These studies determined that repeated stress elicits deficits in glutamatergic activity in the amygdala, neuroplasticity deficits that can be prevented by some but not all antidepressants.

Identification of protein succination as a novel modification of tubulin.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and β-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 μM) inhibited polymerization up to 35% and 59% respectively.

Tianeptine modulates amygdalar glutamate neurochemistry and synaptic proteins in rats subjected to repeated stress.

Stress is a common environmental factor associated with depressive illness and the amygdala is thought to be integral for this association. For example, repeated stress impairs amygdalar neuroplasticity in rodents and these defects parallel amygdalar deficits in depressive illness patients. Because the excitatory neurotransmitter glutamate is important in neuroplasticity, we hypothesized that alterations in amygdalar glutamatergic systems may serve as key players in depressive illness.

Downregulation of hypothalamic insulin receptor expression elicits depressive-like behaviors in rats.

Ongoing epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese. There is a growing appreciation that the complications of obesity extend to the central nervous system (CNS) and may result in increased risk for neurological co-morbidities like depressive illness. One potential mechanistic mediator linking obesity and depressive illness is the adipocyte derived hormone leptin.

Obesity/hyperleptinemic phenotype impairs structural and functional plasticity in the rat hippocampus.

Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese, and there is a growing appreciation that the complications of obesity extend to the central nervous system (CNS). While the vast majority of these studies have focused on the hypothalamus, more recent studies suggest that the complications of obesity may also affect the structural and functional integrity of the hippocampus. A potential contributor to obesity-related CNS abnormalities is the adipocyte-derived hormone leptin.

Obesity/hyperleptinemic phenotype adversely affects hippocampal plasticity: effects of dietary restriction.

Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese and there is a growing appreciation that obesity affects the functional integrity of the central nervous system (CNS). We recently developed a lentivirus (LV) vector that produces an insulin receptor (IR) antisense RNA sequence (IRAS) that when injected into the hypothalamus selectively decreases IR signaling in hypothalamus, resulting in increased body weight, peripheral adiposity and plasma leptin levels. To test the hypothesis that this obesity/hyperleptinemic phenotype would impair hippocampal synaptic transmission, we examined short term potentiation (STP) and long term potentiation (LTP) in the hippocampus of rats that received the LV-IRAS construct or the LV-Control construct in the hypothalamus (hypo-IRAS and hypo-Con, respectively).

Dysregulation of corticosterone secretion in streptozotocin-diabetic rats: modulatory role of the adrenocortical nitrergic system.

An increased activity of the hypothalamo-pituitary-adrenal axis resulting in exaggerated glucocorticoid secretion has been repeatedly described in patients with diabetes mellitus and in animal models of this disease. However, it has been pointed out that experimental diabetes is accompanied by a decreased glucocorticoid response to ACTH stimulation. Because previous studies from our laboratory demonstrate the involvement of nitric oxide (NO) in the modulation of corticosterone production, present investigations were designed to evaluate 1) the impact of streptozotocin (STZ)-induced diabetes on the adrenocortical nitrergic system and 2) the role of NO in the modulation of adrenal steroidogenesis in STZ-diabetic rats.

Insulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent.

In the central nervous system (CNS) insulin mediates a variety of effects including feeding, metabolism and cognition. The cognitive enhancing effects of insulin are proposed to be mediated through activation of insulin receptors in the hippocampus, an important integration center for learning and memory in the mammalian brain. Since less is known regarding insulin signaling events in the hippocampus, the aim of the current study was to determine whether insulin stimulates similar signaling cascades and GLUT4 translocation in the rat hippocampus as has been described in peripheral tissues.

A look inside the diabetic brain: Contributors to diabetes-induced brain aging.

Central nervous system (CNS) complications resulting from diabetes is a problem that is gaining more acceptance and attention. Recent evidence suggests morphological, electrophysiological and cognitive changes, often observed in the hippocampus, in diabetic individuals. Many of the CNS changes observed in diabetic patients and animal models of diabetes are reminiscent of the changes seen in normal aging.

Effects of the AMPA receptor modulator S 18986 on measures of cognition and oxidative stress in aged rats.

Rationale: Development of cognitive-enhancing drugs that delay or halt mild cognitive impairment progression to Alzheimer's disease would be of great benefit.

Objectives: The aim of this study was to examine the ability of (S)-2,3-dihydro-[3,4]-cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S 18986), a positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, to improve behavioral performance and alleviate age-related deficits in oxidative stress status in the prelimbic cortex and hippocampus.

Materials And Methods: Daily administration of S 18986 (0.