Detection of early Alzheimer's disease-like molecular alterations in a mouse model expressing human ApoE4.

The E4 allele of apolipoprotein E (ApoE4) is a key genetic risk factor for late-onset Alzheimer's disease (AD), increasing the risk of developing the disease by up to three-fold. However, the mechanisms by which ApoE4 contributes to AD pathogenesis are poorly understood. Here, we utilize a mouse model expressing either human ApoE3 or human ApoE4 to examine the effects of the E4 allele on a wide range of genetic and molecular pathways that are altered in early AD pathology. We demonstrate that ApoE4-expressing mice begin to show early differential expression of multiple genes, leading to alterations in downstream pathways related to neural cell maintenance, insulin signaling, amyloid processing and clearance, and synaptic plasticity. These alterations may result in the earlier accumulation of pathological proteins such as ß-amyloid that may build up within cells, leading to the accelerated degeneration of neurons and astrocytes as observed in ApoE4-positive individuals. We also examine the metabolic effects associated with a high-fat diet (HFD) in male ApoE4-expressing mice compared with regular chow diet (RD) fed mice at different ages. We found that young ApoE4-expressing mice fed HFD developed metabolic disturbances, such as elevated weight gain, blood glucose, and plasma insulin levels that cumulatively have been observed to increase the risk of AD in humans. Taken together, our results reveal early pathways that could mediate ApoE4-related AD risk and may help identify more tractable therapeutic targets for treating ApoE4-associated AD.

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.

Covid-19 and Priorities for Research in Aging.

This article describes priority areas for research on the impact of the Covid-19 pandemic on older adults that have been identified by the CIHR Institute of Aging (CIHR-IA). The process used by CIHR-IA consists of several iterative phases and thus far has resulted in identification of three key areas for Covid-19 research needs and four cross-cutting thematic areas. The key research priority areas are as follows: response of older adults to disease, vaccination, and therapeutics; mental health and isolation; and supportive care environments. The four cross-cutting themes are equity, diversity, and inclusion (EDI); ethical/moral considerations; evidence-informed practices; and digital health technologies. The priorities outlined in this article will inform CIHR-IA's responses to Covid-19 research needs.

Modulation of sodium-coupled choline transporter CHT function in health and disease.

The sodium-coupled high-affinity choline transporter CHT plays a critical role in acetylcholine (ACh) synthesis by taking up the substrate choline from the synaptic cleft after neurotransmitter release; this conservation mechanism is the rate-limiting step for production of ACh, thereby facilitating communication by subsequent action potentials. Mice carrying a null mutation for CHT die within an hour of birth due to respiratory failure, indicating the essential role of CHT proteins for sustaining cholinergic transmission. Choline uptake activity is regulated dynamically by CHT proteins undergoing rapid trafficking between subcellular compartments and the plasma membrane where they are functionally active. CHT proteins internalize from the cell surface into the endolysosomal pathway by a clathrin-mediated mechanism, but can undergo ubiquitination and proteosomal degradation under conditions such as cellular oxidative stress. Over the years, functionally-relevant CHT polymorphisms have been linked to a range of neurological and psychiatric disorders, including ADHD and depression; the impact of these mutations and the extent to which they alter cholinergic signaling have not been addressed fully. Recent studies have identified compounds that can either promote or diminish cholinergic neurotransmission by modulating CHT function, thus having the potential to serve as pharmacological tools or therapeutic prototypes. Here, we review regulation of CHT activity, trafficking and subcellular disposition of CHT proteins, alteration of transporter function in genetic, neurological and psychiatric diseases, and investigations of compounds that modulate activity of the transporter.

Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter.

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C.

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.

Absence of p75(NTR) expression reduces nerve growth factor immunolocalization in cholinergic septal neurons.

