Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action

Improved method for estimation of inorganic phosphate: Implications for its application in enzyme assays

88-93The conventional method of Fiske and Subba Row for the estimation of inorganic phosphate (Pi) is although rapid, but suffers from the disadvantage that the color is unstable and hence the optical density (OD) measurements have to be carried out within a short time span of 8-12 min. This poses a restriction on the number of samples, which can be handled in a batch. Although, modified procedures involving use of alternate reducing agents/or increasing the concentration of H₂SO₄ in conventional method have been subsequently developed, but the problem of color stability could not be solved. In addition, the use of higher concentrations H₂SO₄ has rendered the methods unsuitable in enzyme assays, especially if the acid labile phosphate containing substrates have been used. In the present study, attempts have been made to suitably modify the method to improve the stability of the color and sensitivity and also for its applicability in enzyme assays, especially when acid labile phosphate containing substrates such as ATP is used. We used the higher concentrations (0.625, 0.8 and 1.0 N) of H₂SO₄ rather than 0.5 N used in the conventional assay procedures. Under these conditions, the reagent blanks do not develop color for up to 24 h, whereas the intensity of the molybdenum blue color in the standard and/or experimental tubes increased with time reaching optimum value at 24 h. Simultaneously, the absorption maximum shifts from 660 nm to 820 nm. The highest concentration of H₂SO₄ (1.0 N) is found to be the most effective in the process of color development. The sensitivity of the method is from 1.7 to 2.1 times higher, as compared to the conventional Fiske and Subba Row method for the measurements carried out at the end of 15 min at 820 nm and with the highest concentration of H₂SO₄ (1.0 N); the sensitivity increased 4.8-fold at the end of 24 h. Presence of glucose and sucrose (1-10 mM), NaCl and KCl (5-100 mM), MgCl₂ (1-10 mM) and BSA (10 to 500 µg per assay tube) do not interfere either with color development or with OD measurements. The extent of ATP hydrolysis is 1.6 to 3.4% for up to 1 h, depending upon the concentration of H₂SO₄ used. Only negligible hydrolysis of G6P is observed under these conditions. These results suggest that the presently modified method is suitable for Pi analysis in the enzyme assays, in the presence of labile phosphate containing substrates

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate's efficacy against bipolar disorder

BACKGROUND: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro. METHODS: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis-Menten kinetics. RESULTS: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4. CONCLUSIONS: VPA’s ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA’s selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA’s efficacy against bipolar disorder

Intranasal delivery of mitochondria targeted neuroprotective compounds for traumatic brain injury: screening based on pharmacological and physiological properties

Abstract Targeting drugs to the mitochondrial level shows great promise for acute and chronic treatment of traumatic brain injury (TBI) in both military and civilian sectors. Perhaps the greatest obstacle to the successful delivery of drug therapies is the blood brain barrier (BBB). Intracerebroventricular and intraparenchymal routes may provide effective delivery of small and large molecule therapies for preclinical neuroprotection studies. However, clinically these delivery methods are invasive, and risk inadequate exposure to injured brain regions due to the rapid turnover of cerebral spinal fluid. The direct intranasal drug delivery approach to therapeutics holds great promise for the treatment of central nervous system (CNS) disorders, as this route is non-invasive, bypasses the BBB, enhances the bioavailability, facilitates drug dose reduction, and reduces adverse systemic effects. Using the intranasal method in animal models, researchers have successfully reduced stroke damage, reversed Alzheimer’s neurodegeneration, reduced anxiety, improved memory, and delivered neurotrophic factors and neural stem cells to the brain. Based on literature spanning the past several decades, this review aims to highlight the advantages of intranasal administration over conventional routes for TBI, and other CNS disorders. More specifically, we have identified and compiled a list of most relevant mitochondria-targeted neuroprotective compounds for intranasal administration based on their mechanisms of action and pharmacological properties. Further, this review also discusses key considerations when selecting and testing future mitochondria-targeted drugs given intranasally for TBI. Graphical Abstrac

Coordinated Gene Expression of Neuroinflammatory and Cell Signaling Markers in Dorsolateral Prefrontal Cortex during Human Brain Development and Aging

