Dominique Arion

Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia

In subjects with schizophrenia, impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC). This dysfunction appears to be due, at least in part, to abnormalities in γ-aminobutyric acid (GABA)-mediated inhibitory circuitry. To test the hypothesis that altered GABA-mediated circuitry in the DLPFC of subjects with schizophrenia reflects expression changes of genes that encode selective presynaptic and postsynaptic components of GABA neurotransmission, we conducted a systematic expression analysis of GABA-related transcripts in the DLPFC of 14 pairs of schizophrenia and age-, sex- and post-mortem interval-matched control subjects using a customized DNA microarray with enhanced sensitivity and specificity. Subjects with schizophrenia exhibited expression deficits in GABA-related transcripts encoding (1) presynaptic regulators of GABA neurotransmission (67 kDa isoform of glutamic acid decarboxylase (GAD67) and GABA transporter 1), (2) neuropeptides (somatostatin (SST), neuropeptide Y (NPY) and cholecystokinin (CCK)) and (3) GABAA receptor subunits (α1, α4, β3, γ2 and δ). Real-time qPCR and/or in situ hybridization confirmed the deficits for six representative transcripts tested in the same pairs and in an extended cohort, respectively. In contrast, GAD67, SST and α1 subunit mRNA levels, as assessed by in situ hybridization, were not altered in the DLPFC of monkeys chronically exposed to antipsychotic medications. These findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SST/NPY-containing and CCK-containing subpopulations of GABA neurons and in the signaling via certain GABAA receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition. In concert with previous findings, these data suggest that working memory dysfunction in schizophrenia is mediated by altered GABA neurotransmission in certain DLPFC microcircuits.

Molecular evidence for increased expression of genes related to immune and chaperone function in the prefrontal cortex in schizophrenia

BACKGROUND Schizophrenia is characterized by complex gene expression changes. The transcriptome alterations in the prefrontal cortex have been the subject of several recent postmortem studies that yielded both convergent and divergent findings. METHODS To increase measurement precision, we used a custom-designed DNA microarray platform with long oligonucleotides and multiple probes with replicates. The platform was designed to assess the expression of > 1800 genes specifically chosen because of their hypothesized roles in the pathophysiology of schizophrenia. The gene expression differences in dorsolateral prefrontal cortex samples from 14 matched pairs of schizophrenia and control subjects were analyzed with two technical replicates and four data mining approaches. RESULTS In addition to replicating many expression changes in synaptic, oligodendrocyte, and signal transduction genes, we uncovered and validated a robust immune/chaperone transcript upregulation in the schizophrenia samples. CONCLUSIONS We speculate that the overexpression of SERPINA3, IFITM1, IFITM2, IFITM3, CHI3L1, MT2A, CD14, HSPB1, HSPA1B, and HSPA1A in schizophrenia subjects represents a long-lasting and correlated signature of an early environmental insult during development that actively contributes to the pathophysiology of prefrontal dysfunction.

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis.

ABSTRACT Nucleoside analog chain terminators such as 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-dideoxy-3′-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

Cortical Deficits of Glutamic Acid Decarboxylase 67 Expression in Schizophrenia: Clinical, Protein, and Cell Type-Specific Features

OBJECTIVE Cognitive deficits in schizophrenia are associated with altered activity of the dorsolateral prefrontal cortex, which has been attributed to lower expression of the 67 kDa isoform of glutamic acid decarboxylase (GAD67), the major γ-aminobutyric acid (GABA)-synthesizing enzyme. However, little is known about the relationship of prefrontal GAD67 mRNA levels and illness severity, translation of the transcript into protein, and protein levels in axon terminals, the key site of GABA production and function. METHOD Quantitative polymerase chain reaction was used to measure GAD67 mRNA levels in postmortem specimens of dorsolateral prefrontal cortex from subjects with schizophrenia and matched comparison subjects with no known history of psychiatric or neurological disorders (N=42 pairs). In a subset of this cohort in which potential confounds of protein measures were controlled (N=19 pairs), Western blotting was used to quantify tissue levels of GAD67 protein in tissue. In five of these pairs, multilabel confocal immunofluorescence was used to quantify GAD67 protein levels in the axon terminals of parvalbumin-containing GABA neurons, which are known to have low levels of GAD67 mRNA in schizophrenia. RESULTS GAD67 mRNA levels were significantly lower in schizophrenia subjects (by 15%), but transcript levels were not associated with predictors or measures of illness severity or chronicity. In schizophrenia subjects, GAD67 protein levels were significantly lower in total gray matter (by 10%) and in parvalbumin axon terminals (by 49%). CONCLUSIONS The findings that lower GAD67 mRNA expression is common in schizophrenia, that it is not a consequence of having the illness, and that it leads to less translation of the protein, especially in the axon terminals of parvalbumin-containing neurons, support the hypothesis that lower GABA synthesis in parvalbumin neurons contributes to dorsolateral prefrontal cortex dysfunction and impaired cognition in schizophrenia.

