GENOMICS OF ACETYLCHOLINESTERASE (AChE) ENZYME AND ITS RELATED RNA CONTENT IN THE BRAIN OF RATTUS NORVEGICUS: A PROSPECTIVE STUDY

Address for Correspondence: Dr. PRK Bhargav MS, MCh, FACS, FIMSA, FAIS, Consultant Endocrine and Metabolic Surgeon, Director and Associate Professor of Endocrine Surgery, Endocare Hospital, Vijayawada, India – 520002, Phone No: +91-866-6617633; +91-9490130798 www.drbhargav.org; endoanswers@gmail.com Inhibition of acetylcholinesterase (AChE), the metabolizing enzyme of acetylcholine is evolving as the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of sex. In the present study, AChE expression was investigated in different brain areas of cerebrum and cerebellum in male and female Rattus norvegicus. On comparing male and female genders, increased AChE activity was seen in cerebrum and cerebellum of female Rattus norvegicus. However, no significant change in AChE activity was found between cerebrum and cerebellum within the same male and female. Thus it appears that sex alters AChE activity in different brain regions (G4 isoform) that may vary in male and female. Sequence analysis revealed that highest divergence was found in between male cerebrum and female cerebrum (11.9) and least divergence was found in between male cerebellum and female cerebellum (6.4) with control AChE NM_EF534897

vital role in cholinergic neurotransmission.The term acetylcholinesterase was introduced in 1949 by Augustintion and Nachmansohn for specific cholinesterase capable of hydrolyzing acetylcholine faster than other esterases.In 1964 the commission of enzymology recommended the name "Acetylcholinesterase" Acetylcholinesterase (AChE) is one of the most efficient enzymes of nervous system which is concentrated at the cholinergic synapses and at neuromuscular synapses where it rapidly hydrolyses the neurotransmitter acetylcholine (Ach) in to choline and acetate thus playing a (Acetylcholine Acetyl hydrolase~ 3.1.1.7)for a true and specific cholinesterase.The distribution of the enzyme in the central and peripheral nerve tissues of different vertebrates demonstrates a high range of variation [1][2][3][4][5][6][7][8][9][10][11][12][13].It has been noted to be localized in non neuronal tissues and Glial cells also [14,15].The enzyme also exhibits molecular diversity with its six different molecular forms and structural dynamics which facilitates its affinity and action with various legends [16][17].In addition, AChE is considered to play several non classical roles independent of its catalytic function i.e. hydrolysis of Ach.These classical and non classical roles of AChE illustrate adequacy about its wide occurrence in neuronal and non neuronal tissues [18][19][20].AChE is widely distributed; it occurs in the central (CNS) and peripheral nervous systems (PNS), and the motor end-plates of the skeletal muscle and the electric organ, but it is also found in many other tissues and in erythrocytes.Therefore such a wide distribution and various functions, molecular forms, structural dynamics etc. of AChE provide adequate base to recall it a versatile enzyme, a detailed knowledge of which, might help to design specific drugs to combat various neurodegenerative diseases associated with this enzyme.2. The highly divergent regions between the AChE sequences when compare with standard sequence are found in male cerebrum where as when compare with male cerebellum, female cerebrum, female cerebellum was less significant, But when compare with male and female cerebrum and cerebellum, the highest divergence was found in between male cerebrum and female cerebrum and least divergence was found in between male cerebellum and female cerebellum (Figure 3).Expression of AChE gene: Expression of Acetylcholinesterase (AChE) was significantly more in female cerebrum and cerebellum when compare with male cerebrum and cerebellum (Figure 4).Also, it was found that Methyl green-pyronin staining on Rattus norvegicus brain regions shows more RNA granules in female cerebrum and female cerebellum (Figure 5).features responsible for their catalysis function.For example, an active site triad (Ser, Glu and His) exist in all the reported AChEs, and the three residues form a plannar array at the bottom of a deep and narrow gorge, which closely resembles the catalytic triad of other a/b hydrolase fold family proteins [36 ].Acetylcholinesterase (AChE; EC 3.1.1.7)in vertebrates was involved in cell development and maturation [37], neuronal development and nerve regeneration [38] and inflammation modulation [39].AChE had also been identified in most invertebrates, including mollusc [31], arthropoda [40], platyhelminthes [41], annelida [42] and nematoda [43].AChE was also reported to be influencing many physiological behaviors in these invertebrates, including locomotion [43,44,45], feeding [43,44], egg laying [43], male mating [43], embryo development [46] and digestive activity [31].However, the immunomodulation, genomics and proteomics of AChE is still unclear in invertebrates.All the above literature evidence on AchE, enthused us to embark on this ambitious study to define structure, topography, influence of gender on distribution of neuronal AchE and finally to look for functional heterogeneity of AChE.In the present study, an AChE gene was studied in male and female galus galus cerebrum and cerebellum.The deduced protein of AChE (NM_205418.1) was composed of 118 amino acids, and it shared 94.6 -97.2% identity with other AChEs of cerebrum and cerebellum of male and female.The importance of AChE in body homeostasis is underscored by the fact that they are the targets of some of the most potent toxins including insecticides, snake venom and chemical weapons [47].Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers.However, AChE activity in brain has not been properly evaluated on the basis of sex.In the present study, AChE activity was investigated in different brain areas cerebrum and cerebellum in male and female Rattus norvegicus.
Females had a significant increase in AChE activity in cerebrum and cerebellum in comparision with male cerebrum and cerebellum.We
All the AChEs share some conserved structural also found that RNA granules are more in female cerebrum and cerebellum it may be one of the reason that affects the expression of AChE gene.It is not possible, at present, to assign a definite factor to explain the pattern of deficit observed in the enzyme activity in male.Among the possibilities, decreased blood flow in brain causing hypoxia has been suggested for decrement in AChE turnover in whole brain of male The variable enzyme activity in different brain areas and sex related changes observed in male and female Rattus norvegicus might be having an important bearing for functional heterogeneity reported for central cholinergic neuronal system and development of specific pharmacotherapy for cognitive disorders.We anticipate that, this study could lay foundation for future AChE studies in vertebrates and humans enroute to discovering novel therapeutic targets based on AChE metabolism.

