Insulin, IGF-1, and muscarinic agonists modulate schizophrenia-associated genes in human neuroblastoma cells.

BACKGROUND: Genes associated with energy metabolism are decreased in schizophrenia brain and human and rodent diabetic skeletal muscle. These and other similarities between diabetes and schizophrenia suggest that an insulin signaling deficit may underlie schizophrenia. We determined with human SH-SY5Y neuroblastoma and astrocyte cell lines whether insulin or other molecules could modulate genes opposite to their change reported in schizophrenia brain. METHODS: Both cell lines were treated with insulin, insulin-like growth factor (IGF)-1, IGF-2, or brain-derived neurotrophic factor (BDNF). Genes whose expression was found with microarrays to be changed by insulin in a reciprocal manner to their change in schizophrenia were used in a 16-gene miniarray to identify small molecules that might mimic insulin. RESULTS: Insulin phosphorylated its receptor in the neuroblastoma cells but not in astrocytes and, like IGF-1, increased ERK1/2 and Akt phosphorylation. Insulin and IGF-1 increased the expression of genes decreased in schizophrenia, including those involved in mitochondrial functions, glucose and energy metabolism, hydrogen ion transport, and synaptic function. These gene effects were confirmed and shown to be dose related with the 16-gene miniarrays. Most of 1940 pharmacologically unique compounds failed to alter gene expression, with the exception of muscarinic agonists, which mimicked insulin and IGF-1, and which were blocked by the muscarinic antagonists atropine and telenzepine. CONCLUSIONS: Stimulation of muscarinic and insulin/IGF-1 receptors alter genes associated with metabolic and synaptic functions in a manner reciprocal to their changes in schizophrenia. Pharmacologic activation of these receptors may normalize genomic alterations in schizophrenia and better address root causes of this disease.

Altered expression of hippocampal dentate granule neuron genes in a mouse model of human 22q11 deletion syndrome.

Hemizygous deletion of a 3 Mb region of 22q11.2 is found in 1/4000 humans and produces 22q11 deletion syndrome (22q11DS). Up to 35% of 22q11DS patients develop schizophrenia, making it the second highest risk factor for schizophrenia. A mouse model for 22q11DS, the Df1/+ mouse, carries a hemizygous deletion in a region syntenic with the human deletion. Df1/+ mice are mostly viable but display deficits in prepulse inhibition and learning and memory, two common traits of schizophrenia thought to result, at least in part, from defects in hippocampal neurons. We used oligonucleotide microarrays and QRT-PCR to evaluate gene expression changes in hippocampal dentate granule neurons of Df1/+ mice versus wild-type littermates (n=12/group). The expression of only 287 genes changed with p value significance below 0.05 by microarray, yet 12 of the 21 Df1 region genes represented on the array showed highly significantly reduced expression compared to wild-type controls (33% on average, p values from 10(-3) to 10(-7)). Variants in two of these genes, COMT and PRODH, have been linked with schizophrenia. Overlap of the 287 genes with the reportedly reduced expression of mitochondrial, ubiquitin/proteasome, and synaptic plasticity genes in schizophrenia dentate granule neurons, was not significant. However, modest increases in expression of mitochondrial electron transport genes were observed in the Df1/+ mice. This perhaps indicates a compensation for mitochondrial dysfunction caused by the strongly reduced expression of the Df1 region-encoded mitochondrial enzymes proline dehydrogenase (Prodh) and thioredoxin reductase 2 (Txnrd2).

Deficient hippocampal neuron expression of proteasome, ubiquitin, and mitochondrial genes in multiple schizophrenia cohorts.

BACKGROUND: Hippocampal dentate granule neurons are altered in schizophrenia, but it is unknown if their gene expressions change in schizophrenia or other psychiatric diseases. METHODS: Laser-captured dentate granule neurons from two groups of schizophrenia and control cases and from major depression and bipolar disease cases were examined for alterations in gene expression using complementary DNA (cDNA) microarrays and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with 24 control cases, the 22 schizophrenia patients in both groups revealed decreases in clusters of genes that encode for protein turnover (proteasome subunits and ubiquitin), mitochondrial oxidative energy metabolism (isocitrate, lactate, malate, nicotinamide adenine dinucleotide [NADH], and succinate dehydrogenases; cytochrome C oxidase; adenosine triphosphate [ATP] synthase), and genes associated with neurite outgrowth, cytoskeletal proteins, and synapse plasticity. These changes were not obtained in 9 bipolar cases or 10 major depression cases and were not associated with age, sex, brain weight, body weight, postmortem interval, or drug history. Brain pH contributed to the variance of some genes but was mostly independent of the disease effect. CONCLUSIONS: Decreases in hippocampal neuron gene expression are consistent with brain imaging and microarray studies of the frontal cortex in schizophrenia. A mitochondrial and ubiquitin-proteasome hypofunctioning of dentate granule neurons may contribute to the deficits of schizophrenia.

The LIM domain-only protein LMO4 is required for neural tube closure.

Nuclear LIM domain-only proteins (LMOs), which consist of two closely spaced 50 amino acid Zn2+-finger protein interaction modules mediate interactions between several classes of transcription factors important for development. LMO2 is necessary for development of the entire hematopoietic system and overexpression of LMO1 or LMO2 results in human acute T cell leukemia. LMO4 is the most widely expressed LMO but its normal function is unknown. During development, LMO4 is expressed in dividing neuroepithelial cells within the ventricular zone along the entire rostrocaudal axis of the nervous system. In telencephalic and spinal cord regions of the CNS, LMO4 is highly expressed in ventral but is low in dorsal proliferating neuroepithelial cells. To understand the role of LMO4 during mouse development, we generated a homozygous null mutation in the gene. We found that LMO4 is required for proper closure of the anterior neural tube. In the absence of LMO4, elevation, bending, and proliferation of the ventral neural epithelium and consequent fusion of the prospective dorsal ends of the neural tube do not occur. LMO4 mutant mice die embryonically and exhibit exencephaly, which is associated with abnormal patterns of cell proliferation and with high levels of apoptotic cell death within the neuroepithelium. LMO4 is thus essential for normal patterns of proliferation and for survival of neural epithelial cells in the rostral neural tube. LMO4 is also expressed in Schwann cell progenitors after these contact neurites, a process mediated in part by neuregulin (Nrg).

Comparison of microarray-based mRNA profiling technologies for identification of psychiatric disease and drug signatures.

The gene expression profiles of human postmortem parietal and prefrontal cortex samples of normal controls and patients with bipolar disease, or human neuroblastoma flat (NBFL) cells treated with the mood-stabilizing drug, valproate, were used to compare the performance of Affymetrix oligonucleotide U133A GeneChips and Agilent Human 1 cDNA microarrays. Among those genes represented on both platforms, the oligo array identified 26-53% more differentially expressed genes compared to the cDNA array in the three experiments, when identical fold change and t-test criteria were applied. The increased sensitivity was primarily the result of more robust fold changes measured by the oligonucleotide system. Essentially all gene changes overlapping between the two platforms were co-directional, and ranged from 4 to 19% depending upon the amount of biological variability within and between the comparison groups. Q-PCR validation rates were virtually identical for the two platforms, with 23-24% validation in the prefrontal cortex experiment, and 56% for both platforms in the cell culture experiment. Validated genes included dopa decarboxylase, dopamine beta-hydroxylase, and dihydropyrimidinase-related protein 3, which were decreased in NBFL cells exposed to valproate, and spinocerebellar ataxia 7, which was increased in bipolar disease. The modest overlap but similar validation rates show that each microarray system identifies a unique set of differentially expressed genes, and thus the greatest information is obtained from the use of both platforms.

Analysis of embryonic motoneuron gene regulation: derepression of general activators function in concert with enhancer factors.

The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. We have characterized how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. We found that non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9 located from -7096 to -6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9 expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord.

Electroconvulsive seizures regulate gene expression of distinct neurotrophic signaling pathways.

Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500-4400 RNA sequences detected in each sample, ECS increased by 1.5- to 11-fold or decreased by at least 34% the expression of 120 unique genes. The hippocampus produced more than three times the number of gene changes seen in the cortex, and many hippocampal gene changes persisted with chronic ECS, unlike in the cortex. Among the 120 genes, 77 have not been reported in previous studies of ECS or seizure responses, and 39 were confirmed among 59 studied by quantitative real time PCR. Another 19 genes, 10 previously unreported, changed by <1.5-fold but with very high significance. Multiple genes were identified within distinct pathways, including the BDNF-MAP kinase-cAMP-cAMP response element-binding protein pathway (15 genes), the arachidonic acid pathway (5 genes), and more than 10 genes in each of the immediate-early gene, neurogenesis, and exercise response gene groups. Neurogenesis, neurite outgrowth, and neuronal plasticity associated with BDNF, glutamate, and cAMP-protein kinase A signaling pathways may mediate the antidepressant effects of ECT in humans. These genes, and others that increase only with chronic ECS such as neuropeptide Y and thyrotropin-releasing hormone, may provide novel ways to select drugs for the treatment of depression and mimic the rapid effectiveness of ECT.

LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions.

LIM homeodomain codes regulate the development of many cell types, though it is poorly understood how these factors control gene expression in a cell-specific manner. Lhx3 is involved in the generation of two adjacent, but distinct, cell types for locomotion, motor neurons and V2 interneurons. Using in vivo function and protein interaction assays, we found that Lhx3 binds directly to the LIM cofactor NLI to trigger V2 interneuron differentiation. In motor neurons, however, Isl1 is available to compete for binding to NLI, displacing Lhx3 to a high-affinity binding site on the C-terminal region of Isl1 and thereby transforming Lhx3 from an interneuron-promoting factor to a motor neuron-promoting factor. This switching mechanism enables specific LIM complexes to form in each cell type and ensures that neuronal fates are tightly segregated.