Dianne A. Cruz

Postnatal development of prefrontal inhibitory circuits and the pathophysiology of cognitive dysfunction in schizophrenia

Abstract: The typical appearance of the clinical features of schizophrenia during late adolescence or early adulthood suggests that adolescence‐related neurodevelopmental events may contribute to the pathophysiology of this disorder. Here the role that GABA‐mediated inhibition in the dorsal lateral prefrontal cortex (DLPFC) plays in regulating working memory, a core cognitive process that matures late and that is disturbed in schizophrenia, is reviewed. Recent studies are summarized that demonstrate (1) that certain pre‐ and postsynaptic markers of GABA neurotransmission in the monkey DLPFC exhibit striking changes during adolescence, and (2) that these same markers are markedly altered in the DLPFC of subjects with schizophrenia. The implications of these findings for treatment and prevention strategies are discussed.

Postnatal development of pre- and postsynaptic GABA markers at chandelier cell connections with pyramidal neurons in monkey prefrontal cortex

The protracted postnatal maturation of the primate prefrontal cortex (PFC) is associated with substantial changes in the number of excitatory synapses on pyramidal neurons, whereas the total number of inhibitory synapses appears to remain constant. In this study, we sought to determine whether the developmental changes in excitatory input to pyramidal cells are paralleled by changes in functional markers of inhibitory inputs to pyramidal neurons. The chandelier subclass of γ‐aminobutyric acid (GABA) neurons provides potent inhibitory control over pyramidal neurons by virtue of their axon terminals, which form distinct vertical structures (termed cartridges) that synapse at the axon initial segment (AIS) of pyramidal neurons. Thus, we examined the relative densities, laminar distributions, and lengths of presynaptic chandelier axon cartridges immunoreactive for the GABA membrane transporter 1 (GAT1) or the calcium‐binding protein parvalbumin (PV) and of postsynaptic pyramidal neuron AIS immunoreactive for the GABAA receptor α2 subunit (GABAA α2) in PFC area 46 of 38 rhesus monkeys (Macaca mulatta). From birth through 2 years of age, the relative densities and laminar distributions of these three markers exhibited different trajectories, suggesting developmental shifts in the weighting of at least some factors that determine inhibition at the AIS. In contrast, from 2 to 4 years of age, all three markers exhibited similar declines in density and length that paralleled the periadolescent pruning of excitatory synapses to pyramidal neurons. Across development, the predominant laminar location of PV‐labeled cartridges and GABAA α2‐immunoreactive AIS shifted from the middle to superficial layers, whereas the laminar distribution of GAT1‐positive cartridges did not change. Together, these findings suggest that the maturation of inhibitory inputs to the AIS of PFC pyramidal neurons is a complex process that may differentially affect the firing patterns of subpopulations of pyramidal neurons at specific postnatal time points. J. Comp. Neurol. 465:385–400, 2003.

The DNA Methylome and Transcriptome of Different Brain Regions in Schizophrenia and Bipolar Disorder

Extensive changes in DNA methylation have been observed in schizophrenia (SC) and bipolar disorder (BP), and may contribute to the pathogenesis of these disorders. Here, we performed genome-scale DNA methylation profiling using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) on two brain regions (including frontal cortex and anterior cingulate) in 5 SC, 7 BP and 6 normal subjects. Comparing with normal controls, we identified substantial differentially methylated regions (DMRs) in these two brain regions of SC and BP. To our surprise, different brain regions show completely distinct distributions of DMRs across the genomes. In frontal cortex of both SC and BP subjects, we observed widespread hypomethylation as compared to normal controls, preferentially targeting the terminal ends of the chromosomes. In contrast, in anterior cingulate, both SC and BP subjects displayed extensive gain of methylation. Notably, in these two brain regions of SC and BP, only a few DMRs overlapped with promoters, whereas a greater proportion occurs in introns and intergenic regions. Functional enrichment analysis indicated that important psychiatric disorder-related biological processes such as neuron development, differentiation and projection may be altered by epigenetic changes located in the intronic regions. Transcriptome analysis revealed consistent dysfunctional processes with those determined by DMRs. Furthermore, DMRs in the same brain regions from SC and BP could successfully distinguish BP and/or SC from normal controls while differentially expressed genes could not. Overall, our results support a major role for brain-region-dependent aberrant DNA methylation in the pathogenesis of these two disorders.

