Kai C. Sonntag

Specific MicroRNAs Modulate Embryonic Stem Cell–Derived Neurogenesis

MicroRNAs (miRNAs) are recently discovered small non‐coding transcripts with a broad spectrum of functions described mostly in invertebrates. As post‐transcriptional regulators of gene expression, miRNAs trigger target mRNA degradation or translational repression. Although hundreds of miRNAs have been cloned from a variety of mammalian tissues and cells and multiple mRNA targets have been predicted, little is known about their functions. So far, a role of miRNA has only been described in hematopoietic, adipocytic, and muscle differentiation; regulation of insulin secretion; and potentially regulation of cancer growth. Here, we describe miRNA expression profiling in mouse embryonic stem (ES) cell– derived neurogenesis in vitro and show that a number of miRNAs are simultaneously co‐induced during differentiation of neural progenitor cells to neurons and astrocytes. There was a clear correlation between miRNA expression profiles in ES cell– derived neurogenesis in vitro and in embryonal neurogenesis in vivo. Using both gain‐of‐function and loss‐of‐function approaches, we demonstrate that brain‐specific miR‐124a and miR‐9 molecules affect neural lineage differentiation in the ES cell– derived cultures. In addition, we provide evidence that signal transducer and activator of transcription (STAT) 3, a member of the STAT family pathway, is involved in the function of these miRNAs. We conclude that distinct miRNAs play a functional role in the determination of neural fates in ES cell differentiation.

Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons

Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor‐1α promoter. The Nurr1‐expression in ES cells lead to up‐regulation of all DA neuronal markers tested, resulting in about a 4‐ to 5‐fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4‐fold increase in the number of DA neurons in Nurr1‐expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic l‐amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.

Transient D1 dopamine receptor expression on prefrontal cortex projection neurons: relationship to enhanced motivational salience of drug cues in adolescence.

Adolescence is a transitional period during development that is associated with a greater likelihood of addiction to drugs than any other age. In the prefrontal cortex (PFC), D1 dopamine receptors mediate motivational salience attribution, which plays a role in addiction. Here, we investigated the relationship of age-related D1 dopamine receptor expression in the PFC with the maturation of cocaine place conditioning. Confocal microscopy revealed that retrogradely traced cortical output neurons to the nucleus accumbens express higher levels of D1 receptors during adolescence compared with younger and older ages. D1 expression does not change on GABAergic interneurons across age. Adolescent differences in D1 expression occur independently of cortical-accumbens connectivity, which proliferates through adulthood. Behaviorally, adolescent rats are more sensitive to cocaine place conditioning than younger and older rats. However, microinjections of the D1 antagonist SCH23390 into the PFC blocked adolescent place preferences, whereas microinjections of D1 agonists dose-dependently increased preferences for cocaine-associated environments previously not preferred by juveniles. These results suggest that the heightened expression of D1 receptors on cortical-accumbens projections may help explain increased sensitivity to environmental events and addictive behaviors during adolescence, whereas the paucity of D1-expressing projections may reduce risk in juveniles.

Analysis of Different Promoter Systems for Efficient Transgene Expression in Mouse Embryonic Stem Cell Lines

Mouse embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the developmental capacity to generate all cell types of the body. Combined with efficient genetic manipulation and in vitro differentiation procedures, ES cells are a useful system for the molecular analysis of developmental pathways. We analyzed and compared the transcriptional activities of a cellular polypeptide chain elongation factor 1 alpha (EF), a cellular‐virus hybrid (cytomegalo‐virus [CMV] immediate early enhancer fused to chicken β‐actin [CBA]), and a viral CMV promoter system in two ES cell lines. When transiently transfected, the EF and CBA promoters robustly drove reporter gene expression, while the CMV promoter was inactive. We also demonstrated that the EF and CBA promoters effectively drove gene expression in different stages of cell development: naïve ES cells, embryoid bodies (EBs), and neuronal precursor cells. In contrast, the CMV promoter did not have transcriptional activity in either ES cells or EB but had significant activity once ES cells differentiated into neuronal precursors. Our data show that individual promoters have different abilities to express reporter gene expression in the ES and other cell types tested.

Parthenogenetic dopamine neurons from primate embryonic stem cells restore function in experimental Parkinson's disease.

The identity and functional potential of dopamine neurons derived in vitro from embryonic stem cells are critical for the development of a stem cell-based replacement therapy for Parkinsons disease. Using a parthenogenetic primate embryonic stem cell line, we have generated dopamine neurons that display persistent expression of midbrain regional and cell-specific transcription factors, which establish their proper identity and allow for their survival. We show here that transplantation of parthenogenetic dopamine neurons restores motor function in hemi-parkinsonian, 6-hydroxy-dopamine-lesioned rats. Exposure to Wnt5a and fibroblast growth factors (FGF) 20 and 2 at the final stage of in vitro differentiation enhanced the survival of dopamine neurons and, correspondingly, the extent of motor recovery of transplanted animals. Importantly for future development of clinical applications, dopamine neurons were post-mitotic at the time of transplantation and there was no tumour formation. These data provide proof for the concept that parthenogenetic stem cells are a suitable source of functional neurons for therapeutic applications.

