K. Wing Leung

The establishment of a sensitive method in determining different neurotransmitters simultaneously in rat brains by using liquid chromatography-electrospray tandem mass spectrometry

An effective way to determine the amount of different neurotransmitters is vital to the study of brain function. Here, a highly sensitive HPLC-MS/MS method was developed to simultaneously measure γ-aminobutyric acid, dopamine, epinephrine, norepinepherine, glutamate and serotonin in one sample. The quantification of the neurotransmitters was achieved by a tandem mass spectrometer using the selected reaction monitoring scan mode. The method validation included selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. For the six neurotransmitters, the linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.991, and the limit of detection (LOD) and the limit of quantification (LOQ) were from 2.5 to 500 pg/mg and 7.5 to 1000 pg/mg, respectively. This method was employed here to reveal different types and amounts of neurotransmitters simultaneously in adult and embryonic rat brains. Here, the change of dopamine concentration in embryonic and adult brain was from 0.071 to 0.760 ng/mg of brain tissue, GABA was from 207.643 to 445.148 ng/mg, glutamate was from 679.535 to 1408.920 ng/mg, serotonin was from 0.058 to 0.485 ng/mg and norepinepherine was from 0.054 to 0.290 ng/mg. For epinephrine, it was only detected in embryonic stage but not in adult, with the concentration at 0.241 ng/mg.

Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase THE SUPPRESSIVE ROLES OF MYOGENESIS AND INNERVATING NERVES

The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChET protein and the enzymatic activity were dramatically increased, but the level of G4 AChE was relatively decreased. This G4 AChE in C2C12 cells was specifically recognized by anti-PRiMA antibody, suggesting the association of this enzyme with PRiMA. Reverse transcription-PCR analysis revealed that the level of PRiMA mRNA was reduced during the myogenic differentiation of C2C12 cells. Overexpression of PRiMA in C2C12 myotubes significantly increased the production of G4 AChE. The oligomerization of G4 AChE, however, did not require the intracellular cytoplasmic tail of PRiMA. After overexpressing the muscle regulatory factors, myogenin and MyoD, the expressions of PRiMA and G4 AChE in cultured myotubes were markedly reduced. In addition, calcitonin gene-related peptide, a known motor neuron-derived factor, and muscular activity were able to suppress PRiMA expression in muscle; the suppression was mediated by the phosphorylation of a cAMP-responsive element-binding protein. In accordance with the in vitro results, sciatic nerve denervation transiently increased the expression of PRiMA mRNA and decreased the phosphorylation of cAMP-responsive element-binding protein as well as its activator calcium/calmodulin-dependent protein kinase II in muscles. Our results suggest that the expression of PRiMA, as well as PRiMA-associated G4 AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).

Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons.

In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChET and PRiMA, PRiMA-linked G4 AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G2 AChE or G4 QN-HC-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G4 AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.

The Assembly of Proline-rich Membrane Anchor (PRiMA)-linked Acetylcholinesterase Enzyme GLYCOSYLATION IS REQUIRED FOR ENZYMATIC ACTIVITY BUT NOT FOR OLIGOMERIZATION

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal “t-peptide” in AChE catalytic subunit (AChET). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChET plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChET mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher Km value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.

Transcriptional regulation of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in neuron: An inductive effect of neuron differentiation

The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form of acetylcholinesterase (G(4) AChE), was revealed in cultured cortical neurons during differentiation. The level of AChE(T) protein, total enzymatic activity and the amount of G(4) AChE were dramatically increased during the neuron differentiation. RT-PCR analyses revealed that the transcript encoding PRiMA was significantly up-regulated in the differentiated neurons. To investigate the transcriptional mechanism on PRiMA regulation, a reporter construct of human PRiMA promoter-tagged luciferase was employed in this study. Upon the neuronal differentiation in cortical neurons, a mitogen-activated protein (MAP) kinase-dependent pathway was stimulated: this signaling cascade was shown to regulate the transcriptional activity of PRiMA. In addition, both PRiMA and AChE(T) transcripts were induced by the over expression of an active mutant of Raf in the cultured neurons. The treatment of a MAP kinase inhibitor (U0126) significantly blocked the expression of PRiMA transcript and promoter-driven luciferase activity as induced by the differentiation of cortical neurons. These results suggested that a MAP kinase signaling pathway served as one of the transcriptional regulators in controlling PRiMA gene expression during the neuronal differentiation process.

The establishment of a highly sensitive method in detecting ketamine and norketamine simultaneously in human hairs by HPLC-Chip-MS/MS

An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of ∼1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.

Restricted localization of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase at the neuromuscular junctions--contribution and expression from motor neurons.

The expression and localization of the proline‐rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), were studied at vertebrate neuromuscular junctions. Both muscle and motor neuron contributed to this synaptic expression pattern. During the development of rat muscles, the expression of PRiMA and AChET and the enzymatic activity increased dramatically; however, the proportion of G4 AChE decreased. G4 AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using western blot and ELISA, both PRiMA protein and PRiMA‐linked G4 AChE were found to be present in large amounts in fast‐twitch muscle (e.g. tibialis), but in relatively low abundance in slow‐twitch muscle (e.g. soleus). These results indicate that the expression level of PRiMA‐linked G4 AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChET and G4 AChE also increased in the spinal cord during development. Such expression in motor neurons contributed to the synaptic localization of G4 AChE. After denervation, the expression of PRiMA, AChET and G4 AChE decreased markedly in the spinal cord, and in fast‐ and slow‐twitch muscles.

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChET and BChET with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (QN-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or QN-GPI always consist of AChET and BChET homodimers. The dimer formation of AChET and BChET depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChET or BChET homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.

PRiMA directs a restricted localization of tetrameric AChE at synapses.

Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.

Calcitonin gene-related peptide induces the expression of acetylcholinesterase-associated collagen ColQ in muscle : a distinction in driving two different promoters between fast-and slow-twitch muscle fibers

The presence of a collagenous protein (ColQ) characterizes the collagen‐tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ‐1 and ColQ‐1a, driven by two promoters: pColQ‐1 and pColQ‐1a, were found in mammalian slow‐ and fast‐twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene‐related peptide (CGRP), a known motor neuron‐derived factor. Application of CGRP, or dibutyryl‐cAMP (Bt2‐cAMP), in cultured myotubes induced the expression of ColQ‐1a transcript and promoter activity; however, the expression of ColQ‐1 transcript did not respond to CGRP or Bt2‐cAMP. The CGRP‐induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP‐responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ‐1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt2‐cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ‐1a at the nmjs of fast‐twitch muscle was governed by a CGRP‐mediated cAMP signaling mechanism.