Protracted Developmental Trajectories of GABAA Receptor α1 and α2 Subunit Expression in Primate Prefrontal Cortex

BACKGROUND In schizophrenia, working memory dysfunction is associated with altered expression of gamma-aminobutyric acid (GABA)(A) receptor alpha1 and alpha2 subunits in the dorsolateral prefrontal cortex (DLPFC). In rodents, cortical alpha subunit expression shifts from low alpha1 and high alpha2 to high alpha1 and low alpha2 during early postnatal development. Because these two alpha subunits confer different functional properties to the GABA(A) receptors containing them, we determined whether this shift in alpha1 and alpha2 subunit expression continues through adolescence in the primate DLPFC, potentially contributing to the maturation of working memory during this developmental period. METHODS Levels of GABA(A) receptor alpha1 and alpha2 subunit mRNAs were determined in the DLPFC of monkeys aged 1 week, 4 weeks, 3 months, 15-17 months (prepubertal), and 43-47 months (postpubertal) and in adult monkeys using in situ hybridization, followed by the quantification of alpha1 subunit protein by western blotting. We also performed whole-cell patch clamp recording of miniature inhibitory postsynaptic potentials (mIPSPs) in DLPFC slices prepared from pre- and postpubertal monkeys. RESULTS The mRNA and protein levels of alpha1 and alpha2 subunits progressively increased and decreased, respectively, throughout postnatal development including adolescence. Furthermore, as predicted by the different functional properties of alpha1-containing versus alpha2-containing GABA(A) receptors, the mIPSP duration was significantly shorter in postpubertal than in prepubertal animals. CONCLUSIONS In contrast to rodents, the developmental shift in GABA(A) receptor alpha subunit expression continues through adolescence in primate DLPFC, inducing a marked change in the kinetics of GABA neurotransmission. Disturbances in this shift might underlie impaired working memory in schizophrenia.

Perisomatic Inhibition and Cortical Circuit Dysfunction in Schizophrenia

Deficits of cognitive control in schizophrenia are associated with altered gamma oscillations in the prefrontal cortex. Paralbumin basket interneurons, which innervate the perisomatic region of pyramidal neurons, appear to play a key role in generating cortical gamma oscillations. In the prefrontal cortex of subjects with schizophrenia, alterations are present in both pre- and post-synaptic markers of the strength of GABA inputs from parvalbumin basket neurons to pyramidal neurons. These alterations may contribute to the neural substrate for impaired gamma oscillations in schizophrenia.

Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia

CONTEXT Disturbances in markers of cortical γ-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of γ-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4. OBJECTIVE To compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects. DESIGN Real-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex. SETTING Human brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiners Office, Pittsburgh, Pennsylvania. PARTICIPANTS Postmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term. MAIN OUTCOME MEASURES Relative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts. RESULTS OXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia. CONCLUSION In schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of γ-aminobutyric acid neurotransmission in the prefrontal cortex.

Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin–proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were <10−7 and <10−5, respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P<10−6) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular–cellular basis of differences in clinical phenotypes.

Molecular markers distinguishing supragranular and infragranular layers in the human prefrontal cortex

The human neocortex is organized into six layers that are differentiated by the size and packing density of their constituent neurons. The gene products that guide the establishment of this lamination have been studied extensively, but the gene expression gradients present across the layers of the adult human neocortex are mostly unknown. As the supragranular (SG) and infragranular (IG) layers of the human prefrontal cortex (PFC) differ in their connectivity and developmental time course, we hypothesized that the SG and IG layers will show distinct differences in their transcriptomes. To test this prediction, we used laser capture microdissection coupled with DNA microarray transcriptome profiling. Sixty‐nine genes exhibited robust and highly consistent expression differences between the SG and IG layers. For six selected markers, in addition to validating the microarray findings, in situ hybridization revealed a complex, subpopulation‐specific neuronal distribution. The markers we identified are likely to be related to the functional differences between the SG and IG layers of the human PFC and can be used for assessing alterations in structure and function of this cortical region in human brain disorders.

The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1

The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3′-azido-3′-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5′-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2′,3′-dideoxynucleoside-5′-monophosphate (2′,3′-ddNMP) analogs that lack a 3′-azido moiety. This led to the hypothesis that the 3′-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZTR) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3′-azido-2′,3′-ddN including 3′-azido-2′,3′-ddA (AZddA), 3′-azido-2′,3′-ddC (AZddC), 3′-azido-2′,3′-ddG (AZddG), AZT, and 3′-azido-2′,3′-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3′-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3′-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3′-azido group on the 3′-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZTR RT for all 3′-azido-ddNMP analogs. Thus, the 3′-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations.