Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Improved repair of dermal wounds in mice lacking microRNA‐155

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155^−/−) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155^−/− mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155^−/− mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

MiR‐155 promotes interleukin‐1β‐induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1‐mediated PI3K/Akt pathway

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation, in which elevated chondrocyte apoptosis and catabolic activity play an important role. MicroRNA‐155 (miR‐155) has recently been shown to regulate apoptosis and catabolic activity in some pathological circumstances, yet, whether and how miR‐155 is associated with OA pathology remain unexplored. We report here that miR‐155 level is significantly up‐regulated in human OA cartilage biopsies and also in primary chondrocytes stimulated by interleukin‐1β (IL‐1β), a pivotal pro‐catabolic factor promoting cartilage degradation. Moreover, miR‐155 inhibition attenuates and its overexpression promotes IL‐1β‐induced apoptosis and catabolic activity in chondrocytes in vitro. We also demonstrate that the PIK3R1 (p85α regulatory subunit of phosphoinositide 3‐kinase (PI3K)) is a target of miR‐155 in chondrocytes, and more importantly, PIK3R1 restoration abrogates miR‐155 effects on chondrocyte apoptosis and catabolic activity. Mechanistically, PIK3R1 positively regulates the transduction of PI3K/Akt pathway, and a specific Akt inhibitor reverses miR‐155 effects on promoting chondrocyte apoptosis and catabolic activity, phenocopying the results obtained via PIK3R1 knockdown, hence establishing that miR‐155 promotes chondrocyte apoptosis and catabolic activity through targeting PIK3R1‐mediated PI3K/Akt pathway activation. Altogether, our study discovers novel roles and mechanisms of miR‐155 in regulating chondrocyte apoptosis and catabolic activity, providing an implication for therapeutically intervening cartilage degradation and OA progression.     

MicroRNA‐155‐3p promotes glioma progression and temozolomide resistance by targeting Six1

The prognosis of glioma is generally poor and is the cause of primary malignancy in the brain. The role of microRNAs has been implicated in tumour inhibition or activation. In several cancers, the Six1 signalling pathway has been found to be aberrant and also relates to the formation of tumours. We analysed the database for expression profiles and clinical specimens of various grades of glioma to assess microRNA‐155‐3p (miR‐155‐3p) expression. The role of miR‐155‐3p in glioblastoma, cell cycle, proliferation, apoptosis and resistance to temozolomide was assessed in vitro through flow cytometry and cell proliferation assays. Bioinformatics analyses, and assays using luciferase reporter, and immunoblotting revealed that miR‐155‐3p targets Six1 and that the relationship between glioma and healthy brain tissues was significantly inverse. In rescue experiments, overexpressed Six1 revoked the changes in cell cycle distribution, proliferation and resistance to temozolomide estimated by apoptosis induced by overexpressed miR‐155‐3p. MiR‐155‐3p inhibition reduced glioma cell growth and proliferation in the brain of a mouse model and increased the survival of mice with gliomas. Thus, miR‐155‐3p modulates Six1 expression and facilitates the progression of glioblastoma and resistance to temozolomide and may act as a novel diagnostic biomarker and a target for glioma treatment.     

Virus‐induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ‐aminobutyric acid metabolic pathway

γ‐Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA‐Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus‐induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA‐Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs‐ and SlGABA‐Ts‐silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH‐silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ‐hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ‐hydroxybutyrate and subsequent ROS accumulation under salt stress.     

Seizure‐induced impairment in neuronal ketogenesis: Role of zinc‐α2‐glycoprotein in mitochondria