<div><p>Background</p><p>Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases.</p><p>Hypothesis</p><p>Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades.</p><p>Methods</p><p>We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes.</p><p>Results</p><p>Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; <i>TLR4</i>, <i>IL1R1</i>, <i>NFKB1</i>, <i>MOBP</i>, <i>PLA2G4A</i>, and <i>PTGS2</i> expression increased in the first years of life, while expression of synaptic genes <i>GAP43</i> and <i>DBN1</i> decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as <i>NFKB1</i>, <i>TRAF6</i>, <i>TLR4</i>, <i>IL1R1</i>, <i>TSPO</i>, and <i>GFAP</i>, but downregulated for neurotrophic and synaptic integrity genes such as <i>BDNF</i>, <i>NGF</i>, <i>PDGFA</i>, <i>SYN</i>, and <i>DBN1</i>.</p><p>Conclusions</p><p>Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate throughout the lifespan underlie different phenotypic processes during Aging compared to Development.</p></div

Intranasal post-cardiac arrest treatment with orexin-A facilitates arousal from coma and ameliorates neuroinflammation

<div><p>Cardiac arrest (CA) entails significant risks of coma resulting in poor neurological and behavioral outcomes after resuscitation. Significant subsequent morbidity and mortality in post-CA patients are largely due to the cerebral and cardiac dysfunction that accompanies prolonged whole-body ischemia post-CA syndrome (PCAS). PCAS results in strong inflammatory responses including neuroinflammation response leading to poor outcome. Currently, there are no proven neuroprotective therapies to improve post-CA outcomes apart from therapeutic hypothermia. Furthermore, there are no acceptable approaches to promote cortical or cognitive arousal following successful return of spontaneous circulation (ROSC). Hypothalamic orexinergic pathway is responsible for arousal and it is negatively affected by neuroinflammation. However, whether activation of the orexinergic pathway can curtail neuroinflammation is unknown. We hypothesize that targeting the orexinergic pathway via intranasal orexin-A (ORXA) treatment will enhance arousal from coma and decrease the production of proinflammatory cytokines resulting in improved functional outcome after resuscitation. We used a highly validated CA rat model to determine the effects of intranasal ORXA treatment 30-minute post resuscitation. At 4hrs post-CA, the mRNA levels of proinflammatory markers (IL1β, iNOS, TNF-α, GFAP, CD11b) and orexin receptors (ORX1R and ORX2R) were examined in different brain regions. CA dramatically increased proinflammatory markers in all brain regions particularly in the prefrontal cortex, hippocampus and hypothalamus. Post-CA intranasal ORXA treatment significantly ameliorated the CA-induced neuroinflammatory markers in the hypothalamus. ORXA administration increased production of orexin receptors (ORX1R and ORX2R) particularly in hypothalamus. In addition, ORXA also resulted in early arousal as measured by quantitative electroencephalogram (EEG) markers, and recovery of the associated behavioral neurologic deficit scale score (NDS). Our results indicate that intranasal delivery of ORXA post-CA has an anti-inflammatory effect and accelerates cortical EEG and behavioral recovery. Beneficial outcomes from intranasal ORXA treatment lay the groundwork for therapeutic clinical approach to treating post-CA coma.</p></div

Similarity matrices (hierarchically clustered heat maps) of Pearson's r correlations of gene expression levels with age in Development (A) and Aging (B) groups.

<p>Red indicates negatively correlated associations; green are positively correlated associations, while black represents non-significant associations between gene pairs. Genes are clustered hierarchically along the left y-axis, which is mirrored above in each heat map.</p

Significant linear regressions of gene expression during both Development and Aging intervals (top), and Aging interval alone (bottom).

<p>Scatterplots illustrating log₂ gene expression over age in years. An increase or decrease of 1 on the log₂ scale (y-axis) represents a two-fold change in gene expression in the positive or negative direction, respectively. Each data point represents observation from one brain (Development: n = 87; Aging: n = 144). Gene name (p-value during Development, p-value during Aging) - <i>CX3CR1</i> (p<0.0001, p<0.0001), <i>NGF</i> (p = 0.006, p = 0.002), <i>MOBP</i> (p<0.0001, p = 0.02), <i>NFκB1</i> (p = 0.01, p = 0.01), SNCA (p<0.0001, p = 0.002). Genes significant in only Aging interval – <i>GFAP</i> (p<0.0001), <i>TSPO</i> (p = 0.006), <i>BDNF</i> (p<0.0001), <i>PTGS2</i> (p = 0.0003), <i>CX3CR1</i> (p = 0.03).</p