Szatmár Horváth

Immune System Disturbances in Schizophrenia

Epidemiological, genetic, transcriptome, postmortem, peripheral biomarker, and therapeutic studies of schizophrenia all point to a dysregulation of both innate and adaptive immune systems in the disease, and it is likely that these immune changes actively contribute to disease symptoms. Gene expression disturbances in the brain of subjects with schizophrenia show complex, region-specific changes with consistently replicated and potentially interdependent induction of serpin peptidase inhibitor, clade A member 3 (SERPINA3) and interferon inducible transmembrane protein (IFITM) family transcripts in the prefrontal cortex. Recent data suggest that IFITM3 expression is a critical mediator of maternal immune activation. Because the IFITM gene family is primarily expressed in the endothelial cells and meninges, and because the meninges play a critical role in interneuron development, we suggest that these two non-neuronal cell populations might play an important role in the disease pathophysiology. Finally, we propose that IFITM3 in particular might be a novel, appealing, knowledge-based drug target for treatment of schizophrenia.

Schizophrenia as a disorder of molecular pathways

Over the last decade, transcriptome studies of postmortem tissue from subjects with schizophrenia revealed that synaptic, mitochondrial, immune system, gamma-aminobutyric acidergic, and oligodendrocytic changes are all integral parts of the disease process. The combined genetic and transcriptomic studies argue that the molecular underpinnings of the disease are even more varied than the symptomatic diversity of schizophrenia. Ultimately, to decipher the pathophysiology of human disorders in general, we will need to understand the function of hundreds of genes and regulatory elements in our genome and the consequences of their overexpression and reduced expression in a developmental context. Furthermore, integration of knowledge from various data sources remains a monumental challenge that has to be systematically addressed in the upcoming decades. In the end, our success in interpreting the molecular changes in schizophrenia will depend on our ability to understand the biology using innovative ideas and cannot depend on the hope of developing novel, more powerful technologies.

Analyzing Schizophrenia by DNA Microarrays

To understand the pathological processes of schizophrenia, we must embrace the analysis of the diseased human brain: we will never be able to recapitulate the pathology of uniquely human disorders in an animal model. Based on the outcome of the transcriptome profiling experiments performed to date, it appears that schizophrenia is associated with a global gene expression disturbance across many cortical regions. In addition, transcriptome changes are present in multiple cell types, including specific subclasses of principal neurons, interneurons, and oligodendrocytes. Furthermore, transcripts related to synaptic transmission, energy metabolism, and inhibitory neurotransmission are routinely found underexpressed in the postmortem brain tissue of subjects with schizophrenia. To put these transcriptome data in biological context, we must make our data publicly available and report our findings in a proper, expanded Minimum Information About a Microarray Experiment format. Cell-type specific expression profiling and sequencing-based transcript assessments should be expanded, with particular attention to understanding splice-variant changes in various mental disorders. Deciphering the pathophysiology of mental disorders depends on integrating data from across many research fields and techniques. Leads from postmortem transcriptome profiling will be essential to generate model animals, perform tissue culture experiments, and develop or evaluate novel drugs to treat this devastating disorder.

The Iowa Gambling Task in depression - what have we learned about sub-optimal decision-making strategies?

Our earlier study found patients with depression to show a preference for larger reward as measured by the Iowa Gambling Task (IGT). In this IGT version, larger rewards were associated with even larger consequent losses. In the light of the clinical markers defining depressive disorder, this finding might appear controversial at first. Performance of depressed patients on various decision-making (DM) tasks is typically found to be impaired. Evidence points toward reduced reward learning, as well as the difficulty to shift strategy and integrate environmental changes into DM contingencies. This results in an impaired ability to modulate behavior as a function of reward, or punishment, respectively. Clinical symptoms of the disorder, the genetic profile, as well as personality traits might also influence DM strategies. More severe depression increased sensitivity to immediate large punishment, thus predicting future decisions, and was also associated with higher harm avoidance. Anhedonic features diminished reward learning abilities to a greater extent, even predicting clinical outcome. Several questions about how these aspects relate remain to be clarified. Is there a genetic predisposition for the DM impairment preceding mood symptoms? Is it the consequence of clinical signs or even learned behavior serving as a coping strategy? Are patients prone to develop an aversion of loss or are they unable to sense or deal with reward or the preference of reward? Does the DM deficit normalize or is a persisting impairment predictor for clinical outcome or relapse risk? To what extent is it influenced by medication effects? How does a long-lasting DM deficit affect daily life and social interactions? Strikingly, research evidence indicates that depressed patients tend to behave less deceptive and more self-focused, resulting in impaired social DM. The difficulty in daily interpersonal interactions might contribute to social isolation, further intensifying depressive symptoms.

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.

Inhibition of parvalbumin-expressing interneurons results in complex behavioral changes

Reduced expression of the Gad1 gene-encoded 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of schizophrenia. GAD67 downregulation occurs in multiple interneuronal sub-populations, including the parvalbumin-positive (PVALB+) cells. To investigate the role of the PV-positive GABAergic interneurons in behavioral and molecular processes, we knocked down the Gad1 transcript using a microRNA engineered to target specifically Gad1 mRNA under the control of Pvalb bacterial artificial chromosome. Verification of construct expression was performed by immunohistochemistry. Follow-up electrophysiological studies revealed a significant reduction in γ-aminobutyric acid (GABA) release probability without alterations in postsynaptic membrane properties or changes in glutamatergic release probability in the prefrontal cortex pyramidal neurons. Behavioral characterization of our transgenic (Tg) mice uncovered that the Pvalb/Gad1 Tg mice have pronounced sensorimotor gating deficits, increased novelty-seeking and reduced fear extinction. Furthermore, NMDA (N-methyl-d-aspartate) receptor antagonism by ketamine had an opposing dose-dependent effect, suggesting that the differential dosage of ketamine might have divergent effects on behavioral processes. All behavioral studies were validated using a second cohort of animals. Our results suggest that reduction of GABAergic transmission from PVALB+ interneurons primarily impacts behavioral domains related to fear and novelty seeking and that these alterations might be related to the behavioral phenotype observed in schizophrenia.

Novel animal models for studying complex brain disorders: BAC-driven miRNA-mediated in vivo silencing of gene expression

In schizophrenia, glutamic acid decarboxylase 1 (GAD1) disturbances are robust, consistently observed, cell-type specific and represent a core feature of the disease. In addition, neuropeptide Y (NPY), which is a phenotypic marker of a sub-population of GAD1-containing interneurons, has shown reduced expression in the prefrontal cortex in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal sub-population might be a core feature of this devastating disorder. However, modeling gene expression disturbances in schizophrenia in a cell type-specific manner has been extremely challenging. To more closely mimic these molecular and cellular human post-mortem findings, we generated a transgenic mouse in which we downregulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for reducing gene expression uses a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP) and a modified intron containing a synthetic microRNA (miRNA) targeted to GAD1. The animals of isogenic strains are generated rapidly, providing a new tool for better understanding the molecular disturbances in the GABAergic system observed in complex neuropsychiatric disorders such as schizophrenia. In the future, because of the small size of the silencing miRNAs combined with our BAC strategy, this method may be modified to allow generation of mice with simultaneous silencing of multiple genes in the same cells with a single construct, and production of splice-variant-specific knockdown animals.

Estrogen effects on arcuate neurons in rat. An in situ electrophysiological study

In acute experiments, the effects of i.p. 17beta-estradiol on the activity of arcuate neurons were studied in ovariectomized rats. 17Beta-estradiol (100 microg/100g, i.p.) increased the spontaneous activity of the observed arcuate neurons with a latency of 20-25 min. In some neurons spontaneous activity could be influenced by stimulation of the olfactory and somatosensory systems. Activation of the trigeminal system significantly increased the spontaneous activity of the studied units, while stimulation of the accessory olfactory bulb decreased it, both with and without 17beta-estradiol treatment. It is suggested that the 20-25 min latency of the 17beta-estradiol effect is based not so much on membrane as on genomic mechanisms. This suggestion is supported by immunocytochemical studies: 17beta-estradiol treatment significantly decreased the number of GABA-positive axo-somatic synapses in the arcuate nucleus.

Facial nerve injury induces facilitation of responses in both trigeminal and facial nuclei of rat

A study was made of the effects of facial nerve transection on trigeminal stimulation- evoked field potentials in the principal trigeminal (Pr5) and facial nuclei (7) in rats. Although the transected branch of the facial nerve contains pure motoric efferents, it resulted in enhanced responses in both Pr5 and 7. These electrophysiological results suggest a functional circuitry involving the whiskers, trigeminal nerve, Pr5 and 7 and the facial nerve as efferent. The disconnection (opening) of this loop results in enhanced responsiveness of the neurons in both Pr5 and 7.

The role of cannabinoid 1 receptor expressing interneurons in behavior.

Schizophrenia is a devastating neurodevelopmental disorder that affects approximately 1% of the population. Reduced expression of the 67-kDa protein isoform of glutamic acid decarboxylase (GAD67) is a hallmark of the disease and is encoded by the GAD1 gene. In schizophrenia, GAD67 downregulation occurs in multiple interneuronal subpopulations, including the cannabinoid receptor type 1 positive (CNR1+) cells, but the functional consequences of these disturbances are not well understood. To investigate the role of the CNR1-positive GABA-ergic interneurons in behavioral and molecular processes, we employed a novel, miRNA-mediated transgenic mouse approach. We silenced the Gad1 transcript using a miRNA engineered to specifically target Gad1 mRNA under the control of Cnr1 bacterial artificial chromosome. Behavioral characterization of our transgenic mice showed elevated and persistent conditioned fear associated with an auditory cue and a significantly altered response to an amphetamine challenge. These deficits could not be attributed to sensory deficits or changes in baseline learning and memory. Furthermore, HPLC analyses revealed that Cnr1/Gad1 mice have enhanced serotonin levels, but not dopamine levels in response to amphetamine. Our findings demonstrate that dysfunction of a small subset of interneurons can have a profound effect on behavior and that the GABA-ergic, monoamine, and cannabinoid systems are functionally interconnected. The results also suggest that understanding the function of various interneuronal subclasses might be essential to develop knowledge-based treatment strategies for various mental disorders including schizophrenia and substance abuse.