In JoVE (1)

Other Publications (20)

Articles by Kai C Sonntag in JoVE

Other articles by Kai C Sonntag on PubMed

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

Stem Cells (Dayton, Ohio). 2002  |  Pubmed ID: 11897870

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 beta-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.

Genetic Engineering of Mouse Embryonic Stem Cells by Nurr1 Enhances Differentiation and Maturation into Dopaminergic Neurons

The European Journal of Neuroscience. Nov, 2002  |  Pubmed ID: 12453046

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-1alpha 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.

Specific MicroRNAs Modulate Embryonic Stem Cell-derived Neurogenesis

Stem Cells (Dayton, Ohio). Apr, 2006  |  Pubmed ID: 16357340

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.

Transient D1 Dopamine Receptor Expression on Prefrontal Cortex Projection Neurons: Relationship to Enhanced Motivational Salience of Drug Cues in Adolescence

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Mar, 2008  |  Pubmed ID: 18322084

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), D(1) dopamine receptors mediate motivational salience attribution, which plays a role in addiction. Here, we investigated the relationship of age-related D(1) 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 D(1) receptors during adolescence compared with younger and older ages. D(1) expression does not change on GABAergic interneurons across age. Adolescent differences in D(1) 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 D(1) antagonist SCH23390 into the PFC blocked adolescent place preferences, whereas microinjections of D(1) agonists dose-dependently increased preferences for cocaine-associated environments previously not preferred by juveniles. These results suggest that the heightened expression of D(1) receptors on cortical-accumbens projections may help explain increased sensitivity to environmental events and addictive behaviors during adolescence, whereas the paucity of D(1)-expressing projections may reduce risk in juveniles.

Juvenile Methylphenidate Modulates Reward-related Behaviors and Cerebral Blood Flow by Decreasing Cortical D3 Receptors

The European Journal of Neuroscience. Jun, 2008  |  Pubmed ID: 18588536

Attention deficit hyperactivity disorder is associated with reduced cortical blood flow that is reversible with exposure to the psychostimulant methylphenidate (MPH). D3 dopamine receptors modulate stimulant-induced changes in blood flow and are associated with reward processing during young adulthood, but their role in the enduring effects of MPH during development is unknown. Rats were given vehicle (VEH) or MPH (2 mg/kg between postnatal days 20-35) and assessed in young adulthood for regional cerebral blood volume (rCBV) after MPH challenge and mRNA expression levels of dopamine receptors. To probe D3 receptor involvement, juvenile subjects were exposed to VEH, MPH, the D3-preferring agonist +/-7-OHDPAT (0.3 mg/kg), the D3 antagonist nafadotride (Naf; 0.05, 0.5 or 5.0 mg/kg) or a Naf (0.05 mg/kg)/MPH combination, and assessed biochemically and behaviorally. Juvenile MPH exposure increased MPH-induced rCBV in the cingulate and medial prefrontal cortex and thalamus in adulthood. Behaviorally, juvenile MPH- or +/-7-OHDPAT-exposed subjects demonstrated an aversion to cocaine-associated environments, which was prevented by juvenile co-treatment with MPH and Naf, or with adult cortical microinjections of +/-7-OHDPAT. Cortical D3 mRNA levels significantly decreased by 23.8 +/- 6.7% in MPH-treated subjects and normalized with combined Naf/MPH treatment, with no change in the other dopamine receptors. Enhanced cortical responsiveness to psychostimulants may occur through a reduction in D3 receptors, which in turn reduces drug-seeking behavior. These data provide evidence for a postnatal sensitive period when juvenile MPH exposure is able to alter cortical development.

Parthenogenetic Dopamine Neurons from Primate Embryonic Stem Cells Restore Function in Experimental Parkinson's Disease

Brain : a Journal of Neurology. Aug, 2008  |  Pubmed ID: 18669499

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 Parkinson's 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.

Gene Expression Profiling of Substantia Nigra Dopamine Neurons: Further Insights into Parkinson's Disease Pathology

Brain : a Journal of Neurology. Jul, 2009  |  Pubmed ID: 19052140

Parkinson's disease is caused by a progressive loss of the midbrain dopamine (DA) neurons in the substantia nigra pars compacta. Although the main cause of Parkinson's disease remains unknown, there is increasing evidence that it is a complex disorder caused by a combination of genetic and environmental factors, which affect key signalling pathways in substantia nigra DA neurons. Insights into pathogenesis of Parkinson's disease stem from in vitro and in vivo models and from postmortem analyses. Recent technological developments have added a new dimension to this research by determining gene expression profiles using high throughput microarray assays. However, many of the studies reported to date were based on whole midbrain dissections, which included cells other than DA neurons. Here, we have used laser microdissection to isolate single DA neurons from the substantia nigra pars compacta of controls and subjects with idiopathic Parkinson's disease matched for age and postmortem interval followed by microarrays to analyse gene expression profiling. Our data confirm a dysregulation of several functional groups of genes involved in the Parkinson's disease pathogenesis. In particular, we found prominent down-regulation of members of the PARK gene family and dysregulation of multiple genes associated with programmed cell death and survival. In addition, genes for neurotransmitter and ion channel receptors were also deregulated, supporting the view that alterations in electrical activity might influence DA neuron function. Our data provide a 'molecular fingerprint identity' of late-stage Parkinson's disease DA neurons that will advance our understanding of the molecular pathology of this disease.