Tolerance to solid organ transplants through transfer of MHC class II genes

Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tgs) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tgs, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts.

Evidence for gender-specific transcriptional profiles of nigral dopamine neurons in Parkinson disease.

Background Epidemiological data suggest that the male gender is one of the risks factors for the development of Parkinson Disease (PD). Also, differences in the clinical manifestation and the course of PD have been observed between males and females. However, little is known about the molecular aspects underlying gender-specificity in PD. To address this issue, we determined the gene expression profiles of male and female dopamine (DA) neurons in sporadic PD. Methodology/Principal Findings We analyzed Affymetrix-based microarrays on laser microdissected DA neurons from postmortem brains of sporadic PD patients and age-matched controls across genders. Pathway enrichment demonstrated that major cellular pathways involved in PD pathogenesis showed different patterns of deregulation between males and females with more prominent downregulation of genes related to oxidative phosporylation, apoptosis, synaptic transmission and transmission of nerve impulse in the male population. In addition, we found upregulation of gene products for metabolic processes and mitochondrial energy consumption in the age-matched male control neurons. On the single cell level, selected data validation using quantitative Real-Time (qRT)-PCR was consistent with microarray raw data and supported some of the observations from data analysis. Conclusions/Significance On the molecular level, our results provide evidence that the expression profiles of aged normal and PD midbrain DA neurons are gender-specific. The observed differences in the expression profiles suggest a disease bias of the male gender, which could be in concordance with clinical observations that the male gender represents a risk factor for sporadic PD. Validation of gene expression by qRT-PCR supported the microarray results, but also pointed to several caveats involved in data interpretation.

A Novel, Multiple Symptom Model of Obsessive-Compulsive-Like Behaviors in Animals

BACKGROUND Current animal models of obsessive-compulsive disorder (OCD) typically involve acute, drug-induced symptom provocation or a genetic association with stereotypies or anxiety. None of these current models demonstrate multiple OCD-like behaviors. METHODS Neonatal rats were treated with the tricyclic antidepressant clomipramine or vehicle between days 9 and 16 twice daily and behaviorally tested in adulthood. RESULTS Clomipramine exposure in immature rats produced significant behavioral and biochemical changes that include enhanced anxiety (elevated plus maze and marble burying), behavioral inflexibility (perseveration in the spontaneous alternation task and impaired reversal learning), working memory impairment (e.g., win-shift paradigm), hoarding, and corticostriatal dysfunction. Dopamine D2 receptors were elevated in the striatum, whereas serotonin 2C, but not serotonin 1A, receptors were elevated in the orbital frontal cortex. CONCLUSIONS This is the first demonstration of multiple symptoms consistent with an OCD-like profile in animals. Moreover, these behaviors are accompanied by biochemical changes in brain regions previously identified as relevant to OCD. This novel model of OCD demonstrates that drug exposure during a sensitive period can program disease-like systems permanently, which could have implications for current and future therapeutic strategies for this and other psychiatric disorders.

Evidence against dopamine D1/D2 receptor heteromers

Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation, because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer, ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout (KO) mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq KO mice, as well as in knock-in mice expressing a mutant Ala286-CaMKIIα that cannot autophosphorylate to become active. Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

Molecular Profiles of Pyramidal Neurons in the Superior Temporal Cortex in Schizophrenia

Abstract Disrupted synchronized oscillatory firing of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30–100 Hz) mediates many of the cognitive deficits and symptoms of schizophrenia. In fact, the density of dendritic spines and the average somal area of pyramidal neurons in layer 3 of the cerebral cortex, which mediate both long-range (associational) and local (intrinsic) corticocortical connections, are decreased in subjects with this illness. To explore the molecular pathophysiology of pyramidal neuronal dysfunction, we extracted ribonucleic acid (RNA) from laser-captured pyramidal neurons from layer 3 of Brodmanns area 42 of the superior temporal gyrus (STG) from postmortem brains from schizophrenia and normal control subjects. We then profiled the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identified 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong to the transforming growth factor beta (TGF-β) and the bone morphogenetic proteins (BMPs) signaling pathways. Disturbances of these signaling mechanisms may in part contribute to the altered expression of other genes found to be differentially expressed in this study, such as those that regulate extracellular matrix (ECM), apoptosis, and cytoskeletal and synaptic plasticity. In addition, we identified 10 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of their predicted gene targets revealed signaling pathways and gene networks that were found by microarray to be dysregulated, raising an interesting possibility that dysfunction of pyramidal neurons in schizophrenia may in part be mediated by a concerted dysregulation of gene network functions as a result of the altered expression of a relatively small number of miRNAs. Taken together, findings of this study provide a neurobiological framework within which specific hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.