Selective ablation of type 3 adenylyl cyclase in somatostatin-positive interneurons produces anxiety-and depression-like behaviors in mice

BACKGROUND Major depressive disorder(MDD)is a highly disabling psychiatric syndrome associated with deficits of specific subpopulations of cortical GABAergic interneurons;however,the underlying molecular mechanism remains unknown.Type 3 adenylyl cyclase(ADCY3,AC3),which is important for neuronal excitability,has been implicated in MDD in a genome-wide association study in humans.Moreover,a study reported that ablation of AC3 in mice caused similar symptoms as MDD patients.AIM To determine if disruption of the AC3 gene in different subtypes of GABAergic interneurons of mice causes depression-like behaviors.METHODS Using immunohistochemistry,we investigated the expression of AC3 in two major subtypes GABAergic interneurons:Somatostatin-positive(SST+)and parvalbumin-positive(PV+)neurons.Genetic manipulations were used to selectively disrupt AC3 expression in SST+or PV+interneurons.A series of behavior tests including rotarod test,open field test(OFT),elevated plus maze test(EPM),forced swimming test(FST),and tail suspension test(TST)were used to evaluate the motor ability,anxiety-and depression-like behaviors,respectively.RESULTS Our results indicate that approximately 90.41%of SST+and 91.22%of PV+interneurons express AC3.After ablation of AC3 in SST+interneurons,the mice spent comparable time in the center area in OFT,but significantly less time in the open arms and low frequency of entries to the open arms in EPM.Furthermore,these mice showed prolonged immobility in FST and more freezing in TST.However,there were no significant changes in these behaviors after specific disruption of AC3 in PV+interneurons.CONCLUSION This study indicates that ablation of AC3 in SST+interneurons of mice increases anxiety-and depression-like behaviors in mice,supporting the general hypothesis that decreased AC3 activity may play a role in human depression.

A Putative Protein with No Known Function in Arabidopsis thaliana Harbors a Domain with Adenylyl Cyclase Activity

Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC261-388) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule. From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.

Effect of Interleukin-1β on the Variation of Adenylyl Cyclase Expression in Rats with Seizures Induced by L-Glutamate

Summary: To explore the mechanism of interleukin-1beta ( IL-1β) in the onset of seizure and the effect of IL-1β on the expression of adenylyl cyclase (AC) in rats with seizure induced by L-glutamate. Experimental rats were first injected with IL-1β and then L-glutamate (a dose under the threshold) was injected into the right lateral ventricle. The rats were sacrificed 4 h after the onset of epileptic activity and examined for changes in behavior, immunohistochemistry and compared with those with seizure induced by L-glutamate alone. It was found that the expression of AC in hippocampal and neocortex of rats with seizure induced by IL-1β and L-glutamate were stronger than that of control group (P<0.05), without significant difference found between the L-glutamate group and IL-1β plus L-glutamate group in the expression of AC, the latent period and the severity of seizure. When IL-ra were given (i.c.v.) first, there was no epileptic activity and the expression of AC did not increase. There were no differences in the expression of AC of rats with IL-1ra and that of control rats. But when 2-methyl-2-(carboxycyclopropyl)glycine (MCCG) was given (i.c.v.) first, the strongest expression of AC, the shortest latent period and the the most serious seizure activities were observed. The results indicated that IL-1β could facilitate the onset of epilepsy induced by L-glutamate through IL-1R, metabotropic glutamate receptors might work with IL-1R and the increased expression of AC might be involved in the process.

G protein b_1λ_2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) b1g2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni ) were used to express the recombinant protein Gb1g2. The cell membrane containing Gb1g2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gb1g2 could signifi-cantly stimulate AC2 activity. The interaction of b1g2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gb1g2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gb1g2 was the same as AC2 activity domain which was stimulated by Gb1g2.

