Differential gene expression between callosal and ipsilateral projection neurons in the monkey dorsolateral prefrontal and posterior parietal cortices.

Reciprocal connections between primate dorsolateral prefrontal (DLPFC) and posterior parietal (PPC) cortices, furnished by subsets of layer 3 pyramidal neurons (PNs), contribute to cognitive processes including working memory (WM). A different subset of layer 3 PNs in each region projects to the homotopic region of the contralateral hemisphere. These ipsilateral (IP) and callosal (CP) projections, respectively, appear to be essential for the maintenance and transfer of information during WM. To determine if IP and CP layer 3 PNs in each region differ in their transcriptomes, fluorescent retrograde tracers were used to label IP and CP layer 3 PNs in the DLPFC and PPC from macaque monkeys. Retrogradely-labeled PNs were captured by laser microdissection and analyzed by RNAseq. Numerous differentially expressed genes (DEGs) were detected between IP and CP neurons in each region and the functional pathways containing many of these DEGs were shared across regions. However, DLPFC and PPC displayed opposite patterns of DEG enrichment between IP and CP neurons. Cross-region analyses indicated that the cortical area targeted by IP or CP layer 3 PNs was a strong correlate of their transcriptome profile. These findings suggest that the transcriptomes of layer 3 PNs reflect regional, projection type and target region specificity.

Differential gene expression in layer 3 pyramidal neurons across 3 regions of the human cortical visual spatial working memory network.

Visual spatial working memory (vsWM) is mediated by a distributed cortical network composed of multiple nodes, including primary visual (V1), posterior parietal (PPC), and dorsolateral prefrontal (DLPFC) cortices. Feedforward and feedback information is transferred among these nodes via projections furnished by pyramidal neurons (PNs) located primarily in cortical layer 3. Morphological and electrophysiological differences among layer 3 PNs across these nodes have been reported; however, the transcriptional signatures underlying these differences have not been examined in the human brain. Here we interrogated the transcriptomes of layer 3 PNs from 39 neurotypical human subjects across 3 critical nodes of the vsWM network. Over 8,000 differentially expressed genes were detected, with more than 6,000 transcriptional differences present between layer 3 PNs in V1 and those in PPC and DLPFC. Additionally, over 600 other genes differed in expression along the rostral-to-caudal hierarchy formed by these 3 nodes. Moreover, pathway analysis revealed enrichment of genes in V1 related to circadian rhythms and in DLPFC of genes involved in synaptic plasticity. Overall, these results show robust regional differences in the transcriptome of layer 3 PNs, which likely contribute to regional specialization in their morphological and physiological features and thus in their functional contributions to vsWM.

Distinct Properties of Layer 3 Pyramidal Neurons from Prefrontal and Parietal Areas of the Monkey Neocortex.

In primates, working memory function depends on activity in a distributed network of cortical areas that display different patterns of delay task-related activity. These differences are correlated with, and might depend on, distinctive properties of the neurons located in each area. For example, layer 3 pyramidal neurons (L3PNs) differ significantly between primary visual and dorsolateral prefrontal (DLPFC) cortices. However, to what extent L3PNs differ between DLPFC and other association cortical areas is less clear. Hence, we compared the properties of L3PNs in monkey DLPFC versus posterior parietal cortex (PPC), a key node in the cortical working memory network. Using patch-clamp recordings and biocytin cell filling in acute brain slices, we assessed the physiology and morphology of L3PNs from monkey DLPFC and PPC. The L3PN transcriptome was studied using laser microdissection combined with DNA microarray or quantitative PCR. We found that in both DLPFC and PPC, L3PNs were divided into regular spiking (RS-L3PNs) and bursting (B-L3PNs) physiological subtypes. Whereas regional differences in single-cell excitability were modest, B-L3PNs were rare in PPC (RS-L3PN:B-L3PN, 94:6), but were abundant in DLPFC (50:50), showing greater physiological diversity. Moreover, DLPFC L3PNs display larger and more complex basal dendrites with higher dendritic spine density. Additionally, we found differential expression of hundreds of genes, suggesting a transcriptional basis for the differences in L3PN phenotype between DLPFC and PPC. These data show that the previously observed differences between DLPFC and PPC neuron activity during working memory tasks are associated with diversity in the cellular/molecular properties of L3PNs.SIGNIFICANCE STATEMENT In the human and nonhuman primate neocortex, layer 3 pyramidal neurons (L3PNs) differ significantly between dorsolateral prefrontal (DLPFC) and sensory areas. Hence, L3PN properties reflect, and may contribute to, a greater complexity of computations performed in DLPFC. However, across association cortical areas, L3PN properties are largely unexplored. We studied the physiology, dendrite morphology and transcriptome of L3PNs from macaque monkey DLPFC and posterior parietal cortex (PPC), two key nodes in the cortical working memory network. L3PNs from DLPFC had greater diversity of physiological properties and larger basal dendrites with higher spine density. Moreover, transcriptome analysis suggested a molecular basis for the differences in the physiological and morphological phenotypes of L3PNs from DLPFC and PPC.

Developmental Expression Patterns of GABAA Receptor Subunits in Layer 3 and 5 Pyramidal Cells of Monkey Prefrontal Cortex.

Cortical pyramidal neuron activity is regulated in part through inhibitory inputs mediated by GABAA receptors. The subunit composition of these receptors confers distinct functional properties. Thus, developmental shifts in subunit expression will likely influence the characteristics of pyramidal cell firing and the functional maturation of processes that depend on these neurons. We used laser microdissection and PCR to quantify postnatal developmental changes in the expression of GABAA receptor subunits (α1, α2, α5, ß2, γ2, and δ) in layer 3 pyramidal cells of monkey prefrontal cortex, which are critical for working memory. To determine the specificity of these changes, we examined glutamate receptor subunits (AMPA Glur1 and NMDA Grin1) and conducted the same analyses in layer 5 pyramidal cells. Expression of GABAA receptor subunit mRNAs changed substantially, whereas glutamate receptor subunit changes were modest over postnatal development. Some transcripts (e.g., GABAA α1) progressively increased from birth until adulthood, whereas others (e.g., GABAA α2) declined with age. Changes in some transcripts were present in only one layer (e.g., GABAA δ). The development of GABAA receptor subunit expression in primate prefrontal pyramidal neurons is protracted and subunit- and layer-specific. These trajectories might contribute to the molecular basis for the maturation of working memory.

Synaptic alterations in pyramidal cells following genetic manipulation of neuronal excitability in monkey prefrontal cortex.

In schizophrenia, layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC) are thought to receive fewer excitatory synaptic inputs and to have lower expression levels of activity-dependent genes and of genes involved in mitochondrial energy production. In concert, these findings from previous studies suggest that DLPFC L3PNs are hypoactive in schizophrenia, disrupting the patterns of activity that are crucial for working memory, which is impaired in the illness. However, whether lower PN activity produces alterations in inhibitory and/or excitatory synaptic strength has not been tested in the primate DLPFC. Here, we decreased PN excitability in rhesus monkey DLPFC in vivo using adeno-associated viral vectors (AAVs) to produce Cre recombinase-mediated overexpression of Kir2.1 channels, a genetic silencing tool that efficiently decreases neuronal excitability. In acute slices prepared from DLPFC 7-12 weeks post-AAV microinjections, Kir2.1-overexpressing PNs had a significantly reduced excitability largely attributable to highly specific effects of the AAV-encoded Kir2.1 channels. Moreover, recordings of synaptic currents showed that Kir2.1-overexpressing DLPFC PNs had reduced strength of excitatory synapses whereas inhibitory synaptic inputs were not affected. The decrease in excitatory synaptic strength was not associated with changes in dendritic spine number, suggesting that excitatory synapse quantity was unaltered in Kir2.1-overexpressing DLPFC PNs. These findings suggest that, in schizophrenia, the excitatory synapses on hypoactive L3PNs are weaker and thus might represent a substrate for novel therapeutic interventions. Significance Statement: In schizophrenia, dorsolateral prefrontal cortex (DLPFC) pyramidal neurons (PNs) have both transcriptional and structural alterations that suggest they are hypoactive. PN hypoactivity is thought to produce synaptic alterations in schizophrenia, however the effects of lower neuronal activity on synaptic function in primate DLPFC have not been examined. Here, we used, for the first time in primate neocortex, adeno-associated viral vectors (AAVs) to reduce PN excitability with Kir2.1 channel overexpression and tested if this manipulation altered the strength of synaptic inputs onto the Kir2.1-overexpressing PNs. Recordings in DLPFC slices showed that Kir2.1 overexpression depressed excitatory (but not inhibitory), synaptic currents, suggesting that, in schizophrenia, the hypoactivity of PNs might be exacerbated by reduced strength of the excitatory synapses they receive.

Infragranular gene expression disturbances in the prefrontal cortex in schizophrenia: signature of altered neural development?

The development of the human neocortex gives rise to a complex cytoarchitecture, grouping together cells with similar structure, connectivity and function. As a result, the six neocortical laminae show distinct molecular content. In schizophrenia, many anatomical and neurochemical changes appear to be restricted to a subset of lamina and/or cell types. In this study, we hypothesized that supragranular (SG; laminae II-III) and infragranular layers (IG; laminae V-VI) of area 46 in the human prefrontal cortex will show distinct and specific transcriptome alterations between subjects with schizophrenia and matched controls. To enhance sample homogeneity, we compared the gene expression patterns of the SG and IG layers of 8 matched middle-aged male subjects with schizophrenia to 8 pairwise matched controls using two replicate DNA microarrays for each sample. The study revealed strong disease-related laminar expression differences between the SG and IG layers. Expression changes were dominated by an overall underexpression of the IG-enriched genes in the schizophrenia subjects compared to normal control subjects. Furthermore, using a diagnosis-blind, unsupervised clustering of the control-derived SG or IG-enriched transcripts, the IG-enriched markers segregated the subjects with schizophrenia from the matched controls with a high degree of confidence. Importantly, multiple members of the semaphorin gene family reported altered gene expression, suggesting that the IG gene expression disturbances in subjects with schizophrenia may be a result of altered cortical development and disrupted brain connectivity.

Mapping Phosphodiesterase 4D (PDE4D) in Macaque Dorsolateral Prefrontal Cortex: Postsynaptic Compartmentalization in Layer III Pyramidal Cell Circuits.

cAMP signaling has powerful, negative effects on cognitive functions of the primate dorsolateral prefrontal cortex (dlPFC), opening potassium channels to reduce firing and impair working memory, and increasing tau phosphorylation in aging neurons. This contrasts with cAMP actions in classic circuits, where it enhances plasticity and transmitter release. PDE4 isozymes regulate cAMP actions, and thus have been a focus of research and drug discovery. Previous work has focused on the localization of PDE4A and PDE4B in dlPFC, but PDE4D is also of great interest, as it is the predominant PDE4 isoform in primate association cortex, and PDE4D expression decreases with aging in human dlPFC. Here we used laser-capture microdissection transcriptomics and found that PDE4D message is enriched in pyramidal cells compared to GABAergic PV-interneurons in layer III of the human dlPFC. A parallel study in rhesus macaques using high-spatial resolution immunoelectron microscopy revealed the ultrastructural locations of PDE4D in primate dlPFC with clarity not possible in human post-mortem tissue. PDE4D was especially prominent in dendrites associated with microtubules, mitochondria, and likely smooth endoplasmic reticulum (SER). There was substantial postsynaptic labeling in dendritic spines, associated with the SER spine-apparatus near glutamatergic-like axospinous synapses, but sparse labeling in axon terminals. We also observed dense PDE4D labeling perisynaptically in astroglial leaflets ensheathing glutamatergic connections. These data suggest that PDE4D is strategically positioned to regulate cAMP signaling in dlPFC glutamatergic synapses and circuits, especially in postsynaptic compartments where it is localized to influence cAMP actions on intracellular trafficking, mitochondrial physiology, and internal calcium release.

GluN2D-mediated excitatory drive onto medial prefrontal cortical PV+ fast-spiking inhibitory interneurons.

Deficits in fast-spiking inhibitory interneurons (FSINs) within the dorsolateral prefrontal cortex (dlPFC) are hypothesized to underlie cognitive impairment associated with schizophrenia. Though representing a minority of interneurons, this key cell type coordinates broad neural network gamma-frequency oscillations, associated with cognition and cognitive flexibility. Here we report expression of GluN2D mRNA selectively in parvalbumin positive cells of human postmortem dlPFC tissue, but not pyramidal neurons, with little to no GluN2C expression in either cell type. In acute murine mPFC slices the GluN2C/D selective positive allosteric modulator (PAM), CIQ(+), increased the intrinsic excitability as well as enhanced NMDAR-mediated EPSCs onto FSINs. This increase in intrinsic excitability with GluN2C/D PAM was also observed in the Dlx 5/6+/- FSIN developmental deficit model with reported FSIN hypoexcitability. Together these data speak to selective modulation of FSINs by a GluN2D PAM, providing a potential mechanism to counter the FSIN-deficit seen in schizophrenia.

Laser capture microdissection-targeted mass spectrometry: a method for multiplexed protein quantification within individual layers of the cerebral cortex.

The mammalian neocortex is organized into layers distinguished by the size, packing density, and connectivity of their constituent neurons. Many neuropsychiatric illnesses are complex trait disorders with etiologic factors converging on neuronal protein networks. Cortical pathology of neuropsychiatric diseases, such as schizophrenia, is often restricted to, or more pronounced in, certain cortical layers, suggesting that genetic vulnerabilities manifest with laminar specificity. Thus, the ability to investigate cortical layer-specific protein levels in human postmortem brain is highly desirable. Here, we developed and validated a laser capture microdissection-mass spectrometry (LCM-MS) approach to quantify over 200 proteins in cortical layers 3 and 5 of two cohorts of human subjects as well as a monkey model of postmortem interval. LCM-MS was readily implementable and reliably identified protein patterns that differed between cortical layers 3 and 5. Our findings suggest that LCM-MS facilitates the precise quantification of proteins within individual cortical layers from human postmortem brain tissue, providing a powerful tool in the study of neuropsychiatric disease.

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.

Transcriptome Alterations in Prefrontal Pyramidal Cells Distinguish Schizophrenia From Bipolar and Major Depressive Disorders.

BACKGROUND: Impairments in certain cognitive processes (e.g., working memory) are typically most pronounced in schizophrenia (SZ), intermediate in bipolar disorder, and least in major depressive disorder. Given that working memory depends, in part, on neural circuitry that includes pyramidal cells in layer 3 (L3) and layer 5 (L5) of the dorsolateral prefrontal cortex (DLPFC), we sought to determine if transcriptome alterations in these neurons were shared or distinctive for each diagnosis. METHODS: Pools of L3 and L5 pyramidal cells in the DLPFC were individually captured by laser microdissection from 19 matched tetrads of unaffected comparison subjects and subjects with SZ, bipolar disorder, and major depressive disorder, and the messenger RNA was subjected to transcriptome profiling by microarray. RESULTS: In DLPFC L3 and L5 pyramidal cells, transcriptome alterations were numerous in subjects with SZ, but rare in subjects with bipolar disorder and major depressive disorder. The leading molecular pathways altered in subjects with SZ involved mitochondrial energy production and the regulation of protein translation. In addition, we did not find any significant transcriptome signatures related to psychosis or suicide. CONCLUSIONS: In concert, these findings suggest that molecular alterations in DLPFC L3 and L5 pyramidal cells might be characteristic of the disease processes operative in individuals diagnosed with SZ and thus might contribute to the circuitry alterations underlying cognitive dysfunction in individuals with SZ.

Altered Expression of ARP2/3 Complex Signaling Pathway Genes in Prefrontal Layer 3 Pyramidal Cells in Schizophrenia.

OBJECTIVE: Lower dendritic spine density on layer 3 pyramidal cells in the dorsolateral prefrontal cortex (DLPFC) appears to contribute to cognitive dysfunction in schizophrenia, whereas psychosis is associated with excessive dopamine release in the striatum. These findings may be related via excitatory projections from the DLPFC to the ventral mesencephalon, the location of dopamine cells projecting to the striatum. Consistent with this hypothesis, deletion of the actin-related protein-2/3 (ARP2/3) complex, which regulates the actin cytoskeleton supporting dendritic spines, produced spine loss in cortical pyramidal cells and striatal hyperdopaminergia in mice. The authors sought to determine whether the ARP2/3 complex is altered in schizophrenia. METHOD: In matched pairs of schizophrenia and comparison subjects, transcript levels of ARP2/3 complex signaling pathway were assessed in laser-microdissected DLPFC layer 3 and 5 pyramidal cells and layer 3 parvalbumin interneurons, and in total DLPFC gray matter. RESULTS: Transcript levels of ARP2/3 complex subunits and of nucleation promotion factors that regulate the ARP2/3 complex were significantly lower in DLPFC layer 3 and 5 pyramidal cells in schizophrenia. In contrast, these transcripts were unaltered, or only modestly changed, in parvalbumin interneurons and DLPFC gray matter. CONCLUSIONS: Down-regulation of the ARP2/3 complex signaling pathway, a common final pathway for multiple signaling cascades that regulate the actin cytoskeleton, would compromise the structural stability of spines, leading to their loss. In concert with findings from deletion of the ARP2/3 complex in mice, these findings support the idea that spine deficits in the DLPFC may contribute to subcortical hyperdopaminergia in schizophrenia.

Presenilin-1-dependent transcriptome changes.

Familial forms of Alzheimer's disease (FADs) are caused by the expression of mutant presenilin 1 (PS1) or presenilin 2. Using DNA microarrays, we explored the brain transcription profiles of mice with conditional knock-out of PS1 (cKO PS1) in the forebrain. In parallel, we performed a transcription profiling of the hippocampus and frontal cortex of the FAD-linked DeltaE9 mutant transgenic (TG) mice and matched controls [TG mice expressing wild-type human PS1 (hPS1)]. When the TG and cKO datasets were cross-compared, the majority of the 30 common expression alterations were in opposite direction, suggesting that the FAD-linked PS1 variant produces transcriptome changes primarily by gain of aberrant function. Our microarray studies also revealed an unanticipated inverse correlation of transcript levels between the brains of mice that coexpress DeltaE9 hPS1+ amyloid precursor protein (APP)695 Swe and DeltaE9 hPS1 single transgenic mice. The opposite directionality of these changes in transcript levels must be a function of APP and/or APP derivatives.

Dysregulated ErbB4 Splicing in Schizophrenia: Selective Effects on Parvalbumin Expression.

OBJECTIVE: Alternative splicing of ErbB4 transcripts is dysregulated in the dorsolateral prefrontal cortex in schizophrenia. ErbB4 regulates the activity of parvalbumin interneurons, and therefore dysregulated ErbB4 splicing could contribute to lower parvalbumin interneuron activity and consequently lower parvalbumin levels in schizophrenia. However, ErbB4 is also present in calretinin interneurons, which are not affected in schizophrenia. Therefore, the authors hypothesized that dysregulated ErbB4 splicing occurs selectively in parvalbumin interneurons and is associated with lower parvalbumin levels in schizophrenia. METHOD: Tissue samples enriched in calretinin and parvalbumin interneurons were laser microdissected from dorsolateral prefrontal cortex layers 2 and 4, respectively, from matched pairs of schizophrenia and comparison subjects. Transcript levels for pan-ErbB4, four ErbB4 splicing variants (JM-a, JM-b, CYT-1, CYT-2), parvalbumin, and calretinin were quantified by quantitative polymerase chain reaction (qPCR) in each layer. Transcript levels for myocardial infarction associated transcript (MIAT), which regulates ErbB4 splicing, were quantified in gray matter by qPCR and in parvalbumin interneurons by microarray. RESULTS: Calretinin and parvalbumin mRNAs were preferentially expressed in layers 2 and 4, respectively. In schizophrenia subjects, lower parvalbumin levels, higher CYT-1 and JM-a levels, and lower CYT-2 and JM-b levels were detected selectively in layer 4. In layer 4, the JM-a/JM-b ratio was inversely correlated with parvalbumin levels in schizophrenia subjects. MIAT levels were preferentially higher in parvalbumin interneurons in schizophrenia subjects. CONCLUSIONS: These findings suggest that elevated MIAT expression alters ErbB4 splicing selectively in parvalbumin interneurons in schizophrenia. Dysregulated ErbB4 splicing in schizophrenia may contribute to lower activity of parvalbumin interneurons and an activity-dependent down-regulation of parvalbumin expression.

Reciprocal Alterations in Regulator of G Protein Signaling 4 and microRNA16 in Schizophrenia.

N-methyl-d-aspartate receptor (NMDAR) hypofunction in the dorsolateral prefrontal cortex (DLPFC) has been implicated in the pathology of schizophrenia. NMDAR activity is negatively regulated by some G protein-coupled receptors (GPCRs). Signaling through these GPCRs is reduced by Regulator of G protein Signaling 4 (RGS4). Thus, lower levels of RGS4 would enhance GPCR-mediated reductions in NMDAR activity and could contribute to NMDAR hypofunction in schizophrenia. In this study, we quantified RGS4 mRNA and protein levels at several levels of resolution in the DLPFC from subjects with schizophrenia and matched healthy comparison subjects. To investigate molecular mechanisms that could contribute to altered RGS4 levels, we quantified levels of small noncoding RNAs, known as microRNAs (miRs), which regulate RGS4 mRNA integrity after transcription. RGS4 mRNA and protein levels were significantly lower in schizophrenia subjects and were positively correlated across all subjects. The RGS4 mRNA deficit was present in pyramidal neurons of DLPFC layers 3 and 5 of the schizophrenia subjects. In contrast, levels of miR16 were significantly higher in the DLPFC of schizophrenia subjects, and higher miR16 levels predicted lower RGS4 mRNA levels. These findings provide convergent evidence of lower RGS4 mRNA and protein levels in schizophrenia that may result from increased expression of miR16. Given the role of RGS4 in regulating GPCRs, and consequently the strength of NMDAR signaling, these findings could contribute to the molecular substrate for NMDAR hypofunction in DLPFC pyramidal cells in schizophrenia.

HIV resistance to zidovudine: the role of pyrophosphorolysis.

Zidovudine-resistant strains of HIV became apparent in many patients soon after advent of zidovudine (AZT) monotherapy. While this resistance could be unequivocally correlated with multiple mutations in HIV reverse transcriptase (D67N, K70R, T215F/Y, K219Q), the mechanism or phenotype for this resistance has remained obscure for more than a decade, despite active investigation. Recent studies indicate that AZT resistance may be related to removal of chain-terminating AZT from the 3'-terminus of the primer, by a process known as pyrophosphorolysis. This process is catalyzed by HIV-1 reverse transcriptase (RT), and is the reverse reaction of DNA polymerization. The D67N/K70R mutations result in a significantly increased rate of RT-catalyzed pyrophosphorolysis at physiological levels of pyrophosphate, which leads to a decrease in the extent of AZT chain termination of nascent viral DNA. The potential replication deficit of an increased reverse reaction during DNA synthesis is compensated by increased DNA synthesis processivity, a phenotype that results from the T215F/Y/K219Q mutations in RT. The net result of these multiple phenotypes imparted by the multiple mutations in RT is the facile synthesis of full-length viral DNA in the presence of AZT. Copyright 1999 Harcourt Publishers Ltd.

Altered expression of CDC42 signaling pathway components in cortical layer 3 pyramidal cells in schizophrenia.