Septal axons provide a cholinergic innervation to the nerve growth factor (NGF)-producing neurons of the mammalian hippocampus. These cholinergic septal afferents are capable of responding to target-derived NGF because they possess trkA and p75(NTR), the two transmembrane receptors that bind NGF and activate ligand-mediated intracellular signaling. To assess the relative importance of p75(NTR) expression for the responsiveness of cholinergic septal neurons to hippocampally derived NGF, we used three lines of mutant and/or transgenic mice: p75(-/-) mice (having two mutated alleles of the p75(NTR) gene), NGF/p75(+/+) mice (transgenic animals overexpressing NGF within central glial cells and having two normal alleles of the p75(NTR) gene), and NGF/p75(-/-) mice (NGF transgenic animals having two mutated alleles of the p75(NTR) gene). BALB/c and C57B1/6 mice (background strains for the mutant and transgenic lines of mice) were used as controls. Both lines of NGF transgenic mice possess elevated levels of NGF protein in the hippocampus and septal region, irrespective of p75(NTR) expression. BALB/c and C57Bl/6 mice display comparably lower levels of NGF protein in both tissues. Despite differing levels of NGF protein, the ratios of hippocampal to septal NGF levels are similar among BALB/c, C57B1/6, and NGF/p75(+/+) mice. Both p75(-/-) and NGF/p75(-/-) mice, on the other hand, have markedly elevated ratios of NGF protein between these two tissues. The lack of p75(NTR) expression also results in a pronounced absence of NGF immunoreactivity in cholinergic septal neurons of p75(-/-) and NGF/p75(-/-) mice. BALB/c, C57B1/6, and NGF/p75(+/+) mice, on the other hand, display NGF immunoreactivity that appears as discrete granules scattered through the cytoplasm of cholinergic septal neurons. Elevated levels of NGF in the hippocampus and septal region coincide with hypertrophy of cholinergic septal neurons of NGF/p75(+/+) mice but not of NGF/p75(-/-) mice. Levels of choline acetyltransferase (ChAT) enzyme activity are, however, elevated in the septal region and hippocampus of both NGF/p75(+/+) and NGF/p75(-/-) mice, compared with control mice. These data indicate that an absence of functional p75(NTR) expression disrupts the normal cellular immunolocalization of NGF by cholinergic septal neurons but does not affect the ability of these neurons to respond to elevated levels of NGF, as determined by ChAT activity.

Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

Nuclear localization of the 82-kDa form of human choline acetyltransferase.

Choline acetyltransferase is the enzyme catalyzing synthesis of the neurotransmitter acetylcholine in cholinergic neurons. In human, transcripts encoding two forms of the enzyme with apparent molecular masses of 69 and 82 kDa are found in brain and spinal cord; the 82-kDa form differs from the 69-kDa enzyme only in terms of a 118-amino acid extension on its amino terminus. Using green fluorescent protein-tagged choline acetyltransferase, we show that the 82-kDa enzyme is targeted to nuclei of cells, whereas the 69-kDa protein is found in cytoplasm. Expression of site-directed and deletion mutants of the 82-kDa isoform reveals that the extended amino terminus contains a nuclear localization signal in the first nine amino acids which targets the protein to nucleus. This represents the first report of a neurotransmitter-synthesizing enzyme that is localized to the cell nucleus.

Molecular mechanisms regulating NGF-mediated enhancement of cholinergic neuronal phenotype: c-fos trans-activation of the choline acetyltransferase gene.

Nerve growth factor (NGF) enhances expression of the cholinergic phenotype observed as increased choline acetyltransferase (ChAT) activity, immunoreactivity, and mRNA. In the present study, treatment of cultured rat embryonic basal forebrain neurons with anti-c-fos, prior to administering NGF, blocked NGF-mediated increases in ChAT activity by 67%; basal ChAT activity was not affected by the antisense oligonucleotide treatment. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that anti-c-fos treatment resulted in not only blockade but enhancement of steady-state ChAT mRNA at different time points. These data suggest that c-fos is an important component in NGF-mediated changes in the cholinergic phenotype and support the hypothesis that c-fos plays a role in the regulation of transcription of the ChAT gene. Elucidation of mechanisms underlying this regulation may aid drug development in neurodegenerative disease.

Optimization of serum-free culture conditions for growth of embryonic rat cholinergic basal forebrain neurons.