Selective alterations in postsynaptic markers of chandelier cell inputs to cortical pyramidal neurons in subjects with schizophrenia.

Markers of GABA neurotransmission between chandelier neurons and their synaptic targets, the axon initial segment (AIS) of pyramidal neurons, are altered in the dorsolateral prefrontal cortex (dlPFC) of subjects with schizophrenia. For example, immunoreactivity for the GABA membrane transporter (GAT1) is decreased in presynaptic chandelier neuron axon terminals, whereas immunoreactivity for the GABAA receptor α2 subunit is increased in postsynaptic AIS. To understand the nature and functional significance of these alterations, we determined the density, laminar distribution, and length of AIS immunoreactive (IR) for ankryin-G and βIV spectrin, two proteins involved in the regulation of synapse structure and ion channel clustering at AIS, in dlPFC area 46 from 14 matched triads of subjects with schizophrenia or major depressive disorder (MDD) and normal comparison participants. The density of ankyrin-G-IR AIS in the superficial, but not in the deep, cortical layers was significantly decreased by 15–19% in the subjects with schizophrenia relative to the other participant groups. In contrast, no group differences were present in the density of βIV spectrin-IR AIS. The length of labeled AIS did not differ across participant groups for either ankyrin-G or βIV spectrin. The density of ankyrin-G-IR AIS was not altered in the dlPFC of macaque monkeys chronically exposed to antipsychotic medications. Given the important role of ankyrin-G in the recruitment and stabilization of sodium channels and other integral membrane proteins to AIS, our findings suggest that these processes are selectively altered in superficial layer pyramidal neurons in subjects with schizophrenia.

Postnatal development of synaptic structure proteins in pyramidal neuron axon initial segments in monkey prefrontal cortex

In the primate prefrontal cortex (PFC), the functional maturation of the synaptic connections of certain classes of γ‐aminobutyric acid (GABA) neurons is very complex. For example, the levels of both pre‐ and postsynaptic proteins that regulate GABA neurotransmission from the chandelier class of cortical interneurons to the axon initial segment (AIS) of pyramidal neurons undergo marked changes during both the perinatal period and adolescence in the monkey PFC. In order to understand the potential molecular mechanisms associated with these developmental refinements, we quantified the relative densities, laminar distributions, and lengths of pyramidal neuron AIS immunoreactive for ankyrin‐G, βIV spectrin, or gephyrin, three proteins involved in regulating synapse structure and receptor localization, in the PFC of rhesus monkeys ranging in age from birth through adulthood. Ankyrin‐G‐ and βIV spectrin‐labeled AIS declined in density and length during the first 6 postnatal months, but then remained stable through adolescence and into adulthood. In contrast, the density of gephyrin‐labeled AIS was stable until approximately 15 months of age and then markedly declined during adolescence. Thus, molecular determinants of the structural features that define GABA inputs to pyramidal neuron AIS in monkey PFC undergo distinct developmental trajectories with different types of changes occurring during the perinatal period and adolescence. In concert with previous data, these findings reveal a two‐phase developmental process of GABAergic synaptic stability and GABA neurotransmission at chandelier cell inputs to pyramidal neurons that likely contributes to the protracted maturation of behaviors mediated by primate PFC circuitry. J. Comp. Neurol. 514:353–367, 2009.

Differential Neuregulin 1 Cleavage in the Prefrontal Cortex and Hippocampus in Schizophrenia and Bipolar Disorder: Preliminary Findings