BACKGROUND: Cognitive dysfunction in schizophrenia is associated with a lower density of dendritic spines on deep layer 3 pyramidal cells in the dorsolateral prefrontal cortex (DLPFC). These alterations appear to reflect dysregulation of the actin cytoskeleton required for spine formation and maintenance. Consistent with this idea, altered expression of genes in the cell division cycle 42 (CDC42)-CDC42 effector protein (CDC42EP) signaling pathway, a key organizer of the actin cytoskeleton, was previously reported in DLPFC gray matter from subjects with schizophrenia. We examined the integrity of the CDC42-p21-activated serine/threonine protein kinases (PAK)-LIM domain-containing serine/threonine protein kinases (LIMK) signaling pathway in schizophrenia in a layer-specific and cell type-specific fashion in DLPFC deep layer 3. METHODS: Using laser microdissection, samples of DLPFC deep layer 3 were collected from 56 matched pairs of subjects with schizophrenia and comparison subjects, and levels of CDC42-PAK-LIMK pathway messenger RNAs were measured by quantitative polymerase chain reaction. These same transcripts also were quantified by microarray in samples of individually microdissected deep layer 3 pyramidal cells from a subset of the same subjects and from monkeys exposed to antipsychotics. RESULTS: Relative to comparison subjects, CDC42EP4, LIMK1, LIMK2, ARHGDIA, and PAK3 messenger RNA levels were significantly upregulated in subjects with schizophrenia in laminar and cellular samples. In contrast, CDC42 and PAK1 messenger RNA levels were significantly downregulated specifically in deep layer 3 pyramidal cells. These differences were not attributable to psychotropic medications or other comorbid factors. CONCLUSIONS: Findings from the present and prior studies converge on synergistic alterations in CDC42 signaling pathway that could destabilize actin dynamics and produce spine deficits preferentially in deep layer 3 pyramidal cells in schizophrenia.

Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation.

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

Lower gene expression for KCNS3 potassium channel subunit in parvalbumin-containing neurons in the prefrontal cortex in schizophrenia.

OBJECTIVE: In schizophrenia, alterations in markers of cortical GABA neurotransmission are prominent in parvalbumin-containing neurons. Parvalbumin neurons selectively express KCNS3, the gene encoding the Kv9.3 potassium channel α-subunit. Kv9.3 subunits are present in voltage-gated potassium channels that contribute to the precise detection of coincident excitatory synaptic inputs to parvalbumin neurons. This distinctive feature of parvalbumin neurons appears important for the synchronization of cortical neural networks in γ-oscillations. Because impaired prefrontal cortical γ-oscillations are thought to underlie the cognitive impairments in schizophrenia, the authors investigated whether KCNS3 mRNA levels are altered in the prefrontal cortex of schizophrenia subjects. METHOD: KCNS3 mRNA expression was evaluated by in situ hybridization in 22 matched pairs of schizophrenia and comparison subjects and by microarray analyses of pooled samples of individually dissected neurons that were labeled with Vicia villosa agglutinin (VVA), a parvalbumin neuron-selective marker, in a separate cohort of 14 pairs. Effects of chronic antipsychotic treatments on KCNS3 expression were tested in the prefrontal cortex of antipsychotic-exposed monkeys. RESULTS: By in situ hybridization, KCNS3 mRNA levels were 23% lower in schizophrenia subjects. At the cellular level, both KCNS3 mRNA-expressing neuron density and KCNS3 mRNA level per neuron were significantly lower. By microarray, KCNS3 mRNA levels were lower by 40% in VVA-labeled neurons from schizophrenia subjects. KCNS3 mRNA levels were not altered in antipsychotic-exposed monkeys. CONCLUSIONS: These findings reveal lower KCNS3 expression in prefrontal cortical parvalbumin neurons in schizophrenia, providing a molecular basis for compromised detection of coincident synaptic inputs to parvalbumin neurons that could contribute to altered γ-oscillations and impaired cognition 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 Examiner's 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.