CONCLUSION Conflicts of Interests: None
Rajesh B, Ramesh B, Et all,.GENOMICS OF ACETYLCHOLINESTERASE (AChE) ENZYME AND ITS RELATED RNA CONTENT IN THE BRAIN OF RATTUS NORVEGICUS: A PROSPECTIVE STUDY.
For the PCR reaction, 4 Hl of cDNA from each synthesis were added to 7 Hl of "2X PCR master mix containing 10X PCR buffer, 10 mM dNTP, 25 mM MgCl2, 5 U of Taq DNA polymerase (Fermentas, USA).Twenty HM of each pair of the primers was added, and the final volume was adjusted to 14 Hl with nuclease free water.The mixtures were denatured at 94 0 C for 3 min.Thirty five cycles of PCR were carried out, with denaturation at 94 0 C for 45 sec, annealing at 57 0 C for 30 sec, and extension at 72 0 C for 1 min, followed by a final extension period of 5 min.PCR products were analyzed by electrophoresis on 1% agarose gels stained with GelStar Nucleic Acid Gel Stain (Cambrex Bio Science Rockland, Inc.).PCR products were cloned into the pGEMT plasmid vector (Promega) and sequenced using forward and reverse primers.Sequencing was performed with the Big DyeTM Terminator Cycle Sequencing Ready Kit, version 3.0 (ABI PrismTM, Perkin Elmer) and an ABI 3700 Applied Biosystems Model automated DNA sequencer.Nucleotide sequences of NWS were analyzed by BLASTN to search for similarities, and sequenceMATERIALS AND METHODSThis prospective study was conducted in Anatomy department of a tertiary care teaching medical school in South India.The study was approved by institutional ethical committee.We ensured that study complied with biomedical ethics guidelines for animal experimentation as laid down by Indian council of Medical Research (ICMR).Rattus norvegicus male and female weighing an average 108 ± 26 (90 -180 grams) were purchased from a local supplier and transported live to the laboratory in aerated tanks.During the acclimatization period, the Rattus norvegicus.were fed daily (Safe feed 7711, Charoen Pokphand Foods PCL, Thailand) weighing about 1% of the body weight, and were then fasted for 24 hours before the experiment.They were sacrificed, the brain was rapidly removed, Rajesh B, Ramesh B, Et all,.GENOMICS OF ACETYLCHOLINESTERASE (AChE) ENZYME AND ITS RELATED RNA CONTENT IN THE BRAIN OF RATTUS NORVEGICUS: A PROSPECTIVE STUDY.alignments was performed with CLUSTAL W (Megalign program, DNASTAR Inc., Madison, WI).Quantitative assessment of RNA by methyl green-pyronin staining: The tissues are fixed in Methacarn solution (methanol: chloroform: glacial acetic Acid = 6:3:1) fixatives for 4 hours for fixation, and then followed by routine histological processing.The 5 to 6 um sections are taken for all tissues and stained with MGP (methyl green pyronin).The Number of RNA granules are estimated by image analysis by used software IMAGE pro 6.2.Stastical analysis: ACHE gene Nucleotide sequences were analyzed by BLASTN to search for similarities, and sequence alignments was performed with CLUSTALW (Megalign program, DNASTAR Inc., Madison, WI).Also analysed methyl green-pyronin staining of RNA granules by image pro 6.2 software.RESULTS AChE cDNA sequences: AChE cDNA sequence was used to investigate difference in male and female cerebrum and cerebellum.The ORF of AChE is comprised of 543 nucleotides (GenBank accession number NM_172009.1),showing significant nucleotide similarity 82.2% and 81.4% respectively with Rattus norvegicus male cerebrum and cerebellum whereas 84.1 % with female cerebrum and cerebellum AChE Figure 1.Peptide sequences of AchE are shown in Figure

Fig. 1 :
Fig. 1: Alignment of nucleotide sequences of Rattus norvegicus AChE from male and female cerebrum and cerebellum with AChE NM_172009.1 Boxes residue differ from the consensus.

Fig. 2 :
Fig. 2: Alignment of peptide sequences of Rattus norvegicus AChE from male and female cerebrum and cerebellum with AChE NM_172009.1 Boxes residue differ from the consensus.

Fig. 3 :
Fig. 3: Percentage identity and divergence of Rattus norvegicus AChE NM_172009.1 nucleotide and amino acid sequences from and Rattus norvegicus AChE of male and female cerebrum and cerebellum.

Fig. 5 :
Fig. 5: Methyl green-pyronin staining on Rattus norvegicus brain regions (A) Female Cerebrum showing maximum numbers of RNA granules in comparison with male cerebrum (B) whereas again RNA granules are more in female cerebellum (C) than that seen with male cerebellum (D) (Magnification 40 X).
[48].Lack of uniformity in profile of AChE activity may be a reflection of functional heterogeneity in central cholinergic system observed by several workers on various parameters[49].Through this study, we inched closer to clarity on structure and neuronal topography of AChE as shown by the results.In conclusion, the present study has provided a profile of AChE activity in major brain areas cerebrum and cerebellum Rattus norvegicus.The activity in cerebrum and cerebellum indicates a possible pattern of distribution of G4 and G1 molecular forms of AChE in brain areas, respectively.It seems that the G4 form is dominant and is affected by sex as compared to G1.
dissected for RNA extraction and sequencing the brain was rapidly removed for RNA extraction followed by reverse transcription and fold induction of gene expression between AChE and 28S rRNA genes, and were then analysized by PCR and Gel analysis.
In this context, we set up to study the distribution, structure of AChE gene and related RNA in neuronal tissues of invertebrate animals weighed, and