Background Neuregulin 1 (NRG1) is a key candidate susceptibility gene for both schizophrenia (SCZ) and bipolar disorder (BPD). The function of the NRG1 transmembrane proteins is regulated by cleavage. Alteration of membrane bound-NRG1 cleavage has been previously shown to be associated with behavioral impairments in mouse models lacking expression of NRG1-cleavage enzymes such as BACE1 and gamma secretase. We sought to determine whether alterations in NRG1 cleavage and associated enzymes occur in patients with SCZ and BPD. Methodology/Principal Findings Using human postmortem brain, we evaluated protein expression of NRG1 cleavage products and enzymes that cleave at the external (BACE1, ADAM17, ADAM19) and internal (PS1-gamma secretase) sides of the cell membrane. We used three different cohorts (Controls, SCZ and BPD) and two distinct brain regions: BA9-prefrontal cortex (Controls (n = 6), SCZ (n = 6) and BPD (n = 6)) and hippocampus (Controls (n = 5), SCZ (n = 6) and BPD (n = 6)). In BA9, the ratio of the NRG1 N-terminal fragment relative to full length was significantly upregulated in the SCZ cohort (Bonferroni test, p = 0.011). ADAM17 was negatively correlated with full length NRG1 levels in the SCZ cohort (r = –0.926, p = 0.008). In the hippocampus we found significantly lower levels of a soluble 50 kDa NRG1 fragment in the two affected groups compared the control cohort (Bonferroni test, p = 0.0018). We also examined the relationship of specific symptomatology criteria with measures of NRG1 cleavage using the Bipolar Inventory of Signs and Symptoms Scale (BISS) and the Montgomery Åsberg Depression Rating Scale (MADRS). Our results showed a positive correlation between ADAM19 and psychosis (r = 0.595 p = 0.019); PS1 and mania (r = 0.535, p = 0.040); PS1 and depression (r = 0.567, p = 0.027) in BA9, and BACE1 with anxiety (r = 0.608, p = 0.03) in the hippocampus. Conclusion/Significance Our preliminary findings suggest region-specific alterations in NRG1 cleavage in SCZ and BPD patients. These changes may be associated with specific symptoms in these psychiatric disorders.

BA11 FKBP5 expression levels correlate with dendritic spine density in postmortem PTSD and controls

Genetic variants of the immunophilin FKBP5 have been implicated in susceptibility to post-traumatic stress disorder (PTSD) and other stress-related disorders. We examined the relationship between mushroom, stubby, thin and filopodial spine densities measured with Golgi staining and FKBP5 gene expression in the medial orbitofrontal cortex (BA11) in individuals diagnosed with PTSD and normal controls (n = 8/8). ANCOVA revealed PTSD cases had a significantly elevated density of stubby spines (29%, P < 0.037) and a trend for a reduction in mushroom spine density (25%, p < 0.082). Levels of FKBP5 mRNA were marginally elevated in the PTSD cases (z = 1.94, p = 0.053) and levels correlated inversely with mushroom (Spearmans rho = −0.83, p < 0.001) and overall spine density (rho = −0.75, p < 0.002) and directly with stubby spine density (rho = 0.55, p < 0.027). These data suggest that FKBP5 may participate in a cellular pathway modulating neuronal spine density changes in the brain, and that this pathway may be dysregulated in PTSD.

Serotonin receptor, SERT mRNA and correlations with symptoms in males with alcohol dependence and suicide

Thompson PM, Cruz DA, Olukotun DY, Delgado PL. Serotonin receptor, SERT mRNA and correlations with symptoms in males with alcohol dependence and suicide.

Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity

Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined.

Concordance of psychiatric symptom ratings between a subject and informant, relevancy to post-mortem research

Investigators are interested in determining whether lifetime behavioral traits and specific mood states experienced close to death affect brain gene and protein expression as assessed in post-mortem human brains. Major obstacles to conducting this type of research are the uncertain reliability of the post-mortem psychiatric diagnoses and clinical information because of the retrospective nature of the information. In this study, we addressed the concordance of clinical information obtained through an informant compared with information obtained through a clinician interview of the subject. To test this, we measured both lifetime and within the week psychiatric symptoms of subjects (n=20) and an informant, their next-of-kin (n=20) who were asked identical questions. We found Diagnostic and Statistical Manual (DSM)-IV axis 1 diagnoses by Mini-International Neuropsychiatric Interview proportion of positive agreement for major depression was 0.97, bipolar disorder was 0.81, whereas proportion of negative agreement was 0.97 for schizophrenia. Symptom scale intra-class correlation coefficients and 95% confidence interval were: Bipolar Inventory of Signs and Symptoms=0.59 (0.23, 0.81), Brief Psychiatric Rating Scale=0.58 (0.19, 0.81), Hamilton Depression Rating Scale=0.44 (0.03, 0.72), Montgomery Asberg Depression Rating Scale=0.44 (0.03, 0.72), Young Mania Rating Scale=0.61 (0.30, 0.82), Barratt Impulsiveness Score=0.36 (−0.11, 0.70) and Childhood Trauma Questionnaire=0.48 (−0.15, 0.83). We show that DSM-IV diagnoses; lifetime impulsivity severity, childhood trauma score and symptom scores were significantly consistent between the subjects and their informants. These data suggest, with some limitations, that both retrospective and informant obtained information can provide useful clinical information in post-mortem research.