Ketone bodies (KBs) were known to suppress seizure. Untraditionally, neurons were recently reported to utilize fatty acids and produce KBs, but the effect of seizure on neuronal ketogenesis has not been researched. Zinc‐α2‐glycoprotein (ZAG) was reported to suppress seizure via unclear mechanism. Interestingly, ZAG was involved in fatty acid β‐oxidation and thus may exert anti‐epileptic effect by promoting ketogenesis. However, this promotive effect of ZAG on neuronal ketogenesis has not been clarified. In this study, we performed immunoprecipitation and mass spectrometry to identify potential interaction partners with ZAG. The mechanisms of how ZAG translocated into mitochondria were determined by quantitative coimmunoprecipitation after treatment with apoptozole, a heat shock cognate protein 70 (HSC70) inhibitor. ZAG level was modulated by lentivirus in neurons or adeno‐associated virus in rat brains. Seizure models were induced by magnesium (Mg²⁺)‐free artificial cerebrospinal fluid in neurons or intraperitoneal injection of pentylenetetrazole kindling in rats. Ketogenesis was determined by cyclic thio‐NADH method in supernatant of neurons or brain homogenate. The effect of peroxisome proliferator–activated receptor γ (PPARγ) on ZAG expression was examined by Western blot, quantitative real‐time polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation qRT‐PCR. We found that seizure induced ketogenesis deficiency via a ZAG‐dependent mechanism. ZAG entered mitochondria through a HSC70‐dependent mechanism, promoted ketogenesis by binding to four β‐subunits of long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (HADHB) and alleviated ketogenesis impairment in a neuronal seizure model and pentylenetetrazole‐kindled epileptic rats. Additionally, PPARγ activation up‐regulated ZAG expression by binding to promoter region of AZGP1 gene and promoted ketogenesis through a ZAG‐dependent mechanism.     

APOE‐modulated Aβ‐induced neuroinflammation in Alzheimer's disease: current landscape,novel data,and future perspective

Chronic glial activation and neuroinflammation induced by the amyloid‐β peptide (Aβ) contribute to Alzheimer's disease (AD) pathology. APOE4 is the greatest AD‐genetic risk factor; increasing risk up to 12‐fold compared to APOE3, with APOE4‐specific neuroinflammation an important component of this risk. This editorial review discusses the role of APOE in inflammation and AD, via a literature review, presentation of novel data on Aβ‐induced neuroinflammation, and discussion of future research directions. The complexity of chronic neuroinflammation, including multiple detrimental and beneficial effects occurring in a temporal and cell‐specific manner, has resulted in conflicting functional data for virtually every inflammatory mediator. Defining a neuroinflammatory phenotype (NIP) is one way to address this issue, focusing on profiling the changes in inflammatory mediator expression during disease progression. Although many studies have shown that APOE4 induces a detrimental NIP in peripheral inflammation and Aβ‐independent neuroinflammation, data for APOE‐modulated Aβ‐induced neuroinflammation are surprisingly limited. We present data supporting the hypothesis that impaired apoE4 function modulates Aβ‐induced effects on inflammatory receptor signaling, including amplification of detrimental (toll‐like receptor 4‐p38α) and suppression of beneficial (IL‐4R‐nuclear receptor) pathways. To ultimately develop APOE genotype‐specific therapeutics, it is critical that future studies define the dynamic NIP profile and pathways that underlie APOE‐modulated chronic neuroinflammation.

     

Renalase contributes to the renal protection of delayed ischaemic preconditioning via the regulation of hypoxia‐inducible factor‐1α

Ischaemic preconditioning (IPC) attenuates acute kidney injury (AKI) from renal ischaemia reperfusion. Renalase, an amine oxidase secreted by the proximal tubule, not only degrades circulating catecholamines but also protects against renal ischaemia reperfusion injury. Here, it has been suggested that the renoprotective effect of renal IPC is partly mediated by renalase. In a model of brief intermittent renal IPC, the increased cortex renalase expression was found to last for 48 hrs. IPC significantly reduced renal tubular inflammation, necrosis and oxidative stress following renal ischaemia reperfusion injury. Such effects were attenuated by blocking renalase with an anti‐renalase monoclonal antibody. We further demonstrated that renalase expression was up‐regulated by hypoxia in vitro via an hypoxia‐inducible factor (HIF)‐1α mechanism. The IPC‐induced up‐regulation of renalase in vivo was also reduced by pre‐treatment with an HIF‐1α inhibitor, 3‐(5′‐Hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole. In summary, the renoprotective effect of IPC is partly dependent on the renalase expression, which may be triggered by hypoxia via an HIF‐1α mechanism. Endogenous renalase shows potential as a therapeutic agent for the prevention and treatment of AKI.     