The objective of the present study was to optimize conditions for culturing embryonic rat basal forebrain neurons in serum-free defined medium to be used in investigations of cholinergic neuron function and responsiveness to neurotrophic factors. It was determined that a combination of neurobasal medium (NB) and DMEM/F12 medium (DM:F12) maintained culture viability, basal choline acetyltransferase (ChAT) activity and responsiveness of these neurons to nerve growth factor (NGF) better than growth of neurons in either medium alone; all media tested contained N2 supplements. While NB which was developed initially for culturing embryonic rat hippocampal neurons supported the growth of basal forebrain neurons, they had reduced ChAT activity and did not respond to NGF with enhanced cholinergic neuronal enzyme activity. On the other hand, DM:F12 did not consistently support survival of the neurons until assay of ChAT activity on day 6 in vitro; surviving cultures were compromised in their cholinergic capacity either under basal or NGF-enhanced conditions. Cultures grown in the combined media responded to brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor (CNTF), at concentrations up to 100 ng/ml with increased ChAT activity as predicted from the literature. These findings suggest that the nutrient composition of the medium is important in promoting expression of the cholinergic neuronal phenotype and that growth factor supplementation alone is insufficient to compensate for inadequate nutrient composition.

NGF-induction of the expression of ChAT mRNA in PC12 cells and primary cultures of embryonic rat basal forebrain.

The objective of this study was to examine the role of nerve growth factor (NGF) in regulation of expression of the cholinergic phenotype. NGF was administered to PC12 cells or primary cultures of embryonic (E17) rat basal forebrain for 2 days, then steady-state levels of choline acetyltransferase (ChAT) mRNA was monitored. Expression of ChAT mRNA isoforms was investigated using reverse transcription-polymerase chain reaction (RT-PCR) to amplify different upstream regions of the ChAT transcripts, and Southern blot analysis was used to verify identity of the PCR products. An NGF-induced increase of 1.8- and 1.5-fold in steady-state level of the ChAT transcript containing the M-exon (M-ChAT) was observed in PC12 cells and embryonic rat basal forebrain neurons, respectively. Also, a 2-fold increase in ChAT protein as determined by western blot analysis was associated with an NGF-mediated increase of 1.7-fold in ChAT activity in rat basal forebrain neurons within the same cultures following 4 days of NGF treatment.

A new twist in an old story: the role for crosstalk of neuronal and trophic activity.

A number of recent findings suggest a reciprocal interaction between neurotransmitters and neurotrophins functioning at the level of the synapse, which may be relevant not only for plasticity changes in the mature nervous system, but also for the development of synaptic connectivity and for survival or maturation of neurons prior to target contact. Thus, neurotrophin-induced attenuation of frequency-dependent depletion of releasable synaptic vesicle pools of neurotransmitter at synapses may participate in Hebbian and non-Hebbian forms of LTP, as a characteristic of mature synaptic contacts. Subsequent to nerve/target contact, neurotrophins also appear to mediate contact-induced enhancement of neurotransmitter release; this may participate in a developmental improvement of synapse efficacy, stabilization of synaptic contacts, and maturation of "conductive" functional synapses. Coincident with a transmitter-induced elevation of cytosolic Ca2+ levels within growth cones, a local neurotrophin-mediated increase in released neurotransmitter occurring subsequent to stabilization of a distinct synaptic contact may then participate in the refinement of synapses with retention of those neurites affected by neurotrophins and withdrawal of those neurites not affected by neurotrophins. Finally, prior to nerve/target contact, Ca2+ channel-generated spontaneous neuronal activity as well as co-expression of neurotrophins and their receptors may play a role in maturational changes.

Measurement of glutamate and glutamine in the medial prefrontal cortex of never-treated schizophrenic patients and healthy controls by proton magnetic resonance spectroscopy.