Evidence for Gender-specific Transcriptional Profiles of Nigral Dopamine Neurons in Parkinson Disease

PloS One. 2010  |  Pubmed ID: 20111594

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.

A Novel, Multiple Symptom Model of Obsessive-compulsive-like Behaviors in Animals

Biological Psychiatry. Oct, 2010  |  Pubmed ID: 20619828

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.

Neuronal Type-specific Gene Expression Profiling and Laser-capture Microdissection

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21761317

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD).

Converging MiRNA Functions in Diverse Brain Disorders: A Case for MiR-124 and MiR-126

Experimental Neurology. Dec, 2011  |  Pubmed ID: 22178324

A growing body of information on the biology of miRNAs has revealed new insight into their roles in normal homeostasis and pathology of disease. miRNAs control all steps of the cellular expression machinery acting through a "single miRNA/multiple targets" or "multiple miRNAs/single target" mechanism. They have profound impact on the regulation of signaling pathways, which govern common and specific functions across different cellular phenotypes. There is increasing evidence that various diseases share similar disturbances in gene expression networks. Since miRNAs have both common and varying effects in different cellular contexts, they might also influence overlapping signaling pathways in different organs and disease entities. Here, we review this concept for two miRNAs highly abundant in the brain, miR-124 and miR-126, and their potential role in diseases of the brain.

Detection of Intranasally Delivered Bone Marrow-derived Mesenchymal Stromal Cells in the Lesioned Mouse Brain: a Cautionary Report

Stem Cells International. 2011  |  Pubmed ID: 22190964

Bone marrow-derived mesenchymal stromal cells (MSCs) hold promise for autologous treatment of neuropathologies. Intranasal delivery is relatively noninvasive and has recently been reported to result in transport of MSCs to the brain. However, the ability of MSCs to migrate from nasal passages to sites of neuropathology and ultimately survive has not been fully examined. In this paper, we harvested MSCs from transgenic mice expressing enhanced green fluorescent protein (cells hereafter referred to as MSC-EGFP) and delivered them intranasally to wild-type mice sustaining mechanical lesions in the striatum. Using fluorescent, colorimetric, and ultrastructural detection methods, GFP-expressing cells were undetectable in the brain from 3 hours to 2 months after MSC delivery. However, bright autofluorescence that strongly resembled emission from GFP was observed in the olfactory bulb and striatum of lesioned control and MSC-EGFP-treated mice. In a control experiment, we directly implanted MSC-EGFPs into the mouse striatum and detected robust GFP expression 1 and 7 days after implantation. These findings suggest that-under our conditions-intranasally delivered MSC-EGFPs do not survive or migrate in the brain. Furthermore, our observations highlight the necessity of including appropriate controls when working with GFP as a cellular marker.

Part-human Animal Research: the Imperative to Move Beyond a Philosophical Debate

The American Journal of Bioethics : AJOB. 2012  |  Pubmed ID: 22881849

Viral Over-expression of D1 Dopamine Receptors in the Prefrontal Cortex Increase High-risk Behaviors in Adults: Comparison with Adolescents

Psychopharmacology. Apr, 2014  |  Pubmed ID: 24408208

Adolescents are often described as "lacking brakes" resulting in an increase in several behaviors associated with risk for addiction. Prefrontal cortex dopamine and cortico-limbic interaction play an important role in addiction, and we have previously shown that the dopamine D1 receptor is elevated on prelimbic prefrontal output neurons in adolescent rats. We hypothesized that a constellation of risk-related behaviors is mediated by prefrontal output neuron expression of D1.

Juvenile Methylphenidate Reduces Prefrontal Cortex Plasticity Via D3 Receptor and BDNF in Adulthood

Frontiers in Synaptic Neuroscience. 2014  |  Pubmed ID: 24478696

Early drug intervention in childhood disorders aims to maximize individual potential in the short- and long-term. Consistently, juvenile exposure to psychostimulants, such as methylphenidate (MPH), reduces risk for substance use in animals and sub-populations of individuals with attention deficit hyperactivity disorder (ADHD). We investigated the effects of MPH on brain plasticity via dopamine receptor D3 (D3R) and brain-derived neurotrophic factor (BDNF) expression in developing rats.

