Human Choline Acetyltransferase Research Articles

An AAV capsid increases transduction of striatum and a ChAT promoter allows selective cholinergic neuron transduction

Adeno-associated virus (AAV) vectors are currently the most efficient option for intracranial gene therapies to treat neurodegenerative disease. Increased efficacy and safety will depend upon robust and specific expression of therapeutic genes into target cell-types within the human brain. In this study, we set out with two objectives: (1) to identify capsids with broader transduction of the striatum upon intracranial injection in mice and (2) to test a truncated human choline acetyltransferase (ChAT) promoter that would allow efficient and selective transduction of cholinergic neurons. We compared AAV9 and an engineered capsid, AAV-S, to mediate widespread reporter gene expression throughout the striatum. We observed that AAV-S transduced a significantly greater area of the injected hemisphere primarily in the rostral direction compared with AAV9 (CAG promoter). We tested AAV9 vectors packaging a reporter gene expression cassette driven by either the ChAT or CAG promoter. Specificity of transgene expression of ChAT neurons over other cells was 7-fold higher, and efficiency was 3-fold higher for the ChAT promoter compared with the CAG promoter. The AAV-ChAT transgene expression cassette should be a useful tool for the study of cholinergic neurons in mice, and the broader transduction area of AAV-S warrants further evaluation of this capsid.

Regional differences of extrinsic and intrinsic cholinergic innervation and alterations by chronic opioid-induced constipation in the porcine colonic enteric nerve system

The colon is richly innervated by extrinsic (ext) and intrinsic (int) cholinergic nerves from central and enteric nervous system (CNS and ENS) modulating colonic motility, secretion and inflammation. However, a challenge lies in our inability to anatomically distinguish such innervation in the colon. The porcine colon is regarded as a useful translational research model due to many similarities with human. The present study developed an approach to differentiate ext- and int-cholinergic innervation of the porcine colon using double immunolabeling with a novel mouse antibody against human peripheral choline acetyltransferase (hpChAT) combined with a rabbit antibody anti-common ChAT (cChAT) respectively. The spatial configuration of ext- and int- cholinergic innervation in the ENS was assessed on 3D images generated from CLARITY-cleared porcine colonic samples. Their densities were computationally quantitated using Imaris 9 within the inner and outer submucosal and myenteric plexuses (ISP, OSP, MP) along the proximal, transverse and distal colonic regions (pC, tC, dC) collected from 18 adult Yucatan minipigs including 6 naïve (3 of each sex), 4 vehicle controls (2 of each sex), 8 treated with loperamide, a μ opioid receptor agonist (LOP, 0.4 or 3 mg/kg/day, po, 4 weeks, 4/group, 2 of each sex). The specificities of cChAT and hpChAT antibodies were confirmed with porcine cervical vagal trunk (CVT), sacral spinal cord (SSC, S2-S4) and colonic ENS. The double labeling showed the strong cChAT immunoreactive (+) fibers in the CVT and neuronal somata and fibers in the SSC, but no hpChAT+ labeling was visualized in these structures. In the colonic MP, the dense hpChAT+ neurons and fibers and varicose cChAT+ fibers were simultaneously visualized. 3D images demonstrated that int- cholinergic neurons (hpChAT+) were closely surrounded by ext- cholinergic varicose fibers and dot like structures (cChAT+), presumably nerve terminals. The density of cChAT+ but not hpChAT+ fibers+somata showed significant differences among three segments (dC>tC>pC) within three enteric plexuses (MP>OSP>ISP) in naïve pigs. Compared to control groups, the high dose LOP induced a 1.3-fold decrease of cChAT+ fiber density in males (p<0.05) and 1.5-fold in females (p<0.05), and 2.2-fold decrease of hpChAT+ fibers+somata density in males (p<0.05) and 3.6-fold in female (p<0.01) with a 1.5-fold greater decrease in females than males (p<0.05) in the MP of the dC. This study demonstrates for the first time the ext- and int- cholinergic innervation, the spatial configuration in 3D structure, regional variations in the porcine colonic ENS and the reduction by chronic opiate use with greater in females than males. These findings provide neuroanatomical evidence of CNS-gut cross-talk via cholinergic innervation of the colon and point to the neuromodulation of this pathway to alleviate functional colonic disorders like chronic opioid-induced constipation. Supported by NIH/SPARC OT2OD024899. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholinergic regulation of vascular endothelial function by human ChAT+ T cells