DYNAMIC CHANGES IN Giα2 LEVELS IN THE RAT HEART ASSOCIATED WITH IMPAIRED CARDIACFUNCTION FOLLOWING ACUTE MYOCARDIAL INFA RCTION

Changes in the functional activity and levels of Gsa and Gia in heart failure have been studied predominantly in the end-stage failing heart.The objective of this study was to determine if levels and function of Gsa and Gia2 in rat hearts change over time following acute myocar(lial infarction (MI) and if so,whether the changes in G proteins are associated with changes in heart function.As compared with sham-operated controls, Giα2, level of MI rats did not change at day l,increased by 64% at day 3 (P<0.01) and 55% at day 9 (P< 0.05)accompanied by reduced adenylyl cyclase activity,and returned to control by day 21. By contrast,Gsa level did not change at any time. Cardiac function in MI animals was markedly impaired at days 1,3 and 9 as evidenced by substantial elevation in LVEDP and reduction in +and -dp/dtmax,and partially restored at day 21. The increased Gia2level in MI rats at days 3 and 9 correlated positively to LVEDP(P< 0.05), and negatively to +and -dp/dtmax (p < 0. 01).The results show a three phase dynamic pattern in Gia2 level following acute MI:a lag phase, an increased expression phase associated with marked impairment of heart function,and a late phase in which the expression retums to control level accompanied by partially restored cardlac function.The results suggest that ② in G protein-mediated pathways,cardiac myocytes respond to MI Via regulating the gene expression of the inhibitory pathway, and ② up-regulation of Gia2 levels is related to the severity of impairment in cardiac function.

Elaborating the Functional Roles of a Leucine-Rich Repeat Protein from Arabidopsis thaliana

Plants, just like any other living organism, naturally get attacked by various pathogenic microorganisms such as bacteria, fungi and viruses. However, unlike animals that utilize their specialized circulatory macrophage system to protect themselves, plants instead use a multi-layered complex system termed the plant innate immunity, which recognizes pathogens and transducing downstream defense responses. They have developed a unique type of trans-membrane receptors or R proteins, which extracellularly, are capable of recognizing pathogen-associated molecular patterns (PAMP) such as flagellin and chitin, while intracellularly, they activate their harbored nucleotide cyclases (NCs) such as adenylyl cyclases (ACs), to generate second messenger molecules such as 3’,5’-cyclic adenosine monophosphate (cAMP), which then propagates and magnifies the defense response. To date, only a single R protein from Arabidopsis thaliana (AtLRR) has been shown to possess AC activity as well as having the ability to defend plants against infection by biotrophic and hemi-biotrophic pathogens. Therefore, in order to further broaden information around the functional roles of this protein (AtLRR), we explored it further, using an array of web-based tools or bioinformatics. These included structural analysis, anatomical expression analysis, developmental expression analysis, co-expression analysis, functional enrichment analysis, stimulus-specific expression analysis and promoter analysis. Findings from structural analysis showed that AtLRR is a multi-domain, trans-membrane molecule that is multi-functional, and thus consistent with all known R-proteins. Findings from anatomical and developmental expression analyses showed that AtLRR is mostly expressed in pollen grains and flowers, senescing leaves as well as during the development of seeds, shoots, roots, seedlings, leaves, flowers, and siliques, linking it to the three key plant physiological processes of reproduction, defense and development respectively. Lastly, findings from co-expression, functional enrichment, stimulus-specific expression and promoter analyses, showed that AtLRR is mostly co-expressed with several other proteins linked to disease resistance, plant reproduction and plant development. Activities and functions of such protein are also commonly regulated by cAMP via a common W-box promoter. So, all in all, our study managed to establish that besides being strongly involved in disease resistance against biotrophic and hemi-biotrophic pathogens, AtLRR also plays key roles in plant development (seed, shoot, root, seedling, leaf, and silique development) and reproduction (flowering, and pollen tube growth and re-orientation), whereby it effects its functions via a W-box or WRKY transcription factor, TTGACY, mediated by cAMP.