Accumulation patterns of endogenous β‐aminobutyric acid during plant development and defence in Arabidopsis thaliana

• We recently discovered that β‐aminobutyric acid (BABA), a molecule known for its ability to prime defences in plants, is a natural plant metabolite. However, the role played by endogenous BABA in plants is currently unknown. In this study we investigated the systemic accumulation of BABA during pathogen infection, levels of BABA during plant growth and development and analysed mutants possibly involved in BABA transport or regulation.
• BABA was quantified by LC‐MS using an improved method adapted from a previously published protocol. Systemic accumulation of BABA was determined by analysing non‐infected leaves and roots after localised infections with Plectosphaerella cucumerina or Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2. The levels of BABA were also quantified in different plant tissues and organs during normal plant growth, and in leaves during senescence. Mutants affecting amino acid transport (aap6, aap3, prot1 and gat1), γ‐aminobutyric acid levels (pop2) and senescence/defence (cpr5‐2) were analysed.
• BABA was found to accumulate only locally after bacterial or fungal infection, with no detectable increase in non‐infected systemic plant parts. In leaves, BABA content increased during natural and induced senescence. Reproductive organs had the highest levels of BABA, and the mutant cpr5‐2 produced constitutively high levels of BABA.
• Synthetic BABA is highly mobile in the receiving plant, whereas endogenous BABA appears to be produced and accumulated locally in a tissue‐specific way. We discuss a possible role for BABA in age‐related resistance and propose a comprehensive model for endogenous and synthetic BABA.

     

β‐arrestin 2 negatively regulates NOD2 signalling pathway through association with TRAF6 in microglia after cerebral ischaemia/reperfusion injury

We previously reported that nucleotide‐binding oligomerization domain‐containing protein (NOD) 2 was involved in the inflammatory responses to cerebral ischaemia/reperfusion (I/R) insult. However, the mechanism by which NOD2 participates in brain ischaemic injury and the regulation of NOD2 in the process are still obscure. Increased β‐arrestin 2 (ARRB2) expression was observed in microglia following cerebral I/R in wild‐type mice besides the up‐regulation of NOD2 and TRAF6. Stimulation of NOD2 by muramyl dipeptide (MDP) in BV2 cells induced the activation of NF‐κB by the phosphorylation of p65 subunit and the degradation of IκBα. Meanwhile, the protein level of Cyclooxygenase‐2 (COX‐2), the protein expression and activity of MMP‐9 were significantly increased in BV2 cells after administration of MDP. Furthermore, overexpression of ARRB2 significantly suppressed the inflammation induced by MDP, silence of ARRB2 significantly enhanced the inflammation induced by MDP in BV2 cells. In addition, we observed endogenous interaction of TRAF6 and ARRB2 after stimulation of MDP or cerebral I/R insult, indicating ARRB2 negatively regulates NOD2‐triggered inflammatory signalling pathway by associating with TRAF6 in microglia after cerebral I/R injury. Finally, the in vivo study clearly confirmed that ARRB2 negatively regulated NOD2‐induced inflammatory response, as ARRB2 deficiency exacerbated stroke outcomes and aggravated the NF‐κB signalling pathway induced by NOD2 stimulation after cerebral I/R injury. These findings revealed ARRB2 negatively regulated NOD2 signalling pathway through the association with TRAF6 in cerebral I/R injury.     

γ‐Aminobutyric acid quantification in small volume biological samples through enzymatically induced electrochemiluminescence

γ‐Aminobutyric acid (GABA) is a well‐known neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system and participates in several processes outside the brain. A reliable quantification method is needed to determine its role in different physiological and pathological conditions. However, GABA measurements have several challenges because GABA is neither fluorescent nor electroactive, and it is difficult to detect using enzymatic reactions because no oxidases or dehydrogenases have been identified. Several methods have been developed to quantify GABA concentrations based on the instrumentation available, the sensitivity required, and the volume of samples analyzed. Most of these methods use high‐performance liquid chromatography (HPLC). Here, we describe a method for quantifying GABA concentrations in small volume samples using enzymatically‐induced electrochemiluminescence with the well‐known GABAse complex, which produces glutamate for use in a luminescent reaction with glutamate oxidase and luminol in an electrochemiluminescence cell. The luminescence obtained was proportional to the GABA concentrations in the micromolar range (1–1000), with linear r² values > 0.95. GABA standards were treated with the enzymatic reactors to generate glutamate (Glu), which was measured simultaneously with an HPLC technique, to validate this new procedure. The assay was further used to determine GABA concentrations in hippocampal extracts. This alternative may be used to quantify GABA levels in fluid samples, such as microdialysates, other perfusates and tissue extracts. Thus, the method presented here is a good alternative for monitoring GABA levels with good sensitivity compared with the traditional methods that are still in use.