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human Tcells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ Tcells in blood. These findings on ChAT+ human Tcells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

A novel transgenic mouse model expressing primate-specific nuclear choline acetyltransferase: insights into potential cholinergic vulnerability

The acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) is an important cholinergic neuronal marker whose levels and/or activity are reduced in physiological and pathological aging. One isoform of ChAT, 82-kDa ChAT, is expressed only in primates and found primarily in nuclei of cholinergic neurons in younger individuals, but this protein becomes mostly cytoplasmic with increasing age and in Alzheimer’s disease (AD). Previous studies suggest that 82-kDa ChAT may be involved in regulating gene expression during cellular stress. Since it is not expressed in rodents, we developed a transgenic mouse model that expresses human 82-kDa ChAT under the control of an Nkx2.1 driver. Behavioral and biochemical assays were used to phenotype this novel transgenic model and elucidate the impact of 82-kDa ChAT expression. The 82-kDa ChAT transcript and protein were expressed predominantly in basal forebrain neurons and subcellular distribution of the protein recapitulated the age-related pattern found previously in human necropsy brains. Older 82-kDa ChAT-expressing mice presented with better age-related memory and inflammatory profiles. In summary, we established a novel transgenic mouse expressing 82-kDa ChAT that is valuable for studying the role of this primate-specific cholinergic enzyme in pathologies associated with cholinergic neuron vulnerability and dysfunction.

A multidimensional computational exploration of congenital myasthenic syndrome causing mutations in human choline acetyltransferase

Missense mutations of human choline acetyltransferase (CHAT) are mainly associated with congenital myasthenic syndrome (CMS). To date, several pathogenic mutations have been reported, but due to the rarity and genetic complexity of CMS and difficult genotype-phenotype correlations, the CHAT mutations, and their consequences are underexplored. In this study, we systematically sift through the available genetic data in search of previously unreported pathogenic mutations and use a dynamic in silico model to provide structural explanations for the pathogenicity of the reported deleterious and undetermined variants. Through rigorous multiparameter analyses, we conclude that mutations can affect CHAT through a variety of different mechanisms: by disrupting the secondary structure, by perturbing the P-loop through long-range allosteric interactions, by disrupting the domain connecting loop, and by affecting the phosphorylation process. This study provides the first dynamic look at how mutations affect the structure and catalytic activity in CHAT and highlights the need for further genomic research to better understand the pathology of CHAT.

Targeted Transgene Expression in Cholinergic Interneurons in the Monkey Striatum Using Canine Adenovirus Serotype 2 Vectors.

The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson’s disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates.

82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions.

The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure.

Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The invitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2h) is substantially reduced compared to WT-ChAT (19.7h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.

Cellular Transplantation as the Treatment of Alzheimer’s Diseas e in Mouse Models

Acetylcholine (Ach) and N-methyl-D-aspartate (NMDA) have been two major therapeutic targets of Alzheimer’s disease (AD) for decade. However, truly effective remedy for AD has not been successfully developed. We previously transplanted neurons derived from human induced pluripotent stem (hiPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. The cell transplantation significantly improved cognitive dysfunction in the dementia model mice. Human choline acetyl transferase (ChAT) positive cholinergic neurons located throughout the cortex of the grafted mice. Human and mouse ChAT positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR) positive neurons significantly increased in the cortex and hippocampus of the grafted dementia mice compared with the vehicle injected dementia mice. Human and mouse vesicular GABA transporter (VGAT) positive neurons distributed mainly in the hippocampus and, though the number was small, human VGAT positive neurons located in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR) positive neurons of both hiPS origin and mouse origin increased significantly compared with those in the vehicle injected mouse cortex. We suggested that positive feedback loops of neurotransmitter secretion of the cortex and hippocampus induced the characteristic distribution of the transplanted neurons. In this review, we summarized current advances in stem cell therapy for dementia model mice, especially to highlight the relationships between major neurotransmitters and host/transplanted neurons.

Cellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells

Cholinergic neuronal loss is a common finding in patients with Alzheimer's disease (AD) and AD model mice. We previously transplanted neurons derived from human induced pluripotent stem (iPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. In the present study, we examined the cellular and molecular mechanisms involved in the alleviation of cognitive dysfunction in transplanted mice.After transplant, mice showed improvement in cognitive function, confirming our previous findings. Human choline acetyltransferase (ChAT)-positive cholinergic neurons were distributed throughout the cortex of the grafted mice. Human and mouse ChAT-positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR)-positive neurons were significantly increased in the cortex and hippocampus of the grafted mice compared with the vehicle-injected mice. In addition, human and mouse vesicular GABA transporter (VGAT)-positive neurons were located mainly in the hippocampus and, though the number was small, human VGAT-positive neurons were observed in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR)-positive neurons of both human origin and mouse origin were significantly increased compared with those in the vehicle-injected mouse cortex. The α7nAChR-positive and GABAR-positive neurons expressed phosphorylated Akt and c-fos in the cortex, suggesting that these receptor-expressing neurons were possibly activated by the neurotransmitters secreted from the grafted neurons.Collectively, the grafted and host neurons may form positive feedback loops via neurotransmitter secretion in both the cerebral cortex and hippocampus, leading to alleviation of cognitive dysfunction in dementia model mice.

Prevention of age-related memory deficit in transgenic mice by human choline acetyltransferase

Choline acetyltransferase (ChAT, acetylCoA:choline O-acetyltransferase, EC 2.3.1.6) is the biosynthetic enzyme of neurotransmitter acetylcholine. Here we showed for the first time that transgenic mice with human ChAT kept excellent learning and memory ability during aging process. Transgenic mice were prepared through microinjection of human ChAT into mouse fertilized eggs, and PCR reaction was used to screen out the transgenic mice. The results of measurements of ChAT activity and acetylcholine level in mouse brain indicated that human ChAT gene was expressed throughout the life of the transgenic mice. The results of step-through test and water maze test suggested that learning and memory ability was improved in transgenic mice compared to that of their age-matched littermates. The results support our idea that supplement of ChAT might serve as a potential therapeutic strategy for cognitive deficit.

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Functional consequences and structural interpretation of mutations of human choline acetyltransferase.

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.

Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.

Redesign of Cosubstrate Specificity and Identification of Important Residues for Substrate Binding to hChAT

In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.

Substrate Binding and Catalytic Mechanism of Human Choline Acetyltransferase,

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.

An open letter to our readers on the use of antibodies

An open letter to our readers on the use of antibodies

Surface-entropy reduction used in the crystallization of human choline acetyltransferase

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

Reduced Density of Cholinergic Interneurons in the Ventral Striatum in Schizophrenia: An In Situ Hybridization Study

The role of the striatum in the pathophysiology of schizophrenia is not understood. In a previous postmortem study, we found a reduction in the density of striatal interneurons that stain immunohistochemically for choline acetyltransferase (ChAT) in schizophrenia. To determine whether this finding represents a specific alteration in ChAT gene expression, we used in situ hybridization to study the striatum of 11 control and 9 schizophrenic subjects with oligonucleotide probes complementary to human ChAT mRNA, preprosomatostatin (PPS) mRNA, and beta-actin mRNA. Densities of ChAT mRNA-positive neurons, ChAT mRNA expression per neuron, PPS mRNA-positive neurons, and beta-actin mRNA expression levels were measured. There were no significant differences between the two groups in densities of PPS mRNA-positive neurons and levels of beta-actin mRNA expression throughout the striatum, or in densities of ChAT mRNA-positive neurons in the caudate nucleus or putamen. However, in the ventral striatum, the mean density of ChAT mRNA-positive neurons was reduced to 26% of control levels in the schizophrenic group. There is a reduction in number or function of the cholinergic interneurons of the ventral striatum in schizophrenia.

Two methods for large-scale purification of recombinant human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 μmol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.