BACKGROUND: Positron emission tomographic and postmortem studies comparing schizophrenic patients with healthy control subjects have found medial prefrontal cortical and anterior cingulate abnormalities that suggest dysfunction in glutamatergic neurons. The glutamate used for nerve signal transduction is predominantly derived from glutamine. After signal transduction, glutamate released into the synapse is converted to glutamine in glial cells, transported back to the presynaptic neuron, and reconverted to glutamate for reuse. In this study, levels of glutamate and glutamine were examined by means of in vivo proton (1H) magnetic resonance spectroscopy. METHODS: Localized in vivo 1H spectra were acquired from a 4.5-cm3 volume in the left medial prefrontal cortex encompassing portions of Brodmann areas 24, 32, and 9 in 10 never-treated schizophrenic subjects and 10 healthy controls of comparable age, sex, handedness, education, and parental education. From each spectrum, metabolite levels were estimated for glutamate and glutamine, as well as 10 other metabolites and 3 macromolecules, by means of a noninteractive computer program that combined modeled in vitro spectra of every metabolite to reconstruct each in vivo spectrum. RESULTS: A significant increase in glutamine level was found in the medial prefrontal cortex of the schizophrenic patients compared with controls. N-acetylaspartate and other measured metabolites and macromolecules were not significantly changed in schizophrenics. CONCLUSION: Increased glutamine levels in the medial prefrontal region most likely reflect decreased glutamatergic activity in this region in never-treated schizophrenic patients compared with healthy controls.

Inhibitors of serine/threonine phosphatases increase membrane-bound choline acetyltransferase activity and enhance acetylcholine synthesis.

The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129 +/- 11% and 137 +/- 10% of control, respectively. High-affinity choline transport was reduced to 77 +/- 6% and 76 +/- 7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73 +/- 6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.

Differential effects of nerve growth factor on expression of choline acetyltransferase and sodium-coupled choline transport in basal forebrain cholinergic neurons in culture.

Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC50 of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.

An in vivo proton magnetic resonance spectroscopy study of schizophrenia patients.

The level of the 1H metabolites in the left dorsolateral prefrontal region of schizophrenia patients at different stages of illness were measured in vivo using a short echo time spectroscopy technique. During both the early onset and chronic stages, normal N-acetylaspartate levels were observed, which suggests that these patients had no significant neuronal cell damage and/or loss. The in vivo measurements of glutamate in the first-episode, drugnaive patients failed to provide convincing evidence for the involvement of the glutamatergic system in the dorsolateral prefrontal region. Significant differences in the glutamine levels were observed in the acutely medicated and chronic patients; however, the interpretation of these differences requires further study.

Identification and partial characterization of the high-affinity choline carrier from rat brain striatum.

[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.

An in vivo study of the prefrontal cortex of schizophrenic patients at different stages of illness via phosphorus magnetic resonance spectroscopy.

BACKGROUND: In this study, phospholipid metabolism of cell membranes, high-energy phosphate metabolism, and intracellular free magnesium concentration in the prefrontal cortex of first-episode drug-naive schizophrenic patients and medicated schizophrenic patients at different stages of illness were compared with those of controls. METHODS: Localized in vivo phosphorus 31 magnetic resonance spectra of the left dorsolateral prefrontal cortex of 11 drug-native, eight newly diagnosed medicated, and 10 chronic medicated patients with schizophrenia were compared with controls of similar gender, education, parental education, and handedness. RESULTS: Significantly decreased levels of phosphomonoesters in drug-native, newly diagnosed medicated, and chronic medicated patients and significantly increased levels of phosphodiesters in drug-native patients were observed when compared with controls. There were no significant differences in the levels of high-energy phosphate metabolites between the groups except for a significant decrease in the inorganic orthophosphate levels of newly diagnosed medicated patients. A significant increase in the intracellular free magnesium concentration was observed in drug-naive, newly diagnosed medicated, and chronic medicated patients compared with controls. There were no correlations between the patients' negative and positive symptoms and the observed phosphorus-containing metabolites. CONCLUSIONS: A reduction in precursors of membrane phospholipid are observed during the early and chronic stages of the schizophrenia illness, and breakdown products of membrane phospholipids are increased at the early stage of illness before medication treatment.