MiR-126 Contributes to Parkinson's Disease by Dysregulating the Insulin-like Growth Factor/phosphoinositide 3-kinase Signaling

Neurobiology of Aging. Jul, 2014  |  Pubmed ID: 24559646

Dopamine (DA) neurons in sporadic Parkinson's disease (PD) display dysregulated gene expression networks and signaling pathways that are implicated in PD pathogenesis. Micro (mi)RNAs are regulators of gene expression, which could be involved in neurodegenerative diseases. We determined the miRNA profiles in laser microdissected DA neurons from postmortem sporadic PD patients' brains and age-matched controls. DA neurons had a distinctive miRNA signature and a set of miRNAs was dysregulated in PD. Bioinformatics analysis provided evidence for correlations of miRNAs with signaling pathways relevant to PD, including an association of miR-126 with insulin/IGF-1/PI3K signaling. In DA neuronal cell systems, enhanced expression of miR-126 impaired IGF-1 signaling and increased vulnerability to the neurotoxin 6-OHDA by downregulating factors in IGF-1/PI3K signaling, including its targets p85β, IRS-1, and SPRED1. Blocking of miR-126 function increased IGF-1 trophism and neuroprotection to 6-OHDA. Our data imply that elevated levels of miR-126 may play a functional role in DA neurons and in PD pathogenesis by downregulating IGF-1/PI3K/AKT signaling and that its inhibition could be a mechanism of neuroprotection.

Molecular Profiles of Parvalbumin-immunoreactive Neurons in the Superior Temporal Cortex in Schizophrenia

Journal of Neurogenetics. Mar-Jun, 2014  |  Pubmed ID: 24628518

Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder.

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

Journal of Neurogenetics. Mar-Jun, 2014  |  Pubmed ID: 24702465

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 Brodmann's 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.

MiR-126 Regulates Growth Factor Activities and Vulnerability to Toxic Insult in Neurons

Molecular Neurobiology. Nov, 2014  |  Pubmed ID: 25407931

Dysfunction of growth factor (GF) activities contributes to the decline and death of neurons during aging and in neurodegenerative diseases. In addition, neurons become more resistant to GF signaling with age. Micro (mi)RNAs are posttranscriptional regulators of gene expression that may be crucial to age- and disease-related changes in GF functions. MiR-126 is involved in regulating insulin/IGF-1/phosphatidylinositol-3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling, and we recently demonstrated a functional role of miR-126 in dopamine neuronal cell survival in models of Parkinson's disease (PD)-associated toxicity. Here, we show that elevated levels of miR-126 increase neuronal vulnerability to ubiquitous toxicity mediated by staurosporine (STS) or Alzheimer's disease (AD)-associated amyloid beta 1-42 peptides (Aβ1-42). The neuroprotective factors IGF-1, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and soluble amyloid precursor protein α (sAPPα) could diminish but not abrogate the toxic effects of miR-126. In miR-126 overexpressing neurons derived from Tg6799 familial AD model mice, we observed an increase in Aβ1-42 toxicity, but surprisingly, both Aβ1-42 and miR-126 promoted neurite sprouting. Pathway analysis revealed that miR-126 overexpression downregulated elements in the GF/PI3K/AKT and ERK signaling cascades, including AKT, GSK-3β, ERK, their phosphorylation, and the miR-126 targets IRS-1 and PIK3R2. Finally, inhibition of miR-126 was neuroprotective against both STS and Aβ1-42 toxicity. Our data provide evidence for a novel mechanism of regulating GF/PI3K signaling in neurons by miR-126 and suggest that miR-126 may be an important mechanistic link between metabolic dysfunction and neurotoxicity in general, during aging, and in the pathogenesis of specific neurological disorders, including PD and AD.

Extinction and Reinstatement to Cocaine-associated Cues in Male and Female Juvenile Rats and the Role of D1 Dopamine Receptor

Neuropharmacology. Mar, 2015  |  Pubmed ID: 25749358

Extinction of behaviors in response to drug-associated cues and prevention of reinstatement are integral for addiction treatment, and can reverse or ameliorate the harmful consequences of drug use. The mechanisms controlling extinction and reinstatement involve prefrontal cortical dopamine receptors, which change in expression and activity during the juvenile and adolescent transitions until they mature in adulthood. Little is known about the role that PFC D1 dopamine receptors play in extinction of drug-paired associations early in life. We used extinction of place preferences for cocaine in juvenile male and female rats following genetic, cell-specific overexpression of D1 on glutamatergic cells in the PFC. All subjects needed to demonstrate cocaine preferences for inclusion in the extinction studies. Here, male juveniles with a preference to 10 mg/kg cocaine took longer to extinguish preferences compared to both male adults and female juveniles. Female juveniles extinguished more rapidly than male juveniles at 20 mg/kg cocaine. Overexpression of D1 in juvenile males significantly facilitated extinction relative to juvenile male controls, whereas D1 prolonged expression of extinction in adults overexpressing D1 and adolescents who naturally have elevated D1 expression. These data suggest that an immature D1 profile in juveniles prevented the learning of new associations, and D1 overexpression may provide sufficient activity to facilitate extinction learning. D1 overexpression reduced reinstatement to a priming dose of cocaine in juvenile males. Together, these data show D1 expression may re-program motivational circuitry to facilitate extinction learning during juvenility that is normally unavailable to juveniles and that sex differences exist.

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